伪狂犬病病毒鄂A株TK基因的克隆及其鉴定

合成了 １ 对能对伪狂犬病病毒（ Ｐｓｅｕｄｏｒａｂｉｅｓ ｖｉｒｕｓ, Ｐ Ｒ Ｖ） Ｔ Ｋ（ｔｈｙｍ ｉｄｉｎｅ ｋｉｎａｓｅ）基因＋ １１９～＋ １ ０７１区进行特异扩增的引物,用猪 Ｐ Ｒ Ｖ 鄂 Ａ 株细胞培养物提取的基因组作模板,扩增出 ９５３ ｂｐ 长的片段,用地高辛标记该片段作探针,通过 Ｓｏｕｔｈ ｅｒｎ 杂交,从克隆有 Ｐ Ｒ Ｖ 鄂 Ａ 株的 Ｂａｍ Ｈ Ｉ片段的重组质粒中钓出含 Ｔ Ｋ 基因的重组质粒 ｐ Ｓ Ｔ Ｋ。对 ｐ Ｓ Ｔ Ｋ 进行酶切分析,绘制了含 Ｔ Ｋ 基因的 Ｂａｍ Ｈ Ｉ片段的图谱,通过测序得出了 Ｔ Ｋ 基因的全序列。将该序列与 Ｐ Ｒ Ｖ Ｎ Ｉ Ａ３ 株 Ｔ Ｋ 基因进行比较,发现鄂 Ａ 株的 Ｔ Ｋ 基因存在变异。     

Cloning and Sequence Analysis of Major Histocompatibility Complex Ⅰ Gene from Goose F1 of Fast-growth Lines

According to the multiple alignments identified major histocompatibility complex Ⅰ (MHC Ⅰ) gene conserved sequence registered in GenBank from the family ducks (Anatidae) anser waterfowl, a pairs of specific primers for the fragments of MHCⅠgene of goose F1 from fast-growth lines were designed and synthesized by Primer Premier 5.0. Using the genome DNA of goose F1 from fast-growth lines, the target gene fragment was obtained by PCR. To conduct sequencing of the fragments of MHCⅠgene of goose F1 from fast-growth lines and make sequence alignment and analysis of protein structure and function by bioinformatics, and research the characteristics of MHCⅠgene of goose F1 and the physicochemical properties of the protein. Bioinformatics was analyzed the nucleic acid data, deduced amino acid sequence and phylogenetic trees. The result of sequence analysis showed that the fragments of MHCⅠgene of goose F1 from fast-growth lines was 1036 bp in length, which coded 96 amino acids polyprotein. The homology were 93% and 83% with MHC Ⅰ gene and coding sequence of Wulong goose in NCBI respectively. There were 72 different bases sequence and 16 amino acids change. There also was higher homology with other poultry, and existed genetic relationship of Siji goose > chickens > ducks.The homology segment sequences corresponding to the fragments of MHCⅠ gene of goose F1 coded 96 amino acids protein, which molecular weight, PI, positively or negatively charged amino acid, estimated half-life, instability index, aliphatic index and average hydrophobicity were 11.342 ku, 5.32, 14, 17, 2.8 h, 34.92, 42.81, -1.066, respectively, and appeared 9 B cell epitopes, but contained no signal peptide. These results indicated that the protein for hydrophilic non-secreted proteins, had the high immunogenicity. In addition, The protein structure study indicated that alpha-helix, beta-sheet, beta-turn and random coil were 31.25%,16.67%, 14.58% and 37.50%, respectively. There existed amino terminal domain and carboxyl terminal domain in the tertiary structure. Therefore, MHC gene had significant difference between species and populations of individuals by the pathogen pressure in environment, and there were the interaction between polymorphism of MHC molecules and the diversity of antigenic peptide. MHC determined the differences of individual susceptibility to disease, and could be treated as a candidate gene for disease resistance.     

广东省部分猪场细环病毒感染状况

细环病毒（TTV）目前在全世界的猪群中广泛存在。为了调查其在广东省猪群流行情况,我们运用PCR方法对广东省不同地方猪场和散养户送检的445份血清进行了检测。结果表明：TTV总的阳性率为68.1%（303/445）,其中TTV1阳性率为43.1%（192/445）,TTV2阳性率为24.9%（111/445）。TTV1的阳性率高于TTV2的阳性率,且猪场送检样品阳性率高于散养户。TTV1和TTV2存在混合感染,猪场样品混合阳性率为23.1%（88/381）,散养户样品混合阳性率为1.6%（1/64）。几份阳性样品测序和进化分析显示,它们分别属于TTV1和TTV2。调查结果表明TTV在广东猪群中已经广泛存在。     

Influence of recombinant duck follistatin protein on embryonic muscle development and gene expressions

Follistatin (FST) acts as a positive regulator of muscle development by inhibiting the activities and expression of myostatin. The recombinant duck FST protein was injected into hatching eggs and was also added to the medium of duck myoblast to study its role on duck embryonic muscle development and gene expressions. Duck embryo weight increased 3.49% (p > 0.05) in FST treatment group as compared with control group, but minor effects were found on leg or breast muscle weights of ducklings at 2 days post‐hatching (p > 0.05). Relative expression of Pax7 was upregulated in both leg and breast muscle tissues (p < 0.05), while MyoD was only upregulated in leg muscle (p < 0.05), and Myf5 was only upregulated in breast muscle (p < 0.05). Relative expression of myostatin was downregulated in both muscle tissues researched (p < 0.05). In vitro studies also showed some maker genes relevant to protein synthesis and degradation, cells’ proliferation and differentiation had significant changes in myoblasts after treated with FST. These results suggested that in ovo feeding of recombinant FST protein to duck hatching eggs had an effect on duck embryo development but have less roles on the duck embryonic muscle development.     

