Identification of selection signatures have become an important tool in population genetics and gene mapping of economically important traits with the explosion of genomic data. Using proper statistical methods and understanding the potential statistical problems in identification of selection signature are of great significance to accurately locate the candidate genes affecting economically important traits and reveal the potential genetic mechanism of adaptive evolution of livestock and poultry. This paper reviews the concept, the category of testing statistics of selection signature, the influencing factors and the related statistical problems in identification of selection signatures. It is expected to provide a reference for further research on identification of selection signatures.

This experiment was conducted to explore the tissue expression profile of HLCS, the polymorphisms in its coding region and the association of different genotypes with residual feed intake in Duroc. Real-time PCR was used to detect HLCS mRNA expression in 8 tissues of Duroc pigs. The polymorphic sites of HLCS were determined in Duroc with high and low RFI lines by PCR and sequencing. Genotypes were detected in Duroc, and their associations with residual feed intake trait were analyzed. Real-time PCR showed that HLCS had the highest expression in backfat, followed by liver, and it had the lowest expression in heart and muscle. Five SNPs were detected in the coding region of HLCS gene. Two of them, c.1408G > C and c.1976A > G were nonsynonymous mutations. The site c.1976A > G was significantly associated with RFI(P<0.05), and the individuals with GG genotype had lower RFI than individuals with AA genotype. These results indicate that HLCS may play an important role in porcine RFI, and c.1976A > G can serve as a useful genetic marker for reducing RFI in swine. This study is valuable to further explore the molecular mechanism of HLCS affecting residual feed intake.

The research was designed to study the activity and the structure of MITF promoter and to provide clues for studying this gene's regulation mechanism in hair follicle. Dual-luciferase expression vectors were constructed and transfected to DF1 cells with lip2000. The dual-luciferase detection kit was used to measure the relative luciferase activity. The 6 expression vectors with different promoter regions of MITF gene and 4 mutant vectors of the core promoter region were constructed in Bashang Long-tail chicken. The core promoter region from -660 bp to +200 bp was identified in chicken MITF gene. The function of -579 bp, -505 bp, -274 bp, -220 bp, -203--202 bp, -98 bp, -46 bp, -14 bp, +1 bp and +28 bp sites in regulating promoter activity was critical. The similarities of transcription factor binding sites including HOX family (HOXA3, HOXD8, HOXD9, HOXD10 and HOX11), NF-1, DBP, TFⅡD and TFⅡB to MITF were changed predicted by software.

This study was conducted to investigate isolation and cultivation of the hair follicle outer root sheath cells (HFORSCs) in vitro, and to estimate the effects of the vascular endothelial growth factor (VEGF) on the proliferation, differentiation and mRNA expression of proliferating cell nuclear antigen(PCNA), keratin 10 (K10) and fibroblast growth factor 5 (FGF 5) genes in the sHFORSCs of Shaanbei White cashmere goat. Skin tissues of goats were collected, and sHFORSCs were obtained through the methods of digestion and then cultured under the condition of 37℃, 5% CO₂ and 100% relative humidity;The 5^th generation of cultured sHFORSCs were selected and treated with different concentration VEGF for 24 and 48 h, respectively. The cell viability was detected by Methyl thiazolyl tetrazolium (MTT), and the cell proliferation was detected by EdU DNA markers;The mRNA expression levels of PCNA, K 10 and FGF 5 in HFORSCs were detected by the real-time fluorescence quantitative PCR. Results showed that:1) sHFORSCs were successfully isolated and cultured in vitro, and cultured cells were positive in the immunohistochemistry test of CK17 and CK15, which were main biomarkers of outer root sheath cells. 2) VEGF could promote the proliferation of sHFORSCs. Treated with same concentration of VEGF, the cell viability of 48 h group was significantly higher than that of 24 h group (P<0.01). Treated with different concentrations of VEGF for 48 h, the cell viability of 100 ng·mL^-1 group was significantly higher than that of control group, 1 ng·mL^-1 group and 10 ng·mL^-1 group (P<0.01), and also significantly higher than that of 50 ng·mL^-1 group (P<0.05). The cell viability of 50 ng·mL^-1 group was significantly higher than that of control group (P<0.05); The proportion of the cell proliferation of 100 and 50 ng·mL^-1 groups were all significantly higher than that of control group, 1 ng·mL^-1 group and 10 ng·mL^-1 group (P<0.01). The proportion of the cell proliferation of 100 ng·mL^-1 group was significantly higher than that of 50 ng·mL^-1 group (P<0.05). 3) The expressions of mRNA of PCNA, K10 and FGF 5 were all detected in HFORSCs. Treated with VEGF for 48 h, the mRNA expression level of PCNA in 100 ng·mL^-1 group was the highest, and was significantly higher than that in control group (P<0.01) and 10 ng·mL^-1 group (P<0.05); The mRNA expression of K10 in control group was the highest, and was significantly higher compared with that in 10 and 100 ng·mL^-1 groups (P<0.01); The mRNA expression of FGF 5 in control group was the highest, and was significantly higher than that in 100 ng·mL^-1 group (P<0.01). In this study, sHFORSCs were successfully isolated from hair follicle and were successfully cultured in vitro; The proliferation of HFORSCs could be promoted and the cell differentiation could be inhibited by VEGF. It inferred that VEGF might play the role of growth promotion of the hair follicle by inhibiting the mRNA expression of FGF 5 in sHFORSCs.

