靶向B7-H3基因RNA干扰慢病毒的构建及人胰腺癌PaTu8988稳定感染细胞株的建立

Construction of B7 H3 RNAi lentiviral vector and establishment of its stably infected human pancreatic cancer PaTu88 cell line

目的构建靶向人B7同源性3（B7-H3）基因的小发夹RNA（shRNA）的慢病毒载体，并建立稳定感染的胰腺癌PaTu8988细胞株。方法根据GenBank提供的B7-H3cDNA序列，设计4条靶向B7-H3的siRNA序列，构建重组干扰质粒pGCSIL-GFP-B7-H3-shRNA。将重组干扰质粒和过表达B7-H3质粒共同转染293T细胞，经蛋白质印迹法筛选出干扰效果最佳的重组干扰质粒。该质粒经慢病毒包装，并感染胰腺癌PaTu8988细胞株，建立稳定低表达B7-H3细胞株。应用实时定量PCR及蛋白质印迹法检测细胞B7-H3基因的表达抑制率。结果携带干扰效果最好的shRNA的慢病毒感染PaTu8988细胞，建立了稳定低表达B7-H3基因的PaTu8988细胞株，其B7-H3mRNA表达的抑制率达96．8％，B7-H3蛋白表达的抑制率达88．1％。结论成功构建了人B7-H3-RNAi的慢病毒载体，并建立了稳定感染的低表达B7-H3基因的PaTu8988细胞株。

Objective To construct the B7-H3-RNAi lentiviral vector and establish a stably infected human pancreatic cancer PaTu88 cell line. Methods According to genetic information of B7 H3 cDNA sequence provided by GenBank, 4 siRNA sequence targeting BT-H3 were designed. The corresponding pGCSIL-GFP recombinant plasmids （pGCSIL-GFP-B7-H3-shRNA） were constructed. Then both recombinant interference,plasmld and over-expressing B7-H3 plasmld （pEGFP-N1-H3-1FLAG-GFP） were transfected into 293T cells. Western blot was used to detect the recombinant interference plasmid with best effect of interference. Then this plasmid underwent lentiviral packaging and was infected into PaTu8988 ceils and a stably low-expression of BT-H3 cell line was established. Real-time quantitative PCR and Western blot were applied to measure the expression inhibition rate of BT-H3. Results Lentiviral with the best gene silencing effect shRNA was infected into PaTu8988 ceil line, and a PaTu8988 cell line, stably low-expressing B7-H3, was established, and the inhibitive rate of B7-H3 mRNA and protein expression were 96.8% and 88.1%, respectively~ Conclusions BT-H3-shRNA lentiviral vector is successively constructed and pancreatic cancer Patu8988 cell line which can stably low-express B7-I-I3 is established.