摘要
目的制备牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）的卯黄免疫球蛋白（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。方法厌氧培养聘国际标准菌株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1g／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2．05g／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10mg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。
Objective To obtain egg yolk antibody in hen eggs laid by hens immunized with the protein of Porphyromonas gingivalis （Pg）. To generate, purify lgY 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 innnunosorbent 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 ： 100 000. Each egg produced more than l0 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.
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2011年第10期586-589,共4页
Chinese Journal of Stomatology
基金
安徽省级重点实验室项目基金（2008）
2010年高校省级自然科学研究项目（KJ2010A159）
安徽省自然科学基金（090413146）
关键词
紫单胞菌
龈
免疫球蛋白类
动物
实验
Porphyromonas gingivalis
Immunoglobulins
Animals,laboratory
