羊种布鲁氏菌wzt基因的原核表达及间接ELISA方法的建立

doi:10.16431/j.cnki.1671-7236.2019.12.030

Abstract

Abstract: In order to establish an indirect ELISA method for detection of Brucella antibody in serum,the wzt gene fragment was amplified from the strain of Brucella melitensis QY1 by PCR and ligated into the pET-30a vector to construct the plasmid pET-30a-wzt.The correct plasmid was transferred to E.coli BL21(DE3) competent cells were expressed by prokaryotic expression system,and the expressed products were analyzed by SDS-PAGE and Western blotting,and then the wzt recombinant protein was purified.The indirect ELISA assay for Brucella was established using wzt recombinant protein as the detection antigen and gradually optimizing the conditions.The results showed that the prokaryotic expression vector of pET-30a-wzt was successfully constructed and expressed in BL21(DE3) host bacteria.SDS-PAGE and Western blotting results showed that the recombinant protein was about 35 ku,which had good reactogenicity.The optimal coating concentration was determined to be 15 μg/mL,the optimal dilution of serum was 1:80,and the optimal dilution of the enzyme-labeled antibody was 1:5 000.The critical value was determined by detecting 24 negative samples,the sample was positive when the D_{450 nm} value was ≥ 0.30,and the sample was negative when the D_{450 nm} value was <0.30.The specificity test results showed that the method did not cross-react;The intra-and inter-assay coefficients of variation were both <10%;120 samples of serum were used in this method,the test was carried out and verified by the Rose-Bengal agglutination test,and the coincidence rate was 96%.This results indicated that the indirect ELISA method established in this experiment provided a reliable technical means for the detection of brucellosis.

Key words: Brucella; wzt gene; prokaryotic expression; indirect ELISA

CLC Number:

S852.61⁺4

ZHAO Lu, GE Zhiyi, LIU Yongsheng, LI Xuerui, CAO Xiaoan. Prokaryotic Expression of wzt Gene in Brucella melitensis and the Establishment of Indirect ELISA[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(12): 3707-3714.

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Prokaryotic Expression of wzt Gene in Brucella melitensis and the Establishment of Indirect ELISA

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