Fibulin-5基因慢病毒表达载体的构建与鉴定

Construction and identification of lentiviral expression vector carrying human Fibulin-5 gene

目的构建Fibulin-5基因的慢病毒表达载体。方法从pBluescript-Fibulin5质粒中克隆出Fibulin-5基因,将其连同慢病毒载体质粒pCDH-CMV-MCS-EF1双酶切,使目的基因连接到载体质粒中,构建成重组慢病毒载体质粒pCDH-F5。对重组慢病毒载体质粒pCDH-F5进行酶切鉴定和测序鉴定后,制备包装病毒并感染膀胱癌细胞系5637,检测细胞中Fibulin-5基因的表达水平。结果构建的重组慢病毒载体质粒pCDH-F5进行PCR及酶切鉴定,获得的克隆和酶切产物与预计的基因片段大小一致。所获得的Fibulin-5基因经测序后与报道序列对比仅有1处突变,且属同义突变,说明pCDH-F5中携带正确的Fibulin-5基因。通过感染5637细胞,发现构建的慢病毒转染效率达95%以上,且能有效表达Fibulin-5基因。结论实验成功构建了Fibulin-5基因的慢病毒表达载体。

Objective To construct lentiviral expression vector carrying human Fibulin-5 gene. Methods Fibulin-5 gene cloned from pBluescript-Fibulin5 plasmid was double restriction digested as well as lentiviral vector plasmid pCDH-CMV-MCS-EF1. Connection of target gene and vector plasmid was followed to generate the lentiviral expression vector plasmid pCDH-F5. After i- dentification with PCR, double restriction digestion and sequencing of recombinant Fibulin-5 gene, the Fibulin-5 lentiviral expression vector was packaged and used to infect bladder cancer cell line 5637, and then the Fibulin-5 mRNA expression was detected. Results PCR of Fibulin-5 from pC- DH-F5 and double restriction digestion of the pCDH-F5 got the same length of gene segment as ex- pected. The sequencing of cloned Fibulin-5 gene was consistent with the described one in the NCBI （NM006329.3） except one mutation, which was further identified to be the same sense mutation. The infective rate of Lenti-F5 to bladder cancer cell line 5637 was more than 95% and the Fibulin-5 gene expression was up-regulated to almost 10-fold level. Conclusions Lentiviral expression vector carrying human Fibulin-5 was successfully constructed.