He Zhi, Zuo Da-ming, Chen Zheng-liang
Department of Immunology, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2006 Apr;26(4):472-5.
To clone the gene coding for the peptidoglycan recognition protein (PGRP) domain (PGRPd) of mouse long PGRP (mPGRP-L) and express the protein in E. coli.
The cDNA fragment encoding PGRPd of mPGRP-L was obtained by RT-PCR from the total RNA of Balb/C mouse liver cells and cloned into pUCm-T vector. The recombinant plasmid were identified by PCR, restriction endonucleases and sequence analysis. The PGRPd gene fragment was amplified by PCR from the recombinant plasmid, inserted into pQE-30 vector and transformed into E. coli strain M15, and the expressed PGRPd protein was purified.
A cDNA fragment of about 500 bp was amplified by RT-PCR and the recombinant plasmid, pmPGRPd, was constructed by linking the fragment to pUCm-T vector. The results of restriction mapping of the recombinant vector were consistent with those of computer analyses. Sequence analysis showed that the cloned gene fragment (518 bp) had identical sequence with the gene encoding PGRPd of mPGRP-L gene in GenBank. The recombinant expression vector pQE-PGRPd was constructed and expressed in E. coli M15. SDS-PAGE showed that the expressed product existed mainly in the lysate supernatant as a soluble protein with relative molecular mass of 29 kD.
The PGRPd cDNA of mPGRP-L has been successfully cloned and expressed in E. coli, which provides the basis for further study of PGRP molecule.
克隆小鼠长型肽聚糖识别蛋白（mPGRP-L）的肽聚糖识别蛋白结构域（PGRPd）编码基因，并在大肠杆菌中表达该蛋白。
通过RT-PCR从Balb/C小鼠肝细胞总RNA中获得编码mPGRP-L的PGRPd的cDNA片段，并克隆到pUCm-T载体中。通过PCR、限制性内切酶和序列分析鉴定重组质粒。从重组质粒中通过PCR扩增PGRPd基因片段，插入pQE-30载体并转化到大肠杆菌菌株M15中，对表达的PGRPd蛋白进行纯化。
通过RT-PCR扩增出约500 bp的cDNA片段，并将该片段与pUCm-T载体连接构建重组质粒pmPGRPd。重组载体的限制性图谱分析结果与计算机分析结果一致。序列分析表明，克隆的基因片段（518 bp）与GenBank中mPGRP-L基因编码PGRPd的基因序列相同。构建了重组表达载体pQE-PGRPd并在大肠杆菌M15中表达。SDS-PAGE显示表达产物主要以可溶性蛋白形式存在于裂解物上清中，相对分子质量为29 kD。
成功克隆了mPGRP-L的PGRPd cDNA并在大肠杆菌中表达，为进一步研究PGRP分子奠定了基础。
