Liu Li-Hong, Xu Lu, Li Yi-Jie, Zhang Fu-Chun, Ma Zheng-Hai
Key Laboratory of Molecular Biology, College of Life Science and Technology, Xinjiang University, Key Laboratory of Xinfiang Biological Resources and Genetic Engineering, Urumqi 830046, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008 Jul;24(7):663-7.
To construct the eukaryotic co-expression vector pcDNA3.1-L1-IRES-L2 for harvesting sufficient amounts of infectious HPV16.
The amplified 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.
The eukaryotic co-expression vector pcDNA3.1-L1-IRES-L2 was successfully constructed and verified by enzymolysis and sequencing.The recombinant plasmid L1 and L2 genes were transcripted 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 protein was expressed in 293T cells.
The pcDNA3h1-L1-IRES-L2 has been constructed successfully, which lays a foundation for in-dept study on the infectious mechanism of HPV16.
构建真核共表达载体pcDNA3.1-L1-IRES-L2，以收获足够量的感染性人乳头瘤病毒16型（HPV16）。
通过聚合酶链反应（PCR）从988质粒中扩增经密码子优化的HPV16衣壳基因，将其克隆到pCR-XL-TOPO载体中，然后亚克隆到真核表达载体pcDNA3.1(+)中。由此构建出能够表达HPV L1基因和L2基因的真核共表达载体pcDNA3.1-L1-IRES-L2。通过酶切和测序对该真核载体进行验证。采用流体动力学转染和脂质体介导转染在体内和体外观察衣壳基因的转录情况。通过蛋白质免疫印迹法检测293T细胞中L1基因的表达。
成功构建真核共表达载体pcDNA3.1-L1-IRES-L2，并经酶切和测序验证。重组质粒L1和L2基因在大鼠肝脏和293T细胞中转录。将pcDNA3.1-L1-IRES-L2转染293T细胞后出现细胞病变效应（CPE），表明两个衣壳基因均表达。蛋白质免疫印迹检测显示重组质粒L1蛋白在293T细胞中表达。
成功构建了pcDNA3h1-L1-IRES-L2，为深入研究HPV16的感染机制奠定了基础。
