本文選題：A型口蹄疫病毒 + 免疫��； 參考：《內(nèi)蒙古大學(xué)》2015年碩士論文

【摘要】：口蹄疫是口蹄疫病毒引發(fā)的一種人畜共患的烈性傳染病,對(duì)現(xiàn)在的畜牧生產(chǎn)有巨大影響�？箍谔阋邌慰寺】贵w技術(shù)對(duì)口蹄疫的檢測(cè),早期預(yù)防等有重要影響,對(duì)研究如何診斷、防治口蹄疫等有重要意義。本研究使用獸用A型口蹄疫滅活疫苗做抗原,免疫健康BALB/c小鼠,讓其體內(nèi)產(chǎn)生抗A型口蹄疫病毒的抗體,通過PEG誘導(dǎo)融合技術(shù)制備出針對(duì)A型口蹄疫病毒的單克隆抗體細(xì)胞株,并針對(duì)雜交細(xì)胞和單克隆抗體的生物學(xué)特性進(jìn)行鑒定。為之后研制開發(fā)應(yīng)用型診斷產(chǎn)品打下基礎(chǔ)。1、口蹄疫病毒單克隆抗體制備條件的探索在進(jìn)行正式單克隆抗體制備前,分別對(duì)SP2/0細(xì)胞、飼養(yǎng)細(xì)胞和脾細(xì)胞在完全培養(yǎng)基和選擇培養(yǎng)基中的生長(zhǎng)特性進(jìn)行了研究；對(duì)融合過程中使用的PEG的分子量和濃度進(jìn)行了優(yōu)選；針對(duì)免疫過程中用到的獸用A型口蹄疫滅活疫苗的免疫劑量進(jìn)行優(yōu)選。結(jié)果表明：1)分子量為4000,濃度為50%的PEG的融合率最高；2)免疫注射0.1mL疫苗時(shí),小鼠血清抗體效價(jià)最好。2、抗A型口蹄疫病毒的單克隆細(xì)胞株的制備,篩選以及克隆化選取健康6-8周齡BALB/c小鼠,每只小鼠經(jīng)過3次腹腔免疫后檢測(cè)血清效價(jià),在融合前3天,尾靜脈加強(qiáng)免疫；在無(wú)菌條件下取出免疫脾細(xì)胞,與SP2/0細(xì)胞融合,經(jīng)HAT條件培養(yǎng)基篩選后,檢測(cè)培養(yǎng)基上清效價(jià),初步確定陽(yáng)性克隆；將抗體陽(yáng)性的細(xì)胞培養(yǎng)孔通過顯微操作法制備出單克隆細(xì)胞,使用間接ELIS A法,測(cè)定獲得的33株單克隆細(xì)胞,根據(jù)OD450nm值,篩選出表達(dá)量較高的細(xì)胞株。結(jié)果表明：1)經(jīng)過三次疫苗注射后,小鼠的抗體效價(jià)達(dá)到1：25600；2)細(xì)胞融合后,通過HAT培養(yǎng)基的篩選,得到雜交細(xì)胞；3)、經(jīng)過顯微操作技術(shù),制備得到33株陽(yáng)性單克隆細(xì)胞,其中3株為高表達(dá)細(xì)胞株：9F3、9F4、10B3。3、抗A型口蹄疫單克隆細(xì)胞株及其抗體的鑒定對(duì)9F3、9F4、10B3的穩(wěn)定性鑒定,結(jié)果顯示：9F3和10B3可以穩(wěn)定表達(dá),9F3細(xì)胞株效價(jià)較高：以9F3作為鑒定目標(biāo),繪制生長(zhǎng)曲線；測(cè)定染色體數(shù)94條；腹水抗體效價(jià)為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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3

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