本文關(guān)鍵詞：抗巖沙�？舅貑慰寺】贵w的制備以及鑒定　出處：《廣州醫(yī)學(xué)院》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 巖沙海葵毒素 單克隆抗體 間接ELISA SDS-PAGE

【摘要】： 目的:巖沙�？舅�(Palytoxin, PTX)是目前已知毒性最強烈的海洋生物毒素之一,具有作用于神經(jīng)、肌肉的電壓門控鈉通道,改變通道的功能,以及對細胞毒性、溶血等多方面的影響。人類在進食經(jīng)PTX污染的食物后,可導(dǎo)致嚴重的肌肉疼痛、腰痛、排黑尿等癥狀,甚至死亡。而目前在我國乃至全球仍未研究出一種針對該毒素的治療藥物,因此PTX的存在對人類的健康安全構(gòu)成相當大的威脅。 本課題通過化學(xué)方法制備出PTX的人工抗原,將其與弗氏佐劑混合后對BALB/c小鼠進行免疫,產(chǎn)生抗PTX抗血清,并以較為成熟的單克隆抗體雜交瘤技術(shù),建立分泌巖沙�？舅氐母咛禺愋詥慰寺】贵w雜交瘤細胞株。該細胞株的建立為日后在實際工作中對PTX的檢測提供有利的工具。 方法:本課題分三部分進行: 1.抗PTX的抗血清的制備 將PTX與載體蛋白KLH、BSA偶聯(lián)成具有免疫功能的PTX-PDP-KLH免疫抗原和PTX-PDP-BSA檢測抗原。用PTX-PDP-KLH免疫雌性6~8周齡BALB/c小鼠,采用眼眶后靜脈叢取血獲得抗PTX抗血清,對其效價進行間接ELISA法檢測,以健康BALB/c小鼠血清為對照組。 2.分泌抗巖沙�？舅豈cAb的雜交瘤制備 將能分泌出抗PTX抗血清的免疫小鼠脾臟細胞通過傳統(tǒng)雜交瘤技術(shù)與NS-1骨髓瘤細胞進行細胞融合,并通過間接ELISA方法篩選克隆陽性雜交瘤細胞株,陽性細胞對其擴大培養(yǎng)并凍存。 3.抗巖沙�？舅貑慰寺】贵w的檢測及生產(chǎn) 將成功分泌抗PTX單克隆抗體的融合細胞進行核型分析、特異性分析后,注射進健康BALB/c小鼠體內(nèi),14天左右產(chǎn)生腹水。腹水經(jīng)硫酸銨沉淀、透析、rProteinA親和層析等方法純化后得到腹水型抗體。 結(jié)果: 1.利用連續(xù)紫外分光光度計檢測出在267 nm時偶聯(lián)物PTX-PDP-KLH出現(xiàn)峰值,而PTX-PDP-BSA在273 nm時出現(xiàn)峰值。將PTX-PDP-KLH對BALB/c小鼠進行皮下、腹部多點免疫,以PTX-PDP-BSA作為檢測抗原進行包被,對其抗血清進行ELISA檢測,所得結(jié)果發(fā)現(xiàn)注射了免疫抗原的小鼠抗血清與對照組有明顯差別(1: 2500時OD7)。 2.雜交瘤細胞融合率為21.43 % ,其陽性克隆率為9.1 % ,經(jīng)過間接ELISA方法反復(fù)篩選,獲得能穩(wěn)定分泌抗PTX單克隆抗體的雜交瘤細胞株A6和B8。 3.核型分析發(fā)現(xiàn),抗PTX單克隆抗體雜交瘤細胞的染色體數(shù)目在94~104之間,特異性檢測中PTX與載體蛋白KLH、BSA沒有發(fā)生交叉反應(yīng),顯示該抗體為抗PTX的特異性抗體。細胞免疫小鼠后產(chǎn)生4~5 ml的腹水,經(jīng)過分離純化后,SDS-PAGE分析表明,抗PTX單克隆抗體細胞株在50 kD和25 kD處各出現(xiàn)一條蛋白條帶,與IgG的重鏈和輕鏈的大小相符。 結(jié)論:1.本研究通過化學(xué)方法成功將PTX和載體蛋白KLH、BSA偶聯(lián),克服了PTX因分子量小而無法刺激機體產(chǎn)生抗體的因素,偶聯(lián)后的PTX-PDP-KLH具有免疫原性,從而誘導(dǎo)小鼠產(chǎn)生抗PTX抗血清。 2.通過雜交瘤技術(shù)獲得A6和B8兩株能穩(wěn)定分泌抗PTX的單克隆抗體的雜交瘤細胞株,該細胞株分泌的單克隆抗體可與PTX特異性結(jié)合,為PTX的鑒定和檢測提供堅實的基礎(chǔ)。
[Abstract]:Objective: palytoxin (Palytoxin, PTX) is one of the most toxic marine biological toxins known strong at present, has the effect on nerve, muscle voltage-gated sodium channel, change the channel function, and the cytotoxicity, hemolysis and other aspects of human. In eating food contaminated with PTX, can lead to severe muscle pain, back pain, black urine and other symptoms, and even death. At present in our country and the world has not yet developed a drug in the treatment of the toxin, so PTX poses a considerable threat to human health and safety.
