摘要
目的原核表达重组人甘露聚糖结合凝集素(MBL)糖识别域(CRD)。方法采用PCR技术,从含汉族人MBL全长编码区cDNA的重组质粒pGEMmbl中扩增出CRD包括颈区的基因片段,将其插入原核表达载体pGEX4T1中,经PCR、限制性酶切和测序确证后,以IPTG诱导其在大肠杆菌中表达。包涵体经变性、复性后以GSTtrap亲和层析柱纯化,融合蛋白经凝血酶切割后回收目的蛋白。分别以Westernblot和ELISA分析表达产物的免疫学和糖结合活性。结果PCR扩增得到长约450bp的目的基因片段,插入pGEX4T1载体所获重组表达载体pGEX4T1CRD的酶切图谱和序列与预期的一致。重组子经诱导表达,表达产物主要以包涵体形式存在。以GSTtrap亲和层析柱纯化获得约Mr43000的GSTCRD融合蛋白,经凝血酶切割后得到约Mr17000的CRD蛋白。纯化的GSTCRD蛋白能与抗人CRD单克隆抗体特异性结合并具糖结合活性。结论获得了具生物学活性的人MBLCRD蛋白,为进一步探索MBL分子CRD的效应功能提供了实验材料。
Objective To express the recombinant carbohydrate-recognition domain （CRD） of human mannan-binding lectin（MBL） in E. coli. Methods The gene fragment of the CRD including the neck region was amplified by PCR from the plasmid pGEM-mbl containing human MBL cDNA, and then inserted into the prokaryotic expression vector pGEX-4T1 and identified by PCR, restriction mapping, and sequencing. The recombinant expression vector was transformed into E. coli strain BL21 （DE3） and induced to express by IFrG. The fusion protein was purified by GSTrap gel affinity chromatography from denatured and renatured products of inclusion bodies, and then digested by thrombin. The immunological and carbohydrate-binding activities of the purified GSY-CRD protein were analyzed by Western blotting and ELISA, respectively. Results A gene fragment of about 450 bp was amplified by PCR and the recombinant expression vector pGEX4T1-CRD was constructed, whose restriction maps and sequence were consistent with those of expected one. The expressed products in BL21 （DE3） existed as inclusion body, from which the GST-CRD fusion protein of Mr 43000 and the CRD protein of Mr 17000 were obtained by GSTmp gel affinity chromatography and thrombin proteolysis, respectively. The purified GST-CRD protein could react with anti-CRD monoclonal antibody and bind to mannan specifically. Conclusion The recombinant MBL GSY-CRD and CRD protein with bioactivities is obtained, which provides the necessary material for studying effective functions of CRD of the MBL molecule.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第6期521-524,共4页
Immunological Journal
关键词
甘露聚糖结合凝集素
糖识别域
融合蛋白
糖结合活性
Mannan-binding lectin
Carbohydrate recognition domain
Fusion protein
Carbohydrate-binding activity
