Construction And Function Study Of The GlnA/ECE1 Gene Knock-Out Mutant Of Streptococcus Suis Serotype 2 And Preparation Of Monoclonal Antibody Against ECE1

Streptococcus suis 2 (S. suis 2) is a very important zoonotic pathogen, associated with meningitis, endocarditis, arthritis and septicemia S. suis 2 has a great harm to the swine and lead to high mortality of swine.In this study,the ECE1 gene was Knockouted from the S. suis 2△GlnA,and a new strain was generated namely S. suis 2△GlnA/ECEl. And hybrid cell lines are acquired stably expressing anti-ECE1. The main contents include the following:1. Construction of the S. suis 2glnA/ECE1 deletion mutantThe plasmid pSET4SAGlnA/ECEl were transformed into S. suis 2 SC19△GlnA. The resultant mutant strain was selected by PCR and Spectinomycin,which was named S. suis 2 SC19△GlnA/ECEl.2. Effect of GlnA/ECEl deletion on the general biological characteristics of S. suis SC19The general biological characteristics of the S. suis 2 SC19 and the AglnA/ECE1 mutant were contrasted under the same conditions. First, the colony, cell morphology and hemolytic activity were validated but, no significant differences between the wild type and mutant strains were confirmed.After passaged in TSB, the△glnA/ECE1 mutant strain is grown stably,but which growth rate was becomed slowly.Compared with sc19 and△glnA, the△glnA/ECE1 mutant was showed a decrease in adhesion and toxicity to the HEp-2 cells. The a-galactosidae was negative in the mutant strains.3. Impact of△glnA/ECE1 mutant on the pathogenicity of S. suis SC19To define the impact of glnA/ECE1 deletion on the pathogenicity of SC19, the experiment carried out three sets of infections in parallel. The pathogenicity of the sc19 and glnA/ECE1 deletion mutant was evaluated in a CD1 mouse model. Results of the experiment displayed that the LD50 of the glnA/ECE1 deletion mutant strain was increased significantly compared with the sc19 and the glnA deletion mutant strain.The mortality and morbidity of CD1 mouse injected the glnA/ECE1 deletion mutant was decreased significantly,and biopsy wasn't showed pathologic changes.4. ECE1 was highly expressed in E. coliECE1 gene of SC19 was cloned into the pET28a vector to construct a recombinant expression plmaid. The recombinant protein was induced by 0.4mM IPTG at 37℃for 4 hours, after the pET28a-ECE1 vector was transformed into E. coli. Analyzed by SDS-PAGE,the result showed that ECE1 could be efficient expressed in supernatant.5. Monoclonal antibodies of ECE1 were produced and verified by western blotBalb/c mice were immunized with ECE1 Freund's complete adjuvant antigen.When the high levels of antibodies were showed in mouse, the spleen cells from mouse immunized were hybridized with SP2/0 bone marrow tumor cells with PEG400. Six hybrid cell lines stably expressing antibody were gained after three times sub-cloning and selection. Then the hybridoma cells were injected into the mice abdomen, and about two weeks ascites could be collected. Results of western blot showed two hybrid cell lines secrete anti-sc19 McAbs namely SE3 and SE6.