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Cloning and Vitro Expression of Human Cytomegalovirus Latency Associated IL-10 Gene
Xiao-Qun Zheng1, Jing-Jing Feng2, Zhi Zeng2, Zhao-Chang Jiang2, Jing-Zhang Ji2, Xiang Li2, Jian-Xin Lü1,2*
1Laboratory Diagnosis Center, the Second Affiliated Hospital of Wenzhou Medical College, Wenzhou 325027, China;2Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical College, Wenzhou 325035, China
Abstract: To provide data and material for the future study on the pathogenic mechanism of human cytomegalovirus latency associated IL-10 (LA-cmvIL-10) and IL-10 encoded by human cytomegalovirus (cmvIL- 10) in the viral infection， we constructed prokaryotic expression vector and eukaryotic expression vector about human cytomegalovirus IL-10 (cmvIL-10) and LA-cmvIL-10 gene. We extracted virus DNA from the positive urine specimens of the patients， overlap extension PCR was applied to amplify the exon of cmvIL-10 and LA-cmvIL-10 gene. PCR products were cloned into the pMal-c2x vector and the pCDNA3.1 vector. The exons of cmvIL-10 and LA-cmvIL-10 gene amplified contain the complete coding region by DNA sequencing. The prokargotic recombinant expression vector pMal-c2x-cmvIL-10 and pMal-c2x-cmvIL-10 had been transformed into E. coli BL21 (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-cmvIL-10 and pCDNA3.1-LAcmvIL-10 were transfected to Hela cell lines. The products identified by Western blot was consistent with our original design.
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