河豚毒素人工抗原的制备及其免疫原性鉴定
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摘要
目的:选择血蓝蛋白(KLH)和牛血清白蛋白(BSA)分别作为载体蛋白与河豚毒素(TTX)进行偶联制备人工抗原,并对合成结果进行鉴定。方法:通过甲醛法将TTX分别与大分子载体蛋白,即血蓝蛋白(KLH)和牛血清白蛋白(BSA)进行偶联。利用紫外扫描法、电泳法对偶联产物进行鉴定,并对6周龄Balb/c雌性小鼠免疫注射检测其免疫原性。结果:紫外扫描显示偶联抗原较载体蛋白有轻微的红移现象出现,且经过琼脂糖凝胶及SDS-PAGE 2种方法电泳检测后发现,载体蛋白较人工抗原之间迁移率均不相同;对免疫小鼠进行间接酶联免疫吸附试验(iELISA)及间接竞争酶联免疫吸附试验(icELISA)联用检测其多抗血清效价及特异性,经过共计6次免疫后的效价能达到1:40w,且能有效抑制TTX。结论:河豚毒素人工抗原制备成功且具备良好的免疫原性。
Ojective: Keyhole Limpet Hemocyanin(KLH) and bovine serum albumin(BSA) were selected as carrier proteins to prepare artificial antigens with tetrodotoxin(TTX), and the synthesis results were identified. Method: In this experiment, TTX was coupled to the macromolecular carrier proteins KLH and BSA by the formaldehyde method, and 6-week-old Balb/c female mice were immunized to obtain mouse polyclonal antibodies. The dual-production was identified by UV-spectroscopy and electrophoresis. Result:The UV-spectroscopy showed that the coupled antigen appeared slightly redshifted with the carrier protein. After electrophoresis by agarose gel and SDS-PAGE, the mobility between carrier protein and artificial antigen was found to be different. The immunized mice were tested by indirect enzyme-linked immunosorbent assay(iELISA) and indirect competitive enzyme-linked immunosorbent assay(icELISA) to detect the multi-antibody serum titer and specificity. The titer after total six immunizations can reach 1:40 w and it can effectively inhibit TTX. Conclusion: Tetrodotoxin artificial antigen was successfully prepared and had good immunogenicity.
Ojective: Keyhole Limpet Hemocyanin(KLH) and bovine serum albumin(BSA) were selected as carrier proteins to prepare artificial antigens with tetrodotoxin(TTX), and the synthesis results were identified. Method: In this experiment, TTX was coupled to the macromolecular carrier proteins KLH and BSA by the formaldehyde method, and 6-week-old Balb/c female mice were immunized to obtain mouse polyclonal antibodies. The dual-production was identified by UV-spectroscopy and electrophoresis. Result:The UV-spectroscopy showed that the coupled antigen appeared slightly redshifted with the carrier protein. After electrophoresis by agarose gel and SDS-PAGE, the mobility between carrier protein and artificial antigen was found to be different. The immunized mice were tested by indirect enzyme-linked immunosorbent assay(iELISA) and indirect competitive enzyme-linked immunosorbent assay(icELISA) to detect the multi-antibody serum titer and specificity. The titer after total six immunizations can reach 1:40 w and it can effectively inhibit TTX. Conclusion: Tetrodotoxin artificial antigen was successfully prepared and had good immunogenicity.
引文
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[14]张伟,汝晓飞,秦海艳,等.马波沙星人工抗原的合成及其免疫原性的鉴定[J].中国畜牧兽医,2018,50(4):66-71.
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[2]Lago J,Rodríguez L P,Blanco L,et al.Tetrodotoxin,an Extremely Potent Marine Neurotoxin:Distribution,Toxicity,Origin and Therapeutical Uses[J].Marine Drugs,2015,13(10):6384.
[3]Vaishali B,Mary L,Madhurima D,et al.Tetrodotoxin:Chemistry,Toxicity,Source,Distribution and Detection[J].Toxins,2014,6(2):693-755.
[4]邓尚贵,彭志英,杨萍,等.河豚毒素研究进展[J].海洋科学,2002,26(10):32-35.
[5]Isbister G K,Kiernan M C.Neurotoxic marine poisoning[J].Lancet Neurology,2005,4(4):219.
[6]刘亚萍.河豚毒素的研究进展[J].食品研究与开发,2008,13(2):691-692.
[7]崔建洲,申雪艳,宫庆礼,等.高效液相色谱-紫外/荧光检测方法测定河豚毒素[J].色谱,2006,24(3):317-317.
[8]刘燕婷,钟青萍,雷红涛,等.基于不同载体免疫原制备河豚毒素抗体的研究[J].卫生研究,2008,37(2):234-236.
[9]Wang R,Huang A,Liu L,et al.Construction of a single chain variable fragment antibody(scFv)against tetrodotoxin(TTX)and its interaction with TTX[J].Toxicon,2014,83(2):22-34.
[10]王磊.抗河豚毒素单链抗体的制备[D].福州:福建农林大学,2010.
[11]刘立才.抗河豚毒素的基因工程单链抗体的制备[D].福州:福建农林大学,2012.
[12]周晓翠.河豚毒素单克隆抗体的制备及ELISA方法的建立[D].长春:吉林大学,2008.
[13]王健伟,王德斌,罗雪云,等.抗河豚毒素单克隆抗体的制备及其特性的初步研究[J].卫生研究,1996,(5):308-311.
[14]张伟,汝晓飞,秦海艳,等.马波沙星人工抗原的合成及其免疫原性的鉴定[J].中国畜牧兽医,2018,50(4):66-71.
[15]Ling S,Xiao S,Xie C,et al.Preparation of Monoclonal Antibody for Brevetoxin 1 and Development of Ic-ELISAand Colloidal Gold Strip to Detect Brevetoxin 1[J].Toxins,2018,10(2):2-11.