PEX慢病毒载体构建及其在293FT细胞中的分泌表达

Construction of and Expression Lentiviral Vector Carrying PEX Gene

目的 构建含人MMP-9信号肽-MMP-2-PEX片段的重组慢病毒,并在293FT细胞中分泌表达PEX蛋白.方法 利用RT-PCR、基因重组等技术,构建含MMP-9信号肽-MMP-2-PEX片段的重组慢病毒表达载体pBPLV-signal-PEX,在脂质体介导下与包装质粒（pLP1、pLP2）、包膜质粒（pLP/VSVG）共转染293FT细胞,包装产生慢病毒并进行滴度测定.慢病毒感染2931;3＂细胞后,Western印迹法检测293FI＂细胞培养上清中PEX的表达.结果 酶切和DNA测序表明慢病毒表达载pBPLV-signal-PEX构建正确,四质粒共转染293FT细胞成功获得慢病毒;慢病毒感染293FT细胞后,在细胞培养卜清中可检测到PEX蛋白的表达.结论 成功构建了含人PEX的重组慢病毒,在体外有效感染293fT细胞并持续分泌表达目的 蛋白,为进一步研究PEX在肿瘤侵袭与转移中的作用提供实验基础.

Objective To construct a lentiviral vector containing MMP-9 signal peptide and non-catalytie carboxyl-terminal hemopexin domain of human MMP-2 gene （PEX） and observe its expression in 293FT cells. Methods Lent/viral vector containing MMP-9 signal peptide and 1V[MP- 2-PEX gene was eonstructed by RT-PCR and recombinant DNA technique. Recombinant lentivirus was produced in packaging 293FF cells transfected with four plasmids of lentiviral system by lipo- fectamine 2000. Expression and secretion of PEX gene in 293FT cells were detected by Western- blot. Results Restriction endonuclease digestion and sequencing analysis verified the correct con- struetion of the recombinant lentiviral expression vector carrying PEX gene and signal peptide. West- ern blot eonfirmed the PEX expression and seeretion in 293FT cells. Conclusion The PEX-lentiviral expression veetor may be applied for further study of PEX effect on neoplastic invasion and metas- tasis.