关键词: 斑点叉尾鮰, C型溶菌酶, 毕赤酵母, 重组DNA表达, 抑菌活性

Abstract: C-type lysozyme is one of various lysozymes in different tissues of all organisms，and is a key immune protein in cells and possesses stable structure and excellent bacteriostatic activity. Thus，it is considered to be promising food preservative and feed additives. In order to develop fish C-type lysozyme，the present study established a preparation method of channel catfish（Ictalurus punctatus）C-type lysozyme，based on Pichia pastoris expression system. A cDNA 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 cDNA encoded a peptide consisting of 127 amino acids. The cflyC fragment was ligated to pPICZαA vector，and a recombinant expression vector pPICZαA-cflyC was constructed，and transformed into competent Pichia pastoris X-33. The yeast transformants containing multi-copy gene insertions were screened using zeocin in different concentrations and PCR identification. The target protein was induced for 144 h with 0.5% methanol at pH 6.0，29℃，and 250 r/min，and the expression product was purified by immobilized metal affinity chromatography（IMAC）. Tricine-SDS-PAGE analysis showed that molecular mass of the purified recombinant protein was about 15.1 kD. MALDI-TOF-TOF analysis demonstrated that it was the expected recombinant cflyC. Folin-reagent method indicated that the expression yield of recombinant cflyC was 2.75 mg/L. Agar well diffusion and activity assays proved that the recombinant cflyC presented antibacterial activity against Bacillus subtilis. The present study firstly realized the recombinant DNA expression of the channel catfish C-type lysozyme in P. pastoris，which provides key basis for its large-scale preparation.

Key words: channel catfish, C-type lysozyme, Pichia pastoris, recombinant DNA expression, bacteriostatic activity