牛巴贝斯虫新疆株MSA-2c基因重组质粒的构建、真核表达及其免疫反应性研究

Construction,Expression and Immunological Reactivity of Xinjiang Babesia bovis MSA-2c Gene Recombination Plasmid

根据GenBank公布的牛巴贝斯虫裂殖子表面抗原MSA-2c基因序列设计引物,采用PCR方法从患病牛的全血基因组中扩增得到798 bp的MSA-2c基因片段,将其克隆至真核表达载体pcDNA3.1(+),测序验证后,小白鼠尾静脉注射瞬时表达MSA-2c基因,取其肝脏提取总RNA,采用反转录聚合酶链式反应(RT-PCR)扩增可得到目的条带;用重组质粒pcDNA3.1(+)-MSA-2c肌肉注射免疫小白鼠4次后,将获得的抗血清作为抗体,纯化的GST-MSA-2c融合蛋白作为抗原进行Western-blot检测,可得到抗原-抗体结合产生的特异性条带,表明重组质粒具有免疫学活性。

The special primers were designed by merozoite surface antigenic gene sequence released in GenBank. A 798 bp MSA-2c gene fragment was obtained from the whole blood genome in the infected cattle by PCR amplification, and transformed into eucaryotic expression vector pcDNA3.1 （＋） in order to construct the recombination plasmid. After sequencing and identifying, the pcDNA3. 1 （＋）-MSA-2c was injected into mice in vena caudalis to conduct the transient expression of MSA-2c. The total RNA was extracted from murine Livers. The target strap was obtained by RT-PCR of the total RNA. The murine antiserum prepared by recombination plasmid pcDNA3.1（＋）-MSA-2c was used as antibody and was injected into mice for four times,and the purified GST-MSA-2c fusion protein as antigen for Western-blotting test. The target strap represented the antigen-antibody complex was found. The result showed that the recombination plasmid pcDNA3.1（＋）-MSA-2c had immunological activity.