鸭疫里默氏杆菌Tet（X）基因原核表达及其功能鉴定.doc

1、鸭疫里默氏杆菌Tet（X）基因原核表达及其功能鉴定打开文本图片集【摘要】：p ：【目的】确定鸭疫里默氏杆菌Tet（_）基因是否与四环素类耐药相关，寻找出抗鸭疫里默氏杆菌药物研发新靶点。【方法】克隆鸭疫里默氏杆菌Tet（_）基因，以大肠杆菌原核表达体系进行诱导表达，利用生物信息学软件分析p 原核表达蛋白的功能和特性，并采用实时荧光定量PCR检测四环素和多西环素对鸭疫里默氏杆菌Tet（_）基因的调控作用。【结果】从鸭疫里默氏杆菌基因组扩增获得的Tet（_）基因编码框全长1167 bp，编码388个氨基酸，其编码蛋白分子量约42.53 kD，理论等电点（pI）为10.073，与AS879615（Yb

2、2）、RIA369（YA-GD）、RAYM8235（RA-YM）、G148_1777（RA-CH-2）、RIA365（YA-GD）、G148-1767（RA-CH-2）、B739035（RA-CH-1）和B739030（RA-CH-1）等8個来于不同种鸭疫里默氏杆菌的蛋白具有较高同性（96100）。利用原核表达体系诱导表达获得的Tet（_）融合蛋白分子量约45.40 kD，且主要以包涵体的形式表达。Tet（_）融合蛋白能有效提高宿主菌株对典型四环素类药物（四环素和多西环素）的最小抑菌浓度（MIC）;实时荧光定量PCR检测结果显示，四环素和多西环素对鸭疫里默氏杆菌Tet（_）基因表达量的调控呈非

3、线性。四环素浓度为04 mg/L时，Tet（_）基因相对表达量随药物浓度的增加而逐渐增加;浓度为412 mg/L时，Tet（_）基因相对表达量随药物浓度的增加而逐渐降低。多西环素药物浓度为03 mg/L时，Tet（_）基因相对表达量随药物浓度的增加而逐渐增加;浓度为36 mg/L时，Tet（_）基因相对表达量随药物浓度的增加而逐渐降低。【结论】Tet（_）可有效提高宿主菌株对四环素类药物的耐受性，在一定的四环素类药物剂量范围内可诱导Tet（_）基因表达。不同鸭疫里默氏杆菌Tet（_）基因具有不同来，说明Tet（_）可能存在基因水平转移，且具有环境扩散的风险。【关键词】：p ： 鸭疫里默氏杆菌;

4、Tet（_）基因;原核表达;功能鉴定中图分类号： S858.32 文献标志码： A 文章编号：2095-1191（20_）04-0844-07Abstract：【Objective】This study was conducted to determine whether the Riemerella anatipestifer Tet（_） gene was associated with tetracycline resistance， and to find new targets of drug development against R.anatipestifer.【Method】R

5、.anatipestifer Tet（_） gene was cloned， and induced by prokaryotic e_pression in Escherichia coli.Its prokaryotic e_pression protein function and characters were analyzed by the bioinformatics software.The regulation effects of tetracycline and do_ycycline on R.anatipestifer Tet（_） gene were analyzed

6、 by real-time fluorescence quantitative PCR.【Result】The full length of the Tet（_） gene obtained from the genome lification of R.anatipestifer was 1167 bp， encoding 388 amino acids.The molecular weight of the encoded protein was about 42.53 kD， and the theoretical isoelectric point （pI） was 10.073.It

7、 had high homology（96-100） with eight proteins from different strains of R.anatipestifer， including AS879615（Yb2）， RIA369（YA-GD）， RAYM8235 （RA-YM）， G148_1777（RA-CH-2）， RIA365（YA-GD）， G148-1767（RA-CH-2）， B739035（RA-CH-1） and B739030 （RA-CH-1）.The molecular weight of the fusion protein of Tet（_） obtai

8、ned by prokaryotic e_pression induction was 45.40 kD， and mainly e_pressed as inclusion body in prokaryotic e_pression system.The Tet（_） fusion protein could effectively increase the minimal inhibitory concentration（MIC） of the host strain against typical tetracyclines（tetracycline and do_ycycline）.

9、The results of real-time quantitative fluorescence PCR showed that a non-linear effect of the etracycline and do_ycycline on the e_pression of Tet（_） gene.When tetracycline concentration was 0-4 mg/L， the relative e_pression of Tet（_） gene increased with the increase of drug concentration.At concent

10、rations ranging from 4 to 12 mg/L， the relative e_pression of Tet（_） gene decreased with increasing drug concentration.When do_ycycline concentration was 0-3 mg/L， the relative e_pression of Tet（_） gene increased with the increase of drug concentration.At concentrations of 3 to 6 mg/L， the relative e_pression of Tet（_） gene decreased with increasing drug concentration.【Conclusion】Tet（_） gene can enhance the tetracycline resistance of host bacteria， and its e_pression level can be induced by tetracycline drugs within a certain