Chen Zhihong, Li Tang, Hu Sujuan, Wang Guiqin
Department of Pediatrics, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, China.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2009 Oct;26(5):1067-71.
This study was aimed to construct the recombinant adenovirus containing rat insulin-like growth factor 1(rIGF-1), and then to investigate its expression in islet beta-cells. RNA was extracted using Trizol from rat livers. rIGF-1 cDNA was obtained using RT-PCR. The purified RT-PCR products and pAdTrack-CMV were digested using Bg1 II and EcoR V and religated by T4 DNA ligase, then transformed into electro-competent JM109 bacteria and selected on Kanamycin LB plates. This plasmid pAd-CMV-rIGF-1 was linearized by PmeI and co-transformed into electro-competent BJ5183 bacteria with pAdEasy-1 and selected on Kanamycin LB plates. After having been screened, the extracted plasmid of positive bacteria was transfected into HEK 293 cells with liposome and was identified by the green fluorescence protein (GFP) expression. The recombinant adenovirus encoding rIGF-1 was named Ad-rIGF-1, and the viral particles were further amplified, purified, and its titer was about 4.0 x 10(8)pfu/ml. Ad-rIGF-1 was transfected into rat pancreatic beta cell lines- RINm5F cells, RT-PCR was carried out to detect the transfer genes, rIGF-1 protein in cells culture supernatants was detected by ELISA method, and its concentration was 91.6 +/- 26.8 ng/ml. rIGF-1 was present in Ad-rIGF-1-infected RINm5F cells as measured by Western blotting. The recombinant adenovirus vector containing rIGF-1 was constructed successfully, and the rIGF-1 protein was expressed by RINm5F cells. This method provided the mechanism of rLGF-1 to prevent beta cell from impairmentand to treat the case of type 1 diabetes.
本研究旨在构建含大鼠胰岛素样生长因子1（rIGF-1）的重组腺病毒，进而研究其在胰岛β细胞中的表达。采用Trizol从大鼠肝脏中提取RNA。通过RT-PCR获得rIGF-1 cDNA。将纯化的RT-PCR产物和pAdTrack-CMV用Bg1 II和EcoR V酶切，并用T4 DNA连接酶连接，然后转化到电感受态JM109细菌中，在含卡那霉素的LB平板上筛选。将该质粒pAd-CMV-rIGF-1用PmeI酶切线性化，并与pAdEasy-1共转化到电感受态BJ5183细菌中，在含卡那霉素的LB平板上筛选。筛选后，提取阳性菌的质粒，用脂质体转染HEK 293细胞，并通过绿色荧光蛋白（GFP）表达进行鉴定。编码rIGF-1的重组腺病毒命名为Ad-rIGF-1，对病毒颗粒进一步扩增、纯化，其滴度约为4.0×10(8)pfu/ml。将Ad-rIGF-1转染到大鼠胰腺β细胞系-RINm5F细胞中，进行RT-PCR检测转移基因，用ELISA法检测细胞培养上清液中的rIGF-1蛋白，其浓度为91.6±26.8 ng/ml。通过蛋白质免疫印迹法检测发现Ad-rIGF-1感染的RINm5F细胞中存在rIGF-1。成功构建了含rIGF-1的重组腺病毒载体，RINm5F细胞表达了rIGF-1蛋白。该方法为rLGF-1预防β细胞损伤和治疗1型糖尿病提供了机制。
