禽多杀性巴氏杆菌ptfa基因壳聚糖纳米DNA疫苗的制备与检测

Preparation and detection of the ptfachitosan nanoparticle DNA vaccine of avian Pasteurella multocida

为制备禽多杀性巴氏杆菌ptfa基因壳聚糖纳米DNA疫苗,PCR扩增获得ptfa基因片段,克隆于真核表达载体pcDNA3.1（＋）中构建重组质粒,以复凝聚法制备ptfa基因的壳聚糖纳米DNA疫苗。通过凝胶阻滞试验确定壳聚糖与DNA分子完全结合时的N/P比值;测定纳米DNA疫苗的包封率;透射电镜观察纳米DNA疫苗的形态;同时检测其抗DNA酶降解的能力及稳定性。结果成功构建禽多杀性巴氏杆菌ptfa基因的裸DNA疫苗并制备出纳米DNA疫苗,该纳米DNA疫苗的N/P比值为2.5,包封率为95.3%,经电镜观察显示其形态大多呈规则的球状,粒径为200nm左右,且具有较好的稳定性,可有效抵抗DNA酶Ⅰ的降解。从而为进一步研究其免疫保护效果奠定了一定的基础。

To prepare the chitosan nanoparticle DNA vaccine of avian Pasteurella multocida, the ptfa gene fragment amplified by PCR from avian Pasteurella multocida genome was cloned into the eukaryotic expression vector pcDNA3.1（＋）, and the recombinant plasmid pcP was obtained. Then complex coacervation was adopted to prepare nanoparticle DNA vaccine of ptfa gene. The N/ P ratio was determined by gel retardation assasy. The encapsulation efficiency was detected and the type of nanoparticle DNA vaccine was observed by transmission electron microscope （TEM） along with determination of stability and the ability of anti DNA enzyme degradation. The results showed recombinant plasmid and the chitosan nanoparticle DNA vaccine were prepared successful- ly. The N/P was 2.5 ： 1 and the encapsulation efficiency was 95.3%. The nanoparticle presented regular sphere shape and the diameter was about 200 nm under the electronic microscope. In addation,the ptfa chitosan nanoparticle DNA vaccine had a satisfactory stability and could resist the degradation of DNase I. This study laid a foundation for further research of immune efficiency of the ptfa chitosan nanoparticle DNA vaccine.