| S.enteritidis is a non-host-specific pathogenic bacterium which can infect and trigger the broiler to die and bring in hidden troubles of food security. Toll-like receptor 4 (TLR4), with the aid of the LBP, CD14 and MD-2, is a recognition receptor that functions as lipopolysaccharide (LPS) sensor and whose activation results in the production of several pro-inflammatory, antiviral and anti-bacterial cy-tokines. In order to study the mechanism of the TLR4/MyD88-dependent signaling transduction pathway and find the functional mutation sites, we performed three experiments.[Trial 1] Study was conducted to get the three-dimensional structure of the exocellular section of the TLR4 and the MD-2. The TLR4-MD-2 heterodimers of the mouse and the human served as tem-plates to simulate the structure of the TLR4-MD-2 heterodimer of the chicken with the ZDOCK pro-gram. Next, we mutated the Asp301 and the Arg611 of the TLR4 with Glu and Gln respectively to analyze the influences of that on the secondary structure of the protein. Results showed that the amino acids made up the interaction interface of the TLR4-MD-2 heterodimers among mouses and chickens and human beings were conservative. The mutations of the TLR4, Asp301Glu and Arg611Gln, brought a remarkable influence on the number of the intramolecular hydrogen bond and the secondary structure around the mutational sites. After mutation, the stability of the protein conformation became worse.[Trail 2] The MyD88 gene of chicken was amplified from spleen cDNA. and subcloned into the pMAl-c5X vector to construct recombinant plasmid pMAL-MyD88. The correct vector was trans-formed into host strain E.coli BL21(DE3) and was induced by 0.2mM IPTG at 30℃for six hours。Based on detection by SDS-PAGE, the result showed the expression of the MBP-MyD88 fusion pro-tein whose molecular weight was 76KD was efficient and the soluble protein was about 75%. The pu-rified MBP-MyD88 protein was mixed with immunological adjuvant and used to immune Balb/c mouse. The hybridoma technique was applied to fusion spleen cells and SP2/0 cells with PEG4000. Positive hybridoma cells were screened by indirect ELISA and 3 times subcloning .Western blot was used to indentify McAb specificity. The hybridoma cells against MBP were rejected and the three McAb against MyD88 were kept and named with 1F7,4G8 and 5E7 respectively. The subclass of 1F7,4G8 and 5E7 was IgG1,IgG1and IgG2a respectively and the light chains wereκequally. The ascite titer reached to at least 1:2×10~5.[Trail 3] Base on the analysis of the influence on the protein conformation brought by the 301and 611 amino acids mutations of the TLR4 protein, the DNA of SPF chickens was genotyped and divided into four groups. The four groups of chickens, whose haplotypes were I(903TT1832GG), II(903TG1832AA), III(903GG1832AA) and IV(903TG1832AG) respectively, were intramuscular in-jected with 0.5mL of the bacterial suspension containing 108 CFU SE at 21 days old. The microbial load and the relative express quantity of MyD88 were determined in 1-5DPI. Results showed that the difference of the microbial load in the spleen, caecum and bursa in 2DPI and 3DPI was significant (P<0.05) or extremely significant(P<0.01); In addition, the relative express quantity of MyD88 of dif-ferent haplotypes in the caecum (1DPI,P<0.01;2DPI,P<0.05), spleen (3DPI,P<0.05) and bursa (2DPI,P<0.01) also showed significant difference. In conclusion, the group of chickens, whose haplo-type were 903TT1832GG, showed much less microbial load and more expression of MyD88 protein and might have stronger resistance and immune defense. |
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