鸡白痢沙门氏菌IPAJ蛋白的表达纯化、多克隆抗体制备及ELISA方法的建立

doi:10.16431/j.cnki.1671-7236.2023.03.036

Abstract

Abstract: 【Objective】 This study was aimed to establish an indirect ELISA method using IPAJ protein encoded by virulence related genes of Salmonella Pullorum as antigen.【Method】 The IPAJ gene was amplified by PCR and connected to pCzn1 expression vector to transform into Escherichia coli BL21 (DE3) competent cells.The recombinant protein of IPAJ gene was induced to express by IPTG and purified,and the reactivity of the protein was verified by Western blotting.The purified IPAJ protein was used to immunize rabbits at 60 days of age for three times to prepare polyclonal antibodies.The antibody titer was detected by ELISA method.ELISA conditions of IPAJ protein as diagnostic antigen were optimized by square titration.An indirect ELISA method using IPAJ protein as diagnostic antigen was established and compared with the results of plate agglutination assay.【Result】 The IPAJ prokaryotic expression vector of Salmonella Pullorum was successfully constructed,and the recombinant protein was purified as inclusion body with good reactivity.Western blotting analysis results showed that IPAJ protein was successfully expressed at 32 ku,indicating good specificity.Indirect ELISA results showed that the titer of polyclonal antibody against IPAJ was 1∶25 600,indicating that polyclonal antibody was successfully prepared.The optimal condition for indirect ELISA using IPAJ protein as diagnostic antigen was preliminarily established as follows:The antigen coating concentration was 5 μg/mL,and 5% skim milk powder was used for 1.5 h,the optimum dilution of the primary antibody was 1∶250 (incubated for 1 h),the optimum dilution of the secondary antibody was 1∶10 000 (incubated for 1 h),and the color was dark for 15 min.Compared with plate agglutination assay results,the coincidence rate was 91.00%.【Conclusion】 The recombinant protein of Salmonella Pullorum IPAJ had good specificity and reactivity.The indirect ELISA method preliminarily established for the diagnosis of Salmonella Pullorum had certain practicability,which could be applied to clinical mass detection,and provide a basis for the establishment of a rapid diagnostic method.

Key words: Salmonella Pullorum; IPAJ protein; polyclonal antibody; indirect ELISA

CLC Number:

S816.48

NIU Xiaojie, XU Mingguo, XIAO Li, LIU Wenhao, CHEN Chuangfu, WANG Yong. Expression and Purification of IPAJ Protein of Salmonella Pullorum, Preparation of Polyclonal Antibody and Establishment of ELISA Method[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1218-1228.

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Expression and Purification of IPAJ Protein of Salmonella Pullorum, Preparation of Polyclonal Antibody and Establishment of ELISA Method

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