小鼠beta防御素2原核表达与抗菌作用的初步研究

Prokaryotic Expression and Purification and Activity Analysis of Mouse Beta Defensins 2

构建小鼠β-防御素-2(mouse beta defensins 2,mBD2)原核表达质粒pET32/mBD2,进行蛋白诱导表达及纯化,测定并纯化蛋白的抗菌活性。旨在为进一步研究其生物学特性奠定基础。通过腹腔注射脂多糖(lipopoly-saccharide,LPS)建立小鼠急性时相反应,采用RT-PCR方法扩增mBD2成熟肽,经KpnI和XhoI双酶切后插入相同酶切的pET-32a(+)载体,构建的重组质粒。将鉴定正确的重组质粒转化大肠杆菌表达菌株BL21(DE3),采用异丙基-D-硫代半乳糖苷(IPTG)诱导融合蛋白的表达。通过镍亲和层析获得纯化的融合蛋白。将融合蛋白采用肠激酶酶切、洗脱并用滤纸片法测定目的蛋白的抗菌活性。成功构建了原核表达质粒pET32a(+)/mBD2,并转化工程菌BL21(DE3)。在0.25 mmol/L IPTG、30℃诱导4 h条件下获得的融合蛋白。采用抑菌试验证实蛋白具有一定的抑制革兰阳性菌及阴性菌生长的作用。本研究成功构建了pET32/mBD2原核表达质粒,得到了在大肠杆菌中稳定表达mBD2蛋白。

The prokaryotic expression vector,pET32a（＋）/mBD2 was constructed and transfed into E.coli BL21,respectively.Then the expression of mBD2 in E.coli BL21 and function was studied.mBD gene was amplified by RT-PCR from sensitive mouse which LPS was injected beforehand.The amplified mBD2 gene fragment was inserted into the plasmid pET-32a（＋）that was digested with Kpn I and Xho I.The recombinant plasmid pET32/mBD2 was transformed into E.coli JM109 and selected with ampicillin.The positive clones containing recombinant plasmid pET32/mBD2 were verified by restriction endonucleases Kpn I and Xho I,and then sequenced.mBD2-TRX fused protein expression in the E.coli BL21（ED3）was identified by SDS-PAGE.The expressed recombinant protein was purifier by His-tag purification kit,digested by enterokinase.Then its antibacterial activity was tested.The pET32a（＋）/mBD2 vectors were constructed successfully and correctly.The BL21（DE3）transformed recombinant plasmid pET32a（＋）/mBD2 had expressed mBD2-TRX fused protein effectively under the conditions of 0.25 mmol/L IPTG and 30℃.The activity assay demonstrated the peptides had ability against three standard stains.