Expression, Purification, Identification And Biological Activity Of Recombinant Multi-epitopes Fusion Protein Of Three Biological

Objective: Because of their susceptivity to crowd, unpredictability and stronglethality, biological terrorist has attracted extensive concerns from countries aroundthe world. There are at least15countries and areas that have plans on biologicalwarfare and most of them are around China. Therefore, it is emergency to develop arapid, sensitive and accurate detection reagent or method against bioterrorism agents.Gold immunochromatography assay (GICA) is simple rapid and sensitive, with nospecial requirments on testers and suitable for rapid on-site monitoring, which hasbecome the research focus on the detection method of bioterrorism agents. To ensurethe high sensitivity and specificity, design and expression of antigen are crucial.Methods: In this study, three biological terrorism factors were chosed as:Brucella, Bacillus anthrax and Botulinum toxin, according to relative literatures fromNCBI, several antigens were chosed for Multi B cell Epitopes of Brucella, Bacillusanthrax and Botulinum toxin, restriction enzyme cutting site,6×His and linker wereintroducted and optimized according to E.coli high-usage codons and transform topET-20b, pET-20b-rOmp, pET-20b-rPA and pET-20b-rHc were construcetd. Thethree fusion proteins are inclusion body form by IPTG inducing. Afterultra-sonication and urea dissolving, the three fusion proteins are expressingcorrectly improved by SDS-PAGE, and in line with expectative protein molecularmass. Ni-NTA affinity chromatography was employed to purify the fusion proteins,SDS-PAGE and Western blot were porfermed to identify the proteins. The biologicalactivities of the three purified fusion proteins: rOmp, rPA and rHc were test byWestern blot. Results: prokaryotic expression vectors of pET-20b-rOmp, pET-20b-rPA andpET-20b-rHc were established, the best expression condition of these three proteinswere1mM IPTG inducing for4h; all three proteins were expressed in Inclusion body,their purity were high after purify. Western blot result showed that the three proteinswere able to cause specified reactions with Brucella monoclonal antibodies, Anthraxprecipitin antibodies and diagnostic serum of botulinum toxin, respectively,suggesting these three recombinant antigens were expressed correctly in engineeringbacteria with outstanding bioactivities.Conclusion: in this essay, three recombinant multi-epitopes fusion proteins ofthree biological terrorism factors were constructed and expressed successfully, andwere proved outstanding bioactivities, which laid foundation for further developmentof three biological terrorism factors colloidal gold immunochromatographic test stripfor rapid detection of synchronization.