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目的制备针对蓝氏贾第鞭毛虫α-4贾第素的特异性抗体,免疫胶体金电镜观察该贾第素的亚细胞定位。方法生物信息学分析并合成α-4贾第素抗原肽,与钥孔血蓝蛋白(KLH)偶联后免疫新西兰白兔,Protein A亲和层析纯化抗血清,采用α-4贾第素重组蛋白和贾第虫滋养体全虫体裂解物通过Western blot验证抗血清结合力和结合特异性,选择高特异性抗体通过免疫胶体金电镜观察α-4贾第素的亚细胞定位。结果筛选出3段可能的抗原肽,合成的抗原肽与KLH偶联后免疫兔得到高效价抗血清,Protein A纯化效果良好;3种纯化抗血清均能与α-4贾第素结合,其中anti-P2具有较高的抗原结合特异性。采用anti-P2进行免疫胶体金电镜,显示α-4贾第素主要定位于鞭毛,胞浆也有少量散在表达。结论制备的α-4贾第素多克隆抗体具有抗原结合特异性。用anti-P2进行免疫胶体金电镜,α-4贾第素主要定位于贾第虫鞭毛。
Objective To prepare specific antibodies to Giardia lamblia α-4 Giardia, and observe the subcellular localization of Giardia by immunogold electron microscopy. Methods Bioinformatics analysis and synthesis of α-4 Giardia antigen peptide, coupled with keyhole limpet hemocyanin (KLH) were immunized New Zealand white rabbits, Protein A affinity chromatography purified antiserum, the use of α-4 Giardin Recombinant protein and giardia trophozoites lysate of all the parasites tested by Western blot antiserum binding and binding specificity, high specificity antibodies were selected by immunohistochemistry gold electron microscopy of α-4 Giardin subcellular localization. As a result, 3 possible antigenic peptides were screened out. After being coupled with KLH, the synthesized antigenic peptides were immunized with rabbit to obtain high titer anti-serum, and the purification results of Protein A were good. All three purified antisera were able to bind to α-4, Anti-P2 has high antigen-binding specificity. Using anti-P2 immunogold gold electron microscopy showed that α-4 Giardin is mainly located in the flagella, cytoplasm also a small amount of scattered expression. Conclusion The prepared α-4 katinin polyclonal antibody has antigen-binding specificity. Anti-P2 immunogold gold electron microscopy, α-4 Jia Ting Su mainly located in Giardia flagellum.