绿色木霉碱性蛋白酶基因TvALP的克隆与原核表达

The cloning and prokaryotic expression of the alkalineprotease gene TvALP from Trichoderma viride

基于绿色木霉中抗菌蛋白质的de-novo质谱测定得到的部分肽段序列,通过BLASTp数据库检索,发现该肽段由碱性蛋白酶基因编码,采取同源克隆技术扩增出目的基因片段。生物信息学分析揭示,TvALP基因(GenBank编号KJ659907)完整开放读码框为1 230 bp,并编码了一个由409个氨基酸组成的蛋白质。该蛋白质的预测分子量约为43 kD,且含有一段由20个氨基酸组成的信号肽。根据分类,该蛋白质属于Peptidase inhibitor_I9超家族,并且是枯草杆菌蛋白酶家族丝氨酸蛋白酶S8家族的一个成员。利用双酶切法将测序正确的质粒连接至原核表达载体pET30a中,在宿主菌BL21(DE3)pLysS中诱导表达。结果在45 kD处显示出1条特异蛋白质条带,与生物性信息预测目的蛋白大小一致,表明切除信号肽的表达质粒pET30a-ΔTvALP(“Δ”表示切除信号肽)在宿主菌BL21(DE3)pLysS中成功表达。经超声波破碎后,蛋白形成了包涵体。但经体外复性技术,未能获得有活性的蛋白进行抗菌机理研究。

A peptide sequence was identified from antimicrobial proteins in Trichoderma viride based on the denovo mass spectrometry determination.Through BLASTp database searching,it was found that this peptide sequence is encoded by a alkaline proteinase gene,and the target gene fragment was amplified using homologybased cloning.Bioinformatics analysis reveals that the TvALP gene(GenBank number KJ659907)has a complete open reading frame of 1230 bp and encodes a protein composed of 409 amino acids.This protein has a predicted molecular weight of about 43 kD,and an isoelectric point of 7.96,and contains a signal peptide composed of 20 amino acids.According to classification,the protein belongs to the peptidase inhibitor_I9 superfamily and is a member of the subtilisin serine protease S8 family. The sequenced plasmid was ligated into the prokaryoticexpression vector pET30a using double digestion, and the expression was induced in the host bacterium BL21(DE3)pLysS. The result showed a specific protein band at 45 kD, which was consistent with the predicted size ofthe target protein, indicating that the expression plasmid pET30a-ΔTvALP ( “Δ” Signaling peptide excision) wassuccessfully expressed in the host bacterium BL21(DE3)pLysS. After sonication, the protein formed inclusionbodies. However, after in vitro renaturation, no active protein was obtained for antibacterial mechanism research.