Experimental Study On Genetic Engineering Vaccine With Peb1 Protein Of Campylobacter Jejuni

Partâ… Reconstruction of Prokaryotic Expressive Vector of peb1A of Campylobacter jejuni and Expression of Target Protein[Objective] To obtain the purified periplasmic solute-binding protein 1(PEB1)of Campylobacter jejuni (CJ) by genetic engineering as the basic work to produce an effective vaccine against CJ infection after constructing the prokaryotic expressive plasmids of peb1A gene from CJ Pen 19. [Methods] (1) Construction and identification of the recombinant plasmids: Total DNA of seven CJ serostrains ( Pen19 and Pen2 both related with GBS; another serostrain Pen19,Pen2 and Pen4,Pen8 as well as Pen21 all associated with enteritis) were as templates respectively, peb1A gene DNA with BamHâ… and Hindâ…¢ sites were amplified by PCR reactions using same primer and some PCR products were sequenced. Because most closely association with GBS, the PCR product of standard Pen19 was selected as target gene. Prokaryotic expression vector pET32a(+),pQE30 and target gene were digested by BamHâ… and Hindâ…¢ respectively. The purified products were used to do ligation reactions; Ligation products wastransformed into E. Coli JM109 and the colonies were selected on LB mediumn with Ampicillin and X-gal/IPTG. The reconstructed plasmids were extracted from white colonies and identified by endonuclease digestions and PCR reactions. With sequencing , the reconstructed plasmids were named as pET32a(+)-peb1A and pQE30-peb1A; (2) Expression and purification of recombinated protein: pET32a(+)-peb1A and pQE30-peb1A were transfered into E. Coli BL21(DE3) and SG13009(pREP4) respectively. The target protein were expressed with induction of IPTG and purified with Ni-NTA resin after ultrasonication. All proteins above were identificated by SDS-PAGE at every step; (3) Identification of fusion protein: Male New England White Rabbits were systematically immunized by inactive CJ Pen19 whole cell to induce specific antiserum. Serum were collected at the2ed,4th,6th,8th and 10th week respectively after the first immunization to detect specific IgG antibody titer by ELISA. Plates were coated with acid extraction of CJ Pen19 and purified expression protein respectively. Anti-CJ antibody was as the first antidody to evaluate reconstructed protein by Western blot test. [Results] (1) A single band at the 780bp site was well shown with PCR reaction in all of the seven CJ strains, sequencing results were shown as high homologies. The peb1A gene have been successfully inserted into the prokaryotic expression vector pET32a(+) and pQE30 , which were identified by endonuclease digestion and a corresponding PCR reaction. Precision of sequencing was demonstrated correctly. (2) TrxA/PEB1 fusion protein in E. Coli BL21(DE3) was succefully expressed in the reconstructed plasmid pET32a(+)-peb1A; (3) Remarkable immunogenicity of the TrxA/PEB1 fusion protein was well identified: A high titer of the specific antibody to CJ Pen19 whole antigen weredemonstrated, which could cross-reacted with purified TrxA/PEB1 fusion protein. The reaction was caused by PEB1 protein but not TrxA protein based on Western blot test. However, it was failure to try to express target protein with reconstructed plasmid of pQE30-peb1A. [Conclusion] (1) Prokaryotic expressive plasmid of peb1A was successfully constructed by genetic engineering. TrxA/PEB1 fusion protein was significantly expressed in E. Coli and purified in qualification;(2) Production amount of TrxA/PEB1 in pET32a(+)-peb1A was high enough so that to be a useful way to acquire massive amount of PEB1 of CJ by genetic engineering; (3) The antigenicity of recombinant TrxA/PEB1 was coincidence with wild strain of CJ Pen19. peb1A gene was relatively conservative for many serostrains of CJ; (4) Recombinant TrxA/PEB1 fusion protein could be a valuable candidate for CJ vaccine with great applied value.[Key Words] Campylobacter jejuni,PEB1,prokaryotic expression,vaccine...