【摘要】 根据毕赤酵母Pichia pastoris密码子偏爱性重编兔防御素基因(Neutrophils peptide-1,pNP-1),设计4条引物搭桥合成.将pNP-1克隆到pPICZα-A载体,使用同尾酶XhoⅠ和SalⅠ在重组载体上构建多价串联体[pNP-1(2×)]和[pNP-1(3×)],分别对这3个基因进行毕赤酵母表达,表达量依次为3.39、6.39和13.05μg/mL.重组蛋白经溴化氢切割后,对得到的活性产物进行抑菌试验,结果表明,重组pNP-1蛋白对苏云金杆菌Bacillus thuringiensis等革兰阳性菌(G+)及大肠埃希菌Escherichia coli等革兰阴性菌(G-)均有抑菌活性,但对白色念珠菌Candida albicans等真菌没有明显的抑菌活性.对pNP-1抑菌机理研究发现,细胞壁和DNA合成抑制剂对pNP-1抑菌活性有协同作用,而多种蛋白质和RNA合成抑制剂对pNP-1抑菌活性有抑制作用.更多还原

【Abstract】 Rabbit defensin gene pNP-1 was reconstructed according to codon preference of Pichia pastoris and then amplified by four specific primers.After pNP-1 cloned to pPICZα-A vector,multiple copies of the pNP-1,[pNP-1(2×)] and [pNP-1(3×)] were constructed by using isocaudarner XhoⅠ and Sal Ⅰ on the recombinant vector.The recombinants pNP-1,[pNP-1(2×)] and [pNP-1(3×)] were expressed by Pichia pastoris,with an expression of 3.39,6.39 and 13.05 μg/mL,respectively.The cleavage of recombinant protein performed by cyanogen bromide,the antibacterial activity of the resultant product was examined by antibacterial test.The results showed that recombinant protein pNP-1 had a remarkable antibacterial activity against Gram-positive and Gram-negative bacteria such as Bacillus thuringiensis and Escherichia coli,but no antibacterial activity against fungi.Further study of the antibacterial mechanism of pNP-1 showed that cell-wall synthesis inhibitors and DNA synthesis inhibitors had obvious synergistic effect on antibacterial activity of pNP-1,while protein synthesis inhibitors and RNA synthesis inhibitors had certain inhibition to antibacterial activity of pNP-1.更多还原