抗钩虫特异性卵黄抗体的制备与鉴定

Preparation and identification of egg yolk immunoglobulin against ancyloma duodenale

目的制备并鉴定特异性抗十二指肠钩虫鸡卵黄免疫球蛋白（IgY），为分析抗钩虫IgY抗体的作用奠定基础。方法制备十二指肠钩虫成虫粗抗原，经皮下和肌肉注射免疫25周产蛋海兰母鸡3次，每只鸡每次的免疫剂量为200ug，初次免疫后28d进行加强免疫，加强免疫间隔时间为10d。水稀释法提取卯黄中的IgY，盐析、透析法纯化IgY，BCA法测定蛋白含量，并进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）和Westernblot分析。ELISA法动态检测抗体效价，观察抗体效价变化。结果免疫后55d的每个鸡蛋能提取40—55mg IgY，经SDS—PAGE和Western blot分析，纯化的IgY有2条主带，分子量分别是35ku和60ku，即IgY的轻链和重链。初次免疫后10d左右出现抗体，至免疫后50～55d为高峰，效价达到1：10000以上。IgV与人血清抗体均可识别30ku处钩虫抗原。结论十二指肠钩虫成虫粗抗原免疫的海兰母鸡能产生高浓度、高效价、特异性的IgY抗体，为分析抗钩虫IgY抗体的作用奠定了基础。

Objective To produce and purify egg yolk immunoglobulin （IgY） against ancyloma duodenale and judge it. In order to lay a foundation that analyse the effect of antibody. Methods 25-week old hens were immunized by subcutaneous injection and intramuscular injection with antigen of hookworms for 3 times. Each hen was immunized with 200 ug antigen per time and the second injection was performed 28 days later. Subsequent injections were performed at 10-day interval. IgY was extracted from the eggs by WD （water-dilution） method. The methods of purification were dialysised and salted out. IgY was measured by BCA method and analyzed by SDSPAGE and Western blot. ELISA method was used to determine the titer of the antiserum. Results 50 - 55 mg IgY was extracted from one egg at the day of 55 after inoculation. SDS-PAGE and Western blot demonstrated that the IgY contained two major protein bands with molecular weight of 35 ku and 60 ku. One was light chain, the other was heavy chain. And the IgY could be recognized by antigen of hookworms. Specific IgY could be detected by SDS- PAGE and ELISA in the eggs laid by the hens from 10 days after the first immunization. The fastigium of immunoactivity was from 50 to 55 day after the primary immunization. The antibody tiler overtoped 1 ： 10 000. IgY and the antibody of patients could be recognized by the molecular weight of 30 ku. Conclusion The hens can produce high concentration and high immunoactivity specific IgY after being immunized with antigen of hookworms and in order to lay a foundation that analyse the effect of IgY