Abstract

The codon-optimized zebrafish β-defensin 3 mature peptide gene mzfDB3 and channel catfish c-type lysozyme gene cflyC were used to design the fusion gene cflyC-mzfDB3. In the fusion protein cflyC-mzfDB3, 4×Gly flexible amino acid linker with an enterokinase cleavage site DDDDK was designed to link the mzfDB3 to the C-terminus of cflyC, which potentially contributes to digestion of the recombinant cflyC-mzfDB3 by endogenous enterokinases. The fusion protein was expressed in Pichia pastoris X-33 using expression vectors harboring 1-, 2-, or 4-copies, respectively, of the expression cassette. The cflyC-mzfDB3 gene copy numbers of P. pastoris transformants were quantified using real-time quantitative PCR. Their expression yields showed that the expression level in the 4-copy cflyC-mzfDB3 transformant was 2.67-fold higher than that in the 1-copy cflyC-mzfDB3 transformant, demonstrating that the increase in the cflyC-mzfDB3 gene copy number resulted in increased protein expression. The culture medium supernatant containing recombinant cflyC-mzfDB3 exhibited antibacterial activity against Gram-positive Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa, indicating that there may be a synergistic effect of mzfDB3 and cflyC on the peptide’s antibacterial activity. In addition, cell-free P. pastoris medium may be a potential candidate for further development as a natural hybrid antimicrobial solution.

中文翻译：

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融合基因cflyC-mzfDB3的多个副本增强了毕赤酵母中的混合抗菌肽的表达。

摘要

使用密码子优化的斑马鱼β-防御素3成熟肽基因mzfDB3和channel鱼c型溶菌酶基因cflyC设计融合基因cflyC-mzfDB3。在融合蛋白cflyC-mzfDB3中，设计了具有肠激酶切割位点DDDDK的4xGly柔性氨基酸接头，将mzfDB3与cflyC的C末端连接，这可能有助于内源性肠激酶消化重组cflyC-mzfDB3。使用分别携带表达盒的1、2或4个拷贝的表达载体，在巴斯德毕赤酵母X-33中表达融合蛋白。P的cflyC-mzfDB3基因拷贝数。巴斯德使用实时定量PCR对转化体进行定量。他们的表达产量表明，在4拷贝cflyC-mzfDB3转化子中的表达水平比在1拷贝cflyC-mzfDB3转化子中的表达水平高2.67倍，表明cflyC-mzfDB3基因拷贝数的增加导致蛋白质增加。表达。含有重组cflyC-mzfDB3的培养基上清显示出对革兰氏阳性单核细胞增生性李斯特菌和革兰氏阴性铜绿假单胞菌的抗菌活性，表明mzfDB3和cflyC对肽的抗菌活性可能具有协同作用。另外，无细胞巴斯德毕赤酵母 培养基可能会作为天然的混合抗菌溶液进一步开发。