Prokaryotic Expression Of Protective Antigen And Lethal Factor Fusion Protein Of Bacillus Anthracis And Establishment Of Indirect ELISA Method

Objective:Anthrax(anthrax)is an acute,virulent zoonotic disease caused by Bacillus anthracis.Since anthrax outbreaks or epidemics have occurred from time to time in recent years,immunization against anthrax in cattle and sheep has been included in the immunization program.Currently,the main anthrax vaccines applied to animals in China are Bacillus anthracis II and Bacillus anthracis without pods,but the current lack of rapid and sensitive antibody detection technology has resulted in the inability to clearly evaluate the immunization effect of anthrax vaccines.Therefore,this experiment was conducted to establish an indirect ELISA assay for Bacillus anthracis antibodies by induced expression of PA-LF1 fusion protein to provide technical support for the detection of antibody potency and antibody elongation after immunization of animals with anthrax vaccine.Method:(1)The genes of Bacillus anthracis weakly virulent strain C40-202 PA and LF1 were amplified separately by PCR,the PA-LF1 fusion gene was obtained by XhoⅠ sticky end ligation,the ligated T vector was cloned and sequenced,the software predicted the structure of PA,LF1 and PA-LF1 protein and compared sequence similarity;the expression vector of p ET32a-PA-LF1 was constructed,IPTG After induced expression,the expressed protein was identified by SDS-PAGE and Western blot,and the solubility of PA-LF1 fusion protein was detected by SDS-PAGE,and the imidazole concentration was optimized for the purification of PA-LF1 fusion protein.The purification of the target protein was performed by Ni Sepharose 6 Fast Flow affinity chromatography column,and the protein concentration was determined.The reactogenicity of PA-LF1 fusion protein was detected by Western blot using mouse anti-His tag monoclonal antibody,sheep serum immunized with Bacillus anthracis vaccine II,and anthrax precipitin as primary antibody,respectively.(2)The expressed PA-LF1 fusion protein was used as the coating antigen,and the indirect ELISA assay was established by tessellation titration method to screen the optimal protein coating concentration,coating conditions,closure time,dilution conditions,primary antibody dilution,primary antibody reaction time,secondary antibody dilution,secondary antibody reaction time,and other conditions,respectively.The sensitivity,specificity and reproducibility of the established indirect ELISA assays were tested.Result:(1)The PA-LF1 gene was successfully obtained with a size of about 2067 bp.The protein structures of PA and LF1 alone and PA-LF1 fusion were unchanged,and the sequence similarity comparison revealed that the PA and LF1 genes of Bacillus anthracis weakly virulent strain C40-202 were consistent with the sequences of PA and LF1 genes of 12 strains of Bacillus anthracis published in the NCBI database.SDS-PAGE showed that the recombinant protein was 96 k Da in size,and the PA-LF1 fusion protein existed mainly as inclusion bodies in the precipitation,and the highest protein expression was obtained at an imidazole concentration of The pure PA-LF1 fusion protein could be obtained at a concentration of 30 m M.Western blot assay confirmed that the PA-LF1 fusion protein could bind specifically to murine anti-His tag monoclonal antibody,sheep serum after immunization with Bacillus anthracis vaccine II and anthrax precipitin,indicating that it has good reactogenicity.(2)The optimal reaction conditions of indirect ELISA screened by tessellation titration were: protein coating concentration of 4 μg/ml,coating condition of 37℃ for 2h,closure time of 37℃ for 2h,antibody dilution of PBST+2%skimmed milk powder,primary antibody dilution of 1:200,primary antibody reaction time of 30 min,secondary antibody dilution of 1:8000,secondary antibody reaction time of 45 min.The sensitivity results showed that B.anthracis weakly virulent C40-202 immunized sheep positive sera 1 and 2 could be detected at a dilution of 1:800,and positive serum 3 could be detected at a dilution of 1:1600,indicating that the indirect ELISA assay for B.anthracis antibodies established in this test has a good sensitivity.The specificity results showed that the indirect ELISA assay was negative for Clostridium perfringens antibody,Clostridium perfringens alpha toxin antibody and Bacillus cereus enterotoxin antibody in sheep,indicating that the indirect ELISA assay has good specificity.The reproducibility results showed that the coefficient of variation of OD450 values ranged from 1.5 % to 5.2 % for intra-batch positive sera,1.2 % to 7.5 % for negative sera,3.9 % for inter-batch positive sera and 4.0 % for negative sera.4.0%.The results of the intraand inter-batch tests showed that the indirect ELISA method for Bacillus anthracis antibodies established in this study is reproducible.Conclusion:(1)We successfully induced the expression and purified PA-LF1 fusion protein,and verified by Western-blot that PA-LF1 fusion protein could bind specifically to sheep anthrax serum and anthrax precipitin,indicating that PA-LF1 fusion protein has good reactogenicity.(2)An indirect ELISA assay for antibodies against Bacillus anthracis was successfully established.