小鼠PGRP-L分子N端基因片段的克隆与原核表达

Cloning and prokaryotic expression of the N-terminal fragment of mouse long type peptidoglycan recognition protein (mPGRP-L)

采用RT-PCR技术,从BALB/c小鼠肝组织总RNA中扩增到长约500bp的小鼠长型肽聚糖识别蛋白(mPGRP-L)分子N端基因片段,将其克隆入pUCm-T载体构建重组质粒pmGN,DNA测序表明该基因片段长530bp,序列与GenBank中的完全一致。应用PCR技术,从重组质粒pmGN中扩增目的基因片段,插入表达质粒pET-28a构建重组表达载体pET-GN,导入大肠杆菌BL21中诱导表达目的蛋白,表达产物以Ni+-NTAagarose层析柱纯化。SDS-PAGE和Westernblot分析发现,表达产物主要以包涵体形式存在,相对分子质量约29000。ELISA表明,重组蛋白能被抗mPGRP-L分子N端单表位多克隆抗体所识别。为mPGRP-L分子的研究奠定了一定基础。

A cDNA fragment of about 500 bp encoding the N-terminal region of mouse long type peptidoglycan recognition protein （mPGRP-L） was amplified by RT-PCR from the total RNA in liver tissues of BALB/c mice, and the recombinant plasmid, pmGN, was constructed through linkage of the DNA fragment with pUCm-T vector. Result of DNA sequencing of a selected clone indicated that the length of the cloned gene fragment was 530 bp and its sequences were identical to the data published in GenBank. The target gene fragment was amplified from recombinant plasmid pmGN by PCR technique, and inserted into expression vector pET-28a to construct the recombinant expression vector pET-GN, and then introduced to E.coli BL21 strain in order to induce the expression of the target proteins. The recombinant expression products were purified by Ni^＋-NTA agarose chromatography. As demonstrated by SDS-PAGE and Western blot analysis, the expressed products existed mainly in the inclusion bodies with a relative molecular mass of 29 000 Mr. These products could be recognized by the mono-epitope multiclonal antibody against N-terminal region of mPGRP-L, as demonstrated by ELISA assay. The results in the present study could provide the foundation for the further studies of PGRP molecule.