人IgE Fc基因的克隆、纯化、真核表达载体的构建及序列测定

Cloning,purification,construction and sequencing of Fc segment of human IgE molecule

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pEGFPN1,T4DNA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p EGFP-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 p EGFP-N1 were extracted and digested with EcoRⅠand BamHⅠ. The linear fragments were purified and retrieved,then ligated to eukaryotic expression vector p EGFP-N1 by T4 DNA ligationase. Ligation production was miniprepared and identified by EcoR Ⅰ and BamH Ⅰ. 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.