蓝氏贾第鞭毛虫胞外核酸酶的表达纯化和活性鉴定

发布时间：2019-05-10 08:11

【摘要】：目的克隆、原核表达蓝氏贾第鞭毛虫(Giardia lamblia,贾第虫)的胞外核酸酶编码区,并对其蛋白产物进行活性鉴定。方法对贾第虫胞外核酸酶(GeNuc)蛋白进行生物信息学分析,根据分析结果以C2株贾第虫基因组DNA为模板扩增获得GeNuc去信号肽段编码区序列,双酶切连入原核表达载体pET-28a(+),将酶切和测序验证正确的重组质粒转化E.coli Rosetta(DE3),经IPTG诱导表达融合蛋白,SDS-PAGE及Western blot鉴定蛋白产物。Ni-NTA亲和层析纯化GeNuc蛋白,经复性后验证其对质粒DNA的水解能力。结果成功克隆了长约800bp的GeNuc编码区并构建了原核表达载体pET-28a(+)-GeNuc,测序结果显示C2株GeNuc序列与WB株相同;在大肠杆菌中诱导表达获得了相对分子量约30.8kDa的融合蛋白;复性后的纯化GeNuc蛋白具有降解双链DNA的能力,但活性较商品化DNaseⅠ低。结论证明了GeNuc的存在,为GeNuc抗体的制备及贾第虫致病机制的研究提供了实验材料。
[Abstract]:Objective to clone and express the extracellular nuclease coding region of Giardia lamblia (Giardia lamblia, in prokaryotic cells, and to identify the activity of its protein product. Methods the extracellular nuclease (GeNuc) protein of Giardia giardia was analyzed by bioinformatics analysis. According to the analysis results, the coding region of GeNuc designaling peptide was amplified by using genomic DNA of C2 strain Giardia as template. The recombinant plasmid was transformed into E.coli Rosetta (DE3) by restriction endonuclease digestion and sequencing, and the fusion protein was induced by IPTG. The protein products were identified by SDS-PAGE and Western blot. GeNuc protein was purified by Ni-NTA affinity chromatography and its hydrolysis ability to plasmid DNA was verified by renaturation. Results the GeNuc coding region of about 800bp was successfully cloned and the prokaryotic expression vector pET-28a ()-GeNuc, was constructed. The results showed that the GeNuc sequence of C2 strain was the same as that of WB strain. The fusion protein with relative molecular weight of about 30.8kDa was induced and expressed in E. coli, and the purified GeNuc protein after renaturation had the ability to degrade double-stranded DNA, but its activity was lower than that of commercial DNase I. Conclusion the existence of GeNuc is proved, which provides experimental materials for the preparation of GeNuc antibody and the study of pathogenic mechanism of Giardia.
【作者单位】： 华北理工大学附属医院检验科;华北理工大学生命科学学院;
【基金】：国家自然科学基金(No.31471954) 河北省青年科学基金(No.C2012401039)联合资助~~
【分类号】：R382.21

【相似文献】

相关期刊论文 前10条

1 权彤彤;;胆汁内查见蓝氏贾第鞭毛虫1例[J];西南国防医药;2009年06期

2 梁毅,曾美琴,吴曼秋;蓝氏贾第鞭毛虫胆道感染16例报告[J];新医学;1974年11期

3 王才中;;滚动玻瓶培养蓝氏贾第鞭毛虫的评价[J];国外医学(寄生虫病分册);1984年01期

4 王又红;蓝氏贾第鞭毛虫基因组序列研究[J];国外医学(寄生虫病分册);1999年03期

5 陈小宁,卢思奇,王f3星,王峰,李春荣,郝祥俊;蓝氏贾第鞭毛虫承德株分子生物学研究[J];承德医学院学报;2000年04期

6 卢思奇,李继红,王凤云,张亚平,文建凡;蓝氏贾第鞭毛虫磷酸丙糖异构酶基因种内差异研究(英文)[J];Chinese Medical Journal;2002年05期

7 温少芳;卢思奇;;蓝氏贾第鞭毛虫遗传多样性研究进展[J];国际医学寄生虫病杂志;2006年01期

8 曹军皓;陈真真;容东宁;李芳;;胸水检出蓝氏贾第鞭毛虫一例[J];检验医学;2006年05期

9 田喜凤;卫茹;杨志宏;卢思奇;;甲硝唑对体外蓝氏贾第鞭毛虫形态结构的损伤[J];中国寄生虫学与寄生虫病杂志;2006年05期

10 杨志宏;田喜凤;卫茹;;蓝氏贾第鞭毛虫的基因及基因型[J];热带病与寄生虫学;2007年01期

相关会议论文 前1条

1 王厚芳;柳杰;孙芾;;隐孢子虫抗原和蓝氏贾第鞭毛虫抗原的快速检测方法及临床应用[A];中华医学会第八次全国检验医学学术会议暨中华医学会检验分会成立30周年庆典大会资料汇编[C];2009年

相关硕士学位论文 前1条

1 巨红妹;蓝氏贾第鞭毛虫三种基因功能研究载体的构建[D];大连大学;2013年

，

本文编号：2473487