Experimental Study On Genetic Engineering Subunit Vaccine Against Bordetella Pertussis

Whooping cough (pertussis) is an severe infectious disease caused by Bordetella pertussis, the incidence of which has been limited due to the relatively high vaccination coverage of whole cell and acellular vaccines; however, traditional vaccines against this disease are inherently reactogenic, side effects associated with these vaccines have decreased their acceptance. Much effort during the last decade has been dedicated to finding an alternative to the reactogenic traditional pertussis vaccine. Following the recent trials, genetic engineering vaccines,especially subunit vaccines have been demonstrated to be able to replace the old pertussis vaccines.Subunit vaccines are superior to traditional vaccines in many aspects. Such as versatility and better specificity, reasonable designation and combination of predominant subunits to induce effective,safe and specific immune response. In recent years, subunit vaccine research has become a new trend in developing vaccines against infectious disease.Screnning of candidate antigens that are safe and effective is the first step in developing subunit vaccines, and expression of candidate antigens in Escherichia coli (E. coli) would be preferable. The most ideal recombinant subunit vaccine candidate antigen would be the smallest, non toxic fragment of the virulence factor capable of eliciting significant protective immunity. Recently, the most suituable virulence factor of B.pertussis for developing subunit vaccine are pertussis toxin (PT),filamentous hemagglutinin (FHA)and Pertactin(Prn). However, all of them are too big in size and thus could not be expressed in E. coli directly. Research is now focused on secrenning of immunogenic fragment from these virulence factor that would elict a protective immunity. Pertussis toxin S1 subunit and filamentous hemagglutinin (FHA) type I domain ( consist of 456 amino acid residue, signed as FHA') are considered to be effective candidate antigens. Researches indicated that the both of them were extremely difficult to express in E. coli alone. In our laboratory, both of them were successfully inserted into prokaryotic expression vector pET22b, 28a, 30a, pTRC99A and pQE30, only the recombinant bacterial pQE30a-S1/M15 was lowly expressed (<10%). So how to gain high expression of these proteins is the key problem we need to solve.In view of these, we are determined to gain high expression of the subunits described above and develope a subunit vaccine against B. pertussis, the method, resule and conclusion are listed as follows.1. Screening of immunogenic antigens from FHA'Using the sofeware DNAstar, wo evaluated the hydrophilicity, flexibility, antigenicity and accessibility of FHA'. we have screened a peptide Fs consisting of 138 acid residues from the C-terminal of FHA', after inserted into pET22b, it was highly expressed (>50%) in E. coli BL21 (DE3). Additionally, it was able to react with an anti-FHA Mab, indicating that the Fs fragment is of good immunogenicity and contains the most immunodominant epitopes of FHA.2 Construction ,expression and purification of candidate antigensWe gained fusion geng Fs-s1'through overlap extension PCR, the two subunit were linked with a linker containing 5amino acid residues. The fusion gene was successfully inserted into pET22b, after induced with IPTG, the amount of target protein Fss1'was about 30% of the total cellular proteins. we further constructed a recombinant protein rFsS1'S1', it was also highly expressed(15%). These proteins were purified from E. coli cell lysates via affinity chromatography and have aa purity of above 95%.3 Biologic activity analysis of candidate antigens.We tested the leukocytosis-promoting and CHO cell clustering activities of both FsS1'and Fs S1'S1', these toxicities were reduced to an acceptable level compared with control(S1,PT)(p<0.01) as reported, so rFsmS1 is safe for vaccine development. We also produced polyclonal antiserum against the FsS1'and FsS1'S1', ELISA indicate that the specific antibody against PT is 1:128 000 and 1:256 000, respectively, and both are 1:256 000 against FHA; These are finally confirmed by double immunodiffusion test.4 Immunoprotection assay of vaccine candidate antigens. These results show that the approach taken in this study to generate a new pertussis vaccine antigen is promising, and we will carry on the study and finally develop a new generation of vaccine against B.pertussis.