This study aimed to identify differentially expressed miRNAs in myogenic differentiation of bovine fetal skeletal muscle derived myoblasts, which would provide important insight for understanding the mechanism of myoblast differentiation at bovine fetal stage. Myoblasts were isolated and cultured from skeletal muscle of 3 bovine fetal on 4 months old, and then induced myogenic differentiation in vitro. miRNAs of myoblast at the initial and terminal stage of myoblast differentiation were sequenced using high throughput sequencing technology. The sequencing data was analyzed to identify the differentially expressed miRNAs. Differentially expressed miRNAs were randomly selected to validate the sequencing accuracy using quantitative RT-PCR. The results revealed 619 miRNAs in 6 libraries, and 150 miRNAs were differentially expressed. Among them, several miRNAs were related to muscle development such as bta-miR-199a-5p. The target genes of differentially expressed miRNAs were predicted and enriched to pathways related to the muscle development such as MAPK pathway. The expression of 8 randomly selected miRNAs were quantified by real-time quantitative PCR for further validation, and their results were consistent with those of sequencing analyses. The study identified 150 differentially expressed miRNAs in the differentiation of bovine fetal muscle derived myoblasts. Our findings will provide valuable insights for further exploring the regulation roles of miRNAs in the development of bovine fetal skeletal muscle.

This study aimed to construct an integrated and controllable transgenic mice model with pGH gene expression, which could reduce the side effects caused by premature expression of GH through controlling the time and quantity of GH gene expression, it would become a meaningful research to improve feed conversion of livestock. The transgenic mice were successfully obtained by prokaryotic microinjection method using a vector of Tet-on regulatory expression system, which was constructed successfully and verified through transfected PK15 cell line. Based on Southern blot results, positive Founders were bred for getting offspring. When we got the transgenic offspring, the induction experiment was carried out with the induction substrate doxycycline(DOX) supplementation in the drinking water of the transgenic mice at 3-10 weeks old. RT-qPCR technology was used to detect the level of exogenous genes expression. The radioactive immunoassay method was used to detect porcine growth hormone content in serum of mice. Body weight was weighed to evaluate the effects of pGH gene on phenotype of transgenic mice. The results showed that the body weight of induced transgenic mice was higher than that of non-induced transgenic mice and non transgenic mice in the same litter(P<0.05); The level of pGH in serum of transgenic mice was significant higher than that of mice in control (P<0.05), which indicated that exogenous GH could effectively express and regulate the growth of mice body. The establishment of model of pGH transgenic mice contributed to explore the effect of GH gene on body development and the molecular mechanism and signal pathway of growth hormone secretion. It would promote the application of GH in livestock production; it also provided a theoretical foundation and technical support for the preparation of safe and efficient transgenic livestock with GH gene.

The objective of this study was to determine the heat sensitivity index for Bama miniature pigs by investigating the effect of ambient high temperature on scrotal temperature distributions and testicular protein expressions related to the antioxidative capacity and cellular apoptosis in the pigs. Twelve 7-month-old mature Bama miniature pigs were selected and randomly allotted to a thermal neutral(TN)group and a heat-treated(HT)group. The HT group was exposed to high ambient temperature, which was changed from 25 to 40℃ and back to 25℃ during each 24 h period for 8 d. At the end of the experiment, testes were collected and measured for the protein expression of nuclear factor erythroid 2-related factor 2(Nrf2), heme oxygenase 1(HO-1)and cleaved caspase 3 (c-caspase 3)using immunohistochemical staining. Results showed that, the HT group pigs presented a significant increase in scrotal surface temperature(P<0.05), which was 3.7℃ higher than the TN group pigs. In the HT group, the testicular protein expression of Nrf2 and HO-1 increased significantly in both the testicular interstitial cells and the germ line cells of seminiferous epithelium(P<0.05). Moreover, prominent nuclear accumulation of Nrf2 protein was observed in the testicular interstitial cells. In the HT group, there was an increased percentage of the c-caspase 3-positive nucleuses in the testicular interstitial cells and germ line cells of seminiferous epithelium(P<0.05), respectively. The percentage of the c-caspase 3-positive nucleuses in the testicular interstitial cells increased more sharply than that in the germ line cells of seminiferous epithelium. In conclusion, the capacity of scrotal thermal regulation was overwhelmed in the 40℃ ambient temperature, which increased the testicular temperature and caused the testicular cell apoptosis. High ambient temperature also activated Nrf2 signal pathway and increased HO-1 expression, indicating that the testicular Nrf2/HO-1 signal pathway might be a potential target for new strategies to protect semen quality from high ambient temperature-induced damage in boars.

