EV71型腸道病毒抗體的制備與鑒定
發(fā)布時(shí)間：2019-01-01 20:18
【摘要】：腸道病毒71型(Enterovirus type71, EV71)是小型RNA病毒科腸道病毒屬成員,其感染性強(qiáng)、致病率高,是導(dǎo)致手足口病的主要病原,同時(shí)能引起多種與神經(jīng)系統(tǒng)相關(guān)的疾病。由于腸道病毒屬成員數(shù)量多、結(jié)構(gòu)和生物學(xué)特性相似,導(dǎo)致其臨床診斷和分類(lèi)鑒定難度加大。目前針對(duì)腸道病毒71型感染多采用抗生素進(jìn)行治療,但是在當(dāng)前病毒耐藥性不斷增加的情況下,尋找一種更安全、更高效的治療特效藥及診斷方法成為一個(gè)急需解決的問(wèn)題。 本論文實(shí)驗(yàn)：(1)制備特異性抗腸道病毒71型卵黃抗體,然后通過(guò)SDS-PAGE凝膠電泳、考馬斯亮藍(lán)染色法、酶聯(lián)免疫吸附試驗(yàn)、免疫雙向瓊脂擴(kuò)散、Western blot、體外病毒中和實(shí)驗(yàn)等方法對(duì)其生物活性進(jìn)行鑒定;(2)通過(guò)建立陽(yáng)性雜交瘤細(xì)胞株及體內(nèi)誘生腹水法制備了大量抗腸道病毒71型VP1蛋白單克隆抗體,辛酸硫酸銨沉淀法純化后通過(guò)SDS-PAGE電泳和紫外分光光度法鑒定抗體純度和含量,間接ELISA法測(cè)定其中和效價(jià),鼠單抗業(yè)型分型試紙鑒定其Ig類(lèi)與業(yè)類(lèi);(3)利用偶聯(lián)劑EDC和Sulfo-NHS將腸道病毒71型VP1蛋白單克隆抗體偶聯(lián)到聚丙烯酸修飾的水溶性核量子點(diǎn)上,并利用酶聯(lián)免疫法、熒光發(fā)射光譜、高性能生物質(zhì)譜等分析方法對(duì)偶聯(lián)物進(jìn)行分析。在此基礎(chǔ)上應(yīng)用卵黃抗體為捕獲抗體,量子點(diǎn)標(biāo)記的單克隆抗體作為檢測(cè)抗體,建立了量子點(diǎn)標(biāo)記免疫熒光檢測(cè)EV71病毒的標(biāo)準(zhǔn)曲線(xiàn)。 實(shí)驗(yàn)結(jié)果：(1)考馬斯亮藍(lán)染色法測(cè)得卵黃中IgY得率達(dá)7.76mg/mL,SDS-PAGE凝膠電泳測(cè)得IgY抗體的純度為94.86%。初免10d后蛋黃中可檢測(cè)到特異性抗EV71IgY抗體,初免40天后ELISA檢測(cè)抗體效價(jià)達(dá)最高1：20480,雙向瓊脂擴(kuò)散檢測(cè)效價(jià)達(dá)1:16。Western blot分析提取的IgY抗體具有良好的免疫反應(yīng)性,能與EV71特異性結(jié)合；同時(shí)制備的特異性抗EV71IgY具有良好的體外中和活性,最低中和溶度為7.9ug/mL,中和效價(jià)為1：128。(2)初步篩選到2株抗腸道病毒71型VPl蛋白的細(xì)胞株,效價(jià)均達(dá)到了106以上；腹水純化后經(jīng)還原性SDS-PAGE電泳可見(jiàn)兩條清晰的IgG重輕鏈,紫外分光光度法測(cè)純化前后蛋白濃度分別為為39.28mg/mL和5.6mg/mL。經(jīng)鼠單抗業(yè)型分型試紙鑒定獲得的單抗類(lèi)型為IgG2b類(lèi),輕鏈為K鏈。(3)由酶聯(lián)免疫法及紫外透射反射觀察結(jié)果可知QDS-MAb同時(shí)擁有MAb的生物活性及QDS的化學(xué)發(fā)光特性。熒光光譜圖中QDS-MAb熒光最大發(fā)射波長(zhǎng)紅移4nm及質(zhì)譜圖中QDS-MAb的分子離子峰比MAb分子離子峰增大了2973這些結(jié)果都證實(shí)抗腸道病毒71型VP1單克隆抗體蛋白成功偶聯(lián)到水溶性量子點(diǎn)上,且結(jié)構(gòu)未受破壞。通過(guò)棋盤(pán)滴定法確定了量子點(diǎn)熒光免疫檢測(cè)EV71法中卵黃抗體最佳包被溶度為4ng/mL,病毒檢測(cè)標(biāo)準(zhǔn)曲線(xiàn)方程為y=301.66x+3307,R2=0.9911,檢測(cè)范圍為1.953×10-3ug/mL至0.03125ug/mL
[Abstract]:Enterovirus 71 (Enterovirus type71, EV71) is a member of the genus of enterovirus in the family RNA, which is highly infectious and highly pathogenic. It is the main cause of hand, foot and mouth disease and can cause many diseases related to the nervous system at the same time. Because of the large number of enterovirus members and the similar structure and biological characteristics, the clinical diagnosis and classification of enterovirus become more difficult. At present, antibiotics are often used to treat enterovirus 71 infection. However, with the increasing drug resistance, it is urgent to find a more safe and efficient method for the treatment and diagnosis of enterovirus 71 infection. In this paper: (1) preparation of specific anti-enterovirus type 71 egg yolk antibody, then through SDS-PAGE gel electrophoresis, Coomassie brilliant blue staining, enzyme-linked immunosorbent assay, immunized two-way Agar diffusion, Western blot, Its biological activity was identified by virus neutralization test in vitro. (2) A large number of monoclonal antibodies against enterovirus 71 VP1 