河豚毒素人工抗原的制备及其免疫原性鉴定

Preparation of tetrodotoxin artificial antigen and identification of its immunogenicity

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.