Construction of fusion gene VH-mms13 and identification of its protein / 中国生化药物杂志

ABSTRACT

Objective To construct the prokaryotic expression vector pET30a( +)-VH-mms13 and identification of its protein after induced with IPTG.Method Heavy chain variable region VH gene of typeⅣcollagenase monoclonal antibody and magnetosome membrane protein gene mms13 were amplified separately,the fusion gene VH-linker-mms13 were synthesized by SOE-PCR technique and inserted into pET30a ( +) plasmid, which was confirmed by restriction enzyme digest and sequencing.Then the recombinant plasmid pET30a ( +)-VH-mms13 was transform into E.coli DE3 and induced with 0.4 mmol/L IPTG.The fused protein was identified by SDS-PAGE and Western blot.Results The length of fusion gene VH-mms13 was 738 bp,and the sequence was correct.After induced with IPTG,the fused protein was found in the inclusion body and Western blot results suggested that the fused protein can bind with His-tag antibody specifically.Conclusion Expression vector pET30a ( +)-VH-mms13 is successfully constructed and the fusion protein has good immunogenicity,which lay the foundation for the development of biomagnetism-targeted drug.