HBV PreS2S/Fc融合基因真核表达载体的构建及表达

Construction and expression of eukaryotic vector bearing fusion gene of HBV PreS2S and Fc fragment

目的:构建含HBV PreS2S和Fc融合基因的真核表达载体pcDNA3 S2S/Fc并在真核细胞中进行表达. 方法:应用重叠延伸剪切技术(splicing by overlapping extension,简称SOE)经两次PCR获得嵌合基因片段S2S/ Fc,回收后克隆到pGEM-T Easy TA克隆载体,获得合适的酶切位点,再采用双粘端连接法转克隆入真核表达载体pcDNA3中,得到真核重组载体pcDNA3 S2S/Fc.然后用脂质体法转染SP2/0细胞. 结果:对重组载体进行了限制性酶切鉴定及测序分析,汪明连接正确;经间接免疫荧光捡测证实该重组载体能在真核细胞中表达插入的外源性基因编码的融合蛋白. 结论:真核表达载体pcDNA3 S2S/Fc的成功构建及在SP2/0 细胞中的有效表达,为进一步探讨HBV感染的特异性免疫治疗提供了实验依据.

AIM: To construct and express a recombinant eukaryotic expression vector bearing fusion gene of HBV S2S and Fc fragment. METHODS: The technique of splicing by overlapping extension and twice PCR were used, and fusion gene fragment was obtained and cloned into pGEM-T Easy TA cloning vector to get suited enzyme sites. Recombinant eukaryotic expression vector pcDNAS S2S/Fc was constructed by double adhesive terminal ligation. Then the recombinant vector was transferred into SP2/0 cells by using Lipofectamine. RESULTS: The recombinant vector was identified by digestion with restriction enzymes and confirmed by DNA sequencing analysis. And the vector bearing fusion gene could be expressed in eukaryotic cells detected by indirect immunofluotescence technique. CONCLUSION: The relative efficient expression of the fusion gene in SP2/0 cells may provide an experimental basis for specific immunotherapy for HBV infection.