恙虫病东方体Karp株47kD蛋白基因的表达及DNA免疫初步研究

Expression of 47kDa protein gene from Orientia tsutsugamushi Karp strain and preliminary studies on its DNA immunization

目的 观察恙虫病东方体Karp株相对分子质量 (Mr)为 47× 10 3 蛋白基因的DNA免疫效果。方法 将恙虫病东方体Karp株Mr 为 47× 10 3 的蛋白基因插入真核表达载体pcDNA3.1( +) ,构建重组质粒pcDNA3.1 47。在证明该重组质粒可在哺乳动物COS7细胞中表达的基础上 ,单独和将其与Mr 为 40× 10 3 重组蛋白联合免疫小鼠。在每次加强免疫之后 10d ,检测小鼠体液和细胞免疫反应。结果 质粒DNA和蛋白联合免疫组所诱导的抗体水平和脾淋巴细胞增殖反应均高于重组质粒DNA和蛋白单独免疫组 ,而第二次加强免疫后重组质粒诱导脾淋巴细胞的增殖情况则显著弱于其它免疫组 (P <0 .0 5 )。结论 重组质粒pcDNA3 .1 47和重组Mr 为 47× 10 3 蛋白在刺激小鼠免疫反应上具有相互加强作用。

Objective To study the immunogenicity of the 47kDa protein gene from Orientia tsutsugamushi Karp strain. Methods The 47kDa antigen gene was cloned into the BamHⅠ/KpnⅠsites of the pcDNA 3.1(+) expression vector (Invitrogen). After verifying that the 47kDa antigen gene could be expressed in eukaryotic cells, 100μg of recombinant plasmids, 50μg 40kDa proteins, or 100μg of recombinant plasmids and 50μg 40kDa proteins (pcDNA3.1/47/40) were administered in three groups of 6-week-old femal BALB/c (10 mice/group); 100μg of control plasmids (pcDNA3.1) or 100μl PBS buffer were administered in two control groups of mice. Thirty and sixty days later, they were boosted twice. Antibody responses and T cell proliferation were then detected. Results The significant IgG rise was found in mice immunized with 40kDa protein, but less than that of the animals immunized with pcDNA3.1/47/40. The spleen cells from pcDNA3.1/47/40 immunized mice showed a better proliferation than rest groups (P<0.05). However, after the second boost immunization, the group of pcDNA3.1/47 showed a less proliferation than other groups (P<0.05). Conclusion Immunizing mice simultaneously with pcDNA3.1/47 and 40kDa protein induce strong immune response. But immunological tolerance may also be induced by pcDNA3.1/47 immunization. [