重组幽门螺杆菌过氧化氢酶脂质体疫苗免疫预防研究  

Evalution of immune prophylaxis of the liposomes-encapsulated recombinant Helicobacter pylori catalase protein vaccine

作　　者：黄文[1] 潘雪[1] 李兆申[1] 白杨[2] 王继德[2] 周殿元[2] 

机构地区：[1]第二军医大学长海医院消化内科,上海200422 [2]南方医科大学南方医院消化研究所

出　　处：《胃肠病学和肝病学杂志》2005年第4期359-362,365,共5页Chinese Journal of Gastroenterology and Hepatology

基　　金：国家自然科学基金(30170890)

摘　　要：目的探讨制备脂质体包裹重组幽门螺杆菌过氧化氢酶(Kat)口服疫苗的方法,并用Helicobacterpylori感染的小鼠模型评价其在预防H·pylori感染中的作用。方法将PET_22(+)/Kat在BL21(DE3)大肠杆菌表达,表达Kat的包涵体经洗涤、变性、复性,采用QSepharoseFastFlow阴离子交换层析和SephacrylS_200凝胶过滤层析分离纯化Kat重组蛋白,用薄膜分散法制备以卵磷脂和胆固醇为膜组分包裹的Kat重组蛋白口服疫苗,并用透射电镜测定其粒径。BALB/c小鼠分为5组,分别通过灌胃方法给予PBS、空白脂质体、Kat重组蛋白+霍乱毒素(CT)、脂质体包裹Kat重组蛋白、脂质体包裹Kat重组蛋白和CT,每周1次共4次,末次攻击2周再用活H·pylori攻击3次,3周后处死小鼠,行胃组织快速尿素酶试验、H·pylori的定植半定量,取脾组织行淋巴细胞增殖试验。结果以包涵体为主的表达产物占全菌总蛋白的24.4%,经纯化获得纯度为95%的重组蛋白,制备的脂质体粒径为0.7±0.4μm。PBS组和空白脂质体组保护率均为0,而Kat重组蛋白+CT组、脂质体包裹Kat重组蛋白组、脂质体包裹Kat重组蛋白+CT组的保护率分别为73.3%、66.7%和86.7%,且均能使免疫小鼠胃黏膜H·pylori感染数目明显减少,三个疫苗组淋巴细胞增殖试验均为阳性。结论口服脂质体能部分代替免疫佐剂的作用,将其作为H·pylori疫苗的免疫佐剂,具有广泛的应用前景。Objective To determine the preparing method of the oral liposome-eneapsulated vaccine with recombinant Helieobaeter pylori eatalase protein （Kat） and investigate the effect of the oral vaccine in prevention of Hp in Hp infected mouse models. Methods The recombinant vector PET-22（ ＋ ）/Kat was transformed into the BL21（DE3） E.eoli. The inelusion t bodies of Kat ware washed, denatured and renatured. The two-step chromatographic procedure （Q sepharose Fast Flow exchange and Sephacryl S-200 gel exclusion） were used for the purfication. The oral lipsome-encapsulated vaccine with phosphatidyl choline （PC） and cholesterols （Chol） was praparated using film method, whose size distribution of folate liposomes was measured by electronic microscopy. BALB/c mice were divided into five groups and immunized by PBS alone, liposome alone, Kat plus CT, liposome-encapsulated Kat and liposome-encapsulated Kat plus CT orally respectively once a week for four weeks. All mice were challenged by alive Hp three times in two weeks after the last immunization and sacrificed in three weeks after the last challeng. Hp was determined by the fast urease test. The bactrerial colonizing density semi-quantitation was observed. Lymph cells from the spleens were served for lymphoproliferation assay. Results The inclusion body expression product accounted for 24.4 % of total bacterial protein. The purity of recombinant protein was about 95 % after purfication. The mean size of folate liposome was 0.7 ＋ 0.4 μm. Protective rates of PBS alone, and liposome alone all were 0, While in growps of Kat plus CT, liposome- encapsulated Kat and liposome - encapsulated Kat plus CT were 73.3% ,66.7% and 86.7% respectively. The mice gastrically inoculated with the latter three immune antigens had significantly less infected with Hp. The lymphoproliferration assay of the three vaccine groups was positive. Conclusion The oral liposome is a potential immunization adjuvant candidate for Hp vaccine in proventing and eradication of Hp infect

关 键 词：幽门螺杆菌 过氧化氢酶 疫苗 脂质体 

分 类 号：R573.3[医药卫生—消化系统]