摘要
目的获得纯化的LMP1CTAR23结构域蛋白。方法用RT-PCR方法从B95-8细胞中扩增EBVLMP1CTAR23结构域的cDNA,将其克隆至PGEM-Teasy载体后进行测序鉴定。亚克隆至原核表达载体PGEX-6P-3,转化大肠杆菌BL21,IPTG诱导重组菌株表达。用SDS-PAGE与Westernblot对表达产物进行鉴定,用Sepharose4B亲和层析柱对表达产物进行纯化,PreScissionProteas酶对融合蛋白进行解离。结果扩增的LMP1CTAR23长度为357bp,测序结果与已知序列吻合。获得PGEX-6P-3-CTAR23原核表达菌株,Westernblot表明重组蛋白能被LMP1单克隆抗体特异结合。获得约42000u的PGEX-6P-3-CTAR23融合蛋白,分离纯化出约13000u的LMP1CTAR23蛋白。结论成功获得EBVLMP1CTAR23蛋白,为研究LMP1致瘤机制,研制生物抗癌药物奠定基础。
Objective To obtain pure EBV LMP1 CTAR23 recombinant protein. Methods EBV LMP1 CTAR23 cDNA which was amplified from B95 8 cell by RT PCR technique was cloned into pGEM T easy vector and sequenced. It was then subcloned into expression vector PGEX 6P 3 and transferred into E.coli BL21. The recombinant protein was expressed and detected by Western blot. The fushion protein was purified by chromatography using Glutathione Sepharose 4B and was digested by the PreScission Proteas. Results The size of amplified LMP1 CTAR23 cDNA was 357bp. DNA sequencing showed the DNA sequence of the cloned gene was the same as the published sequence. The recombinant prokaryotic expression plasmid PGEX 6P 3 CTAR23 was constructed and expressed in E.coli BL21 and the products can be recognized by the anti LMP1 monoclonal antibody using Western blot. The 42000u fusion protein of PGEX 6P 3 CTAR23 and the cleaved 13000u LMP1 CTAR23 protein were purified. Conclusion The pure LMP1 CTAR23 protein provided the basic material for studying the oncogenic role of LMP1 and the development of gene therapy for NPC.
出处
《热带医学杂志》
CAS
2004年第2期117-121,共5页
Journal of Tropical Medicine
基金
国家自然科学基金(No.39970292)
广东省自然科学基金研究项目(No.990183)
