猪骨髓基质抗原2基因的克隆、表达及生物学活性研究

研究目的是克隆猪骨髓基质抗原2(Bone marrow stromal antigen-2,BST-2)基因并进行真核表达,获得具有抗病毒活性的重组BST-2蛋白。研究通过RT-PCR从猪瘟弱毒疫苗和聚肌胞(Poly I:C)刺激的猪肾细胞(PK-15)中扩增出猪BST-2cDNA,克隆至真核表达载体pcDNA3.1/V5-His,构建重组表达质粒pcDNA-BST-2,转染HEK293T细胞,纯化BST-2蛋白并经Bradford法定量,Western blot检测,研究BST-2抗病毒生物学活性。酶切鉴定和核酸序列测定证实pcDNA-BST-2真核表达质粒构建成功,转染HEK293T细胞后,经间接免疫荧光能够检测到绿色荧光。生物学活性测定重组BST-2蛋白具有一定的抗水泡性口炎病毒(Vesicular stomatitis virus,VSV)、H9禽流感病毒(Avian influenza virus,AIV)及猪繁殖与呼吸综合症病毒(Porcine reproductive and respiratory syn-drome virus,PRRSV)的活性。结果表明,重组BST-2具有一定抗病毒生物功能,为进一步研究重组BST-2蛋白的活性以及BST-2抗病毒药物研究奠定了基础。

Molecular Cloning and Expression of Bone Marrow Stromal Antigen-2 and Detection of Its Biological Activity

To clone porcine bone marrow stromal antigen-2(BST-2) gene,construct its recombinant eukaryotic expression plasmid and induce the expression of the fusion antiviral protein,we amplified BST-2 gene by RT-PCR from the total RNA extracted from PK15 cells.The recombinant expression plasmid pcDNA-BST-2 was constructed and then was transfected into HEK293T cells to expresse the BST-2 fusion protein.Western blot and indirect immunofluorescence assay(IFA) were performed,and the biological activity was detected.The results showed that the construction of recombinant plasmid pcDNA-BST-2 was confirmed by restriction enzyme digestion and sequencing.The expressed product had antiviral activity against Vesicular stomatitis virus(VSV),Avian influenza virus(AIV) and Porcine reproductive and respiratory syndrome virus(PRRSV).In conclusion,the research paves the way for further research on bioactivity assayand antiviral medication.