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Cloning and Expression of the Region A Gene of Clumping Factor A of Staphylococcus Aureus from Bovine Mastitis

Author: JiangXiaoJuan
Tutor: HaoYongQing
School: Inner Mongolia Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Mastitis in dairy cattle Staphylococcus aureus Clumping factor A (ClfA) Clone Expression
CLC: S852.61
Type: Master's thesis
Year: 2008
Downloads: 84
Quote: 1
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Abstract


Mastitis cows a common and frequently occurring, the main reason for its occurrence is infected with pathogenic microorganisms. Causing mastitis as many as 80 kinds of pathogenic microorganisms, including bacteria, mycoplasma, fungi and viruses. Staphylococcus aureus dominant bacterial pathogen microorganisms, but over the years there has been no effective measures for the prevention and treatment of Staphylococcus aureus mastitis This paper Staphylococcus aureus cohesion factor AA District gene cloning expression for the developed prevention of Staphylococcus aureus mastitis bioengineering vaccine to provide a basis. ClfA A zone of the GeneBank published NewMan strain primers were designed, the test genotypes were amplified using the PCR method Hohhot surrounding areas exist four major Staphylococcus aureus, Staphylococcus aureus clumping factor A ( ClfA) A region genes. Experiments on four different genotypes of Staphylococcus aureus clumping factor A (ClfA) A region genes were cloned and sequenced, with Genbank standard landing NewMan of strain ClfA A region sequences than genotype 3, type 9 gene, gene 11 the type and NewMan strains homologous, respectively, 97.8%, 96.5%, 98.3%, 85.4% the gene 5 Newman strains homology only. Type 3 gene, gene type 9, type 11 gene between the homology is higher, and gene 5 and three additional genotype homology also lower. The measured sequences the use DNAstar software epitopes analysis, genotype 3 than NewMan strains, type 9 gene in an antigenic sites, selected a genotype that gene 9 type expression. The fragment was cloned into the prokaryotic expression vector pET-32a (), to obtain the recombinant plasmid pET-ClfA-A region. Be transformed into E. coli BL21 (DE3) induced by IPTG, expressed ClfA-A District proteins. Positioning analysis, the target protein exists in a soluble form. His · bind pillar after purification to obtain high purity ClfA-A region protein. The purified protein and Freund's adjuvant immunizing rabbits by three after immunization, high titer antiserum. Anti-adhesion capacity detected by ELISA, tube agglutination test, antibody, engulfed the conditioning test prove that the purpose of protein adhesion activity, homemade hyperimmune serum against Staphylococcus aureus adhesion to the role of bovine fibrinogen swallowed opsonization. The experiments confirmed that the expressed protein to the ClfA-A region protein.

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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
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