摘要
目的:了解内置重组大肠杆菌不耐热肠毒素B亚单位(rLTB)佐剂、幽门螺杆菌(Helicobacter pylori,Hp)尿素酶B亚单位(rUreB)和黏附素(rHpaA)双价基因工程疫苗对Hp SS1株感染BALB/c小鼠的保护作用及其机制.方法:采用NTA-Ni亲和层析法分别收集rUreB、rHpaA、rLTB-rUreB-rHpaA、重组大肠杆菌不耐热肠毒素A亚单位突变体(rLTKA63)、rLTB和霍乱肠毒素B亚单位(rCTB).用Western blot检测各重组蛋白抗原的免疫反应性.用GM1-ELISA检测rLTB、rCTB、rLTB-rUreB-rHpaA的佐剂活性.建立Hp SS1株感染BALB/c小鼠模型,检测不同组合抗原和佐剂的免疫保护效果.采用分离培养和镀银染色法检查感染小鼠胃窦组织中的Hp.建立ELISA,检测免疫小鼠特异性胃液S-IgA和血清IgA.结果:rUreB、rHpaA和rLTB-rUreB-rHpaA可被兔抗Hp全菌抗体识别,并与牛GM1结合.单用rUreB或rHpaA 免疫小鼠,其保护率低于70%;若与rLTB或rCTB合用,其保护率可增至75.0%~83.3%.在不同抗原及佐剂组合中,rLTB-rUreB-rHpaA保护率达100%,其次为rHpaA+rUreB+rCTB+rLTKA63(91.7%)和rUreB+rHpaA+rLTB(90.9%).rLTB和rCTB均有明显的诱生免疫小鼠特异性血清IgA和胃液S-IgA作用,但前者诱生S-IgA的能力强于后者.特异性血清IgA,尤其是胃液S-IgA的阳性率与小鼠免疫保护率基本吻合,rLTB-rUreB-rHpaA免疫小鼠rUreB 和rHpaA 特异性S-IgA阳性率分别高达100%和91.7%.结论:rUreB和rHpaA有良好的免疫反应性和抗原性,rLTB和rCTB有黏膜免疫佐剂活性.内置佐剂UreB和HpaA双价重组疫苗对小鼠高保护率与使用高剂量rLTB、抗原分子量增大和局部高水平特异性S-IgA抗体有关.
To determine the immunoprotective effects of rUreB and rHpaA bivalence genetic engineering vaccine with inner adjuvant of rLTB on BALB/c mice against H. pylori strain SS1 infection. Methods : rUreB, rHpaA, rLTB rUreB-rHpaA, rLTKA63, rLTB and rLTB were collected by NTA-Ni affinity chromatography. Western blot was applied to demonstrate the immunoreactivity of the recombinant protein antigens. Adjuvant activities of rLTB,rCTB and rLTB-rUreB-rHpaA were determined by GM1-ELISA. H. pylori strain SS1-infected BALB/c mouse modal was established to measure immunoprotective effects of different compositions of antigens and adjuvants. H. pylori in gastric biopsy specimens was examined by routine isolation method and silver staining method. Two ELISAs were established to detect specific S-IgA in gastric juices and specific IgA in sera of the immunized mice. Results: rUreB,rHpaA and rLTB-rUreB-rHpaA were recognized by commercial antibody against whole cell of H. pylori and were able to combine to the bovine GM1. The protective rate in the mice immunized with single rUreB or rHpaA was lower than 70% When using rUreB or rHpaA plus rLTB or rCTB, the positive rates increased to 75. 0-83. 3%. With different combination of antigens and adiuvants,the immunoprotective rate of rLTB rUreB rHpaA was as high as 100% ,and then was 91.7% for rUreB +rHpaA+rCTB+rLTKA63 and was 90. 9% for rUreB+rHpaA+rLTB. Both the rLTB and rCTB showed remarkable effects to induce specific IgA in sera and specific S-IgA in gastric juices of the immunized mice,and the former showed stronger S-IgA inducing ability than the latter. The positive rates of specific IgA were basically identical to the corresponding mouse immunoprotective rates. S-IgA positive rates specific to rUreB and rHpaA in rLTB-rUreB-rHpaA immunized mice were 100% and 91. 7%, respectively. Conclusion: The rUreB and rHpaA possess qualified immunoreactivity and antigenicity. The H.TB and rCTB can show adjuvant activity of mucosal immunization. The high immunoprotective rate of Helicohacter pylori UreB and HpaA bivalence recombinant vaccine with inner adjuvant in mice is associated with high dosage of rLTB,larger molecular weight of the antigen and the high levels of local specific S IgA.
出处
《浙江大学学报(医学版)》
CAS
CSCD
2005年第5期405-411,共7页
Journal of Zhejiang University(Medical Sciences)
基金
国家教育部优秀年轻教师基金(教人司1999-5)