CAJ | 학술논문

利用RT－PCR方法从猪肝细胞总RNA中扩增出编码猪α干扰素基因，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的高纯度猪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。
이용RT－PCR방법종저간세포총RNA중확증출편마저α간우소기인，근거대장간균편기성급활성위점개조기인병극륭지원핵표체재체pET21a，전화대장간균BL21（ DE3）진행단백유도표체화감정。중조단백이포함체형식표체，경변－복성급순화처리후，획득다충불동기인형적고순도저α간우소。용세포병변억제법재MDBK／VSV계통상진행항병독활성측정，결과표명경과기인개조적저α간우소（PoIFN－M6）구유경고항병독활성，약위2．97×108 U／mg。본연구위저α간우소기인공정산품적연발급기재림상수의적응용전정기출。
The aim of this study is to obtain porcine interferon-αwith high antiviral activity. Total RNA extracted from swine liver was used for the generation of the porcine interferon-αcDNA. 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.