杜克雷嗜血杆菌血红蛋白受体和其部分蛋白片段的表达纯化及其多克隆抗体建立ELISA检测杜克雷嗜血杆菌的初步研究

Expression and purification of HgbA from Haemophilus ducreyi and its partial fragment for development of a Sandwich ELISA to detect infection of Haemophilus ducreyi using specific polyclonal antibodies

目的:制备纯化杜克雷嗜血杆菌血红蛋白受体(HgbA)及其部分蛋白片段(HgbAF),免疫家兔获得rHgbA和rHgbAF特异性多克隆抗体,用于临床软下疳病原学诊断。方法:采用分子生物学技术克隆HgbA和HgbAF基因,诱导表达并纯化rHgbA和rHgbAF;用rHgbA和rHgbAF免疫家兔制备多克隆抗体,采用Western blot和ELISA方法测定抗体免疫活性;建立杜克雷嗜血杆菌HgbA双抗体夹心ELISA检测系统,对其敏感性、特异性进行初步评价。结果:成功克隆了目的基因,经诱导表达获得纯化杜克雷嗜血杆菌rHgbA及其部分重组蛋白片段;免疫家兔获得rHgbA和rHgbAF特异性抗血清,经饱和硫酸氨盐析后,Western blot实验结果显示其能与rHgbA和rHgbAF特异性结合;建立的杜克雷嗜血杆菌HgbA双抗体夹心ELISA,对杜克雷嗜血杆菌、流感嗜血杆菌及其他7种生殖器溃疡相关的细菌进行检测,发现除杜克雷嗜血杆菌呈现强阳性反应外,其余8种菌均为阴性结果,无交叉反应发生;上述ELISA检测纯化rHgbA灵敏度为1.56 ng/ml,杜克雷嗜血杆菌检测灵敏度为2×105cfu/ml,脓汁模拟样本中杜克雷嗜血杆菌检测的灵敏度为1×106cfu/ml。结论:成功制备了杜克雷嗜血杆菌血红蛋白受体及其部分蛋白片段,免疫家兔所获得的多克隆抗体能与杜克雷嗜血杆菌特异性结合,而与流感嗜血杆菌及其他7种生殖器溃疡相关的细菌无交叉反应,表明建立的ELISA具有较好的特异性。上述ELISA检测方法的敏感性水平不能满足临床所有标本中杜克雷嗜血杆菌的检测,有待于进一步提高,但该方法的建立具有潜在临床应用价值。

Objective： To express and purify hemoglobin receptor （HgbA） and its partial fragment （HgbAF） from Haemophilus ducreyi and to develop a sandwich ELISA for the detection of H. ducreyi infection. Methods： The HgbA, a hemoglobin-binding outer membrane protein of H. ducreyi and its partial fragment （HgbAF） were expressed by cloning the genes of hgbA and its 705bp fragment into pET30a and pET28a respectively, and the expressing products were purified from E. coli BL21 with Ni-NTA-His affinity chromatography. The polyclonal antibodies were developed by immunizing rabbits with the rHgbA and rHgbAF. The anti-rHgbA IgG and anti-rHgbAF IgG were purified respectively by saturated amine sulphate precipitation, and their immunoactivity with rHgbA and rHgbAF was tested by Westen blot and ELISA analysis. A Sandwich ELISA was developed for the detection of chinical infection of H. ducreyi using the specific polyclonal antibodies. Results： The HgbA and its partial fragment, HgbAF of H. ducreyi, were expressed and purified successfully by cloning their genes respectively. The results obtained by Western blot analysis showed that each of the antibodies could react with both antigens, rHgbA and rHgbAF. The results of the ELISA analysis showed that H. ducreyi strain was strongly positive, and all other bacteria, including H. influenzae and the bacteria known to relate to genital ulcers were negative. The results of the ELISA analysis showed that the minimum amount of rHgbA detected was 1.56 ng/ml and the minimum number of CFU of H. ducreyi detected was 2 × 10^5 cfu/ml in buffer and 1 × 10^6 cfu/ml in pus. Conclusion： HgbA and its partial fragment, HgbAF of H. ducreyi are expressed and purified successfully. The pelyclonal antibodies developed by immunizing rabbits using rHgbA and rHgbAF could react not only with rHgbA and rHgbAF, but also with H. ducreyi specifically. They do not react with other bacteria, including H. influenzae and the bacteria known to relate to genital ulcers. So the ELISA based on the polyclonal antibodies was specific for the detection of H. ducreyi. Although the level of sensitivity of the ELISA may not be sufficient to detect H. ducreyi in all clinical specimens, further work to increase the sensitivity could potentially make this assay a valuable tool in areas where chancroid is endemic.