This study investigated the effects of fat deposition on reproductive performance of Beijing-You chickens during the late stage of reproductive period, aiming at providing data support to increase the reproduction efficiency. A total of 65 Beijing-You chickens of 43 weeks of age were selected. The daily egg production was recorded. The incubation performance and egg quality estimation were performed at 53 and 63 weeks of age, respectively. The animals were divided into high, medium and low abdominal fat content groups based on their abdominal fat yield estimated at 65 weeks of age, and the difference of egg number,egg quality and hatchability of egg were analyzed among different groups. The medium abdominal fat yield group produced 17.02% and 8.29% more eggs than the high and low groups, respectively. The hatchability of fertile eggs of the medium abdominal fat yield group was 4.70% and 10.10% higher than that of the high and low groups, respectively. Similarly, the hatchability of setting eggs of the medium abdominal fat yield group was 6.98% and 10.47% higher than that of the high and low groups, respectively. No significant difference on egg weight, yolk weight, shell strength and shell thickness were observed (P>0.05). The egg shape index of the high abdominal fat yield group was significantly higher than that of low group (P=0.05). The results suggested that manners should be taken to regulate the fat deposition during the late stage of reproductive period to gain better egg production, egg quality, and hatchability.

This study was conducted to evaluate the effects of fermented complete feed (FCF) on the growth performance, odor concentration and bacterial community in faeces of growing pigs, and evaluate the application effect of FCF in pigs. Complete feed was anaerobic fermented with Bacillus subtilis and Lactobacillus brevis for 3 days. 90 pigs with the about initial weight 22 kg were randomly allotted into 3 treatment groups (antibiotic-free feed group, antibiotic feed group and FCF group) of 30 each. At the end of the experiment(body weight of pigs was up to about 40 kg), the growth performance of pigs was measured and analyzed. The fresh stool sample was collected from each group to determine the odor concentration by gas chromatography. Additionally, the bacterial community was assayed by using the high-throughout DNA sequencing. The results showed that:1) Fermentation of complete feed significantly increased the fat content in feed (P<0.01) and decreased the calcium content (P<0.01), total phosphorus content (P<0.05) and pH (P<0.01). The number of lactic acid bacteria in the FCF was up to 9.67 lg cfu·g^-1 and lactic acid content was up to 421.67 mmol·kg^-1. 2) Data from the animal experiment indicated that FCF feeding raised the ADFI and ADG of pigs (P<0.05), and reduced the F/G and mortality (P<0.05) compared with the antibiotic-free group. The ADFI of FCF-fed pigs was higher than that of antibiotic-fed pigs (P<0.05). 3) In addition, the indole content in pig faeces was decreased (P<0.05) by the CFC feeding. The skatole and VFA contents showed a similar decreased tendency, however, the p-cresol content showed a rising tendency in FCF group. 4) Importantly, the richness and diversity of faeces bacterial community were improved by the CFC feeding. The relative abundances of phyla including Tenericutes and Saccharibacteria, genera including Clostridium, Turicibacter, Mollicutes, Anaerostipes, Acetitomaculum increased; while the relative abundances of genera including Lactobacillus, Streptococcus, Rikenellaceae, Megasphaera, Oscillospira, Mitsuokella, Prevotella reduced. In conclusion, FCF could improve the growth performance of pigs and the content of p-cresol in faeces, while reduce the content of indole, skatole and VFA in faeces and change the bacterial community in faeces.

