结核分枝杆菌热休克蛋白65与汉坦病毒G2糖蛋白融合基因真核表达载体的构建与表达

Construction and Transient Expression of Recombinant Plasmid of HSP65 Gene of Mycobacterium Tuberculosis and G2 Gene of Hantavirus H8205 Strain in Mammalian Cells

目的构建以结核分枝杆菌热休克蛋白65(HSP65)和汉坦病毒G2糖蛋白重组融合基因为基础的真核表达载体,为进一步研究其在结核病和出血热防治中的作用奠定基础。方法采用聚合酶链反应从结核分枝杆菌H37Rv株基因中,扩增出HSP65的编码基因,从T-H8205G2质粒扩增出G2糖蛋白的编码基因,经相应限制性核酸内切酶消化后,插入真核表达载体pcDNA3.1/His,并将此重组质粒通过脂质体介导转染NIH3T3细胞,用SDS-PAGE、免疫组织化学及免疫荧光方法分别测定表达产物的相对分子量及特异性。结果获得正向插入的pcDNA3.1/His-HSP65-G2重组表达质粒,表达产物的相对分子量为105 kD,与理论预期大小一致,并且可与汉坦病毒H8205株的腹水抗体和HSP65单抗起特异反应。表明构建的pcDNA3.1/His-HSP65-G2能在哺乳动物细胞中表达并具有抗原性。结论编码HSP65和G2抗原基因的重组基因的真核表达载体构建成功。

Objective To construct a nuclcotide vaccine based on the recombinant plasmids of heat shock protein 65 （HSP65） gene of mycobacterium tuberculosis H37Rv Strain and G2 gene segment of Hantavirus H8205 Strain. Methods The gene encoding HSP65 and G2 glycoprotein was amplified from genome of mycobacterium tuberculosis and T-H8205G2 plasmid by polymerase chain reaction （PCR）. After restriction endonuclease digestion correspondingly, the fragments were inserted into the downstream of the vector pcDNA3. 1/His to obtain the recombinant eukaryotic expression plasmid pcDNA3. 1/HisHSP65-G2. The recombinant plasmid peDNA3.1/His-HSP65-G2 was transfected into eukaryotic NIH3T3 cells by lipofectinemediated transfection. SDS-PAGE, immunocytochemistry and indirect FIA analysis were used to detect the molecular weight and specificity of the expressed protein respectively. Results The positive recombinant plasmid harboring the right inserted G2 glyeoprotein gene and HSP65 gene fragment was obtained. The fusion protein in mammalian cells NIH3T3 was about 105 kD, corresponding to the estimated molecular size of the product of recombinanl plasmid and could react with the Hantavirus H8205 Strain hyoer immune mouse ascites and HSP65 antibody of mycobacterium tuberculosis H37Rv Strain. It was suggested the G2 glycoprotein gene and HSP65 gene could be expressed in mammalian cells. Conclusion The successful construction of recombinant eukaryotic expression vector based on HSP65 gene of mycobacterium tuberculosis and G2 gene segment of Hantavirus H8205 Strain lays the foundation for further study on prevention and treatment of tuberculosis and HFRS.