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  

Expression and antibacterial activity of porcine β-defensin 1 in Lactobacillus casei

作　　者：杨兴武[1] 郭奎[1,2] 韩昊莹 赵宇 郑慧华 杨明凡 陈红英[1,3] YANG Xing-wu;GUO Kui;HAN Hao-ying;ZHAO Yu;ZHENG Hui-hua;YANG Ming-fan;CHEN Hong-ying(College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences,Harbin 150069,China;Zhengzhou Major Pig Disease Prevention and Control Laboratory,Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150069 [3]郑州市猪重大疫病防控重点实验室,河南郑州450002

出　　处：《中国预防兽医学报》2019年第2期179-183,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：河南省产学研合作计划项目(152107000003);河南省基础与前沿技术研究计划项目(142300410156)

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.

关 键 词：猪β防御素1 成熟肽基因 干酪乳酸杆菌 表达 

分 类 号：S852.61[农业科学—基础兽医学]