The aim of this study was to investigate the distribution of particulate matter (PM), ammonia (NH₃) and carbon dioxide (CO₂) in an enclosed caged broiler breeder house as well as the component and ultrastructure of fine particulate matter (PM_2.5) collected from the hen house in winter. Detections of temperature, relative humidity, airflow speed, illumination and concentrations of PM, NH₃ and CO₂ were taken from 05:00 to 21:00 in every two hours for 8-day continuous measuring in December 2016 in an enclosed broiler breeder house with 7 monitoring positions inside the house and 1 outside the house. There were 5 626 Youai breeder hens of 57-58 weeks old in the house. Furthermore, particles sampler was fixed at the middle position of the breeder hen house to collect PM_2.5 for 16 h per day which was used to analyze chemical composition and observe ultrastructure. The results showed that:1) The temperature, PM and NH₃ concentrations in forepart of the breeder hen house were markedly lower than middle and back position (P<0.05). The PM_2.5, PM₁₀ and TSP concentrations inside the breeder hen house were significantly higher than outside the breeder hen house(P<0.001). The PM_2.5, PM₁₀ and TSP concentrations all were the highest at feeding time (05:00) in the morning, and these were the lowest after turning off the lights at 21:00. Whereas the ratios of PM_2.5/PM₁₀, PM₁₀/TSP, PM_2.5/TSP were the highest when the chicken flocks were quiet. Airflow speeds showed significant correlation with other microclimatic variables except PM_2.5 (P<0.01). The CO₂ concentration and airflow speed were affected indistinctively by ambient air outside the house. 2) Organic carbon (OC) was the mainly component of PM_2.5 collected from the enclosed breeder hen house, concentrations of NO₃^- and SO₄^2- were also relatively high comparing with other components. It was observed that the PM_2.5 of layer house contained many mineral particles and a part of smoke collection by electron microscopy. Energy spectrum diagrams of PM_2.5 revealed that both mass percent and atomic percent of C and O were the highest. The PM concentrations inside the monitored house were higher than outside. Air quality in the forepart of the house (air inlet) was better than middle and back. Chicken activity mainly caused coarse particles (PM₁₀, TSP) increase. Organic matter and mineral were the major components of PM_2.5 from the enclosed breeder hen house, which mainly come from feed, feces and ground dust.

This study was conducted to study the effects of different dietary N-carbamoylglutamate (NCG) levels on growth performance, nutrient digestibility, nitrogen metabolism and serum biochemical indexes of growing minks. One hundred and fifty healthy weaned Black minks(Mustela vison) with similar body weight were selected and randomly divided into 5 groups with 30 replicates per group and one mink per replicate. These minks were fed with NCG supplementation levels of 0% (group A), 0.02% (group B), 0.06% (group C), 0.10% (group D) and 0.14% (group E), respectively in the experimental diets, NCG was added from June 8^th to September 15^th. The pre-trial lasted for 7 days and the trial for 92 days. From September 12^th, 10 minks (5 male and 5 female) were selected from each group for digestion and metabolism test for 3 days, muck method determination of feed and feces were conducted to measure the contents of dry matter (DM), crude protein (CP) and crude fat (EE) and then to calculate the apparent digestibility and nitrogen metabolism. On the morning of September 15^th, 6 minks(3 male and 3 female) were selected from each group. Blood was collected and the contents of arginine (Arg), nitric oxide (NO), growth hormone (GH), total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase(AST), immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), complement 3 (C3), complement 4 (C4) in serum were determined by ELISA. The results showed that:1) The final weight and average daily gains of female minks fed with NCG were all higher than the control group, that of female minks in group B were the highest, but the differences were not significant between different groups (P>0.05). These indicators of male minks in group B was significantly higher than group A, C and E (P<0.05). The feed/gain of female minks in group B was the lowest, but the differences were not significant between different groups (P>0.05), the feed/gain of male minks in group B was significantly lower than A and E groups (P<0.05). 2) The digestibility of dry matter, crude protein and crude fat of minks fed with NCG were all higher than the control group, but the differences were not significant between different groups (P>0.05). 3) There were no significant differences in nitrogen deposition, net protein utilizable rate and biological value of protein between different groups (P>0.05), but compared with group A, the nitrogen deposition of D group and the net protein utilizable rate, biological value of protein of B group were higher. 4) Compared with group A, the levels of arginine, nitric oxide, growth hormone, total protein, IgA, IgG, IgM, C3 and C4 in serum of minks fed with NCG extremely significantly increased (P<0.01), and group B was all the best, the alanine aminotransferase and aspartate aminotransferase extremely significantly decreased (P<0.01). The results indicated that basal diet supplemented with 0.02% NCG could help the growing minks to gain the average daily weight, reduce the feed/gain, improve the nitrogen deposition, net protein utilizable rate and biological value of protein, and increase the levels of arginine, growth hormone, total protein and immunoglobulin in serum.

