牛环形泰勒虫新疆株Tams1基因真核表达载体的构建及免疫反应性研究  

A Study on Construction of Tams1 Gene Eukaryotic Expression Vector in Xinjiang Bovine Theileria annulata and Immunological Reactivity

作　　者：简子健[1] 苗中秋[1] 马素贞[1] 谷思燚[2] 

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052 [2]辽宁农业职业技术学院,辽宁营口115009

出　　处：《新疆农业科学》2009年第3期651-656,共6页Xinjiang Agricultural Sciences

基　　金：国家自然科学基金项目(3066141)

摘　　要：研究采用PCR方法从焦虫病患牛的全血基因组中扩增得到846 bp的Tams1基因片段,将其克隆至真核表达载pcDNA3.1(+),构建Tams1基因的真核表达质粒。经测序验证后,将Tams1基因的真核表达质粒尾静脉注射小白鼠进行Tams1基因的瞬时表达。取接种小鼠的肝脏提取总RNA,采用RT-PCR扩增目的条带;用重组质粒pcDNA3.1(+)-Tams1皮下注射免疫小白鼠4次后,进行免疫抗体检测。试验结果表明重组质粒pcDNA3.1(+)-Tams1含有完整的Tmas1基因片段。用该重组质粒免疫小鼠56 d后,小鼠血清中抗重组质粒pcDNA3.1(+)-Tams1的抗体效价可达1∶12 000以上,说明该重组质粒具有很强的抗原性。经Western-blot检验可得到抗原-抗体复合物产生的特异性条带,说明该重组质粒pcDNA3.1(+)-Tams1具有免疫原性。A 846 bp Tamsl gene fragment was obtained from the whole blood genome in the infected cattle by PCR amplification and transformed into eukaryotic expression vector pcDNA3.1 （ ＋ ） to construct the recombineafion plasmid.After sequencing and identifying, the transient expression of Tamsl was performed by injecting mice in vena caudalis with pcDNA3.1 （ ＋ ）- Tamsl. The total RNA was extracted from murine livers complaint. The target strap was obtained by RT- PCR from the total RNA. Then the mice were subcutaneouly injected with recombination plasmid pcDNA3.1 （ ＋ ） - Tmasl for four times and the antibody in mirine sera was detected . The results were shown there was complete Tmasl gene fragments in the recombinant plasmid pcDNA3.1 （ ＋ ） - Tmasl. The anti - recombinant plasmid pcDNA3.1 （ ＋ ） - Tmasl antibody titers reached 1 ： 12 000 for 56 d after the injection with recombinant plasmid, suggesting that PeDNA3.1（ ＋ ） - Tmasl had very strong antigenicity. The specific band of antigen - antibody complex was detected by Western - blot test, suggesting the recombinant plasmid pcDNA3.1 （ ＋ ） - Tmasl had the immunogenicity.

关 键 词：牛环形泰勒虫 Tams1基因 表达载体构建 免疫印迹法 

分 类 号：S858.23[农业科学—临床兽医学] S188[农业科学—兽医学]