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

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。

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

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.