CAJ | 학술논문

对鸡沙门氏菌诱导家蝇幼虫构建的抑制性消减文库（ SSH）中筛选得到的家蝇双翅肽 I （ Musca domestica Diptericin I， MdDpt I）基因进行克隆、表达，并对表达产物的抑菌活性进行初步研究。以沙门氏菌诱导家蝇幼虫cDNA为模板，采用cDNA末端快速扩增技术（ RACE），对MdDpt I基因进行扩增，并对扩增产物进行测序和生物信息学分析，进一步去掉信号肽并构建重组表达质粒pET－28a－MdDpt I，转化大肠杆菌BL21（ DE3），IPTG诱导表达，对表达产物进行SDS－PAGE电泳分析。借助亲和纯化获得目的蛋白，并验证目的蛋白的生物活性。 MdDpt I基因全长为419 bp，包含一个300 bp的完整开放阅读框（ ORF），编码99个氨基酸，其cDNA序列与GenBank 中登录号为FJ794602．1的家蝇双翅肽基因同源性为95％。构建了家蝇MdDpt I基因的成熟肽原核表达质粒pET－28a－MdDpt－I，并获得成功表达，表达产物约为12 ku，与预期结果一致。纯化后的目的蛋白表现出一定的抑菌活性。本试验获得了MdDpt I基因的全长序列，构建了原核表达载体，并成功获得表达纯化。
대계사문씨균유도가승유충구건적억제성소감문고（ SSH）중사선득도적가승쌍시태 I （ Musca domestica Diptericin I， MdDpt I）기인진행극륭、표체，병대표체산물적억균활성진행초보연구。이사문씨균유도가승유충cDNA위모판，채용cDNA말단쾌속확증기술（ RACE），대MdDpt I기인진행확증，병대확증산물진행측서화생물신식학분석，진일보거도신호태병구건중조표체질립pET－28a－MdDpt I，전화대장간균BL21（ DE3），IPTG유도표체，대표체산물진행SDS－PAGE전영분석。차조친화순화획득목적단백，병험증목적단백적생물활성。 MdDpt I기인전장위419 bp，포함일개300 bp적완정개방열독광（ ORF），편마99개안기산，기cDNA서렬여GenBank 중등록호위FJ794602．1적가승쌍시태기인동원성위95％。구건료가승MdDpt I기인적성숙태원핵표체질립pET－28a－MdDpt－I，병획득성공표체，표체산물약위12 ku，여예기결과일치。순화후적목적단백표현출일정적억균활성。본시험획득료MdDpt I기인적전장서렬，구건료원핵표체재체，병성공획득표체순화。
The study was done to clone and express gene of MdDpt I ( Musca domestica Diptericin I ) being screened from SSH library by salmonella, in order to lay the foundation for its bioactivity evaluation. Using cDNA end rapid amlification technology( RACE) , MdDpt I gene’ s full-length cDNA was amplified, then recombonant expression plasmid was reconstructed using pET-28a, transformed into E.coli BL21 (DE3)and then induced by IPTG, the recombinant protein was verified by SDS-PAGE. With the aid of affinity to obtain purified fusion protein, the fusion protein was verified. The full-length cDNA of MdDpt I gene was 419 bp, in size, the open reading frame was 300 bp, encoding a 99-aminoacid protein, the homology of this gene’ s cDNA sequence with the MdDpt I from GenBank was up to 95%. The mature peptide recombonant expression plasmid pET-28a-MdDpt I of MdDpt I gene was constructed, and the protein of MdDpt I gene was expressed successfully with the size of 12 ku, The purified fusion protein showed certain antibacterial activity. The result showed that the recombinant plasmid pET-28a-MdDpt I was constructed correctly, which paved the way for further studies on the MdDpt I protein expression and its biological activities.