罗金，杨菁，程琰，张怡，徐望明

(武汉大学人民医院生殖医学中心，武汉　430060)



·实验研究·

以壳聚糖纳米微球为载体的Crisp1-DNA避孕疫苗的制备及体外表达

罗金，杨菁*，程琰，张怡，徐望明

(武汉大学人民医院生殖医学中心，武汉430060)

目的构建以壳聚糖纳米微球为载体的Crisp1 DNA疫苗，对其物理学、生物学活性进行测定，并评估其在COS-7细胞中的表达情况及细胞毒性。方法采用本实验室前期构建的真核表达质粒pcDNA3.1-Crisp1，以复凝法制备载Crisp1 DNA疫苗的壳聚糖纳米粒(CS/DNA NPs)，用透射电镜以及凝胶阻滞分析对其相关物理学、生物学活性进行测定，然后将载Crisp1 DNA疫苗的壳聚糖纳米粒转染至COS-7细胞，检测其细胞毒性，并用间接免疫荧光法鉴定其在细胞内的表达情况。结果透射电镜结果显示纳米粒形态较均一，平均粒径为189.3 nm，Zeta电位约为+0.2 mV。凝胶阻滞分析显示壳聚糖微粒可将DNA疫苗完全阻滞于加样孔中并保护DNA质粒不降解。MTT试验显示壳聚糖组细胞存活率显著高于脂质体组(P<0.01)。间接免疫荧光实验结果显示CS/DNA NPs能在COS-7细胞中有效表达Crisp 1抗原蛋白。结论由壳聚糖纳米微球介导的Crisp1 DNA疫苗，可在真核细胞中有效地表达，且具有较小的细胞毒性，为进一步发展安全有效的DNA避孕疫苗打下了基础。

Crisp-1；避孕；DNA疫苗；壳聚糖；纳米微球

Methods：The eukaryotic expression plasmid pcDNA3.1-Crisp1 was constructed previously and encapsulated in nanoparticles with chitosan by complex coacervation process. Transmission electron microscope (TEM) and Gel retardation assay were used to observe the physical and biological characteristics of the chitosan/pcDNA3.1-Crisp1 nanoparticles. Then，the chitosan/pcDNA3.1-Crisp1 nanoparticles (CS/DNA NPs) were transfected to COS-7 cells，detecting its cytotoxicity and evaluating its expression by indirect immunoflurescence.

Results：TEM showed that chitosan/pcDNA3.1-Crisp1 nanoparticleshad symmetrical shape，with a diameter about 189.3 nm，and zeta potential was+0.2 mV. Gel retardation assay showed that pcDNA3.1-Crisp1 contained in chitosan nanoparticles could be completely retarded and remained intact after DNase I treatment. MTT assay showed that the viability of cells incubated with chitosan-DNA was significantly higher than that of the control Lipofect 2 000 group (P<0.01). Indirect immunouorescence showed that pcDNA3.1-Crisp1 could expressed effectively in COS-7 cells.

Conclusions：Chitosan nanoparticles containing Crisp1-DNA contraceptive vaccine can express effectively in eukaryotic cells and have less cytotoxicity. Chitosan can be used as an effective non-virus DAN vaccine carrier.

(JReprodMed2016，25(10)：947-952)

富含半胱氨酸分泌蛋白-1(cysteine-rich secretory protein-1，Crisp1)是由附睾头部的上皮细胞合成分泌的一种雄激素依赖型糖蛋白。由于在生殖过程中的多个环节起着重要作用，如调控精子的获能、参与精卵融合等，Crisp1被认为是一种极具前景的候选避孕疫苗[1-3]。在前期的研究中，本实验室成功的构建了真核表达载体pcDNA3.1-Crisp1[4]，并证实该疫苗可在小鼠体内诱导特异性免疫反应，但避孕效果不尽理想[5-6]。其原因可能是：(1)由于裸DNA带负电荷，难以通过脂质的细胞膜；(2)在体内易被核酸酶降解而迅速清除[7]；……