The aim of the study is to clone the fulllength coding region of the porcine PNLIPRP2 (pancreatic lipaserelated protein 2) gene, analyze the molecular characteristic, and investigate its expression in the major tissues. The CDS of porcine PNLIPRP2 gene was cloned using the in Silico Cloning and RTPCR methods. The sequence was submitted to the GenBank. Many softwares (e.g., GENSCAN) and online resources (e.g., NCBI) were used for the analysis of bioinformatics and comparative genomics. The expression of PNLIPRP2 gene was investigated by RTPCR in 12 different tissues of 2monthold Large White pig. The fulllength CDS of the porcine PNLIPRP2 is 1 416 bp (EU715062) encoding a protein of 471 amino acids which contains a conserved PLAT domain of pancreatic triglyceride lipase (PLATPL). Porcine PNLIPRP2 is evolutionary more near to human and chimpanzee than other species. Further bioinformatics analysis showed that porcine PNLIPRP2 gene was located on porcine chromosome 14 and contained 12 exons and 11 introns. In addition, porcine PNLIPRP2 mRNA was the most abundant in intestine, followed by the mordrate level in brain and fat, the lowest level in bone marrow and stomach, and almost no expression in other tissues. The CDS fulllength of the porcine PNLIPRP2 gene was cloned. The molecular characteristic and its expression patterns in different tissues were also described. The results indicated that porcine PNLIPRP2 gene may be an important candidate gene in regarding to the fatty deposition and the immunity.

To study the effect of tumor necrosis factor α(TNFα) on lipolysis in differentiated porcine adipocytes and its mechanism, the differentiated porcine adipocytes were treated with TNFα at the concentrations of 1, 10, 100 ng·mL1 for 24 hours and the concentration of 10 ng·mL1 for 0, 1, 6, 12, 24 and 48 hours , respectively. Commercial kits were used to measure the glycerol release; Semiquantitative RTPCR was used to detect the expression levels of ATGL,TGH2,HSL,LPL,MGL and perilipin A genes. The results showed that TNFα promoted the glycerol release in dose and time dependent manners(P<0.05) in differentiated porcine adipocytes，and downregulated the mRNA expression levels of ATGL, TGH2, HSL, LPL, MGL and perilipin A. These results indicated that TNFα could stimulate lipolysis by downregulating the expression level of the perilipin A which was thought to modulate the ATGL and HSL to the surface of the fat droplet.

The purpose of this study is to provide more theoretical basis for the research of evolution of bovine and chromosome electrolocalization of some goat′s coat color genes. The homology of chromosomes among cattle, sheep and goat was analyzed using Phylogenetic Computer Tools and Phylip software based on markers located at their autosomes. Partial coat color candidate genes of goat were mapped using the method of comparative genomics and bioinformatics. The results indicated that similarity of homologous chromosomes among these species was 0.000 0-1.000 0. The coat color candidate genes of goat, Myo5a, Brca1, Sox10, Zic2, kit, Snai2, Wnt3a and Tyrp1 were oriented at chromosome 10, 17, 5, 12, 6, 14, 7, and 8,respectively.

The objective of the study was to clone avian βdefensin10 (AvBD10) gene from duck liver. In addition, expression of recombinant AvBD10 protein in E. coli and determination of its antimicrobial activity were investigated. The mRNA of duck AvBD10 was cloned from liver of duck by RTPCR. The mRNA differential expression of the AvBD10 gene has been demonstrated across a panel of tissues in duck. The cDNA of duck AvBD10 was subcloned into pGEX6p1 vector to construct recombinant plasmid pGEXduck AvBD10. The recombinant plasmid was transformed into E. coli BL21 and the bacteria were induced with IPTG. The recombinant fusion protein was purified by affinity chromatograph. Antimicrobial activity and physiochemical characteristics of the recombinant fusion protein were measured by inhibition zone assay. The results showed that the cDNA of duck AvBD10 consist of 169 bp encoding 55 amino acids. The duck AvBD10 had the highest amino acid homology (85.5%) with the chicken AvBD10. It was shown that the mRNA of duck AvBD10 just expressed in the liver and kidney of ducks at all ages investigated. The molecular weight of recombinant duck AvBD10 protein is about 30 ku (accounted for approximately 34% of the total proteins). The recombinant duck AvBD10 protein exhibit expected antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, and Pasteurella multocida after purified. In conclusion, AvBD10 was cloned from duck liver. It was showed that the mRNA of duck AvBD10 expressed highly in the liver and kidney of duck at all ages investigated. The recombinant duck AvBD10 protein exhibit broadly antimicrobial activity, and the recombinant protein retained high antimicrobial activity under different temperatures and pH.

