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目的克隆小鼠疱疹病毒侵入介体(HVEM)基因,构建其重组腺相关病毒载体,鉴定目的基因在真核细胞中表达。方法用RT-PCR法从小鼠脾脏淋巴细胞克隆出全长HVEM基因,构建携带HVEM基因的穿梭质粒pAAV-IRES-HVEM-hrGFP和辅助质粒pAAV-RC及pAAV-Helper,利用磷酸钙共沉淀法将穿梭质粒共转染入AAV-293细胞中,采用细胞内质粒DNA同源重组法构建重组腺相关病毒rAAV-HVEM。用氯仿-PEG8000法纯化病毒,实时荧光定量PCR测病毒滴度。RT-PCR检测rAAV-HVEM在中国仓鼠卵巢(CHO)细胞中的表达情况。结果成功构建组装重组小鼠HVEM腺相关病毒,纯化后病毒滴度为5×1010v.g/mL,且在CHO转导细胞中能有效表达目的基因。结论构建的重组腺相关病毒载体rAAV-HVEM为组织工程相关细胞转基因构建及临床应用提供先进的载体系统,为今后进一步研究HVEM的功能及其在部分疾病基因免疫治疗中的应用奠定基础。
Objective To clone the gene of mouse herpesvirus invade mediator (HVEM) and construct its recombinant adeno - associated virus vector, and to identify the target gene in eukaryotic cells. Methods The full-length HVEM gene was cloned from mouse spleen lymphocytes by RT-PCR. The shuttle plasmid pAAV-IRES-HVEM-hrGFP carrying the HVEM gene and the helper plasmids pAAV-RC and pAAV-Helper were constructed by using calcium phosphate coprecipitation The shuttle plasmid was co-transfected into AAV-293 cells, and the recombinant adeno-associated virus rAAV-HVEM was constructed by homologous recombination of intracellular plasmid DNA. The virus was purified by chloroform-PEG8000 method and the virus titer was detected by real-time PCR. The expression of rAAV-HVEM in Chinese hamster ovary (CHO) cells was detected by RT-PCR. Results Recombinant mouse HVEM adeno-associated virus was successfully constructed and its titer was 5 × 1010 v.g / mL. The titer of purified virus was 5 × 1010 v.g / mL, and the target gene was expressed efficiently in CHO transduced cells. CONCLUSION: The recombinant adeno-associated virus vector rAAV-HVEM provides an advanced vector system for the construction of transgenic engineering tissue-associated cells and its clinical application, which lays the foundation for further study on the function of HVEM and its application in gene immunotherapy for some diseases.