This research was conducted to predict the intramuscular fat percentage in longissimus muscle of live Beijing Black pig using real-time ultrasound image. The loin muscle area across the 10th to 11th rib, body weight, backfat thickness, loin muscle deepness and two longitudinal realtime ultrasound images were collected across the 9th to 13th rib and 5 cm off-midline on live pigs from 382 Beijing Black pigs. Gray gradient, gray level cooccurrence matrix and wavelet transform parameters whinin a defined region(80×80 pixel region across the 10th to 11th rib) for each ultrasound image were obtained using image analysis software (Matlab). After slaughter, a slice of longissimus muscle from left carcass across the 10th to 11th rib was cut off immediately for determining the intramuscular fat percentage (IMF) by the petroleum ether extraction method. The model to predict longissimus muscle intramuscular fat percentage (PIMF) was developed using linear regression analysis with carcass longissimus muscle intramuscular fat percentage (IMF) as dependent variables and body weight, backfat thickness, loin muscle area, loin muscle deepness and image parameters as independent variables. One hundred and twelve Beijing Black pigs were anew chose for model validation by correlation analysis of real intramuscular fat percentage and predicting intramuscular fat. The result of regression analysis indicated that 9 independent variables containing backfat thickness, loin muscle area and 7 image parameters were significant (P<0.05) in last model. The coefficient of determination and root mean square error for the prediction model were 0.305 8 and 0.006 5. The correlation analysis showed that the Pearson Correlation Coefficients and Spearman Correlation Coefficients were 0.553 4 and 0.627 2 (P<0.000 1). The result indicated that using real-time ultrasound image to predict intramuscular fat percentage in live pig was feasible. And this method can be used in Beijing Black pigs’ breeding work and developing the intramuscular fat effectively.

To investigate whether NMMHC-ⅡA was related to the resistance to PRRSV, the partial region of NMMHC-ⅡA gene 954 bp encoding the C-terminal region of NMMHCⅡA protein including 23 amino acid residues bound to PRRSV in hybrid pigs(Duroc×Yorkshire×Landrace), Laiwu and Dapulian pigs was cloned; then its homology with other species，the differences at nucleotide and amino acid sequences level between pig breeds and mRNA expression pattern in porcine tissues were analyzed. The results showed that NMMHC-ⅡA cDNA sequence was highly conservative among mammalian species, NMMHC-ⅡA had high homology with human, rat, mouse, cattle, monkey and dog, respectively at nucleotide and amino acid levels. Alignment of nucleotide sequence of NMMHC-ⅡA gene among Laiwu, Dapulian and Duroc×Yorkshire×Landrace crossbred commercial pigs revealed three nucleotide changes, however, the amino acid sequence was identical. NMMHC-ⅡA mRNA expression was analyzed in different tissues by RT-PCR, its expression level was high in skeletal muscle, lung, liver, kidney, spleen, bronchial lymph node, tonsil, stomach, large intestine, small intestine tissues, low in heart and cerebrum, and nearly undetectable in cerebellum and uterus. The results reveal that there is no differences in the function of NMMHC-ⅡA gene among Laiwu, Dapulian and Duroc×Yorkshire×Landrace crossbred commercial pigs, and it is expressed in many tissues except cerebellum and uterus. The role of NMMHC-ⅡA in resistance to PRRSV requires further study.

The purpose of this study was to detect the mitochondria distribution and ultrastructure changes that resulted from vitrified porcine MII-stage oocytes with three vitrification techniques. Through transmission electron microscopy (TEM), R-123 (Rhodamine-123) staining and in vitro development, mitochondria distribution and ultrastructure were observed. The result showed that: (1) Given the survival rate of FDA (fluorescein diacetate)-DAPI (4′,6′-diamidino-2-phenylindole dihydrochloride) staining and the cleavage rate after parthenogenetic activation, CLV (Cryoloop vitrification) method (72.00%, 7.22%) got the best results. OPS (Open Pulled Straw,60.00%, 4.85%) and straw (42.22%, 0%) methods were followed by; (2) The rate of normal mitochondria distribution in CLV group (52.24%) was higher than that in the two others groups (OPS, 48.65%; straw, 37.68%) , but there was no significant differences among them (P>0.05); (3) Vague mitochondria in thawed oocytes were observed in the ultrastructure of vitrified oocytes from TEM, and some mitochondrial ridges were reduced or even disappeared. The result indicated that mitochondria was damaged greatly in the freezing process and CLA method could improve the freezing speed and minimize such damages.

