家蝇幼虫Defensin成熟蛋白序列的克隆及在原核中的表达

目的 克隆家蝇幼虫defensin成熟蛋白编码序列并在大肠杆菌中表达，为进一步的研究该基因的功能奠定基础。方法 以家蝇幼虫cDNA文库为模板扩增出defensin基因的成熟蛋白全长编码序列，构建重组原核表达载体pGEX／MDEF，转化的克隆(质粒)通过筛选并测序鉴定。重组的pGEX／MDEF在大肠杆菌BL21(DE3)中表达，SDS-PAGE鉴定其表达情况和分子质量并用抗鼠GST单抗作western blot杂交进一步鉴定融合蛋白的表达。结果 以家蝇幼虫cDNA文库扩增出一条长约为140bp的cDNA片段，经测序证实其为defensin基因成熟蛋白的编码序列，它编码含40个氨基酸的蛋白质，预测其分子质量单位为4kD。被成功克隆入pGEX-4T-1载体的家蝇幼虫defensin基因的cDNA，在重组大肠杆菌BL21(DE3)中诱导可大量生成相对分子质量30KD融合蛋白。结论 成功构建了家蝇幼虫defensin基因原核表达载体，且其成熟蛋白的编码序列在大肠杆菌高效表达。为下一步表达蛋白纯化，以及研究其生物活性提供了材料。

Cloning and expression of defensin gene of Musca domestica larvae.

Objective To amplify defensin gene cDNA of Musca domestica larva, clone it into prokaryotic expression plasmids and express it in Escherichia coli, Methods DNA sequence encoding the defensin mature protein was amplified from Musca domestica larva cDNA library.Then it was cloned into the prokaryotic vector pGEX-4T-1.The transformed clones was screened and identified by sequencing.The recombinant pGEX/MDEF was expressed in E.coli named BL21(DE3). SDS-PAGE was used to identify whether defensin gene was expressed and its molecular weight. Anti-GST monoclonic antibody was used to identify the fusion protein by Western Blot. Results A 140bp length cDNA sequences were obtained from Musca domestica larva cDNA library and identified to be defensin cDNA by sequence analysis.DNA sequence encoding the defensin mature protein encodes a protein of 40 amino acids, the predicted molecular weight of the protein is 4.0 kD.its cDNA was successfully cloned into the pGEX-4T-1 vector,and then.pGEX/ mdef was expressed in BL21(DE3) with molecular weight of about 30kD. Conclusion The defensin gene recombinant prokaryotic expression plasmids was successfully constructed and expressed in E. coli.