胰腺癌CFPAC1细胞增殖诱导配体基因shRNA慢病毒表达载体的构建

Construction of ShRNA lentiviral expression vector targeting APRIL gene in CFPAC1 cell of human pancreatic cancer

目的构建针对人胰腺癌细胞株CFPAC1增殖诱导配体（a proliferation—inducing ligand，APRIL）基因的shRNA慢病毒表达载体。方法应用基因工程技术，筛选出针对APRIL基因的RNAi靶序列，与pGCL—GFP载体连接，构建慢病毒表达载体LV—shAPRIL；将连接产物转化到DH5a感受态细胞，经PCR筛选阳性克隆、测序鉴定。再用LV—shAPRIL、pHelper1．0、pHdper2．0共转染293T细胞，包装产生慢病毒颗粒。重组慢病毒感染CFPAC1细胞，实时定量PCR和Western blotting检测CFPAC1细胞APRIL mRNA和蛋白的表达。结果PCR和测序结果与构建的慢病毒载体的预期结果一致，经包装产生的病毒滴度为5×10^7Tu／ml。构建的慢病毒载体感染CFPAC1细胞后第4天、第4周和第8周，APRIL mRNA表达量较空载体慢病毒感染组分别下降了73％、70％和71％；APRIL蛋白表达量分别下降了66％、63％和62％（P〈0．05）。而各时间段未感染慢病毒的细胞组与空载体组相比无明显差异（P〉0．05）。结论成功构建了APRIL基因的shRNA慢病毒表达载体LV—shAPRIL。

Objective To construct of shRNA lentiviral expression vector targeting APRIL （a proliferation-inducing ligand） gene in CFPAC-1 cell of human pancreatic cancer. Methods We used gene engineering to screen RNA interference targeting sequence of APRIL gene. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCL-GFP vector. The resulting lentiviral vector containing shAPRIL were named LV-shAPRIL. Then it was conformed by PCR and DNA sequencing identification. 293T cells were cotransfected with LV-shAPRIL, pHelper 1.0 and pHelper 2.0 to product lentivirus. The titer of virus was tested according to the expression level of GFP in the 293T cells. After recombinant lentivirus infection into CFPAC-1 cells, we used real-time RT-PCR and Western blotting to examine APRIL mRNA and protein expression at different cell culture period. Results PCR analysis and DNA sequencing conformed that shAPRIL DNA was successfully inserted into the lentiviral vector. The titer of concentrated virus were 5 × 10^7 TU/ml. APRIL expression in CFPAC-1 cells were inhibited significantly at both mRNA and protein level. APRIL mRNA expression were decreased 73%, 70% and 71%, respectively, after the infection of 4 days, 4 weeks and 8 weeks by LV-shAPRIL. APRIL protein expression were decreased 66%, 63% and 62%, respectively , after the infection of 4 days , 4 weeks and 8weeks by LV-shAPRIL. Conclusions ShRNA lentiviral expression vector targeting APRIL gene has been successully constructed, and it can effectively inhibit the expression of APRIL gene in CFPAC-1 cells. This study lays a foundatin for in vivo research APRIL gene scilence in pancreatic cancer cell using the model of nude mice.