家鸡Leptin成熟肽cDNA的克隆、重组蛋白表达及纯化

从 18周龄鸡卵巢组织中抽提总 RNA,使用六聚体随机引物反转录后 ,用鸡 L eptin(瘦素 )特异性引物扩增出鸡L eptin编码区第 5 2～ 4 6 0 bp的长度为 4 0 9bp的 c DNA片段。根据鸡 L eptin的 3′端第 4 6 0～ 4 92 bp序列设计了 3条部分相互重叠并且顺序串联延伸的下游反义引物 ,并在最后 1条引物 3′端连接上 Eco R 切点 ;在以上用于反转录扩增的 5′端引物的 5′端连接一 Bam H 切点。用该 5′端引物分别与 3个 3′端引物配对 ,利用反转录扩增出的 4 0 9bp的L eptin c DNA片段作为第 1模板进行扩增 ,扩增产物再作为模板与下一引物对再次扩增。经 3次扩增得到编码鸡 L ep-tin成熟肽的全长 4 5 1bp的 c DNA序列。将该 4 5 1bp的 L eptin c DNA序列经 Bam H 和 Eco R 双酶切后 ,克隆入表达质粒 p RSET A的 Bam H 和 Eco R 两酶切位点之间 ,构建成表达质粒 p L ep- SCAU。转化有重组表达质粒 p L ep-SCAU的大肠杆菌 BL 2 1(DE3)在 L B培养基中培养后 ,经 IPTG诱导表达出相对分子质量为 2 0 10 0的鸡 L eptin融合蛋白和少量 4 0 2 0 0的 L eptin融合蛋白。L eptin融合蛋白的表达在 IPTG浓度为 0 .0 5 mmol/ L 时达到最高 ,占总菌体蛋白的 32 .6 %。用 Ni- NTA凝胶从 7L 发酵培养菌裂解液中纯化出 180 m g左右     

禽大肠杆菌外膜蛋白基因C(ompC)的序列分析

根据 Gen Bank中人源大肠杆菌 K- 12外膜蛋白基因 C(omp C)的核苷酸序列设计引物 ,应用 PCR方法从禽大肠杆菌 O2 、O78株及它们的融合双价弱毒菌株 O2 ,78(Norr Chlr)中分别扩增得到 omp C基因 ,序列测定及分析比较发现 ,3个菌株的 om p C基因均由 170 2 nt组成 ,核苷酸序列完全相同 ,只有 1个大的开放性阅读框 (ORF) ,长 10 92 bp,编码由 36 3个氨基酸组成的前 Om p C蛋白 ,前 2 1个氨基酸残基组成信号肽 ,成熟的 Omp C蛋白由 342个氨基酸残基组成 ,Mr为 4 0 0 0 0。其氨基酸序列也完全相同。从基因水平上证明了禽大肠杆菌 O2 、O78株及融合双价弱毒菌株 O2 ,78(NorrChlr)存在相同的外膜蛋白 C抗原 ,从而为进一步研究 Omp C蛋白的免疫原性奠定了基础     

表达犬瘟热病毒H基因的重组犬2型腺病毒的构建与鉴定

将犬瘟热病毒 ( canine distemper virus,CDV ) H基因表达盒克隆入转移质粒 p VAXΔE3,构建含 CDV H基因表达盒的转移质粒 p VAXΔ E3L PH,用 Nru 和 Sal 分别酶切转移质粒 p VAXΔ E3L PH和犬 2型腺病毒全基因组质粒p Poly2 - CAV- 2 ,将 CDV H基因表达盒定向克隆入 p Poly2 - CAV- 2质粒中 ,构建 E3区部分缺失的包含 CDV H基因表达盒的犬 2型腺病毒重组质粒 p CAV- 2 / CDVL PH。 Asc 和 Cla 对 p CAV- 2 / CDVL PH进行双酶切 ,释放 CAV- 2 /CDVL PH重组基因组 ,利用脂质体将 CAV- 2 / CDVL PH重组基因组与去除 Sal 和 Nru 片段的 CAV- 2基因组两端片段共转染 DK细胞 ,盲传和重复转染 3次 ,4 d后出现典型 CAV- 2病变 ,获得重组病毒 CAV- 2 / CDVL PH。用 CDV H基因特异性引物经 RT- PCR扩增重组病毒 DK细胞培养物 ,能够扩增出相应的核酸片段 ,表明 CAV- 2 / CDVL PH在DK细胞繁殖的过程中能够转录 CDVL P H的 m RNA,重组病毒免疫犬 ,可以诱导犬产生特异的抗 CAV- 2 HI抗体和抗 CDV中和抗体     

表达口蹄疫病毒P1基因的重组伪狂犬病病毒TK/gG-/PgG-P1的构建

为了构建口蹄疫与伪狂犬病二价基因工程疫苗株 ,将口蹄疫病毒 (FMDV) P1基因插入到伪狂犬病病毒 (PRV)通用载体 p Pg G- uni中 ,得到 PRV转移载体 p Pg G- P1。将转移载体与 Eco R 线性化后的 PRV弱毒疫苗株 TK- /g G- / lac Z 基因组 DNA共转染 PK- 15细胞 ,转染产物经多次空斑纯化和 PCR鉴定 ,获得了纯化的重组 PRV TK- /g G- / Pg G- P1,重组病毒基因组 DNA经酶切鉴定进一步表明 ,FMDV的 P1基因已成功地整合到 PRV弱毒疫苗株的基因组中。Western blot试验表明 ,FMDV P1基因在重组 PRV中得到表达。该研究为进一步研制口蹄疫与伪狂犬病二价基因工程疫苗奠定了坚实的基础     

