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     

三江白猪线粒体DNA-RFLP的分析

采用22种限制性内切酶分析了三江白猪线粒体DNA的多态性。结果表明：在60头个体中检出30种限制性态型．归结为7种基因单倍型；其间的差异来自少数限制性位点的突变。单倍型间平均遗传距离为0．35%，群体遗传多态程度为0．044％。三江白猪遗传多样性非常贫乏，说明三江白猪与地方种猪起源于一个共同祖先，在品种形成早期受到创立者效应的制约。     

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.     

Isolation of two strains of a new Tombusvirus (Havel river virus, HaRV) from surface waters in Germany

Two virus isolates from water samples — one from a small stream in South Western Germany and another one from the Havel river in North Eastern Germany c. 500 km away, proved to be strains, named S and H, respectively, of a new Tombusvirus for which the name Havel river virus (HaRV) had been suggested previously in a brief account. Immunoelectron microscopical decoration tests and sequence comparisons of the coat proteins indicated that the two HaRV strains are only distantly related to known Tombusviruses. The closest relationships were found to Cucumber necrosis virus. Nothing is known about their natural hosts. Because the S strain of HaRV was isolated in a woody area from a small stream close to its origin, they may be pathogens of trees or wild plants in such habitats.     

西安荷斯坦奶牛群5个基因座位遗传多态性的PCR-RFLP分析

应用PCR-RFLP方法对西安荷斯坦牛的κ-en、β-lg、β-lg5′侧翼区、CSN1S2、IGFBP-3共5个基因座位进行了多态性分析。结果表明，在西安荷斯坦牛群中，没有发现携带CSN1S2^P等位基因的个体，其多态信息含量为0。κ-en基因座位呈现低度多态(PIC=0．2366)，β-lg、β-lg5′侧翼区、IGFBP-3基因座位的多态信息含量分别为0．3168、0．3689、0．4439，均呈现中度多态。κ-en、β-lg、β-lg5′侧翼区、IGFBP-3、CSNIS2基因座位的杂合度和DNA多态度分别为0．2742、0．3947、0．4879、0．4891、0和0．0255、0．0116、0．0333、0．0112、0。而且，在西安荷斯坦牛群中，κ-en、β-lg、β-lg5′侧翼区、IGFBP-3共4个基因座位均处于Hardy-Weinberg平衡状态，CSN1S2基因座位处于纯合状态。     

以催乳素受体基因作为撒坝猪部分生产性能候选基因的研究

此文运用PCR-RFLP技术检测催乳素受体(PRLR)基因在撒坝猪群体中的多态性分布,并采用最小二乘分析模型初步统计分析基因多态性与部分生产性能的相关性。结果表明,PRLR基因等位基因B及其基因型BB在群体中占优势,频率分别为0.6594和0.4438;PRLR基因在群体中处于Hardy-Weinberg平衡状态;头胎繁殖性状有利等位基因B对生长性状无不利影响。