| In order to study the relation between PRRSV tropism and cell receptor, and the relation between individual disease resistance and genetic variation or expression of related immunogenes, PBMC from blood of five pig breeds and other tissues were chose in this experiment to investigate the expression of the PRRSV cell receptors gene CD163 and CD169, as well as IL8, IL10, TNFαin different pig breeds and tissues. We also analyzed the gene structure and screened SNP of CD163 and CD169, and then did association analysis between immunal traits and identified SNPs to evaluate DNA maker potential.The main results in this study were listed as follows:1. Developing the method for separating peripheral blood mononuclear cell from porcine small amounts of blood (Using blood heparinized with heparin sodium powder and centrifuged at 3000 r/min for 25min). It could get the best result and be used for RT-PCR and Q-PCR experiments.2.Accessing GeneBank to get mRNA and protein sequence of CD163 and CD169, we analyzed the gene and protein structure to predict the coding domains which are important to the receptors function .To compare the amino acid of CD163 genes of human, cow, mouse and pig, result revealed that they had high homology which reached 85%, but transmembrane domain and cytoplasmic tail varied greatly, the cytoplasmic tail of pig CD163 were composed by the amino acid of 1038-1060. To compare the amino acid of CD169 genes of human, cow, mouse and pig, result revealed that the V-set domain of different species had high homology, the V-set domain and cytoplasmic tail of pig CD169 were composed by amino acid of 20-136 and 1667-1730 respectively. The cytoplasmic tail of pig had 64 amino acid residues which had 20, 32 and 15 amino acid residues more than that of human, mouse and cow.3.We cloned 338bp, 276bp and 1102bp DNA fragments of CD169 were cloned and mutation was not found in these areas. Partial sequence of intron19 and 20 were got by sequencing and sent in NCBI to get the GenBank accession number EU240977 4.293bp, 219bp and 938bp DNA fragments of CD163 were cloned and sequenced. We have found four SNPs : 2439 A>G, 2454 G>C, 2537 G>A and 2685 A>C, and two of them (2537 G>A and 2685 A>C) were chosed to do genotyping and association analysis in immune pig population. 5.applying the method of CRS-PCR- HpaII -RFLP to detect and association analyze of CD163 2537 G>A, it has signification associations (p<0.05) with antibody levels for PRRS, platelet distribution width (PDW), mean platelet volume(MPV), platelet-large cell ratio (P_LCR) of 0-day-old pigs and platelet count (PLT), white blood cell count (WBC) of 17-day-old pigs.6.applying the method of CRS-PCR- BsiYI -RFLP to detect and association analyze of CD163 2685 A>C,it shows signification associations (p<0.05) with wean weight, antibody levels for PRRS (day 0 and day17) and antibody levels for swine fever (day 17)7. We have detected the transcript expression of CD163, CD169, IL8, IL10, TNFαin the PBMC of five pig breeds: Landrace, Yorkshire, Duroc, Meishan and DLY. The difference of IL8 transcript expression between Landrace and Duroc, Meishan , DLY was significant (P<0.05) . The difference of IL10 transcript expression between Yorkshire and Meishan , DLY was significant (P<0.05) .The transcript expression of three cytokines (IL8,IL10 and TNFα) in Meishan were lower than introduced pig.8.We have detected the transcript expression of CD163、CD169、IL8、IL10、TNFαin twelve tissues: heart, liver, spleen, lung, kidney, brain, marrow, lymph, testis, uterus, ovaries and PBMC. CD163、CD169、IL8、IL10 and TNFαwere widely expressed in porcine immune organs and PBMC, receptor gene CD163 and CD169 were widely expressed in the tissues where PRRSV could be detected.9. The radiation hybrid panel (IMpRH) was used for doing the regional assignment of CD163 and CD169 gene in pig. The porcine CD169 gene was located in Sscr 17, result revealed that the most significantly linked marker is END03. |
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