中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2014年
12期
933-940
,共8页
陈泽宇%郭蓉%徐江红%吴娟%薛红刚%范小勇
陳澤宇%郭蓉%徐江紅%吳娟%薛紅剛%範小勇
진택우%곽용%서강홍%오연%설홍강%범소용
肺炎球菌表面黏附素%B 型流感嗜血杆菌%多糖蛋白结合疫苗%免疫原性%免疫保护
肺炎毬菌錶麵黏附素%B 型流感嗜血桿菌%多糖蛋白結閤疫苗%免疫原性%免疫保護
폐염구균표면점부소%B 형류감기혈간균%다당단백결합역묘%면역원성%면역보호
PsaA%Hib%Polysaccharide-protein conjugate vaccine%Immunogenecity%Protection
目的：研究以肺炎球菌表面黏附素（PsaA）为蛋白载体的 b 型流感嗜血杆菌（Hib）多糖结合疫苗的制备及其免疫原性和免疫保护作用。方法用基因工程技术制备 PsaA 基因重组蛋白（rPsaA），通过酰胺缩合法与 Hib 多糖偶联结合，制成结合疫苗（Hib-PsaA）。用此疫苗免疫接种3周龄幼鼠，同时以破伤风类毒素和 B 型流感嗜血杆菌（Hib）多糖结合疫苗（Hib-TT）接种小鼠作为对照及磷酸盐缓冲液（PBS）接种作空白对照。末次免疫后2周观察小鼠对 Hib 多糖和 PsaA 蛋白的免疫原性应答；同时以肺炎球菌分别注入免疫后小鼠和未免疫的对照小鼠听泡，分别于攻击后第3天和第7天观察小鼠中耳内细菌清除率和中耳炎症反应情况，观察新型疫苗的免疫保护作用。结果以基因工程技术成功制备并纯化 rPsaA 蛋白，并运用酰胺缩合法与 Hib 多糖偶联结合，结合物的洗脱峰前移，证明偶联成功。接种该结合疫苗的小鼠产生的抗 PsaA 的抗体 IgG 几何平均滴度（GMT）为4525；抗 Hib 抗体 IgG 的 GMT 为1393。在接种 Hib-TT 疫苗的对照组中，小鼠产生的抗 Hib 抗体 GMT 为1493，两种疫苗的抗 Hib 抗体 GMT 差异无统计学意义，但是 Hib-PsaA 结合疫苗显示出对肺炎球菌的免疫应答，优于以破伤风类毒素为载体的结合疫苗。细菌攻击后第3天实验组小鼠中耳细菌清除率明显高于对照组，组织病理学检查实验组小鼠细菌攻击后第3天和第7天中耳炎症反应明显轻于对照组，显示了 Hib-PsaA 结合疫苗针对肺炎球菌的有效免疫保护作用。结论运用基因工程技术可获得高纯度、高产量的 rPsaA 蛋白，以酰胺缩合法可成功将 rPsaA 蛋白与 Hib 多糖偶联结合，所制备的多糖蛋白结合疫苗在幼小鼠体内可诱导针对 rPsaA 蛋白与 Hib 多糖的免疫应答反应，并且可有效预防肺炎球菌导致的急性中耳炎。提示该疫苗免疫可以在预防 Hib 感染性疾病的同时，对肺炎链球菌导致的急性中耳炎可能有一定的预防作用。
目的：研究以肺炎毬菌錶麵黏附素（PsaA）為蛋白載體的 b 型流感嗜血桿菌（Hib）多糖結閤疫苗的製備及其免疫原性和免疫保護作用。方法用基因工程技術製備 PsaA 基因重組蛋白（rPsaA），通過酰胺縮閤法與 Hib 多糖偶聯結閤，製成結閤疫苗（Hib-PsaA）。用此疫苗免疫接種3週齡幼鼠，同時以破傷風類毒素和 B 型流感嗜血桿菌（Hib）多糖結閤疫苗（Hib-TT）接種小鼠作為對照及燐痠鹽緩遲液（PBS）接種作空白對照。末次免疫後2週觀察小鼠對 Hib 多糖和 PsaA 蛋白的免疫原性應答；同時以肺炎毬菌分彆註入免疫後小鼠和未免疫的對照小鼠聽泡，分彆于攻擊後第3天和第7天觀察小鼠中耳內細菌清除率和中耳炎癥反應情況，觀察新型疫苗的免疫保護作用。結果以基因工程技術成功製備併純化 rPsaA 蛋白，併運用酰胺縮閤法與 Hib 多糖偶聯結閤，結閤物的洗脫峰前移，證明偶聯成功。接種該結閤疫苗的小鼠產生的抗 PsaA 的抗體 IgG 幾何平均滴度（GMT）為4525；抗 Hib 抗體 IgG 的 GMT 為1393。在接種 Hib-TT 疫苗的對照組中，小鼠產生的抗 Hib 抗體 GMT 為1493，兩種疫苗的抗 Hib 抗體 GMT 差異無統計學意義，但是 Hib-PsaA 結閤疫苗顯示齣對肺炎毬菌的免疫應答，優于以破傷風類毒素為載體的結閤疫苗。細菌攻擊後第3天實驗組小鼠中耳細菌清除率明顯高于對照組，組織病理學檢查實驗組小鼠細菌攻擊後第3天和第7天中耳炎癥反應明顯輕于對照組，顯示瞭 Hib-PsaA 結閤疫苗針對肺炎毬菌的有效免疫保護作用。結論運用基因工程技術可穫得高純度、高產量的 rPsaA 蛋白，以酰胺縮閤法可成功將 rPsaA 蛋白與 Hib 多糖偶聯結閤，所製備的多糖蛋白結閤疫苗在幼小鼠體內可誘導針對 rPsaA 蛋白與 Hib 多糖的免疫應答反應，併且可有效預防肺炎毬菌導緻的急性中耳炎。提示該疫苗免疫可以在預防 Hib 感染性疾病的同時，對肺炎鏈毬菌導緻的急性中耳炎可能有一定的預防作用。
목적：연구이폐염구균표면점부소（PsaA）위단백재체적 b 형류감기혈간균（Hib）다당결합역묘적제비급기면역원성화면역보호작용。방법용기인공정기술제비 PsaA 기인중조단백（rPsaA），통과선알축합법여 Hib 다당우련결합，제성결합역묘（Hib-PsaA）。용차역묘면역접충3주령유서，동시이파상풍류독소화 B 형류감기혈간균（Hib）다당결합역묘（Hib-TT）접충소서작위대조급린산염완충액（PBS）접충작공백대조。말차면역후2주관찰소서대 Hib 다당화 PsaA 단백적면역원성응답；동시이폐염구균분별주입면역후소서화미면역적대조소서은포，분별우공격후제3천화제7천관찰소서중이내세균청제솔화중이염증반응정황，관찰신형역묘적면역보호작용。결과이기인공정기술성공제비병순화 rPsaA 단백，병운용선알축합법여 Hib 다당우련결합，결합물적세탈봉전이，증명우련성공。접충해결합역묘적소서산생적항 PsaA 적항체 IgG 궤하평균적도（GMT）위4525；항 Hib 항체 IgG 적 GMT 위1393。재접충 Hib-TT 역묘적대조조중，소서산생적항 Hib 항체 GMT 위1493，량충역묘적항 Hib 항체 GMT 차이무통계학의의，단시 Hib-PsaA 결합역묘현시출대폐염구균적면역응답，우우이파상풍류독소위재체적결합역묘。세균공격후제3천실험조소서중이세균청제솔명현고우대조조，조직병이학검사실험조소서세균공격후제3천화제7천중이염증반응명현경우대조조，현시료 Hib-PsaA 결합역묘침대폐염구균적유효면역보호작용。결론운용기인공정기술가획득고순도、고산량적 rPsaA 단백，이선알축합법가성공장 rPsaA 단백여 Hib 다당우련결합，소제비적다당단백결합역묘재유소서체내가유도침대 rPsaA 단백여 Hib 다당적면역응답반응，병차가유효예방폐염구균도치적급성중이염。제시해역묘면역가이재예방 Hib 감염성질병적동시，대폐염련구균도치적급성중이염가능유일정적예방작용。
