西双版纳茶花鸡HPAI抗性MHC单倍型初筛

利用机体免疫应答水平能较好代表动物抗病力的优点，针对有重要经济价值和公共卫生意义的国家强制免疫病种H5N1高致病性禽流感（HPAI），以抗病主效基因主要组织相容性复合体（MHC）为研究对象，以云南特有较强抗病力原始鸡品种茶花鸡为素材，接种HPAI灭活苗，采用血凝抑制试验、抗原肽特异性酶联免疫斑点法、微卫星扩增分别测定血清特异抗体效价、分泌γ干扰素的效应T细胞数和MHC单倍型，采用改良聚合酶链式反应（PCR+1方法）扩增及克隆MHC等位基因或其功能片段，采用DNA测序调查MHC单倍型内功能性单核苷酸多态性（SNP）位点、数量及频率，应用卡方检验分析免疫应答水平与MHC单倍型频率的关联性，并经重组H5假型病毒中和试验与四聚体分析做功能验证，初选出HPAI抗性MHC单倍型，为通过分子标记辅助选择培育HPAI抗性鸡品种或品系，进而从源头切断由鸡到人的传染途径，提供一种不同于扑杀或免疫手段的新思路。

Taking advantage of in vivo immune response levels well expressing animal disease resistance, this proposal aims to investigate correlations between haplotypes of major histocompatibility complex (MHC), a cluster of main effector genes responsible for disease resistance, of Chahua chicken in Xishuang Banna that is a locally original chicken breed characteristic of stronger disease resistance in Yunnan Province, and their immune responses to H5N1 subserotype influenza virus-caused highly pathogenic avian influenza (HPAI), a disease of economic importance and public health that requires nation-wide vaccination. For this purpose, Chahua chicken will be inoculated with inactivated HPAI vaccine, followed by determination of titers of specific antibodies in the sera by the hemagglutination inhibition test (HIT), counting of the effector T cells that secret gamma interferon by antigen-derived peptides specific enzyme-linked immunosorbent spot assay (ELISPOT), determination of MHC haplotypes using microsatellite amplification, cloning of MHC alleles or their functional fragments in each haplotype by a modified polymerase chain reaction (PCR+1 method), clarification of sites, numbers and frequencies of their functional single nucleotide polymorphisms (SNPs) using DNA sequencing, analysis of association of the MHC haplotypes' frequencies with the immune response levels using the chi-squared test, and further functional confirmation of the putative objective MHC haplotypes by neutralization tests with a pseudotyped virus carrying recombinant H5 and using tetramer analysis. Preliminary screening of HPAI resistant MHC haplotypes will provide a new approach that differs from slaughter or vaccination and may contribute to develop HPAI resistant chicken breeds or lines using molecular marker-assisted selection for ultimately breaking the route of infection from chicken to human.