EB病毒LMP1C端原核表达质粒的构建

Prokaryotic expression plasmid construction of EBV-LMP1 carboxy region

目的通过巢式PCR技术扩增EB病毒LMP1 C端,并构建其原核表达重组质粒,经诱导获得EB病毒LMP1C端蛋白。方法用巢式PCR技术,从B95-8细胞中扩增EBV-LMP1 exon c DNA,克隆至pET-32 a载体,转化入原核表达菌BL21,IPTG诱导重组菌株表达,用SDS-PAGE与W estern b lot对表达产物进行鉴定。结果巢式PCR扩增的LMP1exon c DNA长度为801 bp,测序结果与LMP1 C端cDNA序列吻合,诱导pET-32 a-LMP1 c重组原核表达菌株获得大小约48×103的LMP1 C端融合蛋白,W estern b lot表明重组蛋白能与LMP1单克隆抗体特异结合。结论通过巢式PCR成功构建了原核表达载体,获得的EB病毒LMP1 C端融合蛋白为制备国产LMP1单克隆抗体,研究LMP1致瘤机制奠定了基础。

Objective To amplify EBV-LMP1 carboxy region and express it in recombinant prokaryotic plasmid pET-32a-LMPlc. Methods EBV-LMP1 exon c gene was amplified from the B95-8 cell genomic DNA by nest PCR and cloned into plasmid pET-32a. The recombinant plasmid was then transformed into E. colt. BL21. The EBV-LMP1 C was expressed as a recombinant fusion protein and detected by Western blotting. Resuits The size of amplified LMPlexon c DNA was 801 bp, which was the same as the published EBV-LMP1 carboxy region sequence. The recombinant prokaryotic expression plasmid pET-32a-LMPlc was constructed and 48 × 10^3 fusion protein was expressed in E. colt. BL21. The expressed protein was detected with the anti-LMP1 monoclonal antibody by Western blotting. Conclusion Recombinant prokaryotic expression plasmid was constructed successfully. The expressed recombinant fusion protein provided the basic material to produce our own anti-LMP1 monoclonal antibody and study the oncogenic role of LMP1.