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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。
In order to investigate the role of human cytomegalovirus latent associated interleukin-10 (LA-cmvIL-10) and cytomegalovirus-encoded interleukin-10 (IL-10) in viral infection, we constructed the cmvIL- LA-cmvIL-10 gene prokaryotic and eukaryotic expression system. The viral DNA was extracted from urine samples of children and the cmvIL-10 gene and exon of LA-cmvIL-10 gene were amplified by overlap extension PCR. The product was cloned into prokaryotic expression vector pMal-c2x and eukaryotic expression vector pCDNA3.1 , And the complete coding region was found in the exons of cmvIL-10 and LA-cmvIL-10 amplified by sequencing. The recombinant prokaryotic expression plasmids pMal-c2x-cmvIL-10 and pMal-c2x-LAcmvIL- The strain E. coli BL21 (DE3) was induced by isopropyl-β-D-thiogalactoside (IPTG) to express the target protein in the recipient bacteria. The expression product was immunized in rabbits to obtain specific antiserum. Hela cells were transfected with eukaryotic expression vector pCDNA3.1-cmvIL-10 and pCDNA3.1-LAcmvIL-10, which was identified by Western blot.