protein were prepared by establishing a positive hybridoma cell line and inducing ascites in vivo. The purity and content of antibody were identified by SDS-PAGE electrophoresis and UV spectrophotometry after purification by ammonium octanoate precipitation method. The titer of the antibody was determined by indirect ELISA method. The Ig class and industry class were identified by mouse monoclonal antibody typing test paper. (3) the monoclonal antibody against enterovirus 71 VP1 protein was conjugated to the water-soluble nuclear quantum dot modified by polyacrylic acid by coupling agents EDC and Sulfo-NHS, and the fluorescence emission spectrum was determined by enzyme-linked immunosorbent assay (Elisa). The coupling compounds were analyzed by high performance biological mass spectrometry. On this basis, using yolk antibody as capture antibody and quantum dot labeled monoclonal antibody as detection antibody, the standard curve of EV71 virus detection using quantum dot labeled immunofluorescence was established. The results were as follows: (1) the yield of IgY in yolk by Coomassie brilliant blue staining was 7.76 mg / mL SDS-PAGE and the purity of IgY antibody was 94.86. Specific anti EV71IgY antibody could be detected in egg yolk 10 days after the first immunization, and the titer of ELISA antibody reached 1: 20480 after 40 days, and the titer of IgY antibody extracted by 1:16.Western blot analysis by bidirectional Agar diffusion assay had good immunoreactivity. Can specifically bind to EV71; At the same time, the specific anti- EV71IgY prepared had good neutralization activity in vitro, the lowest neutralization solubility was 7.9 ugmL, and the neutralization titer was 1: 128. (2) two cell lines against enterovirus 71 VPl protein were preliminarily screened. The titers were above 106; After purification of ascites, two clear IgG heavy and light chains were found by reductive SDS-PAGE electrophoresis. The protein concentrations were 39.28mg/mL and 5.6 mg / mL before and after purification by UV spectrophotometry. The type of monoclonal antibody was identified as IgG2b and light chain was K chain. (3) the results of enzyme-linked immunosorbent assay and ultraviolet transmission reflectance showed that QDS-MAb possessed the bioactivity of MAb and the chemiluminescence characteristics of QDS at the same time. The maximum emission wavelength of QDS-MAb red shift 4nm in fluorescence spectrum and the molecular ion peak of QDS-MAb in mass spectrogram are 2973 larger than that of MAb. These results confirm that the monoclonal antibody protein against enterovirus 71 VP1 has been successfully coupled. Connected to water-soluble quantum dots, And the structure is not damaged. By means of chessboard titration, the best coating solubility of egg yolk antibody in EV71 was determined to be 4ng / mL, the standard curve equation of virus detection was yang301.66x 3307R2O0.9911, and the detection range was from 1.953 脳 10-3ug/mL to 0.03125ug/mL.