This study was designed to investigate the role of chicken A-FABP gene in lipid metabolism of adipocytes and the regulation relationship between A-FABP gene and the genes related to lipid metabolism. The shRNA interference vector with A-FABP gene and eukaryotic expression vector were used. Chicken adipocytes with short hairpin RNA-mediated knockdown and A-FABP cDNA overexpression were used. The genes expression level in chicken adipocytes were detected at 24, 36, 48, 60 and 72 h after transfection with the interference and overexpression plasmids of A-FABP. The results showed that the expression levels of PPARγ and perilipin genes were downregulated at 24 and 36 h and upregulated at 48 h in the A-FABP interference group. The expression levels of E-FABP genes was downregulated at 48 and 72 h in the A-FABP interference group. The expression levels of PPARγ and perilipin genes were upregulated at 24 and 36 h and downregulated at 48 h in the A-FABP overexpression group. The expression levels of E-FABP genes was downregulated at all detected time pionts in the A-FABP overexpression group. These results indicated that A-FABP might affect expression of PPARγ, perilipin and E-FABP in chicken adipocytes.

Seven muscle and adipose related miRNAs, including miR-1, miR-133, miR-24, miR-122, miR-103, miR-143 and let7 were selected to analyze their expression and target expression profiles. Muscles RNA from back and hind leg muscle of 4 cashmere goats were extracted, after reversetranscription, SYBR TaqMan PCR was used to detect gene expression, and miRNA target expression profiles were got by PAGE electrophoresis and MiRNA Targets Figure Print (MTFP) technique. The result showed that the expression of miRNA were as follows:miR-1>miR-133>miR-24>let7>miR-143>miR-103>miR-122, the expression patterns of miR-1 and miR-133 were similar,and the expression levels of all miRNAs in adult goat were higher than that in lamb; moreover, the difference of miRNAs expression levels were also affected by gender, and they had relationship with number and expression levels of target genes. 10 target genes in 5 miRNAs were sequenced and Blast annotation, which coded proteins related to signal transduction, cell cycle, and so on. The expression analysis of 7 miRNAs and their target genes showed differences and similarities between muscle development related miRNAs and adipose development related miRNAs in cashmere goat skeletal muscle, which were favorable to the research for meat quality related genes in cashmere goat.

 The aim of this study was to construct expression vector containing Lactoferricin gene and interferon-α gene which was transfected into the bovine fetal fibroblasts. The transgenic cells got would be used as the donor cells for SCNT to produce the transgenic bovine that would resist mastitis disease and FMD. The vector with a bovine Lactoferrincin B cassette containing a goat β-casein regulatory sequence, a human interferon-α cassette containing the immediate early promoter of CMV, the EGFP gene as reporter and the neo gene as positive selection marker, was constructed. FuGENE Transfection Reagent and Amaxa Nucleofector were used in transfection. After 48 hours，the cells were selected with G418 and detected by PCR. The expression of interferon-α protein was detected by Western blotting. The transgenic bovine fetal fibroblast cell lines were constructed in which Lactoferricin and interferon-α genes were integrated and the expression of interferon-α protein was detected.

The aim of this study was to study the change of DNA methylation around STAT5-binding region in the far upstream promoter and its effect on gene expression of the bovine CSN1S1 during Staphylococcus aureus mastitis in dairy cattle. The methylation status of the promoter and the expression of CSN1S1 mRNA were detected by the bisulphite modification technique and the real-time quantitative PCR at 0,12,24,36 and 196 h after intramammary inoculation with Staphylococcus aureus.The result showed that the level of methylation gradually increased with time within 24 h,while the expression of CSN1S1 mRNA were significantly down-regulated in infected mammary tissue（P<0.01）, at 36 and 196 h after infection, the methylation status and gene expression levels were no significant difference from that at 24 h.The results suggest that DNA re-methylation of mammary tissue can be caused by Staphylococcus aureus mastitis, DNA re-methylation may be a general regulation of the acute phase of bovine mastitis.

