钙网蛋白融合HBsAg基因重组腺病毒新型载体疫苗的构建与鉴定

Construction and characterization of a novel therapeutic vaccine of recombinant adenovirus vector containing calreticulin/HBsAg fusion gene

目的:构建表达钙网蛋白(calreticulin,CRT)与乙型肝炎病毒表面抗原(hepatitis B surface antigen,HBsAg)融合基因重组腺病毒载体(Ad-CRT/HBsAg),为研发新型乙型肝炎病毒(hepatitis B virus,HBV)治疗性疫苗奠定基础。方法:采用腺病毒表达系统(ViraPowerTM Adenoviral Expression System)构建重组腺病毒表达载体。首先利用RT-PCR的方法扩增CRT基因,并进一步构建CRT与HBsAg基因融合重组的pJW4303表达载体,在构建过程中给融合基因加上特定的CACC接头,再克隆入载体pEN-TR/D-TOPO以获得入门克隆,经PCR及测序鉴定正确后,用重组酶(LR ClonaseTMⅡEnzyme Mix)进行入门克隆与表达载体(pAd-CMV/V5-DEST)间的重组反应,以获得表达克隆Ad-CRT/HBsAg。表达克隆鉴定后,用限制性内切酶PacⅠ线性化后转染HEK293A包装细胞得到重组腺病毒。经过扩增后,用极限稀释法检测病毒滴度,用Western blot法检测Ad-CRT/HBsAg载体是否能正确表达目的蛋白。结果:构建的含有CRT/HBsAg融合基因的腺病毒表达克隆,经PCR和测序鉴定构建正确。重组表达克隆转染HEK293A细胞并扩增,获得的病毒滴度为2.68×1011 pfu/ml,且这个重组病毒载体能正确表达CRT/HBsAg融合蛋白。结论:成功构建了CRT/HBsAg融合基因重组腺病毒载体(rAd-CRT/HBsAg),为此重组载体用于治疗HBV慢性感染以及HBsAg阳性肝癌奠定基础。

Objective：To generate recombinant adenoviral vector containing CRT-HBsAg fusion gene for developing a safe,effective and HBsAg-specific therapeutic vaccine.Methods：The fusion of CRT and HBsAg gene was constructed by using polymerase chain reaction（PCR）,endonuclease digestion and ligation methods,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（Ad-CRT/HBsAg）containing CRT-HBsAg fusion gene was constructed by homologous recombinantion.The linearized DNA plasmid of the recombinant adenoviral vector was transfected into human embryo kidney（HEK 293A） cells to package and amplify recombinant adenovirus.The recombinant adenovirus titer was characterized by using the End-dilution assay.The expression of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg transfected 293A cells was detected by Western blot.Results：The CRT-HBsAg fusion gene was characterized by using PCR,and sequencing result revealed that the length and sequence were accurate.The recombinant adenoviral vector,Ad-CRT/HBsAg,was generated successfully.The titer of Ad-CRT/HBsAg was characterized as 2.68×1011 pfu/ml.The CRT-HBsAg fusion protein was expressed by HEK 293A cells correctly.Conclusion：Recombinant replication-defective adenovirus expression vector containing CRT/HBsAg fusion gene was constructed successfully,and this study has provided an experimental basis for further research of HBV gene therapy.