This experiment was conducted to study the effects of tumor progression locus 2 (TPL2) on replication of foot-and-mouth disease virus (FMDV) in baby hamster kidney (BHK-21) cells. BHK-21 cells were infected by FMDV, the mRNA level of TPL2 was detected by qRT-PCR. TPL2 eukaryotic expression plasmid and RNAi sequences were designed and synthesized based on TPL2 sequences published in GenBank (NM007746.2). The effects of TPL2 on FMDV replication in BHK-21 cells were evaluated by qRT-PCR and Western blotting. The results indicated that the transcriptional level of TPL2 was significantly up-regulated during FMDV infection; Overexpression of TPL2 facilitated the replication of FMDV and downregulation of endogenous TPL2 inhibited FMDV replication. These results revealed that TPL2 promoted the proliferation of FMDV in BHK-21 cells. This research further expanded our understanding of the relationship between FMDV and the innate immune mechanism and had accumulated scientific data for the relevant research of TPL2.

The objective of this work was to establish a SLAM-Vero cell line and a SLAM-BHK21 cell line that stanbly expressing SLAM receptor, and then to compare their sensitivities to the canine distemper virus (CDV) wild strains, aiming at providing another tools for the rapid isolation of CDV for further study. The eukaryotic expression vector Pcag-SLAM, which contains the SLAM gene, was transfected into the Vero cells and BHK-21 cells, respectively. Then the cells which harbor the SLAM gene were subsequently screened with G418 combined with end point dilution, RT-PCR, immunofluorescence assay, and Western blot. The two cell lines were subsequently applied to isolate virus from 5 different tissues in 3 clinical cases. The isolated CDV was further identified by RT-PCR and Indirect Immunofluorescence assay. The results showed that the SLAM-Vero cells lines and SLAM-BHK21 cells lines were constructed successfully. SLAM-Vero inoculated virus for 12-24 h presented a typical CDV-induced cell fusion of CPE. By using SLAM-Vero cells, two strains of CDV were obtained from three CDV positive canine lungs and spleens, while SLAM-BHK21 cell lines, parental Vero cells and parental BHK21 cells failed to isolate the virus. The results indicated that CDV was more readily to replicate and proliferate in SLAM-Vero cells and easier to produce visible CPE than that in SLAM-BHK21 cells. Canine lungs and spleens are more ideal candidates for isolation of CDV when using the newly established SLAM-Vero cell lines in this report.

GP5 protein of porcine reproductive and respiratory virus (PRRSV) is considered to be the main protein that inducing production of neutralizing antibodies. In order to investigate the ability of the protective immunity induced by the GP5 protein, the full-length GP5 and the truncated GP5 lacking of the signal peptide (aa 1-31) and transmembrane region (aa 66-125) were expressed using baculovirus and prokaryotic expression systems. Additionally, the truncated GP5 lacking of the signal peptide(aa 1-31) and transmembrane region (aa 66-125) were expressed using E. coli expression systems. Three anti-sera (ELISA assay antibody titre:800, 1 600, and 200 respectively) specific for each recombinant protein were derived from mice immunized with three recombinant proteins with Freund's Incomplete Adjuvant. PAM cells used in neutralization assay were obtained from PRRSV-negative healthy pigs by bronchoalveolar lavage after necropsy. PRRSV HeN-3 (the heterologous strain) and VR2332 (the homologous strain) strains were isolated in Marc-145 cells. The real-time quantitative PCR was used to detect PRRSV RNA levels in infected PAM cells. It was considered positive that virus blocking rate was higher than 70% which had been established in our laboratory. Results were as follows:1) The results showed that three anti-sera specific for three recombinant proteins did not have neutralizing activities against two strains of PRRSV (titers were < 1:2). 2) The anti-sera of PRRSV VR2332 had neutralizing activity against PRRSV VR2332 strain (titers were 1:8), and hadn't neutralizing activity against PRRSV HeN-3 strain but rather enhanced infection. Our current work showed the main neutralizing epitopes of GP5 were probably conformational epitopes and the neutralizing epitopes of PRRSV were not fully presented in the GP5 protein. The positive anti-sera of PRRSV could provide immune protection, but the immune protection occurs only in the same strain. It would mediate the ADE effect on heterologous strains on the contrary.