This study was conducted to investigate the effects of concentrateroughage ratio in diets on concentration of VFA in rumen liquid, pH and digestive enzyme activities in different sections of alimentary canal. The twenty four crossbred weaned male lambs of Poll Dorset (♂)×(Tansheep × Small Tailed Han sheep) (♀) weighing (25.83±6.14) kg at the age of 3–5 months were divided similarly into three groups. They were fed on the diets with concentrateroughage ratio of 65∶35（A）,50∶50（B）and 35∶65（C）（digestible energy and crude protein were at 1.0, 0.9 and 0.8 times of NRC recommendation） respectively during the 40 days of regular experimental periods. Six lambs from each group were slaughtered for sampling at the end of regular feeding periods. The results showed that: (1) Concentration of total VFA (TVFA) in rumen liquid of lambs from group A was obviously higher than those from group B and C (P<0.05). (2) The pH of rumen liquid, homogenates of mucosa (7.03, 7.31 and 7.35) and contents (7.19, 7.30 and 7.58) in behindjejunum，and of contents in ileum(7.16, 7.33, 7.57) were affected significantly by the concentrateroughage ratio of diets（P<0.05）. (3)Molar proportions of acetic acid and propionic acid were ranged from 0.612 to 0.654 and from 0.185 to 0.190 respectively, the acetic / propionic ratios varied ranging from 3.38 to 3.57. (4) There were nonuniform pattern ranked of activities of various digestive enzyme along small intestine with concentrateroughage ratio of the diets. (5)The highest activities of chymotrypsin in content and those of lipase in mucosa homogenate and content occurred in the middlejejunum, activity of trypsinase in contents of behindjejunum was the highest, and the highest activities of αamylase were noted in content of duodenum and behindjejunum. It was concluded that still acetic acid pattern of rumen fermentation was kept in lambs fed total mixed diet with higher concentrateroughage ratio; the pH of contents in rumen and abomasums, of mucosa and contents in behind section of small intestine were affected by the concentrateroughage ratio of the diets; there was also higher activity of αamylase in duodenum.

The influence of maternal different dietary protein levels on muscle fiber and expression of Myostatin gene of offspring of two broiler lines was investigated. The maternal different dietary protein levels were 80%, 100% and 120% NRC level for maternal broiler breeder. The offspring was feed normal protein level diet. The results showed that: ⑴The maternal high protein diets increased the body weight of 1 day broilers significantly(P<0.05), but the body weight of 56 day broilers had no difference among treatments(P>0.05). ⑵ With the maternal protein levels decreased, muscle fiber diameter of breast of offspring was decreased, low protein group was significant lower than the normal and high protein groups(P<0.05), but there was no significant difference between the two lines. ⑶ With maternal protein levels decreased, muscle fiber density of breast of offspring was increased, low protein group was higher than the normal and high protein groups significantly(P<0.05), but there was no significant difference between
the two lines. ⑷The high protein diets of maternal broiler has a trend to decrease the expression level of offspring Myostatin gene（P>0.05）, compared to the normal and low protein groups, the expression level of Myostatin gene of lean line broilers breast of high protein group in 21 day was decreased significantly（P<0.05）.The results demonstrated that the maternal high protein diet increased the body weight of 1 day broilers significantly, and there are a trend that expression level of Myostatin gene was decreased in high protein group, the maternal low protein diet declined the muscle fiber diameter and increased the muscle fiber density, but there are no significant difference between the two lines（P>0.05）.

The objective of this study was to construct infectious clone of encephalomyocarditis virus (EMCV). The genomic cDNA of EMCV BJC3 was amplified by three overlapped segments using RTPCR, and cloned into lowcopy plasmid pWSK29 to construct fulllength cDNA clone pWSKBJC3/w. The pWSKBJC3/w was in vitro transcribed and transfected into BHK21 cells to rescue the virus. The results showed that the fulllength cDNA clone was infectious and the virus could be rescued on BHK21 cells. The rescued virus designated rVBJC3W was identified by RTPCR and indirect immunofluorescence assay (IFA). The rescued virus had similar growth characteristics with its parental virus BJC3 and remained the pathogenicity for mice. Our results indicated that the first infectious cDNA clone of EMCV in China was successfully established and provided an essential tool for investigating the molecular basis of pathogenicity of EMCV.

