【摘要】 【目的】克隆人巨细胞病毒(HCMV)UL123基因cDNA全长(ie1)及其外显子4(ie1-exon 4),构建原核表达重组质粒,诱导立即早期蛋白1(IE1)及其C-端86-491氨基酸(IE1-C86~491)表达,并鉴定表达产物。【方法】分别用RT-PCR和PCR法将ie1和ie1-exon 4从感染了HCMV的人胚肺成纤维细胞(HEL)中扩增出来,再分别克隆入原核表达载体pTWIN1上intein2的N端,转化大肠杆菌,经PCR、限制性酶切和测序鉴定阳性重组子,在低温和低IPTG浓度下诱导重组子表达可溶性重组融合蛋白rIE1/intein2和rIE1-C86~491/intein2,用SDS-PAGE和Western Blotting对表达产物进行鉴定。【结果】经PCR、限制性酶切和测序鉴定重组质粒pTWIN1/ie1和pTWIN1/ie1-exon 4构建成功,SDS-PAGE和Western Blotting分析表明融合蛋白在大肠杆菌中表达。【结论】成功克隆并表达了ie1和ie1-exon 4。更多还原

【Abstract】 【Objective】To clone and express the cDNA total length(ie1) and exon 4 of human cytomegalovirus(HCMV) UL123 gene.【Methods】ie1 and ie1-exon 4 amplified respectively by RT-PCR and PCR from human embryonic lung fibroblast infected with HCMV were cloned respectively into the N-terminal of interin 2 of prokaryotic expression vector pTWIN1.Then they were transformed into E.coli ER 2566,and the positive recombinants were identified by PCR,restriction enzyme digestion and sequencing.Under low temperature and low concentration of IPTG,the soluble fusion proteins were expressed in E.coli ER 2566,and analyzed by SDS-PAGE and Western blotting.【Results】The results showed that ie1 and ie1-exon 4 were successfully cloned into vector pTWIN1 and expressed in E coli ER 2566.【Conclusion】The iel and iel-exon 4 are successfully cloned and expressed.更多还原