基于重组毕赤酵母的草鱼C型溶菌酶生物合成及其抑菌活性

doi:10.13560/j.cnki.biotech.bull.1985.2021-0256

摘要/Abstract

摘要：

C型溶菌酶（Chicken-type lysozyme）作为草鱼（Ctenopharyngodon idella）内源性免疫系统中的重要蛋白质类免疫因子,能在草鱼抵抗病原微生物侵染的过程中发挥重要作用,亦是开发绿色饲料添加剂或生物类抗菌剂的佳选。本研究通过逆转录PCR（reverse transcription PCR,RT-PCR）克隆草鱼C型溶菌酶的编码基因“CilyC”,再经二次PCR在其5'和3'端添加各种必需位点。将“CilyC”与表达载体“pPICZαA”连接后转入工程菌毕赤酵母（Pichia pastoris）X-33中,获得重组菌株“X-33/pPICZαA-CilyC”。经含高浓度博莱霉素的培养基筛选得到高拷贝重组菌株后,对其最适蛋白质表达条件进行优化和筛选。通过镍离子亲和层析法纯化重组菌株的表达产物,并对纯化产物进行Western blot分析和LC-MS/MS质谱鉴定。此外,经平板涂布法和最小抑菌浓度（MIC）法考察重组菌株表达产物的抑菌活性。结果表明:X-33/pPICZαA-CilyC在29℃、250 r/min、1%甲醇浓度、96 h的发酵培养条件下,能产13.7 mg/L的重组蛋白;该重组蛋白经结构鉴定为预期分子量14.5 kD的CilyC蛋白。抑菌试验结果显示:重组CilyC具有明显的抗革兰氏阳性的金黄色葡萄球菌（Staphylococcus aureus）、枯草芽孢杆菌（Bacillus subtilisi）、单增李斯特菌（Listeria monocytogenes）和蜡样芽孢杆菌（Bacillus cereus）以及革兰氏阴性的铜绿假单胞菌（Pseudomonas aeruginosa）和沙门氏菌（Salmonella）的生物学活性。本研究构建的重组毕赤酵母菌株“X-33/pPICZαA-CilyC”能有效合成草鱼C型溶菌酶,为鱼类来源C型溶菌酶的大规模制备奠定了良好基础。

关键词: 草鱼, C型溶菌酶, 重组毕赤酵母, 抑菌活性

Abstract:

C-type（Chicken-type）lysozyme,as an important protein immune factor in the endogenous immune system of grass carp（Ctenopharyngodon idella）,can play an important role in the resistance of grass carp to pathogenic microorganisms,it is also a good choice for the development of green feed additives or biological antimicrobial agents. In the present study,the cDNA encoding grass carp C-type lysozyme（CilyC）was cloned by reverse transcription PCR（RT-PCR）. Subsequently,two times of PCR were performed to add various necessary sites to its 5' and 3' ends. The CilyC was connected to the expression vector “pPICZαA” and then transferred into the engineering strain Pichia pastoris X-33;thus,the recombinant P. pastoris strain X-33/pPICZαA-CilyC was constructed. The recombinant strains with multiple copies of gene inserts were screened by using a medium containing high concentration of zeocin. Protein expression conditions for the recombinant strains were optimized and screened. The expression products were purified by nickel ion affinity chromatography,the purified protein was subjected to Western blot analysis and LC-MS/MS identification. In addition,the antibacterial activity of the recombinant strain expression products was detected by plate coating and MIC（minimum inhibitory concentration）method. The results showed that the X-33/pPICZαA-CilyC produced 13.7 mg/L recombinant protein under the optimal fermentation conditions（29^oC,250 r/min,96 h,and 1% methanol）. The recombinant protein was structurally identified as an expected CilyC protein with a molecular weight of 14.5 kD. The results of bacteriostatic tests showed that the recombinant CilyC had obvious biological activities against Gram-positive Staphylococcus aureus,Bacillus subtilis,Listeria monocytogenes and Bacillus cereus,as well as Gram-negative Pseudomonas aeruginosa and Salmonella. The recombinant P. pastoris strain “X-33/pPICZαA-CilyC” constructed in this study may effectively synthesize the grass carp C-type lysozyme,which lays a good foundation for the large-scale preparation of fish-derived C-type lysozyme.

Key words: Ctenopharyngodon idella, C-type lysozyme, recombinant Pichia pastoris, antibacterial activity

杨悦, 陶妍, 谢晶, 钱韻芳. 基于重组毕赤酵母的草鱼C型溶菌酶生物合成及其抑菌活性[J]. 生物技术通报, 2021, 37(12): 169-179.

