PFP、GrB真核共表达载体的构建及表达分析

Construction of co-expression vector of pVAX1-PIG and expression of granzyme B and perforin in human Hep-2 cell line

目的:体外扩增出PFP、GrB基因的全片段并构建共表达重组体pVAX1-PIG(即pVAX1-PFP-IRES-GrB),分析其在人喉癌细胞Hep-2中的表达情况。方法:利用RT-PCR的方法从人的喉癌组织浸润淋巴细胞中扩增全长PFP、GrB的cDNA片段并构建共表达重组体pVAX1-PIG。利用脂质体LipofectamineTM2000将重组真核表达载体pVAX1-PIG转染入人的Hep-2细胞株中,采用RT-PCR、间接免疫荧光法分析其在人Hep-2中的表达情况。结果:经双酶切、测序法证实已成功扩增PFP、GrB基因的全长cDNA,并构建共表达重组体pVAX1-PIG,在荧光显微镜下可见已转染pVAX1-PIG的人Hep-2细胞的胞浆发出苹果绿色荧光。结论:成功扩增PFP、GrB全片段、构建共表达重组体pVAX1-PIG,并在人Hep-2细胞中检测到PFP、GrB蛋白的表达,穿孔素(PFP)/颗粒酶B共同表达能够介导细胞的凋亡,为其在喉癌治疗中的应用研究奠定了基础。

Objective： To amplify flail-length expressing sequence of human granzyme B （ GrB ） and pefforin （PFP） firstly, then to construct a co-expression vector of pVAX1 -PiG and to analyze the expression of human GrB and PFP in Hep-2 cells. Method： The human PFP and GrB expressing sequences were amplified by RT-PCR. The expression vector pVAX1-PIG was constructed. The recombinant vector was transfected into human Hep-2 cells and their expressions were detected by RT-PCR and indirect immunofluorescent antibody assay. Results：The GrB and PFP cDNA fragment were cloned into the vector of pVAX1 in the fight direction. The target proteins were detected in the transfected Hep-2 cells. Condusion： The vector of pVAX1-PIG was constructed successfully and expressed in Hep-2 cells line. The perforin/granzyme B protein can induce cell apoptosis. These results provide some foundation in gene therapy for tumor by making use of PFP and GrB gene.