The study of optimization of prescription is an important link in the research of Chinese medicine prescription and a key step in the industrialization and promotion of Chinese medicine. This article introduces the concept of formulating traditional Chinese medicine, the principle, purpose, significance and guiding ideology of formulating formulas, and summarizes the methods used in formulating formulas in recent years are mainly divided into two categories, compared the experimental designs based on modern mathematical models (orthogonal design, homogeneous design, baseline proportional design, Plackett-Burman design, central composite design, Box-Behnken, Doehlert design), and analyzed the advantages and disadvantages in experimental arrangements, data analysis and fitting models, the scope of application and practicability in the field of formulation optimization, and put forward the prospect of future method design.

Capsular polysaccharide (CPS) is a carbohydrate that is widely present on the surface of bacteria, mycoplasma, and some fungi. At the same time, CPS can help the bacteria resist the adverse environment such as dry and low temperature, and block the killing of host complement and phagocytosis by forming a physical barrier on the surface of bacteria. Under long-term multiple stress environments, pathogens have evolved a variety of immune escape mechanisms and promote host infection; among non-pathogenic microorganisms, CPS can positively regulate host immune function, and antagonize immune factors, to protect the host from inflammatory diseases caused by pathogens. This article will combine the relevant research works of our team to review the structure, synthesis regulatory mechanisms, biological function, immune evasion mechanism and pathogenic mechanism of CPS, especially the positive regulation of the host immune system by CPS and its application potential, to provide references for the research on pathogenic mechanisms of pathogenic bacteria and effective prevention and control of epidemic diseases.

The microbial communities of humans and animals are mainly distributed in mucosal organs. Recent studies have shown that bacteriophages can adhere to mucosal surfaces and provide an antimicrobial defense. In the meantime, bacteriophages translocate across the mucosa into the body and migrate with the blood to other tissues and organs, giving rise to the body's immune response and affecting systemic immunity. However, these endogenous bacteriophages can be eliminated through phagocytosis or specific antibodies. At present, there is still a lack of research on phage translocation and interaction with the immune system after entering the body. Here we review recent researches on bacteriophages adhering to the mucosa and translocating into the circulatory system, as well as the effect of endogenous bacteriophages on systemic immunity. This review may provide a reference for more extensive and in-depth studies in the future.

The study aimed to identify the polymorphism of RXRB gene, screen SNPs significantly associated with growth, fattening and reproductive traits, and provide novel genetic markers for the genetic improvement of pigs. In this study, the blood genomic DNA of 962 healthy Large White pigs (459 American Large White pigs and 503 French Large White pigs) were sampled. The SNPs of RXRB gene were detected by direct sequencing, and PCR-RFLP methods and used to perform the genetic polymorphism analysis. The mixed linear model (Mixed) in SAS 8.0 software was used to analyze the association between the SNPs of RXRB gene and phenotypic traits. The Haploview 4.2 software was used to analyze the linkage disequilibrium of the SNPs. The results showed that a total of 10 SNPs were detected in the two strains of Large White pigs, of which there were 5 SNPs in the American Large White pigs and 5 SNPs in the French Large White pigs. According to the result of χ² test, the 10 SNPs were all under the Hardy-Weinberg equilibrium status (P>0.05). The results of association analysis showed that rs340542491, rs330162688 and rs326226767 were significantly associated with days at 100 kg body weight (P<0.05). The rs340542491, rs330162688, rs336609453 and rs332169586 were significantly associated with backfat thickness (P<0.05). The rs340542491, rs325538588, rs691889709 and rs323107853 were significantly associated with birth weight (P<0.05). For body length, only the individuals with genotype GA at rs324141460 were significantly larger than those with GG genotype (P<0.05). The rs325538588 and rs80789331 were significantly associated with the loin muscle area (P<0.05). The rs340542491 and rs330162688 were extremely significantly associated with number of left nipples (P<0.01). The rs324141460 was extremely significantly associated with number of left and right nipples (P<0.01). The rs336609453 and rs332169586 were significantly associated with the number of right nipples (P<0.05). The results of linkage disequilibrium analysis showed that rs326226767-rs325538588 was in a strong linkage disequilibrium status (D'=0.96, r²=0.91>0.33), rs330162688-rs324141460 was in a fully linkage status (D'=1), and rs324141460-rs340542491 was in a fully linked status (D'=1) in American Large White pigs. The results of association analysis of haplotype combinations were consistent with the results of the association analysis of single SNP; they were extremely significant (P<0.01) or significant (P<0.05) associated with days at 100 kg body weight, backfat thickness, birth weight and the number of nipples. In French Large White pigs, rs691889709-rs323107853 was in a fully linkage status (D'=1, r²=1). The associations between haplotype combinations and the body length, number of nipples reached extremely significant (P<0.01) or significant (P<0.05) levels. The results suggest that the 10 SNPs of RXRB gene can be used as molecular markers for pig breeding improvement, and provide the basis for the in-depth study of the function of RXRB.

The study aimed to explore the relationship between diarrhea of weaned piglets of hybrid combination and genetic variation of α-1,2-fucosyltransferase 1 (FUT1) gene. In this experiment, 35-day-old Dudian, Duzang and Duroc weaned piglets were selected as the research objects, and were divided into 3 groups according to breeds, with 113, 165 and 87 replicates, respectively. FUT1 gene polymorphism was detected by sequencing to analyze the correlation between gene polymorphism and diarrhea rate of weaned piglets. The results showed that there were 4 polymorphic sites in FUT1 gene, including T18C, C229T, C2418A and A306G, all of which were in moderate polymorphism and heterozygosity. C229T, C2418A and A306G were missense mutations, which might be key sites affecting porcine diarrhea. The degree of diarrhea was relatively serious in the hybrid combination Dudian, Duzang pig with TT genotype at T18C, and 3 pig populations with CC genotype at C229T, CA genotype at C2418A and GG genotype at A306G, which showed dominant inheritance. The result suggest that the effects of breeds and FUT1 gene polymorphisms on weaned piglets diarrhea have certain differences and correlations.

