[牙龈卟啉单胞菌fimA基因的克隆及其在大肠杆菌中的表达与纯化]

[Cloning of the fimA gene of Porphyromonas gingivalis and its expression and purification in Escherichia coli].

作者信息

Liu Wei, Yu Fei, Chen Wei-Min, He Wei

机构信息

Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

出版信息

Hua Xi Kou Qiang Yi Xue Za Zhi. 2009 Dec;27(6):614-7.

PMID:20077894

Abstract

UNLABELLED

OBJECTIVE; To clone the fimA gene of Porphyromonas gingivalis (P. gingivalis) and detect its expression in Escherichia coli (E. coli).

METHODS

The fimA gene was obtained by PCR from the genome of P. gingivalis to construct a prokaryotic expression plasmid pT-BAD/fimA. pT-BAD/fimA was transformed into E. coli BL21 (DE3) competent cells and the recombination protein was characterized by means of matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. The bound protein was eluted with different concentrations of imidazole (250, 200, 150, 100, 50 micromol x L(-1)) respectively.

RESULTS

DNA sequencing showed that the fragment was 99.9% consistent with that of the published. After induction with L-arabinose, a new 3.8 x 10(4) protein appeared on SDS-PAGE gel. The protein was further identified by MALDI-TOF-MS. Purity of 95% of the target protein was purified by Ni-NTA Purification System after eluted with 100 micromol x L(-1) imidazole.

CONCLUSION

The fimA gene of P. gingivalis was cloned successfully and its protein was expressed correctly in E. coli. A high purity of protein FimA was obtained and it could be applied for follow-up researches.

摘要

未标记

目的；克隆牙龈卟啉单胞菌（牙龈卟啉单胞菌）的fimA基因并检测其在大肠杆菌（大肠杆菌）中的表达。

方法

通过聚合酶链反应（PCR）从牙龈卟啉单胞菌基因组中获得fimA基因，构建原核表达质粒pT-BAD/fimA。将pT-BAD/fimA转化到大肠杆菌BL21（DE3）感受态细胞中，并用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）分析对重组蛋白进行鉴定。分别用不同浓度的咪唑（250、200、150、100、50 μmol·L⁻¹）洗脱结合蛋白。