Development And Application Of Indirect Elisa And Indirect Hemagglutination Assay For Antibodies Detection Against Strptococcus Suis Serotype 2 Strains

Streptococcus suis is an important swine pathogen that causes a. number ofPathological conditions such as meningitis, endocarditis, arthritis, pneumonia andsepticemia. There are currently 35 seroypes of Streptococcus suis, among which type 2 isthe most popular and pathogenic. But Streptococcus suis type 2 often combined infectionwith some other etiological agents as Actinobacillus suis, Bacillus erysipelatous andPRRS virus, It results in tremendous difficulty by clinical diagnosis and antibodiesdetection. Thus the topic purpose of this study:1 To establish indirect ELISA detection method of Streptococcus suis type 2 usedcapsular bolysaccharides as antigen.2 To construct the prokaryotic expression vectors for suilysin gene and examine itsimmunoreactivity of expression product and establish indirect hemagglutination assay(IHA) detection method of Streptococcus suis used Suilysin protein as antigen.3 Detect clinical serums by two above-mentioned kinds of method to check thecoincidence of two kinds of method and analyze the epidemiology characteristic ofStreptococcus suis type 2 in lately one year in our courtry.Streptococcus suis type 2 can create many kinds of virulence factor, for examplecapsular bolysaccharides(CPS), Muramidase-released protein (MRP),Extracellular factor (EF), Suilysin (SLY) and so on. Among the total, capsularbolysaccharides is most early corroborative virulence factor of Streptococcus suis type 2,it is also the base of typing. This test extracts capsular bolysaccharides by modifiedkodana method to establish indirect ELISA detection method and optimizes the bestworking condition to manufacture indirect ELISA detection kit Otherwise, Streptococcussuis type 2 has a high proportional to display suilysin activity, so Suilysinis knownas the virulence factor. On the base of the suquence of suilysin gene, The suilysin gene ofStreptococcus suis type 2 isolated LT-1 was amplified by PCR from bacterial genomicDNA, cloned into pMD18-T vector and then sequenced. The sequencing result showedthat the PCR product was composed of 1494 nucleotides encoding a polypeptide of 497amino acids. Streptococcus suis suilysin gene was cloned into a prokaryotic expression plasmid pET-28a, and the recombinant plasmid was then identifid by PCR and restrictionenzyme digestion and screened the positive clones. Suilysin expression was analyzed bySDS-PAGE and Western blot. Suilysin was expressed in the recombinant strains andreactive to positive serum as shown by immnuno-blotting. The sheep erythrocyte(SE) wasallergized by the expressed SLY protein and optimized all round conditions, then theindirect hemagglutination assay was established of Streptococcus suis.To detect 3 991 shares serum products by indirect ELISA detection method inHenan, Anhui, Jiangxi, Hunan, Hubei, Fujian, Guangdong, Guangxi and Beijing, the rateof positive was 33.75 %, 41.6% and so on. The two kinds of method established bythis research operate simplely, specificity highly, sensitivity highly and refered todetecting antibody and epidemiological survey agaist Streptococcus suis.