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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 investigate the construction method of polyvalent vaccine pVAX1 / SjHGPRT.SDISP against Schistosoma japonicum, and to verify the success of this method based on gene and protein levels. Methods: SjHGPRT and SjSDISP were amplified by PCR using pcDNA3.0 / SjHGPRT and pcDNA3.0 / SjSDISP as templates respectively. The full length SjHGPRT.SDISP was amplified by overlap method. The purified products SjHGPRT.SDISP and pVax1 plasmid using Kpn I and Xba I double digestion, 1% low melting point gel recovery of the target DNA fragment and digested Pvax1 vector fragment double glue. The ligation product was transformed into DH5α cells. Positive clones were selected and double-digested and sequenced to identify if they were successfully constructed at the genetic level. The constructed products were immunized to observe the animals, and immunohistochemistry was used to determine whether the vaccine pVAX1 / SjHGPRT.SDISP was successfully constructed from the protein level. Results: The results of enzyme digestion and sequencing showed that the inserted sequences of the recombinant plasmids were respectively identical with the target gene. Immunohistochemical staining of muscular tissue of Kunming mice showed strong brown granules in the muscle cells of immunized mice and negative Myocytes in the control group were negative. CONCLUSION: The eukaryotic recombinant plasmid pVAX1 / SjHG-PRT.SDISP was successfully constructed and expressed well in Kunming mouse muscle cells.