Development Of An Indirect ELISA Method With The Main Antigentic Region Of Mycoplasma Ovipneumoniae P130 Protein Expressed In E.coli

Mycoplasma pneumoniae(Mycoplasma ovipneumoniae),mainly through the respiratory tract,causes infectious pleuropneumonia in sheep.The clinical symptoms are large lobar pneumonia,the mycoplasma is widely distributed,and the damage is covered by many countries and regions.Xinjiang,Inner Mongolia,Shandong and other areas are the main areas for the development of sheep raising industry.In recent years,the epidemic trend and scope of Mycoplasma pneumoniae have gradually expanded,which has brought great impact to the sheep industry.We should adopt the principle of prevention and treatment as the secondary factor,and establish a rapid detection method to prevent and control the disease.As far as we are concerned,the Mycoplasma pneumoniae is still very weak in our country.Therefore,this study has identified the isolated Mycoplasma pneumoniae strains,and then the isolation and purification of the strains for follow-up tests.In order to establish a simple and rapid detection method,indirect ELISA antibody detection method was established.Mycoplasma pneumoniae membrane protein P130-3 was used as the envelope antigen.According to the gene function prediction annotation,the P130-3 protein of Mycoplasma sheeppneumoniae(Mo)showed a coding size of about 130.5ku.This experiment took the whole genome of Mycoplasma ovum as a template,amplified P130-3 gene fragments by PCR technology,selected the main P130-3 domain P130-3(693bp),and constructed the primary antigen table of the main antigen domain of the P130-3 protein.The carrier,PET32a-P130-3,was transformed into BL21,and the induced expression conditions were 0.8mMol/L IPTG and 37 C,and SDS-PAGE and Western blot were identified by ultrasonic crushing,centrifugation and purification.In this experiment,an indirect ELISA detection method based on P130-3 was established to determine the optimized indirect ELISA conditions: the antigen inclusion concentration was 0.5 mu g/mL,4 centigrade was overnight,the sealing solution was 1% BSA,37 centigrade closed 1H,and the optimum concentration of antiserum was 1:100,and the optimum reaction condition was 37 C and 1H;two was the donkey anti sheep horseradish peroxidase.The optimal working concentration of labelled antibody enzyme labeled two was 1:6000,and the best reaction time was 37 C and 1H.The results showed that the size of the expressed P130-3 protein was 43 ku,which was consistent with the expected value.The established indirect ELISA method has good sensitivity,specificity and reproducibility.Indirect ELISA was used to identify negative serum by 30 kits,and the critical value of yin and Yang was determined by 0.238.The coincidence rate of the total bacterial protein and the indirect ELISA method established by this test was detected by 150 clinical samples.The results showed that the coincidence rate was 90.67%.To sum up,the indirect ELISA method established in this experiment is simple and fast,and is cheap compared with other detection methods.It can be used to diagnose Mycoplasma pneumoniae disease and epidemiological investigation.