摘要
目的构建含乙型肝炎病毒(HBV)囊膜蛋白的前前-S至前S2区酵母细胞表达质粒,以重组质粒转化酵母细胞后的自激活作用来探讨靶区域表达蛋白是否具有反式激活作用。方法用多聚酶链反应法扩增含前前-S区的前-S2基因序列,定向克隆于pDEST32载体,经序列测定,重组质粒命名为pDEST32-pS2。用乙酸锂转化法将重组质粒转化酵母菌MaV203,经Western blot法验证重组质粒在酵母细胞中的表达。按酵母双杂交法将重组质粒和载体pDEST22共同转入酵母细胞MaV203,并在SC/-Leu/-Trp/-His三缺培养基及不同浓度梯度3AT培养基中生长,判断靶区域编码蛋白是否具有自激活功能。结果重组质粒pDEST32-pS2经序列测定含有HBV自前前-S至前-S2基因序列,Western blot法证实转染重组质粒的酵母细胞可表达前S1和前S2蛋白。将pDEST32-pS2与pDEST22共转染MaV203,证实被转染酵母细胞不能在浓度为28mM的3AT培养基中生长。结论构建了pDEST32-pS2表达载体,pDEST32-pS2可在酵母细胞中表达部分囊膜蛋白,前前-S区至前S2区域编码的部分表面抗原可能具有较弱的反式激活作用,可以被3AT所抑制。
Objective Reconstructing the yeast expression vector aimed patial HBV envelope gene from pre-pre-S to pS2 as the target region. After yeast two-hybrid,auto-activation was used to explore the transactivation function of the expressed protein. Methods Pre-pre-S to prsS2 region coding region containing Not I and Sal I endoenzyme sites was obtained by PCR method. After enzyme digestion,the PCR products were conducted in yeast expression vector pDEST32. Reconstituted plasmids were sequenced by 3730 sequenator and were named as pDEST32-pS2. Reconstituted plasmid was transformed into the yeast cell MaV203 by Liac-mediated transformation method. The partial HBV surface protein expressed in the yeast cell was tested by Western blot analysis. According to yeast two-hybrid protocol,we transformed both pDEST32-pS2 and pDEST22 into MaV203 and tested the autoactivation in the yeast under a series of 3AT concentrations in SC/-Leu/-Trp/-His plates. Results Reconstituted plasmid pDEST32-pS2 including the anticipated fragment of pS2 gene was proved by DNA sequencing. Western blot analysis showed that plasmid pDEST32-pS2-transformed yeast cell did express pS1 and pS2 protein. Yeast two-hybrid test show that MaV203 transformed by pDEST32-pS2 and pDEST22 did not grow in SC/- Leu/-Trp/-His plates with 3AT at concentration more than 28mM. Conclusion MaV203 transformed by pDEST32-pS2 can express pS2 protein. The leading region of HBsAg might have slight trans-activation ability.
出处
《实用肝脏病杂志》
CAS
2008年第6期361-363,369,共4页
Journal of Practical Hepatology
基金
厦门市首批重大疾病科研攻关项目(WKZ0501)
厦门市卫生局医学科研立项项目(WSK0506)
厦门大学引进人才科研启动基金(Z03109)
福建青年科技人才创新项目(2006F3127)
福建高校新世纪人才创新资助项目
