人IgE Fc基因的克隆、纯化、真核表达载体的构建及序列测定-现代医学

人IgE Fc基因的克隆、纯化、真核表达载体的构建及序列测定

单位：承德医学院附属医院皮肤科, 河北承德 067000

分类号：Q782

出版年·卷·期（页码）：2019·47·第六期（631-636）

摘要：

目的：构建重组质粒pEGFP-N1/IgE Fc，为建立一种以IgE分子Fc段为佐剂的高效安全的抗肿瘤免疫治疗奠定基础。方法：分离急性荨麻疹病人的外周静脉血淋巴细胞，Trizol一步法提取总RNA，甲醛变性琼脂糖凝胶电泳证实所提RNA没有明显降解后，在AMV逆转录酶作用下进行逆转录反应，Pfu DNA聚合酶作用下进行聚合反应得到目的片断。提取质粒pEGFP-N1，将pEGFP-N1及IgE Fc基因的cDNA分别用限制性内切酶EcoRⅠ、BamHⅠ双酶切，电泳分离，纯化回收试剂盒回收IgE Fc基因cDNA片断和质粒pEGFP-N1，T₄ DNA连接酶连接两片断，连接产物转化感受态细胞Top10，小量提取质粒，限制性内切酶EcoRⅠ、BamHⅠ初步酶切鉴定，重组质粒送大连宝生物工程有限公司测序。结果：酶切及测序结果表明pEGFP-N1/IgE Fc真核表达质粒构建成功。结论：成功构建重组质粒pEGFP-N1/IgE Fc，为进一步研究其功能及探索新的抗肿瘤方向奠定了基础。

Objective:To constructe recombinant plasmid pEGFP-N1/IgE Fc and to establish a basis for the efficien and safe anti-tumor immunotherapy with Fc segment of IgE molecule as an adjuvant. Methods:The peripheral venous blood lymphocytes of patients with acute urticaria were isolated. Total RNA was extracted by Trizol one-step method to obtain IgE Fc cDNA with AMV reverse transcriptase and Pfu DNA polymerase after RNA was verified that there was no obvious degradation. IgE Fc and Plasmids pEGFP-N1 were extracted and digested with EcoRⅠand BamHⅠ.The linear fragments were purified and retrieved, then ligated to eukaryotic expression vector pEGFP-N1 by T₄ DNA ligationase. Ligation production was miniprepared and identified by EcoRⅠand BamHⅠ. The recombinant vector with insert fragment was sequenced by TaKaRa Biotechnology of Dalian. Results:The recombinant plasmid pEGFP-N1/IgE Fc was constructed successfully. Conclusion:The recombinant plasmid pEGFP-N1/IgE Fc is successfully constructed, which sets a foundation for further study of its function and exploration of new anti-tumor direction.

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