Recombinant GrB and PFP Co-expression in Hep-2 Cells

• Published in: Chinese journal of cancer research Vol. 20; no. 2; pp. 105 - 109
• Main Author: 李秀英 肖瑛 赖延东
• Format: Journal Article
• Language: English
• Published: Heidelberg Chinese Anti-Cancer Association 01.06.2008; Jinan University College of Pharmacy,Guangzhou,510632%Institute for Chemical Carcinogenesis,Guangzhou Medical College,Guangzhou 510182
• Subjects: Hep-2细胞; Medicine; Medicine & Public Health; 基因克隆; 基因表达; 淋巴细胞; 蛋白质; Granzyme B; Gene expression; Lymphocytes; Q812; Gene clone; Pore forming protein; Granzyme B:Pore forming protein
• ISSN: 1000-9604; 1993-0631
• DOI: 10.1007/s11670-008-0105-1

Objective： To achieve the co-expression of GrB and PFP in Hep-2 cells and analyze the growth inhibiting effects on Hep-2 cells. Methods： Lymphocytes were separated from human laryngeal carcinoma tissue, complete Exon fragments of GrB and PFP were amplified by RT-PCR via extracting lymphocytes total RNA, and they were recombined to the downstream of T7 promoter in the vector pVAX1. The recombinant plasmid pVAX1-PIG was transfected into Hep-2 cells with Lipofectamine 2000. The expression of proteins was identified by RT-PCR, MTT and western blot assay. Results： The gene sequence of the RT-PCR products of GrB and PFP were consistent with the data of GenBank by DNA sequencing analysis. The GrB and PFP cDNA fragment were cloned into the vector of pVAX1 in the right direction and the open reading fragment of GrB and PFP were maintained. The target proteins were detected in the transfected Hep-2 cells, and the inhibitive effect of PFP and GrB on Hep-2 cells growth were studied by thiazolyl blue （MTT） test. Conclusion： The pVAX1-PFP-IRES-GrB plasmid was successfully constructed and expressed, and the expression of PFP and GrB could inhibit the growth of Hep-2 cells.