抗日本血吸虫pVAX1/SjHGPRT·SDISP多价疫苗的构建方法探讨

Construction Method of Multivalent DNA Vaccine pVAX1/SjHGPRT·SDISP Against Schistosoma Japonicum

目的:探讨抗日本血吸虫生殖产卵编码基因多价疫苗pVAX1/SjHGPRT.SDISP的构建方法并从基因与蛋白水平验证该构建方法是否成功。方法:分别以pcDNA3.0/SjHGPRT、pcDNA3.0/SjSDISP为模板,设计引物扩增SjHGPRT、SjSDISP,采用overlap方法扩增全长SjHGPRT.SDISP。将纯化后的产物SjHGPRT.SDISP以及pVax1质粒采用Kpn I和Xba I双酶切,1%低熔点胶回收目的 DNA片段以及酶切后Pvax1载体片段双胶连。连接产物转化至DH5α细胞。挑选阳性克隆采用双酶切以及测序方法从基因水平鉴定是否构建成功。将构建产物免疫观察动物,采用免疫组化方法从蛋白水平鉴定疫苗pVAX1/SjHGPRT.SDISP是否构建成功。结果:酶切方法以及测序结果均显示重组质粒的插入序列分别与目的基因完全一致,昆明小鼠肌肉组织酶免疫组织化学染色显示免疫组小鼠肌细胞中呈现较强的棕黄色颗粒,而阴性对照组肌细胞呈阴性。结论:真核重组质粒pVAX1/SjHG-PRT.SDISP构建成功,且在昆明鼠肌肉细胞内能够获得良好的表达。

Objective： To explore the method to construct the multivalent DNA vaccine pVAX1/SjHGPRT-SDIS and certify whether this method was successful at both gene and protein level. Methods＂ SjHGPRT and SjSDISP were amplified with the templates of pcDNA3.0/SjHGPRT and pcDNA3.0/SjSDISP. Full-length SjHGPRT-SDISP were amplified by overlap method. The purified products SjHGPRT. SDISP and plasmid pVaxl were digested with Kpn I and Xba I. 1% low melting Gel was used to collect the fragments of DNA and make the carriers＇ fragments of Pvaxl glue together after digestion. The ligated product was transformed into DH5α cell. Positive clones were screened to confirm whether the construction is successful at gene level with the method of digestion and sequencing. Use the products of construction to immune and observe animals and confirm whether the construction of vaccine pVAX1/SjHGPRT. SDISP was successful with the method of immunohistochemical staining. Results： Restriction enzyme digestion and sequencing results showed that the inserted sequence of the recombinant plasmid is completely the same with the target gene. Result of immunohistochemistry showed intense brown particles appeared in the muscle cells of Kunming mise immuned with the multivalent vaccine, while no specific fluorescence or brown particles appeared in muscle of the control groups. Conclusions： The pVAX1/SjHGPRT. SDISP was successfully constructed, which may be well expressed in the muscle of vaecined mice.