To further study the regulatory mechanisms of Cholesterol 7α-hydroxylase (CYP7A1) in bile acid synthesis and cholesterol metabolism, the complete cDNA of CYP7A1 gene(2 279 bp) was cloned by RT-PCR and RACE from goose (Anser anser) liver. The CDS of CYP7A1 gene was sub-cloned into pET-28a to construct pET-28a-cyp7α1 plasmid. The recombinant plasmid pET-28a-cyp7α1 was transformed into E.coli BL21 (DE3) and the recombinant protein expression was induced by IPTG. The recombinant protein His-CYP7A1 was purified with His-Bind Purification Kit. The recombinant protein was immunized into the Bal b/c mice to obtain the polyclonal antibodies. Antigenic of His-CYP7A1 and the specificity of mouse anti-goose CYP7A1 polyclonal antibodies were detected by Western blotting. The amino acid sequence of goose CYP7A1 protein has high homology with that of chicken(93%), rat(66%), human(67%), monodelphis domestica(68%) and mouse(66%). The SDSPAGE analysis result of His-CYP7A1 showed that the recombinant protein molecular size was about 59 ku, consistent with the predicttion. Highpurity His-CYP7A1 protein was obtained through His-tag affinity purification.The mouse anti-goose CYP7A1 polyclonal antibodies were obtained with high sensitivity (1∶10⁴). The mouse anti-his and anti-goose CYP7A1 antibodies could recognize antigens of His-CYP7A1 by Western blotting with mouse anti-goose CYP7A1 polyclonal antibodies with better specificity. The current results contribute to further understanding of gene structure and function of CYP7A1 and provide an important molecular tool for the functional study of CYP7A1.

This study was conducted to investigate the effect of Metformin on PRKAG3 gene expression, growth performance and pork quality in pigs. 10 DLY pigs (80 kg) were randomly divided into control and experimental groups, pigs in experimental group fed diet supplemented with Metformin at dose of 400 mg·kg^-1 body weight. After 2 weeks, all pigs were slaughtered to determine growth performance, AMPK activity, PRKAG3 gene expression, AMPKγ3 protein levels and pork quality traits. The results showed that, compared with control group, experimental group had 13.92% higher AMPK activity (P<0.05), 4.39-fold PRKAG3 gene expression (P<0.05) and 35.53% higher AMPKγ3 protein level (P<0.05). Pigs in experimental group had 13.80% lower feed intake (P<0.05), 13.14% lower average daily gain and 6.67% higher F/G than that in control group. Experimental group reduced dressing percentage, lean percentage, eye lean area, and backfat thickness（P>0.05）, and significantly reduced carcass length(P<0.05). Compared with control group, experimental group had lower pH2, drip loss, a values, higher pH1, shear force and cooked meat percentage, but there was no significant difference between control and experimental groups（P>0.05）. Experimental group significantly increased L and b values (P<0.01). These results suggested that supplementary 400 mg·kg^-1 body weight Metformin in diet activated PRAKAG3 gene expression, which affected the growth performance and pork quality.

