载淋球菌PorB基因壳聚糖纳米粒的制备及其诱导小鼠的免疫应答
发布时间：2019-01-21 18:03
【摘要】：目的:以壳聚糖为基质材料,制备淋球菌porB基因缓释微球,通过鼻黏膜途径及肌注途径免疫BALB/c小鼠,分析其免疫活性,探讨其能否增强特异性体液免疫和细胞免疫应答水平,为壳聚糖纳米粒作为淋球菌DNA疫苗载体的应用提供实验依据。 方法:采用复凝聚法制备CS-pcDNA3.1(-)/porB微球,分别用透射电镜,激光粒度分析仪观察微球形态、大小、zeta电位;核酸酶研究其保护性; DNA电泳阻滞分析CS-pcDNA3.1(-)/porB纳米粒结合能力;释放实验评价CS-pcDNA3.1(-) /porB的稳定性;用MTT法评价CS-pcDNA3.1(-)/porB的细胞毒性高低。将壳聚糖-pcDNA3.1(-)/porB纳米粒和裸质粒pcDNA3.1(-)/porB纳米粒通过鼻饲和肌注途径免疫雌性BALB/c小鼠,ELISA法测定小鼠体液免疫和细胞免疫应答水平。 结果: (1)壳聚糖与pcDNA3.1(-)/porB质粒形成稳定的微球,包封率大于90%,多数成球形,直径在100-300nm之间。 (2)壳聚糖纳米粒能有效抵抗核酸酶的降解作用,4℃保存45d,壳聚糖纳米粒仍能较稳定地和porB质粒结合。MTT毒性试验证明壳聚糖-pcDNA3.1(-)/porB纳米粒在高剂量才出现低细胞毒性。 (3) CS-pcDNA3.1(-)/porB与裸质粒pcDNA3.1(-)/porB组通过鼻黏膜免疫及肌肉注射免疫均能诱导小鼠产生有效的免疫应答。CS-pcDNA3.1(-)/porB诱导的血清特异性IgG和生殖道灌洗液特异性sIgA抗体水平随免疫时间呈上升趋势,与裸质粒pcDNA3.1(-)/porB组相比差异显著(P0.05),同时CS-pcDNA3.1(-)/porB鼻饲和肌注免疫组小鼠脾淋巴细胞经特异性抗原刺激后,脾淋巴细胞诱生的IFN-γ和IL-4含量明显升高,与裸质粒pcDNA3.1(-)/porB组、PBS和壳聚糖对照组之间差异具有显著性(P0.01);脾淋巴细胞刺激指数(分别为1.54±0.28和1.63±0.27)明显高于裸质粒pcDNA3.1(-)/porB组、壳聚糖组和PBS组(P 0.05 );鼻饲途径的CS-pcDNA3.1(-)/porB组、裸质粒pcDNA3.1(-)/porB组血清IgG亚类IgG2a/IgG1比值分别为1.441、1.745 ,肌注途径的CS-pcDNA3.1(-)/porB组、裸质粒pcDNA3.1(-)/porB组血清IgG亚类IgG2a/IgG1比值分别为1.387、1.761。 结论: (1) porB基因可与壳聚糖形成稳定的缓释微球,并能在293T细胞内表达; (2)壳聚糖纳米粒能增强pcDNA3.1(-)/porB核酸疫苗的鼻饲及肌注免疫效果; (3) pcDNA3.1(-)/porB核酸疫苗主要诱导Th1型倾向性免疫应答。
[Abstract]:Objective: to prepare sustained release microspheres of porB gene from Neisseria gonorrhoeae and immunize BALB/c mice by nasal mucosal pathway and intramuscular injection. To explore whether it can enhance specific humoral immunity and cellular immune response, and to provide experimental basis for the application of chitosan nanoparticles as DNA vaccine carrier of Neisseria gonorrhoeae (Neisseria gonorrhoeae). Methods: CS-pcDNA3.1 (-) / porB microspheres were prepared by complex coacervation method. The morphology, size and zeta potential of CS-pcDNA3.1 (-) / porB microspheres were observed by transmission electron microscope (TEM) and laser particle size analyzer respectively. The binding ability of CS-pcDNA3.1 (-) / porB nanoparticles was analyzed by DNA electrophoresis, the stability of CS-pcDNA3.1 (-) / porB was evaluated by release assay, and the cytotoxicity of CS-pcDNA3.1 (-) / porB was evaluated by MTT assay. Chitosan-pcDNA3.1 (-) / porB nanoparticles and bare plasmid pcDNA3.1 (-) / porB nanoparticles were used to immunize female BALB/c mice by nasal feeding and intramuscular injection. Humoral and cellular immune responses were measured by ELISA method. Results: (1) chitosan and pcDNA3.1 (-) / porB plasmids formed stable microspheres, the encapsulation efficiency was greater than 90, most of them were spherical, and the diameters were between 100-300nm. (2) chitosan nanoparticles could effectively resist the degradation of nuclease and were stored at 4 鈩，
本文编号：2412891
[Abstract]:Objective: to prepare sustained release microspheres of porB gene from Neisseria gonorrhoeae and immunize BALB/c mice by nasal mucosal pathway and intramuscular injection. To explore whether it can enhance specific humoral immunity and cellular immune response, and to provide experimental basis for the application of chitosan nanoparticles as DNA vaccine carrier of Neisseria gonorrhoeae (Neisseria gonorrhoeae). Methods: CS-pcDNA3.1 (-) / porB microspheres were prepared by complex coacervation method. The morphology, size and zeta potential of CS-pcDNA3.1 (-) / porB microspheres were observed by transmission electron microscope (TEM) and laser particle size analyzer respectively. The binding ability of CS-pcDNA3.1 (-) / porB nanoparticles was analyzed by DNA electrophoresis, the stability of CS-pcDNA3.1 (-) / porB was evaluated by release assay, and the cytotoxicity of CS-pcDNA3.1 (-) / porB was evaluated by MTT assay. Chitosan-pcDNA3.1 (-) / porB nanoparticles and bare plasmid pcDNA3.1 (-) / porB nanoparticles were used to immunize female BALB/c mice by nasal feeding and intramuscular injection. Humoral and cellular immune responses were measured by ELISA method. Results: (1) chitosan and pcDNA3.1 (-) / porB plasmids formed stable microspheres, the encapsulation efficiency was greater than 90, most of them were spherical, and the diameters were between 100-300nm. (2) chitosan nanoparticles could effectively resist the degradation of nuclease and were stored at 4 鈩，
本文编号：2412891
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