HPV16-E7 H2P融合钙网蛋白基因重组腺病毒载体疫苗的构建与免疫学鉴定  

CONSTRUCTION AND CHARACTERIZATION OF A NOVEL THERAPEUTIC VACCINE OF CALRETICULIN/HPV16-E7H2P FUSION GENE RECOMBINANT ADENOVIRUS VETOR

作　　者：刘瑞林 马永臻 张永东 马春玲 

机构地区：[1]临沂市中医院检验科,山东临沂276002 [2]山东医学高等专科学校

出　　处：《山东医学高等专科学校学报》2012年第5期321-324,F0003,共5页Journal of Shandong Medical College

基　　金：山东省医药卫生科技发展计划项目(No.2009 HW087);临沂市科技发展计划项目(No.201013067)

摘　　要：目的构建表达钙网蛋白(CRT)与乳头瘤病毒16型E7基因H2P突变型(HPV16-E7 H2P)融合蛋白重组腺病毒载体(Ad-CRT/HPV16-E7 H2P),为研发新型乳头瘤病毒治疗性疫苗奠定基础。方法首先利用RT-PCR的方法扩增CRT基因,并进一步构建CRT与HPV16-E7 H2P基因融合重组的pJW4303表达载体,将目的基因加上特定的CACC接头后克隆入载体pENTR/D-TOPO以获得入门克隆。经过入门克隆与表达载体(pAd-CMV/V5-DEST)间的重组反应获得表达克隆Ad-CRT/HPV16-E7 H2P。表达克隆线性化后转染HEK293A包装细胞,得到重组腺病毒。结果构建的Ad-CRT/HPV16-E7 H2P经PCR和测序鉴定构建正确;转染HEK293A细胞并扩增后获得的病毒滴度为1.95×1011 pfu/mL,该重组病毒载体能正确表达CRT/HPV16-E7 H2P融合蛋白。结论成功构建了Ad-CRT/HPV16-E7 H2P。Objective To generate recombinant adenoviral vector containing CRT-HPV16-E7H2P fusion gene for developing a safe,effective and Human papilloma virus（HPV）-specific therapeutic vaccine.Methods The fusion of CRT and HPV16-E7H2P gene was constructed by using polymerase chain reaction（PCR）,and then the fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA（CACC） sequence was added to the 5′end.Adenoviral expression vector containing CRT-HPV16-E7H2P fusion gene was constructed by homologous recombination.The linearized DNA plasmid of the recombinant adenoviral vector transfected human embryo kidney（HEK） 293A cells to package and amplify recombinant adenovirus.Resu ltsThe CRT-HPV16-E7H2P fusion gene was characterized by using PCR,and the sequencing result revealed that the length and sequence were accurate.The construction of CRT-HPV16-E7H2P fusion gene recombinant pENTR/D-TOPO vector was successful,and the recombinant adenoviral vector,Ad-CRT/HPV16-E7H2P,was generated successfully.The titer of Ad-CRT/HPV16-E7H2P was characterized as 1.95×10^11pfu/ml.The CRT-HPV16-E7H2P fusion protein was expressed by HEK 293A cells correctly.ConclusionCRT/HPV16-E7H2P fusion gene recombinant replication-defective adenovirus expression vector was constructed successfully,and this study has provided an experimental basis for further research of HPV gene therapy.

关 键 词：钙网蛋白 乳头瘤病毒 腺病毒表达载体 治疗性疫苗 

分 类 号：R392[医药卫生—免疫学]