耐碱细菌NTT36耐碱基因的克隆及表达产物的分析

CLONE AND SUBCOLNE OF THE ALKALOPHILIC GENE FROM ALKALOPHILIC Bacillus sp.NTT36 AND ANALYSIS OF EXPRESSED PRODUCTS IN E.coli

耐碱芽孢杆菌ＮＴＴ３６总ＤＮＡ经ＥｃｏＲＩ酶不完全消化，与质粒ｐＵＣ１８的ＥｃｏＲＩ酶切产物在体外重组后，转化大肠杆菌ＨＢ１０１．在碱性平板上直接筛选耐碱转化子，转化子经检测具有氨苄青霉素（Ａｍｐｒ）抗性，分析表明转化子具有１５ｋｂ大小的重组质粒．用此重组质粒转化大肠杆菌ＨＢ１０１、ＤＨ５α和ＸＬ１－Ｂｌｕｅ，均产生大量同时具有耐碱性和Ａｍｐ抗性的转化子．通过Ｓｏｕｔｈｅｒｎ杂交和点杂交证明此重组质粒（定名为ｐＧＣＡ）由ｐＵＣ１８和ＮＴＴ３６总ＤＮＡ的片段组成．分别提取上述３种克隆转化子和ＮＴＴ３６细胞膜蛋白，进行ＳＤＳ－ＰＡＧＥ梯度凝胶电泳，发现在ｐＨ１０．６条件下培养的转化子和供体菌ＮＴＴ３６与在中性培养基中生长的转化子和受体菌相比，有５条多肽带有明显差异．表明这５条多肽带与耐碱性有关．

The total DNA of alkalophilic Bacillus sp.NTT36 and the vector plasmid pUC18(Amp r) were digested with restriction enzyme EcoRI, respectively. The pUC18 EcoRI fragment was ligated with the digested total DNA in vitro by using T 4 DNA ligase. The ligated products were transformed into E.coli HB101.The transformants were obtained from alkaline plates. About a 15 kb of recombinant plasmid(pGCA) was detected. To make sure, the plasmid pGCA was transformed into E.coli XL1 blue and DH5α again, and the alkalophilic transformants resistant to ampicillin were obtained in the transformants of E.coli XL1 blue and DH5. The recombinant plasmid was proved to composed of plasmid pUC18 and DNA fragmant from NTT36 by southern and blot hybridization method. The membrane proteins of transformants cultured at pH10.6 and 7.0, NTT36 at pH10.6, E.coli HB101,XL1 blue and DH5α at pH7.0, were extracted and run gradient SDS PAGE .Transformants cultured at pH10.6 have five special polypeptide bands which NTT36 also has, while E.coli and the transformants cultured at pH7.0 didn't have these five bands. The result showed that the alkalophilic gene from NTT36 was cloned and expressed in E.coli.