HABP1基因慢病毒干扰和表达载体的构建及表达

Construction and identification of the lentivirus vectors which interfere or increase the expression of HABP1 gene

摘要:

摘要：目的：构建慢病毒介导的透明质酸结合蛋白1基因（HABP1）干扰载体及表达载体，建立HABP1降表达及过表达的稳定肾癌细胞系786-0。 方法：合成人HABP1基因短发夹RNA(shRNA)干扰序列，与慢病毒载体pLKO.1-TRC连接产生重组质粒pLKO.1-shHABP1;双酶切质粒pCDNA3.1(-)-HABP1-Flag，将目的基因片段连接到pCDH-CMV-MCS-EF1-Puro载体上，组成重组表达载体pCDH-HABP1；酶切鉴定及DNA测序后，转染293T细胞，收集慢病毒颗粒感染786-0细胞，嘌呤霉素筛选稳定细胞系，western blot验证HABP1蛋白的表达水平。 结果：构建成干扰载体pLKO.1-shHABP1及表达载体pCDH-HABP1；与随机序列对照组比较pLKO.1-shHABP1转染的786-0细胞HABP1表达水平(0.30±0.01)明显降低(t=25.98,P<0.05)，pCDH-HABP1转染786-0细胞后，HABP1的表达水平（3.20±0.40）明显升高(t=10.34,P<0.05)。 结论：成功构建HABP1基因的干扰载体及表达载体，且786-0细胞可稳定降表达及过表达HABP1。

关键词:透明质酸结合蛋白1;肾癌细胞;慢病毒载体

Abstract:

Abstract: Objective：To construct the lentivirus vectors which interfere or increase the expression of hyaluronan binding protein 1 ( HABP1 ) gene, and establish the human renal carcinoma cell lines 786-0 cells which stably down express or over-express the HABP1 protein. Methods：The interfering shRNA of the human HABP1 gene was synthesized, and inserted into the lentiviral vector pLKO.1-TRC to produce the recombinant plasmid pLKO.1-shHABP1. The HABP1 gene was obtained from the pCDNA3.1(-)-HABP1-Flag plasmid by the enzyme digestion, and inserted into the pCDH-CMV-MCS-EF1-Puro plasmid to produce the recombinant express plasmid pCDH-HABP1. The two constructed plasmids were identified by the enzyme digestion and DNA sequencing, and then transfected into 293T cells to obtain the lentivirus suspension, respectively. The renal carcinoma cell line 786-0 cells infected with the lentivirus suspension were collected, and screened with puromycin, and the levels of HABP1 protein in them were detected by western blot. Results：The plasmids pLKO.1-shHABP1 and pCDH-HABP1 were constructed successfully. western blot showed that the levels of HABP1 protein in 786-0 cells infected with pLKO.1-shHABP1 (0.30±0.01, t=25.98, P<0.05) and with pCDH-HABP1 (3.20±0.40, t=10.34, P<0.05) were significantly lower and higher, respectively, than that in the control. Conclusion：The vectors interferring or over-expressing HABP1 gene were constructed successfully, and the established renal carcinoma cell lines 786-0 cells stably down-expressed or over-expressed HABP1 protein.

Key words:hyaluronan binding protein 1; renal carcinoma cell; lentivirus vector