YANG Yue, TAO Yan, XIE Jing, QIAN Yun-fang. Biosynthesis of Ctenopharyngodon idella C-type Lysozyme Based on Recombinant Pichia pastoris and Its Antibacterial Activity[J]. Biotechnology Bulletin, 2021, 37(12): 169-179.

图/表 11

表1 引物序列

Table 1 Primer sequences

Primer name	Primer sequence（5'-3'）	Size/bp
CF	TCTTCAGATAGCAGAAATTGACAGT	25
CR	TAGGTAATTAAAGATCCCTCAGGCAA	26
CP1	CATCATCATCATCATCATCGCACAATGGG	29
CP2	TCTAGATTATCAGTGCGATTCGCA	24
CP3	CTCGAGAAAAGACATCATCATCATCATCATC	31
3'AOX1	GCAAATGGCATTCTGACATCC	21
5'AOX1	GACTGGTTCCAATTGACAAGC	21

图1 目的基因CilyC的核苷酸及其推断的氨基酸序列粗体表示6×His标签;斜体表示Kex2信号肽酶切割位点;下划线表示Xho I和Xba I限制性内切酶位点;阴影表示半胱氨酸残基;方框表示活性位点残基

Fig. 1 Nucleotide and deduced amino acid sequences of the target gene CilyC Bold letters refer to 6×His;italics refer to Kex2 signal peptidase cleavage site. Underlines refer to Xho I and Xba I restriction endonuclease sites. Shaded letters refer to conserved cysteine residues. Boxes refer to active site residues

图2 CilyC蛋白的三维结构标注不同数字的Cys表示半胱氨酸残基;红色、绿色、黄色和蓝色表示4对二硫键;橙色和粉色球体表示2个活性位点残基

Fig.2 Three-dimensional structure of the CilyC protein Different numbers of Cys refer to cysteine residues. Red,green,yellow and blue refer to 4 pairs of disulfide bonds. Orange and pink refer to residues of the active site

图3 重组表达载体pPICZαA-CilyC的菌落PCR（A）和双酶切鉴定（B） M:DNA分子量标准;1-9:菌落PCR产物;10:双酶切产物

Fig. 3 Identification of recombinant expression vector pPICZαA-CilyC by colony PCR（A）and restriction endonuclease digestion（B） M:DNA ladder. 1-9:Colony PCR products. 10:Products digested by restriction endonucleases

图4 重组毕赤酵母菌株基因组DNA的PCR鉴定 M:DNA分子量标准;1-8:PCR 产物

Fig. 4 PCR identification for the genome DNA of recom-binant P. pastoris strain M:DNA ladder. 1-8:PCR products

图5 重组菌株预表达产物的Tricine-SDS-PAGE分析 M:超低蛋白质分子量标准;0:X-33/pPICZαA的表达产物;1-3:X-33/pPICZαA-CilyC的表达产物

Fig. 5 Tricine-SDS-PAGE analysis for pre-expressed products of the recombinant strains M:Ultra-low molecular weight protein marker. 0:Expression product of the X-33/pPICZαA. 1-3:Expression products of X-33/pPICZαA-CilyC

图6 甲醇浓度（A）和表达时间（B）对重组菌株表达产物的影响相同字母项之间无显著差异,不同字母项之间有显著差异（P < 0.05）;数据为平均值±标准差,n=3;M:超低蛋白质分子量标准

Fig. 6 Effects of methanol concentration（A）and expression time（B）on the expression products of the recombinant strain There is no significant difference among the same letter items,and there is a significant difference among different letter items（P <0.05）. Data are in format of mean ±SD,n=3. M:Ultra-low molecular weight protein marker

图7 纯化蛋白质的Tricine-SDS-PAGE（A）和Western blot（B）分析 M:彩虹预染蛋白质分子量标准;1:X-33/pPICZαA的表达产物;2:纯化蛋白质

Fig. 7 Tricine-SDS-PAGE（A）and Western blot（B）analysis for the purified protein M:Rainbow pre-stained protein marker. 1:Expression product of the X-33/pPICZαA. 2:Purified protein

图8 纯化蛋白质的LC-MS/MS质谱分析大写英文字母表示氨基酸残基

Fig. 8 LC-MS/MS analysis for the purified protein Capital letters refer to amino acid sequences of the peptide fragments