The objective of this experiment was to evaluate the effect of ambient ammonia and humidity on antioxidant capacity and meat quality of broiler chickens. One hundred and ninety-two 21-day-old AA male broiler chicks were randomly allotted to two equal groups exposed to a high ambient ammonia (70 mg·kg^-1, H) and a low ambient ammonia (30 mg·kg^-1, L). Half of the broilers from each group were treated with relative humidity 60% (Control, C) and 35% (Treatment, T). Thus, four treatment groups named C+L, C+H, T+L and T+H, respectively. Growth performance of birds was recorded from 21 d to 42 d; The antioxidant capacity of blood and meat, meat quality were also determined. The results showed that average final BW, ADG, ADFI, T-AOC of blood and pectoralis muscle, and a^* value in pectoralis muscle at postmortem 45 min were significantly lower (P<0.05), while TBARS at postmortem 5 days, drip loss ratio, b^* value (P<0.05) and L^* value (P=0.054) at postmortem 45 min and shear force of pectoralis muscle(P=0.075) were increased when birds exposed to high level of ambient ammonia(70 mg·kg^-1) . Average final BW, ADFI (P<0.05), and ADG (P=0.072) as well as activity of SOD and GSH-Px in pectoralis muscle (P<0.05) and L^* value 45 min after slaughtering (P=0.053) were declined,while TBARS at postmortem 5 and 7 days and shear force of pectoralis muscle(P=0.057) were increased when birds exposed to low RH (35%) . Compared with C+L, the average final BW, ADG, ADFI, T-AOC of blood, activity of SOD and GSH-Px in pectoralis muscle, a^* value at postmortem 45 min were significantly lower (P<0.05),while TBARS at postmortem 5 and 7 days, drip loss ratio and shear force of pectoralis muscle were increased (P<0.05) when birds exposed to T+H. The results of this research suggested that high level of ammonia and low relative humidity in poultry house had adversely effects on growth performance, antioxidant capacity and meat quality of broilers，and the adversely effects of high level of ammonia could be enhanced under low relative humidity.

The study was conducted to investigate the effects of different levels of the whole cottonseed taking the place of the cottonseed cake in the diets on meat performance in fattening Limmonsin crossbred steers. Forty healthy steers with similar weight (454.37±42.42)kg were randomly allocated to four treatments with ten replicates each and one steer in each replicate, precise feeding concentrates with different levels of the whole cottonseed during the 90 days finishing periods. The results showed that the different levels of the whole cottonseed in diet had no signification influence on feed intake, the ratio of bone and meat, the percentage of the fine quality beef, pH, gossypol residue in muscle, kidney and lung, serum biochemical indexes(P>0.05). With the levels of the whole cottonseed in concentrates changed, the average daily again(ADG), carcass weight, dressing percentage, net meat percentage, LM area, backfat thickness, feed conversion efficiency, shear stress, gossypol residue in liver and heart, meat color had changed significantly(P<0.05). When the content of the whole cottonseed reached 22.0%, the carcass weight, dressing percentage, net meat percentage, ratio of meat and bone, LM area, high percentage of the beef, the intermuscular fat was the lowest. In conclusion, the whole cottonseed replacing cottonseed cakes and oil in fattening crossbred steers’ diets had no effect on rapidly fattening, but it depressed carcass quality.

To research the mechanism of porcine reproductive and respiratory syndrome virus (PRRSV) infection mediated by immune complex, we investigated the effects of immune complex on the factors which affect the multiplication of PRRSV. In this study, 200 TCID₅₀ was inoculated in PAM cells, with an equal volume rabbit anti-pig IgG-IgG complex, which final concentration was 1.30 mg·mL^－1，and was added to the PAM cells before, after or with PRRSV infection. Cells and the supernatant were collected at 12, 24, 36 and 48 hours, respectively. While we also set the control group with PRRSV infection, immune complexes group and the healthy cells group, apart. The mRNA levels of IFN-α, IL-10 and TNF-α were detected by establishing relative quantitative PCR method. The PRRSV RNA copies of 48 hours were detected by establishing absolute quantitative PCR method. And the quantitative data was analysed. At the same time, the protein quantity of IFN-α was detected by the ELISA method. The immune complex could enhance the multiplication of PRRSV in PAM absolutely (P<0.05). At the same time, after the infection of 48 hours, the immune complex could raise the mRNA levels of IFN-α and TNF-α, but before the 36 hours, it had no evident regulation, while in all the times, the immune complex could decrease the mRNA level of IL-10. The protein level of INF-α is showed as a "down-up-down" tendency in healthy PAM cells during 12 to 48 hours. The result indicated that the expression and transcription of IFN-α was not happened at the same time. While PRRSV infected at the early stage, the immune complex could suppress the transcription levels of antiviral cytokines induced by PRRSV infection, and the virus' copy was promoted. But at the later, the enhancement of PRRSV was inhibited owing to the high concentration of antiviral cytokines. And the transcription of TNF-α induced by the immune complex had no obvious regulation. It might be reasoned for that the immune complex could combine with both activated and inhibitory FcγR to jointly regulate the cytokines transcription induced by PRRSV.

 Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of the ongoing “porcine high fever syndrome” in China, is capable of genetic and antigenic mutations at high frequency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great interest. In this study, based on an infectious cDNA clone of an attenuated TypeⅡ PRRSV strain pAPRRS and the highly pathogenic PRRSV cDNA clone pJX143, we replaced the coding sequence of pAPRRS nsp2 with those of the HP PRRSV to develop a series of chimeric clones. Upon transfection of chimeric clones into MA104 cells, typical PRRSV cytopathic effects were observed. This study provided a valuable tool to develop the chimeric PRRSV as vaccine candidate offering cross-protection to HP PRRSV strains. Furthermore, the infectious chimeric cDNA clone provides a powerful tool to molecular dissection of the mechanism of pathogenesis of the increasing-virulence of the on-going prevalent PRRSV in China.

The aim of this study was to establish a diplex xMAP array, which can detect porcine parvovirus (PPV) and porcine circovius type 2 (PCV2) simultaneously, to meet the requirements of the immigration quarantine and clinical high-throughput detection. According to the structural protein VP2 gene of PPV and PCV2 genome sequences in GenBank, specific probe, primers and probe’s reverse complement sequence of PPV and PCV2 were designed after multiple sequence alignment analysis. The probe were coupled to microspheres, and the reverse primers, probe’s reverse complement sequence were labeled with biotin. The target sequences were amplified by diplex asymmetry PCR, then products of PCR were hybridized with the probes which coupled verify successful, hybridization results were detected by the xMAP instrument, then the diplex xMAP array, which was established through optimizing the reaction conditions, were used to detect clinical samples. The diplex xMAP array, for detecting PPV and PCV2, was established and had a good specificity and sensitivity, and limit of detection were 1.53×10⁴ and 2.58×10² in gene copy respectively. The detection results of 56 clinical samples showed that this method was 100% consistent with PCR. This study established the diplex xMAP array for detecting PPV and PCV2 successfully; the methods could be used for the immigration quarantine and veterinary clinical detection.

The Porcine Bocavirus was found in Sweden in 2009, which belongs to Parvovirus subfamily, Parvoviridae with singlestranded DNA. As a new kind of virus, the studies on Porcine Bocavirus is in the stage of preliminary exploration by humans. The paper has described the source, discovery, classification, genome and epidemiology of Porcine Bocavirus. According to the GenBank including partial and complete gene sequences of 25 Porcine Bocaviruses, we put analysis of arranging the group of homology on NS, NP, VP and complete gene sequences about PBoV with analysis software of DNAStar, arithmetic Jotun-Hein. And we established the PBoV system of Phylogenetic tree. The result revealed that the PBoV includes two large branches and many hereditary clusters. But there are some differences in the nucleotides sequences of different hereditary clusters containing strains.

The objective of this study was to investigate the positive rate for antigen P27 of Avian leukosis virus in eggs from different breeder groups and its correlation with the incidence of avian leukemia both in the breeder chickens and its next generation. It is hoped that this study will be helpful for the breeders to estimate the risk of avian leukosis virus infection. Eggs or sera samples from 7 domestic breeder groups from Tianjin, Shandong, Hubei, Hebei province were collected and detected for antigen P27 of ALV and antibodies to ALV-A/B and ALV-J respectively. Some samples with tumors were isolated for exogenous ALV. The tumor report and complaint for the next generation of each breeder were tracted and analyzed. The positive rate of antigen P27 in eggs of different breeders is quite different and the positive rate range from 0 to 12%, positive rate for antigen P27 in eggs closely related to the incidence of ALV infection both for the breeders and its next generation. That is, the higher it is, the more serious for ALV infection and the more complaints from customers. The positive rate for antigen P27 in eggs of breeders conform well to virus isolation and tumor incidence and can be used as an important index to estimate the risk of ALV infection.

