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靶向Atp6i和T细胞免疫应答cDNA7的小干扰RNA载体构建及其腺相关病毒重组颗粒的制备

Construction of small interfering RNA vector targeting Atp6i and T cell immune response cDNA7 and preparation of its recombinant adeno-associated virus particle

摘要:

目的 构建靶向Atp6i和T细胞免疫应答cDNA7(TIRC7)的RNA干扰重组体,包装成腺相关病毒(AAV)重组颗粒,为使用该干扰重组体进行体内外基因治疗相关疾病提供依据.方法 设计特异性针对Atp6i和TIRC7基因共同区域的小干扰RNA序列,连接到经Xba Ⅰ和BglⅡ酶切线性化的pAAV.H1载体上并转化DH5α菌株,提取质粒行酶切鉴定后进行测序分析.将酶切及测序分析鉴定成功的小干扰RNA序列与pAAV-RC、pHelper采用磷酸钙沉淀法共转染AAV-293细胞,复制、包装组成AAV.H1Atp6i重组病毒颗粒,以磷酸盐缓冲液(PBS)作为空白对照组,AAV.H1Luc作为阴性对照组,荧光显微镜观察转染效率.Western免疫印迹检测AAV.H 1Atp6i重组病毒颗粒对TIRC7蛋白表达的影响.结果 成功构建靶向Atp6i和TIRC7的RNA干扰重组体.重组质粒、pAAV-RC及pHelper成功转染AAV-293细胞,转染效率近100%,成功包装成AAV.H1Atp6i重组病毒颗粒.Western免疫印迹显示TIRC7蛋白在空白对照组、阴性对照组及AAV.H1Atp6i处理组均有表达,相对分子质量为75 000,AAV.H1Atp6i处理组TIRC7蛋白表达较空白对照组和阴性对照组均下降了80% (0.271 ±0.072比0.988±0.042、0.992±0.053,均P<0.05).结论 成功获得靶向Atp6i和TIRC7的RNA干扰重组体以及AAV.H1Atp6i重组病毒颗粒,可以干扰TIRC7蛋白的表达.

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abstracts:

Objective To construct the recombinant RNA interference vector targeting Atp6i and T cell immune response cDNA7 (TIRC7),and to prepare its recombinant AAV particle in order to provide evidence for gene therapy for diseases using the recombinant RNA interference vector in vivo and in vitro.Methods Sequences of small interference iRNA targeting the common region of Atp6i and TIRC7 genes were designed and connected to the pAAV.H1 vector linearized by enzyme digestion of Xba Ⅰ and Bgl Ⅱ and then transformed into DH5α where plasmid was identified using enzyme digestion for sequencing analysis.The identified siRNA sequences,pAAV-RC and pHelper were used to transfect AAV-293 cells by calcium phosphate precipitation method for recombinant AAV.H1Atp6i particle,with phosphate buffer solution(PBS)used in blank control group and AAV.H1Luc in negative control group.The transfection efficiency was then observed by fluorescence microscopy.Western blotting was used to detect the effects of the recombinant AAV.H1Atp6i particle on TIRC7 protein.Results The recombinant siRNA targeting Atp6i and TIRC7 was successfully constructed.Furthermore,AAV-293 cells were successfully transfected by the recombinant particle,pAAV-RC and pHelper,with the transfection efficiency being almost 100%.And the recombinant AAV.H1Atp6i particle was successfully packed and obtained.Western blotting showed the expressions of TIRC7 protein in blank control,negative control and AAV.H1Atp6i treatment groups,with the molecular weight being 75 000.Nevertheless,the TIRC7 expression was decreased by 80% in AAV.H1Atp6i treatment group as compared with that in blank control and negative control groups (0.271±0.072 vs 0.988±0.042 and 0.992±0.053,all P<0.05).Conclusion The recombinant siRNA targeting Atp6i and TIRC7 and recombinant AAV.H1Atp6i particle are both successfully obtained,which may exhibit interference on TIRC7 expression.

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