To investigate the effect of GP5, the most important structural protein encoded by porcine reproductive and respiratory syndrome virus (PRRSV) ORF5, on virus replication and it's mechanism, a recombinant plasmid (named as pPB-GP5) was constructed using PiggyBac Transposon System Vectors and transfected into Marc-145 cells. Marc-145 cell line stably expressing GP5 was obtained by puromycin resistance screening and three times sub-cloning. GP5 was confirmed stable expression in Marc-145 cells by RT-PCR, Western blot and IFA detection and the cells was named as Marc-145-GP5. On the basis that GP5 expression does not affect cell proliferation, the effect of GP5 stable expression on virus replication was investigated by N gene copies, N protein expression, and viral titers in supernatant after PRRSV SD16 infecting Marc-145-GP5 cells. IFN-α, IFN-β, and IFN-γ protein levels were analyzed using monkey IFN ELISA kit. Then three pairs specific small interfering RNA (siRNA), siRNA GP5-471, siRNA GP5-404 and siRNA GP5-525, targeting ORF5 and one pair negative control siRNA, scrambled siRNA, were designed and synthesized. siRNAs were transfected into Marc-145 cells and then the cells were infected with 0.1 multiplicity of infection (MOI) PRRSV SD16 strain at 6 h or 12 h after transfection. The cells were collected at 24 h after infection and total RNA or protein were extracted. GP5 mRNA level was detected using real time PCR (qPCR) and N protein level was analyzed using Western blot, respectively. RT-PCR, Western bolt and IFA detection results showed that GP5 was stably expressed in Marc-145 cells and did not affect cell proliferation. But GP5 expression in Marc-145 cells promoted the replication of PRRSV in early infection time through down-regulating of IFN production. Transfection with specific siRNAs targeting ORF5 inhibited PRRSV replication and siRNA GP5-471, at a concentration of 100 nmol·L^-1, showed the most effective inhibition and had highly significant difference compared to negative control siRNA treated group. These findings suggest that GP5 plays a regulatory role in PRRSV replication.

The aim of this study was to explore the biological characteristics and pathogenic of a NADC30-like PRRSV. Thirty days old piglets confirmed to be free of PRRSV and PCV2 were inoculated with a NADC30-like PRRSV FJZ03 strain and a HP-PRRSV FJLYDX strain, respectively. The clinical symptoms, rectal temperature, virus excretion, virus loads in the sera, and tissues lesions were monitored and recorded. We found that FJZ03 strain could be rapidly replicated and developed higher titer of viremia in piglets. Moreover, the time of rectal temperatures rose and virus excretion in FJZ03 infected piglets were earlier than that of FJLYDX infected. Furthermore, piglets infected with FJZ03 displayed severe interstitial pneumonia and spleen atrophy. Cytokine test results showed that the cytokines concentrations of sera of FJZ03 infected piglets increased at different degrees. The IL-10 and TNF-α concentration in FJZ03 group was obvious higher than that of the FJLYDX group. Therefore, our results indicated that FJZ03 is pathogenic for piglets.

The aim of this study was to construct a double gene fusion plasmid of the membrane (M) and nucleocapsid (N) genes of variant porcine epidemic diarrhea virus (PEDV) and analyze the immunogenicity of the expressed proteins. The M and N genes were amplified by PCR from PEDV variant isolate, FJFQ2014(GenBank No.KJ646580). First, the M gene was cloned into pEASY-Blunt E2 (pE2) vector to construct pE2-M plasmid. Then the amplified N gene and E2-M gene of expression vector pE2-M were cloned into the prokaryotic expression vector, resulting a recombinant plasmid pE2-M-N. The plasmid was transformed to Transetta (DE3) for protein expression. The expressed product was identified by SDS-PAGE and Western blot. The BLAB/c mice were immunized with the purified PEDV M-N protein and the antibody titer was determined by ELISA. The results indicated that PEDV M-N fusion protein was successfully expressed in Transetta (DE3) and had immunogenicity, and induced anti-PEDV antibody in mice. The pE2-M-N plasmid of variant PEDV was constructed and expressed successfully. This study lays a foundation for the development of the serodiagnostic methods and immunology research for PEDV.

In order to obtain polyclonal antibody against porcine deltacoronavirus (PDCoV) spike-1(S1) protein, we cloned the gene (1-1 566 bp), and then expressed the Sa protein (106-1 290 bp) of S1 gene epitope prediction region successfully. The S1 gene epitope prediction region Sa (106-1 290 bp) was cloned into the prokaryotic expression vector pET22b(+) to construct recombinant plasmid pET22b-Sa by using the bioinformatics software to analyze the S1 nucleotide and the amino acid sequence. The recombinant expression vector was transformed into Rosetta (DE3)competent strain, and the recombinant protein was acquired by induction of IPTG. The recombinant protein was purified by ultrafiltration column concentration and immobilized Ni ion affinity chromatography. The activity of the protein Sa obtained was examined by Western blot. New Zealand rabbits were immunized with the Sa protein 4 times to prepare polyclonal antibody. The results showed that the open reading frame of S1 gene of CHN-2015 strain was 1 566 bp (GenBank No.:KY398009), encoding 522 amino acids. It is a non-transmembrane protein with hydrophilicity and 15 potential B cell epitopes Bit;The recombinant protein was expressed in prokaryotic expression. The protein was mainly expressed in soluble form at 37℃ for 5 h at the final concentration of 0.8 mmol·L^-1 IPTG. Immunoblotting results showed that the expressed Sa protein had good immunogenicity in the supernatant and inclusion bodies. The rabbit anti-Sa antibody titer was up to 1:10 240. The results showed that the expressed Sa protein contained antigen epitope recognition region of porcine Deltacoronavirus, and had a good immunogenicity.