This study aimed to clone the full-length cDNA of the porcine ADAR2 gene and explore its expression pattern in different tissues of pigs. Rapid-amplification of cDNA ends (RACE) was used to clone the full-length cDNA sequence of the ADAR2 gene in Large White pigs and the sequence was analyzed by bioinformatics. Real-time PCR was used to detect the ADAR2 mRNA expression in the heart, liver, lung, kidney, spleen, brain, small intestine, muscle and backfat of 35-day-old pigs. Porcine ADAR2 cDNA sequence of 6 305 bp was cloned, which contained 12 exons and encoded 704 amino acids. The nucleic acid and amino acid sequences shared a high identity (>84%) with that of other mammals including human, chimpanzee, macaque, gibbon, cow, goat and sheep. The deduced ADAR2 had two double-stranded RNA binding motifs and an adenosine deaminase domain. Real-time PCR results showed that the ADAR2 expressed in all the detected tissues, and had the highest expression in the lung. The full-length cDNA sequence of porcine ADAR2 gene was successfully cloned, and it was widely expressed in tissues of pigs. These findings provide a solid foundation for further function study of ADAR2.

The study aimed to explore the function of Apob gene in the lipid metabolism of chicken liver. The physical and chemical properties of chicken Apob protein were analyzed; RT-qPCR was used to detect the expression of Apob gene in the tissues of 4-week-old yellow broiler chickens, 3 replicates were set up in each group and 3 parallel experiments were carried out. Three pairs of sgRNA were designed according to the sequence of key domains of chicken Apob protein to construct Cas/gRNA vectors; T7 endonuclease I (T7EI) digestion method and TA cloning sequencing method were used to screen knockout active sites and calculate gene knockout efficiency in cells after the Cas/gRNA vector was transfected into DF-1 cells; The mRNA expression of Apob gene was detected in subcloned cells using RT-qPCR after gene knockout. The results showed that the relative molecular mass of chicken Apob was 523.356 ku and the average hydrophilicity was -0.300, which was a stable protein. And the Apob gene mainly expressed in chicken liver, kidney and small intestine tissues. T7EI digestion result showed that Cas/gRNA vectors could knock out the gene effectively. TA clone sequencing results showed that knockout efficiency of 3 active sites(Cas/gRNA6, Cas/gRNA7 and Cas/gRNA8) were 33.3%, 65% and 80%, respectively. At the same time, RT-qPCR results showed that the expression level of Apob genes in transfected Cas9/gRNA7, Cas9/gRNA6, Cas9/gRNA8 cells were down-regulated by about 99.96% (P<0.01), 85%(P<0.01),47%(P<0.05), respectively. In summary, we revealed the expression characteristics of Apob gene in chicken tissues and the physicochemical properties of the protein; Then we contructed Cas/gRNA knockout vectors, screened the optimal knock sites, and established Apob knockout subclonal cells successfully, which laid a foundation for exploring the function of Apob gene in chicken liver.

To investigate the effects of sheep Musclin on glucose metabolism and insulin function, the structure properties of sheep Musclin protein were predicted by using various bioinformatics softwares and the eukaryotic expression vector of Musclin gene was constructed. The sheep fetal myoblasts were divided into 4 groups and treated as follows:mock-vector group (pcDNA3.1), Musclin-overexpressed group (pcDNA3.1-Musclin), mock-vector + insulin group (pcDNA3.1 + Insulin) and Musclin-overexpressed + insulin group (pcDNA3.1-Musclin + Insulin). Then the cells were collected at 48 and 72 h after differentiation induction, and the effects of Musclin on glucose metabolism in normal or insulin-treated differentiating myoblasts were analyzed and the changes of the related gene expression were detected by real-time PCR. The restriction endonuclease digestion and sequencing results indicated that the expression vector (pcDNA3.1-Musclin) was successfully constructed. The results of bioinformatics analysis showed that the sheep Musclin protein contained a signal peptide and had a restriction site at the 28th amino acid. The subcellular localization showed that it was in the extracellular space, and belonged to the secretory protein. Its secondary and tertiary structure mainly included alpha helixes and random coils. The cellular assays showed that the overexpression of sheep Musclin gene significantly reduced the glycogen content in the normal or insulin-treated sheep differentiating myoblasts and increased the glucose content in medium. The overexpression of Musclin inhibited the expressions of IRS1, AKT1, AKT2, GLUT1, GLUT4 mRNA, and promoted the expression of GSK3β mRNA in the Musclin-overexpressed group and Musclin-overexpressed + insulin group sheep myoblasts induced differentiation for 72 h, while only the expression of GLUT4 mRNA was inhibited after cells were induced differentiation for 48 h. In conclusion, the overexpression of the sheep Musclin gene affected the glucose metabolism and attenuated the functions of insulin in sheep differentiating myoblasts, and they coordinated with each other to maintain the homeostasis of glucose metabolism. The functional mechanisms of sheep Musclin were involved in the expression changes of the above genes and were related to the differentiation state of sheep myoblasts.

