Construction of human YPEL5 gene eukaryotic expression vector and its expression in esophageal carcinoma cells
10.3969/j.issn.1673-4130.2018.02.001
- VernacularTitle:人YPEL5基因真核表达载体的构建及在食管癌细胞中的表达
- Author:
Changjun NIE
1
;
Xiaohui QIN
;
Qingyan ZHONG
;
Qiuhua WANG
;
Ning TANG
;
Ren CAI
;
Dingyuan ZENG
Author Information
1. 柳州市出生缺陷预防与控制重点实验室
- Keywords:
YPEL5 gene;
molecular cloning;
real-time fluorescent quantitative polymerase chain reaction
- From:
International Journal of Laboratory Medicine
2018;39(2):129-132
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct human yippee-like 5(YPEL5) gene eukaryotic expression recombinant plasmid and to express in esophageal carcinoma EC9706 cells .Methods The cDNA from human normal tissue was taken as a template and amplified to YPEL5 gene coding sequence with 366 bp in length .Then this se-quence was inserted into the multiple cloning site areas of eukaryotic expression vector pCDH-CD513B for ob-taining the eukaryotic expression vector pCDH-CD513B-Flag-YPEL5 .After the bacterial colony PCR identifi-cation ,it was sent to the corporation for testing the sequence .The successfully constructed recombinant plas-mid was transfected into human esophageal carcinoma EC9706 cells .The expression of PEL5 gene in EC9706 cells was detected by QRT-PCR and Western Blot .Results The YPEL5 gene segment with 366 bp in length was successfully amplified .pCDH-CD513B-Flag-YPEL5 recombinant plasmid was obtained by double enzyme digestion ,connection ,conversion and screening .The gene sequencing identification showed that the inserted gene sequence in recombinant plasmid was consistent with that in the GenBank .After 2 d of transfecting into EC9706 cells ,the QRT-PCR and Western Blot revealed that YPEL5 gene expression was significantly up-reg-ulated .Conclusion The pCDH-CD513B-Flag-YPEL5 eukaryotic expression vector is successfully constructed and is expressed in esophageal squamous cancer cell line EC9706 ,thus which lays a foundation for studying its function in the progression of esophageal cancer .