Objective To prepare a conjugate vaccine by linking Haemophilus influenzae type b (Hib)polysaccharide to PsaA protein carrier and evaluate the immunogenicity and efficacy of the conjugate vaccine. Methods A recombinant protein rPsaA,expressed by using the genetic engineering technology, was used as a protein carrier to prepare conjugate vaccine together with Hib polysaccharide. Ten mice at age of 3 weeks were immunized with the conjugate vaccine,while another 10 age-matched mice were immunized with Hib-tetanus toxoid(Hib-TT)vaccine which was produced formerly as a control. The mice treated with equal volume of PBS were set up as the negative control. The IgG antibodies in serum samples against PsaA and Hib polysaccharide were detected in two weeks after the final immunization. A suspension of Pneumococ-cus was injected into the middle ears of mice from experiment and control group. Histopathological analysis was performed to measure the clearance of bacteria in the middle ears and the severity of infection on days 3 and 7 after bacterial challenge. Results The rPsaA protein was prepared by the genetic engineering tech-nology and purified successfully with anion-exchange column. The Hib polysaccharide-PsaA protein conju-gate vaccine was prepared through a series of amide condensation reactions. The detection of IgG antibodies against PsaA protein and Hib polysaccharide in the immunized mice demonstrated that there was no signifi-cant difference with the titer of IgG against Hib polysaccharide between the mice immunized with the Hib-PsaA conjugate vaccine and those immunized with the Hib-TT vaccine. Less Pneumococcus strains were de-tected in the middle ears of mice immunized with the conjugate vaccine than those mice immunized with the Hib-TT vaccine three days after challenge. The mice from control group showed severe inflammation in the middle ears than those from experiment group. The Hib polysaccharide-PsaA protein conjugate vaccine im-proved protection against Pneumococcus infections as compared with the Hib-TT vaccine. Conclusion The rPsaA protein could be produced by genetic engineering technology and purified by anion-exchange column. The Hib polysaccharide was successfully conjugated with the rPsaA protein through amide condensation reac-tion. Both anti-PsaA and anti-Hib immune responses were induced in young mice by the injection of Hib pol-ysaccharide-PsaA protein conjugate vaccine. Apart from providing protection against Hib infection,the con-jugate vaccine might also be used for the prevention of acute otitis media caused by Pneumococcus infection.
