HBeAg真核表达载体的构建及其在中国仓鼠卵巢细胞中的表达

杨军; 刘妮; 卫阳; 靳耀锋; 王军宁; 康安静; 苏宝山; 李宗芳

HBeAg真核表达载体的构建及其在中国仓鼠卵巢细胞中的表达

1. 西安交通大学医学院第二附属医院病理科
2. 西安交通大学医学院第二附属医院科研实验中心
3. 西安交通大学医学院第二附属医院外科

基金项目:

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)；

详细信息

中图分类号: R512.62
计量
- 文章访问数: 2754
- HTML全文浏览量: 13
- PDF下载量: 651
- 被引次数: 0
出版历程
- 出版日期: 2012-11-20

Construction of a pcDNA-HBeAg expression vector to express HBeAg in CHO cells

Research funding:

摘要

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）。P...
- 肝炎病毒,乙型 /
- 肝炎e抗原,乙型 /
- pcDNA3.1（+）真核表达载体
Abstract:
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.
- hepatitis B virus /
- hepatitis B e antigen /
- pcDNA3.1 (+) eukaryotic expression vector

HTML全文

参考文献(14)

[1]Lau G, Marcellin P, Peters M.Chronic hepatitis B:a global healthproblem requiring coherent worldwide treatment strategies[J].Hep-atol Int, 2007, 1 (2) :316-325.

[2]陈大明, 肖宏.全球乙型肝炎疫苗研发分析[J].第二军医大学学报, 2006, 27 (7) :774-777.

[3]Block TM, Guo H, Guo JT.Molecular virology of hepatitis B virus forclinicians[J].Clin Liver Dis, 2007, 11 (4) :685-706

[4]Lang T, Lo C, Skinner N, et al.The hepatitis B e antigen (HBeAg) targets and suppresses activation of the toll-like recep-tor signaling pathway[J].J Hepatol, 2011, 55 (4) :762-769.

[5]Purvina M, Hoste A, Rossignol JM, et al.Human hepatitis B virale antigen and its precursor P20 inhibit T lymphocyte proliferation[J].Biochem Biophys Res Commun, 2012, 417 (4) :1310-1315.

[6]Chen MT, Billaud JN, Sllberg M, et al.A function of the hep-atitis B virus precore protein is to regulate the immune re-sponse to the core antigen[J].Proc Natl Acad Sci USA, 2004, 101 (41) :14913-14918.

[7]Chen M, Sllberg M, Hughes J, et al.Immune tolerancesplit between hepatitis B virus precore and core proteins[J].J Virol, 2005, 79 (5) :3016-3027.

[8]杨志勇, 杨茜, 肖贵宝, 等.HBeAg与乙型肝炎相关性肝细胞癌关系的临床观察[J].临床肝胆病杂志, 2012, 28 (4) :270-272.

[9]Fattovich G, Bortolotti F, Donato F.Natural history of chronichepatitis B:special emphasis on disease progression andprognostic factors[J].J Hepatol, 2008, 48 (2) :335-352.

[10]Liaw YF.HBeAg seroconversion as an important end point inthe treatment of chronic hepatitis B[J].Hepatol Int, 2009, 3 (3) :425-433.

[11]Lau GK.Current treatments for patients with HBeAg-positivechronic hepatitis B virus infection:a comparison focusing onHBeAg seroconversion[J].Liver Int, 2010, 30 (4) :512-520.

[12]周倩, 核小维, 罗志刚, 等.重组乙型肝炎病毒e抗原的研究进展[J].中国生物工程杂志, 2008, 28 (6) :108-112.

[13]Roseman AM, Berriman JA, Wynne SA, et al.A structuralmodel for maturation of the hepatitis B virus core[J].ProcNatl Acad Sci USA, 2005, 102 (44) :15821-15826.

[14]Walsh R, Nuttall S, Revill P, et al.Targeting the hepatitis Bvirus precore antigen with a novel IgNAR single variable do-main intrabody[J].Virology, 2011, 411 (1) :132-141.

施引文献

资源附件(0)

访问统计