發(fā)布時(shí)間：2018-06-07 23:47

  本文選題：創(chuàng)傷弧菌 + 鞭毛蛋白��； 參考：《細(xì)胞與分子免疫學(xué)雜志》2013年10期

【摘要】：目的制備創(chuàng)傷弧菌鞭毛蛋白單克隆抗體(mAb)并建立雙抗夾心ELISA。方法采用差速離心法提取創(chuàng)傷弧菌ATCC 1.1758菌株的鞭毛蛋白,并以此為抗原免疫BALB/c小鼠,抗血清效價(jià)達(dá)到1∶32 000時(shí),取脾細(xì)胞與生長(zhǎng)良好的對(duì)數(shù)期Sp2/0骨髓瘤細(xì)胞進(jìn)行融合。采用雜交瘤技術(shù)和ELISA制備并篩選分泌mAb的雜交瘤細(xì)胞株,有限稀釋法對(duì)陽(yáng)性孔克隆化培養(yǎng)。制備腹水,純化以得到抗體。結(jié)果獲得5株持續(xù)、穩(wěn)定分泌抗創(chuàng)傷弧菌鞭毛蛋白mAb的雜交瘤細(xì)胞株,命名為VVNo.1~VVNo.5,抗體效價(jià)高達(dá)1∶(2×106)。SDS-PAGE結(jié)果顯示,提取出的鞭毛蛋白相對(duì)分子質(zhì)量(M r)為44 000并且純度較高。采用VVNo.5抗體建立了創(chuàng)傷弧菌雙抗夾心ELISA,該方法的檢測(cè)靈敏度達(dá)到103CFU/mL菌液,通過采用12株創(chuàng)傷弧菌和130株非創(chuàng)傷弧菌進(jìn)行特異性實(shí)驗(yàn),12株創(chuàng)傷弧菌呈陽(yáng)性反應(yīng),130株非創(chuàng)傷弧菌呈陰性反應(yīng),結(jié)果表明VVNo.5抗體具有良好的特異性。在基質(zhì)添加試驗(yàn)中,通過增菌,最低檢出限為2 CFU/25 g樣品。結(jié)論成功制備了創(chuàng)傷弧菌鞭毛蛋白單克隆抗體,并將其應(yīng)用于雙抗夾心ELISA,該方法的檢測(cè)靈敏度達(dá)到1×103CFU/mL菌液,與所測(cè)試的非目標(biāo)菌沒有交叉反應(yīng)。
[Abstract]:Objective to prepare the monoclonal antibody (mAb) of Vibrio vulnificus flagellin (mAb) and establish a double anti sandwich ELISA. method to extract flagellin of Vibrio vulnificus ATCC 1.1758 by differential centrifugation, and to immunize BALB/c mice with antigen, and when the antiserum titer reached 1: 32000, the splenocytes were taken with the logarithmic Sp2/0 myeloma cells with good growth. Hybridoma technique and ELISA were used to prepare and screen hybridoma cell lines secreting mAb. A finite dilution method was used to culture positive kork. The ascites were prepared and purified to obtain antibodies. The results obtained 5 hybridoma cell lines that secreted the flagellin mAb of Vibrio vulnificus steadily and were named VVNo.1~VVNo.5. The antibody titer was up to 1: (2 * 106). .SDS-PAGE results showed that the relative molecular mass (M R) of the extracted flagellin was 44000 and the purity was high. Using VVNo.5 antibody, the double anti sandwich ELISA of Vibrio vulnificus was established. The detection sensitivity of this method reached 103CFU/mL liquid. The specific experiment was carried out by using 12 strains of Vibrio vulnificus and 130 strains of Vibrio vulnificus, and 12 strains of Vibrio vulnificus were presented. The positive reaction, 130 non traumatic Vibrio showed negative reaction, the results showed that the VVNo.5 antibody had good specificity. In the matrix addition test, the minimum detection limit was 2 CFU/25 G by adding bacteria. Conclusion the monoclonal antibody of Vibrio vulnificus flagellin was successfully prepared, and it should be used in the double resistance sandwich ELISA. The sensitivity of the method was reached. Up to 1 x 103CFU/mL, there was no cross reaction with the tested non target bacteria.
【作者單位】： 北京出入境檢驗(yàn)檢疫局;北京農(nóng)學(xué)院;河北出入境檢驗(yàn)檢疫局;北京大學(xué);
【基金】：國(guó)家質(zhì)檢總局公益性行業(yè)科研專項(xiàng)(201110034)
【分類號(hào)】：R392.11

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本文編號(hào)：1993354