Balb/c小鼠MBL-C糖识别域的原核表达及其多克隆抗体的制备

目的 原核表达Balb／c小鼠肝型甘露聚糖结合凝集素（mMBL-C）糖识别域（CRD）并制备其多克隆抗体。方法应用PCR从含Baib／c小鼠MBL-C全长编码区基因的质粒pmMBL-C中扩增目的基因片段，构建重组表达载体，在大肠杆菌中表达。分离纯化表达产物并免疫动物，制备mMBL-C CRD的抗血清，采用ELISA和Western blot鉴定其效价和特异性。结果从pmMBL-C中扩增到约450bp的DNA片段，构建重组载体pET-CKS-mMBL-C-CRD和pET32a-mMBL-C-CRD，经酶切和测序鉴定，证实重组表达载体构建成功。将其导入大肠杆菌BL21（DE3）中表达，表达产物主要以包涵体的形式存在，相对分子质量（Mr）分别为44000和34000。利用pET-CKS载体表达产物免疫家兔，分离免疫血清后，使用pET32a（＋）载体表达产物进行ELISA及Westem blotting，显示多克隆抗体能够与mMBL-C CRD蛋白特异结合。结论获得了重组mMBL-CCRD蛋白及其多克隆抗体，为mMBL-C分子以及CRD的研究奠定了一定的基础。

Prokaryotic expression of the carbohydrate recognition domain of Balb/c mouse MBL-C and preparation of its polyclonal antibody

Objective To express the carbohydrate recognition domain(CRD) of Balb/c mouse MBL-C gene in E.coli and prepare its polyclonal antibody.Methods The target gene fragment was obtained by PCR from plasmid pmMBL-C which contains mouse MBL-C(cDNA.) The recombinant expression vectors were constructed by inserting the gene into prokaryotic expression vectors and expressed in E.coli,and then the product was purified.Rabbits were immunized with the protein and the antiserum was obtained.The titers and specificity of the antibodies were analyzed by ELISA and Western blotting.Results A DNA fragment about 450 bp was amplified by PCR and the recombinant plasmids were constructed,which named pET-CKS-mMBL-C-CRD and pET32a-mMBL-C-CRD.The result of restriction maps and sequencing of the selected clones were consistent with those expected by computer analyses.The expression products in E.coli BL21(DE3) existed mainly as inclusion body,whose relative molecular mass(M_r) were about 44 000 and 34 000,respectively.The protein expressed by pET-CKS vector was injected into rabbits to prepare polyclonal antibody.The antiserum could bind to the mMBL-C CRD protein specifically.Conclusion The expression and antibody preparation of mouse MBL-C CRD gene provide a base for studying mMBL-C molecule and CRD.