肠出血型大肠埃希菌O157:H7 EspF蛋白多克隆抗体的制备和初步纯化

Preparation and primary purification of polyclonal antibodies against EspF of enterohaemorrhagic Escherichia coli O157:H7

目的制备肠出血型大肠埃希菌(EHEC)O157:H7EspF蛋白多克隆抗体并初步纯化。方法生物信息学方法分析EHECO157:H7EspF蛋白的柔韧性、亲水性、表面可能性及抗原表位,选取1条由15个氨基酸残基组成的多肽为半抗原,在其C端偶联钥孔血蓝蛋白(KLH),免疫新西兰大白兔制备抗血清。ELISA测定抗血清效价,免疫印迹法鉴定其特异性,辛酸-硫酸铵法初步纯化抗血清,SDS-PAGE检测抗体纯度。结果选择EspF蛋白抗原指数最高的肽段72-TPSRPAPPPPTSGQA-86(0.506)为半抗原,合成抗原多肽经高效液相色谱鉴定,纯度为95.78%,经质谱分析其分子量为1563.76Mr,与目的多肽分子量一致;加强免疫3次后,EHECO157:H7EspF蛋白的抗血清效价达1:2048000;该血清对EHECO157:H7野生株和espF突变株(△espF)的EspF蛋白均有特异性,并经辛酸-硫酸铵法获得一定纯度的抗体。结论成功地制备了效价高、特异性强的EHECO157:H7EspF蛋白多克隆抗体。

Objective To obtain antibodies against the protein Espf of EHEC O157：H7. Methods The flexibility, hydrophilcity, surface probability and the epitope of B cell were analyzed by bioinformatic tools. Hapten with 15 amino acids was coupled with KLH. Subsequently rabbits were immunized to prepare anti-EspF polyclonal antibody. Valence of antibody was detected by ELISA,specificity was tested by Western blot. Polyclonal antiserum was purified by caprylic acid-ammonium sulfate precipitation,then the purity of antibodies was detected by SDS-PAGE. Results Polyclonal antiserum against the protein EspF of EHEC O157：H7 was obtained, and the titer of the antiserum obtained in this experiment was up to 1：2 048 000, and the specificity was confirmed by Western blot. Conclusion We have obtained specific antibodies with high-titer and speciticity against the protein EspF of EHEC O157：H7.