【作者】 林彤；

【导师】 ?蒈?李克生； 薛慧文；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 利用杂交瘤细胞技术，以表达的Asial型口蹄疫病毒VP1蛋白免疫BALB／c小鼠，无菌条件下取小鼠脾细胞与SP2／0细胞进行细胞融合。经多次克隆和间接ELISA筛选，获得三株能分泌抗Asial型口蹄疫的单克隆杂交瘤细胞株，并对其特性作了鉴定。在此基础上，利用胶体金免疫层析技术制备了Asial型口蹄疫病毒单抗胶体金诊断试纸条。经单克隆抗体亚类鉴定，三株单克隆抗体依次为IgG1、IgG2a和IgG2b。杂交瘤细胞株的染色体计数为104条，在杂交瘤细胞染色体范围内。用间接ELISA对腹水和细胞上清液中单抗效价进行测定，三株单抗的腹水中单抗效价≥1∶10⁶，而细胞上清中单抗效价≥1∶10⁴。用叠加实验对3株单抗的抗原位点进行了分析，发现三株单抗针对2个不同的抗原位点，其中2株单抗作用于同一抗原表位。对所获得的三株单抗经SDS-PAGE电泳，结果显示，所有单抗重链的分子量均为45kD左右，轻链的分子量为25kD左右。三株杂交瘤细胞和注射小鼠腹腔内的杂交瘤细胞经过5次连续传代后，用间接ELISA对每代次的细胞上清中的单抗效价进行了检测，传代细胞每代的单抗效价稳定在1∶10⁴左右，腹水的抗体效价稳定于1∶10⁵～10⁶。用Asial单抗分别与O、A、C、Asial型病毒抗原和Asial重组抗原进行反应，结果三株单抗均与Asial型口蹄疫病毒和表达VP1蛋白有特异的反应，而与C、O和A型口蹄疫抗原不发生交叉反应。利用获得的单抗成功研制出了检测Asial型口蹄疫病毒抗原的金标快速诊断试纸条。用该试纸条分别对A、O、C和Asial型口蹄疫病毒抗原以及猪水泡病病毒抗原等87份田间样品进行了检测，发现检测Asial型口蹄疫病毒抗原的试纸条出现2条明显的条带，而A、O、C型口蹄疫病毒抗原以及猪水泡病病毒抗原只出现对照线，检测线没有条带，说明该试纸条不口蹄疫病毒A、O、C型以及猪水泡病病毒抗原发生反应，特异性强。用该试纸条对口蹄疫细胞毒液(10^-7TCID50)的10倍系列稀释液进行了检测，最低可以检测到大约10^-4TCID₅₀。初步实验确定该试纸条在4℃、37℃和室温下的有效期为4个月。对于Asial型口蹄疫病毒金标试纸条的特性和应用Asial型口蹄疫病毒单克隆抗体的诊断价值还有待进一步的研究。更多还原

【Abstract】 The monoclonal antibody（McAb） has strong specific and repeat characters, and it can be make into productions of detection and be used in research fields. The purposes of this paper were for obtain monoclonal antibodies to Asial type Foot-and-Mouth Disease Virus（FMDV）, and for make them into detective productions of FMDV antigen with a rapid, specific, sensitive and convenient mean.The BALB/C mice were immunized 4 times with Asial type FMDV. VP1 protein。The myeloma cell line SP2/0 was fused with the spleen of immunized BALB/C by PEG-1000, and by several limiting dilution, three specific monoclonal antibodies were screened by indirect ELISA. The three specific monoclonal antibodies cell strains named 1B8, 5E1 and 5E2, respectively.Among the three cell strains, 1B8 belonged to IgG₁ subclass of Ig, 5E1 and 5E2 belonged to IgG2b and IgG2a subclass of Ig,. The results of specificity cross test indicated that the three cell strains’ monoclonal antibodies were not specific reaction with the A, C or O type of FMDV. Their titers in cell culture supematant were 1∶10⁴, Their titers of abdomen liquor were 1∶4×10⁶, respectively.The purified protein of monoclon by SDS-PAGE showed the molecular weights of the heavy chain and light chain were about 45.0 kD and 25.0 kD. The number of chromosomal to SP2/0 cell strains were 50, to hybridoma cell strains were 104。By mean of indirect ELISA, the antigenic epitopes was detected and recognized by1B8, 5E1 and 5E2. The results indicated that the epitopes recognized 1B8 overlapped partly that of 5E2, and the epitopes recognized 1B8 is different to that of 5E1.In the appliance research test, lemon acid was made to colloidal gold and marked it with monoclonal antibody. The strips can recognize Asial type FMDV and VP1 protein, can detective 10^-4 TCID₅₀ virus particle, can store three months in different temperature condition。through experiment we find these strips have highly specificity、sensitivity and stability。In this whole experiment, we obtained monoclonal antibodies to FMDV, and had a significance trial for make them into detective productions of FMDV antigen in the future.更多还原