【學(xué)位授予單位】：廣東工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R392
本文編號(hào)：2398065
[Abstract]:Enterovirus 71 (Enterovirus type71, EV71) is a member of the genus of enterovirus in the family RNA, which is highly infectious and highly pathogenic. It is the main cause of hand, foot and mouth disease and can cause many diseases related to the nervous system at the same time. Because of the large number of enterovirus members and the similar structure and biological characteristics, the clinical diagnosis and classification of enterovirus become more difficult. At present, antibiotics are often used to treat enterovirus 71 infection. However, with the increasing drug resistance, it is urgent to find a more safe and efficient method for the treatment and diagnosis of enterovirus 71 infection. In this paper: (1) preparation of specific anti-enterovirus type 71 egg yolk antibody, then through SDS-PAGE gel electrophoresis, Coomassie brilliant blue staining, enzyme-linked immunosorbent assay, immunized two-way Agar diffusion, Western blot, Its biological activity was identified by virus neutralization test in vitro. (2) A large number of monoclonal antibodies against enterovirus 71 VP1 protein were prepared by establishing a positive hybridoma cell line and inducing ascites in vivo. The purity and content of antibody were identified by SDS-PAGE electrophoresis and UV spectrophotometry after purification by ammonium octanoate precipitation method. The titer of the antibody was determined by indirect ELISA method. The Ig class and industry class were identified by mouse monoclonal antibody typing test paper. (3) the monoclonal antibody against enterovirus 71 VP1 protein was conjugated to the water-soluble nuclear quantum dot modified by polyacrylic acid by coupling agents EDC and Sulfo-NHS, and the fluorescence emission spectrum was determined by enzyme-linked immunosorbent assay (Elisa). The coupling compounds were analyzed by high performance biological mass spectrometry. On this basis, using yolk antibody as capture antibody and quantum dot labeled monoclonal antibody as detection antibody, the standard curve of EV71 virus detection using quantum dot labeled immunofluorescence was established. The results were as follows: (1) the yield of IgY in yolk by Coomassie brilliant blue staining was 7.76 mg / mL SDS-PAGE and the purity of IgY antibody was 94.86. Specific anti EV71IgY antibody could be detected in egg yolk 10 days after the first immunization, and the titer of ELISA antibody reached 1: 20480 after 40 days, and the titer of IgY antibody extracted by 1:16.Western blot analysis by bidirectional Agar diffusion assay had good immunoreactivity. Can specifically bind to EV71; At the same time, the specific anti- EV71IgY prepared had good neutralization activity in vitro, the lowest neutralization solubility was 7.9 ugmL, and the neutralization titer was 1: 128. (2) two cell lines against enterovirus 71 VPl protein were preliminarily screened. The titers were above 106; After purification of ascites, two clear IgG heavy and light chains were found by reductive SDS-PAGE electrophoresis. The protein concentrations were 39.28mg/mL and 5.6 mg / mL before and after purification by UV spectrophotometry. The type of monoclonal antibody was identified as IgG2b and light chain was K chain. (3) the results of enzyme-linked immunosorbent assay and ultraviolet transmission reflectance showed that QDS-MAb possessed the bioactivity of MAb and the chemiluminescence characteristics of QDS at the same time. The maximum emission wavelength of QDS-MAb red shift 4nm in fluorescence spectrum and the molecular ion peak of QDS-MAb in mass spectrogram are 2973 larger than that of MAb. These results confirm that the monoclonal antibody protein against enterovirus 71 VP1 has been successfully coupled. Connected to water-soluble quantum dots, And the structure is not damaged. By means of chessboard titration, the best coating solubility of egg yolk antibody in EV71 was determined to be 4ng / mL, the standard curve equation of virus detection was yang301.66x 3307R2O0.9911, and the detection range was from 1.953 脳 10-3ug/mL to 0.03125ug/mL.
【學(xué)位授予單位】：廣東工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 崔愛(ài)利,許文波,李秀珠,胡家瑜,凌華,唐偉,楊智宏,張燕,陳立,Hiroyuki Shimizu;腸道病毒71型的RT-PCR診斷及基因特征[J];病毒學(xué)報(bào);2004年02期
2 楊帆,金奇,何雅青,李良成,侯云德;腸道病毒71型中國(guó)分離株全基因組核苷酸序列分析[J];中國(guó)科學(xué)(C輯:生命科學(xué));2001年02期
3 張紹蘭;;雞卵黃抗體研究進(jìn)展[J];西南軍醫(yī);2008年03期
4 寧明哲,童明慶;稀土元素銪標(biāo)記技術(shù)的應(yīng)用研究[J];臨床檢驗(yàn)雜志;2004年04期
5 井慶川,魏祥法,史勇;卵黃抗體制備及在畜牧業(yè)中的應(yīng)用[J];中國(guó)禽業(yè)導(dǎo)刊;2004年16期
6 譚翠燕,梁汝強(qiáng),阮康成;量子點(diǎn)在生命科學(xué)中的應(yīng)用[J];生物化學(xué)與生物物理學(xué)報(bào);2002年01期
7 鐘青萍,楊榮鑒,熊勇華,楊寧生;雞卵黃免疫球蛋白研究動(dòng)態(tài)[J];生命科學(xué);1998年05期
8 黃華偉;杜美菊;;免疫熒光分析的研究進(jìn)展[J];應(yīng)用化工;2007年04期
9 黃莉,趙德明;卵黃抗體的研究進(jìn)展[J];實(shí)驗(yàn)動(dòng)物科學(xué)與管理;2003年03期
10 馬貴軍;韓素娟;剡根強(qiáng);周霞;;應(yīng)用辛酸硫酸銨法提取純化兔IgG[J];上海畜牧獸醫(yī)通訊;2007年04期
相關(guān)碩士學(xué)位論文 前4條
1 張彩霞;雪卡毒素單克隆抗體的制備及鑒定[D];廣東工業(yè)大學(xué);2011年
2 趙玉龍;牛病毒性腹瀉病毒（BVDV）卵黃抗體制備及其對(duì)病毒中和活性研究[D];石河子大學(xué);2009年
3 林柏玲;黃曲霉毒素B_1快速檢測(cè)試劑盒的研制[D];天津商業(yè)大學(xué);2007年
4 吳揚(yáng);草丁膦單克隆抗體的制備[D];華中師范大學(xué);2007年
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