[1]杨兴武?,郭 奎?,韩昊莹,等.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[J].中国预防兽医学报,2019,(02):179-183.[doi:0.3969/j.issn.1008-0589.201709020]
 YANG Xing-wu?,GUO Kui?,HAN Hao-ying,et al.Expression and antibacterial activity of porcine β-defensin 1in Lactobacillus casei[J].Chinese journal of preventive veterinary medicine,2019,(02):179-183.[doi:0.3969/j.issn.1008-0589.201709020]
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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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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年02
页码:
179-183
栏目:
免疫学
出版日期:
2019-02-15

文章信息/Info

Title:
Expression and antibacterial activity of porcine β-defensin 1in Lactobacillus casei
文章编号:
1008-0589(2019)02-0179-05
作者:
 

杨兴武1?郭 奎12?韩昊莹1赵 宇1郑慧华1杨明凡13陈红英13*

 (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)
Author(s):
 

 YANG Xing-wu1? GUO Kui12? HAN Hao-ying1 ZHAO Yu1 ZHENG Hui-hua1 YANG Ming-fan13 CHEN Hong-ying13*

 (1. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China;
2. Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China;
3. Zhengzhou Major Pig Disease Prevention and Control Laboratory, Henan Agricultural University, Zhengzhou 450002, China)
关键词:
猪β防御素1成熟肽基因干酪乳酸杆菌表达
Keywords:
porcine β-defensin 1  mature peptide gene  Lactobacillus casei  expression
分类号:
S852.61
DOI:
0.3969/j.issn.1008-0589.201709020
文献标志码:
A
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。
Abstract:
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-SP- PBD1 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 5.8ku. 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.

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                                   (本文编辑:李 娜)

更新日期/Last Update: 2019-03-06