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Identification and Analysis of the in Vivo Induced Antigen of Haemophilsu Parasuis SH0165

Author: ZhangShu
Tutor: ChenHuanChun
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Parasuis Vivo induced antigen technology Genomic expression library
CLC: S852.61
Type: Master's thesis
Year: 2011
Downloads: 66
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Abstract


Parasuis (Haemophilus parasuis, HPS) is one of the main pathogens causing porcine respiratory. Haemophilus its cause of pig disease, also known as swine Glasser's disease (Glasser's disease) symptoms of multiple serositis and arthritis, while also cause meningitis. But mostly in vitro simulation of parasuis virulence genes naturally infected with their environment, to carry on its virulence or pathogenicity factors and there is no exact reports. Vivo induced antigen technology is a high throughput immunological screening method does not require the animal model. Have been infected by using the serum of the host of the target pathogen to study the antigen gene in the host body-specific expression of pathogenic genes of pathogenic microorganisms can be expressed in a simple and efficient identification of disease infection process. This technology can be used for the identification of acute and chronic infection in vivo specific expression of an immunogenic antigen, outer membrane proteins and secreted proteins can be simultaneously captured, can detect the different stages and different pathogenic path infections etc.. In many pathogens, especially in human disease pathogen has important applications, screening to many meaningful pathogenic factor for the development of novel drug targets, the development of efficient vaccines, the establishment of diagnostic methods, and provides a theoretical basis. The subject parasuis study places the main internal and external differentially expressed genes, the application of in vivo induced antigen technology for the screening and identification of the gene in vivo expression of local isolates of H. parasuis SH0165. Successfully constructed a genomic expression library of the strain, rehabilitation prepared porcine serum screening to 24 individuals differentially expressed genes using immunological techniques, and its validation. Main contents are as follows: 1.HPS genomic expression library construction sequencing: Select SH0165 strains, genomic DNA was extracted and recovered with Sau3AI digested 0.5 ~ 3kb fragments were cloned into the prokaryotic expression vector pQE80L/81L/82L transformed into E. coli, a genomic expression library was constructed. Library reorganization coverage greater than 80%, the insert size by restriction enzyme digestion and PCR. Library to meet quality requirements, screening studies can be carried out. 2 Preparation of serum antibody probes: SH0165 strain challenged pigs, collected convalescent sera, parasuis and E. coli bacterial cell antigen, crushing supernatant and secreted antigens to the nitrocellulose membrane as the carrier its for adsorption. ELISA detection of serum adsorption effect rehabilitation titer is significantly reduced, and can be used for library screening. 3 Screening of the in vivo induced antigen: Western blot screening library after IPTG induction, after twice Filter to obtain 39 positive clones. By sequencing analysis, to determine the open reading frame (ORF), including a total of 80 ORFs. Selected which contains a plurality of the ORF of positive clones were subcloned and used again by Western blot screening of each ORF, final 24 individual expressed genes. The in vivo expression of the gene validation: Preparation parasuis SH0165 immune serum. After E. coli antigen adsorbed sera and convalescent sera, respectively as an anti-immunoglobulin identify 24 individual expressed genes, found that 75% of the gene and the immune serum does not react with the convalescent sera reaction proved these genes are expressed in a host in vivo specific . And select one of the nucleic acid of the encoded host enzyme inhibiting protein genes the GAM and coding cells RODS to determine protein gene mreC prokaryotic expression and protein purification, the characteristics of the in vivo expression of these two genes was verified from the expression levels by Western blot. Vivo induced antigen technology screening to parasuis genes belonging to several aspects of metabolism, regulation, adhesion, transporters, cell structure, as well as resistant. Some genes may parasuis potential virulence factors, some genes may have a better immunogenicity as vaccine candidate genes, but also need further validation. Some genes are more sensitive to distinguish between immune serum and infected serum can be used as a diagnostic antigen, the establishment of the method of differential diagnosis.

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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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