Pertactin (PRN) is identified as one of the most important protective immunogen of Bordetella bronchiseptica (Bb). To study the immunogenicity of region 1 (R1) of PRN, the complete coding sequence (2 040 bp) and its 5′terminal 1 173 bp fragment of the prn gene were separately cloned into the prokaryotic expression vector pGEXKG, and expressed in the E. coli BL21 (DE3) with induction by IPTG. The concentration of purified expression products, glutathione Stransferase (GST)R1 and GSTPRN were 312.6 and 207.3 μg·mL1, and the purity of them were 95.2% and 92.4%, respectively. The GSTR1 and GSTPRN showed the similar immunological reactivity in Westernblot analysis. Mice, immunized subcutaneously with two doses of purified GSTR1 or GSTPRN proteins mixed with an equal volume of Freund′s adjuvant, produced robust PRNspecific IgG antibody levels. In these two groups, all 9 mice survived intranasal challenge with three times the 50% lethal dose (LD50) of virulent Bb HH0809, whereas 3 of 9 and 7 of 9 mice survived challenge with ten times LD50, respectively. Furthermore, complete protection (10/10) against intraperitoneal (i.p.) challenge with ten times the LD50 of virulent HH0809 strain was seen in mice that were injected i.p. with 0.5 mL rabbit antiGSTR1 or antiGSTPRN serum, whilst there were no survivors in group of mice that received PRNabsorbed rabbit antiGSTR1 (0/5) or antiGSTPRN serum (0/5). In this study, we have shown that the recombinant GSTR1 protein had strong immunogenicity against Bb infection, which built a good foundation for the further research of highly efficient vaccine against bordetellosis.

To study the virulence characteristics of H. parasuis, we determined the 50% lethal doses (LD50) of H. parasuis serotype 5 strain in guinea pigs with the improved the Karber method. The LD50 was 1.33×109CFU, the 95% confidence range of the LD50 was 1.35×109-1.30×109CFU. Then guinea pigs were infected with 1 LD50 doses of H. parasuis by the nasal cavity, trachea, lungs, muscles, the abdominal cavity and subcutaneous inoculation respectively to explore pathogenic differences among different inoculation route. Guinea pigs have been 4/8, 2/8 and 5/8 deaths in 3 experimental groups by the trachea, lungs and the abdominal cavity inoculation, respectively. The tissue distribution of H. parasuis in infected guinea pig was tested by using paraffin sections and immunohistochemical techniques. After trachea infection with 1 ×LD50dose of H. parasuis, brain, heart, liver, lung, spleen and kidney tissues of guinea pigs were collected. Lung tissue showed positive result by immunohistochemistry staining from 6 h post trachea inoculation, then after 8 h, the abovementioned tissues presented positive. The results showed that H. parasuis can induce disease in guinea pig through 4 inoculation routes, such as the trachea, lungs, muscles and the abdominal cavity inoculation, and the H. parasuis can infect lungs, heart, liver, spleen and kidney tissues, and can lead to brain infection by breaking through bloodbrain barrier.

Abstract:The histological characteristics, distribution and variation discipline of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) during skin′s development of goat fetus were studied with histological methods and immunohistochemistry. The results showed that the epidermis formed at the embryonic 6th week, and then the thickness increased gradually because of transition from simple epithelium to stratified epithelium, and decreased after the 15th week. The structure of dermis occurred at the 10th week, and the derivates in dermis appeared after the 11th week. As the process of prenatal development, all structures of skin matured gradually. Low expression of EGF and EGFR could be observed at the 6th week. Afterwards, the expression of EGF and EGFR increased as growth. Before the 11th week, EGF positive reaction mainly located in cytoplasm of basal layer cells of epidermis, hair follicular epithelia cells and fibroblasts, and EGFR mainly located on cell membrane of these cells. From 11th to 16th week, the expressional range of EGF and EGFR extended from basal layer cells, prickle cells, hair follicular epithelia cells and fibroblasts to vascular endothelial cells, epithelial cells of sweat gland and arrector pili muscle. EGF mainly distributed in cytoplasm of these cells, and EGFR were also located on membrane of these cells. From 17th week to birth, EGF mainly located in basal layer cells and hair follicular epithelia cells as the skin thinned, the positive intensity continued to increase. The expressed amount of EGF and EGFR trended to increase during the whole development stage just accompanied by a hysteresis in expression of EGFR. There were extreme significant correlations between expressed amount of EGF and EGFR. The results demonstrated that EGF and EGFR played important roles in the development of skin and its derivates.

The experiment was conducted with the objective of examining the effect of dietary high fluorine on the antioxidant function and ultrastructure of liver in chickens. Three hundred onedayold Avian chickens were randomly divided into four groups, and fed on diets as follows: controls(F 23 mg·kg1) and high fluorine (F 400 mg·kg1，high fluorine group Ⅰ；F 800 mg·kg1, high fluorine group Ⅱ；F 1 200 mg·kg1, high fluorine group Ⅲ) for 42 days. The liver and serum superoxide dismutase and glutathione peroxidase activities were decreased（P<0.01 or P<0.05）,and the malondialdehyde and free fatty acid contents were increased（P<0.01 or P<0.05）in high fluorine groups Ⅱ and Ⅲ in comparison with those of control group. Ultrastructure of hepatocyte was changed in high fluorine groups Ⅱ and Ⅲ, and the number of hepatocyte glycogen pellets were obviously decreased. The mitochondria were enlarged and its crista broken or/and disappeared. The endoplasmic reticulum was dilated. The results showed that dietary fluorine in 800—1 200 mg·kg1 decreased hepatic antioxidant function and damaged hepatocytes. The hepatic function was finally impaired in chickens.