Study On Biological Function Of Recombinant Vaccines Of Bordetella Pertussis Toxin S1 And Its Mutant

Bordetella pertussis is the causative agent of whooping cough,and is a Gram-negative aerobic coccus. The current pertussis vaccines,although efficacious,in some instances produce undesirable side effects. Molecular engineering of pertussis toxin,the major protective antigen,could provide a safer,new generation of vaccines against whooping cough. As a first critical step in the development of such a vaccine,the complete nucleotide sequence of the pertussis toxin gene was determined and the amino acid sequences of the individual subunits were deduced.Its major virulence factor is the pertussis toxin,an A/B exotoxin that mediates both colonisation and toxaemic stages of the the disease. Recombinant,inactive forms of the 5 subunits that make up the toxin have proven to be good vaccines. The S2 and S3 subunits of the toxin form part of the "B" moiety. They are responsible for binding the whole toxin to host cells prior to invasion,and are classed as adhesins. S2 attaches to a host receptor called lactosylceramide. It has also been speculated that the S3 unit may preferentially bind phagocytes.All five subunits are coded by closely linked cistrons. A promoter-like structure was found in the 5'-flanking region , suggesting that the toxin is expressed through a polycistronic messenger RNA. The order of the cistrons is S1,S2,S4,S5,and S3. All subunits contain signal peptides of variable length. The calculated molecular weights of the mature subunits are 26 024 for S1,21 924 for S2,21 873 for S3,12 058 for S4,and 11 013 for S5. Subunits S2 and S3 share 70% amino acid homology and 75% nucleotide homology. Subunit S1 contains two regions of eight amino acids homologous to analogous regions in the A subunit of both cholera and Escherichia coli heat labile toxins.Therefore,it is necessary to make more investigation on the S1 gene and the proteins encoded. We construct protein vaccines and fusion gene DNA vaccines with S1 fragments,feed mice with them,observe different immune responses and protective efficacy develop new B.pertussis protein vaccine antigens. In view of these,this study investigated biological function of B.pertussis rS1 from the following aspects:1. Cloning and expression of S1.The gene encoding B.pertussis S1 were amplified from B.pertussis PT chromosome DNA by PCR techniques,and the 702bp PCR product was cloned into vector pMD-18T by restriction endonucleases. The bioinformatics software DNAssist and GenBank were employed to analyze the diversity of the B.pertussis S1 gene. With different vectors and induce conditions to abtain the best way .The recombinant vectors were transformed and expression in E.coli,under induction of IPTG. The recombinant protein was purified with AKTA-explore 100 system. The expression products were analyzed by Tris-Tricine. Western blot and immunodiffusion assay determined the immunity of rS1.2. Cloning and expression of EspA- S1. Linker is designed in espA's downstream primers and S1's upstream primers .By PCR amplification EspA and S1 fragment were obtained. Then pET28a (+)-espA- S1 recombinant plasmid was transformed into E.coli BL21 and the target protein induced by IPTG was detected by SDS-PAGE. As a result,the fusion protein EspA- S1 expressed in prokaryotic expression system successfully and the preliminarily purified fusion protein has been gotted.3.Cloning and expression of rS1.The gene encoding B.pertussis rS1 were amplified from B.pertussis PT chromosome DNA by PCR techniques,and the 702bp PCR product was cloned into vector pQE-30 by restriction endonucleases. The recombinant vectors were transformed and expression in E.coli M15 , under induction of IPTG. The recombinant protein rS1 purified with AKTA-explore 100 system. The expression products were analyzed by Tris-Tricine. Western blot and immunodiffusion assay was employed to determined the immunity of rS1.4. Study on biological function of B.pertussis rS1 proteinConstructing the in vitro cell model of CHO. After bening incubated in HEPES-DMEM to OD600≈0.6,CHO cell adherence grown on the cover glass was incubated with the bacterium together for 4 hours with 5% CO2 at 37℃.At last,adherence effect was observed by Giemsa's staining and electron microscope.Statistic analysis showed no significant difference betwen the effect of S1 antibody block clutering and PT. rS1 was significantly different compared with PT and S1.The lymphocytosis-promotion factor(LPF) activities of S1and rS1 were neasured in 5-to 8-week-old female BALB/c mice.0.5μg of PT in 0.2ml of PBS was injected ip into ten mice, and white blood cell(WBC)count was determined with a Coulter on 20μl of blood obtained from a tail vein 5 days later. The results of this study showed that S1's WBC was not significant difference compared with PT. rS1 was significant difference (P<0.01) compared with PT and S1.5. Study on immunoprotection of rS1 as antigen of B.pertussis vaccineImmunoprotection test of rS1 recombination protein and S1 fusion protein to host mammal. In this study,BALB/c mice were injected with the preliminarily purified recombination protein rS1 and S1. Titer of antiserum IgG of the mice to rS1 and S1 increased evidently.In a word,the rS1 and S1 recombination protein were expressed and a toxin cell model on CHO has been constructed successfully. The anti-rS1 had immunoprotection effect by different means. It is important to study rS1 in developing engineering vaccine against B.pertussis.