本文選題：A型口蹄疫病毒 + 結(jié)構(gòu)蛋白VP1��； 參考：《中國農(nóng)業(yè)科學(xué)院》2010年碩士論文

【摘要】： 口蹄疫(Foot-and-mouth Disease,FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus, FMDV)所引起的一種偶蹄動(dòng)物的急性高度接觸性傳染病,被世界動(dòng)物衛(wèi)生組織(OIE)列為A類傳染病之首,給流行國家和地區(qū)的政治、經(jīng)濟(jì)和社會(huì)問題帶來了不利影響,而單克隆抗體以其特異性強(qiáng)、重復(fù)性好、穩(wěn)定性高的優(yōu)點(diǎn)已成為口蹄疫診斷和研究的焦點(diǎn)。 本研究利用重組菌pGEM-VP1為模板成功克隆出VP1基因,將其連接到表達(dá)載體上誘導(dǎo)表達(dá)出融合蛋白�？扇苄苑治霰砻鞅磉_(dá)的融合蛋白是以包涵體的形式存在,超聲裂解菌體后利用鎳離子親和層析的方法進(jìn)行蛋白純化,SDS-PAGE分析顯示得到了較純的融合蛋白,并以此作為免疫原免疫6周齡的雌性BALB/c小鼠,加強(qiáng)免疫后取其脾臟和骨髓瘤細(xì)胞(Sp2/0)進(jìn)行融合,通過間接ELISA法初步篩選,有限稀釋法進(jìn)行亞克隆,最終獲得了4株能穩(wěn)定分泌單克隆抗體的雜交瘤細(xì)胞株,分別命名為7B11、7H11、8G2、8H4。利用SDS-PAGE、間接ELISA法、Western blot分析和間接免疫熒光法進(jìn)行了生物學(xué)特性的分析和鑒定。實(shí)驗(yàn)結(jié)果表明獲得的4株單抗具有較高的抗體效價(jià)和特異性,抗體亞類鑒定結(jié)果顯示,7B11和7H11均屬于IgG1,8H4和8G2屬于IgG2a,雜交瘤細(xì)胞的染色體數(shù)目均在98-108條之間,辛酸-飽和硫酸銨法純化的單抗只有抗體的重鏈和輕鏈兩條鏈,大小分別為50ku和25ku,相對(duì)親和力為8G28H47H117B11。 用純化的單抗與乳膠致敏,初步建立了快速檢測A型FMDV抗原的方法,對(duì)致敏抗體濃度、致敏時(shí)間和溫度等條件進(jìn)行優(yōu)化。確定包被單抗(7B11,7H11)的稀釋倍數(shù)為1:40,37℃致敏4h能達(dá)到較好的實(shí)驗(yàn)效果。特異性試驗(yàn)顯示,該方法與O型、Asia1型FMDV病毒的反應(yīng)均呈陰性。 本研究成功制備了針對(duì)A型FMDV的單克隆抗體,并對(duì)其生物學(xué)特性進(jìn)行了分析和鑒定,在此基礎(chǔ)上建立了反向乳膠凝集法檢測A型口蹄疫病毒抗原的方法,這為口蹄疫的診斷和研究提供了平臺(tái)。
[Abstract]:Foot-and-mouth disease (FMD) is an acute highly contact infectious disease of cloven-hoofed animals caused by foot-and-mouth Disease Virus, FMDV). It is listed as the first class A infectious disease by the World Organization of Animal Health (OIE), and it is given to the politics of endemic countries and regions. Economic and social problems have brought adverse effects, and monoclonal antibodies have become the focus of diagnosis and research of foot-and-mouth disease because of their strong specificity, good reproducibility and high stability. In this study, the recombinant strain pGEM-VP1 was used as the template to clone the VP1 gene and ligated to the expression vector to induce the expression of the fusion protein. Soluble analysis showed that the expressed fusion protein existed in the form of inclusion body. The purified protein was purified by nickel ion affinity chromatography after ultrasonic lysis, and a pure fusion protein was obtained by SDS-PAGE analysis. Female BALB/c mice of 6 weeks old were immunized by this method. The spleen and myeloma cell line Sp2 / 0 were harvested after enhanced immunization and then subcloned by indirect ELISA method, and then subcloned by limited dilution method. Finally, four hybridoma cell lines which can secrete monoclonal antibody stably were obtained and named as 7B117H117H118G2O8H4. The biological characteristics were analyzed and identified by SDS-PAGE, indirect ELISA blot analysis and indirect immunofluorescence assay. The results showed that the antibody titers and specificity of the four McAbs obtained were higher. The results of antibody subclass identification showed that both IgG1B11 and 8G2 belonged to IgG2a, and the chromosome number of hybridoma cells ranged from 98-108. The monoclonal antibody purified by octanoic acid-saturated ammonium sulfate method had only two chains of antibody, heavy chain and light chain, with the size of 50ku and 25ku. the relative affinity was 8G28H47H117B11. A method for rapid detection of type A FMDV antigen was established by sensitizing the purified McAb with latex. The sensitized antibody concentration, sensitizing time and temperature were optimized. It was determined that the dilution ratio of the coated McAb was 1: 4037 鈩，

本文編號(hào)：1896713