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构建含有鼠胰岛素样生长因子1(rIGF-1)重组腺病毒,研究其在胰岛β细胞的表达。Trizol一步法提取大鼠肝脏组织总RNA,RT-PCR法合成鼠胰岛素样生长因1(rIGF-1)cDNA;PCR纯化产物和pAdTrack-CMV分别经BglⅡ和EcoRⅤ双酶切后纯化,T4DNA连接酶连接,转化JM109感受态细菌,卡那霉素平板筛选阳性克隆扩增培养,抽提质粒;经BglⅡ和EcoRⅤ双酶切及测序鉴定后,将正确重组体pAd-CMV-rIGF-1转化包含腺病毒骨架质粒pAdEasy-1的BJ5183菌。同源重组后用选择性培养基筛选阳性克隆,提取质粒用脂质体介导转染293细胞,获得重组腺病毒Ad-rIGF-1,病毒滴度为4.0×108pfu/ml。Ad-rIGF-1感染大鼠胰岛β细胞株-RINm5F细胞,RT-PCR法对重组腺病毒进行鉴定,培养上清ELISA法测鼠IGF-1蛋白为91.6±26.8ng/ml,Westernblotting分析感染细胞有特异性rIGF-1条带。本研究成功构建Ad-rIGF-1重组腺病毒,并在胰岛β细胞有效表达,为下一步研究rIGF-1因子对胰岛β细胞的保护及基因治疗1型糖尿病奠定了基础。
The recombinant adenovirus containing murine insulin-like growth factor 1 (rIGF-1) was constructed and its expression in pancreatic β cells was studied. Trizol was used to extract the total RNA of rat liver tissue. The rIGF-1 cDNA was synthesized by RT-PCR. The purified product of PCR and pAdTrack-CMV were digested with BglII and EcoRV respectively, and the T4DNA ligase The recombinant plasmid pAd-CMV-rIGF-1 was transformed into E.coli containing adenovirus JM109 competent cells, kanamycin plate screening positive clones expansion culture, plasmid extraction; Bgl Ⅱ and EcoRⅤ double enzyme digestion and sequencing identification, the correct recombinant pAd- Virus backbone plasmid pAdEasy-1 of BJ5183 bacteria. After homologous recombination, the positive clones were screened by selective medium. The plasmid was extracted and transfected into 293 cells by lipofectamine. The recombinant adenovirus Ad-rIGF-1 was obtained. The virus titer was 4.0 × 108pfu / ml. Ad-rIGF-1 infected rat pancreatic β-cell line - RINm5F cells, RT-PCR method for the identification of recombinant adenovirus, culture supernatant ELISA assay mouse IGF-1 protein was 91.6 ± 26.8ng / ml, Western blotting analysis of infected cells There are specific rIGF-1 bands. In this study, Ad-rIGF-1 recombinant adenovirus was constructed successfully and efficiently expressed in islet β cells, which laid the foundation for the further study on the protection of rIGF-1 on islet β cells and gene therapy for type 1 diabetes.