水痘带状疱疹病毒重组腺病毒载体疫苗的构建及评价
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摘要
目的构建表达水痘带状疱疹病毒(varicella-zoster virus,VZV)gE抗原的重组腺病毒载体疫苗,并对其免疫原性进行评价。方法采用AdEasy系统构建携带gE基因的重组腺病毒质粒pAdEasy-gE,PCR及Western blot法鉴定AD293细胞系包装的重组腺病毒rAd-gE,采用阴离子和复合介质Capto Core 700两步法对其进行纯化。以3. 3×10~6、1×10~7、3×10~7ifu剂量的rAd-gE分别单针肌内注射免疫NIH小鼠,检测小鼠血清抗体滴度。结果 PCR及PacⅠ酶切鉴定表明,携带gE基因的pAdEasy-gE构建成功;PCR及Western blot分析显示,AD293细胞系成功包装可表达gE糖蛋白的rAd-gE;经两步法纯化,可回收18. 2%的rAd-gE。1×10~7和3×10~7 ifu剂量的rAd-gE可引起小鼠的体液免疫响应。结论已成功构建表达高度糖基化gE糖蛋白的rAd-gE,可有效引起小鼠的体液免疫响应,为VZV新型重组腺病毒疫苗的进一步研发奠定基础。
Objective To construct a recombinant adenovirus expressing glycoprotein E(gE)gene against varicella zoster virus(VZV)and evaluate its immunogenicity. Methods Recombinant adenovirus plasmid pAdEasy-gE carrying gE gene was constructed by using AdEasy system. The recombinant adenovirus rAd-gE packed in AD293 cells was identified by PCR and Western blot and purified by anion exchange and Capto Core 700 chromatography. NIH mice were injected i. m.with rAd-gE at dosages of 3. 3 × 10~6,1 × 10~7 and 3 × 10~7 ifu respectively,of which the serum antibody titer was determined. Results PCR and restriction analysis with PacⅠproved that pAdEasy-gE was constructed correctly. PCR and Western blot proved that rAd-gE was packaged successfully in AD293 cells. The recovery of rAd-gE purified by anion exchange and Capto Core 700 chromatography was 18. 2%. Humoral immune response was induced in mice by r AD-gE at dosages of 1 × 10~7 and 3 × 10~7 ifu. Conclusion The rAd-gE highly expressing gE gene was successfully constructed,which induced effective humoral immunity and laid a foundation of further development of recombinant adenovirus vaccine against VZV.
Objective To construct a recombinant adenovirus expressing glycoprotein E(gE)gene against varicella zoster virus(VZV)and evaluate its immunogenicity. Methods Recombinant adenovirus plasmid pAdEasy-gE carrying gE gene was constructed by using AdEasy system. The recombinant adenovirus rAd-gE packed in AD293 cells was identified by PCR and Western blot and purified by anion exchange and Capto Core 700 chromatography. NIH mice were injected i. m.with rAd-gE at dosages of 3. 3 × 10~6,1 × 10~7 and 3 × 10~7 ifu respectively,of which the serum antibody titer was determined. Results PCR and restriction analysis with PacⅠproved that pAdEasy-gE was constructed correctly. PCR and Western blot proved that rAd-gE was packaged successfully in AD293 cells. The recovery of rAd-gE purified by anion exchange and Capto Core 700 chromatography was 18. 2%. Humoral immune response was induced in mice by r AD-gE at dosages of 1 × 10~7 and 3 × 10~7 ifu. Conclusion The rAd-gE highly expressing gE gene was successfully constructed,which induced effective humoral immunity and laid a foundation of further development of recombinant adenovirus vaccine against VZV.
引文
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[12]THOMSSON E,PERSSON L,GRAHN A,et al.Recombinant glycoprotein E produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology[J].J Virol Methods,2011,175(1):53-59.
[13]LIU J,CHEN C,ZHU R,et al.Evaluation of immunity to varicella zoster virus with a novel double antigen sandwich enzyme-linked immunosorbent assay[J].Appl Microbiol Biotechnol,2016,100(21):9321-9329.
[2]MICHALIK D E,STEINBERG S P,LARUSSA P S,et al.Primary vaccine failure after 1 dose of varicella vaccine in healthy children[J].J Infect Dis,2008,197(7):944-949.
[3]SUENAGA T,MATSUMOTO M,ARISAWA F,et al.Sialic acids on varicella-zoster virus glycoprotein B are required for cell-cell fusion[J].J Biol Chem,2015,290(32):19833-19843.
[4]ALONSO-PADILLA J,PAPP T,KAJAN G L,et al.Development of novel adenoviral vectors to overcome challenges observed with HAdV-5-based constructs[J].Mol Ther,2016,24(1):6-16.
[5]EWER K J,LAMBE T,ROLLIER C S,et al.Viral vectors as vaccine platforms:from immunogenicity to impact[J].Curr Opin Immunol,2016,41(1):47-54.
[6]ERTL H C.Viral vectors as vaccine carriers[J].Curr Opin Virol,2016,21(1):1-8.
[7]UUSI-KERTTULA H,HULIN-CURTIS S,DAVIES J,et al.Oncolytic adenovirus:strategies and insights for vector design and immuno-oncolytic applications[J].Viruses,2015,7(11):6009-6042.
[8]MIEST T S,CATTANEO R.New viruses for cancer therapy:meeting clinical needs[J].Nat Rev Microbiol,2014,12(1):23-34.
[9]HOFACRE A,WODARZ D,KOMAROVA N L,et al.Early infection and spread of a conditionally replicating adenovirus under conditions of plaque formation[J].Virology,2012,423(1):89-96.
[10]BAO L D,WEI G M,GAN H M,et al.Immunogenicity of varicella zoster virus glycoprotein E DNA vaccine[J].Exp Ther Med,2016,11(5):1788-1794.
[11]LUO J,DENG Z L,LUO X,et al.A protocol for rapid generation of recombinant adenoviruses using the AdEasy system[J].Nat Protoc,2007,2(5):1236-1247.
[12]THOMSSON E,PERSSON L,GRAHN A,et al.Recombinant glycoprotein E produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology[J].J Virol Methods,2011,175(1):53-59.
[13]LIU J,CHEN C,ZHU R,et al.Evaluation of immunity to varicella zoster virus with a novel double antigen sandwich enzyme-linked immunosorbent assay[J].Appl Microbiol Biotechnol,2016,100(21):9321-9329.