抗A型口蹄疫病毒单克隆抗体的制备

发布时间：2018-04-23 12:40

本文选题：A型口蹄疫病毒 + 免疫　；参考：《内蒙古大学》2015年硕士论文

【摘要】：口蹄疫是口蹄疫病毒引发的一种人畜共患的烈性传染病,对现在的畜牧生产有巨大影响。抗口蹄疫单克隆抗体技术对口蹄疫的检测,早期预防等有重要影响,对研究如何诊断、防治口蹄疫等有重要意义。本研究使用兽用A型口蹄疫灭活疫苗做抗原,免疫健康BALB/c小鼠,让其体内产生抗A型口蹄疫病毒的抗体,通过PEG诱导融合技术制备出针对A型口蹄疫病毒的单克隆抗体细胞株,并针对杂交细胞和单克隆抗体的生物学特性进行鉴定。为之后研制开发应用型诊断产品打下基础。1、口蹄疫病毒单克隆抗体制备条件的探索在进行正式单克隆抗体制备前,分别对SP2/0细胞、饲养细胞和脾细胞在完全培养基和选择培养基中的生长特性进行了研究；对融合过程中使用的PEG的分子量和浓度进行了优选；针对免疫过程中用到的兽用A型口蹄疫灭活疫苗的免疫剂量进行优选。结果表明：1)分子量为4000,浓度为50%的PEG的融合率最高；2)免疫注射0.1mL疫苗时,小鼠血清抗体效价最好。2、抗A型口蹄疫病毒的单克隆细胞株的制备,筛选以及克隆化选取健康6-8周龄BALB/c小鼠,每只小鼠经过3次腹腔免疫后检测血清效价,在融合前3天,尾静脉加强免疫；在无菌条件下取出免疫脾细胞,与SP2/0细胞融合,经HAT条件培养基筛选后,检测培养基上清效价,初步确定阳性克隆；将抗体阳性的细胞培养孔通过显微操作法制备出单克隆细胞,使用间接ELIS A法,测定获得的33株单克隆细胞,根据OD450nm值,筛选出表达量较高的细胞株。结果表明：1)经过三次疫苗注射后,小鼠的抗体效价达到1：25600；2)细胞融合后,通过HAT培养基的筛选,得到杂交细胞；3)、经过显微操作技术,制备得到33株阳性单克隆细胞,其中3株为高表达细胞株：9F3、9F4、10B3。3、抗A型口蹄疫单克隆细胞株及其抗体的鉴定对9F3、9F4、10B3的稳定性鉴定,结果显示：9F3和10B3可以稳定表达,9F3细胞株效价较高：以9F3作为鉴定目标,绘制生长曲线；测定染色体数94条；腹水抗体效价为1：25600。
[Abstract]:Foot-and-mouth disease (FMD) is a severe infectious disease caused by foot-and-mouth disease virus (FMDV). Anti-foot-and-mouth disease monoclonal antibody technique plays an important role in the detection and early prevention of foot-and-mouth disease. It is of great significance to study how to diagnose and prevent foot-and-mouth disease. In this study, animal inactivated foot-and-mouth disease (FMD) type A vaccine was used as antigen to immunize healthy BALB/c mice to produce antibodies against FMDV A, and monoclonal antibody cell lines against FMDV A were prepared by PEG induction fusion technique. The biological characteristics of hybrid cells and monoclonal antibodies were identified. To lay the foundation for the development of the applied diagnostic product. 1. To explore the conditions for preparation of monoclonal antibodies against foot-and-mouth disease virus (FMDV). Before the preparation of McAbs, SP2/0 cells were treated separately. The growth characteristics of feeder cells and spleen cells in complete and selective medium were studied, and the molecular weight and concentration of PEG used in fusion were optimized. The immune dose of animal type A foot-and-mouth disease inactivated vaccine was selected. The results showed that the highest fusion rate of PEG with molecular weight of 4 000 and concentration of 50% was obtained when injected with 0.1mL vaccine, the titer of serum antibody was the best in mice, and the monoclonal cell line against FMDV A was prepared. Healthy BALB/c mice aged 6-8 weeks were selected for screening and cloning. The serum titers of each mouse were tested after three times of intraperitoneal immunization. The caudal vein was immunized three days before fusion, and spleen cells were removed under sterile conditions and fused with SP2/0 cells. After screening by HAT conditioned medium, the titer of supernatant of medium was detected, and the positive clone was preliminarily determined. Monoclonal cells were prepared by micromanipulation and indirect ELIS A method was used. According to the OD450nm value, 33 monoclonal cells were screened out with high expression. The results showed that the antibody titer of mice reached 1: 25600-2 after three times of vaccine injection. After the fusion of the cells, the hybrids were obtained by HAT medium screening, and 33 monoclonal positive cells were prepared by micromanipulation. Three of them were highly expressed cell lines: 1 9F3, 9F4, 10B3.3. Monoclonal cell lines and their antibodies against type A foot-and-mouth disease (FMD) were identified. The results showed that the stability of 9F3F3 and 10B3 could be stably expressed in 9F3 cell lines. 9F3 was used as the target for identification. The antibody titer of ascites was 1: 25600.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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