海藻糖合酶基因在大肠杆菌中的克隆、表达及酶学性质分析
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摘要
应用PCR技术从芝田硫化叶菌(Sulfolobus Shibatea)B12中扩增出麦芽寡糖基海藻糖合酶(maltooligosyl trhalose synthase,MTSase)基因,通过T4DNA连接酶将其连接于T—easy—vector上,通过电转化法将重组质粒转化到大肠杆菌受体菌DH5 Q中,蓝白斑筛选阳性菌落,通过菌落PCR、质粒PCR扩增及质粒酶切鉴定、测序,用Nde I和BamH I同时双酶切重组质粒和原核表达载体PET一21a,通过T4DNA连接酶将目的基因片段克隆入表达载体中,酶切鉴定,利用热击法将重组质粒PET-21a-MTSase转化入BL21(DE3)大肠杆菌中,经过IPTG诱导表达MTGase基因蛋白,SDS—PAGE电泳检测其表达蛋白的分子量为74KD,表达量占菌体总蛋白的16.8%,酶活为38.53U/克湿菌体。并进行MTSase基因工程菌的酶活测定方法及发酵条件的优化。
研究结果表明,通过对其基因序列的测定,得到了这个酶基因的全序列, mTSase的基因由2,187bp组成,编码728个氨基酸;其结果与同属的产酶菌株硫矿硫化叶菌KMl的核苷酸序列比较,同源性为96%。
The genes encoding for maltooligosyl trehalose synthase(MTSase)were amplified from the total DNA of Sulfolobus shibatae B12 by the polymerase chain reaction(PCR),ligated the fragment with T-easy—vector by T4 DNA ligase and transformemed the competent cells Ecoli DH5α.The transformants were screened on amplicillin/Xgal-IPTG on plate.Identifying the fragment by clony PCR、plasmid amplifying PCR、restriction enzyme digest analysis and sequencing.Restriction endonucleases Nde I and BamH 1 were used to cleave both T-easy—vector-MTSase and PET-21 a,cloned the object gene fragment to expression vector by , T4 DNA ligase,identified by enzyme digest,transformed recombinant plasmid to E.coli BL21(DE3)by heat shock,induced the recombinant plasmids PET-21a-GT by the addition of IPTG,expressed MTSase protein.
The recombinant protein were shown to be 16.8%of the total bacterial protein. The molecular weight of expressed MTSase detected by SDS-PAGE was 74 kDa..The enzyme activity was 38.53U of the total wet bacterial substance.The conditions of affecting the recombinant enzyme expression were optimized. Findings showed:The nucleotide sequence of the MTSase gene consisted of 2,187bp open reading frame which encoded for a protein with 729 amino acid.The overall nucleotide sequence and amino acid sequence homologies between MTSase of Sulfolobus shibatae B12 and Sulfolobus solfataricus KMl is 96%.
研究结果表明,通过对其基因序列的测定,得到了这个酶基因的全序列, mTSase的基因由2,187bp组成,编码728个氨基酸;其结果与同属的产酶菌株硫矿硫化叶菌KMl的核苷酸序列比较,同源性为96%。
The genes encoding for maltooligosyl trehalose synthase(MTSase)were amplified from the total DNA of Sulfolobus shibatae B12 by the polymerase chain reaction(PCR),ligated the fragment with T-easy—vector by T4 DNA ligase and transformemed the competent cells Ecoli DH5α.The transformants were screened on amplicillin/Xgal-IPTG on plate.Identifying the fragment by clony PCR、plasmid amplifying PCR、restriction enzyme digest analysis and sequencing.Restriction endonucleases Nde I and BamH 1 were used to cleave both T-easy—vector-MTSase and PET-21 a,cloned the object gene fragment to expression vector by , T4 DNA ligase,identified by enzyme digest,transformed recombinant plasmid to E.coli BL21(DE3)by heat shock,induced the recombinant plasmids PET-21a-GT by the addition of IPTG,expressed MTSase protein.
The recombinant protein were shown to be 16.8%of the total bacterial protein. The molecular weight of expressed MTSase detected by SDS-PAGE was 74 kDa..The enzyme activity was 38.53U of the total wet bacterial substance.The conditions of affecting the recombinant enzyme expression were optimized. Findings showed:The nucleotide sequence of the MTSase gene consisted of 2,187bp open reading frame which encoded for a protein with 729 amino acid.The overall nucleotide sequence and amino acid sequence homologies between MTSase of Sulfolobus shibatae B12 and Sulfolobus solfataricus KMl is 96%.
引文
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