摘要
目的:发展壳聚糖-卵透明带口服DNA粘膜免疫避孕疫苗.方法:首先利用基因工程技术构建含卵透明带特异抗原pZP3αDNA的真核质粒表达载体pVAX1-pZP3α,然后用壳聚糖C390包被pVAX1-pZP3α重组质粒制备免疫微囊,测定免疫微囊对质粒DNA的包裹率,用原子力显微镜(AFM)观察壳聚糖-DNA微囊的形态结构,并体外转染HeLa细胞,用间接免疫荧光法(IIF)检测pZP3α的瞬时表达.结果:pZP3αDNA正确插入到载体pVAX1中并与壳聚糖C390形成纳米免疫微囊,对重组质粒pVAX1-pZP3α的包裹率达90%以上.AFM成像技术显示这些微囊的直径为100nm到300nm不等,平均为224 6nm,形状为球形、椭圆形等.体外转染HeLa细胞用IIF法检测到了抗原pZP3α在细胞内的表达.结论:壳聚糖-卵透明带口服DNA纳米免疫微囊制备成功,为进一步发展更安全、方便、有效的卵透明带DNA避孕疫苗打下了基础.
Aim:To develop the oral immunocontraceptive DNA vaccines with chitosan which easily forms nannoparticles. Methods: The cDNA for porcine zona pellucida-3α(pZP3α), without the N-terminal signal sequence and C-terminus transmembrane-like domain, was cloned in mammalian expression vector pVAX1. The recombinant pVAX1-pZP3α was encapsulated with chitosan C390 to nanoparticles. The morphology of chitosan/pVAX1-pZP3α nanoparticles was determined by Atomic Force Microscope(AFM). Encapsulated efficiency of chitosan/pVAX1-pZP3α nanoparticls was also determined. pVAX1-pZP3α plasmid DNA encapsulated with chitosan was transfected into HeLa cells in vitro for determining transient expression by indirect immunofluorescence. Results: The pZP3α DNA had been inserted into pVAX1 correctly. The imaging by AFM demonstrated that the diameter of the particles is different from 100 nm to 300 nm(the mean diameter is 224.6 nm)and the shape is spherical or elliptical et al. The result of encapsulated efficiency of chitosan/pVAX1-pZP3α nanoparticls shows that more than 90% pVAX1-pZP3α DNA is encapsulated with chitosan. Transfected Hela cell can react with anti-pZP3 antibody, showing bright-green fluorescence in the indirect immunofluorescence(IIF)assay which indicates the expression of recombinant pZP3α protein in vitro. Conlusion: Chitosan/zona pellucida DNA nanoparticles have been preparated successfully, which will provide new ideas and ways for research for more effective, more convenient oral immunocontraceptive vaccines.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
2004年第1期102-109,共8页
Journal of Jinan University(Natural Science & Medicine Edition)
关键词
卵透明带
DNA疫苗
壳聚糖
纳米微囊
免疫避孕
zona pellucida(ZP)
DNA vaccine
chitosan
nanoparticles
immunocontraception
