细菌素Paracin 1.7在乳酸乳球菌NZ9000中的异源表达及抑菌性质的研究

doi:10.11924/j.issn.1000-6850.casb2021-0463

中国农学通报 ›› 2021, Vol. 37 ›› Issue (26): 15-23.doi: 10.11924/j.issn.1000-6850.casb2021-0463

细菌素Paracin 1.7在乳酸乳球菌NZ9000中的异源表达及抑菌性质的研究

潘月¹^,²(), 杨志馨¹^,², 杨晓丽¹, 王旭¹^,², 宋刚¹^,²()

¹黑龙江大学农业微生物技术教育部工程研究中心,哈尔滨 150080
²黑龙江大学生命科学学院黑龙江省普通高校微生物重点实的验室,哈尔滨 150080

收稿日期:2021-04-30 修回日期:2021-06-02 出版日期:2021-09-15 发布日期:2021-09-30
通讯作者: 宋刚
作者简介:潘月,女,1996年出生,黑龙江齐齐哈尔人,硕士研究生,研究方向为微生物学。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号黑龙江大学,Tel：0451-86609016,E-mail： 15145068391@163.com。
基金资助:
国家自然科学基金“产细菌素乳酸菌对东北酸菜发酵体系微生物区系功能的影响及其分子机制的解析”(31770544)

The Heterologous Expression and Antibacterial Properties of the Bacteriocin Paracin1.7 in Lactococcus Lactis NZ9000

Pan Yue¹^,²(), Yang Zhixin¹^,², Yang Xiaoli¹, Wang Xu¹^,², Song Gang¹^,²()

¹Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150080
²Key Laboratory of Microbiology, College of Heilongjiang Province, School of Life Sciences, Heilongjiang University, Harbin 150080

Received:2021-04-30 Revised:2021-06-02 Online:2021-09-15 Published:2021-09-30
Contact: Song Gang

摘要/Abstract

摘要：

为了获得抑菌效果更好的细菌素,本试验采用异源表达的方法表达假定为细菌素的蛋白57AA、62AA、77AA、84AA,表达宿主为乳酸乳球菌(Lactococcus lactis)NZ9000,表达载体为pNZ8148,检测异源表达产物的抑菌效果。结果表明,4种假定的蛋白57AA、62AA、77AA、84AA对B. subtilis均有抑菌效果,57AA、62AA、77AA、84AA的抑菌活性与副干酪乳杆菌HD1.7产生的Paracin 1.7相比分别提高了7.57倍、4.53倍、5.89倍（差异极显著P<0.01）和1.20倍（差异显著P<0.05）。结论为异源表达的假定为细菌素的蛋白57AA、62AA、77AA、84AA的抑菌效果更好。

关键词: 细菌素, Paracin1.7, 乳酸乳球菌, 异源表达, 抑菌性质

Abstract:

To obtain the bacteriocin with better antibacterial effect, this experiment used heterologous expression method to express the presumed bacteriocin protein 57AA, 62AA, 77AA, 84AA, taking the expression host of Lactococcus lactis NZ9000 and the vector of pNZ8148 to detect the antibacterial effect of heterologous expression products. The results show that the four hypothetical protein 57AA, 62AA, 77AA, and 84AA have antibacterial effects on B. subtilis. Compared with Paracin 1.7 produced by Lactobacillus paracasei HD1.7, the antibacterial activities of 57AA, 62AA, 77AA, and 84AA were improved by 7.57 times, 4.53 times, 5.89 times (very significant difference P<0.01) and 1.20 times (significant difference P<0.05), respectively. In conclusion, the heterologously expressed protein 57AA, 62AA, 77AA, and 84AA, which are putatively expressed as bacteriocins, have better antibacterial effects.

Key words: bacteriocin, Paracin 1.7, Lactococcus lactis, heterologous expression, antibacterial properties

中图分类号:

S182

潘月, 杨志馨, 杨晓丽, 王旭, 宋刚. 细菌素Paracin 1.7在乳酸乳球菌NZ9000中的异源表达及抑菌性质的研究[J]. 中国农学通报, 2021, 37(26): 15-23.

Pan Yue, Yang Zhixin, Yang Xiaoli, Wang Xu, Song Gang. The Heterologous Expression and Antibacterial Properties of the Bacteriocin Paracin1.7 in Lactococcus Lactis NZ9000[J]. Chinese Agricultural Science Bulletin, 2021, 37(26): 15-23.

