CAJ | 학술논문

为了获得高效抗病毒活性的猪α-干扰素蛋白,采用PCR法扩增获得猪α-干扰素基因完整的开放阅读框(ORF),构建重组表达质粒pPICZαC-IFN,电击转化毕赤酵母感受态细胞X33.挑阳性菌落诱导表达,离心取上清,SDS-PAGE和 Western-blotting分析,可见1条相对分子质量约19 400的清晰蛋白条带,与理论值相符,表明表达产物为猪α-干扰素.在Vero细胞上检测该干扰素抗VSV的活性为4.04×10~6 IU/L.
위료획득고효항병독활성적저α-간우소단백,채용PCR법확증획득저α-간우소기인완정적개방열독광(ORF),구건중조표체질립pPICZαC-IFN,전격전화필적효모감수태세포X33.도양성균락유도표체,리심취상청,SDS-PAGE화 Western-blotting분석,가견1조상대분자질량약19 400적청석단백조대,여이론치상부,표명표체산물위저α-간우소.재Vero세포상검측해간우소항VSV적활성위4.04×10~6 IU/L.
In order to get alpha-interferon(INF-α) with high level secretive expression and high antiviral activities,the porcine alpha-interferon gene was amplified by PCR,and a DNA fragment of 501 bp,the open reading framework (ORF) was obtained.Then,the target gene and pPICZαC were digested with EcoRI/XbaI and were linked.The recombinant plasmid of pPICZαC-IFN was linearized by SacI and electroporated into Pichia pastoris X-33.PCR assay was used to identify colonies.The high copy recombinat strains were screened and induced by regulation of methanol utilization.IFN-α protein was detected by SDS-PAGE and Western-blotting analysis.The result showed the IFN-α protein with a raletive molecular mass of 19 400 was expressed in Pichia pastoris X-33.The antiviral activity of IFN-α against vesicular stomatitis virus(VSV) was investigated on the Vero cell and the result indicated that IFN-α could inhibit VSV and the activity was 4.04×10~6 IU/L.