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Construction and Function Study of the GlnA/ECE1 Gene Knock-Out Mutant of Streptococcus Suis Serotype 2 and Preparation of Monoclonal Antibody Against ECE1

Author: XuHaiJun
Tutor: XiaoShaoBo
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Streptococcus suis type 2 Endothelin converting enzyme a Monoclonal antibodies
CLC: S852.611
Type: Master's thesis
Year: 2011
Downloads: 27
Quote: 0
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Abstract


Suis type 2 (Streptococcus suis serotype 2 S. suis 2) can lead to human and pig meningitis, arthritis, septicemia, endocarditis, is a zoonotic pathogen. Type 2 Streptococcus suis great harm pig herd incidence and high mortality. Constructed on the basis of this study glutamine synthetase gene △ GlnA deficient strains of Streptococcus suis type 2 suis type 2 △ GlnA / ECEl double gene deletion strains, and screened stable secretion ECE1 monoclonal antibody hybridization tumor cell lines, attenuated vaccine for Streptococcus suis genetic engineering research and pigs streptococcal disease diagnostic methods establish a reference base, the main contents: 1.2 suis △ GlnA / ECEl double knockout strains screening with the The recombinant plasmid was transformed on the downstream homology arms in ECE1 S. suis SC19 △ GlnA the competent cells resistant medium screening and PCR identification, the last successful gene deletion strains of S. suis SC19 △ GlnA/ECE1 double. 2.2 suis △ GlnA / biological characteristics of ECEl double gene deletion strains experimental select S. suis SC19, S. suis SC19 △ GlnA S. suis SC19 △ GlnA/ECE1 three strains at the same time carried out the experiments under the same conditions. Results show that △ GlnA/ECE1 double gene deletion strains in colony morphology on TSA solid medium size did not change, no change in hemolysis on TSA plus sheep blood medium, and stable growth in TSB medium passaged . Determination of the three strains of the bacterium change over time, changes in absorbance values ??and draw the curve, the results show the △ GlnA deletion mutant growth rate slows down the △ GlnA/ECE1 deletion strains with △ GlnA deletion mutant compared to no change. Detection using the French Merieux API20 Strep standard internationally recognized biochemical characteristics of the three strains, the results showed the △ GlnA/ECE1 Deletion Mutant and △ GlnA deletion mutant no difference compared to the a-galactosidae detection becomes negative only with the wild strain. The deletion strains △ GlnA/ECE1 Hep-2 adhesion and toxicity, △ GlnA deletion mutant contrast slightly decreased, but decreased significantly compared with the wild strain. 3.2 suis △ GlnA/ECE1 double knockout strains CD1 mice virulence changes in selected S. suis SC19 S. suis SC19 △ GlnA S. suis SC19 △ GlnA/ECE1 three strains at the same time under the same conditions animal experiments. The experimental results showed that the infected CD1 mice LD50 of S. suis SC19 △ GlnA/ECE1 double knockout strains was significantly higher than the two parental strains, its infection-induced morbidity and mortality compared with the two parental strains also fell. The graph of the amount of change over time in different tissues of mice after infection carrier was not △ GlnA/ECE1 double knockout strains infected mice tissue contaminated than the wild strain infection group decreased a lot with the the △ GlnA gene deletion mutant infection group compared to slightly lower. △ GlnA/ECE1 mutant infected mice brain and lung tissue sections observed under the microscope are normal, substantially no visual pathological damage; while the △ GlnA gene knockout mutant infected group brain tissue substantially no significant pathological changes, but the lung tissue inflammatory cells oozing lesions; mouse brain and lung tissue of wild strain-infected group were serious pathological changes. Endothelin converting enzyme 1 efficient expression in E. coli system based on the GenBank SSU05 0 153 gene sequences to design PCR amplification of the full-length gene sequence primers endothelin converting enzyme 1, was successfully constructed. expression vector pET-28a ECE1, and then transformed into E. coli BL21 after restriction endonuclease correct save spare. Experiment by groping under different conditions, and ultimately determine the induced expression 4 hours in the 37 ° C shaker IPTG (0.4mmol/mL) ECE1 expression conditions, and determine the soluble expression product with the inclusion body proteins have generated facilitate experiments purified The purified soluble expression of the protein. Purified ECE1 5. Endothelin converting enzyme 1 Preparation of monoclonal antibodies will be induced to express protein as antigen Babl / C mice to produce immune spleen cells for cell fusion with SP2 / 0 myeloma cell fusion. ECE1 protein coating antigen indirect ELISA screening fusion-positive cell lines, and the final six hybridoma cell lines stably secreting antibodies after three cloned by limiting dilution method. Were respectively the SC19 strain bacterial and △ GlnA/ECE1 of mutant bacteria as antigen prepared mouse ascites monoclonal antibody is an anti-western blot validation experiments, the results show that there are two monoclonal antibody cell lines SC19 strain reaction was positive , named SE3 and SE6. All cell lines the mAb with △ GlnA / ECEl mutant reaction was negative.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Pathogenic bacteria > Cocci
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