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目的:构建人HIF-1α基因的shRNA慢病毒载体、包装生产重组慢病毒颗粒,病毒途径高效感染胃癌细胞株BGC-823,并验证基因沉默效率。方法:在NCBI数据查找人HIF-1α基因序列,然后使用siRNA在线设计软件,设计针对基因CDS区的3条siRNA序列及Negative序列。根据设计好的siRNA序列设计双链互补的shRNA-Oligo DNA,退火形成双链后与线性化载体链接,构建shRNA重组表达载体。经由293TN细胞包装shRNA重组慢病毒颗粒,随后使用重组病毒感染BGC-823,通过荧光标记蛋白GFP确定感染效率后收集细胞样本,分别采用Real-time PCR和Western blot方法检测靶基因在mRNA和蛋白质水平的沉默效率。结果:shRNA重组表达载体测序结果与设计序列完全一致,包装病毒后滴度达到1×104 ifu/μL。慢病毒感染BGC-823细胞取得了极高的基因转导效率。mRNA检测结果显示,siRNA3对于对与目的基因的沉默效果最好,较阴性对照序列组相比较,mRNA的表达量下降了92%,Western blot检测的结果与mRNA检测结果完全相符合。结论:通过慢病毒途径,可以在BGC-823细胞中高效的进行HIF-1α基因的沉默。
OBJECTIVE: To construct shRNA lentiviral vector with human HIF-1α gene and packaging recombinant lentivirus vector. The virus pathway was highly effective to infect gastric cancer cell line BGC-823 and verify the gene silencing efficiency. METHODS: The human HIF-1α gene sequence was searched on the basis of NCBI data, and then 3 siRNA sequences and Negative sequences targeting the CDS region of the gene were designed using siRNA online design software. The double-stranded complementary shRNA-Oligo DNA was designed according to the designed siRNA sequence, annealed to form a double strand, and then ligated with a linearized vector to construct a recombinant shRNA expression vector. The 293TN cells were packaged with shRNA recombinant lentivirus particles, and then infected with recombinant virus BGC-823, the infection efficiency was determined by fluorescently labeled protein GFP cell samples were collected, respectively, Real-time PCR and Western blot were used to detect the target gene mRNA and protein levels The silence efficiency. Results: The sequencing result of shRNA recombinant expression vector was exactly the same as that of the designed sequence, and the virus titers reached 1 × 104 ifu / μL. Lentiviral infection BGC-823 cells achieved a very high gene transduction efficiency. The results of mRNA analysis showed that siRNA3 had the best silencing effect on the target gene. Compared with the negative control group, the expression of siRNA3 was decreased by 92%. The result of Western blot was in good agreement with the result of mRNA. CONCLUSIONS: HIF-1α gene silencing can be efficiently performed in BGC-823 cells by the lentiviral pathway.