丙型肝炎病毒HLA-A2限制性复合多表位基因重组腺病毒的包装、筛选及鉴定

韦三华; 任继鸿; 刘丽; 贾战生; 董轲; 林芳; 王希; 张利军; 刘昕阳; 张惠中

丙型肝炎病毒HLA-A2限制性复合多表位基因重组腺病毒的包装、筛选及鉴定

1. 第四军医大学唐都医院临床实验与检验输血科
2. 第四军医大学唐都医院全军感染病诊疗中心

基金项目:

国家自然科学基金项目(30700760)；国家“863”计划专题(2007AA02Z441)；

详细信息

中图分类号: R392
计量
- 文章访问数: 3847
- HTML全文浏览量: 13
- PDF下载量: 834
- 被引次数: 0
出版历程
- 出版日期: 2011-01-20

Packaging, selection and identification of recombinant adenoviruses carrying HCV HLA-A2 restricted compound multi-epitopes antigen

Research funding:

摘要

摘要:
目的构建丙型肝炎病毒（HCV）HLA-A2限制性复合多表位基因的重组转移质粒pAdtrack-CMV-Ub-Mep,转染HEK293细胞,包装重组腺病毒rAd-Ub-Mep。方法复合表位Ub-Mep基因克隆到pAdTrack-CMV穿梭质粒,得到重组的穿梭质粒pAdtrack-CMV-Ub-Mep,将穿梭质粒pAdtrack-CMV-Ub-Mep和腺病毒骨架质粒pAdEasy1共转染HEK293细胞,产生重组腺病毒rAd-Ub-Mep,通过检测绿色荧光蛋白（GFP）的表达和PCR扩增目的基因的方法鉴定重组腺病毒,并测定重组腺病毒滴度。结果经酶切鉴定、PCR鉴定和荧光分析,均证实得到重组的腺病毒。测定病毒滴度为1.2×1011cfu/L。结论成功包装出重组腺病毒rAd-Ub-Mep,构建了HCV HLA-A2限制性多表位基因的重组腺病毒疫苗,为进一步研究HLA-A2限制性复合多表位基因诱导的细胞免疫应答奠定了基础。
- 肝炎病毒属 /
- 表位 /
- 腺病毒,人
Abstract:
Objective To construct recombinant shuttle plasmid pAdtrack-CMV-Ub-Mep carrying HCV HLA-A2 restricted compound multi-epitopes gene, and transfect to HEK293 cells, to obtain recombinant adenoviruses rAd-Ub-Mep.Methods The compound antigen gene Ub-Mep was cloned into shuttle plasmid pAdTrack-CMV for pAdtrack-CMV-Ub-Mep.The recombinant adenoviruses rAd-Ub-Mep was packed in HEK293 cells by co-transfection with pAdtrack-CMV-Ub-Mep and frame plasmid pAdEasy1.The recombinant adenoviruses rAd-Ub-Mep was identified by PCR and expression of GFP, and the virus titer was determined by TCID50.Results The recombinant adenoviruses rAd-Ub-Mep was confirmed by enzyme cutting, PCR and fluorometric analysis.The virus titer was 1.2×1011cfu/L.Conclusion Success to construct recombinant adenoviruses vaccine, is the base for the study of cellular immunity induced by HCV HLA-A2 restricted compound multi-epitopes gene.
- hepacivirus /
- epitopes /
- adenoviruses

HTML全文

参考文献(12)

[1]韦三华, 尹文, 雷迎峰, 等.HCV多表位基因重组BCG的筛选及其诱导的免疫应答研究[J].中华微生物学和免疫学杂志, 2007, 27 (11) :1041-1045.

[2]Wei SH, Yin W, An QX, et al.A novel HCV vaccine approach using recombinant BCG expressing multi-epitope antigen[J].Archieve of viology, 2008, 153 (6) :1021-1029.

[3]韦三华, 董轲, 林芳, 等.HCV HLA-A2限制性复合多表位基因的构建、克隆表达及其免疫特性分析[J].中国免疫学杂志, 2001, 33 (1) :73-75.

[4]Houshton M, Abrignani S.Prospects for a vaccine against the hepatitis C virus[J].Nature, 2005, 436 (7053) :961-966.

[5]Kawai T, Akira S.Innate immune recognition of viral infection[J].Nat Immunol, 2006, 7 (2) :131-137.

[6]Folgori A, Capone S, Ruggeri L, et al.A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees[J].Nat Med, 2006, 12 (2) :190-197.

[7]Form X, Bukh J, Purcell R H.The challenge of developing a vaccine against hepatitis C virus[J].J Hepatol, 2002, 37 (5) :684-695.

[8]Gao M, Wang HP, Wang YN, et al.HCV-NS3 Th1 minigene vaccine based on invariant chain CLIP genetic substitution enhances CD4+Th1 cell responses in vivo[J].Vaccine, 2006, 24 (26) :5491-5497.

[9]Ward S, Lauer G, Isba R, et al.Cellular immune responses against hepatitis C virus:the evidence base 2002[J].Clin Exp Immunol, 2002, 128 (2) :195-203.

[10]Yu H, Huang H, Xiang J, et al.Dendritic cells pulsed with hepatitis C virus NS3 protein induce immune responses and protection from infection with recombinant vaccinia virus expressing NS3[J].J Gen Virol, 2006, 87 (Pt1) :1-10.

[11]Arribillaga L, de Cerio AL, Sarobe P, et al.Vaccination with an adenoviral vector encoding hepatitis C virus (HCV) NS3 protein protects against infection with HCV-recombinant vaccinia virus[J].Vaccine, 2002, (21) :202-210.

[12]Hao CQ, Li Gy, Zhou YX, et al.Construction, package and identification of replication-deficient recombinant adenovirus expression vector of HCV C[J].World Chin J Digestol, 2003, 11 (2) :144-147.

施引文献

资源附件(0)

访问统计