Effect of different levels of black cumin (Nigella sativa L.) on performance,intestinal Escherichia coli colonization and jejunal morphology in laying hens

This study was conducted to investigate the effects of different levels of black cumin seeds (Nigella sativa L.) on performance, intestinal Escherichia coli count and morphology of jejunal epithelial cells in laying hens. A total of 100 Leghorn laying hens (Hy‐Line W‐36) of 49 weeks old were randomly distributed among five cage replicates of five birds each. Experimental diets consisted of different levels (0%, 1%, 2% and 3% of diet) of dietary black cumin inclusion. The experimental period lasted for a total of 10 weeks, and egg quality indexes and laying hens' performance were measured as two 35‐day trial periods. At the final day, two hens per replicate were slaughtered to investigate the influence of dietary treatments on intestinal E. coli colonization and morphology of jejunal cells. Although dietary black cumin in all supplementation levels decreased (p < 0.05) the enumeration of ileal E. coli, the morphological and histological alterations in small intestine such as enhancement of villus height to crypt depth ratio, increased goblet cell numbers and proliferation of lamina propria lymphatic follicles were observed after dietary supplementation with at least 2% black cumin. Dietary treatments decreased (p < 0.05) the concentration of serum cholesterol and triglycerides and increased (p < 0.05) serum HDL concentration and relative weight of pancreas; however, the egg yolk cholesterol was not influenced by dietary treatments. In addition, dietary supplementation with black cumin improved (p < 0.05) eggshell quality and Haugh unit. The best feed conversion ratio was obtained when diets were supplemented with 2% black cumin. This improvement was due to the increase (p < 0.05) in egg mass and contemporaneous decrease (p < 0.01) in feed consumption. The present results indicated that regardless of supplementation level, dietary inclusion of black cumin decreased E. coli enumeration in ileal digesta and improved serum lipid profile and eggshell quality, whereas the best intestinal health indices and haying hens' performance were obtained by at least 2% black cumin seeds.     

犬新孢子虫NcSRS2基因原核表达质粒的构建

根据已发表的犬新孢子虫NcSRS2基因序列,设计1对含有EcoRⅠ和NotⅠ酶切位点的引物。以提取犬新孢子虫虫体基因组DNA为模板,应用PCR扩增获得NcSRS2 ORF基因片段,将此基因片段克隆到pMD18-T Simple载体上,用EcoRⅠ和NotⅠ双酶切该片段,回收得到含有两个酶切位点黏端的Nc-SRS2 ORF基因,将此基因片段克隆至相同酶切回收后的PGEM-4T-2原核表达载体中,获得重组质粒pGEX-NcSRS2,经PCR鉴定,限制性内切酶分析和克隆片段序列测定比较,证实了重组质粒的正确性。     

伪狂犬病毒GDSH株的分离鉴定及gE基因的序列分析

从广东四会某猪场分离到一株疑为猪伪狂犬病病毒(PRV)的病毒,病毒在猪肾细胞上出现细胞变圆、拉网、融合等典型病变,并具有细胞泛嗜性特点。在MDCK细胞上测得其TCID50值为10-8/0.1mL,能被伪狂犬病病毒标准阳性血清中和。将0.1mL病毒液接种小鼠后发生奇痒并麻痹致死,接种猪3天后发病,7天死亡,从攻毒病死猪的脑组织病理切片上观察到典型的病毒性脑膜脑炎及血管套现象。通过PCR扩增到PRVgD基因,由此进一步证明所分离病毒为猪伪狂犬病毒,并命名为GDSH株。根据GenBank中发表的序列,设计一对扩增PRVgE基因的特异性引物,建立可以区分PRV野毒株与疫苗株的PCR诊断方法。以此方法对病毒的细胞培养液进行检测,结果证实所分毒株为PRV野毒株,经克隆测序后与GenBank收录的其它PRVgE基因序列进行比较,发现所测毒株的核苷酸序列与其它PRV毒株的同源性介于98.3%～99.9%之间,其中与PRVEa株的亲缘关系最近为99.9%。     

粗纤维和赖氨酸水平对生长期豫南黑猪的影响

本试验旨在探讨饲粮中不同粗纤维和赖氨酸水平对生长期豫南黑猪生长性能、血清生化指标及营养物质表观消化率的影响.试验选取162头体重为30 kg左右的豫南黑猪,采用3（CF：4％、5.5％、7％）×3（lys：0.75％、0.85％、0.95％）两因子交叉完全随机设计,共设9个处理,每个处理3个重复,每重复6头猪.通过测定其生长性能、血清生化指标及营养物质表观消化率,确定生长期豫南黑猪的最佳粗纤维和赖氨酸水平.结果表明：不同粗纤维和赖氨酸水平对生长期豫南黑猪平均日采食量（ADFI）、平均日增重（ADG）和料重比（F/G）均无显著影响（P＞0.05）;粗纤维和赖氨酸水平分别为7％、0.75％时,料重比最低（P＜0.05）;粗纤维和赖氨酸水平分别为5.5％、0.85％时血清中游离三碘甲状腺原氨酸（FREET3）水平最高（P＜0.05）.饲粮中添加粗纤维使营养物质表观消化率下降,但通过适量比例的粗纤维和赖氨酸的合理添加,对饲粮中能量、粗蛋白、粗脂肪、粗纤维、Ca和P的表观消化率无显著影响（P＞0.05）.上述结果提示,30～60 kg生长期豫南黑猪的饲粮适宜粗纤维水平和赖氨酸水平分别为7％和0.75％.