The study aimed to analyze the genetic structure of parental lines of Chinese Simmental beef cattle and Chinese local cattle by the 770K SNP markers covering the whole genome of cattle and trait-related SNP markers, and predict heterosis of different hybrid combinations in growth, carcass and meat quality traits by genetic distance (GD) of parental lines. One thousand two hundred and twenty-two Chinese Simmental beef cattle in Ulgai of Xilin Gol League, Inner Mongolia and 8 Chinese local cattle breeds including 190 were selected to form 8 hybrid combinations, and the genetic structure of parental lines were analyzed. Parent populations were genotyped using Illumina BovineHD SNPs array(770K). The QTLs corresponding to the target traits were screened by cattle QTL database and the SNP loci of the whole genome were mapped to obtain trait-related SNP markers. The state homology matrix was constructed by using SNP markers, and the genetic distance between the parent lines of each hybrid combination was calculated. All parent lines were clustered into 3 groups, Chinese Simmental beef cattle was clustered into one category, northern cattle (Menggu cattle, Xizang cattle and Chaidamu cattle) and southern cattle (Zhaotong cattle, Pingwu cattle, Nandan cattle, Wenshan cattle and Liangshan cattle) were clustered into one category, respectively. Chinese Simmental beef cattle and northern breeds of Chinese local cattle were relatively closer on the PCA map, indicating that the genetic relationship between them was relatively closer, and the difference in the genetic background was smaller. Among all the traits, the parent combinations with the largest genetic distance were Chinese Simmental beef cattle and Nandan cattle. The genetic distance between Chinese Simmental beef cattle and Zhaotong cattle was the smallest for marbling score, and the smallest genetic distance of other traits was all between Chinese Simmental beef cattle and Pingwu cattle. In summary, the possible combination with better heterosis in all traits was between Chinese Simmental beef cattle and Nandan cattle.

This study aimed to use resequencing technology to analyze the differences in Shitou goose gene Indel and to explore its egg production regulatory pathway. Five adult Shitou geese and five Jilin white geese with different egg production were selected, and the whole genome resequencing method was used to compare and analyze the indel mutations at the genome level of the two breeds. The results showed that 350.35 Gb data was obtained by sequencing, among which the average values of Q20 and Q30 reached 96.74% and 92.19%, respectively. Annotation analysis found 748 821.2 Indel mutation sites. The distribution trend and the number of Indel loci in the coding region (CDS) of 10 experimental geese were similar to those of the reference genome. Among them, 11 737 Indel loci resulted in insertion or deletion. On the basis, egg-laying related pathways were further analyzed and screened, and 15, 1, 1, 1, and 18 variant genes in the GnRH, estrogen, oxytocin, prolactin signaling pathways and progesterone-mediated oocyte maturation pathway, respectively involved in the regulation of goose egg-laying were found. The results suggest that the GnRH, estrogen, oxytocin, prolactin signaling pathways and progesterone-mediated oocyte maturation pathway are all related to Shitou goose egg-laying, and egg-laying related genes were further screened. These data will provide an effective basis for studying the reproductive performance of Shitou geese.

The purpose of the study was to establish methods for isolation, culture, purification, differentiation and identification of chicken skeletal muscle satellite cells (MSC) in vitro. Selecting SPF chicken embryos of 12 embryonic age, integrating the principles of loose muscle fiber and free fiber matrix layer cells, the enzymes used to separate cells, cell purification and differentiation induction methods were optimized, and the MSC was identified from the aspects of morphology, cell differentiation ability, growth curve, immunofluorescence detection, RT-PCR identification, etc. Therefore, a method of digesting, separating and culturing chicken skeletal muscle satellite cells with mixed enzymes was proposed. The results showed that the separated and purified cells had strong refractive index and adhered to the wall after 24 hours, showing a spindle shape; after induced differentiation, it could form neatly arranged multinucleated myotubes; the cell growth curve measured by using CCK-8 method showed a typical "S" shape; after the optimization, the cell survival rate was (90.82±1.294)%, the cell purity was (90.44±1.264)%; immunofluorescence detection of the marker genes Pax7 and Desmin were positive; RT-PCR detection of the marker genes Pax7, MyHC, MyoD1 were positive; and the expression of the marker gene Pax7 before differentiation was 1.705 times that after differentiation, and the expression of the marker gene MyHC after differentiation was 13.073 times that before differentiation. This study established a quick and simple method for culturing chicken skeletal muscle satellite cells in vitro, which provides a good cell model for studying the biological mechanism of chicken skeletal muscle cells, optimization of broiler breeds, and cell transplantation repair.

The purpose of this study was to clone the full-length cDNA sequence of deleted in azoospermia like (DAZL) from Banna mini-pig inbred line (BMI), obtain its multi-tissue mRNA expression patterns, characterize the protein structure, and acquire the subcellular localization of DAZL. The tissue samples were collected from 10-month-old adult BMI boar and the full-length cDNA of DAZL gene was cloned from BMI using RACE and RT-PCR technologies. The multi-tissue mRNA transcription levels of DAZL were analyzed using qPCR. The structural characteristics and conserved domains of DAZL protein were analyzed using online tools. The subcellular localization of DAZL protein in the ST cell was detected by in vitro cell transfection technology. The results showed that the full-length cDNA sequence of DAZL was 2 985 bp (KU705632) with an 888 bp CDS encoding 295 amino acids (AOC89050). The BMI DAZL gene was located on chromosome 13 of BMI genome and contained 11 exons. Multi-tissue qPCR results showed that DAZL mRNA was exclusively and highly expressed in the testis. Further bioinformatics analysis showed that DAZL protein contained two conserved domains, RMP and DAZ, that were homologous with other mammals, with random coil structures more than 50%. Evolutionary analysis showed that the amino acid sequence of BMI DAZL was highly conserved and shared the closest genetic relationship with Bovidae. Fluorescence co-localization results of pEGFP-C1-DAZL transfected ST cells showed that DAZL protein was localized in the nucleus. The sequence characterization, mRNA expression, protein structure and location of BMI DAZL gene were clarified at DNA, mRNA and protein levels, respectively. This study will lay the foundation for further functional studies of DAZL during spermatogenesis in BMI.

