牛乳铁蛋白N-叶在毕赤氏酵母中的高效表达及抑菌性

High expression of bovine lactoferrin N-lobe in Pichia pastoris and its antibacterial activity

摘要:

【目的】本研究将牛乳铁蛋白的N-叶(BLF-N)克隆至毕赤氏酵母菌基因组中,通过密码子优化和发酵条件优化,实现BLF-N的异源高效表达,研究重组BLF-N的抑菌功能。【方法】本文以BLF基因为模板,按照毕赤氏酵母的密码子偏好性进行密码子优化,构建重组表达载体pPIC9K-UBLF-N,电击转化到Pichia pastoris GS115中,获得重组菌P. pastoris GS115/pPIC9K-UBLF-N。通过高拷贝策略和发酵条件优化,结合SDS-PAGE分析BLF-N蛋白表达情况,研究重组的BLF-N蛋白对革兰氏阳性和革兰氏阴性菌的抑菌活性。【结果】SDS-PAGE结果表明,BLF-N在P. pastoris GS115中实现了可溶性高效表达,分子量为37 kDa左右,与其理论分子量一致。通过高拷贝筛选,获得了在3 mg/mL遗传霉素G418抗性平板上生长的转化子;通过发酵条件优化,获得最优蛋白表达条件为:0.2%(V/V)甲醇添加量、30℃、pH 5.0,在此条件下,重组BLF-N的表达量为50.5 mg/L。抑菌实验结果表明,重组BLF-N对大肠杆菌和金黄色葡萄球菌均有较强的抑菌活性,且重组BLF-N较商业BLF抑菌活性更高。【结论】通过蛋白表达和发酵条件优化策略,本研究成功实现了BLF-N在毕赤氏酵母中的高效表达,且重组BLF-N的抑菌活性较BLF显著提升。该研究为牛乳铁蛋白的高效绿色制备及产业化生产奠定了研究基础,同时为后续研究BLF和BLF-N的结构-功能关系差异提供了理论指导。

Abstract:

[Objective] We cloned the bovine lactoferrin N-lobe (BLF-N) into the genome of Pichia pastoris and enabled the heterologous expression of BLF-N through the optimization of its gene codon and fermentation conditions.Furthermore,we studied the antibacterial activity of the recombinant protein.[Methods] With BLF gene as template,BLF-N gene was optimized according to the codon bias of P.pastoris.On this basis,the recombinant expression vector pPIC9K-UBLF-N was constructed and transformed into P.pastoris GS115 by electroporation to yield the recombinant P. pastoris GS115/pPIC9K-UBLF-N.Through high-copy screening and optimization of fermentation conditions,BLF-N expression was improved and then evaluated by SDS-PAGE.The inhibition of the recombinant BLF-N on Gram-positive and Gram-negative bacteria was explored.[Results] SDS-PAGE results showed the efficient soluble expression of BLF-N with molecular weight of about 37 kDa (consistent with the theoretical molecular weight) in P. pastoris GS115.A transformant resistant to 3 mg/mL genomycin G418 was obtained through high-copy screening.The highest recombinant BLF-N yield (50.5 mg/L) was achieved under the following optimized fermentation conditions:0.2%(V/V) methanol,30℃,and pH 5.0.Recombinant BLF-N demonstrated strong inhibition on Escherichia coli and Staphylococcus aureus and higher antibacterial activity than commercial BLF.[Conclusion] Through high-copy screening and optimization of fermentation conditions,we achieved the efficient expression of BLF-N in P. pastoris and the recombinant BLF-N presented significantly higher antibacterial activity than the commercial BLF.This study lays a foundation for the efficient and green preparation and industrial production of BLF-N and provides theoretical guidance for research on the difference in structure-function relationship between BLF and BLF-N.