[Construction and eukaryotic expression of recombinant porcine single-chain interleukin-12].
OBJECTIVE
To clone the p40 and p35 subunit cDNA of porcine IL-12(pIL-12) and construct the fusion gene of recombinant porcine single-chain interleukin-12 (pscIL-12).


METHODS
The total RNAs were extracted from porcine peripheral blood mononuclear cells (PBMCs) and porcine splenic lymphocytes for cloning pIL-12 p35 and p40 cDNA by RT-PCR. A hydrophobic polypeptide linker (Gly4Ser)3 was used for splicing two different gene fragments (pIL-12) p40+linker+p35 (pscIL-12) by recombinant PCR to construct pscIL-12 fusion gene. The pscIL-12 fusion gene was then inserted into pcDNA3.1(+) eukaryotic expression plasmid, and the resulted pcDNA3.1(+)-pscIL-12 was transfected into CHO-K1 cells via lipofectin. The expression of pscIL-12 mRNA in the transfected cells was identified by RT-PCR.


RESULTS
The sequence of the cloned porcine IL-12 p40 and p35 cDNA and the constructed pscIL-12 fusion gene were verified by PCR, restriction enzyme digestion and sequencing. The mRNA of pscIL-12 fusion gene was detected in the transfected CHO-K1 cells by RT-PCR.


CONCLUSIONS
The constructed pcDNA3.1(+)-pscIL-12 eukaryotic expression plasmid allows expression of pscIL-12 in CHO-K1 cells, thus facilitating further study of the biological activity and adjuvant effect of pscIL-12 fusion protein.
Department of Microbiology and Parasitology, Bengbu Medical College, Bengbu, China. wangxuem0508@sina.com
Xue-mei
XM
Wang
Yuan-ying
YY
Yuan
Qiang
Q
Fang
Hui
H
Xia
Xin
X
Sun
Bai-qing
BQ
Li
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000145:classification / Q000235:genetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013552:Swine
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D018664:Interleukin-12