弓形虫P30抗原单克隆抗体的制备、鉴定及初步应用

发布时间：2018-04-02 00:29

本文选题：弓形虫　切入点：P30抗原　出处：《南华大学》2007年硕士论文

【摘要】： 目的制备抗弓形虫P30抗原的单克隆抗体,建立弓形虫循环抗原检测法,评价抗P30抗原单克隆抗体的早期诊断价值。方法重组质粒pET28b(+)-P30经BL21(DE3)表达,使用Ni-NTA亲和层析柱纯化重组P30蛋白,经透析、复性后,定量P30蛋白。以复性的P30蛋白免疫BALB/c小鼠,分离免疫小鼠的脾细胞,在聚乙二醇的作用下与骨髓瘤细胞SP2/0融合,融合后的细胞进行HAT选择培养,10~20天后筛选阳性克隆。用重组P30包板,间接酶联免疫试验检测杂交瘤细胞培养上清,阳性克隆经亚克隆建株。降植烷预处理小鼠后,腹腔注射状态良好的杂交瘤细胞,体内诱生腹水法制备抗体,7~10天后即可收获腹水。对腹水中的McAb先用硫酸铵沉淀法进行粗提,然后用免疫亲和层析法进一步纯化。快速定性试纸法测定其亚类, ELISA法测定腹水和纯化后抗体的效价,进行杂交瘤细胞核型分析、SDS-PAGE和Western blotting分析,ELISA法测定亲和力。以纯化McAb 2H9包板,9D7为标记抗体建立双单抗夹心ELISA法,检测感染鼠血清中的弓形虫CAg。结果纯化、复性后P30蛋白纯度90%,浓度512ng/mL。筛选出2株抗P30抗原的单克隆抗体,命名为9D7、2H9。用体内诱生腹水法制备抗体,小鼠腹水阳性形成率为90%以上,腹水收获量平均为6.5mL/只。9D7、2H9重链分别为IgG2a、IgG1,轻链均为κ型。9D7和2H9腹水平均效价分别在1∶8 000和1∶10 000以上,纯化后效价均在1∶3 000以上,纯化后McAb蛋白含量在0.8～1.6 mg/mL。Western blotting分析显示2株McAb均能识别弓形虫速殖子天然P30抗原和重组P30抗原。细胞核型分析显示,2株杂交瘤细胞染色体均在100条以上。9D7和2H9识别不同抗原表位,亲和力常数分别为6.13×10~7 M~(-1)和7.64×10~9M~(-1)。双单抗夹心ELISA检测鼠血清中的CAg,第4、5、6天阳性率分别为20%、50%、60%。结论 (1)获得了2株稳定分泌抗P30抗原单克隆抗体的杂交瘤细胞株。 (2)所得到的单克隆抗体均有较好的特异性和亲和力。 (3)抗P30抗原的单克隆抗体可用于弓形虫病早期诊断研究。
[Abstract]:Objective to prepare monoclonal antibody against P30 antigen of Toxoplasma gondii and to establish a method for detection of circulating antigen of Toxoplasma gondii, and to evaluate the early diagnostic value of monoclonal antibody against P30 antigen of Toxoplasma gondii.Methods the recombinant plasmid pET28b (pET-P30) was expressed by BL21DE-3. The recombinant P30 protein was purified by Ni-NTA affinity chromatography. After dialysis and renaturation, the P30 protein was quantified.BALB/c mice were immunized with renatured P30 protein. Spleen cells of immunized mice were isolated and fused with SP2/0 of myeloma cells under the action of polyethylene glycol. Positive clones were screened by HAT selective culture for 20 days.Indirect enzyme linked immunosorbent assay (Elisa) was used to detect the culture supernatant of hybridoma cells and the positive clones were subcloned.Mice pretreated with deplantane were injected intraperitoneally with hybridoma cells in good condition. The ascites could be harvested 10 days after the preparation of antibodies by inducing ascites in vivo.The McAb in ascites was extracted by ammonium sulfate precipitation method and then purified by immunoaffinity chromatography.The subclasses were determined by rapid qualitative test paper method, the titers of ascites and purified antibodies were determined by ELISA method, and the affinity was determined by SDS-PAGE and Western blotting analysis.A double monoclonal antibody sandwich ELISA method was established to detect Toxoplasma gondii in sera of infected mice by using purified McAb 2H9 inclusion plate 9D7 as labeled antibody.Results the purity of P30 protein was 90%, the concentration of P30 protein was 512 ng / mL.Two monoclonal antibodies against P30 antigen were screened and named 9D7H9.The content of purified McAb protein was 0.810. 6 mg/mL.Western blotting analysis showed that the two strains of McAb could recognize Toxoplasma gondii Tachyzoites natural P30 antigen and recombinant P30 antigen.The karyotype analysis showed that the chromosomes of the two hybridomas were above 100. 9D7 and 2H9 recognized different antigenic epitopes. The affinity constants were 6.13 脳 10 ~ (7) M ~ (-1) and 7.64 脳 10 ~ (9) M ~ (-1) ~ (-1), respectively.Double monoclonal antibody sandwich ELISA was used to detect CAg in rat serum. The positive rates of CAG in serum of mice on day 5 ~ 6 were 20 ~ (50) and 60 ~ (th), respectively.ConclusionTwo hybridoma cell lines secreting monoclonal antibodies against P30 antigen were obtained.The monoclonal antibodies obtained have good specificity and affinity.3) Monoclonal antibodies against P30 antigen can be used for early diagnosis of toxoplasmosis.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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