In order to understand the epidemic status and phylogenetic relationship of porcine circovirus type 3 in healthy slaughter pigs, 181 lymph nodes collected from slaughterhouse in Guangxi were detected for PCV3 pathogens. PCV3 infection in healthy slaughter pigs was detected by PCR method. The positive rate of PCV3 was 24.31%, which suggest that the infection of PCV3 in healthy pigs is more severe. Six PCV3 complete genome sequences were amplified with PCR in the positive samples.Compared with PCV3 complete genome in GenBank and the results showed the nucleotide homology of PCV3 sequences was 98.6%-99.9% and genes of PCV3 were very conservative and similar. Genetic evolution analysis of PCV3 ORF2 gene showed that PCV3 strains could be divided into two major types. Analyses of the phylogenetic relationship of PCVs Cap and suggesting cross protection between PCV2 and PCV3 is impossible.

To study the relationship of autophagy and apoptosis after PK-15 cells were infected by pseudorabies virus (PRV), porcine host anti-apoptotic family proteins (Bcl-2 and Bcl-xL) interacted with Beclin1 were further analyzed. Western blotting (WB) was taken to measure the expression of LC3 and P62 proteins; and the level of apoptosis in PK-15 cells infected by PRV was investigated by Flow cytometry. Finally, Co-immunoprecipitation (Co-IP) was used to investigate the interaction between Beclin1 and Bcl-2/Bcl-xL. The expression of LC3-Ⅱ significantly increased, while that of P62 and p-AKT (S473) significantly decreased. In addition, the results of Flow cytometry showed that apoptosis was induced by PRV. Meanwhile, cleaved Caspase-9/8 significantly increased compared to control group detected by WB. Moreover, Co-IP results showed that the infection of PRV prevented the association between Beclin1/Bcl-2 and Beclin1/Bcl-xL. PRV induces autophagy which may depend on PI3K-I/AKT and apoptosis which simultaneously active Caspase-9 and Caspase-8, respectively. Autophagy might be induced through the interactions of Beclin1/Bcl-2 or Beclin1/Bcl-xL.

Up to now, the roles of cathepsins in the life cycle of Porcine reproductive and respiratory syndrome virus (PRRSV) remain unclear. This study was conducted to characterize the mechanisms of cathepsins involved in PRRSV life cycle including adsorption, invasion, uncoating, biosynthesis, assembly and release. The Chlorocebus sabaeus embryonic kidney epithelial cells Marc145 (PRRSV can be propagated) derived from MA-104 was selected. Each cathepsin shRNA clone was constructed within the lentivirus plasmid vector pLKO.1, and the shRNA library consists of sequence verified shRNA lentiviral plasmid DNA against all the cathepsin genes that allow for high throughput loss-of-function screens. Puromycin was used to select inserts in mammalian cells. Finally, there were 45 clones involving 15 cathepsin genes in this cathepsin-shRNA library, and the expression of cathepsin mRNA in the stable cell lines was markedly downregulated. These stable cell lines will lay the foundation for the further study on the roles of cathepsins in PRRSV life cycle.

The study was conducted to investigate the effect of LuxS/AI-2 quorum-sensing system on the biological characteristics of Salmonella enterica Serovar Enteritidis. A luxS mutant strain was constructed by Red homologous recombination system, and its ΔluxS gene stability, growth features, antimicrobial susceptibility, adherence and invasion, as well as biofilm formation were determined and compared with the wild type. Our results showed that the ΔluxS gene exhibits stability in ΔluxS mutant. The growth of ΔluxS mutant was slightly faster than the wild strain in vitro. Biofilm formation of the mutant strain was significantly enhanced as well as the adhesion and invasion ability to DF-1 cells and Caco-2 cells. Deletion of luxS resulted in a 128-to 256-fold increase in susceptibility to fluoroquinolones and moderate increases (2-to 8-fold) in susceptibility to ampicillin, tetracycline, doxycycline, chloramphenicol, and florfenicol. These results suggest that LuxS/AI-2 system affects biological characteristics of SE including biofilm formation, adhesion and invasion to cells, moreover, the AI-2/LuxS sensing system affect antimicrobial susceptibility of SE through certain resistance mechanisms.

