不可分型流感嗜血杆菌外膜蛋白P6基因真核质粒的保护性研究

Research on the protective immunity of the eukaryotic vector for the gene encoding the outer membrane protein P6 of the nontypeable Haemophilus influenzae

目的:探讨不可分型流感嗜血杆菌(nontypeable Haemophilus influenzae,NTHi)外膜蛋白P6(outer membrane proteinP6,P6)真核质粒的免疫保护性及其可能的保护机制。方法:构建pcDNA3.1/HisA-P6核酸疫苗,转染HeLa细胞;将P6质粒免疫BALB/c小鼠后,ELISA法检测小鼠脾淋巴细胞IFN-γ和IL-4产生水平;CCK-8法分析脾淋巴细胞增殖情况,并建立小鼠NTHi感染模型,检测小鼠鼻咽部灌洗液NTHi数目变化;HE染色法分析鼻黏膜病理变化。结果:pcDNA3.1/HisA-P6在HeLa细胞中有目的蛋白的表达,免疫后pcDNA3.1/HisA-P6组小鼠脾淋巴细胞产生的IFN-γ及刺激指数均高于pcDNA3.1/HisA、PBS对照组(P<0.05),而IL-4的表达无差异;P6组鼻咽部NTHi清除率也显著高于对照组(P<0.01);病理显示P6组小鼠鼻黏膜组织结构基本正常,而对照组鼻黏膜紊乱、脱落。讨论:P6核酸疫苗可诱导小鼠产生明显的抗NTHi保护效应,其可能的保护机制包括细胞免疫、体液免疫及黏膜免疫。

Objective To discuss the protective immunity of the eukaryotic vector for the gene encoding the outer membrane protein P6 （P6）of the nontypeable Haemophilus influenzae （NTHi） and its possible protective mechanism. Methods pcDNA3.1/HisA-P6 was constructed and transfected into HeLa cells.Then the plasmids was used to immunize BALB/c mice,IFN-γand IL-4 producted by spleen lymphocyte was detected by ELISA; The proliferation of spleen lymphocyte was analysed by CCK-8 ; The NTHi infection model was also established, then the nasopharyngeal clearance of NTHi and the nasal mucosa pathological changes were assessed. Results The mice spleen lymphocyte immunized with pcDNA3.1/HisA-P6 evoked a higher production of IFN-γ and stimulation index than controls which were immunized by pcDNA3.1/HisA or PBS （P 〈 0.05）, while there were no significant difference on IL-4 among them; The nasopharyngeal clearance of NTHi immunized with pcDNA3.1/HisA-P6 was also higher than controls （P 〈 0.01 ） ; Pathological mechanism showed the nasal mucosa immunized with pcDNA3.1/ HisA-P6 were nearly normal while the nasal mucosa of controls disordered and fell off. Conclusion These data display the eukaryotic vector for P6 induces obviously protective effect against NTHi in the BALB/c mice model, the possible protection mechanism may include both cellular immunity and humoral immunity.