摘要
为获得具有高冰核活性的基因工程菌,从冰核细菌Erwinia ananas 110扩增冰核基因iceA,将其克隆到pMD19–T载体上,转化大肠杆菌DH5α,单、双酶切鉴定并测序;阳性克隆目的片段亚克隆到表达载体pET–23a(+)上,转化大肠杆菌DH5α,单、双酶切鉴定重组质粒;阳性重组质粒转化大肠杆菌BL21(DE3)pLysS,并经IPTG诱导表达。SDS–PAGE电泳检测表明,冰核基因iceA能够并以包涵体形式表达,相对分子质量约为180 000。冰核活性测定结果表明,重组菌BL21(DE3)pLysS/pET–ice的冰核活性与野生冰核细菌Erwinia ananas 110在–5、–4、–3、–2℃下无明显差别。
To obtain recombinant strain with high ice nucleation activity, iceA gene were amplified by PCR from ice nucleation active bacteria Erwinia ananas 110 and cloned into vector pMD 19-T which was transformed into E. coli DH5α The recombinant clones were screened by single and double digestion before sequenced. From the positive recombinant strain, iceA gene was subcloned into prokaryotic expression vector pET-23a（＋）, resulting in recombinant plasmid pET 23a（＋）-ice which was transformed into E.coli BL21（DE3）pLysS and induced by IPTG. SDS-PAGE indicated that ice nucleation active protein was expressed as inclusion bodies with molecular weight of about 180 000. Ice nucleation activity test showed there was no difference in ice nucleation activity under -5, -4, -3, and -2℃ between recombinant E.coli BL21 （DE3）pLysS and wild ice nucleation active bacteria Erwinia ananas 110.
出处
《湖南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2013年第4期354-358,共5页
Journal of Hunan Agricultural University(Natural Sciences)
基金
湖南省杰出青年基金(10JJ1005)
关键词
冰核细菌
原核表达
冰核蛋白
冰核活性
ice nucleation active bacteria
prokaryotic expression
ice nucleation active protein
ice nucleation activity
