猪endoglin胞外段真核表达载体的构建及其诱导抗小鼠自身endoglin抗体的实验研究(英文)

Construction of plasmid DNA vaccine encoding extracellular domain of porcine endoglin and its induction of autoantibodies in mice

目的分别扩增猪和小鼠endoglin胞外段cDNA,构建真核表达载体作为DNA疫苗,了解猪en-doglin DNA疫苗是否能够在小鼠体内诱导产生抗小鼠endoglin的自身抗体。方法利用RT-PCR技术,从猪和小鼠胚肝中分别扩增出猪和小鼠的endoglin胞外段,用内切酶消化后分别将其插入真核表达质粒pcDNA3.1(+)中,经琼脂糖凝胶电泳和测序鉴定后,体外转染真核细胞COS-1,用RT-PCR和免疫印迹鉴定是否能正确表达。然后大量扩增和提取相应质粒,将这些质粒作为DNA疫苗,肌注免疫小鼠,通过ELISA、免疫印迹和ELISPOT测定抗自身endoglin的抗体和脾脏中分泌特异性抗endoglin抗体的B淋巴细胞。结果琼脂糖凝胶电泳和测序鉴定证实RT-PCR扩增的cDNA及构建的重组真核表达质粒正确,重组的真核表达质粒能在COS-1细胞中正确表达,猪endoglin胞外段真核质粒DNA疫苗免疫小鼠可以诱导产生抗小鼠endoglin的自身抗体。结论重组猪endoglin胞外段真核表达载体可作为DNA疫苗用于抗肿瘤血管生成的基因治疗。

Objective: To explore the possibility that a plasmid DNA vaccine encoding the extracellular domain of porcine endoglin induces production of autoantibodies against self-endoglin in mice. Methods: The cDNAs of extracellular domains of porcine and murine endoglins were amplified by RT-PCR respectively. These cDNAs were digested by restriction endonuclease and inserted into the eukaryotic-expression vector pcDNA 3.1(+). The resultant recombinant plasmids were confirmed by agarose gel electrophoresis analysis and sequencing, then transfected into eukaryotic cell COS-1 by Lipofectin. RT-PCR and Western blot were used to certify whether the purpose proteins were correctly expressed in COS-1 cells. Thereafter, the recombinant porcine plasmid was used as a DNA vaccine to explore its capability of induction of autoantibodies against self-endoglin in BALB/c mice, which was determined by ELISA, Western blot analysis and ELISPOT assay. Results: The cDNAs of porcine and murine endoglins were correctly amplified. The recombinant eukaryotic plasmids were successfully constructed, and they could be correctly expressed in the COS-1 cells. The recombinant porcine plasmid used as a DNA vaccine could induce autoantibodies against self-endoglin in mice. Conclusion: The recombinant plasmid encoding the porcine endoglin can be used as a DNA vaccine to induce autoantibodies against mouse-endoglin and provide a basis for the gene therapy of endoglin-associated tumor angiogenesis.