CAJ | 학술논문

为了获得高效的中性蛋白酶基因，试验用PCR方法从解淀粉芽孢杆菌中扩增中性蛋白酶基因（npr），通过BamHⅠ、SalⅠ双酶切后，将该基因与表达载体PET-28a连接，同时转入到大肠杆菌BL21中表达，通过SDS-PAGE电泳技术对构建的工程菌的基因表达产物进行分析。获得特异 npr基因序列含1566 bp，编码522个氨基酸，含有完整的ORF，同源性达到100%。蛋白酶的分子量为57.4 kDa。IPTG能诱导中性蛋白酶基因在重组菌进行表达，通过改变IPTG诱导浓度、诱导温度及诱导时间等因素，得出在添加剂IPTG至终浓度为0.8 mmol·L-1，30℃诱导4 h为最佳诱导条件，获得最高酶活为450 U·mL-1。
위료획득고효적중성단백매기인，시험용PCR방법종해정분아포간균중확증중성단백매기인（npr），통과BamHⅠ、SalⅠ쌍매절후，장해기인여표체재체PET-28a련접，동시전입도대장간균BL21중표체，통과SDS-PAGE전영기술대구건적공정균적기인표체산물진행분석。획득특이 npr기인서렬함1566 bp，편마522개안기산，함유완정적ORF，동원성체도100%。단백매적분자량위57.4 kDa。IPTG능유도중성단백매기인재중조균진행표체，통과개변IPTG유도농도、유도온도급유도시간등인소，득출재첨가제IPTG지종농도위0.8 mmol·L-1，30℃유도4 h위최가유도조건，획득최고매활위450 U·mL-1。
To obtain the efficient neutral protease gene,the neutral protease gene(npr)was amplifier from Bacillus amyloliquefaciens by using PCR technology. After the double enzyme by BamHⅠand SalⅠ,the gene was linked with the express vecter PET-28a and transformed into Escherichia coliBL21. The gene expression product of gene engineering bacteria was analyzed by SDS-PAGE electrophoresis. The length of the amplified specific gene sequences was 1 566 bp,and the gene encoded 522 amino acids and contained the complete ORF. The sequence homology of the gene reached 100%,and the protein molecular weight was 57.4 kDa. By IPTG induction,the neutral protease gene was expressed highly in recombinant strain. By changing the IPTG induction factors such as concentration,temperature and induction time,the best induction conditions were additive IPTG 0.8 mmol·L-1,temperature 30℃and induction 4 h,and the highest enzyme activity was 450 U·mL-1.