YANG Yu-cheng,WANG Yi-qin,QIAN Di,HE Yun,HUANG Jiang-ju,HONG Su-ling.Prokaryotic expression plasmid construction of EBV-LMP1 carboxy region[J].J Amry Med Univ (J Third Mil Med Univ),2007,29(01):56-58.
YANG Yu-cheng,WANG Yi-qin,QIAN Di,HE Yun,HUANG Jiang-ju,HONG Su-ling.Prokaryotic expression plasmid construction of EBV-LMP1 carboxy region[J].J Amry Med Univ (J Third Mil Med Univ),2007,29(01):56-58.DOI:
Prokaryotic expression plasmid construction of EBV-LMP1 carboxy region
从B95-8细胞中扩增EBV-LMP1 exon c DNA,克隆至pET-32a载体,转化入原核表达菌BL21,IPTG诱导重组菌株表达,用SDS-PAGE与Western blot对表达产物进行鉴定。 结果 巢式PCR扩增的LMP1 exon c DNA长度为801 bp,测序结果与LMP1 C端 cDNA序列吻合,诱导pET-32a-LMP1c重组原核表达菌株获得大小约48×10
3
的LMP1 C端融合蛋白
Western blot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。
Abstract
Objective To amplify EBV-LMP1 carboxy region and express it in recombinant prokaryotic plasmid pET-32a-LMP1c. 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. coli. BL21. The EBV-LMP1 C was expressed as a recombinant fusion protein and detected by Western blotting. Results The size of amplified LMP1exon c DNA was 801 bp
which was the same as the published EBV-LMP1 carboxy region sequence. The recombinant prokaryotic expression plasmid pET-32a-LMP1c was constructed and 48×10
3
fusion protein was expressed in E. coli. BL21. The expresse
d 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.
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