【作者】 陈恩林；

【导师】 王泽霖； 陈陆；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 根据我国当前禽流感H9亚型的变异和流行现状，为了研究不同H9N2亚型毒株HA抗原表位的变异性和多态性，本试验将1998～2005年间河南农大禽病研究所分离的25株H9亚型毒株经有限稀释法纯化作进一步鉴定（特异性和RT-PCR鉴定）后与禽病研究所研制、保存的5株H9N2血凝素单抗：A4、C9、F6、H6、C2和1株H9N2亚型非血凝素血单抗G4进行交叉HI反应和双夹心ELISA试验。在HI试验中25株H₉N₂毒株均不与非血凝素单抗G4反应，与5株血凝素单抗的HI结果可以分为二类，第一类2个毒株即致病性偏强的A／Chicken／Henan／A3／98（H9N2）（3#）株和A／Chicken／Henan／C4／02（H9N2）（18#）株，与所有5株血凝素单抗都不发生反应；另一类除3#和18#外的23个毒株，与所有5株单抗都发生反应，HI值为2¹5～2¹7。双夹心ELISA试验也显示了类似的结果，中和试验表明3#和18#毒株与5株血凝素单抗都不发生反应（P／N值1～2），与1株非血凝素单抗G 4弱反应（P／N比值为3）；其余23个毒株与上述6株单抗都发生反应，但不同毒株间有差异，1#毒反应较弱（P／N比值为2．1～3．3），多数毒株反应较强（P／N比值为5．3～9．3）。5株H9N2血凝素单抗不与3#和18#毒株反应，对3#和18#毒株无中和活性，说明在3#和18#毒株的血凝素表位上已经缺失了该单抗作用位点，或已发生了较大变异。不同H9N2亚型禽流感病毒血凝素抗原上的变异特性及致病性上的明显差异，进一步证实了H9N2亚型禽流感病毒（AIV）在抗原性上的变异和多态性。从25个H9N2亚型毒株中挑选出包含3#和18#在内的函盖不同抗原性和致病性的7个代表株进行了非结构（NS）蛋白基因扩增，并把扩增的基因片段克隆到pGEM-T载体中，转化大肠杆菌JM-109，阳性菌液送生物公司测序，获得了NS1和NS2蛋白的完整编码序列。序列分析表明7株H9N2亚型毒株的NS基因的核苷酸同源性在95．7％～99．8之间，氨基酸同源性在93．2％～99．6％之间，与中国大陆的其它分离株同源性高、进化稳定且高度保守；在基因系统发育进化树中都处于等位基因A类的亚洲禽一猪群分枝。7株H9N2亚型毒株与中国大陆其它分离株一样，NS1蛋白的C末端核定位信号区都缺失13个氨基酸。10#，17#，24#的39位碱基由C突变为T。8#，10#，15#，18#，24#的64位碱基由A突变为G；139位，225位由G突变为A；236位，375位由T突变为C；399位由C突变为T。8#，10#，15#，17#，18#，24#的114位，453位碱基由A突变为G；267位由C突变为T；681位由T突变为C。这些核苷酸及氨基酸的变化对其抗原性和致病性的影响，尚不清楚。但18#H9N2分离株NS1基因编码区的第460位核苷酸由G突变成T，与其它5株H9N2分离株相比，致使NS1蛋白的C末端核定位信号区有64个氨基酸缺失。18#毒株在25个毒株中致病性最强(ELD₅010^-8.7、MDT为69．9、ICPI为0．44、IVPI为0．51、)，且在血凝素表位上已经缺失了上述单抗的作用位点，这些致病性上和抗原性上变化很可能与NS1基因碱基的缺失有一定关系，但确切的机理和解释尚需进一步研究。本研究从生物学特性和分子水平上研究了H9N2亚型禽流感病毒在抗原性和致病性的遗传变异，可为我国禽流感的防制提供可靠的科学依据，为H9N2禽流感免疫和疫苗株的选择提供理论上的指导。更多还原

【Abstract】 According to the variation and epidemic status of H9N2 subtype AIV in our country, in order to study the variability and multiformity of HA antigenic epitope of different H9N2 subtype strains,in this reseach we purified 25 H9N2 AIV isolated during 1998-2005,then identified by speciality and RT-PCR methods. Five HA MoAb（A4,F6,H6,C9,C2,G4） that searched and saved were used to do the cross HI test and double layer ELISA test, in HI test ,25 H9N2 AIV strains don’t have reactivity to G4 which is not aim at HA,according to the result of HI test of five HA MoAb,we classified them to two kinds,the first kind is having strong virulence: A/Chicken/Henan /A3/98（H9N2）（3#） and A/ Chicken / Henan /C4/02（H9N2）（18#）, they don’t have reactivity to five HA MoAb;the other kind is 25 strains except 3# and 18# which having reactivity to five HA MoAb,the HI value is between 2¹⁵ and 2¹⁷.And the conclusion of the double layer ELISA test is identical,3# and 18# don’t have reactivity to five HA MoAb（P/N value is between 1 and 2）,have low reactivity to G4（P/N value is 3）;The other 23 strains have reactivity to 6 MoAb,but have difference among them,l# have low reactivity（P/N value is between 2.1 and 3.3）,most strains have more highly reactivity （P/N value is between 5.3 and 9.3）.Five HA MoAb don’t have reactivity to 3# and 18#,it shows that the HA epitope of 3# and 18# have site loss aim at the five MoAb,or have bigger variation. The variable characteristic and obvious different virulence confirm that there is variation in antigenity and multiformity of H9N2 AIV.RT-PCR was employed to amplify the cDNA of 7 strains（including 3# and 18#） which are representative（cover different antigenicity and virulence）,then linked to pGEM-T vector,transformed into E.coli JM-109,sequenced positive recombinant plasmid. NS gene sequence analysis shows that the nucleotide homology of 7 H9N2 strains is between 95.7% and 99.8%,deduced amino acid homology is between 93.2 % and 99.6%.It shows that the NS gene in china is high stable,belongs to the avian-swine branch of A allele. Like other isolates in china mainland ,there are 13 amino acide loss in the signal area of C’end of 7 H9N2 subtype strains .The nucleotide acide at 39 site of 10#,17#,24# mutate from C to T; The nucleotide acide at 64 site of 8#,10#,15#,18# mutate from A to G;The 139 and 225 site mutate from G to A,236 and 375 site mutate from T to C;339 site mutate from C to T. 8#, 10#, 15#, 17#, 18#, 24#’s 114 and 453 site mutate from A to G;267 site mutate from C to T;681 site mutate T to C. The effect of the nucleotide acide and amino acide’s change still need further reseach. But 460 site of 18#’s NS1 coding area mutate from G to T,make the signal area of of NS1 protein’s C’end lose 64 amino acid.l8# has more stroner virulence(ELD₅₀ is 10^-8.7、MDT is 69.9、ICPI is 0.44、IVPI is 0.51),and has loss of MoAb action site on HA epitope,the change of virulence and antigenicity may have certain relation to the loss of NS1 gene,but the mechanism and explaination still need further reseach. In this reseach,we study the variation of H9N2 subtype AIV on antigenicity and virulence,it will provide gist to the prevention and cure H9N2 AI ,provide guidance to the inoculation and the selection of H9N2 strain used to produce vaccine.更多还原