基于pgk蛋白的犬嗜血支原体ELISA诊断方法的建立

The Establishment of pgk-based ELISA for the Diagnosis of Mycoplasma haemocanis

为了开发一种准确、快速的检测犬嗜血支原体的方法,建立了一种基于犬嗜血支原体磷酸甘油酸激酶(PGK)的间接ELISA方法.从已发表的NCBI犬嗜血支原体全基因组序列库中获得了其磷酸甘油酸激酶(PGK)基因序列.通过选择原核生物首选的密码子优化基因序列,将化学合成的新基因序列pgk插入质粒中,成功构建了原核表达载体pet32a(+)-pgk,并在大肠杆菌BL21中表达.经SDS-PAGE证实,该蛋白的相对分子量约为60 ku.以纯化的PGK重组蛋白作为抗原进行包被,建立了基于PGK蛋白的间接ELISA方法,并进一步对条件进行优化.结果表明,抗原的最佳包被浓度为2.5μg/mL,血清的最佳稀释倍数为1∶200,封闭剂的最佳工作浓度为5%脱脂乳,最佳封闭时间为45 min,待检血清的最佳作用时间为60 min,酶标二抗的最佳工作浓度为1∶5000,酶标二抗的最佳作用时间为30 min.基于以上,建立了一种检测犬嗜血支原体的间接ELISA方法,并选择临床阴性血清计算其临界值.采用该方法检测166份临床样本,阳性率为21.1%,与荧光定量PCR检测结果一致.本研究为犬嗜血支原体的临床监测提供了一种有效的检测方法,并为进一步预防和治疗该病提供了研究依据.

In order to develop an accurate and rapid method for the detection of Mycoplasma haemocanis,an indirect ELISA method based on phosphoglycerate kinase(PGK)of Mycoplasma haemocanis was established.The phosphoglycerate kinase(PGK)gene sequence of Mycoplasma haemocanis was obtained from the published whole genome sequence of Mycoplasma haemocanis in NCBI.By selecting the codon optimized gene sequence preferred by prokaryotes and inserting the chemically synthesized new gene sequence pgk into the plasmid,the prokaryotic expression vector pet32a(+)-pgk was successfully constructed and expressed in E.coli BL21.The relative molecular weight of the protein was confirmed to be about 60 ku by SDS-PAGE.Using purified PGK recombinant protein as coating antigen,an indirect ELISA method based on PGK protein was established and further optimized.The results showed that the optimum coating concentration of antigen was 2.5μg/mL,the optimum dilution multiple of serum was 1∶200,the optimal working concentration of sealant was 5%skim milk,the optimal sealing time of sealant was 45 min,the optimal action time of serum to be tested was 60 min,the optimal working concentration of enzyme labeled second antibody was 1∶5000,and the optimal action time of enzyme labeled second antibody was 30min.Based on this,an indirect ELISA method for canine Mycoplasma haemocanis was established,and the clinical negative serum was selected to calculate the critical value.This method was used to detect 166 clinical samples,and the positive rate was 21.1%,which was consistent with the result of fluorescence quantitative PCR.The completion of this experiment provides an effective detection method for clinical monitoring of Mycoplasma haemocanis in dogs and provides a research basis for further prevention and treatment of the disease.