Construction and prokaryotic expression of His-tagged expression vector of human IP-10 and identification of its activity
- VernacularTitle:人IP-10融合蛋白表达载体的构建和表达及活性鉴定
- Author:
Ziyun SHAO
;
Zhifeng LIU
;
Yi PENG
;
Jia XU
;
Qinghe QIN
;
Peng DENG
;
Yong JIANG
- Publication Type:Journal Article
- Keywords:
Interferon-?-inducible protein 10;
Fusion proteins;
Cell movement
- From:
Chinese Journal of Pathophysiology
1989;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To construct prokaryotic expression vector of His-tagged human IP-10 for further study of its biological function in the inflammatory response. METHODS: The coding sequence of IP-10 lacking signal peptide was amplified from human lung cDNA library by polymerase chain reaction (PCR) and the fragment was cloned into pET-14b plasmid for the construction of His-tagged fusion protein expressing vector, pET-14b/IP-10. After being identified by enzyme digestion and sequencing, the recombinant vector was transformed into a strain of E. coli, BL21 (DE_3). The expression of His-tagged fusion protein was induced with IPTG and purified with Ni+-NTA affinity chromatography. Then the chemotactic activity of IP-10 was determined by transwell migration assay on THP-1 cells. RESULTS: The construction of pET-14b/IP-10 recombinant vector was proved by enzyme digestion and sequencing. The fusion protein IP-10, which was purified by a routine Ni+ affinity method, had an activity on the induction of cell migration of THP-1. CONCLUSION: We successfully construct IP-10 fusion protein expressing vector and get the fusion protein with high bioactivity, which provides essential materials for the future studies on IP-10.
