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[Cloning and prokaryotic expression of human secreted IL-16 gene]
Chuan-ping Si1, Shu-ling Li, Jia Fu
1Department of Immunology, Jining Medical College, Jining 272067, China. chipsi@163.com
Aim:
To clone human secreted IL-16 cDNA, construct its prokaryotic expression vector and express it in E.coli DH5α.
Methods:
The secreted IL-16 gene fragment from the cDNA library of human peripheral blood mononuclear cells (PBMC) was amplified by PCR. After purified, the product was cloned into pUC18 T-vector. The recombinant plasmid was confirmed by PCR, endonuclease digestion and sequencing analysis and then subcloned into prokaryotic expression vector pMAL-C2. Then the recombinant expression plasmid pMAL-IL-16 was transformed into E.coli DH5α. The expression of secreted hIL-16 was induced with IPTG and identified by SDS-PAGE and Western blotting.
Results:
Using PCR, we obtained the human secreted IL-16 cDNA fragment which was 393 bp and encoded 130 amino acids. The prokaryotic expression vector pMAL-IL-16 we constructed was successfully transformed into E.coli DH5α, and under the induction of IPTG, we found the expression of the recombinant fusion protein with relative molecular weight (M(r);) being 56 000 as expected and confirmed by SDS-PAGE and Western blotting.
Conclusion:
We successfully cloned the human secreted IL-16 cDNA and constructed its prokaryotic expression vector and expressed it in E.coli, which is helpful for further purification of human secreted IL-16 protein.
