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  本文選題：弓形蟲(chóng)　切入點(diǎn)：P30抗原　出處：《南華大學(xué)》2007年碩士論文

【摘要】： 目的制備抗弓形蟲(chóng)P30抗原的單克隆抗體,建立弓形蟲(chóng)循環(huán)抗原檢測(cè)法,評(píng)價(jià)抗P30抗原單克隆抗體的早期診斷價(jià)值。 方法重組質(zhì)粒pET28b(+)-P30經(jīng)BL21(DE3)表達(dá),使用Ni-NTA親和層析柱純化重組P30蛋白,經(jīng)透析、復(fù)性后,定量P30蛋白。以復(fù)性的P30蛋白免疫BALB/c小鼠,分離免疫小鼠的脾細(xì)胞,在聚乙二醇的作用下與骨髓瘤細(xì)胞SP2/0融合,融合后的細(xì)胞進(jìn)行HAT選擇培養(yǎng),10~20天后篩選陽(yáng)性克隆。用重組P30包板,間接酶聯(lián)免疫試驗(yàn)檢測(cè)雜交瘤細(xì)胞培養(yǎng)上清,陽(yáng)性克隆經(jīng)亞克隆建株。降植烷預(yù)處理小鼠后,腹腔注射狀態(tài)良好的雜交瘤細(xì)胞,體內(nèi)誘生腹水法制備抗體,7~10天后即可收獲腹水。對(duì)腹水中的McAb先用硫酸銨沉淀法進(jìn)行粗提,然后用免疫親和層析法進(jìn)一步純化。快速定性試紙法測(cè)定其亞類(lèi), ELISA法測(cè)定腹水和純化后抗體的效價(jià),進(jìn)行雜交瘤細(xì)胞核型分析、SDS-PAGE和Western blotting分析,ELISA法測(cè)定親和力。以純化McAb 2H9包板,9D7為標(biāo)記抗體建立雙單抗夾心ELISA法,檢測(cè)感染鼠血清中的弓形蟲(chóng)CAg。 結(jié)果純化、復(fù)性后P30蛋白純度90%,濃度512ng/mL。篩選出2株抗P30抗原的單克隆抗體,命名為9D7、2H9。用體內(nèi)誘生腹水法制備抗體,小鼠腹水陽(yáng)性形成率為90%以上,腹水收獲量平均為6.5mL/只。9D7、2H9重鏈分別為IgG2a、IgG1,輕鏈均為κ型。9D7和2H9腹水平均效價(jià)分別在1∶8 000和1∶10 000以上,純化后效價(jià)均在1∶3 000以上,純化后McAb蛋白含量在0.8～1.6 mg/mL。Western blotting分析顯示2株McAb均能識(shí)別弓形蟲(chóng)速殖子天然P30抗原和重組P30抗原。細(xì)胞核型分析顯示,2株雜交瘤細(xì)胞染色體均在100條以上。9D7和2H9識(shí)別不同抗原表位,親和力常數(shù)分別為6.13×10~7 M~(-1)和7.64×10~9M~(-1)。雙單抗夾心ELISA檢測(cè)鼠血清中的CAg,第4、5、6天陽(yáng)性率分別為20%、50%、60%。 結(jié)論 (1)獲得了2株穩(wěn)定分泌抗P30抗原單克隆抗體的雜交瘤細(xì)胞株。 (2)所得到的單克隆抗體均有較好的特異性和親和力。 (3)抗P30抗原的單克隆抗體可用于弓形蟲(chóng)病早期診斷研究。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 劉明如;弓形蟲(chóng)單克隆抗體的制備及生物學(xué)活性的初步鑒定[D];廣西大學(xué);2012年

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本文編號(hào)：1698003