不可分型流感嗜血杆菌 LPD 基因分布及其重组表达产物免疫保护作用

摘要

目的：了解无荚膜型流感嗜血杆菌（NTHi）临床菌株中外膜脂蛋白 D（LPD）基因分布及其序列保守性，确定重组表达的 LPD（rLPD）免疫原性和免疫保护性。方法采用 PCR 检测 NTHi临床菌株中 LPD 基因，T-A 克隆后测序。构建 LPD 基因原核表达系统，采用 Ni-NTA 亲和层析法提纯表达的 rLPD。采用 SDS-PAGE 和 Bio-Rad 凝胶图像分析系统检测 rLPD 表达情况及其产量。采用ELISA 和 Western blot 检测 rLPD 的抗原性和免疫反应性。采用小鼠感染模型了解 rLPD 对 NTHi 致死性感染的免疫保护效果。结果所有 NTHi 菌株均检出 LPD 基因，其核苷酸和氨基酸序列相似性分别高达98.0％~99.4％和98.5％~100％。所构建的原核表达系统能高效表达 rLPD。rLPD 免疫家兔可产生高效价抗体，NTHi 全菌兔抗血清及 NTHi 感染患儿血清能识别 rLPD 并与之结合。ELISA 结果显示，93.6％（58/62）和53.2％（32/62）NTHi 感染患儿血清分别 rLPD-IgM 和 rLPD-IgG 阳性。100μg或200μg rLPD 对 NTHi 致死性感染小鼠的免疫保护率分别为60.0％或73.3％。结论 LPD基因在 NTHi 菌株中广泛分布且序列保守，其重组表达产物 rLPD 具有良好的免疫原性及一定的免疫保护作用，可作为 NTHi 基因工程疫苗候选抗原之一。%Objective To investigate the distribution and sequence conservation of genes encoding the outer membrane lipoprotein D(LPD)of nontypeable Haemophilus influenzae(NTHi)isolates and to ana-lyze the immunogenicity and the immunoprotective effects of the expressed recombinant LPD(rLPD). Meth-ods PCR analysis was used to detect the genes encoding LPD of NTHi isolates. The PCR products were se-quenced after T-A cloning. A prokaryotic expression system for genes encoding LPD was established to ex-press the rLPD. Ni-NTA affinity chromatography was used for purification. SDS-PAGE and Bio-Rad Gel Im-age Analyzer were used to detect the expression and the yield of rLPD. The antigenicity and immunoreactivity of rLPD were detected by ELISA and Western blot assay. The immunoprotective effects of rLPD against lethal dose of NTHi were evaluated in a mouse model. Results All of the tested NTHi isolates were positive for the genes encoding LPD. They shared 98. 0% -99. 4% homologies in nucleotide sequences and 98. 5% -100% homologies in amino acid sequences. The established prokaryotic expression system expressed rLPD with a high yield. High levels of antibody in rabbits were induced by the rLPD. The anti-NTHi antiserum samples from rabbits and children could recognize and react with the rLPD. The result of ELISA indicated that 93. 6%(58 / 62)and 53. 2%(32 / 62)of the serum samples from children with NTHi infection were positive for rLPD-IgM and rLPD-IgG,respectively. The rLPD at concentrations of 100 μg and 200 μg could respectively protect 60. 0% and 73. 3% of mice from lethal NTHi infection. Conclusion The genes enco-ding LPD were extensively distributed in NTHi isolates with high sequence conservation. The expressed rLPD could be used as a potential candidate antigen in the development of genetic engineering vaccine against NTHi infection considering its high immunogenicity and immunoprotective effects.