The objective of this study was to understand the dynamic distribution pattern of G. anatis in chicken and the effect of chicken infectious bronchitis virus (IBV) on the distribution pattern. In the present study, SPF layer chickens were inoculated with G. anatis or/and chicken infectious bronchitis virus (IBV). Quantitative PCR (qPCR) with SYBR Green I was used to detect the DNA of G. anatis in different organs at different time point from chicken after artificial infection. In G. anatis group, G. anatis DNA was firstly detected in the trachea, heart, spleen, ovary and kidney of chickens 12 hours post inoculation (PI), in the liver, duodenum and oviduct 24 hours PI, in the lung 48 hours PI, and in the palate cleft 72 hours PI, respectively. In-mixed infection group, G. anatis DNA was detected 12 hours PI in the ovaries, and 24 hours PI in all the organs. The highest content of G. anatis DNA was noticed in ovary and the highest detection rate was found in trachea of birds. Mixed-infection group had significantly higher qPCR detection rate of all organs than G. anatis group. G. anatis could cause systemic infection of the birds, and coinoculation of G. anatis and IBV increased the systemic infection. IBV promoted the spread of G. anatis, moreover, IBV benefited the multiplication and pathogenicity of G. anatis in the duodenum and thus resulted in clinical diarrhea. It was further confirmed that the pathogenicity of G. anatis mainly focus on the respiratory and reproductive systems. Trachea and ovaries were the target organs of G. anatis.

This study aims to develop a new kind of bivalent genetic engineering vaccine used to control Streptococcus suis (SS) disease and Haemophilus parasuis (HPS) disease, and to evaluate the vaccine's immune effect. Four proteins, Streptococcus suis serotype 2 proteins SLY, HP0197, and Haemophilus parasuis outer membrane proteins D15 and HPS06257 are selected, recombinant expressed, purified and added adjuvant of certain proportion to be made with different components of subunit vaccines. In the present study, the Streptococcus suis serotype 2-Haemophilus parasuis (SS2-HPS) bivalent subunit vaccine and their single vaccines could elicit a significant humoral antibody response and confer significant protection against challenge with lethal dose of SS2 or HPS in mice, respectively. In addition, the hyperimmune sera against these vaccines could efficiently kill the bacteria in the opsonized phagocytosis test and confer significant protection against SS2 infection in the experiment of passive immunization. The study shows this SS-HPS bivalent subunit vaccine has good immune protection in mice.

The aim of this study was to investigate the taxonomic relationships of ascarid nematodes from Ailuropoda melanoleuca, Ailurus fulgens, Ursus maritimus, Ursus arctos pruinosus, Ursus thibetanus mupinensis, Ursus arctos lasiotus, Pan troglodytes and Hylobates hoolock. The mitochondrial cytochrome coxidase subunitsⅠ(COXⅠ) and Ⅱ (COXⅡ) were amplified by PCR and sequenced directly. Then, sequence analyses was carried out based on COXⅠ and COXⅡ sequences from ascarid nematodes acquired in our study and 12 homologous sequences from GenBank. The lengths of COXⅠ and COXⅡ in ascarid nematodes from the eight species of captive animals were all 393 bp and 582 bp, respectively. The COXⅠ and COXⅡ of ascarid nematodes from giant panda shared 94.8%-95.0% and 94.9%-95.5% sequence identity with these from A. fulgens, U. maritimus, U. arctos pruinosus, U. thibetanus mupinensis and U. arctos lasiotus, respectively. Additionally, the COXⅠand COXⅡ genes of ascarid nematodes from P. troglodytes shared 99.8% and 99.5% identity with the nematodes from H. hooloc, respectively. Phylogentic analysis showed that ascarid nematodes from A. melanoleuca, A. fulgens and four Ursids species belonged to the genus Baylisascaris, while roundworms from H. hoolock and P. troglodytes clustered under the genus Ascaris. These results showed that a co-evolutionary relationship may link the ascarid nematodes and their corresponding hosts together.

This study was designed to seek the activation effect on T lymphocytes by dendritic cells pulsed with recombinant VP4 protein of foot-and-mouth disease virus (FMDV). To this end, we have successfully constructed the prokaryotic expression vector of pET32a-VP4. The recombinant VP4 protein was expressed with induction of IPTG. VP4 protein was purified by SDS-PAGE and electro-elution approaches and identified with Western blot. Bone morrow-derived dendritic cells (BMDCs) were generated in vitro and pulsed with purified VP4 protein. The VP4-loaded BMDCs were co-cultured with lymph node T cells. Culture supernatants were harvested at indicated time points and the levels of IFN-γ were assayed with ELISA. The results showed that the IFN-γ levels of test groups were higher than that of control groups significantly at 3, 9, and 48 hours after co-culture. These data indicate that BMDCs pulsed with FMDV VP4 protein are able to efficiently stimulate T lymphocyte activation and consequently initiate Th1-like response with enhanced secretion of IFN-γ.

