人颗粒酶B基因的克隆、蛋白纯化及表达分析

TIL具有抗肿瘤的活性 ,其中颗粒酶B在杀伤肿瘤细胞的过程中起了最重要的作用。本实验拟扩增GrB全序列 ,构建GrB的原核表达载体 ,从而建立其原核表达体系 ,获得高效表达的重组GrB ,纯化蛋白。用淋巴细胞分离液分离肿瘤组织中的淋巴细胞 ,并抽提RNA ,RT PCR方法获得GrB的全长 ,并重组到pGEX 4T 1中 ,用限制性内切酶酶切和DNA测序法进行鉴定 ,IPTG诱导pGEX GrB转化的BL2 1菌 ,大量纯化表达产物 ,并通过SDS PAGE、Westernblot分析表达产物 ,用MTT法体外检测GrB对Hep2细胞的作用。结果 :限制性内切酶酶切和DNA测序分析证实成功获得GrB基因片段 ,GrB准确克隆入pGEX 4T 1 ,成功的构建pGEX GrB ,经诱导表达出与GST融合的蛋白 ,经凝血酶酶切后获得与GrB相符的条带 ,并能与GrB特异性抗体结合。体外实验表明 ,GrB能抑制Hep2细胞的增殖。成功构建了pGEX GrB ,并成功表达、大量纯化GrB蛋白 ,其能够抑制Hep2细胞的增殖。

Cloning,Purification and Prokaryotic Expression of Human Granzyme B Gene

Granzyme B plays an important role in the anti-tumor activity of tumor-infiltrating lymphocytes(TIL). In this study, the gene encoding the entire sequence(GrB)was amplified, and the prokaryotic expression vector was constructed, purified and identified in order to obtain the recombinant GrB with high efficiency. Lymphocytes were separated from human laryngeal carcinoma tissues, and the GrB fragment was amplified by RT-PCR through extracting the total RNA from lymphocytes. After identification by DNA sequencing, the gene was inserted into BL21 expression vector pGEX-4T-1. The pGEX-GrB was thus constructed and transformed into BL21. The expression products were purified and identified by SDS-PAGE and Western blotting, and the in vitro proliferation inhibition activity of GrB on Hep2 cells was determined by MTT method. It has been proved that sequence of the PT-PCR products was totally consistent with the data of GenBank by DNA sequencing analysis. The cDNA fragment of GrB was cloned in the vector of pGEX-4T-1 in right direction, and the open reading frame(ORF)of GrB was maintained. The target protein molecules could be detected in BL21, and a GST fusion protein was expressed after induction, which could be recognized by GrB-protein-specific antibody. As demonstrated by in vitro experiments, the purified pro GrB protein could inhibit the proliferation of Hep2 cells. It is concluded that the pGEX-GrB plasmid was successfully constructed and expressed in the present study, and it was shown that the GrB protein could inhibit the proliferation of Hep2 cells.