幽门螺杆菌热休克蛋白60脂质体疫苗的制备及其免疫预防作用

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (05): 531-534.

幽门螺杆菌热休克蛋白60脂质体疫苗的制备及其免疫预防作用

黄文¹, 白杨¹, 王继德¹, 武金宝¹, 李国锋², 张卫民¹, 周殿元¹

1. 南方医科大学南方医院消化病研究所, 广东, 广州, 510515;
2. 南方医科大学南方医院药学部, 广东, 广州, 510515

出版日期:2005-05-20 发布日期:2005-05-20
基金资助:
收稿日期:2004-9-28。
基金项目:国家自然科学基金(30170890)
作者简介:黄文(1966- ),男,2003年毕业于第一军医大学,博士后,副教授,副主任医师,现工作单位:第二军医大学长海医院消化科,电话:021-25074838,E-mail:huangsh666@263.net

Preparation oral liposome-encapsulated recombinant Helicobacter pylori heat shock protein 60 vaccine for prevention of Hp infection

HUANG Wen¹, BAI Yang¹, WANG Ji-de¹, WU Jin-bao¹, LI Guo-feng², ZHANG Wei-ming¹, ZHOU Dian-yuan¹

1. 南方医科大学南方医院消化病研究所, 广东, 广州, 510515;
2. 南方医科大学南方医院药学部, 广东, 广州, 510515

Online:2005-05-20 Published:2005-05-20

摘要/Abstract

摘要： 目的探讨制备脂质体包裹重组幽门螺杆菌(Hp)热休克蛋白60(Hsp60)口服疫苗的方法,并用Hp感染的小鼠模型评价其在预防Hp感染中的作用.方法将PET-22(+)/Hsp60在BL21(DE3)大肠杆菌表达,Ni-NTA琼脂糖树脂纯化Hsp60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Hsp60重组蛋白口服疫苗,并用透射电镜测定其粒径.75只BALB/C小鼠分为5组,分别通过灌胃方法给予PBS、空白脂质体、Hsp60重组蛋白+霍乱霉素(CT)、脂质体包裹Hsp60重组蛋白、脂质体包裹Hsp60重组蛋白+CT,每周1次共次,末次攻击2周再用活Hp攻击3次,3周后处死小鼠,行胃组织快速尿素酶试验、Hp的定植半定量、炎症程度及其炎症活动度的评分.结果可溶性表达产物占全菌总蛋白的27%,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.)μm.PBS组和空白脂质体组保护率均为0,而Hsp60重组蛋白+CT组、脂质体包裹Hsp60重组蛋白组、脂质体包裹Hsp60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粘膜Hp感染数目明显减少,炎症反应减轻.结论口服脂质体能分地代替免疫佐剂,作为Hp疫苗的免疫佐剂,将具有广泛的应用前景.

Abstract: Objective To prepare oral liposome-encapsulated recombinant Helicobacter pylori (Hp) heat shock protein 60 (Hsp60) vaccine and investigate its effect against Hp infection in mice.Methods The recombinant vector PET-22(+)/Hsp60 was transformed into BL21(DE3) E.coli. The recombinant protein was purified with Ni-NTA agrose resin and the oral liposome-encapsulated vaccine was prepared with phosphatidyl choline and cholesterols using film method, with the size distribution of the folate liposomes measured by transimssion electronic microscopy. BALB/c mice were divided into 5 groups and immunized by intragastric administration of PBS, liposome, rHsp60 plus choleratoxin (CT), liposome-encapsulated rHsp60, and liposome-encapsulated rHsp60 plus CT, respectively, given once a week for 4 weeks. All the mice were challenged by Hp for 3 times within two weeks following the last immunization and sacrificed 3 weeks after the last challenge. Hp detection was performed by fast urease test. Semi-quantitative assessment of the bacterial colonization density observation of the inflammation severity and gastric histopathology were carried out. Results The soluble expression product accounted for 27% of the total bacterial protein. The purity of recombinant fusion protein was about 95% after purification. The mean size of the folate liposomes was 0.7±0.4 μm. PBS or liposome alone showed no immune-enhancing effect, and rHsp60 plus CT, liposome-encapsulated rHsp60 and liposome-encapsulated rHsp60 plus CT had the protective rates against Hp infection of 73.3%, 66.7% and 86.7%, respectively. The latter 3 preparations effected significantly reduced Hp infection and alleviated the inflammation in the gastric mucosa of the mice challenged with Hp.Conclusion The oral liposome may serve as a potential adjuvant for Hp vaccine in preventing Hp infection.

