应用毕赤酵母表达中国株HIV-1核心蛋白Gag  

Expression of HIV-1 Gag Protein (Chinese Strain) in Pichia Pastoris

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

机构地区：[1]解放军军需大学基因工程重点实验室,长春130062 [2]吉林大学生命科学学院,长春130023 [3]黑龙江省青岗县第3人民医院,161100

出　　处：《中国生物制品学杂志》2002年第2期72-74,共3页Chinese Journal of Biologicals

摘　　要：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。[Abstract] Objective To construct a recombinant plasmid pPICGAG for the expression of whole length of HIV - 1 Gag gene in Pichia pastoris . Methods The plasmid pKSGAG containing the whole length of HIV - 1 Gag gene was digested with Not I and Xhol and cloned into expression vector pPIC9. The constructed plasmid pPICGAG was linarized with Sac I and transformed to Pichia pastoris GS 115.The positive transforma-nts were identified by PCR and expressed in BMGY and BMMY media.The expressed products were analyzed by SDS - PAGE. Results The integration rate of transformant was 72.7% . SDS - PAGE showed a relative molecular weight of expressed protein of about 55000, and Western blot showed specific reaction of it with McAb. Conclusion The HIV - 1 Gag protein with good reactogenicity and specificity was successfully expressed in Pichia pastoris.

关 键 词：HIV-1 核心蛋白 毕赤酵母 表达 中国株 

分 类 号：R373.9[医药卫生—病原生物学] R512.91[医药卫生—基础医学]