猪PBD1成熟肽基因在干酪乳酸杆菌中的表达及其抑菌活性的检测
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摘要
为获得一株能够稳定表达猪β防御素-1 (PBD1)成熟肽蛋白的重组干酪乳酸杆菌,本研究将PBD1成熟肽基因克隆至含有短小干酪乳酸杆菌信号肽的pUCK-SP载体中,再将SP-PBD1亚克隆到干酪乳酸杆菌整合性表达质粒pMJ67的Lac启动子下游,获得重组质粒pMJ67-SP-PBD1,电转化入干酪乳酸杆菌,经红霉素抗性筛选和基因组DNA PCR测序鉴定,证实猪PBD1基因整合到了干酪乳酸杆菌中。采用半定量RT-PCR分析显示,经2%乳糖诱导18 h后的重组菌中猪PBD1 mRNA含量最高。Western blot检测显示,经乳糖诱导的重组菌表达了约5.8 ku的目的蛋白,与PBD1成熟蛋白理论分子量相符。抑菌试验结果显示重组PBD1对葡萄球菌具有明显抑制作用。表明猪PBD1基因在重组干酪乳酸杆菌中获得了表达,且具有抑菌活性。本研究为进一步研发具有广谱抗菌作用的微生态制剂奠定了基础。
To construct recombinant Lactobacillus casei stably expressing porcine β-defensin 1(PBD1), the PBD1 mature peptide gene was cloned into pUCK-SP vector containing L.casei signal peptide, and then SP-PBD1 was cloned into lac promoter downstream of L.casei integrated expression plasmid pMJ67 to produce a recombinant plasmid pMJ67-SP-PBD1. The pMJ67-SPPBD1 was transformed by electroporation into L.casei competent cells, and PBD1 mature peptide gene was successfully integrated into L.casei, as revealed by the erythromycin resistant selection, PCR of genomic DNA and PCR products sequencing. The highest mRNA level of PBD1 in the recombinant L.casei CECT5276 strain containing the integrative vector pMJ67-SP-PBD1 was identified by semi quantitative RT-PCR analysis after induction for 18 hrs with 2% lactose. Western blot analysis showed that the recombinant strain expressed the target protein of 5.8 ku. The recombinant PBD1 had obvious inhibitory effect on Staphylococcus.These results indicated that the PBD1 was able to be expressed in the recombinant Lb.casei CECT5276 and had antibacterial activity. This lays a foundation for further research and development of probiotics with broad-spectrum antibacterial activity.
To construct recombinant Lactobacillus casei stably expressing porcine β-defensin 1(PBD1), the PBD1 mature peptide gene was cloned into pUCK-SP vector containing L.casei signal peptide, and then SP-PBD1 was cloned into lac promoter downstream of L.casei integrated expression plasmid pMJ67 to produce a recombinant plasmid pMJ67-SP-PBD1. The pMJ67-SPPBD1 was transformed by electroporation into L.casei competent cells, and PBD1 mature peptide gene was successfully integrated into L.casei, as revealed by the erythromycin resistant selection, PCR of genomic DNA and PCR products sequencing. The highest mRNA level of PBD1 in the recombinant L.casei CECT5276 strain containing the integrative vector pMJ67-SP-PBD1 was identified by semi quantitative RT-PCR analysis after induction for 18 hrs with 2% lactose. Western blot analysis showed that the recombinant strain expressed the target protein of 5.8 ku. The recombinant PBD1 had obvious inhibitory effect on Staphylococcus.These results indicated that the PBD1 was able to be expressed in the recombinant Lb.casei CECT5276 and had antibacterial activity. This lays a foundation for further research and development of probiotics with broad-spectrum antibacterial activity.
引文
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[2]Thomma B P,Cammue B P,Thevissen K.Plant defensins[J].Planta,2002,216(2):193-202.
[3]Pruthviraj D R,Usha A P,Venkatachalapathy R T.Identification of a novel single nucleotide polymorphism in porcine Betadefensin-1 Gene[J].Asian-Australas J Anim Sci,2016,29(3):315-320.
[4]罗刚,魏泓.猪防御素基因PBDI在大肠杆菌中的重组和融合表达[J].遗传,2003,25(2):146-150.
[5]姜丽华,卢海蓉,黄德新,等.猪β防御素1基因在毕赤酵母中的分泌表达[J].生物工程学报,2006,22(6):1036-1039.
[6]江学斌,陈嘉蔚,杨军,等.猪源抗菌肽PBD-1在毕赤酵母中的表达及鉴定[J].中国畜牧兽医,2016,43(3):644-649.
[7]Ogawa M,Shimizu K,Nomoto K,et al.Inhibition of in vitro growth of Shiga toxin-producing Escherichia coli O157:H7 by probiotic Lactobacillus strains due to production of lactic acid[J].Int J Food Microbiol,2002,74(1-2):167.
[8]Liu Di-qiu,Wang Xiang-qing,Ge Jun-wei,et al.Comparison of the immune responses induced by oral immunization of mice with Lactobacillus casei-expressing porcine parvovirus VP2 and VP2 fused to Escherichia coli,heat-labile enterotoxin B subunit protein[J].Comp Immunol Microbiol Infect Dis,2011,34(1):73-81.
[9]Hou Xi-lin,Yu Li-yun,Liu Jian-zhu,et al.Surface-displayed porcine epidemic diarrhea viral(PEDV)antigens on lactic acid bacteria[J].Vaccine,2007,26(1):24-31.
[10]Wei Chun-hua,Liu Jian-kui,Hou Xi-lin,et al.Immunogenicity and protective efficacy of orally or intranasally administered recombinant Lactobacillus casei expressing ETEC K99[J].Vaccine,2010,28(24):4113-4118.
[11]Yang Xi,Cheng Yu-ting,Tan Mei-fang,et al.Overexpression of porcine beta-defensin 2 enhances resistance to Actinobacillus pleuropneumoniae infection in pigs[J].Infect Immun,2015,83(7):2836-2843.
[12]Peng Zi-xin,Wang An-ru,Xie Lin-qi,et al.Use of recombinant porcineβ-defensin 2 as a medicated feed additive for weaned piglets[J].Sci Rep,2016,6:26790.
[13]Yang De,Liu Zhen-hua,Tewary P,et al.Defensin participation in innate and adaptive immunity[J].Curr Pharm Des,2007,13(30):3131-3139.
[14]吴冬,刘尚全,袁媛,等.不同信号肽对重组胰高血糖素样肽-1载体表达效率的影响[J].安徽医科大学学报,2013,48(7):764-768.
[15]王怡,郭躬德,孔祥增.基于数据划分和集成的方法预测信号肽[J].计算机工程与应用,2012,48(36):238-244.