【作者】 张馨玉； 马一介； 金华利； 杨若耶； 肖冲； 张富春； 王宾；

【Author】 ZHANG Xin-Yu MA Yi-Jie JIN Hua-Li YANG Ruo-YeXIAO Chong ZHANG Fu-Chun WANG Bin( State Key Laboratory for Agrobiotecbnology, College of Biological Science, China Agricultural University, Beijing 100094, China Key Laboratory for Bioresources and Bioengineering, College of Life Science and Technology,Xinjiang University, Urmuqi 830046, China)

【机构】 中国农业大学生物学院农业生物技术国家重点实验室； 新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室；

【摘要】 壳聚糖(chitosan)是一种具有高密度正电荷并能够与质粒DNA形成颗粒,并在体外携带DNA转染真核细胞的多糖类物质。又由于其具有粘膜粘附性,保护DNA免受Dnase降解的性质,使其成为DNA疫苗粘膜免疫发送介导的选择之一。为了检测小分子壳聚糖能否提高DNA疫苗的粘膜免疫效果,用亚硝酸钠将壳聚糖进行了解聚得到解聚后的小分子壳聚糖(DCH)。经琼脂糖电泳检测DCH与质粒DNA成功包裹形成颗粒,经过小鼠胃液的消化,认为DCH可以一定程度的保护DNA。利用DCH包裹pCMVβ经滴鼻和口服途径进行小鼠的体内表达检测,结果显示DCH可以提高pCMVβ的表达,滴鼻途径效果好于CHI。将CHI和DCH与口蹄疫病毒(foot and mouth disease virus)的DNA疫苗pcD-VPl包裹形成纳米颗粒,通过滴鼻(i.n.)和口服(i.o.)两种不同的粘膜途径在0、14、28 d免疫C57小鼠,分别检测小鼠的特异性针对口蹄疫的生殖道部位的粘膜IgA抗体水平。结果显示经过滴鼻免疫DCH-DNA组的抗体水平显著高于CHI-DNA组和裸DNA组,但是在口服途径DCH-DNA不能显著提高抗体水平,而CHI-DNA组可以显著提高抗体水平。更多还原

【Abstract】 Chitosan (CHI) is a polysaccharide and has been demonstrated as a potential gene encapsulation and delivery system. Due to its adhesion to the mucosa surface and ability to protect the DNA from digestion by Dnase, CHI was researched as a nonviral gene delivery system for DNA vaccine. To invesitgate whether chitosan oligomers(DCH) can serve for a better gene delivery system, we first depolymerized CHI to DCH with NaNO2 and subsequently analyzed its ability of encapsulation using agarose gel. Results showed that DCH could encapsulate the plasmid DNA to form DCH-DNA complexes. To test the stability of these complexes, they were treated with gastric juice. The result indicated that DCH had the ability to protect plasmid from the digestion by gastric juice. To test the expression of the plasmid encapsulated by DCH in vivo, KM mice were intranasally and intraorally administered with DCH-pCMV-β. 7 days after the administration, the expression of plasmid pCMV-β was observed in the tissues stained by X-gal. Mice were immunized intranasally and intraorally with DCH-pcD-VP1 nanoparticles on 0, 14 and 28 d. Plasmid pcD-VP1 is a DNA vaccine against Foot-and-mouth disease virus. The Vaginal IgA was tested on 7, 21, 28 and 35 d. The IgA antibody level of the DCH-DNA group was significantly higher than that of the CHI-DNA and naked DNA group receiving intranasal administration while the IgA antibody level of DCH-DNA group did not increase receiving intraoral administration.更多还原