The study aimed to analyze the proteomics of chicken seminal plasma and screen the differential proteins related to asthenospermia. The experimental roosters were selected from Ningdu yellow chicken hatched in the same batch, and semen quality was examined every 3 days,3 times in total. Four individuals with forward sperm ratio <20% and straight forward sperm ratio <10% were selected as asthenospermia group. Four individuals with forward sperm ratio >80% and straight forward sperm ratio >40% were selected as control group. Using liquid chromatography-mass spectrometry (LC-MS/MS) technology, the proteomics of chicken seminal plasma was studied and differential proteins related to asthenospermia were preliminary screened. The results showed that the sperm motility and density of roosters in asthenospermia group were extremely significantly lower than those in control group(P<0.01). A total of 78 differentially expressed proteins were screened out between the two groups. Compared with control group, 5 differential proteins were up-regulated and the others were down-regulated in the asthenospermia group. It is important to note that the expression of keratin 9 (KRT9) and selenoprotein P-2 (SEPP2) were only detected in asthenospermia group, and the expression of 4-hydroxy-2-oxoglutaracturonase (HOGA1), phenylalanine hydroxylase (PAH) and lecithin cholesterol acyltransferase (LCAT) were only detected in the control group. According to GO and KEGG analysis, the functions of differential proteins were mainly focused on metabolism, glycolysis, carbon metabolism, amino acid synthesis pathways, and so on. The differentially expressed proteins in seminal plasma of Ningdu yellow chickens in asthenospermia and control groups screened in this study could be used as candidate targets for screening and treatment of asthenospermia in chickens. The further functional analysis and verification will provide a basis for elucidating the pathogenesis of asthenospermia.

This study aimed to explore the effect of 17β-hydroxysteroid dehydrogenase 12 (HSD17B12) in Hu sheep pituitary on the secretion of pituitary hormones. Three healthy Hu sheep rams at sexually mature stage (9-month-old) with similar weight (about 40 kg) were selected. And the pituitary tissues were collected for HSD17B12 gene CDS region amplification and protein homology analysis to determine the CDS region sequence. The location of HSD17B12 in sexually mature male Hu sheep pituitary was detected by immunohistochemistry. In addition, pituitary cells were isolated in vitro, and after down-regulated the expression of HSD17B12 using RNA interference and cell transfection, the expression levels of hormone-related genes were measured by qPCR. Moreover, the changes of cell proliferation, apoptosis and cycle were analyzed by flow cytometry. The results showed that the CDS region of HSD17B12 gene was 939 bp, and it was highly conserved among species. And the immunohistochemistry analysis indicated that HSD17B12 was positively expressed in most cells of pituitary tissue. After RNA interference of HSD17B12 in vitro, a significant decrease in cell proliferation efficiency (P<0.05), a significant increase in cell apoptosis rate (P<0.05), and significant change in cell cycle (P<0.05) were detected. In addition, the expression levels of gonadotropin synthesis-related genes FSHβ, LHβ and growth hormone gene GH were significantly reduced (P<0.05). The results suggested that interfering the expression of HSD17B12 in Hu sheep pituitary cells could significantly reduce the secretion of gonadotropins and growth hormone by affecting cell proliferation and apoptosis. This study preliminarily proved the important role of HSD17B12 in Hu sheep pituitary, and provided a basis for further exploration of its mechanism.

This study aimed to investigate the effect of inflammatory factor (interleukin-10, IL-10) on the expression of genes related to volatile fatty acid (VFA) absorption in bovine rumen epithelial cells. The experiment adopted a randomized block design. Under the conditions of in vitro culture of rumen epithelial cells, the effect of 50 ng·mL^-1 IL-10 on genes expression of nuclear factor (NF-κB), putative anion transporter 1 (PAT1), anion exchanger 2(AE2), monocarboxylate transporter 1(MCT1), monocarboxylate transporter 4 (MCT4), Na^-/H⁺exchanger 1 (NHE1) in bovine rumen epithelial cells was detected. The results showed that, under neutral conditions, IL-10 addition significantly down-regulated the expression of NF-κB, PAT1, MCT1, NHE1 genes (P<0.05), and significantly up-regulated the expression of AE2 gene (P<0.05), while had no significant effect on the gene expression of MCT4. Under acidic conditions, the expression of NF-κB gene in the IL-10 addition group was significantly inhibited (P<0.05), while the expression of PAT1, MCT1, and MCT4 genes were significantly promoted (P<0.05). Compared with the condition of pH7.2 group, the NF-κB gene expression in the pH5.5 group was significantly increased (P<0.05). The results suggest that, under inflammatory conditions, interleukin-10 as an anti-inflammatory factor can alleviate the inflammatory response of rumen epithelial cells and promote the genes expression of VFA absorption-related protein.

