Expression Of Protective Antigen Of Bacillus Anthracis And Establishment And Preliminary Application Of Indirect ELISA Method

Bacillus anthracis is a gram-positive corynebacterium,the pathogen of causing anthrax mainly in cattle,horses,sheep and other herbivores.It can infect humans through the contact of infectious material or animals.After the discovery of bacillus anthracis,it has posed health burden to humans and animals.However,the detection of this pathogen is underdeveloped,thus it is an urgent need to develop a fast,convenient,and sensitive method for detection of clinical samples.In this study,we attempted to develop an indirect ELISA method for the detection of antibodies of bacillus anthracis using recombinant PA83 and PA63 proteins The experiment design and results are summaried below.（1）Plasmid construction and protein expressionProkaryotic expression plasmids p ET-32a-PA83 and p ET-32a-PA63 were constructed by codon optimization of the PA83 and PA63 gene sequences published in NCBI database.The plasmids with correct sequence were transformed into E.coil BL21 and induced for expression by IPTG.SDS-PAGE showed that recombinant PA83 was 103k D,with the purified protein concentration was 2 mg/m L;PA63 protein was 83k D and protein concentration was 0.5 mg/m L.Western blot analysis showed that PA83 and PA63 had good reactivity with His Tag Mouse Monoclonal Antibody.（2）Comparative immunization effect of PA83 protein and in-use vaccineNew Zealand white rabbits were immunized with purified PA83 protein and two traditional in-use vaccines（Anthrax Spore Vaccine,Nonencapsulated Strain）and Anthrax Spore Vaccine,Live（Strain No.2）).The serum antibody levels,cytokines,and lymphocyte proliferation of immunized rabbits were measured and analyzed.The results showed that PA83 protein immunization elicited serum antibody with endpoint titer up to 1:122880,significantly higher than that of the two traditional vaccines（p<0.01）.However,no significant difference in the cytokines after immunization of PA83 and traditional vaccines（p>0.05）.In addition,there was no detectable change in lymphocyte growth and proliferation after immunization of PA83and vaccines.These results showed that PA83 protein immunization elicited high level of humoral response but no cellular immunity.（3）Establishment of indirect ELISA assay based on recombinant PA83 and PA63 proteinsTo develop ELISA for detection of bacillus anthracis in bovine serum base on PA83 and PA63 proteins,we optimized the reaction conditions.ELISA plate was coated with PA63 protein of 2μg/m L at 4℃overnight,blocked with 1%BSA at 37℃for 2h.Bovine serum samples were diluted by1%BSA（1:200）and incubated at room temperature for 1h;Rabbit-Anti-Bovine Ig G/HRP antibody was diluted（1:1000）and incubated at room temperature for1h.The development was performed by incubation with TMB Single-Component Substrate solution at room temperature for 15 min.The reaction results were detected at OD_{450 nm}with an enzyme marker.The Cut-off value was 0.332.This method had no cross reaction with bovine foot-and-mouth disease type A,O,sub1,bovine viral diarrhea,and brucedisease,as all such samples were negative in the testing.The sensitivity of testing was 1:400.The intra-lot and inter-lot repetition coefficients were less than 10%.The method showed ressemble the results of commercial kit by 80%in the testing of 15 immunized bovine sera.In the testing of 576 clinical bovine sera samples,this method showed positive rate of 2.4%（14/576）,and re-evaluation of positive samples with commercial kit showed coincidence rate of 85%.Unfortuately,the indirect ELISA based on PA83 protein showed low sensitivity compared to PA63,thus it was not further developed.The indirect ELISA method established in this study has high sensitivity,specificity and reproducibility for testing bovine serum for anthrax bacillus infections,thus lay a solid foundation for further development.