The aim of the present study was to express goat mature interleukin-4 gene in E. coli and to test the bioactivity of the recombinant protein. The mature GIL-4 (mGIL-4) was amplified using pMD18-T-pGIL-4 (pGIL-4, goat IL-4 complete sequence) as template by PCR. The recombinant plasmid of pET-32a(+) containing mGIL-4 gene was transformed into E. coli BL21, and induced by IPTG. The recombinant protein expressed in E. coli was purified by modified method and refolded. Lymphocyte transformation test was performed to detect the bioactivity of recombinant protein to stimulate the proliferation of T lymphocytes in goat’s peripheral blood mononuclear cells (PBMC). Sequence analysis of mGIL-4 demonstrated an open reading frame (ORF) of 339 base pairs encoding for 112 amino acids. The recombinant protein was approximately 31.3 kD and was mainly existed in the inclusion body. Through improved purification methods, highly purified recombinant protein was obtained. The bioactivity test proved that purified recombinant protein was able to significantly stimulate the proliferation of goat peripheral blood T lymphoblasts in a dose-dependent manner and the recombinant protein of 8 μg·mL^-1 presented the highest stimulation. The data provided foundations for the scale production and the application as adjuvant of the goat recombinant interleukin-4.

 To explore the antagonism mechanism of the specificity antagonist for miniature pigs combined anaesthetic, the effect of the specificity antagonist on the c-jun gene transcription in the hippocampus of rats anesthetized with XFM was studied in this experiment. Ninety adult SD rats were divided randomly into three groups: XFM group, XFM add saline group and XFM add the specificity antagonist group. Each group consists of five subgroups, and samples were collected at different time points. The transcription of c-jun gene mRNA in every sample was detected with real time PCR. The result showed that The c-jun gene mRNA contents in each time point of the XFM group were obviously increased as compared with 0 min (P<0.01). There were no significant effect of injection of saline after XFM administrated on the c-jun gene mRNA transcription (P>0.05). The level of c-jun gene mRNA transcription after the specificity antagonist administrated was significantly lower than XFM group (P<0.01 or P<0.05). These results indicated that the specificity antagonist for miniature pigs combined anaesthetic attenuates the c-jun gene mRNA transcription in the hippocampus of rats induced by XFM, this may be related to the antagonism mechanism of the specificity antagonist for miniature pigs’ combined Anaesthetic.

This experiment was conducted to obtain the coding sequence of Banna Mini-pig Inbred Line (BMI) growth hormone (GH) gene and to study its prokaryotic expression. BMI GH gene sequence was cloned from pituitary by RT-PCR and inserted into pMD 18-T vector for sequencing and bioinformatics analysis. The length of GH cDNA sequence was 690 bp, which contained a CDS of 651 bp. GH CDS encoded 216 amino acids with 26 amino acids of signal peptide. It was found that the GH amino acids sequence shared 100%, 100%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 98% and 98% identity with those of Wuzhishan pig, Ningxiang pig, Tibetan pig, Xiang pig, Dawu pig, Taihu pig, Chenghua pig, Neijiang pig, Rongchang pig, Landrace, Yorkshire, Yanan pig and Duroc pig, respectively. Subcloned the GH encoding mature peptide sequence into the prokaryotic expressing vector pET-32a (+) directionally to constitute the recombinant plasmid pET-32a-GH, then it was transformed into the E.coli Rosseta bacteria for expression under induction of IPTG. SDS-PAGE and Western blotting analysis showed that recombinant plasmid pET-32a-GH was successfully expressed in high level. The fusion protein expressed in form of inclusion body and the molecular weight was approximate 40.6 ku. These results will lay the foundation for further studies of the effects of GH on the dwarfism of Banna Mini-pig Inbred Line.