斑点叉尾鮰C型溶菌酶在毕赤酵母中的表达及其抑菌活性

Expression of Channel Catfish C-type Lysozyme in Pichia pastoris and Its Bacteriostatic Activity

C型溶菌酶是存在于各种生物组织中的数种溶菌酶成员中的一员,是重要的细胞内免疫蛋白,具有稳定的空间结构和显著的抑菌功能,被认为是良好的食品保鲜剂和饲料添加剂。为了开发鱼类来源的C型溶菌酶,研究建立了基于毕赤酵母(Pichia pastoris)表达系统的斑点叉尾鮰(Ictalurus punctatus)C型溶菌酶的制备方法。首先通过PCR获得5'和3'端分别添加Xho I和Xba I酶切位点的编码斑点叉尾鮰C型溶菌酶的c DNA(cflyC),其编码由127个氨基酸残基组成的多肽;将该cflyC与表达载体p PICZαA连接以构建重组表达载体p PICZαA-cflyC;转化至毕赤酵母X-33后,通过不同浓度的博来霉素和对酵母基因组DNA的PCR鉴定,筛选得到高拷贝的酵母转化子;经0.5%甲醇诱导,在p H6.0、29℃、250 r/min下培养144 h,得到的表达产物经固化金属离子亲和层析(IMAC)纯化,得到重组蛋白;经Tricine-SDS-PAGE分析,表明重组蛋白的分子量为15.1 k D;进一步通过MALDI-TOF-TOF质谱鉴定,证明该重组蛋白为预期的重组cflyC。通过福林酚法测得重组cflyC的表达量为2.75 mg/L。琼脂糖凝胶扩散法和酶活力测定证明,重组cflyC对枯草芽孢杆菌具有抑菌活性。本研究首次实现了斑点叉尾鮰C型溶菌酶在毕赤酵母中的重组DNA表达,为其大规模制备奠定了基础。

C-type lysozyme is one of various lysozymes in different tissues of all organisms,and is a key immune protein in cells andpossesses stable structure and excellent bacteriostatic activity. Thus,it is considered to be promising food preservative and feed additives. Inorder to develop fish C-type lysozyme,the present study established a preparation method of channel catfish（Ictalurus punctatus）C-typelysozyme,based on Pichia pastoris expression system. A c DNA fragment（cflyC）added with the Xho I and Xba I restriction sites at 5 ‘and3＇ends respectively,encoding the C-type lysozyme of channel catfish,was obtained by PCR. This c DNA encoded a peptide consisting of 127 amino acids. The cflyC fragment was ligated to p PICZαA vector,and a recombinant expression vector p PICZαA-cflyC was constructed,andtransformed into competent Pichia pastoris X-33. The yeast transformants containing multi-copy gene insertions were screened using zeocin indifferent concentrations and PCR identification. The target protein was induced for 144 h with 0.5% methanol at p H 6.0,29℃,and 250 r/min,and the expression product was purified by immobilized metal affinity chromatography（IMAC）. Tricine-SDS-PAGE analysis showedthat molecular mass of the purified recombinant protein was about 15.1 k D. MALDI-TOF-TOF analysis demonstrated that it was the expectedrecombinant cflyC. Folin-reagent method indicated that the expression yield of recombinant cflyC was 2.75 mg/L. Agar well diffusion andactivity assays proved that the recombinant cflyC presented antibacterial activity against Bacillus subtilis. The present study firstly realized therecombinant DNA expression of the channel catfish C-type lysozyme in P. pastoris,which provides key basis for its large-scale preparation.