图9 重组菌株培养液上清对革兰氏阳性和阴性细菌的抑菌活性 A:用X-33/pPICZαA的培养液上清处理的细菌;B:用X-33/pPICZαA-CilyC的培养液上清处理的细菌

Fig. 9 Antibacterial activities of the recombinant strain culture supernatant against Gram-positive and Gram-negative bacteria A:Bacteria treated with the culture supernatant of X-33/pPICZαA. B:Bbacteria treated with the culture supernatant of X-33/pPICZαA-CilyC

表2 重组CilyC对受试菌株的最小抑菌浓度

Table 2 Minimum inhibitory concentration（MIC）of the recombinant CilyC against different test bacteria

受试菌株 Test strain	MIC/（µg·mL^-1）
革兰氏阳性细菌Gram-positive
金黄色葡萄球菌Staphylococcus aureus	60
枯草芽孢杆菌Bacillus subtilis	80
单增李斯特菌Listeria monocytogenes	80
蜡样芽孢杆菌Bacillus cereus	80
革兰氏阴性细菌Gram-negative
沙门氏菌Salmonella	80
铜绿假单胞菌Pseudomonas aeruginosa	120

参考文献 25

[1]	Xie JW, Cheng CH, Ma HL, et al. Molecular characterization, expression and antimicrobial activities of a c-type lysozyme from the mud crab, Scylla paramamosain[J]. Dev Comp Immunol, 2019, 98:54-64. doi: 10.1016/j.dci.2019.04.002 URL
[2]	Gao FY, Qu L, Yu SG, et al. Identification and expression analysis of three c-type lysozymes in Oreochromis aureus[J]. Fish Shellfish Immunol, 2012, 32(5):779-788. doi: 10.1016/j.fsi.2012.01.031 URL
[3]	Yu LP, Sun BG, Li J, et al. Characterization of a c-type lysozyme of Scophthalmus maximus:expression, activity, and antibacterial effect[J]. Fish Shellfish Immunol, 2013, 34(1):46-54. doi: 10.1016/j.fsi.2012.10.007 URL
[4]	Wu TT, Jiang QQ, Wu D, et al. What is new in lysozyme research and its application in food industry? A review[J]. Food Chem, 2019, 274:698-709. doi: 10.1016/j.foodchem.2018.09.017 URL
[5]	Hester PY. Enrichments in cages[M]// Egg Innovations and Strategies for Improvements. Amsterdam:Elsevier, 2017:77-88.
[6]	Wei S, Huang Y, Cai J, et al. Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides[J]. Fish Shellfish Immunol, 2012, 33(2):186-196. doi: 10.1016/j.fsi.2012.03.027 URL
[7]	Li SW, Wang D, Liu HB, et al. Expression and antimicrobial activity of c-type lysozyme in Taimen(Hucho taimen, Pallas)[J]. Dev Comp Immunol, 2016, 63:156-162. doi: 10.1016/j.dci.2016.06.003 URL
[8]	Yang D, Wang Q, Cao R, et al. Molecular characterization, expression and antimicrobial activities of two c-type lysozymes from Manila clam Venerupis philippinarum[J]. Dev Comp Immunol, 2017, 73:109-118. doi: 10.1016/j.dci.2017.03.018 URL
[9]	杨杰, 丛丽娜, 夏涛, 等. 日本对虾c型溶菌酶的高效重组表达及产物分析[J]. 生物技术通报, 2012(6):99-105.
	Yang J, Cong LN, Xia T, et al. High level recombinant expression of c-type lysozyme from Marsupenaeus japonicus and product analysis[J]. Biotechnol Bull, 2012(6):99-105.
[10]	瞿兰, 叶星, 田园园, 等. 罗非鱼3种C型溶菌酶重组蛋白的制备及与几种鱼虾溶菌酶溶菌谱的比较[J]. 生物技术通报, 2012(11):161-166.
	Qu L, Ye X, Tian YY, et al. Preparation of three C-type recombinant lysozymes of Oreochromis aureus and comparation of their bacteriolytic spectrum with several other lysozymes[J]. Biotechnol Bull, 2012(11):161-166.
[11]	Neshani A, Eydgahi MRA, Zare H, et al. Extended-spectrum antimicrobial activity of the low cost produced tilapia piscidin 4(TP4)marine antimicrobial peptide[J]. Journal of Research in Medical and Dental Science, 2018, 6(5):327-334.