The objective of this study was to investigate the developmental changes of ileal microbiota and fatty acid binding proteins and its correlation with fat deposition in Jinhua pigs. Four healthy Jinhua pigs at 45(D45), 90(D90), 150(D150)and 270 (D270) days of age were selected, respectively to measure their body weight and backfat thickness. The ileal mucosa and contents were collected. The expression of fatty acid binding protein genes in the ileal mucosa was measured by fluorescent quantitative PCR, and ileal microbiota were analyzed using high-throughput sequencing based on 16S rRNA gene. The results showed:1) The weight and backfat thickness of Jinhua pigs increased significantly with age increasing (P<0.05); 2) The expression of fatty acid binding protein genes FABP1, FABP2 and FABP4 in the ileal mucosa showed significant increase with age increasing (P<0.05); 3) Firmicutes and Bacteroidetes were the predominant phyla in the ileum of Jinhua pigs. At the genus level, Clostridium sensu stricto 1, Lactobacillus,Terrisporobacter, Romboutsia and Streptococcus were the dominant bacteria; With age increasing, the relative abunance of Clostridium sensu stricto 1 and Streptococcus increased at first and then decreased while Lactobacillus, Terrisporobacter and Romboutsia decreased at first and then increased. PCoA analysis showed that there were significantly different clusters at each age. 4) Spearman correlation analysis showed that the correlation coefficients of the expression levels of fatty acid binding proteins FABP1, FABP2 and FABP4 with backfat thickness were 0.480 1, 0.597 9 and 0.795 3, respectively; The correlation coefficients of Epulopiscium and Mycoplasma with fatty acid binding proteins FABP1, FABP2 and FABP4 in the ileum and backfat thickness of Jinhua pigs were 0.38-0.76 and 0.45-0.64, respectively. It is concluded that the developmental changes were observed for the ileal microbiota and the expression of fatty acid binding protein, which were asscociated with fat deposition in Jinhua pigs.

This study aimed to explore the nuclease activity of extracellular products from Salmonella Choleraesuis and its effect on the formation of macrophages extracellular traps (METs). Agar culture assay, agarose gel electrophoresis, and agar diffusion method were used to investigate the nuclease activity of extracellular products of S. Choleraesuis. Effects of temperature, pH, and metal ions on the nuclease activity of extracellular products were explored by agarose gel electrophoresis. The effect of the extracellular products on METs induced by Candida albicans was measured. The results showed that λDNA was degraded by the extracellular products from S. Choleraesuis. The nuclease activity was inhibited under a high temperature and acidic conditions. The optimum temperature and pH of the nuclease activity were 42℃ and pH 9.0, respectively. The nuclease of the extracellular products was inactivated under a high temperature of 60℃ or higher. Na⁺, K⁺, Ca²⁺, and Ni²⁺ did not affect the nuclease activity. The high ionic strength of Ba²⁺, Mg²⁺, and Mn²⁺ with 5.00 and 10.00 mmol·L^-1 enhanced the nuclease activity of extracellular products. The activity of cleave λDNA promoted with the concentrations of 0.01-1.00 mmol·L^-1 of Zn²⁺, Cu²⁺, and Fe³⁺; conversely, low concentrations of 0.01-1.00 mmol·L^-1 of Co²⁺ inhibited the nuclease activity. Fluorescence microscope and quantification analyses revealed that the METs induced by Candida albicans were degraded by the extracellular products from S. Choleraesuis. This study confirmed that the extracellular products from S. Choleraesuis have nuclease activity, which provides a reference for further study of the role of extracellular products in the interaction between S. Choleraesuis infection and the host immune.

T6SS (type VI secretion system) is a common secretion system in Gram-negative bacteria. And its mechanism of effector protein Hcp2b is still unclear. The Hcp2b protein of avian pathogenic Escherichia coli (APEC) is the main subject of this study, which aims to explore the role and mechanism of Hcp2b protein in the process of APEC infection of chicken tracheal mucosa. The Red recombination method was used to construct the hcp2b-deleted strain by using the plasmid pKD3 as the template. And the pSTV-28 plasmid was connected to the hcp2b target fragment to construct the recombinant vector, which was introduced to the competent Δhcp2b strain to construct the hcp2b-reverted strain. Then, we measured the growth curve of Δhcp2b strain. Seven-day-old chicks were infected with the hcp2b-deleted strain and the wild strain by tracheal injection. The infected tracheal mucosal cells were collected at 12 and 24 h after infection and subjected to transcriptomics sequencing and bioinformatics analysis. The results showed that the Δhcp2b strain and Chcp2b strain were successfully constructed. And the deletion of hcp2b gene did not affect the growth performance of the strain. The expression profile of the mRNA of the tracheal mucosa changed after the hcp2b gene-deleted strain infected. Twelve hours after infection, the expression of 144 genes changed (87 differential genes were up-regulated and 57 were down-regulated); Twenty-four hours after infection, 135 gene expressions changed (79 differential genes up-regulated and 56 down-regulated). Bioinformatics analysis found that differential genes were enriched in signaling pathways such as Cytokine-cytokine receptor interaction, Protein processing in endoplasmic reticulum, etc. The deletion of hcp2b gene does not affect the growth characteristics of APEC. After the deletion of hcp2b gene, it affects the change of mRNA transcription level of Cytokine-cytokine receptor interaction pathway gene in chick tracheal mucosa. And the expression of IL-1β and IL-12b are all up-regulated. This study provides a theoretical basis for the study of the pathogenic mechanism of APEC.

