rAAV2／1-Acrp30病毒的纯化、扩增与活性检测

Clearing, amplification and activity detection of the recombinant adeno-associated virus vector2/1 with adiponectin

目的将大鼠脂联素基因（Acrp30）克隆到腺相关病毒（AAV）载体中，经包装、纯化、扩增后获得rAAV2／1-Acrp30病毒，并进行活性检测。方法筛选阳性克隆获得pSNAV2．0-Acrp30，EcoRⅠ／SalⅠ双酶切鉴定，并行测序。转染BHK21细胞，G418筛选培养，获得抗药克隆细胞株。HSV1-rc／△UL2感染，包装此细胞株并收获病毒载体rAAV2／1-Acrp30。行DNA斑点杂交法测定病毒滴度，SDS-PAGE分析判断病毒纯度，Western blot法检测目的蛋白脂联素在HEK293细胞中的表达活性。结果PCR电泳及酶切鉴定表明，pSNAV2．0-Acrp30重组成功，基因测序显示装入pSNAV2．0质粒中的Acrp30基因正确。rAAV2／1-Acrp30病毒的大致滴度为1．0×10^12μg／ml，HEK293细胞分泌蛋白浓度为50ng／ml，病毒载体纯度在95％以上。结论实验获得的脂联素病毒载体滴度高、感染性好，可试用于GK（Cow—Kakizaki）大鼠脂联素转基因治疗。

Objective To clear, amplify and detect the activity of the recombinant adeno-associated virus vector with adiponectin（rAAV2/1-Acrp30）. Methods Recombinant plasmid pSNAV2.0-Acrp30 was obtained. The recombinant plasmid was then transfected into BHK21 cells using LipofectAMINE^TM 2000. The G418 resistant cells were obtained consequently. These cells were infected with HSV1-rc/ △UL2 which has the function of packaging and copying recombinant AAV. After purification, the construction of recombinant rAAV2/1-Acrp30 was collected. Results The construction of recombinant pSNAV2.0-Acrp30 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequencing showed that the recombinant pSNAV2.0-Acrp30 was correct. The virus titer was about 1.0 × 10^12μg/ml. The purity of the recombinant AAV2/1 was fairly high using the SDS-PAGE method. Conclusion With this method, rAAV2/1-Acrp30 with high virus titers and purity can be acquired successfully and it can meet the demands of the experimental study of Acrp30 gene therapy of GK rats.