Interleukin10Gene Partial Sequence Cloning, Genetic Variation And Its Associations With Immune Traits Of Rabbit

Interleukin10(IL-10) was a special cytokine, which could inhibit the activation of mononuclear/macrophage system and the synthesis of monokines and inflammatory factors. IL-10has important immunoregulatory activities, also, it was closely related to disease susceptibility, severity and the development process of diseases. This study used New Zealand white rabbits as the experimental materials and made clonal analysis to IL-10promoters. Besides, we used New Zealand white rabbits, Fujian yellow rabbits and their reciprocal cross hybrid groups as a research object, then determined the relevant immune parameters and made correlation analysis between polymorphism of5exons of IL-10gene and immune parameters. The main results of this study are as follows:1. This study cloned partial sequences of5’ flanking region of IL-10gene in rabbits, and compared them with human IL-10promoter region, which was published in Genbank. As a consequence, we found that, rabbit and human IL-10gene promoter had some high similarities in partial sequences. Then, we predicted the promoter related components of cloned sequences through computer software. Basically, we were sure that this block of sequence was one part of the rabbit IL-10gene promoter. The sequence of human IL-10gene promoter,which was published in Genbank, was4018bp-long and it located at the upstream-29～-4047bp of start codons. By comparison, the region of upstream-29～-348bp of start codons in human IL-10gene was Consistent with5’ flanking region of IL-10gene in rabbits. So we supposed that, the same gene promoter sequence in different species had some conservatism, while partial sequences also had large variabilities.2. The four rabbit groups used in this study respectively were:New Zealand white rabbits, Fujian yellow rabbits, hybrids of New Zealand white rabbits (♂) and Fujian yellow rabbits (♀) named Xinhuang rabbit (N-Y rabbit for short) and hybrids of New Zealand white rabbits (♀)and Fujian yellow rabbits (♂) named Huangxin rabbit (Y-N rabbit for short). This study used RT-SSCP to detect the polymorphism of5exons of IL-10gene in four rabbit groups. Results showed that, there was no genetic diversity in exons1,2,5; we had found three alleles in exon3and six kinds of genotype, which were AA, BB, CC, AB, AC and BC. Then, we found two mutable sites, A1435G and G1519A, which were both samesense mutation that would not cause amino acid changes. Besides, there were two alleles in exon4and three kinds of genotype, which were OO, TT and TO respectively. In exon4, we detected an inserted base (T) between position2532and2533, which lead to the changes of amino acid sequence after this location. According to statistics, in exon3, the frequency of allele A was the highest, while the the frequency of allele C was the lowest, and we only found one individual of CC genotype in New Zealand white rabbit group. In exon4, the frequency of allele O was far higher than that of allele T, and among all detected samples, there was only one individual of TT genotype. In the X2test of different genotype distribution in exon3and exon4, there was significant difference(P<0.05) or extremely significant difference(P<0.01) between New Zealand white rabbit group and the others.3. This study used enzyme-linked immuno assay (ELISA) kit to detect partial immune traits of the four rabbit groups. Results showed that, the level of IL-10and IgG in the blood of Fujian yellow rabbits was significantly higher than the other three groups(P<0.05). The content of INF-y in Xinhuang rabbits and Fujian yellow rabbits was the highest, and there was no significant difference(P>0.05) between these two groups. In terms of white blood counts(WBC), New Zealand white rabbit group was significantly lower than the others(P<0.05).4. The correlation analysis between different genotypes and immune parameters suggested that, the immune parameters corresponding to each genotype in exon3of the four groups except for Xinhuang rabbit group had some correlations. The WBC in New Zealand white rabbits with AC genotype was significantly higher than that with BB、BC genotype(P<0.05). The level of IgG in Huangxin rabbits with AB genotype was significantly higher than that with AA genotype(P<0.05), besides, the IL-10level was the highest in BB genotype rabbits. The level of IgG in Fujian yellow rabbits with AA genotype was significantly lower than that with other genotypes(P<0.05), besides, the IL-10level was the highest in BB genotype rabbits, and there was no significant difference between these two genotypes. In addition, the immune parameters corresponding to each genotype in exon4of New Zealand white rabbits, Xinhuang rabbits and Huangxin rabbits showed no significant difference. We only detected that, the WBC in Fujian yellow rabbit group with TO genotype was significantly higher than that with OO genotype. For the comprehensive analysis of the genetic effects with two diverse exons, we also need to combine the two exon genotypes of Fujian yellow rabbits, and analyze the differences of WBC in combined and individual genotypes.