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伪结核棒状杆菌磷脂酶D蛋白的表达及 间接ELISA 方法的建立与应用

文献类型： 中文期刊

关键词： 结核棒状杆菌;;磷脂酶D;;间接ELISA;;抗体检测

期刊名称：中国预防兽医学报

ISSN： 1008-0589

年卷期： 2025 年

页码：

收录情况： 北大核心（2023版） ; ; CSCD（2025-2026年度） ; ; 科技核心（2024版） ; ; 农林核心（2024版）

摘要： To develop a rapid detection method for Corynebacterium pseudotuberculosis (Cp), the phospholipase D (PLD) gene was amplified from the Cp reference strain ATCC19410 using PCR and cloned into the pET-28a vector to generate the recombinant plasmid pET-28a-PLD. After verifying its sequence, the plasmid was transformed into BL21(DE3) cell, the expression of recombinant PLD protein (rPLD) was induced with IPTG, and the protein was purified from gel slices. The expression and purification of rPLD were evaluated by SDS-PAGE, and its immunoreactivity was detected by western blot. The results showed the presence of rPLD at 33ku with good purification, and Western blot demonstrated good immunoreactivity. Using the purified rPLD as coating antigen, the reaction conditions were optimized using a matrix method, and an indirect ELISA method for detecting Cp serum antibodies was preliminarily established. The results showed that the optimal antigen concentration was 100 ng per well, the optimal serum dilution was 1􀏑50, the serum incubation time was 0.5 hours, the optimal blocking solution was 2% gelatin, the blocking time was 2 hours, the optimal dilution ratio of rabbit anti-goat horseradish peroxidase (HRP)-IgG was 1􀏑20000, the secondary antibody incubation time was 1 hours, and the cutoff value of 0.432. The ELISA method was used to detect positive sera against Cp, Capripoxvirus, Mycoplasma mycoides subsp. Mycoides, Mycobacterium tuberculosis, Mycobacterium avium subsp. Paratuberculosis, Peste des petits ruminants virus and Brucella melitensis, and it showed that all were negative except for Cp, indicating a strong specificity. After diluting Cp positive serum by a certain ratio, the ELISA method was used for detection, and the results showed that the detection limit of this method was 1􀏑640, indicating high sensitivity. The repeatability test which was conducted using 5 Cp positive sera and 3 Cp negative sera revealed a maximum coefficient of variation of 5.53% within batches and 7.79% between batches, indicating good repeatability. In a study of 50 clinical serum samples, the ELISA method identified 27 positive and 23 negative samples, while the bacterial agglutination inhibition test identified 24 positive and 26 negative samples, resulting in positive and negative agreement rates of 88.9% and 88.5%, respectively. A survey of 451 serum samples from sheep farms in Heilongjiang Province showed a 51% positive rate for Cp antibodies (230/451), demonstrating a high infection rate of Caseous lymphadenitis. These findings suggest that the indirect ELISA method established in this study is a promising tool for detecting Cp serum antibodies, providing strong support for the early diagnosis and epidemiological research of Cp infection.

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