融合表达淋巴细胞脉络丛脑膜炎病毒和卵清白蛋白CD8+T细胞表位的减毒鼠伤寒沙门氏菌的构建与鉴定

依据淋巴细胞脉络丛脑膜炎病毒(LCMV)主要保护性抗原CD8 T细胞表位VRRPQASGVYMGNLTAQ和卵清白蛋白(OVA)CD8 T细胞表位SIINFEKL,设计、合成两条编码LCMV和OVA CD8 T细胞表位的寡核苷酸片段,经退火后,克隆入绿色荧光蛋白表达质粒pYAGFP,经PCR扩增和序列测定分析,证实成功构建T细胞表位与绿色荧光蛋白融合表达的重组质粒pYAGFPL-O,将此重组质粒pYAGFPL-O转化减毒鼠伤寒沙门氏菌X4550,获得重组沙门氏菌X4550(pYAGFPL-O).用SDS-PAGE电泳测得重组菌表达的融合蛋白约为30kD.同时,荧光显微镜下观察到X4550(pYAGFPL-O)发出黄绿色荧光.这些结果表明LCMV和OVA T细胞表位已成功表达.重组菌X4550(pYAGFPL-O)的获得为研究沙门氏菌载体携带外源抗原的T细胞应答规律及调控机理打下了重要基础.     

抗鸡传染性喉气管炎重组鸡痘病毒基因工程疫苗同居感染试验

疫苗的接触传播是疫苗免疫接种需要考虑的重要因素,为了检测重组鸡痘病毒载体疫苗水平传播的能力,对隔离条件下饲养的SPF鸡用重组鸡痘病毒基因工程疫苗接种,同时设立非免疫对照鸡,饲养期间特意延长清粪时间以增加感染的机会,1个月之后攻击传染性喉气管炎WG株强毒和鸡痘102株强毒,疫苗免疫鸡全部获得保护,而非免疫鸡则全部发病.在试验动物饲养场的自然条件下,将免疫鸡和试验对照两组鸡饲养在同一个鸡舍内,让疫苗毒的传播更接近自然条件.在每个月的攻毒试验中,对照鸡都没有获得对鸡痘和传染性喉气管炎强毒的保护.在疫苗免疫期间进行连续5个月的跟踪检测,同居未免疫鸡没有检测到抗传染性喉气管炎病毒gB抗体.这些实验结果表明抗鸡传染性喉气管炎重组鸡痘病毒基因工程疫苗不能通过接触传播.     

Myofibrillar proteolysis in chick muscle cell cultures during heat stress

The effect of heat stress on protein oxidation and myofibrillar proteolysis in chick myotubes was investigated. Myotubes were incubated at 37 or 41°C for 6 and 24 h. Protein carbonyl content, as an index of protein oxidation, increased more at 41°C than at 37°C for 6 and 24 h incubations. N^τ‐methylhistidine release as an index of myofibrillar proteolysis also increased more at 41°C than at 37°C for 6 and 24 h incubations. Proteasome activity also increased more under those same conditions. Calpain and cathepsin D but not B + L activities showed a greater increase at 41°C than at 37°C for 24 but not the 6 h incubation. These results indicate that heat stress increases protein oxidation and proteasome activity, resulting in an increase in myofibrillar proteolysis for short‐term incubation and, for long‐term incubation, it increases calpain, proteasome and cathepsin D activities, finally accelerating myofibrillar proteolysis in chick myotubes.     

C-reactive protein concentrations in canine acute pancreatitis

Objective: To determine if C‐reactive protein (CRP) concentration is elevated in spontaneously occurring canine acute pancreatitis (AP), and to measure changes in CRP during the course of hospitalization. Design: Prospective study. Setting: Tufts University School of Veterinary Medicine Foster Hospital for Small Animals. Animals: Sixteen client‐owned dogs with AP and 16 healthy controls. Interventions: Blood samples were obtained from the AP group on the day of diagnosis (Day 1), and on Days 3 and 5, unless the dog died or was discharged from the hospital. Blood was obtained from the control dogs once. Measurements and main results: Serum CRP was measured using a commercial immunoassay for each dog with AP and for healthy controls. Day 1 CRP concentrations were significantly higher in the AP group (56.1±12.7 μg/mL) compared with controls (2.8±1.3 μg/mL; P<0.001). For the 7 dogs that had samples collected on all 3 days, the mean CRP concentrations decreased significantly (P=0.043) over the 5 days of measurement. Of the 16 dogs with AP, 14 were discharged from the hospital and 2 were euthanized. Conclusions: Serum CRP concentrations were elevated in this group of 16 dogs with spontaneously occurring AP. In the 7 dogs that had measurements on all 3 days, the mean CRP concentration decreased from the day of diagnosis to the measurement made 5 days later.     

Expression of the tobacco β-1,3-glucanase gene, PR-2d, following induction of SAR with Peronospora tabacina

Systemic acquired resistance (SAR) is induced following inoculation of Peronospora tabacina sporangia into the stems of Nicotiana tabacum plants highly susceptible to the pathogen. Previous results have shown that accumulation of acidic β-1,3-glucanases (PR-2's) following induction of SAR by P. tabacina may contribute to resistance to P. tabacina. We showed that up-regulation of the PR-2 gene, PR-2d, following stem inoculation with P. tabacina, is associated with SAR. Studies using plants transformed with GUS constructs containing the full length promoter from PR-2d or promoter deletions, provided evidence that a previously characterized regulatory element that is involved in response to salicylic acid (SA), may be involved in regulation of PR-2d following induction of SAR with P. tabacina. This work provides evidence that regulation of PR-2 genes during P. tabacina-induced SAR may be similar to regulation of these genes during infection of N-gene tobacco by TMV or following exogenous application of SA, and provides further support for the role of SA in regulation of genes during P. tabacina-induced SAR.