[Cloning and expression of human interleukin-26 in Escherichia coli].
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.
Liu
Yi-Qing
YQ
Medical Research Center, Shandong Provincial Hospital, Shandong University, Jinan 250021, China.
Chen
Zi-Jiang
ZJ
Zhang
Xue
X
Wang
Lai-Cheng
LC
Jiao
Yu-Lian
YL
Zhang
Jie
J
Ma
Chun-Yan
CY
Cui
Bin
B
Gao
Xin-Pu
XP
Liu
Zheng-Min
ZM
Wu
Kan
K
Zhao
Yue-Ran
YR
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007378:Interleukins / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
C406767:IL26 protein, human; D007378:Interleukins; D011994:Recombinant Proteins