人免疫缺陷病毒1(HIV-1)膜蛋白基因片段在酵母中的克隆表达

Cloning and expression of the envelope gene fragment from human immunodeficiency virus 1 in Saccharomyces cerevisiae

目的为获得足够量的膜糖蛋白，以便于对不同ＨＩＶ分离株膜糖蛋白的结构与功能进行进一步的研究。方法从人免疫缺陷病毒１（ＨＩＶ－１）ＨＸＢ２分离株原病毒基因组的重组质粒ｐＨＸＢ２中克隆了两段膜糖蛋白基因（ＥＮＶ）片段。以酵母穿梭诱导表达质粒ｐＹＥＳ２为载体，构建了两个相应的重组表达质粒ｐＹＥＮＶ１和ｐＹＥＮＶ２；进一步利用大肠杆菌β－半乳糖苷酶基因（β－ｌａｃＺ）构建了ＨＩＶ－１膜外糖蛋白ＤＮＡ片段与β－ｌａｃＺ基因的融合表达质粒。将此３种质粒分别转化单细胞真核生物酿酒酵母ＢＪ１９９１，得到的转化子经半乳糖诱导表达后进行菌体全蛋白的ＳＤＳ－ＰＡＧＥ分析。结果克隆的基因片段在酿酒酵母中产生了分子质量为５０×１０３的特异性诱导蛋白；对含此融合表达质粒的酵母转化子半乳糖诱导后表达产物的免疫检测表明，与对照菌株相比，融合表达产物具有和ＨＩＶ－１阳性血清抗体反应的抗原性。结论可通过β－半乳糖苷酶活性的测定直接指示抗原片段的表达；

Objective To acquire enough envelope glycoproteins so as to facilitate a further study of the structure and function of envelope glycoproteins from various kinds of virus isolates. Methods Two envelope glycoprotein gene fragments were cloned from the recombinant plasmid pHXB2 containing the human immunodeficiency virus 1(HIV 1) proviral genome of HXB2 isolate. Two corresponding expression plasmids pYENV1 and pYENV2 were constructed using a yeast shuttle inducible expression vector pYES2 Furthermore plasmid pYENVG12 with the fused gene cassette between external envelope gene fragment and β lacZ gene was constructed. These three plasmids were transformed into the unicellular eukaryotic S. cerevisiae BJ1991 and the galactose induced transformants were analyzed with the SDS PAGE. Results The result showed that a 50kD specific protein induced from the transformant containing the fused plasmid was shown to have antigenic reactivity with HIV 1 antibodies positive sera by immunodetection. Conclusions Therefore not only could the expression of the antigenic fragment be directly indicated by the β galactosidase activity, but also it laid some foundation for further purification of the fragment of the expressed envelope glycoproteins.