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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.
OBJECTIVE: To develop an oral DNA mucosal immunization contraceptive vaccine for chitosan-zona pellucida.Methods: The eukaryotic plasmid expression vector pVAX1-pZP3α containing zona pellucida-specific antigen pZP3αDNA was constructed by genetic engineering and then coated with chitosan C390 pVAX1-pZP3α recombinant plasmids were used to prepare the immune microcapsules. The encapsulation efficiency of the immunized microcapsules to the plasmid DNA was determined. The morphological structure of the chitosan-DNA microcapsules was observed with atomic force microscope (AFM). HeLa cells were transfected with in vitro immunofluorescence (IIF) was used to detect the transient expression of pZP3α.Results: The pZP3α DNA was correctly inserted into vector pVAX1 and formed nano-immunized microcapsules with chitosan C390.The encapsulation rate of recombinant plasmid pVAX1-pZP3α was more than 90% .The results of AFM imaging showed that these The diameter of microcapsules ranged from 100nm to 300nm with an average of 224 6nm.The shape of the microcapsules was spherical and oval.The expression of pZP3α in cells was detected by IIF method in HeLa cells.Conclusion: The successful preparation of orally immunized DNA nanocapsules has laid the foundation for the further development of safer, more convenient and effective zona pellucida DNA contraceptive vaccines.