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�й����ﻯѧ���������ѧ�� 2004, Vol. 20 Issue (06 ):738-738     DOI:
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Molecular Cloning of Cu,Zn-Superoxide Dismutase Gene from Bacillus cereus M22 and Its Expression
SHANG Yu-lei 1), ZHANG Bing-xin 1), WANG Qi 2)��, MEI Ru-hong 2) ( 1)College of Agriculture & Biotechnology,Zhejiang University,Hangzhou\ 310029,China; 2)College of Agronomy & Biotechnology,China Agricultural University,Beijing\ 100094,China)

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ժҪ ����PCR���� ,������ѿ��˾�M2 2������DNA�������� 132 0bp�Ļ���Ƭ�� .��Ƭ�κ����� 179��������л�Ŀ����Ķ��� ,�ƶ����������뱨����BacillusanthracisCu ,Zn SOD������96 %ͬԴ�� ,����N�� 16��������л��ƶ�Ϊ�ź������� .��Cu ,Zn SOD�������������pET 2 2b(+ )�����������pET 2 2b sodC ,ת��E .coliBL2 1(DE3) ,IPTG�յ��ںϵ��ױ�� .SDS PAGE��ʾ ,�ںϵ��ױ�۷�������Լ 2 4kD ,ռ�����ѽ�Һ���ܵ��׵� 2 1 3% .���˱������ת��SODȱ���ʹ󳦸˾�PN132 ,�����˸þ����paraquat�Ŀ��� .NBT�����Ʒ��ⶨSOD������ʾ ,��PN132��SOD������� ,��������IPTG�յ�ǰSOD���Լ��� (1 79U/mg) ,�յ�����Ըߴ� 6 9 76U/mg .�DZ��Ե�Ӿ�����ʾ ,��ø���Բ�ͬ�̶ȵ��ܵ� 5mmol LH2 O2 �� 5mmol LKCN������
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Abstract�� A 1320 bp Cu,Zn-superoxide dismutase(SOD) gene fragment containing the coding region which consisted of 179 amino acids and 5��- and 3��-flanking sequence, was cloned from genomic DNA of the endophytic bacterium Bacillus cereus M22 by PCR.The deduced amino acid sequences showed a high homology to other bacterial Cu,Zn-SOD.The first 16 amino acids were predominantly either hydrophobic or basic,and may serve as a signal peptide for this putative lipoprotein.The gene was inserted into the expression vector pET-22b(+)between EcoR�� and Xho�� sites with a correct open reading frame,and the recombinant pET-22b-sodC was transformed into E.coli BL21(DE3).After induction about 3 hours by 1 mmol/L IPTG,a special protein band appeared near the position of molecular weight 24 kD and was more than 21.3% of the total lysate proteins.The pET-22b-sodC was also transformed into E.coli PN132.The recombinant protein protected E.coli SOD null strain PN132,which could not grow on the LB plate containing 10 ��mol/L paraquat.The SOD activity was up to 69.76 U/mg in the lysate of PN132(pET-22b-sodC) with IPTG induction,and was judged to be a Cu,Zn-SOD on the basis of inhibition by potassium cyanide(KCN) and hydrogen peroxide(H\-2O\-2).
Keywords�� Bacillus cereus   Cu   Zn-superoxide dismutase   cloning and sequencing   prokaryotic expression      
Received 2003-12-26;
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������.����ѿ��˾�M22ͭп���������绯ø����Ŀ�¡����� [J]  �й����ﻯѧ���������ѧ��, 2004,V20(06 ): 738-738
.Molecular Cloning of Cu,Zn-Superoxide Dismutase Gene from Bacillus cereus M22 and Its Expression [J]  Chinese Journal of Biochemistry and Molecular Biol, 2004,V20(06 ): 738-738
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http://cjbmb.bjmu.edu.cn/CN/     ��     http://cjbmb.bjmu.edu.cn/CN/Y2004/V20/I06 /738