Sub-cloning and soluble indution of CILP-MBP recombinant protein by tempera-ture variable / 中国免疫学杂志

ABSTRACT

Abstract Objective: To construct the Second half of CILP(C2) MBP fusion protein by sub-cloning technology. Methods: Recombinantfusion proteins, which contain the fragments within the C2 region(designated C2F1, C2F2 and C2F3) of the non-porcine nucleotide pyrophos-phohydrolase-homologous region of CILP, were prepared using pMAL-eHis vector. The recombinat genes are induced by different temperatures(22℃,30℃,37℃ ). Results: Expression using pMAL-eHis system can be induced chemically by adding IPTG. 37℃ temperature prmotes in-soluble inclusion-body formation,but 22℃ temperature can not induce the enough expression of recombinant protein. Onl 30℃ temperaturecan induce enough amount of soluble recombinant protein. The characers of fusion proteins that they carried 6 straight histidines, (His)6, at tbeC-terminus of multiple cloning sites for affinity purification were assessed by sodium dodecy1 sulfate-polyacylamide gel electrophoresis(SDS-PAGE) and Western blot. Nucleotide sequences of the insertion genes were confirmed by dideoxy sequencing. Conclusion: C2F1, C2F2,C2F3-MBP fnsion proteins were constructed successfully.These recombinant proteins may provide important roles in the future study on CILP.