图/表 18

序号	名称	用途	来源
1	乳酸乳球菌(Lactococcus lactis)NZ9000	异源表达宿主	黑龙江大学微生物重点实验室保藏
2	副干酪乳杆菌(Lactobacillus paracasei )HD1.7	原初宿主
3	枯草芽胞杆菌(Bacillus subtilis)ATCC 11774	抑菌试验指示菌
4	大肠杆菌(Escherichia coli )DH5α	克隆宿主

表1. 供试菌株

序号	名称	特征	大小/bp	用途
1	pNZ8148	Cm ^r携带乳链菌肽诱导型启动子P _nisA	3165	表达载体
2	pMD18-T	—	2692	克隆载体

表2. 试验用质粒

图1. pNZ8148质粒图谱

图2. pMD18-T质粒图谱

序号	名称	序列(5’-3’)	片段长度	用途
1	57AA-up	GCTCTAGAACGGTGGTTTATGGT	451 bp	扩增全长
1	57AA-down	GGAAGCTTGATGTTGTCCTCCT	451 bp	扩增全长
2	62AA-up	GCCTCTAGATTCGTCGTATTTTCA	423 bp	扩增全长
2	62AA-down	GCAAGCTTATCCTTTCTTCCTGTT	423 bp	扩增全长
3	77AA-up	GCTCTAGACCAAGCAAGGAGATTT	542 bp	扩增全长
3	77AA-down	GCAAGCTTACACCCACTAGGTTTA	542 bp	扩增全长
4	84AA-up	GCGTCTAGATAGGCAGCAAAGAG	516 bp	扩增全长
4	84AA-down	GGAAGCTTCGCCAATAATAACG	516 bp	扩增全长

表3. 引物序列

试剂	管号
试剂	0	1	2	3	4	5
BSA标准溶液/mL	0.0	0.2	0.4	0.6	0.8	1.0
蒸馏水/mL	1.0	0.8	0.6	0.4	0.2	0.0
蛋白质含量(mg/mL)	0.0	1.0	2.0	3.0	4.0	5.0

表4. 牛血清白蛋白梯度溶液设计(0~5.0 mg/mL)

图3. L. paracasei HD1.7基因组DNA M：DNA Marker DL15000;1：基因组DNA

图4. paracin1.7基因的PCR扩增 M：250 bp DNA Ladder;1、2、3、4：57AA、62AA、77AA和84AA基因的PCR产物

图5. Paracin1.7基因阳性克隆的PCR验证 M：DNA Marker DL2000;1、2、3、4：pT-57AA、62AA、77AA和84AA的PCR产物

图6. pT- Paracin1.7质粒的双酶切验证 M：250 bp DNA Ladder;1、2、3、4：pT-57AA、62AA、77AA和84AA的双酶切产物

图7. pNZ8148质粒的双酶切 M：250 bp DNA Ladder;1、2、3、4：pNZ8148-57AA、62AA、77AA和84AA的PCR产物

图8. paracin1.7基因阳性克隆的PCR验证 M：250 bp DNA Ladder;1：pNZ8148质粒;2：pNZ8148质粒的双酶切产物

图9. pNZ8148-Paracin1.7质粒的双酶切验证 M：250 bp DNA Ladder;1、2、3、4：pNZ8148-57AA、62AA、77AA和84AA的双酶切产物

图10. 57AA、62AA、77AA和84AA基因表达蛋白的SDS-PAGE检测（分离胶浓度15%） M：彩色预染蛋白Marker 5-245KDa;1：L. paracasei HD1.7的上清液SDS-PAGE检测结果;2：含pNZ8148质粒的L. lactis NZ9000上清液SDS-PAGE检测结果;3、4、5、6：57AA、62AA、77AA和84AA基因表达蛋白的SDS-PAGE结果

图11. Paracin1.7的标准效价曲线

图12. 牛血清白蛋白(BSA)标准曲线(0~5.0 mg/mL)

图13. 57AA、62AA、77AA、84AA基因表达蛋白的抑菌效果（指示菌为B. subtilis） A：L. paracasei HD1.7的上清液抑菌效果;B：含pNZ8148质粒的L. lactis NZ9000上清液抑菌效果;C、D、E、F：57AA、62AA、77AA、84AA基因表达蛋白的抑菌效果。

样品	蛋白含量/(mg/mL)	抑菌圈直径/(mm)	细菌素效价/(AU/mL)
L. paracasei HD1.7产生的Paracin1.7	1.14±0.11	12.04±0.98	52.46±1.12
57AA	2.51±0.08	27.31±1.12	397.19±0.89
62AA	2.37±0.16	26.11±1.07	238.23±2.09
77AA	2.43±0.12	26.73±2.08	309.03±1.20
84AA	2.27±0.15	15.13±1.15	62.95±1.14

表5. 57AA、62AA、77AA、84AA的蛋白表达情况

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细菌素Paracin 1.7在乳酸乳球菌NZ9000中的异源表达及抑菌性质的研究

The Heterologous Expression and Antibacterial Properties of the Bacteriocin Paracin1.7 in Lactococcus Lactis NZ9000

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