融合蛋白 NrCAM-Fc的重组体的构建

目的：构建神经原相关的细胞粘附分子（NrCAM）和免疫球蛋白Fc片段融合基因的重组质粒并通过Baculovirus载体转染到昆虫细胞。方法：利用RT－PCR方法从神经母细胞瘤细胞系SK－N－SN获得NrCAM的信号肽区和跨膜区片段并直接克隆到pGEMT载体，经多次酶切、连接、转化、筛选获得NrCAM-Fc的重组副合基因，测序后将此基因通过Baculovirus载体转染至昆虫细胞。结果：多种酶切和测序结果表明NrCAM-Fc的序列与原序列符合率达98％以上，NrCAM与Fc连接处序列保证了Fc的读码框架（ORF）。结论：Baculovirus载体转染效率高，检测方法简便易行。

Construction of Recombinant Fusion Protein NrCAM-Fc

Objective: The aim was to construct the recombinant of neuro related cell adhesion molecule(NrCAM) and immunoglobin Fc fragment and transfect it into insect cells. Methods: RT PCR was used to get the fragments of signal peptide and transmembrane domain. RT PCR products were cloned directly by pGEMT vector. After several times of digestion,DNA purification from gel,ligation and transformation,we obtained construct of NrCAM Fc which was transfected into insect cells by pBlueBac4.5 vector(Baculovirus vector). Results:The coincidence rate of the sequence between the recombinant and the original was over 98%. The link of the NrCAM and Fc was kept in exact open reading frame(ORF). Conclusion:Baculovirus vector is highly efficient for transfection. It is easy to detect after transfection.