【作者】 张怀宇；

【导师】 王泰健； 赵启祖；

【作者基本信息】 中国兽医药品监察所 ， 预防兽医学， 2007， 硕士

【摘要】 采用蔗糖密度梯度离心法纯化灭活AsiaⅠ型（新疆毒株）口蹄疫病毒146S抗原，免疫BALB／C小鼠，融合脾细胞与SP2／0骨髓瘤细胞，使用间接夹心ELISA方法筛选，获得4株可稳定分泌抗AsiaⅠ型口蹄疫病毒单克隆抗体的杂交瘤细胞株。经鉴定，其中1^#G₃C₂、2^#G₆B₉、10^#C₂G₁细胞培养上清抗体效价可达1：20000以上，腹水抗体效价可达1：10⁶以上；8^#E₃C₁₀细胞培养上清抗体效价也可达1：5，120以上，腹水抗体效价可达1：81，920以上。四株单抗的亚型分别是1^#G₃C₂、8^#E₃C₁₀、10^#C₂G₁均为IgG₁亚类；2^#G₆B₉为IgM亚类。经SDS-PAGE和免疫组化染色确定1^#G₃C₂、2^#G₆B₉、10^#C₂G₁单抗针对构象型表位，而8^#E₃C₁₀单抗针对线型表位。其中8^#E₃C₁₀腹水单抗不纯，需要进行亚克隆，以提高纯度。四株单抗均有中和能力，其中8^#E₃C₁₀单抗中和能力最弱。均具有很好的型特异性。以上述1^#G₃C₂、2^#G₆B₉、10^#C₂G₁单抗的混合抗体为基础，建立了间接夹心ELISA方法。使用该方法测定了AsiaⅠ型病毒灭活抗原、O型病毒灭活抗原、正常BHK₂₁细胞培养液，结果表明该方法特异性好，所测定OD值和AsiaⅠ型抗原稀释倍数呈明显线性关系。采用该方法对11批AsiaⅠ型、O型单价或双价成品疫苗破乳抗原进行了测定，结果表明不同批次不同企业生产的疫苗完整病毒颗粒抗原含量有差异，进一步完善有望用于成品AsiaⅠ型口蹄疫疫苗中有效颗粒抗原含量的测定，从而发展成为一种疫苗效力检验替代方法。采用RT-PCR技术，扩增测定了制苗种毒（新疆株和LC株）VP1基因序列，对生产企业制苗种毒进行了“身份”鉴定，结果表明各企业均以规程批准的制苗毒株生产疫苗。同时与我国目前AsiaⅠ型主要流行毒株（江苏株）VP1核苷酸序列及推导氨基酸序列进行了比较，结果表明，新疆株对江苏株的核苷酸同源性为84.4％，氨基酸同源性为89.6％；LC株与江苏株的核苷酸同源性为84.4％，氨基酸同源性为90.0％；新疆株与LC株的核苷酸同源性为83.4％，氨基酸同源性为87.2％。通过对两个疫苗毒株的VP1基因及推导氨基酸序列的分析，发现两个疫苗株与流行株（江苏株）不属于同一基因型（VP1核苷酸序列同源性＞85％为同一基因型），新疆株和LC株的VP1G-H环140～160位氨基酸和羧基端203～211位氨基酸残基内的分别有9个、7个变异位点，但两者的RGD基序均未发生改变，其利用整联蛋白途径来识别宿主细胞位点能力也未发生变化。因此使用这两个疫苗种毒株制备出的疫苗能否完全保护当前主要流行毒株（江苏株），还有待进一步的研究。更多还原

【Abstract】 Taking sucrose desity gradient centrifugation method to pure 146S antigen of type Asia I （xinjiang vaccine strains） inactivated foot-and-mouth disease virus, then immunized mouse（blab/c） and fuse the SP2/0 myeloma cells with the spleen cells, after screening by IS-ELISA, four hybridoma cell line secreting stably monoclonal antibody against type Asia I FMDV were prepared. By identifving, the IS-ELISA antibody titers of culture supernatant and ascities to 1^#G₃C₂, 2^#G₆B₉ and 10^#C₂G₁ were not less than 1:20000, 1:10⁶ respectively, the IS-ELISA antibody titers of culture supernatant and ascities to 8^#E₃C₁₀ were 1:5120, 1:81920 respectively. The immunoglobulin subclass of the McAb to 1^#G₃C₂, 8^#E₃C₁₀、10^#C₂G₁、2^#G₆B₉ were IgG₁, IgG₁, IgG₁, IgM respectively By SDS-PAGE and immunihistochemistry staining, we identified that the McAbs of 1^#G₃C₂、2^#G₆B₉、10^#C₂G₁ recognized discontinuous epitopes, and the McAbs of 8^#E₃C₁₀ recognized continuous epitope. All are the neutralizing McAbs, but the McAb of 8^#E₃C₁₀ have low PD₅₀. All have highly specific.A IS-ELISA based on the mixtue of three different monoclonal antibodys（1^#G₃C₂、2^#G₆B₉、10^#C₂G₁） has been developed and it was applied to the testing of the type Asia I FMDV inactivated antigens, type O FMDV inactivated antigens and BHK₂₁ cell surpernatant. The experiment result demonstrated that the array have highly specific and there is a better correlation between our determined OD value and type Asia I antigen diluted multiple. We had studied totally 11 batch the antigen yields of the formulation of FMD vaccines series, including serotype Asia I, O and bivalent （O-Asia I）, the data indicated that the whole virions of commercial vaccines is different in differ corporation production and differ batch. Then it is expected to apply in developing the testing of effective antigen yields in type Asia I FMD commercial vaccines and it will become a alternative proposal for assessment of vaccine potency.In order to identified the identity of viral seeds in manufacture plant, we amplified and secquenced the VP1 gene of two FMD vaccine strains（lc vaccine strain and xinjiang vaccine strain） by using RT-PCR. The result indicated that all corporation have taken FMD vaccine with standard procedures. At the same time, we compared the VP1 nucleotide acid sequence and deduced amino acid sequence of two vaccine strains with jiangsu strain, which is the main prevalence strain of type Asia I in China. The homology analysis showed that xinjiang strain to jiangsu strain’s nucleotide acid sequence majority is 84.4%, amino acid majority is 89.6%; LC vaccine strain to jiangsu strain’s nucleotide acid sequence majority is 84.4%, amino acid majority is 90.0%; xinjiang strain to LC vaccine strain’s nucleotide acid sequence majority is 83.4%, amino acid majority is 87.2%. Through the analysis of VP1 gene and deduced amino acid sequence, we found that two vaccine strains and jiangsu strain are not belong to one genotype（VP1 nucleotide acid sequence majority＞85% classified to the same genotype）. There are 9 and 7 mutation respectively in VP1 G-H loop amino acid 140 to 160 and carboxyl extremity amino acid 203 to 211 for xinjiang vaccine strain and LC vaccine strain, but both arginine-glycine-aspartic acid （RGD） sequence didn’t change, and they didn’t lose the ability for using integrin approach to recognize epitopes of host cell. So we didn’t know the vaccine come fi-om FMDV mxinjiang strian and LC strain whether could protect host from attacking of prevail strain （jiangsu strain） or not. It need to research further.更多还原