密码子优化HPV16衣壳基因真核共表达载体的构建及细胞转染

Construction of codon-optimized HPV16 capsid genes in the eukaryotic co-expression vector and transfection of cells

目的:构建密码子优化的HPV16衣壳基因真核共表达载体pcDNA3.1-L1-IRES-L2。方法:用PCR技术从988载体中获得L1-IRES-L2片段,将该片段克隆到pCR-XL-TO-PO载体,然后定向亚克隆到pcDNA3.1(+)真核表达载体中,从而构建真核共表达载体pcDNA3.1-L1-IRES-L2;通过水动力转染技术(hydrodynamics-based transfection)和脂质体细胞转染法(liposome-mediated transfection of cells),检测衣壳基因的体内、外转录情况;重组质粒转染后293T细胞后观察其形态变化,用Western blot方法检测293T细胞中L1衣壳蛋白的表达。结果:酶切和测序结果表明真核共表达载体pcD-NA3.1-L1-IRES-L2构建正确。重组质粒中的L1和L2基因在小鼠肝脏、293T细胞中均发生转录。重组质粒转染293T细胞后出现CPE(cytopathic effect)现象,表明衣壳基因在细胞中已表达。Western blot方法检测发现L1蛋白在293T细胞中表达。结论:成功地构建了pcDNA3.1-L1-IRES-L2共表达真核载体,为进一步研究HPV16感染机制奠定基础。

AIM： To construct the eukaryotic co-expres- sion vector pcDNA3. 1-L1-IRES-L2 for harvesting sufficient amounts of infectious HPV16. METHODS： The amplifed codon-optimized HPV16 capsid genes from 988 plasmid by PCR were cloned into pCR-XL-TOPO vector and then subcloned into eukaryotic expressing vector pcDNA3.1 （ ＋ ）. Thus, eukaryotic co-expression vector pcDNA3.1-L1- IRES-L2 capable of expressing HPV L1 gene and L2 gene was constructed. This eukaryotic vector was verified by enzymolysis and sequencing. The transcription of capsid genes was observed in vivo and in vitro by hydrodynamics-based transfection and liposome-mediated transfection. The expression of L1 gene in 293T cells was examined by Western blot. RESULTS： The eukaryotic co-expression vector pcD- NA3. 1-LI-IRES-L2 was successfully constructed and verified by enzymolysis and sequencing. The recombinant plasmid L1 and L2 genes were transcdpted in the livers of the rats and 293T cells. Cytopathic effect （CPE） occurred after the transfected with pcDNA3.1-L1-IRES-L2 into 293T cells, indicating that the two capsid genes were expressed. Western blot detection showed the recombinant plasmid L1 pro- tein was expressed in 293T cells. CONCLUSION： The pcD- NA3. 1-LI-IRES-L2 has been constructed successfully, which lays a foundation for in-dept study on the infectious mechanism of HPV16.