摘要
为筛选茶花鸡H5N1高致病性禽流感(HPAI)抗性MHC单倍型,本实验对293只脱温茶花鸡先后两次接种同一HPAI灭活疫苗(0.4 m L/只),二次免疫前、后采集抗凝外周血,分别应用病毒血凝抑制(HI)试验和夹心酶联免疫吸附试验(ELISA)测定血浆特异性抗体效价和IFN-γ水平;通过PCR扩增分析微卫星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。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。结果显示,应用PCR扩增LEI0258获得50种长度多态性片段,包括已有命名的20种和新发现的30种;二免后茶花鸡实验群体特异性抗体效价和IFN-γ水平均极显著高于二次免疫前(p<0.01); 249 bp、307 bp、319 bp携带组和B17单倍型纯合子组二次免疫后抗体效价和IFN-γ水平均显著高于其它组(p<0.05)。本实验初步判定249 bp、307 bp、319 bp携带者和B17单倍型纯合子具有较高的HPAI免疫应答水平,提示其可以作为进一步鉴定HPAI抗性的候选MHC单倍型,有望为建立HPAI控制新策略提供思路。
In order to screening MHC haplotypes of H5N1 highly pathogenic avian influenza(HPAI)resistant from Chahua chickens,a total of 293 birds were inoculated twice with 0.4mL dose of the same inactivated HPAI vaccine,Peripheral blood with anticoagulant was collected before and after the 2nd immunization and the specific antibody titers and IFN-γlevels in the plasma were examined by viral hemagglutination inhibition(HI)assay and ELISA,respectively.Microsatellite LEI0258 and MCW0371were analyzed by PCR,individual MHC haplotypes and genotypes were determined according to the length polymorphism of microsatellite.Finally,those MHC haplotypes and genotypes with a frequency no fewer than 5 were selected and subjected to one-way analysis of variance for possible association with antibody titers or IFN-γlevels.Consequently,the PCR amplification of LEI0258 resulted in 50 length polymorphism fragments,of which 20 had been given nomenclature and the other 30 were newly discovered.Both specific antibody titer and IFN-γlevel were significantly higher post booster immunization than those prior to it(p<0.01)in the bird population.One-way ANOVA revealed that chicken groups bearing 249bp,307bp,319bp and B17homozygous produced significantly higher antibody titers and IFN-γlevel than the other groups after 2nd immunization(p<0.05).These findings preliminarily demonstrate that 249bp,307bp,319bp carriers and B17 homozygotes had stronger acquired immune response to HPAI,indicating their role as candidates for identification of HPAI-resistant MHC haplotypes in further,it is expected to provide ideas for establishment of a new strategy for HPAI control.
作者
郑玉芳
简宗辉
段锐锐
鲁琼芬
刘建平
陈培富
ZHENG Yu-fang;JIAN Zong-hui;DUAN Rui-rui;LU Qiong-fen;LIU Jian-ping;CHEN Pei-fu(College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China;College of Animal Science and Technology,Yunnan Agricultural University,Kunming 650201,China;Dai Autonomous Prefecture Animal Husbandry Technology Extension workstation,Xishuangbanna 666100,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2019年第1期63-68,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金地区项目(31260516)
云南农业大学研究生科技创新项目(2016ykc12)