【摘要】 目的制备具有生物学活性的重组人甘露聚糖结合凝集素三肽链亚单位（trhMBL）。方法我们先前已构建了N端缺失的重组人甘露聚糖结合凝集素（rhMBL△N）基因的原核表达载体并在大肠杆菌中高效表达rhMBL△N融合蛋白。本实验利用胶原蛋白的3条肽链依其自身性质而相互缠绕成三股螺旋、装配成三级结构的原理,首先以凝血酶酶切rhMBL融合蛋白,将获得的单链rhMBL蛋白在50 mmol/LPBS（pH7.2）和ddH₂O中交替反复透析使之自动装配成trhMBL,最后以配体结合试验和C4d沉淀试验对终产物trhMBL进行生物学活性分析。结果 rhMBL融合蛋白经凝血酶酶切,获得相对分子质量约20 000的rhMBL单链蛋白;将其反复透析后得到相对分子质量约50 000的rhMBL三肽链亚单位trhMBL;活性分析表明,该MBL亚单位的配体结合活性比rhMBL△N肽链高,并获得了激活补体凝集素途径的活性,但这些活性比天然MBL低。结论成功获得了具有生物学活性的重组人-MBL三肽链亚单位;这种三肽链组织形式不仅是MBL的结构亚单位,而且是其功能亚单位。更多还原

【Abstract】 Objective To prepare the trimeric subunits of recombinant human mannan-binding lectin（MBL） with biological activities.Methods A prokaryotic expression vector containing human MBL N-terminal deletant（rhMBL A N） gene we previously constructed was transformed into E.coli for efficient expression of rhMBL A N fusion protein.Based on the principle that the collagen polypeptides tend to self-assembly into the tertiary structure of proteins by forming a triple helix due to the characteristic properties of the collagen proteins,rhMBL A N fusion protein was limitedly hydrolyzed with thrombin.The obtained rhMBL△N polypeptide was repeatedly dialyzed in 50 mmol/L PBS（pH7.2） and ddH₂O,and the final product was analyzed for its bioactivities using a ligand-binding assay and a C4d deposition assay.Results rhMBL A N polypeptide with a relative molecular mass of about 20 000 was obtained by limited proteolysis of rhMBL A N fusion protein with thrombin.Repeated dialyses of rhMBL A N polypeptides in 50 mmol/L PBS and ddH₂O resulted in the isolation of the trimeric subunit trhMBL A N（with a relative molecular mass of about 50 000）,which contained a collagen-like helix.The trhMBL AN protein had a higher ligand-binding activity than rhMBL AN polypeptide,and acquired the activity to initiate the lectin pathway of complement activation,but the activities were lower than those of natural MBL.Conclusion We have successfully obtained the bioactive trimeric subunit of rhMBL,trhMBL A N,and this structural subunit is also the functional subunit of the MBL molecule.更多还原