preS1-preS2-HBsAg真核表达载体的构建及在293细胞中的表达

目的研究含有前S1抗原、前S2抗原的乙肝病毒外膜蛋白(前S1蛋白+前S2蛋白+S蛋白)的跨膜特性。方法依据乙肝病毒大外膜蛋白的计算机模拟图将其分为分别命名为HBsAg1(1-174),HBsAg2(1-245),HBsAg3(1-294),HBsAg4(1-341),HBsAg5(1-373),HBsAg6(1-400),最终累加成一条连续的长链状态,贯穿在细胞内外形成四次跨膜区,并以此6个片段为模板设计6对引物。从含有乙肝病毒外膜蛋白基因的质粒pcDNA3.1-preS1-preS2-HBsAg中扩增出前S1,S2基因,采用重叠延伸PCR方法连接人尿激酶原信号肽+6×组氨酸(His)+前S1蛋白、前S2蛋白+HBsAg(1～6)+血细胞凝集素(HA);PCR产物经XhoⅠ和EcoRⅠ双酶切后插入经同样处理的真核表达载体pIRES2-EGFP,构建含有Pro-UK,6×His,preS1,preS2,HBsAg跨膜序列及HA的表达载体pIRES2-EGFP/Pro-UK-6×His-preS1-preS2-HBsAg(1～6)-HA,该表达载体应能够共表达乙肝病毒外膜蛋白和绿色荧光蛋白。采用阳离子脂质体法将构建好的表达载体转染293细胞,经G418加压筛选阳性克隆,并进一步通过荧光观察、蛋白质免疫印迹、免疫双标等方法检测目的蛋白在293细胞的表达。结果 PCR结果显示,电泳片段大小符合HBsAg1～HBsAg6片段的600,810,957,1098,1194和1275 bp理论值。酶切鉴定正确的重组质粒测序结果与预期序列完全一致。转染和单克隆筛选后得到转染成功的HBsAg1～HBsAg6的293细胞,呈亮绿色,细胞形态良好,荧光表达强度高;Western印迹结果显示,在相对分子质量31 000,34 000,44 000,47000,54 000和60 000处有明显的阶梯状蛋白信号,条带清晰并与理论值相一致。结论成功构建了含有前S1、前S2蛋白的乙肝外膜蛋白基因的真核表达载体,并获得了能共表达乙肝外膜蛋白跨膜序列和绿色荧光蛋白的293细胞系。

Construction of preS1-preS2-HBsAg of eukaryotic expression vector and its transmembrane property in 293 cells

OBJECTIVE To study the transmembrane properties of preS1-preS2-HBsAg of hepatitis B virus. METHODS Devided the large envelope protein(L protein) into six fragments according to its computer simulation structure model to design six pairs of primers (named HBsAg1-6). Final accumulation into a continuous length of chain which could form the four transmembrane domain in the cells inside and outside. The preS1 and preS2 fragments were amplified from the plasmid pcDNA3.1-preS1-preS2-HBsAg. And we linked pro-urokinase signal peptide(pro-UK) with 6×histidine(6×His), HBsAg(1-6) and hemagglutinin (HA) by overlapping extension PCR method. The PCR products were double digested with XhoⅠ and EcoRⅠand cloned into the expression vector pIRES2-EGFP. The constructed expression vectors were pIRES2-EGFP/Pro-UK-6×His-preS1-preS2-HBsAg(1-6)-HA, and they could coexpress preS1-preS2-HBsAg(1-6) , GFP and HA theoretically. The vectors were transfected into 293 cells respectively using Lipofectamine method. The positive clones were screened by G418 pressure. The expression of preS1-preS2-HBsAg(1-6) was detected using fluorescence observation, Western blotting and immune double labeling method indirectly. RESULTS PCR results showed that, the size of electrophoresis fragment in line with the theoretical value of HBsAg 1-HBsAg 6 fragments which are 600, 810, 957, 1098, 1194 and 1275 bp. The restriction endonuclease digestion of recombinant plasmid sequencing results was entirely consistent with the expected sequence.Get 293 cells which were successfully transferred of HBsAg 1-HBsAg 6 after transfection and monoclonal antibody screening. The cells were bright green, in good shape, and with high fluorescence expression intensity. Western blotting results showed that, there was ladder-like protein signaling on relative molecular mass of 31 000, 34 000, 44 000, 47 000, 54 000 and 60 000. Clear bands were consistent with the theoretical value.CONCLUSION Eukaryotic expression vectors are successfully constructed, the vectors contain Pro-UK, 6×His, preS1, preS2, HBsAg(1-6) and HA genes. And the 293 cell lines which could coexpress HBsAg(1-6), GFP and HA proteins are obtained.