本文關(guān)鍵詞：布魯氏菌多克隆抗體和單克隆抗體的制備與鑒定　出處：《河北農(nóng)業(yè)大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

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【摘要】： 布魯氏菌是一種革蘭氏陰性、兼性細(xì)胞內(nèi)寄生菌,它可以感染人和多種動(dòng)物,導(dǎo)致人和動(dòng)物的布魯氏菌病。對(duì)于反芻動(dòng)物,通常引起流產(chǎn)及不孕不育。人患布魯氏菌病通常是由于接觸患病動(dòng)物所引起,人與人之間不能傳播。過去,布魯氏菌病曾一度在全世界范圍內(nèi)流行,造成嚴(yán)重的經(jīng)濟(jì)損失�，F(xiàn)在布魯氏菌病在歐、美等一些發(fā)達(dá)國(guó)家,已得到了有效的控制,但在廣大發(fā)展中國(guó)家仍普遍流行。在我國(guó),布魯氏菌病曾得到過有效的控制,但九十年代后,布魯氏菌病有回升的趨勢(shì),特別是近幾年來,人和動(dòng)物布魯氏菌病的患病數(shù)迅速升高,這也使得對(duì)于布魯氏菌的研究和布魯氏菌病的診斷顯得尤為重要。 用牛種布魯氏菌A19疫苗皮下多點(diǎn)注射免疫兩只新西蘭兔,兩周一次。在第二次免疫兩周后用試管凝集試驗(yàn)測(cè)定其血清效價(jià)為1:1600,達(dá)到所需標(biāo)準(zhǔn),然后兔心臟采血并分離血清,用硫酸銨沉淀法粗提IgG,再用Sephadex-G25層析柱脫鹽,Bradford法測(cè)定蛋白含量,最后SDS-PAGE電泳試驗(yàn)鑒定。結(jié)果顯示成功得到了初步純化的兔抗牛布魯氏菌A19多克隆IgG。 采用雜交瘤細(xì)胞技術(shù),以布魯氏菌全菌為抗原,以不同劑量和不同免疫方法免疫8周齡BALB/c小鼠,每次免疫間隔時(shí)間為3周,在融合前3d進(jìn)行最后一次加強(qiáng)免疫。取小鼠脾細(xì)胞和SP2/0細(xì)胞經(jīng)PEG-4000按10:1比例進(jìn)行融合。采用布魯氏菌細(xì)胞壁抗原作為篩選抗原,用自己建立的間接酶聯(lián)免疫吸附法(Enzyme-Linked Immunosorbent Assay, ELISA)檢測(cè)雜交瘤細(xì)胞培養(yǎng)液上清,篩選出的強(qiáng)陽性孔雜交瘤細(xì)胞用有限稀釋法克隆培養(yǎng),直到陽性率達(dá)到100%.經(jīng)過3～4次亞克隆后,得到了2株能穩(wěn)定分泌抗布魯氏菌抗體的雜交瘤細(xì)胞株,其產(chǎn)生的單抗分別命名為3E7和1C6。采用ELISA方法檢測(cè)細(xì)胞上清和腹水的效價(jià),其中3E7細(xì)胞上清和腹水的效價(jià)分別為1:1600和1:20480,而1C6分別為1:800和1:10240。通過疊加試驗(yàn)對(duì)這兩種單抗進(jìn)行抗原位點(diǎn)分析,結(jié)果表明2株單抗分別針對(duì)不同的抗原位點(diǎn)。在單抗的穩(wěn)定性試驗(yàn)中,兩株細(xì)胞在四次克隆化培養(yǎng)的細(xì)胞上清的OD450均有上升趨勢(shì),四代腹水的抗體效價(jià)均在1:104～1:105之間,說明有較好的穩(wěn)定性。對(duì)硫酸銨純化后的3E7和1C6單抗進(jìn)行SDS-PAGE電泳實(shí)驗(yàn),結(jié)果顯示,2株單抗的重鏈分子質(zhì)量為50kD左右,輕鏈分子質(zhì)量為25kD左右。用這兩種單抗分別與布魯氏菌細(xì)胞壁抗原進(jìn)行Western-blot分析,結(jié)果顯示兩株單抗都能夠和布魯氏菌細(xì)胞壁抗原結(jié)合,且抗原抗體反應(yīng)條帶分別出現(xiàn)在分子質(zhì)量為31kD和55kD的位置,提示這兩種單抗可能是由分子質(zhì)量為31kD和55kD的抗原產(chǎn)生。
[Abstract]:brucella is a gram - negative , and is also a parasitic fungus in a sex cell . It can infect humans and various animals . It can infect humans and animals . It usually causes abortion and infertility . In the past , the disease of brucella has been effectively controlled due to contact with diseased animals . In China , the disease of brucella has been effectively controlled . In China , the disease of brucella has recovered rapidly , especially in recent years . Two New Zealand rabbits were immunized subcutaneously with an A19 vaccine . Two weeks after the second immunization , the serum titer was 1 : 1600 , the required standard was reached , then the serum was isolated from the rabbit heart . The serum was extracted with ammonium sulfate precipitation method . The protein content was determined by using Sephadex - G25 chromatography column , and the protein content was determined by SDS - PAGE electrophoresis . The results showed that the purified rabbit anti - bovine brucella abortus A19 polyclonal IgG was successfully obtained . A hybridoma cell line with monoclonal antibody of 3E7 and 1C6 was prepared by ELISA . The results showed that both monoclonal antibodies were able to bind to the cell wall antigen of brucella . The results showed that the two monoclonal antibodies were able to bind to the cell wall antigen of brucella . The results showed that the two monoclonal antibodies were able to bind to the cell wall antigen of brucella . The results showed that the two monoclonal antibodies could be produced by the antigen of 31kD and 55kD respectively .

【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前3條

1 鐘旭;硫酸雌酮多克隆和單克隆抗體的制備及鑒定[D];湖南農(nóng)業(yè)大學(xué);2011年

2 吳兵;斑點(diǎn)叉尾洶病毒單克隆抗體及血清免疫球蛋白多克隆抗體的制備及初步應(yīng)用[D];華中農(nóng)業(yè)大學(xué);2011年

3 張慧慧;羊種布氏桿菌單克隆抗體的制備及其對(duì)臨床分離株的檢測(cè)[D];吉林大學(xué);2013年

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本文編號(hào)：1402454