人巨细胞病毒潜伏相关IL-10基因克隆及体外表达

Cloning and Vitro Expression of Human Cytomegalovirus Latency Associated IL-10 Gene

为了研究人巨细胞病毒潜伏相关白细胞介素-10(LA-cmvIL-10)及巨细胞病毒编码的白细胞介素-10(cmvIL-10)在病毒感染中的作用,构建了cmvIL-10基因和LA-cmvIL-10基因的原核及真核表达体系。从患儿尿液标本中提取病毒DNA,应用重叠延伸PCR扩增cmvIL-10基因和LA-cmvIL-10基因外显子,产物克隆至原核表达载体pMal-c2x和真核表达载体pCDNA3.1上,测序显示扩增的cmvIL-10基因和LA-cmvIL-10基因外显子包含了完整的编码区;重组的原核表达质粒pMal-c2x-cmvIL-10和pMal-c2x-LAcmvIL-10转化受体菌E.coli BL21(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,在受体菌内表达目的蛋白。将表达产物免疫家兔,获得特异性抗血清。真核表达载体pCDNA3.1-cmvIL-10和pCDNA3.1-LAcmvIL-10转染Hela细胞,经Western blot鉴定与设计相符。

To provide data and material for the future study on the pathogenic mechanism of human cytomegalovirus latency associated IL-10 （LA-cmvlL-10） and IL-10 encoded by human cytomegalovirus （cmvlL- 10） in the viral infection, we constructed prokaryotic expression vector and eukaryotic expression vector about human cytomegalovirus IL-10 （cmvlL-10） and LA-cmvlL-10 gene. We extracted virus DNA from the positive urine specimens of the patients, overlap extension PCR was applied to amplify the exon of cmvlL-10 and LA-cmvlL-10 gene. PCR products were cloned into the pMal-c2x vector and the pCDNA3.1 vector. The exons of cmvlL-lO and LA-cmvlL-10 gene amplified contain the complete coding region by DNA sequencing. The prokargotic recombinant expression vector pMaI-c2x-cmvIL-10 and pMal-c2x-cmvlL-10 had been transformed into E. coli B L21 （DE3）. By isopropyl-β-D -thiogalagtoside （IPTG） induction, our protein was expressed in receptor strain. The purified recombinant protein was used to immune rabbit for preparing polyclonal antibody with specificity. The eukaryotic expression vectors pCDNA3.1-cmvlL-10 and pCDNA3.1-LAcmvlL-10 were transfected to Hela cell lines. The products identified by Western blot was consistent with our original design.