Construction Of Truncated Mutants Of Gene Encoding Bovine Doppel And Their Expression In E.Coli

【Objective】Doppel protein (Dpl) is a paralog of the cellular form of the prion. Research of the struction and function of Dpl is helpful to reveal the pathogenesis of prion disease. Monoclonal antibody (Mab) is a powerful tool to study protein. Several Mabs against bovine Dpl have been prepared by our laboratory. In order to identify the epitopes recognized by these Mabs, truncated mutants of genes encoding bovine Dpl were developed through gene cloning and the corresponding recombinant proteins were generated by their expression in E.coli. The prepared fusion proteins could provide materials for the analysis of epitopes of the Mabs.【Methods】Primers for cloning 9 truncated mutants were designed based on the analysis of the genes encoding bovine mature Dpl and deduced characteristics of the protein struction. The truncated mutants were cloned by polymerace chain reaction (PCR) and then transformed into E.coli DH5αafter ligation with prokaryotic expression vector pET-30a(+). The recombinant plasmids identified by double-enzyme digestion and sequencing were introduced into host strain E. coli JM109 (DE)3 and E.coli BL21(DE)3 and then induced to express with isoproplyl-β-D-thiogalactoside (IPTG). The molecular weight, relative expression level and reaction with polyclonal antibody against Dpl of the fusion proteins were detected by SDS-PAGE, Western blotting and indirect ELISA.【Results】The results of double-enzyme digestion and sequencing of the recombinant plasmids demonstrated that 9 truncated mutants of gene encoding bovine Dpl were inserted in-frame into expression vector pET-30a(+). The results of SDS-PAGE, western blotting and indirect ELISA showed that 6 out of 9 truncated mutants were expressed at high level by E. coli JM109(DE)3 or E. coli BL21(DE)3 and the 6 mutants were boDPL(61-124),boDPL(72-155),boDPL(87-155),boDPL(58- 155),boDPL(27-144) and boDPL(27-155), respectively. The expression of the other three mutants were not satisfactory and further research would be needed.【Conclusion】9 truncated mutants of gene encoding bovine Dpl were successfully constructed. 6 out of 9 mutants were expressed at high level in E. coli with the induction of IPTG and could well react with the specific antibody againt Dpl. The work would lay material foundation for the analysis of epitopes recognized by the Mab prepared by our laboratory.