This study aims to evaluate and compare the immunogenicity and protective efficacy of virulence proteins, SrtA, SeM and EAG of Streptococcus equi subsp. equi (S. equi). These 3 recombinant proteins were expressed, purified, and then were used alone or in combination to immunize mice. The mice serum antibody titer, antibody subtype, and immune molecules were detected after immunization. Challenge protection rate, bacterial loading of liver, spleen, kidney, and lung were detected after challenge. In addition, histopathological changes of liver, spleen, kidney, and lung were observed. The ELISA results showed that IgG, IgG1 and IgG2b levels induced by the combined immunization group were higher than those immunized alone group and SrtA immunization group induced higher IgG2a levels than SeM and EAG group. The results of challenge protection rate, bacterial load and histopathological observation showed that the combined immunization group was superior to the single immunization group. The fluorescence quantitative PCR results of immune related molecules showed that the transcription levels of MHCⅠ, TCR, TLR2, TLR3 and TLR4 induced by SrtA were higher than those induced by SeM,EAG immunized group and control group. The MHC Ⅰ and TLR3 induced by combined-immunized group were higher than those of single-immunized group. In conclusion, the SrtA was a better immune stimulation and induction antigen and three proteins immunized in combination has better immunogenicity and provide efficacious immune protection. This data laid theory foundation and experiment basis for the subunit vaccine development of strangles.

Isolation of an appropriate lytic phage against Salmonella Typhimurium will help to provide a biological for controlling infection or pollution caused by this pathogen. A strain of Salmonella Typhimurium with multidrug resistance which had been isolated from bulk milk was used as host bacteria. Artificial induction method was employed. Three chickens were fed daily with the host bacterium suspension for a week. After 7 days, the phage isolation and culture was conducted from chicken feces by using double agar plate method. The titer, nucleic acid type, host spectrum, thermal and acid-base stability and therapeutic effect in Salmonella Typhimurium infected mice using the phage were analyzed. A lytic phage against Salmonella Typhimurium was isolated and purified, named KM104. This phage efficiently lysed the homologous host strain and another Salmonella Typhimurium strains in vitro, producing clear and circular plaques with a diameter of about 1 mm. The viral nucleic acid type was DNA and its genome size was 28 kb or so. The highest titer of the virus was 4.6×10⁸ PFU·mL^-1. The optimal multiplicity of infection (MOI) was 1:10, the incubation period was approximately 6 min, and it reached the highest titer at 70 min. The phage maintained high viability under the condition of 20-60℃ and a pH value range of 2.0-8.0. The phage effectively reduced the injury in duodenal tissue of experimental mice caused by Salmonella Typhimurium. The results here indicated that KM104 can efficiently lyse Salmonella Typhimurium in vivo and in vitro with rapid proliferation and strong adaptability to the environment. Its acid-fast character is expected to help resist the degradation by gastric acid, making the phage have a potential application value in preventing and controlling Salmonella Typhimurium infection or pollution.

The aim of this study was to investigate the effect of porcine deltacoronavirus(PDCoV) infected neonatal piglet on small intestinal goblet cells (GC). Six neonatal piglets without colostrum were randomly divided into infection group (n=3) and control group (n=3). After acclimation for 48 h, the infected group was inoculated orally with a PDCoV-CHN-HG-2017 preparation with a titer of 1×10⁵TCID₅₀·mL^-1 (5 mL per pig), while the mock-infected group was inoculated with 5 mL of maintenance medium. Clinical signs were monitored hourly. The infected piglets developed typical symptoms, such as diarrhea, dehydration and lethargy, from 22 to 70 hours post inoculation (hpi). Piglets were euthanized at 22, 39 and 70 hpi after the onset of clinical symptoms. At the same time point, one piglet was treated in the control group. HE staining, PAS staining and AB staining were used to observe the histopathological changes and the number of GC in the small intestine of piglets. Fluorescent quantitative PCR and ELISA were used to detect the transcriptional quantity and content of mucoprotein-2 (MUC2) and hairy and enhancer of split-1 (Hes1) in small intestine of neonatal piglets. The results showed that piglets in the infected group had different degree of damage to the mucosal structure of small intestine compared with piglets in the control group, the villus length were significantly decreased (P<0.05), the ratio of VH:CD significantly decreased (P<0.05). Compared with the control group, the number of GC in each segment of small intestine of infected piglets was significantly reduced. The results of PAS staining and AB staining were consistent. Compared with the control group, the transcription and content of Hes1 mRNA in each segment of the small intestine of piglets in the infected group were all increased, and there were significant differences in the transcription of Hes1 in jejunum and ileum (P<0.01 or P<0.05), and there were significant differences in the content of Hes1 mRNA in the duodenum and ileum (P<0.01). Compared with the control group, the transcription and content of MUC2 mRNA in each segment of the small intestine of infected piglets were reduced, and the differences were significant in the duodenum and ileum (P<0.05, P<0.01 or P<0.001). In summary, the number of small intestinal GC was significantly reduced after PDCoV infection in neonatal piglets. PDCoV infection in piglets may inhibit the formation and secretion of GC in the small intestine by activating the Notch signaling pathway in the intestine to enhance the expression of target gene Hes1, resulting in a decrease in the number of GC and the expression of MUC2.

