| Whooping cough, an acute respiratory infectious disease caused by Bordetella pertussis,especially occurs in infants and young children. The pathogen can produce a number ofvirulence factors during the invasion to the host, including pertussis toxin and adenylatecyclase toxin (ACT or CyaA), which play important roles in bacteria colonization andpathogenesis. ACT possesses a series of a glycine-and aspartate-rich nonapeptide repeatsthat belongs to the RTX (repeat in toxin) family of bacterial cytolysins. Thebio-functional toxin molecule consists of an N-terminal~400-residue-long adenylatecyclase (AC) enzyme domain and a C-terminal~1300-residue-long hemolytic activity.Although the previous studies demonstrated that ACT was involved in the immuneregulation during invasion to the host, the exact mechanism is still unclear. Furthermore,it is recommended to monitor the ACT content in the manufacturing process of pertussisvaccine according to the current European Pharmacopoeia (EP). However, the relevanttesting method is unavailable till now in China. Therefore, in the present study, therecombinant ACT with different gene mutation forms were constructed, expressed andpurified. The cytotoxicity and AC enzyme activity of the recombinant protein wasanalyzed and its function on regulating innate immune response was further investigated.In addition, the anti-ACT monoclonal and polyclonal antibodies were prepared and anenzyme immunoassay for detection of the adenylate cyclase toxin was established andvalidated.Construction, expression and purification of recombinant ACTThe primers were designed based on the published gene sequences of ACT andcyclolysin activating lysine acyltransferase (CyaC) in GenBank. The coding DNAsequences of ACT and CyaC were amplified by PCR, digested and ligated with theexpression vector, pETduet-1. The constructed plasmid, pETduet-1/cyaA-cyaC, wastransformed into E.coli BL21(DE3), and the positive clone was identified by ampicillinresistance selection, PCR, restriction endonuclease analysis and DNA sequencing.Recombinant ACT was expressed by induction with isopropyl-β-D-thiogalactoside(IPTG) and successively purified by ammonium sulphate precipitation and DEAEion-exchange chromatography. It was shown that the purity of recombinant ACT proteinwas about90%. Results from ELISA and Western Blot assays revealed that the purified recombinant ACT can specifically react with the mice immune sera immunized withwhole cell pertussis vaccine (WPV) and co-purified acellular pertussis vaccine (APV).The endotoxin in the protein was removed by affinity chromatography to a level of only145.5pg endotoxin per μg protein.Characterization and function analysis of recombinant ACTThe cytotoxic effect of recombinant ACT on murine monocyte-macrophages J774A.1was determined by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assay and Lactate dehydrogenase (LDH) assay. The intracellular cyclic adenosinemonophosphate (cAMP) level was also detected to evaluate the AC activity of therecombinant ACT. Results showed that ACT could induce the increase of theconcentration of intracellular cAMP and release of LDH with the dose dependentresponse and lead to the cell death, but such effect was not observed for the mutant ACT(CyaA*). A morphological change of Baby Hamster Kidney (BHK)21cells caused byACT, transformation from the bipolar fibroblast morphology to the aberrant stellateshapes was found, which further illustrated the AC enzyme activity of the recombinantprotein.To evaluate the immune regulation function of ACT, mice peritoneal macrophages weretreated with ACT, lipopolysaccharide (LPS), and ACT plus LPS, respectively. Thenintracellular kinases were determined by Western Blot, and the result showed thatp-NF-κB, and p-p-38MAPK and p-JNK were enhanced in different degrees for cellstreated with ACT plus LPS. Cytokines in the supernatant were determined by ELISA. Itshowed that IL-1β, IL-6and IL-10secreted by cells treated with ACT plus LPS, wereincreased but the TNFα and IL-12production of the same cells were inhibited, andcomparing with the cells treated by LPS only, the difference was significant (P＜0.01,n=6). And a high level of IL-6was observed for cells stimulated by ACT only, and therewas no significant difference with the co-treated group (P＞0.05, n=6).Preparation of anti-ACT antibodies and establishment of testing method of ACTFour hybridoma cell strains (1A1,1B8,3F9and4D5) stably secreting anti-ACTmonoclonal antibody (McAbs) were obtained. Each of them was administrated to miceby intraperitoneal injection to produce the ascetic fluid in which the anti-ACT McAbswith a titer of up to1:104existed. The four strains of ascetic fluid were collectedseparately and purified by caprylic acid-ammonium sulfate precipitation. At the sametime, rabbit immune sera with high titer anti-ACT polyclonal antibodies (pAbs) wereprepared by three times of booster immunization, and purified by protein A affinitychromatography. The result of SDS-PAGE showed that the purity of McAbs and pAbs were above90%. Western Blot andSlit Blot hybridization assays revealed goodspecificity of the antibodies, which did not react with other antigens from B. pertussis.The purified McAbs were labelled by HRP, and McAbs recognized different antigenicepitopes were screened out by competitive ELISA assay. By pair-matching experiment,two anti-ACT McAbs that recognized different antigenic epitope (McAb3F9andHRP-lablled McAb1B8) were selected as the capture antibody and detecting antibodyrespectively, to establish a sandwich ELISA for measurement of ACT. The optimumworking concentration was5μg/ml of the capture antibody3F9and2μg/ml of thedetecting antibody HRP-labeled1B8(about1:5000dilution). The minimum detectionlimit of the assay was1.17μg/L. The intra-and inter-assay coefficients of variation (CV)were9.93%and10.24%, respectively. The average recovery rate was103.24%. Goodspecificity of the method was showed for none of pertussis toxin (PT), pertactin (PRN),filamentous hemagglutinin (FHA)，fimbriae (FIM) and tracheal colonization factor (TCF)was detected by this method.In conclusion, a co-expression prokaryotic system of ACT and its co-translationalmodification protein CyaC was constructed, and purified recombinant ACT protein wasobtained. These data will provide a good foundation for later studies on ACT. A series ofassays on bioactivity of the recombinant protein were conducted by which thecytotoxicity and AC activity was confirmed. Function assays showed that secretion ofIL-12and TNFα induced by LPS in mice peritoneal macrophages could be inhibited byACT, while secretion of IL-10could be enhanced. These results offered furtherevidences on that ACT could regulate the innate immune response through signalpathway mediated by Toll-Like Receptor (TLR), especially TLR4. Previous studies onACT and its interacting protein, Toll-interleukin1Receptor (TIR) domain containingadaptor protein (TIRAP) may reveal the specific mechanism. High quality anti-ACTantibodies were produced and the sandwich ELISA for quantitative detection of ACTwas established. These studies will facilitate the quality control of the acellular pertussisvaccine. |
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