Clone and expression of APH(3′′)-Ⅰand AAC(2′)-Ⅰ gene of Stenotrophomonas maltophilia
10.3969/j.issn.1673-4130.2016.15.018
- VernacularTitle:嗜麦芽窄食单胞菌产氨基糖苷类修饰酶基因 APH（3′′）-Ⅰ和A AC（2′）-Ⅰ的克隆与表达
- Author:
Xiaoshan GUAN
;
Ruili GUAN
;
Huamin ZHONG
;
Qiulian DENG
;
Yongqiang XIE
;
Zhenwen ZHOU
- Publication Type:Journal Article
- Keywords:
Stenotrophomonas maltophilia;
AM Es;
clone;
prokaryotic expression
- From:
International Journal of Laboratory Medicine
2016;37(15):2099-2101
- CountryChina
- Language:Chinese
-
Abstract:
Objective To perform the amplification ,sequencing and prokaryotic expression of APH (3′′)‐Ⅰ and AAC (2′)‐Ⅰgenes from the clinically isolated gzch810 strain(SM gzch810)of Stenotrophomonas maltophilia to provide the basic materials for the next step functional test .Methods The SM gzch810 genome chromosome was extracted ,the APH (3′′)‐Ⅰ ,AAC (2′)‐Ⅰ whole genes were amplified by PCR and sequenced after being cloned into pMD18‐T vector .The recombination were subcloned into pGEX‐4T‐1 vector and the expression of the recombinant APH (3′′)‐Ⅰ and AAC (2′)‐Ⅰ were analyzed by SDS‐PAGE .Results The 800bp and 550bp DNA fragments of APH(3′′)‐Ⅰ ,AAC(2′)‐Ⅰ gene were amplified from SM gzch810 by PCR and sequenced ;the sequence comparison analysis showed that DNA and amino acid sequence identities of APH (3′′)‐Ⅰand AAC (2′)‐Ⅰ genes with other strains were 91% and 95% respectively .The sequence of APH (3′′)‐Ⅰand AAC(2′)‐Ⅰ of SM gzch810 were submitted to GenBank(accession number :HQ315852 and HQ315853);two major protein bands corresponding to the expected recombinant GST‐TP fusion proteins (56 × 103 and 46 × 103 respectively) were identified by SDS‐PAGE .Conclusion APH(3′′)‐Ⅰand AAC(2′)‐Ⅰgene of SM gzch810 are successfully cloned and expressed ,which lays a good foundation for further detecting corresponding antibi‐otic resistance and functional evaluation of above two kinds of recombinant E .coli .
