中国株HIV-1结构蛋白基因gag与gp120在巴斯德毕赤酵母中的融合表达  

Fusion expression of structural protein gene gag and gp120 of HIV-1_(CN) in Pichia pastoris

作　　者：江文正[1] 金宁一[1] 王宏伟[1] 张应玖[2] 金洪涛[1] 

机构地区：[1]解放军军需大学基因工程实验室,吉林长春130062 [2]吉林大学生命科学学院,吉林长春130023

出　　处：《细胞与分子免疫学杂志》2002年第5期426-428,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家杰出青年基金资助 ;No .3982 5 119

摘　　要：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 gp12Aim To express fusion protein of HIV-1 CNB gag and gp120 in Pichia pastoris. Methods Using the yeast expression plasmid pPIC9 as the vector, the recombinant yeast expression plasmid pPICGP containing gag and gp120 chimeric genes of HIV-1 was constructed. After being linearized by Sac I, the plasmid pPICGP was electrotransformed into Pichia pastoris strain GS115. The positive yeast transformants were identified by PCR, and fusion protein was expressed in Pichia pastoris under the induction of methanol. The expressed products were analyzed by SDS-PAGE and Western blot, and the genetic stability of the positive strains were also studied. Results Of twelve yeast colonies, 8 transformants were positive, indicating that the integration rate was 66.7%. The SDS-PAGE and Western blot analysis showed that there was a specific protein band at the site of Mr 57 000, which reacted with anti-p24 mAb and anti-gp120 mAb. The yeast transformants still had the foreign gene after ten times of passage in YPD medium. Conclusion The chimeric gene of gag and gp120 of HIV-1 was successfully expressed in Pichia pastoris, but the relative molecular mass(Mr) of expressed protein is smaller than the expected value, suggesting partial expression of gp120 gene in the fused gene.

关 键 词：HIV-1 结构蛋白 毕赤巴斯德酵母 融合表达 人工型 免疫缺陷病毒 

分 类 号：R373.9[医药卫生—病原生物学]