猪α干扰素基因突变、表达及其高效抗病毒活性筛选研究

The Mutations, Expression and Antiviral Activity Screening of Porcine Interferon-α

关键词:  猪α干扰素  表达  基因型  抗病毒活性。

Key words:  Porcine Interferon-α  expression  genotype  antiviral activity.

摘要:

利用RT-PCR方法从猪肝细胞总RNA中扩增出编码猪α干扰素(Interferon,IFN)基因,根据大肠杆菌偏嗜性及活性位点改造基因并克隆至原核表达载体pET21a,转化大肠杆菌BL21(DE3)进行蛋白诱导表达和鉴定。重组蛋白以包涵体形式表达,经变复性及纯化处理后,获得多种不同基因型高纯度猪α干扰素。用细胞病变抑制法在MDBK/VSV系统上进行抗病毒活性测定,结果表明经过基因改造的猪α干扰素(PoIFN-M6)具有更高抗病毒活性,约为2.97×10₈ U/mg。本研究为猪α干扰素基因工程产品的研发及其在临床兽医的应用奠定基础。

Abstract:

The aim of this study is to obtain porcine interferon-α with high antiviral activity. Total RNA extracted from swine liver was used for the generation of the porcine interferon-α cDNA. Here we optimized the codon usage of the gene for E.coli. Then the reconstructed fragments were cloned into vector pET21a to obtain the different genotypes of interferon-α, and expressed in E.coli BL21 (DE3). The results showed that the protein was expressed as inclusion body. After dissolution, re-naturation and purification, we gained the pure protein. The effect of the reconstructed porcine Interferon-α on reduction of viral cytopathic effect(CPE) was detected in MDBK cells infected with vesicular stomatitis virus (VSV). As a result, the protein PoIFN-M6 was verified to have a high antivital activity by CPE inhibition test, which was about 2.97×108 U/mg. The research laid the foundation for the genetic expression of porcine interferon-α and the clinical use of it.