组织激肽释放酶、绿色荧光蛋白双基因共表达载体的构建及其对平滑肌细胞增殖的影响

Construction of a coexpression vector carrying both Kallikrein 1 and green fluorescent protein gene and its effects on proliferation of vascular smooth muscle cells

目的构建人组织激肽释放酶(HK1)和增强型绿色荧光蛋白(EGFP)的逆转录病毒双基因共表达载体并探讨其对血管平滑肌细胞(SMCs)增殖的影响。方法采用聚合酶链反应(PCR)技术扩增人HK1基因,通过基因重组构建HK1和EGFP逆转录病毒双基因共表达载体,经酶切及测序鉴定正确后,包装成为重组逆转录病毒rRV-HK1-IRES-EGFP,病毒感染SMCs细胞后,荧光显微镜和Western blot检测HK1基因的表达情况。结果成功构建了带有EGFP的HK1基因的逆转录病毒表达载体,并将其转入SMCs细胞,感染后SMCs增殖受到抑制。结论构建的HK1基因逆转录病毒可在体外高效表达,为HK1基因治疗的研究奠定了基础。

Objective To construct a retroviral coexpression vector-pLHK1SN-IRES-EGFP, cartying both Human KaUikrein 1 （HK1） and enhanced green fluorescent protein （EGFP）, and to examine its effects on proliferation of vascular smooth muscle cells. Methods HK1 gene was obtained by PCR amplification. The retroviral coexpression vector carrying both HK1 and EGFP gene was constructed using ge- netic recombination technique,then identified and transferred to the packaging cell PA317 to pack the virus. This new recombinant virus rRV-HKI-IRES-EGFP was used to transfect SMCs. The expression of exogenous HK1 gene was detected by fluorescence microscopy and Western blot. Results The recombinant retroviral vector carrying exogenous HK1 and EGFP genes was constructed, and the recombinant virus was transfected into SMCs. After transfection,the growth ability of SMCs was profoundly inhibited. Conclusion The recombinant retrovirus carrying HK1 and EGFP was successfully constructed and the transgene SMCs expressed these two exogenous ~ene in vitro.which provided a sound basis of HK1 gene therapy.