This topic through chemical methods for preparation of artificial antigen PTX, which mixed with Freund's adjuvant in BALB/c mice were immunized with anti PTX antiserum, and mature monoclonal antibody hybridoma technology, establish palytoxin highly specific monoclonal antibody hybridoma cell lines. The establishment of the cell line the day after the tool for the detection of PTX in practical work is beneficial.
Methods: the subject was divided into three parts:
Preparation of antiserum of 1. anti PTX
The PTX and the carrier protein KLH, BSA antigen and PTX-PDP-BSA coupling to PTX-PDP-KLH antigen detection with immune function. PTX-PDP-KLH was used to immunize female 6~8 BALB/c mice with orbital venous plexus blood obtained anti PTX antiserum was detected by indirect ELISA on its titer, healthy BALB/c mice serum as the control group.
Preparation of hybridoma with 2. secreted anemone McAb
Mice spleen cells secreting anti PTX antisera were fused with NS-1 myeloma cells by traditional hybridoma technology, and then screened positive hybridoma cell lines by indirect ELISA method. The positive cells were expanded and cryopreserved by positive cells.
Detection and production of monoclonal antibodies against 3. anti sand sea anemone toxin
The fusion cells which successfully secreted anti PTX monoclonal antibodies were analyzed by karyotype. After specific analysis, they were injected into healthy BALB/c mice, and ascites was produced for about 14 days. Ascites was purified by ammonium sulfate precipitation, dialysis and rProteinA affinity chromatography, and then ascites type antibody was obtained.
Results: 1. using the continuous ultraviolet spectrophotometer detected at 267 nm conjugate PTX-PDP-KLH peak, and PTX-PDP-BSA peak at 273 nm. The PTX-PDP-KLH of BALB/c mice abdominal subcutaneous, immunization, PTX-PDP-BSA as detection antigen was detected by ELISA, the results showed that the antiserum. Injection of immune antigen in mice antiserum and the control group had significant difference (1: 2500 OD7).
2., the fusion rate of hybridoma cells was 21.43%, and the positive clone rate was 9.1%. After repeated screening by indirect ELISA, hybridoma cell lines A6 and B8. secreting anti PTX monoclonal antibodies could be obtained steadily.
3. karyotype analysis showed that chromosome number of anti PTX monoclonal antibody hybridoma cells between 94~104, PTX and carrier protein KLH specific BSA detection, no cross reaction, showed that the antibody specificity of anti PTX antibody. Cells in mice immunized with 4~5 ml ascites after purified, SDS-PAGE analysis showed that that anti PTX monoclonal antibodies in 50 kD and 25 kD to the emergence of a protein band, consistent with the heavy chain and light chain of IgG size.
Conclusion: 1.. In this study, PTX was successfully coupled with the carrier protein KLH and BSA by chemical methods. It overcomes the fact that PTX can not stimulate the body to produce antibodies because of its small molecular weight. The PTX-PDP-KLH after coupling has immunogenicity, and induces the production of anti PTX antiserum in mice.
2. through hybridoma technology, we obtained two hybridoma cell lines, A6 and B8, which can secrete monoclonal antibodies against PTX. The monoclonal antibodies secreted by this cell line can be specifically combined with PTX, providing a solid foundation for identification and detection of PTX.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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