本文選題：黃曲霉毒素B1　切入點：單克隆抗體　出處：《福建農(nóng)林大學(xué)》2013年碩士論文

【摘要】：黃曲霉毒素（Aflatoxin，AFT）主要是由黃曲霉（Aspergillus flavus）和寄生曲霉（Aspergillus parasiticus）產(chǎn)生的毒性極強(qiáng)的次生代謝產(chǎn)物，黃曲霉毒素是一類結(jié)構(gòu)類似的化合物的總稱。在黃曲霉毒素家族中，常見的有黃曲霉毒素B1、B2、G1、G2、M1和M2等。早在1988年黃曲霉毒素B1被國際腫瘤研究機(jī)構(gòu)（IARC）定為一級致癌物。傳統(tǒng)的檢測方法不但耗時耗力，成本偏高，而且還需要專業(yè)技術(shù)人員對分析儀器進(jìn)行操作。采用免疫化學(xué)分析法進(jìn)行檢測則可以克服以上缺點。本文旨在篩選出能分泌抗AFB1的高親和力的單克隆抗體細(xì)胞株，為建立免疫化學(xué)檢測方法打下基礎(chǔ)。 首先用碳二亞胺法（EDC）構(gòu)建了兩種完全抗原AFB1-BSA和AFB1-OVA，其中AFB1-BSA作為免疫原免疫兩只8周齡BALB/C雌性小鼠四次，AFB1-OVA作為包被原包被酶標(biāo)板，iELISA法測定免疫小鼠血清效價。加強(qiáng)免疫后，兩只小鼠的效價分別達(dá)到了128000和32000。采用PEG（4000）法將SP2/0骨髓瘤細(xì)胞和免疫小鼠脾臟細(xì)胞進(jìn)行融合，HAT篩選培養(yǎng)基篩選培養(yǎng)，經(jīng)過3~4輪篩選得到三株能穩(wěn)定分泌抗AFB1單克隆抗體的雜交瘤細(xì)胞株：1B7、5A8、5B8。其中，1B7和5A8細(xì)胞株分泌的單克隆抗體為IgM亞型，5B8細(xì)胞株分泌的抗體為IgG1亞型。為便于后期純化，本次實驗采用5B8細(xì)胞株進(jìn)行后續(xù)實驗。經(jīng)測定，5B8細(xì)胞株分泌的單克隆抗體效價達(dá)到20,0000，親和力為7.96×108。將該細(xì)胞株進(jìn)行擴(kuò)大培養(yǎng)并注射到13周齡雌性Balb/c小鼠腹腔中，一周后取腹水，采用辛酸-硫酸銨法純化腹水，經(jīng)SDS-PAGE檢測可以看到明顯的重鏈和輕鏈條帶，，證明單克隆抗體純化成功。
[Abstract]:Aflatoxin (AFT) is a highly toxic secondary metabolite produced by Aspergillus flavusand Aspergillus parasiticus. Aflatoxin is a class of compounds with similar structure.Among the aflatoxin families, aflatoxin B _ (1) B _ (2) G _ (1) G _ (2) G _ (2) M _ 1 and M _ 2 are common.As early as 1988, aflatoxin B1 was designated as a carcinogen by the International Cancer Research Institute (IARC).The traditional detection method is not only time-consuming and expensive, but also needs professional technicians to operate the analytical instrument.The above shortcomings can be overcome by immunochemical analysis.The aim of this study was to screen monoclonal antibody cell lines which secreted high affinity to AFB1, which laid the foundation for the establishment of immunocytochemical assay.Two complete antigens (AFB1-BSA and AFB1-OVA) were constructed by carbodiimide method. Two 8-week-old BALB/C female mice were immunized with AFB1-BSA for four times.The titers of the two mice reached 128000 and 32000 respectively after immunization.The SP2/0 myeloma cells and the spleen cells of immunized mice were screened and cultured by the method of PEGN 4000. after 3 rounds of screening, three hybridoma cell lines were obtained which could stably secrete the monoclonal antibody against AFB1, namely: 1B7A8A8A8B8B8.The monoclonal antibody secreted by 5A8 cell line was IgG1 subtype of IgM subtype 5B8 cell line.In order to facilitate the later purification, 5B8 cell line was used to carry out the follow-up experiment.The titer of monoclonal antibody secreted by the cell line was 20: 00000.The affinity was 7.96 脳 10 ~ (8).The cell line was cultured and injected into the abdominal cavity of 13 week-old female Balb/c mice. Ascites were extracted one week later, and ascites were purified by caprylic acid-ammonium sulfate method. The heavy chain and light chain band could be seen by SDS-PAGE detection.It was proved that the monoclonal antibody was purified successfully.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 馬健;林清;;黃曲霉毒素及其致病機(jī)理簡述[J];中國牛業(yè)科學(xué);2012年01期

2 蘇建家,李瑗,班克臣,覃柳亮,王輝云,楊春,歐超,嚴(yán)瑞琪;動態(tài)研究HBV和AFB_1誘發(fā)樹鼠句肝細(xì)胞癌過程中一些基因的表達(dá)[J];廣西醫(yī)科大學(xué)學(xué)報;2001年02期

3 涂文升;翟紅艷;黃天壬;;廣西肝癌高發(fā)區(qū)壯族人群血清中游離黃曲霉毒素B_1含量的HPLC測定[J];廣西醫(yī)科大學(xué)學(xué)報;2010年06期

4 莊振宏;張峰;李燕云;袁軍;楊燕凌;林玲;汪世華;;黃曲霉毒素致癌機(jī)理的研究進(jìn)展[J];湖北農(nóng)業(yè)科學(xué);2011年08期

5 白璐;;黃曲霉毒素的危害[J];吉林農(nóng)業(yè);2012年01期

6 陳福生,羅信昌,周啟,李根久;黃曲霉毒素B_1的免疫檢測Ⅰ.抗原的制備[J];菌物系統(tǒng);1999年03期

7 李佐卿,謝東華,孫大為,康繼韜,俞雪鈞;免疫親和柱HPLC快速測定蜂蜜中黃曲霉毒素B_1、B_2、G_1、G_2[J];寧波高等�？茖W(xué)校學(xué)報;2001年S1期

8 周思祥,劉福成;農(nóng)藥人工抗原的合成研究進(jìn)展[J];農(nóng)藥;2005年08期

9 柳其芳;;酶聯(lián)免疫吸附法和薄層色譜法聯(lián)合分析黃曲霉毒素B_1的研究[J];中國熱帶醫(yī)學(xué);2006年02期

10 莊振宏;李蘭;孫憲連;張新艷;單超;汪世華;王宗華;;抗黃曲霉毒素B1單鏈抗體(scFv)庫的建立和篩選[J];熱帶作物學(xué)報;2009年01期

相關(guān)博士學(xué)位論文 前1條

1 孫秀蘭;食品中黃曲霉毒素B_1金標(biāo)免疫層析檢測方法研究[D];江南大學(xué);2005年

相關(guān)碩士學(xué)位論文 前2條

1 王磊;黃曲霉毒素B_1免疫學(xué)快速檢測技術(shù)研究[D];河南科技大學(xué);2011年

2 蔣廣明;黃曲霉毒素B_1單抗的制備及初步應(yīng)用[D];揚(yáng)州大學(xué);2007年

本文編號：1716897