Cloning and expression of human interleukin-26 in Escherichia coli.
- Author:
Yi-Qing LIU
1
;
Zi-Jiang CHEN
;
Xue ZHANG
;
Lai-Cheng WANG
;
Yu-Lian JIAO
;
Jie ZHANG
;
Chun-Yan MA
;
Bin CUI
;
Xin-Pu GAO
;
Zheng-Min LIU
;
Kan WU
;
Yue-Ran ZHAO
Author Information
1. Medical Research Center, Shandong Provincial Hospital, Shandong University, Jinan 250021, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Humans;
Interleukins;
biosynthesis;
genetics;
Recombinant Proteins;
biosynthesis;
genetics;
Reverse Transcriptase Polymerase Chain Reaction
- From:
Chinese Journal of Biotechnology
2006;22(3):413-417
- CountryChina
- Language:Chinese
-
Abstract:
To clone human interleukin-26 (hIL-26) and express it in E. coli efficiently. Two pairs of primers were synthesized according to the hIL-26 gene reported on GenBank. The hIL-26 gene was cloned by nest PCR following the first round RT-PCR from human peripherial blood monocytes total RNA, and then the PCR product was cloned into pMD18-T vector. Colony PCR, restriction analysis and sequence analysis showed that the gene cloned was the same as the reported hIL-26. The recombinant was cut with BamHI and EcoR I to obtain the hIL-26 fragment, and then the fragment was inserted into pBV220 which was cut with the same enzymes. The recombinant expression vector was induced to express hIL-26 at 42 degrees C, SDS-PAGE analysis showed that the recombinant protein accounted for up to 20% of the whole protein of E. coli, and the protein was also confirmed by Western blotting. Purity of the protein was found to be above 90% after purified with molecular sieve. After renaturalized with glutathione buffer, the promoting effect of it on the production of IFN-y in PBMC was detected by RT-PCR. A recombinant bacterial strain for expressing hIL-26 with biological activity was constructed successfully.
