Isolation,Identification Of Streptococcus Equi Subsp.Equi-MDMC0316 And Establishment Of An Indirect ELISA Method For RSeM Protein

Strangles is a common upper respiratory tract infectious disease of equine animals caused by Streptococcus equi subsp.equi all over the world,which is characterized by mucous purulent nasal fluid secreted from nasal cavity and abscess formed in submandibular lymph nodes or retropharyngeal lymph nodes.In some areas of Inner Mongolia,the epidemic is serious,and spring and autumn are the main epidemic seasons.There is no effective vaccine,and the drug treatment effect is unstable.Eighteen samples were collected from the head and neck lymph nodes of horses suspected of suffering from Strangles in a racecourse in Xilingol,Inner Mongolia,and the pathogenic bacteria were isolated,purified and identified by routine methods;A pair of specific primers for Se M gene were designed by primer 5.0.The Se M gene product was amplified by PCR and cloned into the E.coli expression vector p ET-32 a,and the recombinant plasmid p ET-32a-Se M was constructed.The r Se M protein was induced by IPTG;Using r Se M protein as antigen protein,mice were immunized and mouse anti-Se M polyclonal antibody were prepared.An indirect ELISA assay for epidemiological investigation of Strangles was established,and 264 clinical horse serum were detected and analyzed.1.A pathogens strain was successfully isolated by sheep blood agar plate line separation method.Species identification and genetic evolution analysis of the isolated strain were carried out by physiological and biochemical experiments and molecular biological identification methods.It was found that the isolated strain was consistent with the standard physiological and biochemical tests of typical S.equi;According to BLAST analysis of 16 S r RNA gene,it was found that the sequence homology between the isolated strain and S.equi registered in NCBI was 100%.The genetic phylogenetic tree was drawn by MEGA 7.0,which indicated that the isolated strain was closest to XJALT-10-M2(number of JQ975921.1);in Xin Jiang and located in the same branch.The isolated strain was named S.equi strain and registered in Gen Bank system(number:OK254140.1).2.Se M gene of SEE with a complete open reading frame of 1198 bp was successfully amplified in this experiment.The target gene was cloned into p MD-19 T vector,and the correctness of ORF gene sequence was validated by sequencing.The recombinant p ET-32a-Se M was successfully purified by affinity chromatography after IPTG induced expression.Final concentration of recombinant fusion protein modulated to 325 μg/m L.3.The fused r Se M protein was used as the antigen protein and serum was collected from the orbits of the mice after three immunizations to reach a potency of 1:4400 and identified as a mouse anti-r Se M protein polyclonal antibody.The immunoreactivity of r Se M protein was confirmed by SDS-PAGE and Western boltting test.Using mouse anti-r Se M polyclonal antibody as the first antibody and r Se M protein as the coating antigen,the detection method of Indirect ELISA was preliminarily established,the best coating concentration of antigen was 5 μg/m L,the best dilution concentration of serum sample was 1:400,and the best dilution concentration of enzyme-labeled antibody was1:5000;Specificity test results were,The lysis products of four strains were selected as encapsulated antigens,and the results were all negative,and the assay showed high specificity;The results of the sensitivity test showed that the established indirect ELISA method achieved a sensitivity range within 1:4000.ELISA positive coincidence test was carried out with known positive horse serum samples and negative horse serum samples.The test results were analyzed by Graph Pad Prism 9,which showed that the test results were reliable and the coincidence rate was 100%.This method can be used to detect a large number of horse serum clinical samples.4.The indirect ELISA method established in this test was applied to 264 clinical horse serums for the detection of Strangles Se M Protein Antibodies.The results showed positive serums count of 147 and negative serums count of 117,a detection rate of 56%.A validated determination range was developed for the assay results,all data were imported into The R Programming Language for principal component analysis,and PCA scatter plots were drawn.Which was consistent with the pre-design of the experiment,which that the similarity of samples in the group was very high and the repeatability was good.This indirect ELISA method can accurately detect the production of Strangles specific antibodies in horses and can provide reference data to accurately determine whether the horse being tested is infected with Strangles or has a past history of Strangles.This experiment provides a reliable detection method for the epidemiological investigation of Strangles and the exclusion study of horses with previous history of the Strangles,and provides theoretical and technical support for the preventive control,early screening and traceability study of Strangles in Inner Mongolia.