HIV核心抗原p24原核系统的高效表达、纯化及活性鉴定  

Prokaryotic expression and purification of human immunodeficiency virus p24 antigen

作　　者：侯俊[1] 貌盼勇[1] 洪世雯[1] 胡燕[1] 杨健洋[1] 沈宏辉[1] 朱雷[1] 

机构地区：[1]解放军第三○二医院病毒研究室,北京100039

出　　处：《中华实验和临床病毒学杂志》2005年第1期28-31,共4页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的　在原核表达系统中对HIVp2 4抗原基因进行克隆和高效表达、纯化并鉴定其活性。方法　利用PCR技术从HIV-1全基因质粒 (B2N)中扩增p2 4抗原基因 ,并克隆入T载体中。通过酶切消化后连接到表达载体pRSET上 ,用此连接产物转化大肠埃希菌BL2 1,经IPTG诱导 ,表达p2 4抗原。利用固定化金属离子 (Ni2 + )配体亲和层析技术从表达蛋白中纯化目的蛋白。并运用双酶切技术、SDS-PAGE电泳、WesternBlot(WB)及ELISA法分别对插入基因片段的正确性、表达产物的活性及特异性进行检测。结果　PCR产物约为 6 90bp ,与预期p2 4抗原全基因片段大小一致。重组质粒T-p2 4和pRSET-p2 4经BamHⅠ和HindⅢ双酶切 ,其插入的外源基因片段均为 6 90bp。将纯化前与纯化后的蛋白作SDS PAGE电泳 ,均可见一条约 2 4× 10 3的外源基因表达带 ,与计算的相对分子质量相符。经WB和ELISA试验 ,证明基因工程表达的p2 4抗原具有较高的特异性及活性。结论　成功构建了HIVp2 4表达载体pRSET-p2 4 ,并在原核细胞中高效表达 。Objective To express and purify the HIV p24 gene in E.coli cells,and identify p24 antigen activity. Methods The full length gene fragment of HIV p24 was amplified by PCR and inserted into the pRSET vector in order to construct the pRSET-p24 recombined vector. After transforming into E.coil, the purified p24 protein was prepared by metal-ligand affinity chromatography (IMAC). The accuracy of inserted gene and activity, specificity of HIV p24 proteins were detected by two enzymes digestion technology, SDS-PAGE, Western Blot (WB) and ELISA. Results The length of the HIV p24 gene fragment was 690 bp after digesting the recombinant plasmid T-p24 and pRSET-p24 with BamH Ⅰ and Hind Ⅲ. The expressed proteins had a single expected band of about 24×10~3 in SDS-PAGE. The specificity and activity of p24 protein were tested by WB and ELISA. Conclusion The HIV p24 sequence from HIV-1 gene plasmid has been expressed in E.coil. This protein possessed good specificity and activity.

关 键 词：HIV 核心抗原 高效表达 纯化 特异性 活性 原核系统 表达载体 酶切 外源基因表达 

分 类 号：R392[医药卫生—免疫学]