牛支原体重组蛋白P48原核表达、多克隆抗体制备及间接ELISA抗体检测方法的建立

Prokaryotic Expression,Polyclonal Antibody Production and Development of an Indirect ELISA Antibody Detection Method of Recombinant Protein P48 of Mycoplasma bovis

【目的】建立牛支原体抗体间接ELISA检测方法,为牛支原体灭活疫苗的效力检测提供方法。【方法】构建pET-32a-P48原核表达质粒,转化大肠杆菌BL21(DE3)Plyss感受态细胞筛选阳性质粒并进行双酶切验证,用终浓度为1 mmol/L的IPTG诱导重组蛋白表达,使用镍柱进行蛋白纯化,经SDS-PAGE验证蛋白纯度及分子质量大小,采用Western blotting检测重组P48蛋白特异性和反应原性;将重组P48蛋白免疫家兔制备多克隆抗体。建立牛支原体间接ELISA抗体检测方法并进行条件优化,确定阴阳性临界值。对建立的方法进行敏感性、稳定性、特异性及与平板凝集试验的符合率试验检测。【结果】经SDS-PAGE验证,P48蛋白分子质量大小为62.68 ku,纯度在90%以上;Western blotting检测结果显示,P48蛋白仅与相应抗体结合呈现单一条带,该蛋白特异性和反应原性良好;琼脂扩散方法测得阳性血清效价为1∶64。建立的间接ELISA检测方法条件优化结果为:抗原包被浓度为0.3μg/mL,1%BSA 37℃封闭120 min,待检血清按1∶1280稀释,37℃孵育60 min,二抗稀释度为1∶5000,37℃孵育60 min,邻苯二胺常温显色10 min。结果判定标准为:D 492 nm值>0.3196为阳性;D 492 nm值<0.3101为阴性;0.3101≤D 492 nm值≤0.3196为可疑。敏感性试验结果显示,当阳性样品稀释度为1∶218时,阳性血清判读结果仍为阳性。稳定性试验结果显示,3批次P48蛋白包被的96孔板检测结果的变异系数均<10%;与牛溶血性曼氏海姆杆菌、牛巴氏杆菌阳性血清无交叉反应。本试验建立的间接ELISA抗体检测方法与平板凝集试验阳性血清符合率为100%(18/18),阴性血清符合率为97.5%(39/40)。【结论】本研究建立的牛支原体P48间接ELISA抗体检测方法具有良好的敏感性、特异性及稳定性,为牛支原体抗体检测及牛支原体疫苗效力检测提供了方法。

【Objective】The purpose of the test was to establish an indirect ELISA method for detection of antibodies to Mycoplasma bovis,and provide a method for testing the efficacy of inactivated vaccine against Mycoplasma bovis.【Method】The prokaryotic expression plasmid pET-32a-P48 was constructed and transformed into E.coli BL21(DE3)Plyss competent cells to screen the positive plasmid and perform double digestion verification.The recombinant protein was induced to express with IPTG with a final concentration of 1 mmol/L,and purified with nickel column.The protein purity and molecular weight were verified by SDS-PAGE,and the specificity and reactogenicity of the recombinant P48 protein were detected by Western blotting.The polyclonal antibodies serum was prepared by immunizing rabbits with the recombinant P48 protein.An indirect ELISA antibody detection method for Mycoplasma bovis was established and the conditions of the method were optimized,the critical value was determined.The sensitivity,stability,specificity and coincidence rate of the established method with plate agglutination test were tested.【Result】The molecular weight of P48 protein was 62.68 ku and its purity was over 90%by SDS-PAGE.Western blotting test results showed that P48 protein was only bound to the corresponding antibody and showed a single band,which showed good specificity and reactivity.The positive serum titer was 1∶64 by agar diffusion method.The optimized results of the established indirect ELISA detection method were as follows:The antigen-coated concentration was 0.3μg/mL,1%BSA was blocked at 37℃for 120 min,the serum to be tested was diluted at 1∶1280,incubated at 37℃for 60 min,the secondary antibody dilution was 1∶5000,incubated at 37℃for 60 min,and o-phenylenediamine was colored at room temperature for 10 min.D 492 nm value>0.3196 was positive,D 492 nm value<0.3101 was negative,0.3101≤D 492 nm value≤0.3196 was suspicious.The results of sensitivity test showed that when the dilution of positive sample was 1∶218,the interpretation of positive serum was still positive.The stability test results showed that the coefficient of variation of the 96-well plates of 3 batches of P48 protein coated was less than 10%.There was no cross reaction with positive serum of Mannheimia haemolytica and Pasteurella.Compared with plate agglutination test,the coincidence rate of positive serum and negative serum was 100%(18/18)and 97.5%(39/40),respectively.【Conclusion】The indirect ELISA antibody detection method for Mycoplasma bovis P48 established in this study had good sensitivity,specificity,and stability,providing a detection method for Mycoplasma bovis antibody detection and Mycoplasma bovis vaccine efficacy detection。