中图分类号:

R378.3

黄文¹, 白杨¹, 王继德¹, 武金宝¹, 李国锋², 张卫民¹, 周殿元¹. 幽门螺杆菌热休克蛋白60脂质体疫苗的制备及其免疫预防作用[J]. 南方医科大学学报, 2005, 25(05): 531-534.

HUANG Wen¹, BAI Yang¹, WANG Ji-de¹, WU Jin-bao¹, LI Guo-feng², ZHANG Wei-ming¹, ZHOU Dian-yuan¹. Preparation oral liposome-encapsulated recombinant Helicobacter pylori heat shock protein 60 vaccine for prevention of Hp infection[J]. Journal of Southern Medical University, 2005, 25(05): 531-534.

参考文献

[1] 白杨,张亚历,王继德,等幽门螺杆菌热休克蛋白60基因的克隆、表达及免疫原性研究[J].第一军医大学学报,2002,22(1):3-5.Bai Y, Zhang YL,Wang JD, et al. Study on the cloning expression and the immunogenicity of Helicobacterpylori heat shock protein 60 gene[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2002,22(1): 3-5.
[2] 白杨,张亚历,王继德,等.幽门螺杆菌4种黏附素基因保守区的克隆、序列及其生物信息学分析[J].第一军医大学学报,2002,22(10):869-71.Bai Y, Zhang YL, Wang JD, et al. Conservative region of the genes encoding four adhesions of Helicobacterpylori: cloning, sequence analysis and biological information analysis [J].J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2002, 22(10): 869-71.
[3] 白杨,张兆山,王继德,等.幽门螺杆菌粘附素基因hpaA的克隆及表达[J] 第一军医大学学报,2000,20(3):210-3.Bai Y, Zhang ZS,Wang JD, et al. Cloning and expression of adhesion gene hpaA of Helicobacter pylori [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2000, 20(3): 210-3.
[4] Gunawan E, Tsuji S, Tsujii M, et al. Influences of Helicobacter pylori on gastric angiogenesis and ulcer healing in mice [J]. J Gastroenterol Hepatol, 2002, 17(9): 960-5.
[5] 郭刚,王毅超,刘开云,等蒙古沙鼠感染幽门螺杆菌后的胃部病理学变化研究[J].中国人兽共患病杂志,2002,18(3):25-28.Guo G, Wang YC, Liu KY, et al. Establishment ofa Mogolian gerbil model infected steadily with Helicobacterpylori [J]. Chin J Zoonotic, 2002, 18(3): 25-8.
[6] Yamaguchi H, Osaki T, Kai M, et al. Immune response against a cross-reactive epitope on the heat shock protein 60 homologue of Helicobacter priori[J]. Infect Immun, 2000, 68(6): 3448-54.
[7] 李明峰,何志勇,凌贞,等.幽门螺杆菌HspA融合蛋白口服疫苗的构建[J].生物学与生物物理学报,2001,33(3):360-4.Li MF, He ZY, Ling Z, et al. A candidate oral vaccine to Helicobacterpylori: fusion protein of HspA and CtxB [J]. ACTA Biochem Biophys, 2001, 33(3): 360-4.
[8] Deepe GS Jr, Gibbons RS. Cellular and molecular regulation of vaccination with heat shock protein 60 from Histoplasma capsulatura [J]. Infect Immun, 2002, 70(7): 3759-67.
[9] Gregoriadis G, Bacon A, Caparros-Wanderley W, et al. A role for liposomes in genetic vaccination [J]. Vaccine, 2002, 20(Suppl 5):B1-9.
[10] Ninomiya A, Ogasawara K, Kajino K, et al. Intranasal administration of a synthetic peptide vaccine encapsulated in liposome together with an anti-CD40 antibody induces protective immunity against influenza A virus in mice [J]. Vaccine, 2002, 20(25-26): 3123-9.
[11] Wright JC, Williams JN, Christodoulides M, et al. Immunization with the recombinant PorB outer membrane protein induces a bactericidal immune response against Neisseria meningitidis [J].Infect Immun, 2002, 70(8):4028-34.
[12] 陈晓光,杨培梁,龚娅,等.适当比例的DNA:脂质体混合物能显著增强DNA疫苗的免疫效果[J].中国人兽共患病杂志,2002,18(1):12-6.Chen XG, Yang PL, Gong Y, et al. Lipid DNA complex with suitable ratio can increase the immunological efficiency of DNA vaccine [J]. Chin J Zoonotic Dis, 2002, 18(1): 12-6.