北京生物医学工程

人ＴＲＡＭ基因真核表达载体的构建

Construction of eukaryotic expression vector of human TRAM gene

作者：高建伟叶峰蒋宏峰

单位：首都医科大学附属北京安贞医院-北京市心肺血管疾病研究所药理室（北京 100029） <p>通信作者：蒋宏峰，教授。E-mail:jhf@pku.edu.cn</p> <p> </p>

分类号：R318 ；Q786

出版年·卷·期（页码）：2022·41·3（307-310）

摘要：

目的构建TRAM基因真核表达载体，为研究TRAM的功能提供研究工具。方法首先采用PCR方法扩增人TRAM蛋白相应的编码序列，将扩增的特定片段克隆至pCMV-N-Flag真核表达载体。然后，对重组载体进行DNA测序，确认序列正确后将重组载体转染至HEK-293T细胞，并用Western blot方法检测TRAM蛋白的表达以评价载体是否构建成功。结果菌落PCR电泳可检测到800 bp附近出现目的条带，质粒DNA测序显示载体插入705 bp的核苷酸序列，其序列与TRAM完全一致。Western blot检测到HEK-293T细胞高表达带Flag标签的TRAM蛋白。结论基于PCR扩增、双酶切、酶连接扩增片段和载体成功构建带Flag标签的TRAM真核表达载体。构建的质粒可为进一步研究干扰素的调控和抗病毒免疫治疗提供一种研究工具。

Objective To construct the eukaryotic expression vector of TRAM gene ，and to provide a research tool for studying the function of TRAM protein. Methods Firstly, the corresponding coding sequence of human TRAM protein was amplified by PCR, and the amplified specific fragment was cloned into pCMV-N-Flag eukaryotic expression vector. Then the recombinant vector was sequenced. After confirming the correct sequence, the recombinant vector was transfected into HEK-293T cells. The expression of TRAM protein was detected by Western blot to evaluate whether the vector was successfully constructed. Results The target band near 800bp could be detected by bacterial colony PCR electrophoresis. Plasmid DNA sequencing showed that 705 bp nucleotide sequence was inserted into the vector, which was completely consistent with TRAM. Western blot detected that HEK-293T cells highly expressed TRAM protein with Flag tag. Conclusions The eukaryotic expression vector TRAM with Flag tag is successfully constructed based on the methods of PCR amplification, double enzyme digestion, and enzyme linking amplified fragments and vectors. The constructed plasmid can provide a research tool for further study of interferon regulation and antiviral immunotherapy.

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