论文部分内容阅读
目的 构建含HIV-1Gag全长基因的重组酵母表达质粒pPICGAG，并在毕赤酵母中进行表达。方法用Not I和XhoI将含HIV－1 Gag全长基因的质粒pKSGAG双酶切后，克隆到酵母表达载体pPIC9中，构建了重组表达质粒 pPICGAG。pPICGAG用 SacI线性化后，电转化毕赤酵母 GS115，PCR鉴定阳性酵母转化子，并分别将PCR阳性的酵母转化子在BMGY和BMMY培养基中进行诱导表达，表达产物进行SDS-PAGE电泳分析。结果 酵母转化子的整合率为72．7％。SDS-PAGE结果显示表达蛋白的相对分子质量为55000左右，与预计计算的值相同。Western blot结果显示表达蛋白能与单克隆抗体发生特异性反应。结论 在毕赤酵母中成功地表达了HIV-1核心蛋白Gag，且表达的蛋白具有良好的反应原性和特异性。
Objective To construct a recombinant yeast expression plasmid pPICGAG containing the full-length HIV-1 Gag gene and express it in Pichia pastoris. Methods The plasmid pKSGAG containing the full-length HIV-1 Gag gene was digested with Not I and XhoI and cloned into the yeast expression vector pPIC9 to construct the recombinant plasmid pPICGAG. pPICGAG was linearized with SacI and transformed into Pichia pastoris GS115 by electroporation. The positive yeast transformants were identified by PCR. The PCR positive yeast transformants were induced in BMGY and BMMY medium, and the expressed products were analyzed by SDS-PAGE. Results The integration rate of yeast transformants was 72.7%. The SDS-PAGE results showed that the relative molecular mass of the expressed protein was about 55000, which was the same as expected. Western blot results showed that the expressed protein reacted specifically with monoclonal antibodies. Conclusion The HIV-1 core protein Gag was successfully expressed in Pichia pastoris and the expressed protein has good reactivity and specificity.