几丁质酶表达载体的构建及在类产碱假单胞菌中的表达研究

Construction of Chitinase Gene Expression Vector and Expression in Pseudomonas pseudoalcaligenes

作者利用类产碱假单胞菌(Pseudomonaspseudoalcaligenes)的强基因启动子PA7的部分片段LQZ,将环状芽孢杆菌(Bacilluscirculans)C 2中克隆的几丁质酶基因编码区序列片段CSP连接在LQZ启动子阅读框翻译起始位点atg之后,插入pBluescriptsk(-)成功构建P.pseudoalcaligenes表达载体pBPL1.用假单胞菌转化法转化,利用几丁质平板进行筛选,转化子pBPLC1在几丁质平板上能够产生水解圈,在P.pseudoalcaligenes中成功表达.同时对Tm值相差大的引物进行PCR扩增研究,发现添加5%～10%DMSO能提高引物与模板序列特异性复性,和扩增反应效率.

LQZ fragment was amplificated by PCR using strong promoter PA7 of Pseudomonas pseudoalcaligenes as template.CSP fragment(Bacillus circulans C-2 chitinase gene) was connected to LQZ fragment after translation initiation site ATG with T_4 ligase, the connection fragment was inserted into pBluescript sk(-). Chitinase gene expression vector pBPLC1 of P.pseudoalcaligenes was constructed successfully.Recombinant PBPLC1 could produce circle of hydrolytic chitin on chitin plate.The author also found that (5%～10%)(v/v)DMSO could improve efficiency of PCR amplification though enhancing specific annealing of the primers and themplete.