[12]	Meng DM, Zhao JF, Ling X, et al. Recombinant expression, purification and antimicrobial activity of a novel antimicrobial peptide PaDef in Pichia pastoris[J]. Protein Expr Purif, 2017, 130:90-99. doi: 10.1016/j.pep.2016.10.003 URL
[13]	冯亚东, 陶妍, 李雯, 等. 斑点叉尾鮰C型溶菌酶在毕赤酵母中的表达及其抑菌活性[J]. 生物技术通报, 2017, 33(7):195-202. doi: 10.13560/j.cnki.biotech.bull.1985.2017-0094
	Feng YD, Tao Y, Li W, et al. Expression of channel catfish C-type lysozyme in Pichia pastoris and its bacteriostatic activity[J]. Biotechnol Bull, 2017, 33(7):195-202.
[14]	王强厚, 陶妍, 崔旭, 等. 三疣梭子蟹C型溶菌酶在毕赤酵母中的表达及其抑菌活性[J]. 生物技术通报, 2018, 34(10):135-142. doi: 10.13560/j.cnki.biotech.bull.1985.2018-0241
	Wang QH, Tao Y, Cui X, et al. Expression of swimming crab C-type lysozyme in Pichia pastoris and its bacteriostatic activity[J]. Biotechnol Bull, 2018, 34(10):135-142.
[15]	颜倩倩, 陶妍, 李雯, 等. 鲤鱼c型溶菌酶在毕赤酵母中的重组表达及其抑菌活性[J]. 农业生物技术学报, 2019, 27(11):1912-1922.
	Yan QQ, Tao Y, Li W, et al. Recombinant expression of common carp(Cyprinus carpio)c-type lysozyme in Pichia pastoris and its antibacterial activity[J]. J Agric Biotechnol, 2019, 27(11):1912-1922.
[16]	农业农村部渔业渔政管理局, 2018中国渔业统计年鉴[M]. 北京: 中国农业出版社, 2018:5-8.
	Ministry of Agriculture and Rural Affairs of PRC, Fisheries and Fisheries Administration. China Fishery Statistical Yearbook[M]. Beijing: China Agriculture Press, 2018:5-8.
[17]	农业部渔业渔政管理局, 2014中国渔业统计年鉴[M]. 北京: 中国农业出版社, 2014.
	Ministry of Agriculture and Rural Affairs of PRC, Fisheries and Fisheries Administration. China Fishery Statistical Yearbook[M]. Beijing: China Agriculture Press, 2020:4-25.
[18]	Ye X, Zhang LL, Tian YY, et al. Identification and expression analysis of the g-type and c-type lysozymes in grass carp Ctenopharyn-godon idellus[J]. Dev Comp Immunol, 2010, 34(5):501-509. doi: 10.1016/j.dci.2009.12.009 URL
[19]	陈佩. 维氏气单胞菌对草鱼溶菌酶表达的影响及其免疫效果的研究[D]. 长沙:湖南师范大学, 2020.
	Chen P. Study on the expression change of lysozymes and its immune effect in grass carp(Ctenopharyngodon idella)after infected with Aeromonas veronii[D]. Changsha:Hunan Normal University, 2020.
[20]	金虹, 李海帅, 帖金凤, 等. 应用微量稀释法测定消毒剂最小抑菌浓度方法的建立[J]. 中国消毒学杂志, 2018, 35(11):801-804, 808.
	Jin H, Li HS, Tie JF, et al. Establishment of a broth microdilution MIC testing method for disinfectants[J]. Chin J Disinfect, 2018, 35(11):801-804, 808.
[21]	Leśnierowski G, Yang T. Lysozyme and its modified forms:a critical appraisal of selected properties and potential[J]. Trends Food Sci Technol, 2021, 107:333-342. doi: 10.1016/j.tifs.2020.11.004 URL
[22]	Lu XM, Jin XB, Zhu JY, et al. Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli[J]. Appl Microbiol Biotechnol, 2010, 87(6):2169-2176. doi: 10.1007/s00253-010-2606-3 URL
[23]	Zhao H, Tang J, Cao L, et al. Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system[J]. Enzyme Microb Technol, 2015, 77:61-67. doi: 10.1016/j.enzmictec.2015.06.001 URL
[24]	Wang T, Xu Y, Liu W, et al. Expression of Apostichopus japonicus lysozyme in the methylotrophic yeast Pichia pastoris[J]. Protein Expr Purif, 2011, 77(1):20-25. doi: 10.1016/j.pep.2011.01.002 URL
[25]	Zhou XY, Yu Y, Tao JJ, et al. Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation[J]. J Biosci Bioeng, 2014, 118(4):420-425. doi: 10.1016/j.jbiosc.2014.03.009 URL