南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (04): 472-475.

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小鼠PGRP-L分子PGRP结构域基因的克隆及其在大肠杆菌中的表达

何智; 左大明; 陈政良;   

  1. 南方医科大学免疫学教研室; 南方医科大学免疫学教研室 广东 广州 510515; 广东 广州 510515;
  • 出版日期:2006-04-20 发布日期:2006-04-20

Cloning and prokaryotic expression of the gene encoding PGRP domain of mouse long peptidoglycan recognition protein

HE Zhi, ZUO Da-ming, CHEN Zheng-liang Department of Immunology, Southern Medical University, Guangzhou 510515, China   

  1. 南方医科大学免疫学教研室; 南方医科大学免疫学教研室 广东 广州 510515; 广东 广州 510515;
  • Online:2006-04-20 Published:2006-04-20

摘要: 目的克隆小鼠长型肽聚糖识别蛋白(PGRP-L)的PGRP结构域基因并在大肠杆菌中表达。方法采用RT-PCR技术,从Balb/C小鼠肝组织总RNA中扩增PGRP-L的PGRP结构域基因片段,将其克隆入pUCm-T载体,以PCR、酶切和测序进行鉴定。以PCR从含PGRP结构域基因的重组质粒中扩增目的基因片段,插入表达质粒pQE-30构建重组表达质粒,转化大肠杆菌M15,诱导表达并纯化目的蛋白。结果 RT-PCR扩增得到约500bp的基因片段,将其与pUCm-T 载体连接构建成重组质粒pmPGRPd,其酶切图谱与计算机分析结果一致,该基因片段长518 bp,序列与Genbank中相应基因序列相同。构建成重组表达载体pQE-PGRPd,在大肠杆菌M15中表达,表达产物主要存在于菌体裂解液上清中,为一相对分子质量约29 000的可溶性蛋白。结论成功克隆并原核表达了小鼠PGRP-L分子PGRP结构域基因,为 PGRP-L分子的研究奠定了一定基础。 

Abstract: Objective 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. Methods 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. Results 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. Conclusion 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. 

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