CAJ | 학술논문

利用 RT-PCR技术扩增获得猪瘟病毒NS3基因片段，将其克隆至酵母双杂交系统诱饵载体pGBKT7中，经酶切，测序验证其正确插入后，将重组诱饵质粒转化酵母菌Y2H Gold ，检测其在酵母中毒性和自我激活作用，并利用Western blot分析诱饵载体在酵母中的转化情况。结果表明，pGBKT7-NS3诱饵载体在Y2H Gold酵母细胞中可以表达，此载体在酵母细胞Y2H Gold中无毒性和自我激活能力，研究结果为使用酵母双杂交技术筛选相互作用蛋白奠定了基础。
이용 RT-PCR기술확증획득저온병독NS3기인편단，장기극륭지효모쌍잡교계통유이재체pGBKT7중，경매절，측서험증기정학삽입후，장중조유이질립전화효모균Y2H Gold ，검측기재효모중독성화자아격활작용，병이용Western blot분석유이재체재효모중적전화정황。결과표명，pGBKT7-NS3유이재체재Y2H Gold효모세포중가이표체，차재체재효모세포Y2H Gold중무독성화자아격활능력，연구결과위사용효모쌍잡교기술사선상호작용단백전정료기출。
The NS3 gene of classical swine fever virus was amplified by RT-PCR and confirmed by sequen-cing and enzyme digestion .The NS3 gene was then subcloned into pGBKT7 to construct the bait vector ac-cording to the reading frame .The reconstructed bait vector pGBKT7-NS3 was transformed into Y2H Gold yeast strain ,its self-activation and toxic effect were tested by the phenotype assay .The expression of pG-BKT7-NS3 was analysed by Western blot .As a result ,the successfully reconstructed bait vector pGBKT7-NS3 would not self-activitate the reporter gene and had no toxic effect to the yeast ,which laid the founda-tion for using yeast two-hybrid assay to screen interaction proteins .