重组人骨形成蛋白4基因腺相关病毒载体的构建

目的 构建重组人骨形成蛋白4（human bone morphogenetie protein4，hBMP-4）基因腺相关病毒载体。方法 根据hBMP4的基因序列设计引物，上游引入EcoRⅠ位点，下游引入Sal Ⅰ位点，以EX-A0242M01hBMP4为模板扩增目的基因。将hBMP-4基因克隆入pUC18载体中，获得重组质粒pUCl8hBMP-4。然后用EcoRⅠ与Sal Ⅰ酶切pUC18-hBMP-4及质粒pSNAV，回收酶切片段，T4DNA连接酶16C过夜，转化大肠杆菌DH5a感受肽细胞，筛选阳性克隆获得pSNAV-hBMP-4，EcoRⅠ／SalⅡ双酶切鉴定，并行全基因测序。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AAV-hBMP-4。行DNA点杂交法测定病毒滴度，SDS-PAGE分析判断病毒纯度。结果 PCR电泳及酶切鉴定表明，pSNAV-hBMP-4重组成功，基因测序显示装入pSNAV质粒中的hBMP-4基因正确；AAV-hBMP-4病毒的大致滴度为1．5×10^12μg／ml，分泌蛋白浓度为0．124mg／ml，病毒载体纯度在95％以上。结论 实验获得的病毒载体滴度高、感染性好，可以满足骨组织工程的需要。

CONSTRUCTION OF RECOMBINANT ADENO-ASSOCIATED VIRUS VECTOR WITH HUMAN BONE MORPHOGENETIC PROTEIN 4 GENE

OBJECTIVE: To construct the recombinant adeno-associated virus vector with human bone morphogenetic protein 4 gene (AAV-hBMP-4). METHODS: The hBMP-4 gene primer was designed basing on the corresponding gene sequence in GenBank. EcoR I site was introduced into the upstream of the primer and Sal I site into downstream. The hBMP-4 gene was amplified with the template of EX-A0242-M01-hBMP-4, then was cloned into pUC18 vector to construct recombinant plasmid pUC18-hBMP-4. The plasmids pUC18-hBMP-4 and plasmid pSNAV cut by EcoR I and Sal I enzyme, the fragments were collected and linked with T4 DNA ligase at 16 C over night, recombinant plasmid pSNAV-hBMP-4 was obtained. The recombinant plasmid was then transfected into BHK21 cells using Lipofectamine TM2000. The G418 resistant cells were obtained consequently. These cells were infected with HSV1-rc/deltaUL2 which has the function of packaging and copying the recombinant AAV. After purification, the construction of recombinant AAV-hBMP-4 was completed. RESULTS: The construction of the recombinant pSNAV-hBMP-4 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequence in the recombinant pSNAV-hBMP-4 was correct. The virus titer was about 1.5 x 10(12) microg/ml. The purity of the virus was more than 95% using the SDS-PAGE method. CONCLUSION: With this method, high virus titers and purity of AAV-hBMP-4 can be acquired successfully and it is useful to bone tissue engineering.