Bovine mastitis is the inflammatory response in bovine mammary gland, it is the most prevalent disease in dairy cattle worldwide. Many cytokines are inflammatory mediators. But the key cytokines associated with bovine mastitis have not been elucidated. Allograft inflammatory factor-1 (AIF-1) plays a central role in immune regulation and it is over-expressed in a vast array of inflammatory diseases. Therefore, we explored the possible role of bovine AIF-1 related to bovine mastitis in the present study. First, bovine AIF-1 gene was cloned by RT-PCR and the AIF-1protein was purified by a Ni-NTA spin column. Then, bovine mammary gland epithelial cell MAC-T line was treated with AIF-1 for 48 h. The concentrations of TNF-α, IL-6, AIF-1 and monocyte chemoattractant protein 1 secretion were measured by ELISA assay kits. Finally, phosphorylation of IκBα was detected by Western blot. The results showed that the average concentration of AIF-1 in milks suffering from mastitis was significantly higher than that in the healthy cows, while its value decreased in cows recovered from mastitis. Furthermore, bovine AIF-1 gene was cloned and recombinant bovine AIF-1 protein was prepared. The protein up-regulated TNF-α, IL-6 and monocyte chemoattractant protein 1 secretion from bovine mammary epithelial cells with NF-κB activating, then NF-κB signaling inhibitor BAY 11-7085 abolished the increase of these inflammatory cytokine secretion induced by AIF-1. The present study indicated that AIF-1 induced inflammatory mediator production from bovine mammary epithelial cells via NF-κB signaling.

As the key factors related to neuroprotective function in the nervous system of vertebrates, neuroglobin (NGB) and hypoxia inducible factor-1α (HIF-1α) play important physiological functions in the process of hypoxic adaptation. This study aimed to explore the relationship between expression and distribution of NGB and HIF-1α in the process of adaptation to hypoxia of yak (Bos grunniens) hindbrain which plays an important regulatory function in animal movement, breathing, sensory and other physiological activities. RT-qPCR, Western blot and immunohistochemical methods were used to investigate the expression and localization characteristics of NGB and HIF-1α in different regions of yak hindbrain. The results showed that the expression trend characteristics between NGB and HIF-1α in yak hindbrain were basically consistent, the highest expression level was found in the archicerebellum which was significantly higher than those in the medulla oblongata, pons and other parts of cerebellum (P<0.05), the expression level in the cerebellar hemisphere cortex was less than that in the archicerebellum and the lowest expression level was found in the palaeocerebellum, there were also some differences in the expression levels of the two factors in other regions. The distribution characteristics of NGB and HIF-1α positive products were similar, and the overall intensity of the HIF-1α protein immunopositive response was higher than that of NGB protein. NGB and HIF-1α were mainly expressed in the cytoplasm of neurons in the medulla oblongata and pons. Scattered NGB and HIF-1α positive cells could be found in the molecular layer of the cerebellar cortex, archicerebellum, neocerebellum and palaeocerebellum. There were also a small number of positive expressions in the basket cells. The dendritic cell cytoplasm of Purkinje cells in the Purkinje cell layer showed a strong positive expression of NGB and HIF-1α. Strong positive expression of NGB and HIF-1α were also observed in the granular cell cytoplasm. The regions of white matter (medulla) showed rarely NGB and HIF-1α positive expression in the cytoplasm of the neurons. No expression was observed in the negative control. The above results suggested that there were selective differences between the expression of NGB and HIF-1α in different regions of yak hindbrain during the long-term adaptation to hypoxic environment. The archicerebellum has a higher tolerance to hypoxia, followed by the cerebellar hemisphere, and the weakest tolerance to hypoxia in the palaeocerebellum. The reason for the differences might be related to the specific functions undertaken by the relevant regions of the hindbrain in yak and the adaptive function of hypoxia in each region mainly depends on its specific neuronal structure.

The mechanisms of chronic postsurgical pain (CPSP) are complicated and still eludes explanation. In this study, a surgery-related animal model in rat was used to search the metabolites on the transition from acute to chronic pain, aiming to identify the potential biomarkers associated postsurgical pain. GC-MS-based metabolomics technique were performed to investigate the different metabolites between acute to chronic postsurgical pain. It showed that 224 metabolites were screened, while 35 of which were significant difference (VIP>1,P<0.05,∣log₂FC∣ ≥ 2). These metabolites were mainly involved in 45 metabolic pathways through pathway enrichment analysis. The results showed that metabolites such as N-acetylaspartic acid, aspartic acid, pantothenic acid, beta-alanine, 3-hydroxybutyric acid and alanine/aspartate/glutamate metabolism,β-alanine metabolism, pantothenate and CoA biosynthesis, regulating sugar metabolic pathways are closely related with the transition from acute to chronic pain. To dissertate the key substances of the transition of acute pain changed into chronic pain can not only help us to understand underlying mechanisms of CPSP, but also provide a new insight and method for recognizing and managing postsurgical pain.

Based on network pharmacology and molecular docking technology, this study explored the anti-PRRSV mechanism of matrine from the perspective of anti-inflammatory. PharmMapper server and Genecards disease database were used to obtain the potential targets of matrine; STRING online analysis platform combined with Cytoscape software was used to construct the target protein-protein interaction (PPI) network and perform topological analysis to screen the key targets; DAVID database was used to perform gene ontology GO enrichment analysis and KEGG pathway enrichment analysis of the targets, and Cytoscape software was used to construct the "drug-target-pathway" network diagram; Autodock Vina software was used for molecular docking to select the best binding targets. The results of network analysis showed that there were 23 potential targets of matrine; the protein interaction network suggested that IGFⅠ, MAPK8, CASP3, and NR3C1 may be the core targets of matrine; 116 cell biological processes were obtained by GO enrichment analysis, and 47 related signaling pathways were obtained by KEGG pathway enrichment analysis; molecular docking showed that MAPK8 had a good affinity to matrine and maybe the main target of matrine exerting its effect. Matrine may play an anti-inflammatory role by acting on the key protein of MAPK8, thus achieving anti-PRRSV effect.

