摘要
目的克隆HBeAg基因,构建重组HBeAg真核表达载体,并在中国仓鼠卵巢细胞(CHO细胞)中进行表达。方法采用PCR法从HBeAg阳性乙型肝炎患者血清HBV DNA中扩增HBeAg基因,克隆入pcDNA3.1(+)真核表达载体中,构建重组pcDNA-HBeAg真核表达载体,经PCR、双酶切、测序鉴定后,将其转染入CHO细胞,G418筛选,用PCR、免疫斑点、Western Blot、免疫细胞化学方法检测HBeAg在CHO细胞中的表达。结果成功克隆到HBeAg基因,并构建了重组pcDNA-HBeAg真核表达载体;基因测序证实克隆的HBeAg基因中共有12个位点发生单碱基置换突变(C1819G,A2007T,C2046T,C2061G,G2106A,C2109A,C2146T,T2172C,C2203T,A2235G,G2253A,C2298T),1个位点发生缺失突变(2346 del T);成熟HBeAg蛋白中149位缬氨酸(valine,V)突变为苯丙氨酸(phenylalanine,F)(V149F),在HBeAg蛋白羧基端融合有一段长11个氨基酸的多肽(RLESRGPVZTR)。PCR、免疫斑点、Western Blot和免疫细胞化学方法证实重组pcDNA-HBeAg真核表达载体可在CHO细胞中表达分泌型HBeAg蛋白。结论重组HBeAg真核表达载体的构建和表达为HBeAg的临床诊断和深入研究提供了条件。
Objective To clone the hepatitis B e antigen（HBeAg） gene from the hepatitis B virus（HBV） genome into a eukaryotic expression vector so that the HBeAg gene may be efficiently expressed in the Chinese hamster ovary（CHO） cell line for in vitro analyses.Methods The HBeAg gene was PCR amplified from HBV DNA extracted from the serum of an HBeAg-positive patient and using primers based on the HBV strain FMU013.The purified amplicon was inserted into the pcDNA3.1（＋） eukaryotic expression vector.The resultant recombinant plasmid,pcDNA-HBeAg,was verified by restriction enzyme digestion and PCR sequencing,and then transformed into cultured CHO cells.Expression of the HBeAg protein was confirmed by PCR,enzyme-immunodotting assay,Western blotting,and immunocytochemistry.Results The HBV HBeAg gene was successfully amplified and cloned to generate the pcDNA-HBeAg eukaryotic expression vector.Nucleotide sequencing analysis of the full-length HBeAg gene clone revealed 12 base substitution mutations（C1819G,A2007T,C2046T,C2061G,G2106A,C2109A,C2146T,T2172C,C2203T,A2235G,G2253A,and C2298T） and 1 deletion mutation（2346 del T）.Amino acid sequence analysis of the mature HBeAg protein revealed only one mutation,which was a valine（V） to phenylalanine（F） substitution at site 149（V149F）.The V149F mutation formed a peptide（RLESRGPVZTR） that was fused to the carboxyl terminus of the HBeAg protein.The fusion HBeAg protein was expressed in CHO cells,and was detected by PCR,enzyme-immunodotting,Western blotting,and immunocytochemistry tests.Conclusion The recombinant pcDNA-HBeAg vector successfully expresses the fusion HBeAg protein in CHO cells.This newly developed in vitro system is a potentially useful and convenient tool for further study of HBeAg.
出处
《临床肝胆病杂志》
CAS
2012年第11期845-848,共4页
Journal of Clinical Hepatology
基金
陕西省科技统筹创新工程计划项目(2011KTCL03-14)
教育部新教师基金(编号:2007-0698106)
教育部新世纪优秀人才计划(编号:NCET-10-0647)
