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目的 将中国流行株B亚型HIV 1结构蛋白基因gag和 gp12 0在巴斯德毕赤酵母中进行融合表达。 方法 以酵母分泌型表达质粒 pPIC9为载体 ,构建含HIV 1gag和gp12 0嵌合基因的重组酵母表达质粒pPICGP。用SacI将pPICGP线性化后 ,电转化巴斯德毕赤酵母GS115 ,用PCR的方法鉴定阳性酵母转化子。阳性转化子在巴斯德毕赤酵母中用甲醇进行诱导表达 ,表达产物以SDS PAGE和West ernblot进行分析 ,并对阳性菌株的遗传稳定性进行研究。结果 12个酵母转化子中共筛选到了 8个阳性酵母转化子 ,其整合率为 6 6 .7%。SDS PAGE和Westernblot分析显示 ,在相对分子质量 (Mr)为 5 70 0 0处有 1条特异蛋白带 ,且能与抗p2 4单抗 (mAb)和抗 gp12 0mAb发生反应。酵母转化子在YPD培养基上传代 10次后 ,其外源基因未丢失。结论 在巴斯德毕赤酵母中成功地表达了HIV 1gag gp12 0嵌合基因 ,且表达蛋白具有特异性 ,但其Mr较预计计算的值要小 ,说明嵌合基因中的 gp12 0基因只有部分进行了表达
OBJECTIVE: To construct the fusion protein of HIV-1 structural gene gag and gp12 0 of Chinese subtype B in Pichia pastoris. Methods The recombinant yeast expression plasmid pPICGP containing HIV 1 gag and gp12 0 chimeric genes was constructed by using yeast secreting plasmid pPIC9 as a vector. After linearization of pPICGP with Sad, Pichia pastoris GS115 was electroporated, and positive yeast transformants were identified by PCR. The positive transformants were induced with methanol in Pichia pastoris and the expressed products were analyzed by SDS PAGE and West ernblot. The genetic stability of the positive strains was also studied. Results A total of 8 positive yeast transformants were screened from 12 yeast transformants, with an integration rate of 6.6%. SDS PAGE and Western blot analysis showed that there was one specific protein band at a molecular weight of 5 700 and could react with anti-p2 4 mAb and anti-gp12 0 mAb. Yeast transformants in YPD medium after passage 10 times, the foreign gene is not lost. Conclusion The HIV 1 gag gp12 0 chimeric gene was successfully expressed in Pichia pastoris and the expressed protein was specific but its Mr value was smaller than predicted, indicating that only part of the gp12 0 gene in the chimeric gene Expressed