Fibulin-5真核表达载体的构建及稳定转染人膀胱癌细胞的研究

Construction of Fibulin-5 eukaryotic expression vector and establishment of its stable transfected bladder cancer cell line

目的构建Fibulin-5真核表达载体并转染人膀胱癌5637细胞。方法经聚合酶链反应（PCR）扩增Fibulin-5cDNA，然后将其与pMD-19T载体连接。pMD-19T—Fibulin-5重组体经过限制性内切酶XhoI和EcoRⅠ的消化后产生XhoⅠ-Fibulin5-EcoRⅠ片段。将该片段插入增强型绿色荧光蛋白载体（p-EGFPN1）质粒的相似位点并最终合成p-EGFP—F5质粒重组体，并通过BgⅢ单酶切鉴定。通过脂质体介导法将p-EGFP-F5质粒转染至人膀胱癌5637细胞株内。结果PCR扩增片段长度为1429bp。XhoⅠ和EcoRⅠ双酶切的片段经DNA测序显示Fibulin-5cDNA插入正确。p-EG—FP—F5质粒重组体经BgⅢ单酶切出一约450bp的条带。转染48h后，pEGFP—Fibulin-5和pEGFP—N1转染成功的细胞均有不同程度的绿色荧光表达。结论成功构建Fibulin-5绿色荧光蛋白真核表达载体并转染至人膀胱癌细胞内。

Objective To construct eukaryotic expression vector of Fibulin-5 and establish stable human bladder cancer cell line. Methods Fibulin-5 cDNA in hDANCE_CFLAG plasmid was amplified by polymerase chain reaction （PCR）. The amplified products were cloned into pMD-19T simple vector. The pMD-19T-Fibulin-5 vector was cut by Xho Ⅰ and EcoR I to generate a Xho I-FibulinS-EcoR I fragment that was ligated into enhanced green fluorescent protein carrier （ p-EGFP-N1 ） plasmid to synthesize p-EGFP- Fibulin-5 plasmid, which was then cut by Bg Ⅲ. Finally, the p-EGFP-Fibulin-5 plasmid was transfected into bladder cancer cell line 5637 by liposomes. Results The length of PCR products was 1429 bp. The results of DNA sequencing for the fragments by Xho Ⅰ and EcoR Ⅰ suggested the correct insertion of Fibulin- 5 eDNA. A band with about 450 bp was generated after p-EGFP-Fibulin-5 plasmids were digested by BgⅢ. The successfully transfected cells by pEGFP-Fibulin-5 or pEGFP-N1 emitted green fluorescence. Conclusion Fibulin-5 eukaryotie expression vector was successfully constructed and transfected to human bladder cancer cell line.