猪链球菌自溶素的原核表达及其抗体间接ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2022.09.025

Abstract

Abstract: 【Objective】 This study was aimed to establish a rapid and accurate indirect ELISA method for detecting Streptococcus suis autolysin (atl) antibody.【Method】 A pair of primers was designed according to the Streptococcus suis atl gene sequence registered in GenBank, and the atl gene sequence was obtained from Streptococcus suis by PCR.The recombinant plasmids pET-32a-atl and pGEX-4T-1-atl were constructed, and then were transferred into BL21 competent cells for induction expression.The purified atl-His fusion protein was used as immunogen to immunize New Zealand White rabbits to prepare polyclonal antibody.The reactivity of atl-GST fusion protein was detected by Western blotting, using the purified atl-GST fusion protein as the coating antigen and the positive serum of Streptococcus suis infection as the standard serum, an indirect ELISA method for the detection of Streptococcus suis autolysin antibody was established.184 serum samples were detected by the ELISA method established in this test and the commercial Streptococcus suis type 2 ELISA antibody detection kit at the same time, and the clinical samples were detected by the method established in this test.【Result】 atl gene was successfully amplified from Streptococcus suis.The constructed recombinant plasmids was induced, expressed and purified to obtain 40 ku atl-His and 48 ku atl-GST fusion proteins.Western blotting result showed that rabbit anti atl-His antiserum had good reactivity with atl-GST fusion protein.The results of indirect ELISA showed that the critical value of negative serum and positive serum was 0.318, and the positive serum was still positive when diluted to 1:1280.The intra-batch and inter-batch coefficients of variation were both less than 10%, indicating that the method had good reproducibility and sensitivity. 96 positive samples were detected by the ELISA method and 66 positive samples were detected by the commercial Streptococcus suis type 2 ELISA antibody test kit, with a compliance rate of 71.7%.A total of 458 pig serum samples from different feeding stages of some pig farms in Jiangxi were detected by the established method, of which 277 positive samples were detected, and the positive rate was 60.4%.【Conclusion】 The indirect ELISA method for detecting Streptococcus suis autolysin antibody infection was successfully established and preliminarily applied, which laid a foundation for the seroepidemiological investigation of Streptococcus suis.

Key words: Streptococcus suis; autolysin; induced expression; indirect ELISA

CLC Number:

S852.61⁺1

HAN Rui, YANG Xing, ZHANG Wenbo, CHEN Jialing, ZHOU Xiaoli. Prokaryotic Expression of Autolysin from Streptococcus suis and Establishment of an Indirect ELISA Method for Detection of Its Antibody[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(9): 3508-3519.

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Prokaryotic Expression of Autolysin from Streptococcus suis and Establishment of an Indirect ELISA Method for Detection of Its Antibody

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