猪肺炎支原体Mhp271蛋白的多克隆抗体制备及在抗猪肺炎支原体感染中的应用

Preparation of polyclonal antibodies against Mhp271 and their application in combating Mycoplasma hyopneumoniae infections

摘要:

本研究旨在制备猪肺炎支原体(Mycoplasma hyopneumoniae, Mhp)膜蛋白Mhp271的多克隆抗体(polyclonal antibody, pAb)并系统评价其免疫学特性及潜在应用价值。首先通过PCR技术扩增获得mhp271基因片段(3 156 bp)，将其克隆至原核表达载体pMAL-c5x中构建重组表达质粒pMAL-c5x-mhp271。经PCR鉴定和DNA测序验证正确后，将重组质粒转化至大肠杆菌BL21(DE3)感受态细胞中，通过IPTG诱导表达重组Mhp271蛋白(rMhp271)。Western blotting结果显示，rMhp271在160 kDa处呈现特异性条带，表明该重组蛋白成功表达。将纯化的rMhp271蛋白乳化后经3次免疫BALB/c小鼠，并在第3次免疫1周后采血，分离血清获得抗Mhp271蛋白pAb。为评价该pAb的反应原性，建立Mhp体外感染猪肺泡巨噬细胞(porcine alveolar macrophages, PAMs)模型，感染12 h后分别采用Western blotting和间接免疫荧光试验(indirect immunofluorescence assay, IFA)进行检测。Western blotting结果显示，在Mhp感染的PAMs细胞裂解物中可见118 kDa特异性条带，而未感染对照组未检测到相应条带；IFA检测结果显示，Mhp感染组细胞中出现明显的绿色荧光信号，未感染对照组则无荧光信号。进一步通过体外封闭试验评估抗Mhp271蛋白pAb的抗Mhp感染潜力。将Mhp分别与100倍稀释的抗Mhp271蛋白pAb (实验组)或阴性血清(对照组)预孵育30 min后，接种至PAMs细胞并培养12 h，TaqMan实时荧光定量PCR检测结果显示，实验组Mhp载量较对照组显著降低；IFA检测发现实验组荧光信号强度明显减弱。本研究制备的抗Mhp271蛋白pAb具有良好的反应原性和抗感染活性，可用于Western blotting、IFA等免疫学检测方法。

Abstract:

The objective of this study was to prepare polyclonal antibodies (pAb) against the Mycoplasma hyopneumoniae (Mhp) membrane protein Mhp271 and systematically evaluate their immunological characteristics and potential applications. The mhp271 gene (3 156 bp) was amplified by PCR and cloned into the prokaryotic expression vector pMAL-c5x to construct the recombinant plasmid pMAL-c5x-mhp271. After verification by PCR and DNA sequencing, the construct was transformed into Escherichia coli BL21(DE3) competent cells, and the expression of recombinant Mhp271 (rMhp271) was induced with IPTG. Western blotting revealed a specific band at approximately 160 kDa, confirming successful expression of rMhp271. Purified rMhp271 was emulsified and used to immunize BALB/c mice three times. Serum samples were collected one week after the final immunization, and anti-Mhp271 specific pAb was isolated. To assess the reactivity of the anti-Mhp271 pAb, we used porcine alveolar macrophages (PAMs) to establish a cell model of Mhp infection. After Mhp infection for 12 h, Western blotting and indirect immunofluorescence assay (IFA) were employed to assess protein expression. Western blotting results showed a specific band at approximately 118 kDa in the lysate from Mhp-infected PAMs, while no corresponding band was detected in the uninfected control group. IFA demonstrated distinct green fluorescence signals in infected cells, whereas no fluorescence was observed in the uninfected control group. Furthermore, the potential of anti-Mhp271 pAb to inhibit Mhp infection was evaluated through in vitro blocking assays. Mhp was pre-incubated with either 100-fold diluted anti-Mhp271 serum (experimental group) or negative serum (control group) for 30 min before being inoculated into PAMs for 12 h. TaqMan real-time quantitative PCR indicated a significant reduction in Mhp load in the experimental group compared with the control group, which was further confirmed by the weakened fluorescence in IFA. Overall, the prepared anti-Mhp271 pAb demonstrated good reactogenicity and anti-infective activity, being suitable for immunological detection methods such as Western blotting and IFA.