家蝇防御素在大肠杆菌中的表达、纯化与抗体制备

Recombinant expression in E. coli,purification and polyclonal antibody preparation of a defensin from the housefly

家蝇防御素是从家蝇中克隆得到的1种抗菌肽。为了进一步研究家蝇防御素的功能和制备特异性抗体,采用大肠杆菌表达外源蛋白的方法, 进行了家蝇防御素原核表达的研究。根据克隆到的家蝇防御素基因(Mdde) 的cDNA序列, 设计特异性引物, PCR 扩增成熟肽的cDNA片段, 将成熟肽序列重组到表达载体pGEX 4T 1中, 构建m Mdde/pGEX 4T 1重组表达载体, 在大肠杆菌BL21 中诱导表达, 重组表达的融合蛋白GST Mdde占菌体总蛋白的33 4%。纯化得到GST Mdde后, 再用凝血酶将其从特定位点切开, 得到表达的m Mdde。液体抑菌实验结果初步表明, 表达的融合蛋白GST Mdde对细菌生长有一定的抑制作用。利用纯化的GST Mdde融合蛋白, 制备了抗血清。

Defensin is a kind of cationic, inducible antimicrobial peptides that have been found in large range of living organisms and contribute to host defense by disrupting the cytoplasmic membrane of microorganisms. A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDNA library of the housefly Musca domestica. To further study the function and to prepare the anti-Mdde polyclonal antibody, we chose the prokaryotic expression system (pGEX-4T-1) to express the mature Mdde as a foreign protein. Mature defensin gene was amplified from cDNA library using specific primers by PCR. The DNA fragment was modified by adding EcoRⅠsite at 5′ end and Xho Ⅰsite at 3′ end respectively, and then cloned into expression vector pGEX-4T-1. The recombinant mMdde/pGEX-4T-1 was identified by sequencing. The E.coli BL21 was transformed by recombinant mMdde/pGEX-4T-1 and the positive clones (mMdde/pGEX-4T-1/BL21) were screened by PCR. 1 mmol/L IPTG can induce a high level of expression of GST-mdde which consists of 26 kD Glutathione S-Tranferase (GST) and 4 kD of Mdde. The fusion protein GST-Mdde was purified by GST-affinity chromatograph and cleaved by thrombin for getting Mdde. The recombinant Mdde showed low antibacterial activities by liquid growth inhibition method .