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目的构建丙型肝炎病毒(HCV)构象性表位AR3与乙型肝炎病毒S抗原(HBs)嵌合的真核表达质粒,并在293T细胞中表达鉴定。方法采用重叠拼接PCR法,将HCV AR3表位的基因插入pSVB-24H质粒中,构建pSVB-24H-AR3重组质粒;再用PCR法扩增出全长的AR3-S嵌合基因,将其克隆到pCMV-HA载体中,构建pCMV-HA-AR3-S真核表达质粒。脂质体和PEI转染法导入293T细胞,Western blot和ELISA法分别检测重组蛋白的表达及分泌情况。结果 PCR法分别扩增出pSVB-24H部分载体、HCV AR3及HBs部分基因共3条基因片段;利用两步重叠拼接PCR,扩增得到1 118bp的三片段拼接产物,双酶切鉴定重组质粒pSVB-24H-AR3位置正确,测序分析表明插入序列及开放读码框正确,但细胞表达产物难以被抗-HBs识别;将完整的1 128bp的AR3-S基因插入带HA标签的表达载体中,酶切鉴定和测序分析表明重组质粒pCMV-HA-AR3-S构建正确,Western blot结果显示重组AR3-S蛋白可与HA抗体特异性反应;ELISA结果也显示细胞内能检测到较高水平的AR3-S重组蛋白。结论成功构建pSVB-24H-AR3和pCMV-HA-AR3-S嵌合表达质粒并在细胞中表达,为后续开展基于HBs的病毒样HCV颗粒疫苗研究提供了实验依据。
Objective To construct the eukaryotic expression plasmid of hepatitis C virus (HCV) conformational epitope AR3 chimeric with hepatitis B virus S antigen (HBs) and identify it in 293T cells. Methods The gene of HCV AR3 epitope was inserted into pSVB-24H plasmid by overlapping splicing PCR to construct pSVB-24H-AR3 recombinant plasmid. The full-length AR3-S chimeric gene was amplified by PCR and cloned PCMV-HA vector to construct pCMV-HA-AR3-S eukaryotic expression plasmid. 293T cells were transfected with liposome and PEI. Western blot and ELISA were used to detect the expression and secretion of recombinant protein respectively. Results A total of 3 fragments of pSVB-24H partial vector, HCV AR3 and partial HBs gene were amplified by PCR. Two-step overlapping splicing PCR was used to amplify the 1 118bp three-segment spliced product. Double enzyme digestion identified the recombinant plasmid pSVB The correct position of -24H-AR3 was confirmed by sequencing analysis. The inserted sequence and open reading frame (ORF) were correct but the expressed products were difficult to recognize by anti-HBs. The complete 128 bp AR3-S gene was inserted into HA tagged expression vector, Western blot analysis showed that the recombinant AR3-S protein reacted specifically with HA antibody. The results of ELISA also showed that higher levels of AR3-S protein were detected in the cells by Western blot analysis. S recombinant protein. Conclusion The chimeric expression plasmids of pSVB-24H-AR3 and pCMV-HA-AR3-S were successfully constructed and expressed in the cells, which provided an experimental basis for further study on HBs-based virus-like particle vaccine.