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目的:构建钙整合素结合蛋白(CIB)的逆转录病毒表达载体,并建立包装细胞系。方法:质粒pet32-CIB经EcoRI和XhoI双酶切后亚克隆至逆转录病毒载体pLXSN,构建重组逆转录病毒表达载体pLXSN-CIB,PCR及双酶切鉴定后,通过脂质体转染导入包装细胞pA317,G418稳定筛选后获得抗性克隆,NIH3T3细胞进行逆转录病毒颗粒滴度测定。结果:重组逆转录病毒表达载体pLXSN-CIB质粒测序结果与GenBank中序列一致,经PCR及酶切鉴定证实,获得了大约574bp的基因片段,且正确插入pLXSN-CIB载体中;建立了pA317-CIB包装细胞系,采用NIH3T3细胞测定病毒滴度为6.81×105CFU/mL。结论:pLXSN-CIB结构正确,成功构建了CIB基因的逆转录病毒表达载体并建立了pLXSN-CIB包装细胞系。
Objective: To construct retroviral expression vector of calcium integrin binding protein (CIB) and to establish a packaging cell line. METHODS: The plasmid pet32-CIB was double-digested with EcoRI and XhoI and subcloned into the retroviral vector pLXSN. The recombinant retroviral vector pLXSN-CIB was constructed and identified by PCR and restriction enzyme digestion. Resistant clones were obtained after stable screening of cells pA317 and G418, and NIH3T3 cells were used for determination of retroviral particle titer. Results: The recombinant plasmid pLXSN-CIB was sequenced according to the sequence of GenBank and confirmed by PCR and restriction enzyme digestion. The gene fragment of about 574bp was obtained and inserted into pLXSN-CIB vector correctly. The pA317-CIB The cell line was packaged and the titer of the virus was determined to be 6.81 × 10 5 CFU / mL using NIH3T3 cells. Conclusion: The pLXSN-CIB was constructed correctly and the retroviral expression vector of CIB gene was successfully constructed and the pLXSN-CIB packaging cell line was established.