EV71型腸道病毒抗體的制備與鑒定
發(fā)布時間：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、致病率高,是導致手足口病的主要病原,同時能引起多種與神經(jīng)系統(tǒng)相關的疾病。由于腸道病毒屬成員數(shù)量多、結(jié)構(gòu)和生物學特性相似,導致其臨床診斷和分類鑒定難度加大。目前針對腸道病毒71型感染多采用抗生素進行治療,但是在當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急需解決的問題。 本論文實驗：(1)制備特異性抗腸道病毒71型卵黃抗體,然后通過SDS-PAGE凝膠電泳、考馬斯亮藍染色法、酶聯(lián)免疫吸附試驗、免疫雙向瓊脂擴散、Western blot、體外病毒中和實驗等方法對其生物活性進行鑒定;(2)通過建立陽性雜交瘤細胞株及體內(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法測定其中和效價,鼠單抗業(yè)型分型試紙鑒定其Ig類與業(yè)類;(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)譜等分析方法對偶聯(lián)物進行分析。在此基礎上應用卵黃抗體為捕獲抗體,量子點標記的單克隆抗體作為檢測抗體,建立了量子點標記免疫熒光檢測EV71病毒的標準曲線。 實驗結(jié)果：(1)考馬斯亮藍染色法測得卵黃中IgY得率達7.76mg/mL,SDS-PAGE凝膠電泳測得IgY抗體的純度為94.86%。初免10d后蛋黃中可檢測到特異性抗EV71IgY抗體,初免40天后ELISA檢測抗體效價達最高1：20480,雙向瓊脂擴散檢測效價達1:16。Western blot分析提取的IgY抗體具有良好的免疫反應性,能與EV71特異性結(jié)合；同時制備的特異性抗EV71IgY具有良好的體外中和活性,最低中和溶度為7.9ug/mL,中和效價為1：128。(2)初步篩選到2株抗腸道病毒71型VPl蛋白的細胞株,效價均達到了106以上；腹水純化后經(jīng)還原性SDS-PAGE電泳可見兩條清晰的IgG重輕鏈,紫外分光光度法測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型為IgG2b類,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同時擁有MAb的生物活性及QDS的化學發(fā)光特性。熒光光譜圖中QDS-MAb熒光最大發(fā)射波長紅移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)到水溶性量子點上,且結(jié)構(gòu)未受破壞。通過棋盤滴定法確定了量子點熒光免疫檢測EV71法中卵黃抗體最佳包被溶度為4ng/mL,病毒檢測標準曲線方程為y=301.66x+3307,R2=0.9911,檢測范圍為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.
【學位授予單位】：廣東工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392
本文編號：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.
【學位授予單位】：廣東工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392
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