Cloning, Prokaryotic Expression, Purification And Identifying Of Bordetella Pertussis Adenylate Cyclase Toxin Gene

Objective: Recombinant plasmids pET30a/cyaA and pET30a/cyaC were constructed via amplifying the gens of Adenylate Cyclase Toxin (CyaA, ACT) and cyaC from Bordetella pertussis, strain CS. Then screning and save clones bacteria after the plasmid was being transformed into E.coli DH5α. Then recombinant double gens plasmid, pET30a/cyaCA, was constructed. The plasmid pET30a/cyaA was transformed into E.coli BL21 (DE3), and the CyaA was induced expressing by isopropyl-β-D-thiogalactoside (IPTG). Purify the interesting protein and obtain the recombinant protein which's purity was about 90%, and identify it. These are for laying a foundation of further application study on the recombinant CyaA.Methods: Chromosomal DNA was prepared from Bordetella pertussis, strain CS, by using a Wizard Genomic DNA Purification Kit. The primers were designed according to the cyaA and cyaC sequences that indexed in GenBank, and synthesized. Gens were amplified by PCR with Chromosomal DNA being as template. The amplified fragment and the plasmid, pET30a, were digested with enzymes, and then the DNA was ligated to the plasmid. The ligation mixture was transformed into competent E.coli DH5α. The bacteria were plated onto LB agar supplemented with kanamycin, and cultured in 37℃overnight. Some of colonies were picked up and clutured, certified to be proper by PCR, and then extracted their plasmids to perform double digestion and sequencing eventually. The result of sequencing was analysed and spliced by DNAStar software. The spliced sequence was contrasted by Blast in NCBI. The complete cyaA with T7 promotor of pET30a fragment, being amplified by PCR with pET30a/cyaA being as template, was ligated to pET30a/cyaC. The recombinant plasmid pET30a/cyaCA had been constructed according to the above method.The clones'plasmid pET30a/cyaA was extracted and transformed into competent E.coli BL21 (DE3). The bacteria were plated onto LB agar supplemented with kanamycin, and cultured in 37℃overnight. Some of colonies were picked up and clutured, certified to be proper by PCR, then extracted their plasmids to perform double digestion to certify it's proper. The proper bacterium was cultured in a bit of LB medium, and induced with IPTG to express the CyaA. Then the interesting protein expression was checked by SDS Agarose gel electrophoresis. The proper bacterium was cultured in massive LB medium, and induced with IPTG to express the CyaA, after that it was processed as following: cells were broken by sonication, insoluble debris were dissolved, and the interesting protein's form of expression was checked by SDS agarose gel electrophoresis. The liquid to be purified was dialysised against dialysate. It was loaded on a column of DEAE-sephacel. The column was washed with A buffer (containing 2M urea, 20mM Tris, pH 8.0) until the level of A280 to baseline and then with 9% B buffer (2M urea, 2M NaCl, 20mM Tris, pH 8.0) to elute the additional proteins. Eventually, the interesting protein was eluted with 40% B buffer. Collecte the peak eluted liquid. Check the interesting protein with SDS Agarose gel electrophoresis. The purified recombinant protein was concentrated and analysised its purity.NIH mices were immunized with whole cell pertussis vaccines (wPV), acellular pertussis vaccines (aPV), and saline, respectively. Sera of these mices were obtained via taking hearts blood. 96-well plates were coated with purified recombinant CyaA which was deluted in phosphate-buffered saline (PBS). The reactivity for recombinant CyaA of Bordetella pertussis was checked via Enzyme-linked immunosorbant assay (ELISA). Besides, recombinant CyaA was shifted to nitrocellulose membrane after it was carried out SDS agarose gel electrophoresis. The reactivity of recombinant CyaA was further checked by Western blotting (WB) being acted with the immunization mice serum as the first antibody. Forthermore, recombinant CyaA was tested by ELISA and WB with the preschool children serum immunized with wPV as the first antibody.Results: The Bordetella Pertussis cyaA gen was successfully cloned, and its sequence was submitted to GenBan and indexed, GQ370813. Recombinant CyaA was expressed in prokaryotic cell and purified; the purity of it was about 90%. The concentration of concentrated recombinant CyaA was 0.83 mg/ml. The antibody contentration of wPV immuned mice serum group was significantly higher than that of aPV and saline immuned mice serum group (p<0.05). While the antibody concentration of aPV immuned mice serum group was not higher clearly than that of saline immuned mice serum group (p>0.05). The result of western blotting showed color bands by recombinant CyaA reacting with mice serum immunized with whole cell pertussis vaccines (wPV) and acellular pertussis vaccines (aPV) respectively, the color of former was obverously than the latter. But the mice serum immunized with saline showed no color band. Recombinant CyaA and preschool children serum immunized with wPV had strong reaction in ELISA and WB testes.Conclusion: The Bordetella Pertussis cyaA gen was successfully cloned in Escherichia coli, and its prokaryotic expression system was constructed. Purification conditions of the recombinant CyaA protein were probed. The activity of the recombinant CyaA was good via ELISA and Western blotting identifying, which provided a basis for its further application study.