The aims of this study were to identify genetic variants and construct haplotypes of polymorphic sites of ACTN3 gene exon in Yili horse, and determine different haplotypes effects on the mRNA expression level and protein secondary stucture of ACTN3, which would provide a theoretical basis for further researching the influence of the gene expression on athletic performance. Direct sequencing was taken to detect the polymorphic sites of of ACTN3 of 38 Yili horse and analyze its genotypes. The haplotypes and bioinformation were analyzed by haploview 4.2 and online software (RNAfold and sopm), respectively. A total of 8 SNPs were identified from the ACTN3 gene exon, only T9059G was the missense mutant, and the other 7 were synonymous mutants. Based on the 8 SNPs, linkage analysis showed that the strong linkage between A6206G and A11517G, and the different linkage relationship among the other SNPs. There was 14 haplotypes by the 8 SNPs, H1(GGATCCCG) was the dominant haplotype, and the different haplotypes could cause the change of mRNA secondary structure and minimum free energy of ACTN3 gene. The missense mutation of T9059G changed the secondary structure of ACTN3 protein. The result indicate that the T9059G is one of the improtant functional sites affecting athletic performance, which affect the secondary structure of ACTN3 protein.

The study aimed to investigate the expression and localization of annexin2 (ANXA2) and endoplasmic reticulum protein 29 (ERp29) genes in mammary gland tissue of normal and clinical mastitis sheep (Ovis aries). In this study, mammary gland tissues of 3 normal and 3 clinical mastitis sheep (Ovis aries) were sampled. qRT-PCR, Western blot (WB) and immunohistochemistry (IHC) were used to detect the expression and distribution of ANXA2 and ERp29 mRNA and proteins in mammary gland tissue of normal and clinical mastitis sheep.The results showed that the expression of ANXA2 mRNA in the mammary gland tissue of clinical mastitis sheep was significantly higher than that of normal group (P<0.01), while the expression of ANXA2 protein was significantly lower than that of normal group (P<0.01). The expression of ERp29 mRNA and protein in the mammary gland tissue of clinical mastitis sheep were significantly higher than that of normal group (P<0.01). ANXA2 and ERp29 proteins had positive reactions in mammary gland tissues of normal and clinical mastitis sheep respectively and mainly located in mammary epithelial cells. The expression of ANXA2 and ERp29 were significantly different in the mammary gland tissue of normal and clinical mastitis sheep, which may be involved in the regulation of the occurrence and development of sheep mastitis.

In order to diagnose and detect porcine circovirus type 3 (PCV3), in this research, specific primers were designed according to the conserved sequence of ORF2 gene. By optimizing the concentration of Bst DNA polymerase, the ratio of primers, Mg²⁺, dNTPs, betaine and reaction condition, the loop-mediated isothermal nucleic acid amplification (LAMP) was established. The results manifested that ladder strips were observed after 36min with a constant temperature, and the product could be judged by SYBR Ⅰ staining. The detection limit of this way was 1.0×10¹ copies·μL^-1 which had no cross reaction with porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine circovirus type 1 (PCV1) and type 2 (PCV2). From 68 lymph nodes of diseased pigs in respiratory system, the positive rate of 30.9% tested by LAMP had a high coincidence rate of 95.6% (65/68) with that by PCR detection. Compared with traditional technology, this method is more sensitive, simpler and can be used for detection and clinical diagnosis of PCV3.

To detect the Porcine Circovirus type 3 (PCV3) sensitively, rapidly and specifically, four pairs of primers were designed targeting the conserved region of PCV3 ORF3 gene. SYBR-based quantitative PCR results showed that Real-time PCR with all primer pairs can detect viral genome efficaciously. Then a TaqMan probe were designed according to the PCR products intersection sequence, and a real-time quantitative PCR method was established by optimizing the reaction conditions and systems. The method presented a good linear relation with the Pmd18-t-Cap vector(Recombinant plasmid harboring PCV3 ORF2 gene) ranging from 1.29×10²-1.29×10⁹ copies·μL^-1. The assay was highly specific for PCV3, without cross-reaction with other porcine virus and bacteria, such as Porcine circovirus 2(PCV2), Porcine reproductive and respiratory syndrome virus(PRRSV), Porcine rotavirus(PoRV), Haemophilus parasuis(Hps), Actinobacillus pleuropneumoniae(App). And the limit detection of this assay was 1.29×10² copies·μL^-1, more sensitive than the conventional PCR. The CV (Coefficient of Variation) of intra-assay and inter-assay were less than 2%, showed that the assay was reproducible. The real-time qPCR was further used to detect the DNA sample extracted from clinical sample collected from 2016 to 2017, the PCV3 positive ratio was 8.06% (10/124), and the co-infection rate of PCV3/PCV2 was 8.06% (10/124). The real-time qPCR assay provides a high sensitivity and specificity, repeatability method for detection of PCV3.