CAJ | 학술논문

目的制备牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)的卵黄免疫球蛋白( immunoglobulin yolk,IgY)并评价其理化性质,以期为采用被动免疫抗体防治慢性牙周炎寻找新途径.方法厌氧培养Pg国际标准菌株ATCC33277,收集细菌,超声振荡至菌体完全破碎,离心,收集上清液,即为全菌可溶性蛋白抗原.罗曼母鸡隔离饲养至5个月龄用于抗体的制备.以质量浓度为1 g/LPg全菌可溶性蛋白抗原1ml与等量弗氏完全佐剂吹打混匀成油包水状注射剂,经皮下和肌肉多点注射免疫母鸡,共注射免疫4次,每次免疫间隔时间为10d.初次免疫即开始收集鸡蛋,标记后4℃保存.提纯免疫后不同时间的抗Pg-IgY,二喹啉甲酸总蛋白定量法测定蛋白浓度,SDS聚丙烯酰胺凝胶电泳分析鉴定,间接酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法进行IgY 抗体效价变化和理化性质的检测.结果通过免疫诱导出质量浓度为2.05 g/L的特异性IgY,经SDS聚丙烯酰胺凝胶电泳测得相对分子质量分别为65000的重链和25000的轻链,与理论值一致；间接ELISA法结果显示初次免疫后5d左右出现抗Pg-IgY,免疫后50～55d达峰值；抗体效价达到1∶100000以上；一个鸡蛋可以生产超过10 mg的IgY,纯度高达95％.结论 Pg全菌蛋白免疫产蛋母鸡可使鸡蛋产生高效价、高浓度的特异性IgY,该抗体纯度高、耐热、耐酸碱,理化性质稳定,适合口腔环境.
목적 제비아간계람단포균(Porphyromonas gingivalis,Pg)적란황면역구단백( immunoglobulin yolk,IgY)병평개기이화성질,이기위채용피동면역항체방치만성아주염심조신도경.방법 염양배양Pg국제표준균주ATCC33277,수집세균,초성진탕지균체완전파쇄,리심,수집상청액,즉위전균가용성단백항원.라만모계격리사양지5개월령용우항체적제비.이질량농도위1 g/LPg전균가용성단백항원1ml여등량불씨완전좌제취타혼균성유포수상주사제,경피하화기육다점주사면역모계,공주사면역4차,매차면역간격시간위10d.초차면역즉개시수집계단,표기후4℃보존.제순면역후불동시간적항Pg-IgY,이규람갑산총단백정량법측정단백농도,SDS취병희선알응효전영분석감정,간접매련면역흡부측정(enzyme-linked immunosorbent assay,ELISA)법진행IgY 항체효개변화화이화성질적검측.결과 통과면역유도출질량농도위2.05 g/L적특이성IgY,경SDS취병희선알응효전영측득상대분자질량분별위65000적중련화25000적경련,여이론치일치；간접ELISA법결과현시초차면역후5d좌우출현항Pg-IgY,면역후50～55d체봉치；항체효개체도1∶100000이상；일개계단가이생산초과10 mg적IgY,순도고체95％.결론 Pg전균단백면역산단모계가사계단산생고효개、고농도적특이성IgY,해항체순도고、내열、내산감,이화성질은정,괄합구강배경.
Objective To obtain egg yolk antibody in hen eggs laid by hens immunized with the protein of Porphyromonas gingivalis(Pg).To generate,purify IgY against Pg(anti-Pg-IgY) and identify its specificity.Methods PgATCC33277 was cultured under standard anaerobic conditions and harvested after proliferation.Then Pg was extracted by sonication until the cell pellets were shattered completely.After centrifugatiton,the supernatant was collected.Five-month-old Roman hens were immunized for egg antibody production.The antibody was inoculated intramuscularly and subcutaneously in the breast from multiple spot with 1.0 ml of a vaccine consisting of oil-adjuvant protein which was mixed with 1 ml protein of Pg and 1 ml Freund's adjuvant complete every 10 days,for 4 times.The eggs were collected after the first immunization and stored at 4 ℃.The anti-Pg-IgY was extracted and purified.The protein concentration was tested by bicinchoninic acid( BCA),the specificity of IgY analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE),the titre of IgY and its physicochemical character were evaluated by indirect enzyme-linked immunosorbcnt assay.Results The concentration of obtained anti-Pg-IgY was 2.05 g/L.SDS-PAGE analysis of the anti-Pg-IgY showed that the molecular weight of IgY was consistent with the theoretical value.Protein of anti-Pg-IgY appeared approximately 5 days after the first immunization,and reached the peak at 50-55 days.Antibody titres reached 1 ∶100000.Each egg produced more than 10 mg IgY,and its purification was up to 95％ as well.Conclusions Layer hens immuned by Pg may provide specific IgY of high titre and high concentration.The antibody has high purity and is heat,acid and alkali-resistant.