犬细小病毒VP2蛋白在真核细胞中的分泌表达及特性

Secreting expression and characterization of Canine parvovirus VP2 protein in eukaryotic cells

【目的】利用真核细胞分泌表达犬细小病毒VP2蛋白和研究其特性。【方法】为构建犬细小病毒(Canineparvovirus,CPV)VP2基因的真核分泌型表达载体,首先通过酶切从含有人CD5信号肽序列的质粒中将CD5信号肽基因片段切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2基因,并将其插入到pcDNA3.1-CD5sp载体中CD5信号肽的下游,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经磷酸钙介导转染293T细胞,使其在真核细胞中进行分泌表达,并通过ELISA检测表达的VP2蛋白与犬转铁蛋白受体(TfR)结合的活性。【结果】序列分析结果表明,本实验构建的犬细小病毒VP2基因真核分泌型表达载体结构正确,将该表达载体转染的293T细胞,在培养基中通过Western-blot检测到有VP2重组蛋白的存在。经ELISA检测表明表达的重组VP2蛋白具有与犬转铁蛋白受体结合的活性。【结论】利用人的CD5信号肽实现了犬细小病毒VP2蛋白在真核细胞中的分泌表达,表达的VP2蛋白具有与犬转铁蛋白受体结合的活性。

[Objective] To study and characterize secretive expression of canine parvovirus capsid protein 2 （VP2） gene in eukaryotic cells. [ Methods] To construct secreting expression vector of VP2 gene, we obtained CD5 signal peptide （SP） DNA fragment from plasmid containing human CD5 SP DNA sequence and inserted the fragment into multiple clone site of eukaryotic expression vector pcDNA3.1A. The canine parvovirus VP2 gene was amplified by PCR and inserted into expression vector pcDNA3.1-CD5sp down stream of CD5 SP. The recombinant pcDNA-CD5sp-VP2 plasmids were transfected into HEK293T cells mediated by calcium phosphate. VP2 binding activity for canine transferrin receptor was analyzed by ELISA method. [ Results] Recombinant pcDNA-CD5sp-VP2 plasmid proved to be correct by sequencing. VP2 proteins were detected by Western-blot in the culture medium of transfected 293T ceils, which indicated that the expressed VP2 protein could be secreted into the medium mediated by human CD5 SP. VP2 protein had the activity to bind canine transferrin receptor（TfR）. [ Conclusion] The secreting expression of VP2 in eukaryotic cells was achieved by using human CD5 SP. Recombinant VP2 showed the ability to bind canine TfR.