The study aimed to determine immune enhancement activities of ethanolic extract from wild C. deserticola with dual Th₁ and Th₂ responses by promoting DCs maturation in mice. OVA was used as antigen, three different doses of EEWCD (low, medium, high) were combined with OVA in 0.9% NaCl. ICR mice were randomly separated into seven groups, 0.9% NaCl group, EEWCD group, OVA group, OVA-EEWCD-L group, OVA-EEWCD-M group, OVA-EEWCD-H group, and OVA-Alum group. ICR mice were subcutaneously injected twice with a 2-week interval. After vaccination, IgG titers, IgG subclasses were detected by ELISA, splenocyte proliferation was performed with MTT method, cytokine, DCs surface markers and Treg frequency were tested by FACS. The data showed that EEWCD significantly enhanced the serum IgG titers and IgG subclasses (P<0.05). Meanwhile, EEWCD significantly promoted OVA-specific splenocyte proliferation and T/B-proliferation (P<0.05). EEWCD also could significantly promote the level of CD4⁺IL-4,CD4⁺IFN-γ and CD8⁺IFN-γ (P<0.05). Furthermore, the expression levels of CD40, CD80, CD86 and MHC-Ⅱ in DCs were significantly improved (P<0.05), whereas the frequency of Treg was significantly decreased (P<0.05). EEWCD-M showed remarkable immunomodulatory activity. No abnormal behavior and significant difference in mice body weight were observed (P>0.05). The data showed that subcutaneous administration of EEWCD significantly enhanced Th₁/Th₂ immune responses by promoting DCs maturation and reducing the frequency of Treg.

The study aimed to investigate the effects of different concentrations of allicin on the expression of microphthalmia-associated transcription factor (MITF) and its effects on the proliferation and migration of B16F10 cells, which provide a new idea for the treatment of animal melanoma in pet and animal husbandry. B16F10 cells were divided into control group (0 μg·mL^-1) and experimental group (5, 10, 15, 20 μg·mL^-1) supplemented with different concentrations of allicin. The expression of MITF mRNA in B16F10 cells was detected by qRT-PCR and the expression of MITF protein was detected by Western blot. The cell growth rate was calculated by using the absorbance of cells at 515 nm, and then the inhibition rate of different concentrations of allicin on cell proliferation was determined. Wound healing was used to evaluate the effects of different concentrations of allicin on the migration of B16F10 cells after 42 hours. The results showed that the expression of MITF mRNA decreased gradually with the increase of allicin concentration, while Western blot results showed that the expression of MITF protein decreased in a concentration-dependent manner. Allicin at the concentrations of 5, 10, 15, 20 μg·mL^-1 could significantly inhibit the proliferation of B16F10 cells (P<0.001), and the inhibition rate increased with the increase of allicin concentration. Wound healing results showed that allicin significantly inhibited the cell migration at the concentration of 10 μg·mL^-1, 15 μg·mL^-1 and 20 μg·mL^-1. This experiment showed that allicin could affect the expression of the melanoma-specific marker gene MITF and inhibit the proliferation and migration of B16F10 cells.

The objective of this study was to prepare monoclonal antibodies against the S273R protein of African swine fever virus (ASFV). BALB/c mice were immunized with purified recombinant S273R protein expressed by prokaryotic expression system and hybridoma cells were obtained by fusion of spleen cells and myeloma cells. An indirect ELISA based on the purified S273R protein was developed. Four hybridoma cell lines stably secreting MAbs against the S273R protein were harvested after screening and subcloning, and their sensitivity and specificity were detected by ELISA, Western blot, and immunofluorescence assay (IFA). Four MAbs heavy chains belong to IgG1, and light chains were κ. This research provided technical support for further study of biological functions of ASFV pS273R, as well as its gene-deletion based vaccine development.

This study aimed to investigate the epidemic status and characteristics of equid herpesvirus-1(EHV-1) infection in Xinjiang. One thousand six hundred and fifty-seven samples of horse sera were collected from Urumqi, Yili, Changji, Bazhou, from 2019 to 2020. Enzyme-linked immunosorbent assay (ELISA) was performed to detect EHV-1 antibody. Statistical analysis of the positive rate of EHV-1 antibody in different regions, breeds, genders and ages. The detection rate of Urumqi, Yili, Changji and Bazhou were 39.34%, 22.56%, 90.52% and 5.00%; The positive rate of Thoroughbred horse, Half-bred horse, Hasake horse, Yili horse, Yanqi horse, Przewalski's horse were 20.34%, 32.93%, 44.92%, 19.35%, 5.00%, 90.52%, respectively; The positive rate of Przewalski's horse and domestic horse were 90.52% and 29.32%, respectively. The positive rate of local variety and introduced variety were 31.70% and 25.50%; The positive rate of female and male were 38.84% and 22.00%; The positive rate of infancy, youth, adult, old age were 35.37%, 33.00%, 34.31% and 23.81%, respectively. The results indicated that EHV-1 infection was widespread in large-scale stud-farms in Urumqi, Changji, Yili and Baizhou of Xinjiang. The infection rates of EHV-1 in different regions, breeds, genders and ages were significantly different (P<0.05). This study provides survey data for the research and prevention of EHV-1 infection in the region.