刚地弓形虫缓殖子期特异抗原BSR4的重组表达与免疫特性的初步研究

Recombinant Expression and Immunologic Identification of Bradyzoite-specific Antigen BSR4 of Toxoplasma gondii

目的重组表达刚地弓形虫(Toxoplasma gondii)Prugniaud(PRU)株缓殖子期特异抗原(BSR4)基因,并检测其免疫特性。方法将已构建的重组表达质粒pET28a(+)-BSR4转化至大肠埃希菌(E.coli)BL21中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达并纯化。分别以慢性感染PRU株刚地弓形虫小鼠血清和健康小鼠血清为一抗,用蛋白质印迹(Western blottting)鉴定BSR4重组蛋白的免疫反应性。用3H-TdR掺入法检测慢性感染PRU株刚地弓形虫小鼠脾淋巴细胞对不同浓度BSR4重组蛋白的特异性增殖水平,计算刺激指数(SI)。以BSR4重组蛋白为抗原,用ELISA法检测急性(抗弓形虫IgG-IgM+)和慢性(抗弓形虫IgG+IgM-)弓形虫病患者血清,以及健康人血清,各20份,评判该蛋白的免疫反应性。结果重组质粒pET28a(+)-BSR4经IPTG诱导后表达重组蛋白BSR4,经变性、复性和纯化后获得相对分子质量(Mr)45 000的可溶性蛋白。Western blotting分析结果显示,该蛋白能被慢性弓形虫感染小鼠血清识别。脾淋巴细胞增殖试验表明,1、5和25μg/ml重组蛋白BSR4刺激感染弓形虫小鼠脾淋巴细胞的SI分别为1.13、0.88和1.17,显著高于未感染组(分别为0.46、0.24和0.49,均P<0.01)。ELISA检测结果显示,BSR4抗原能被慢性弓形虫病患者血清(IgG+IgM-)特异性识别(20/20),而不能被急性弓形虫病患者血清(IgG-IgM+)识别(0/20)。结论重组BSR4蛋白具有特异的免疫原性和免疫反应性。

Objective To express the recombinant BSR4 protein of Toxoplasma gondii Prugniaud （PRU） strain and study its immunologic characteristics. Methods The recombinant plasmid pET28a（＋）-BSR4 was transformed into E. coli BL21, followed by expression of BSR4 induced by IPTG and its purification. The immunoreactivity of the recombinant protein BSR4 was analyzed by Western blotting with the sera from mice infected by PRU strain of T. gondii or normal mice as the first antibody. The 3H-TdR incorporation assay was performed to determine the proliferation of splenocytes in mice infected by PRU strain stimulated by BSR4, and the stimulation index （SI） was calculated. ELISA was used to evaluate the immunoreactivity of BSR4 protein, in which 20 sera from each of acute （anti-T. gondii IgG-IgM~） and chronic （IgGqgM-） toxoplasmosis patients, and healthy people were tested. Results The recombinant BSR4 was induced and expressed. After denaturation, renaturation and purification, the soluble protein （Mr 45 000） was obtained and detected by anti-T, gondii serum with Western blotting. BSR4 in 1, 5, and 25 μg/ml induced the proliferation of splenocytes in mice infected by PRU strain with higher SI （1.13, 0.88, and 1.17） than that of control （0.46, 0.24, and 0.49, respectively, P〈0.01）. ELISA showed that the recombinant BSR4 specifically reacted with the sera of toxoplasmosis patients （IgGqgM-, 20/20）, not with that of the acute cases （IgG-IgM, 0/20）. Conclusion The recombinant BSR4 shows specific immunogenicity and immunoreactivity.