Cloning and expression of human interleukin-26 in Escherichia coli / 生物工程学报
Chinese Journal of Biotechnology
; (12): 413-417, 2006.
Article
em Zh
| WPRIM
| ID: wpr-286274
Biblioteca responsável:
WPRO
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.
Texto completo:
1
Índice:
WPRIM
Assunto principal:
Proteínas Recombinantes
/
Interleucinas
/
Clonagem Molecular
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
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Escherichia coli
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Genética
/
Metabolismo
Limite:
Humans
Idioma:
Zh
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2006
Tipo de documento:
Article