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Cloning and expression of gene of hepatitis B virus e antigen in yeast
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摘要: 为探讨乙型肝炎病毒 （HBV）e抗原 （HBeAg）的功能 ,用多聚酶链反应 （PCR）的方法以HBVayw亚型全序质粒pCP10为模板扩增HBeAg基因 ,克隆到pGEM -T载体中扩增并测序 ,鉴定符合GenBank报告序列。用EcoRI和PstI双酶切后回收片段 ,连接到真核表达载体pGBKT7中并转化酵母AH10 9,在色氨酸缺陷型培养基 （SD/-Trp）上筛选阳性菌落。提取阳性酵母菌的蛋白质 ,进行十二烷基磺酸钠 -聚丙烯酰胺凝胶电泳 （SDS -PAGE）和Western免疫印迹分析 ,显示HBeAg基因在酵母细胞中表达 ,表达产物在胞内存在 ,相对分子质量 （Mr）为 43 0 0 0左右Abstract: To investigate the biological functions of HBeAg protein, PCR was performed to amplify the gene of HBeAg from the plasmid pCP10 containing the whole fragment of ayw subtype of HBV.The PCR product was cloned into pGEM-T vector and was identified by DNA sequencing.Then, the gene of HBeAg was digested from pGEM-T vector and cloned into yeast expression plasmid pGBKT7. The reconstructed plasmid pGBKT7-HBeAg was transformed into yeast cell AH109 and screened on the synthetic dropout nutrient medium, (SD/-Trp) .The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.The results of SDS-PAGE and Western blotting assay showed the HBeAg was expressed in yeast cells and relative molecular weight of the expressed product was about 43000 Da.
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Key words:
- Hepatitis B virus e antigen /
- Yeast genetics /
- Gene exoression
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