壳聚糖季铵盐(HTCC)载HSV-2型DNA疫苗pgD诱导小鼠免疫应答的初步研究

Preliminary research of 3-chlorine-2-hydroxypropyl trimethyl chitosan(HTCC)-pgD nanoparticles on immune responses to HSV-Ⅱ DNA vaccine in mice

目的:制备了壳聚糖季铵盐-DNA疫苗(pgD)纳米粒复合物,并对其诱导BALB/c小鼠产生免疫应答的能力做了初步研究。方法:通过复凝聚法制备了HTCC-pgD纳米粒复合物并对其进行表征。60只BALB/c小鼠随机分成5组:①HTCC10对照组;②pcDNAkan空载体对照组;③重组质粒pgD组;④HTCC∶pgD=5∶1组;⑤HTCC∶pgD=10∶1组。各组小鼠后腿肌肉注射,注射剂量为100μg/(只.次),隔2周免疫1次,共免疫2次。末次免疫2周后,通过流式细胞仪检测外周血中CD4+、CD8+百分率,ELISA法检测血清中IgG抗体效价、IFN-γ、IL-4含量这4个方面来研究该纳米粒复合物对小鼠免疫反应的影响。结果:HTCC能与质粒pgD很好结合成纳米复合物,保护DNA免受核酸酶的降解。小鼠经免疫2次后,流式细胞仪检测外周血中CD4+T淋巴细胞亚群结果显示HTCC-pgD纳米粒复合物组的CD4+百分率要高于其他对照组(P<0.05)。血清ELISA检测IgG结果显示与对照组相比,HTCC-pgD纳米粒复合物可诱导小鼠机体产生较高滴度的IgG抗体。HTCC-pgD纳米粒复合物组的小鼠血清中IFN-γ水平与对照组相比有所上调,IL-4水平有下降。结论:通过复凝聚法制备的HTCC-pgD纳米粒复合物可诱导BALB/c小鼠产生特异性的细胞免疫和体液免疫,为HSV-2型DNA疫苗研制提供科学依据。

Objective：The DNA vaccine expressing herpes simplex virus type II（ HSV -2） glycoprotein D （gD） and 3 -chlo- rine -2 -hydroxypropyl trimethyl chitosan （HTCC） was prepared as a new nanoparticle composite, and its ability to induce im- mune response in mice was investigated. Methods： The nanoparticle composites were synthesized by complex coacervation meth- od with HTCC and plasmid DNA and the characteristics were evaluated. Female BALB/c mice were randomly divided into five groups （HTCC10 control group, pcDNAkan group, pgD group, HTCC： pgD =5：1 group and HTCC： pgD = 10：1 group） and im- munized respectively with once intramuscular injection every 3 weeks for twice. Two weeks after the last injection, blood samples were collected and assessed with ELISA to detect IgG. CD4^＋ and CD8^＋ T cells after immunization were detected by flow cytometry. ELISA was used to detect cytokine level in serum, such as IFN -γ and IL - 4. Results： HTCC could form nanoparticle compos- ites with plasmid DNA. HTCC - pgD nanoparticles renders a significant protection of the plasmid. Compared with the control groups, the mice inoculated with HTCC - pgD nanoparticles could produce higher levels of antibody. The HTCC - pgD nanoparti- cles groups also elicited higher levels of CD4 at 6 weeks after immunization（P 〈 0.05 ）. Serum IFN - γ level was higher in HTCC -pgD nanoparticle composites group than that in control group, while serum IL- 4 level was conversed. Conclusion： HTCC - pgD nanoparticles can both enhance the cell - mediated immunity and humoral immunity in BALB/c mice. The present study could provide a scientific basis for the HSV -2 DNA vaccine research.