E-cadherin重组慢病毒载体的构建及表达  

Construction and expression of recombinant lentiviral vectors for E- cadherin overexpression and RNA interfer-ence

作　　者：陈栋[1] 吴众[1] 罗林杰[1] 刘杰[1] 李少华[1] 

机构地区：[1]同济大学附属第十人民医院骨科一区创伤骨科,上海200072

出　　处：《中国矫形外科杂志》2014年第23期2169-2175,共7页Orthopedic Journal of China

基　　金：国家自然科学基金资助项目(项目批准号:81271990)

摘　　要：[目的]构建E-cadherin过表达和RNAi慢病毒载体,转染A549肿瘤细胞,观察E-cadherin的表达。[方法]根据Gene Bank E-cadherin的序列,选择设计两条序列和一条无关RNAi序列作为对照,通过Mlu I和Nde I两个酶切位点,将设计好的siRNA片段插入包装质粒PHR-SIN-CSGW-H1a(PHR)构建E-Cadherin RNAi慢病毒载体PHR-EA、PHR-EB。PCR扩增E-cadherin基因与pc DNA3.1质粒使用Bam HI、Xho I双酶切,连接鉴定后,亚克隆至慢病毒载体PHR,构建-E-cadherin过表达慢病毒载体PHR-E-cadherin。慢病毒包装后,感染A549肿瘤细胞,观察表达。[结果]成功构建E-cadherin过表达和RNAi慢病毒载体,并分别经PCR和质粒酶切鉴定。RTPCR检测E-cadherin的干扰和过表达情况,两个E-cadherin的RNAi病毒感染细胞后,都能使目的基因表达下调,而E-cadherin过表达慢病毒转染后细胞的E-cadherin表达明显提高了。Western Blot检测证实E-cadherin的表达确实被慢病毒所上调和干扰。[结论]成功构建出E-cadherin基因过表达和RNAi慢病毒载体,并在A549肿瘤细胞中成功表达,为进一步转染神经干细胞观察其生物学影响及对脊髓损伤修复的效果奠定基础。[Objective] To construct recombinant lentiviral vectors for E- cadherin overexpression and RNA interference（ RNAi）,to observe its expression in A549 cells. [Methods] The rat E- cadherin gene sequence was obtained from GenBank,and two different E- cadherin- specific short hairpin RNAs were designed and inserted into the PHR- SIN- CSGW-H1 a vector,generating E- Cadherin RNAi lentiviral vectors,PHR- EA and PHR- EB,respectively. The pc DNA3. 1- E-cadherin construct was also inserted into the lentiviral vector to generate the PHR- E- Cadherin overexpression vector. The recombinant lentiviral vectors were harvested from 293 T cells and were used to infect A549 cells. The expression of E- cadherin mRNA and protein was detected in A549 cells using reverse transcriptase polymerase chain reaction（ RT- PCR） and western blot,respectively. [Results] Two recombinant RNAi lentiviral vectors and an overexpression lentiviral vector were successfully constructed,as confirmed using DNA sequencing. E- cadherin- targeting RNAi expressed from the 1entiviral vectors had obvious inhibitory effects on E- cadherin mRNA and protein expression in the A549 human tumor cells,whereas the over- expression vector had the opposite effect. [Conclusion] The recombinant rat E- cadherin lentiviral vectors were successfully constructed and packaged,laying the foundation for neural stem cell（ NSC） transfection and subsequent application of E- cadherin- NSCs to the treatment of spinal cord injuries at the gene level.

关 键 词：E-CADHERIN基因 RNA干扰 过表达 神经干细胞 

分 类 号：R651.2[医药卫生—外科学]