抗岩沙海葵毒素单克隆抗体的制备以及鉴定

发布时间：2018-01-04 18:01

本文关键词：抗岩沙海葵毒素单克隆抗体的制备以及鉴定　出处：《广州医学院》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:岩沙海葵毒素(Palytoxin, PTX)是目前已知毒性最强烈的海洋生物毒素之一,具有作用于神经、肌肉的电压门控钠通道,改变通道的功能,以及对细胞毒性、溶血等多方面的影响。人类在进食经PTX污染的食物后,可导致严重的肌肉疼痛、腰痛、排黑尿等症状,甚至死亡。而目前在我国乃至全球仍未研究出一种针对该毒素的治疗药物,因此PTX的存在对人类的健康安全构成相当大的威胁。本课题通过化学方法制备出PTX的人工抗原,将其与弗氏佐剂混合后对BALB/c小鼠进行免疫,产生抗PTX抗血清,并以较为成熟的单克隆抗体杂交瘤技术,建立分泌岩沙海葵毒素的高特异性单克隆抗体杂交瘤细胞株。该细胞株的建立为日后在实际工作中对PTX的检测提供有利的工具。方法:本课题分三部分进行: 1.抗PTX的抗血清的制备将PTX与载体蛋白KLH、BSA偶联成具有免疫功能的PTX-PDP-KLH免疫抗原和PTX-PDP-BSA检测抗原。用PTX-PDP-KLH免疫雌性6~8周龄BALB/c小鼠,采用眼眶后静脉丛取血获得抗PTX抗血清,对其效价进行间接ELISA法检测,以健康BALB/c小鼠血清为对照组。 2.分泌抗岩沙海葵毒素McAb的杂交瘤制备将能分泌出抗PTX抗血清的免疫小鼠脾脏细胞通过传统杂交瘤技术与NS-1骨髓瘤细胞进行细胞融合,并通过间接ELISA方法筛选克隆阳性杂交瘤细胞株,阳性细胞对其扩大培养并冻存。 3.抗岩沙海葵毒素单克隆抗体的检测及生产将成功分泌抗PTX单克隆抗体的融合细胞进行核型分析、特异性分析后,注射进健康BALB/c小鼠体内,14天左右产生腹水。腹水经硫酸铵沉淀、透析、rProteinA亲和层析等方法纯化后得到腹水型抗体。结果: 1.利用连续紫外分光光度计检测出在267 nm时偶联物PTX-PDP-KLH出现峰值,而PTX-PDP-BSA在273 nm时出现峰值。将PTX-PDP-KLH对BALB/c小鼠进行皮下、腹部多点免疫,以PTX-PDP-BSA作为检测抗原进行包被,对其抗血清进行ELISA检测,所得结果发现注射了免疫抗原的小鼠抗血清与对照组有明显差别(1: 2500时OD7)。 2.杂交瘤细胞融合率为21.43 % ,其阳性克隆率为9.1 % ,经过间接ELISA方法反复筛选,获得能稳定分泌抗PTX单克隆抗体的杂交瘤细胞株A6和B8。 3.核型分析发现,抗PTX单克隆抗体杂交瘤细胞的染色体数目在94~104之间,特异性检测中PTX与载体蛋白KLH、BSA没有发生交叉反应,显示该抗体为抗PTX的特异性抗体。细胞免疫小鼠后产生4~5 ml的腹水,经过分离纯化后,SDS-PAGE分析表明,抗PTX单克隆抗体细胞株在50 kD和25 kD处各出现一条蛋白条带,与IgG的重链和轻链的大小相符。结论:1.本研究通过化学方法成功将PTX和载体蛋白KLH、BSA偶联,克服了PTX因分子量小而无法刺激机体产生抗体的因素,偶联后的PTX-PDP-KLH具有免疫原性,从而诱导小鼠产生抗PTX抗血清。 2.通过杂交瘤技术获得A6和B8两株能稳定分泌抗PTX的单克隆抗体的杂交瘤细胞株,该细胞株分泌的单克隆抗体可与PTX特异性结合,为PTX的鉴定和检测提供坚实的基础。
[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.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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