[Soluble expression of chicken interferon-gamma and antiviral activity of purified expression product].
OBJECTIVE
We cloned and expressed chicken interferon-gamma gene (chIFN-gamma), and detected the bioactivity of chIFN-gamma expressed in Escherichia coli (E. coli).


METHODS
The chIFN-gamma mature protein gene was amplified by RT-PCR from spleen lymphocytes of chicken which were stimulated with concanavalin A (ConA) and then cloned into the prokaryotic expression vector pET-32a ( + ). Recombinant expression plasmid of pET-32a ( + )-chIFN-gamma was constructed and then transformed into the competent E. coli BL21 (DE3) cells for expression with IPTG induction. Purified soluble rchIFN-gamma proteins were obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blot assay. The antiviral activity was detected by MDCK-VSV system.


RESULTS
A 456 bp cDNA encoding chIFN-gamma mature protein gene was cloned and successfully expressed in E. coli with approximate molecular weight of 31.0 kDa, which could be recognized by anti-His mAb and rabbit polyclonal antibody against chlFN-gamma. The recombinant chIFN-gamma proteins were expressed to form inclusion bodies with a portion of soluble protein. The soluble rchIFN-gamma proteins were purified by Ni-NTA column under a native condition with the yield of 3.0 mg/L. The purified recombinant chIFN-gamma (rchIFN-gamma) proteins by 1:32 dilution could resist 100 TCID50 VSV virus attack.


CONCLUSIONS
The purified rchIFN-gamma proteins by Ni-NTA column under a native condition had better antiviral activity, which will establish a basis for further developing new type antiviral interferon praeparatum.
Qi
Jing
J
Institute of Animal and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Shandong Key Laboratory of Animal Disease Control and Breeding, Jinan 250100, China. qj-happy@tom.com
Du
Yijun
Y
Zhu
Xiaoling
X
Hu
Beixia
B
Sun
Shouli
S
Zhang
Xiumei
X
Liu
Yuqing
Y
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology; D002645:Chickens; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007371:Interferon-gamma / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D008214:Lymphocytes / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000166:cytology
D000998:Antiviral Agents; D011994:Recombinant Proteins; D007371:Interferon-gamma