西双版纳茶花鸡HPAI抗性MHC单倍型初筛

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.

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。

H5N1高致病性禽流感（HPAI）是严重危害养禽业和人类健康的人兽共患病，但现行的疫苗接种、扑杀等防控措施并不能完全控制该病。有国外学者报道红色原鸡（Gallus gallus）对HPAI具有较强抵抗力，但与已知决定动物抗病力的关键遗传因素主要组织相容性复合物（MHC）的具体关系尚不明确。茶花鸡是由红色原鸡驯养而成的半野生鸡品种，并与其存在自然交配和基因交流，故可能具有类似的遗传抗病特性。基于获得性免疫应答水平可视为动物抗病力的间接选择指标，对接种HPAI疫苗的茶花鸡测定其保护性免疫应答水平，有望找到代表HPAI抗性的MHC单倍型或基因型，为发展不同于或可配合疫苗接种的HPAI控制策略提供新思路。本项目对西双版纳茶花鸡保种场提供的293只茶花鸡，于35和53日龄接种同一HPAI灭活疫苗（0.4 mL/只），在二次免疫前、后采集翅下静脉抗凝全血约2 mL，分离血浆和血细胞。随后应用病毒HI试验和夹心法ELISA分别测定血浆特异性抗体效价和效应T细胞干扰素（IFN-γ）水平；提取基因组DNA，通过PCR扩增、TA克隆和DNA测序分析微卫星LEI0258和MCW0371，根据扩增产物的长度多态性确定个体MHC单倍型及基因型。最后选取频数不少于5的MHC单倍型或基因型，对其与抗体效价和IFN-γ水平的关联性分别做单因素方差分析。结果：获得LEI0258长度多态性片段50种，包括已有命名20种和新发现30种，并以205-bp频率最高（49.66%）；分出MHC单倍型纯合子43种，包括B15.2、B17、B23、BW3、BW11和B13.2共6种单倍型纯合子20只，未命名或新发现37种单倍型纯合子55只，其中以B17和205-bp（LEI0258）/204-bp（MCW0371）频率最高，均为3.74%，并从中检测到单核苷酸多态性（SNP）位点；二免后鸡群抗体效价和IFN-γ水平均极显著高于二免前（P﹤0.01）；二免后，249-bp、319-bp携带组和B17单倍型纯合子组抗体效价显著高于其他单倍型组或基因型组（P﹤0.05），这3组及307-bp携带组血浆IFN-γ水平显著高于其他组（P﹤0.05）。结果表明，B17单倍型较205-bp/204-bp单倍型对HPAI疫苗保护性免疫应答水平高，可作为HPAI抗性候选单倍型，相关个体可用于组建MHC单倍型纯系做活体抗病功能验证。