绵羊肺炎支原体P6058aa-928aa蛋白的原核表达及间接ELISA方法的建立

【目的】 建立基于绵羊肺炎支原体表面脂蛋白P60的间接ELISA方法。【方法】 利用生物信息学软件对P60蛋白进行抗原性分析，筛选出抗原性最佳的区域，扩增目的基因后构建重组表达载体并进行基因测序，将测序正确的质粒转化大肠杆菌BL21(DE3)感受态细胞，用IPTG诱导抗原性最佳蛋白表达并进行诱导时间优化;利用SDS-PAGE分析该蛋白分子质量大小及其表达形式。以重组蛋白作为抗原，绵羊肺炎支原体阳性血清作为抗体，通过Western blotting方法分析其反应原性。以重组蛋白为包被抗原，建立抗原性最佳蛋白的间接ELISA抗体检测方法，用该方法对20份阴性血清进行检测，计算阴阳临界值;检测该方法的特异性、敏感性和重复性，并用建立的间接ELISA方法与间接血凝法对92份绵羊临床血清样本进行检测。【结果】 经DNAStar分析，P60蛋白第58－928位氨基酸区域的平均抗原指数在0.8~1.7之间，亲水指数为0~2.0，证明该区域(P60_58aa-928aa)抗原性和亲水性较强;通过PCR扩增得到P60_58aa-928aa基因，并构建重组表达载体，通过基因测序证明该重组表达载体与预期结果一致。SDS-PAGE结果显示，IPTG诱导浓度为1 mmol/L，诱导10 h时蛋白表达量较高，蛋白分子质量为42 ku，在大肠杆菌中以包涵体的形式表达;Western blotting结果表明，构建的重组蛋白具有良好的反应原性。对ELISA各条件进行优化结果显示:抗原包被浓度为0.5 μg/mL，一抗最佳稀释浓度为1∶100，最佳孵育时间为1.5 h，酶标二抗最佳稀释倍数为1∶4 000，判定阴阳性血清的临界值为0.36。对口蹄疫病毒(FMDV)、小反刍兽疫病毒(PPRV)、布氏杆菌(Brucella)、牛支原体(Mb)、丝状支原体山羊亚种(Mmc)的标准阳性血清的检测均为阴性，证明该方法特异性较强;敏感性试验结果表明，血清稀释度为1∶2 048时仍为阳性;批内重复试验和批间重复试验的变异系数均<10%，证明该方法具有较好的重复性。利用所建立的间接ELISA方法与间接血凝法对92份羊血清检测结果表明，二者的阳性符合率为81.6%，阴性符合率为92.6%，总符合率为88.0%。【结论】 本研究建立的间接ELISA方法可用于临床样本的检测，为绵羊肺炎支原体的疫苗免疫效果评价和疾病诊断提供了较为可靠的方法。

Prokaryotic Expression and Indirect ELISA Method Establishment of P6058aa-928aa Protein of Mycoplasma ovipneumoniae

【Objective】 The purpose of this study was to establish an indirect ELISA method based on lipoprotein P60 of Mycoplasma ovipneumoniae.【Method】 Bioinformatics softwares were used to analyze the antigenicity of P60 encoded proteins,and the region with the best antigenicity was screened out.After the target gene was amplified,the recombinant expression vector was constructed and the gene was sequenced,the correctly sequenced plasmid was transformed into Escherichia coli BL21(DE3) competent cells.The expression of the best antigenity protein was induced by IPTG and the induction time was optimized.SDS-PAGE was used to analyze the molecular weight of the protein and its expression form.Using recombinant protein as antigen and positive serum of Mycoplasma ovipneumoniae as antibody,Western blotting was used to analyze its reactivity.An indirect ELISA method was established to detect the best antigenicity protein by using the recombinant protein as the coating antigen.Twenty negative serum samples were detected by this method,and the critical value of positive and negative was calculated.The specificity,sensitivity and repeatability of this method were tested.The established indirect ELISA method and indirect hemagglutination were used to detect 92 sheep clinical serum samples.【Result】 According to DNAStar analysis,the average antigenic index of 58-928 amino acid region of P60 protein ranged from 0.8 to 1.7,and hydrophilic index ranged from 0 to 2.0,indicating that the region P60_58aa-928aa had strong antigenicity and hydrophilicity.The P60_58aa-928aa gene was amplified by PCR,and the recombinant expression vector was constructed.Gene sequencing proved that the recombinant expression vector was consistent with the expected results.SDS-PAGE results showed that when IPTG was induced at the concentration of 1 mmol/L and the induction time was 10 h,the protein expression level was higher,the molecular weight of protein was 42 ku,and it was expressed as inclusion body in Escherichia coli.Western blotting results confirmed that the recombinant protein had good reactivity.The optimal ELISA conditions showed that the concentration of antigen coating was 0.5 μg/mL,the optimal dilution concentration of primary antibody was 1∶100,the optimal incubation time was 1.5 h,and the optimal dilution ratio of enzyme-conjugate secondary antibody was 1∶4 000.The critical value of positive and negative serum was 0.36.The standard positive serum of Foot-and-mouth disease virus (FMDV),Peste des petits ruminants virus (PPRV),Brucella,Mycoplasma bovis (Mb) and Mycoplasma filamentum goat subspecies (Mmc) were all negative,which proved that the method had high specificity.In the sensitivity test,the result was still positive at the serum dilution ratio of 1∶2 048.The coefficient of variation of intra-batch and inter-batch repeated tests were all less than 10%,which proved that the method had good repeatability.The established indirect ELISA method and indirect hemagglutination method were used to detect 92 sheep serum samples,and the results showed that the positive coincidence rate was 81.6%,the negative coincidence rate was 92.6%,and the total coincidence rate was 88.0%.【Conclusion】 This indirect ELISA method could be used to detect clinical samples,and providing a reliable method for the evaluation of vaccine immune effect and disease diagnosis of Mycoplasma ovipneumoniae.