丝胶抗冻肽在大肠杆菌中的重组表达及其抗冻活性初探

Recombinant Expression of Sericin Antifreeze Peptide in E.coli and Its Antifreeze Activity

为了高效制备抗冻肽,研究一种丝胶抗冻肽目的基因SerD在大肠杆菌BL21菌株中的重组表达,并分析表达产物的抗冻活性。首先合成SerD基因片段,经KpnI和XhoI双酶切后定向插入质粒载体Pet32a中,构建表达质粒Pet32a-SerD,然后电转化入大肠杆菌BL21(DE3),在IPTG诱导下进行目的基因表达,利用镍琼脂糖亲和层析对目的重组蛋白进行纯化,并对其进行抗冻活性分析。结果表明,最佳表达条件为重组菌在20℃诱导16 h;通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis,SDS-PAGE)和蛋白质免疫印迹试验(Western-Blot)鉴定重组蛋白表达成功且His-SerD融合蛋白表达的分子量在25～35 kDa之间;胞内表达His-SerD融合蛋白的大肠杆菌BL21-SerD复苏后的生长活性明显高于PBL空载菌;添加His-SerD融合蛋白可明显降低溶液中冰晶颗粒大小,具有较好的重结晶抑制效果。本文通过基因工程方法在大肠杆菌中成功构建丝胶肽抗冻肽的重组表达系统,并最终获得具有抗冷冻胁迫保护作用的His-SerD融合蛋白。

In order to obtain antifreeze peptides efficiently, the recombinant expression of serin antifreeze peptide target gene SerD in E.coli BL21 （ DE3 ） was studied, and the antifreeze activity of the expressed product was analyzed subsequently.The gene encoding SerD was synthesized, directionally inserted into vector Pet32a after Kpnl and XhoI double enzyme digestion, and constructed the expression vector Pet32a-SerD.Electroporation was converted the expression vector to E.coli BI21 （ DE3）.IPTG induced the expression of target genes, and the target protein was purified by nickel sepharose affinity chromatography. The results indicated that the optimum expression condition was 16 h at 20 ℃. SDS-PAGE and Western-Blot identified the successful expression of recombinant protein, and the molecular weight of His-SerD fusion protein was between 25-35 kDa. The activity of E.coli expressing His - SerD fusion protein after freezing showed that the growth activity of BI21 - SerD expressing His-SerD fusion protein was significantly higher than that of PBL airborne bacteria.The results of recrystallization inhibition activity of His-SerD fusion protein showed that the addition of His-SerD fusion protein could obviously reduce the size of ice crystal particles in the solution,indicated that it had a good recrystallization inhibition activity. This study showed that the recombinant expression system of sericin peptide antifreeze peptide could be successfully constructed in E. coli by genetic engineering method, and the His-SerD fusion protein with antifreezing stress protection could be obtained.