藏绵羊溶菌酶1基因的克隆、原核表达及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2015.01.007

Abstract

Abstract: The experiment was aimed to study the expression of the Tibetan sheep lysozyme (LZM) 1 gene in rumen, omasum, abomasum, trachea, liver and kidney and the evolutionary relationship between LZM1 and other lysozymes, and its antibacterial activity.The LZM1 gene cDNA sequence was cloned by RT-PCR method, the expression of LZM1 gene was detected by qPCR.Amino acids sequence alignment was performed between LZM1 and cattle stomach lysozyme, Tibetan sheep stomach lysozyme 2, human stomach lysozyme, et al.And phylogenetic tree was constructed based on Neighbor-Joining, the cloned gene was inserted into pET-32a vector to construct a prokaryotic expression vector pET-32a-LZM1.Recombinant expression vector was transformed into E.coli BL21 (DE3) followed by IPTG (isopropyl-β-D-thiogalactoside) inducing.Heterologous expressed LZM1 was analyzed by SDS-PAGE.The bacteriolytic activity of recombinant protein was observed by turbidimetry and agarose plate diffusion experiment.The results showed that LZM1 gene was successfully cloned from rumen, omasum, abomasums, trachea, liver and spleen of Tibetan sheep.The LZM1 gene was expressed in rumen, omasum, abomasums, trachea, liver and spleen, and the mRNA level of LZM1 in abomasus was higher than in other tissues.The highest amino acids sequence similarity (96%) was observed between LZM1 and cattle stomach lysozyme (No:NM_001080339.1), the recombinant protein LZM1 was correctly expressed, with the molecular mass of 35 ku.The bacteriolytic activity of recombinant protein was 400 U／mL.The result of the agarose diffusion assay indicated that the recombinant protein possessed a certain antibacterial activity against Staphylococcus aureus.

Key words: Tibetan sheep; lysozyme; clone; prokaryotic expression; sequence analysis

CLC Number:

S826

JIANG Wei-hua, ZHU Lian-lian, LIU Yi-li, LIN Zhong-li, JIANG Ming-feng. Cloning, Prokaryotic Expression and Bioinformatic Analysis of Stomach Lysozyme 1 Gene from Tibetan Sheep[J]. , 2015, 42(1): 44-52.

References

[1] 李亚男,刘洪臣,杨丽,等.蛋清溶菌酶对人单纯胞疹病毒Ⅰ型抑制作用的实验研究[J].军医进修学院学报,2008,29(6):517-519.
[2] 荣晓花,凌沛学.溶菌酶的研究进展[J].中国生化药物杂志,1999,20(6):319-320.
[3] Callewaert L,Vanoirbeek K G,Lurquin I,et al.The Rcs two-component system regulates expression of lysozyme inhibitors and is induced by exposure to lysozyme[J].J Bacteriol,2009,191(6):1979-1981.
[4] Deckers D,Vanlint D,Callewaert L,et al.Role of the lysozyme inhibitor Ivy in growth of survival of Escherichia coli and Pseudomonas aeruginosa bacteria in hen egg white and in human saliva and breast milk[J].Appl Environ Microbiol,2008,74(14):4434-4439.
[5] Fleming A.On a remarkable bacteriolytic element found in tissues and secretions[J].Proceedings of the Royal Society,1922,93(653):306-317.
[6] 王镜岩.生物化学[M].第3版.北京:高等教育出版社,2007.
[7] Prager E M.Adaptive evolution of lysozyme:Changes in amino acid sequence,regulation of expression and gene number[M].Basel:Birkhuser Verlag,1995.
[8] 孙国庆,哈福,苗永旺,等.动物溶菌酶基因的研究进展[J].中国牛业科学,2012,38(3):51-54.
[9] 船津胜,鹤大典.溶菌酶[M].济南:山东科学技术出版社,1982.
[10] Dobson D E,Prager E M,Wilson A C.Stomach lysozymes of ruminants.Ⅰ.Distribution and catalytic properties[J].J Biol Chem,1984,259(18):11607-11616.
[11] Jollès P,Schoentgen F,Jollès J,et al.Stomach lysozymes of ruminants.Ⅱ.Amino acid sequence of cow lysozymes 2 and immunological comparisons with other lysozymes[J].J Biol Chem,1984,259(18):11617-11625.
[12] Stewart C B,Wilson A C.Sequence convergence and functional adaptation of lysozymes from foregut fermenters[J].Cold Spring Harbor Symp Quant Biol,1987,52:891-899.
[13] Stewart C B,Schilling J W,Wilson A C.Adaptive ecolution in stomach lysozymes of foregut fermenters[J].Nature,1987,330:401-404.
[14] Cooper C A,Brundige D R,Reh W A,et al.Lysozyme transgenic goats'milk positively impacts intestinal cytokine expression and morphology[J].Transgenic Res,2011,20:1235-1243.
[15] Jackson K A,Berg J M,Murray J D,et al.Evaluating the fitness of human lysozyme transgenic dairy goats:Growth and reproductive traits[J].Tansgenic Res,2010,19:977-986.
[16] 朱莲莲,江明锋,张鹏,等.藏绵羊胃溶菌酶基因的克隆、定量分析及其蛋白性质和结构的预测[J].中国畜牧杂志,2013,40(23):12-17.
[17] 钱曦,华雪铭,黄旭雄,等.异育银鲫c型溶菌酶全长cDNA序列的生物信息学分析及其组织表达分析[J].中国生物化学与分子生物学报,2008,24(4):337-347.
[18] Irwin D M,Wilson A C.Multiple cDNA sequences and the evolution of bovine stomach lysozymes[J].J Biol Chem,1989,264(19):11387-11393.
[19] Jollès J,Prager E M,Alnemri E S,et al.Amino acid sequences of stomach and nonstomach lysozymes of ruminants[J].J Mol Evol,1990,30(4):370-382.
[20] Irwin D M,Wilson A C.Conceaed evolution of ruminant stomach lysozymes.Characterization of lysozyme cDNA clones from sheep and deer[J].J Biol Chem,1990,265(9):4944-4952.
[21] Ito Y,Yamada H,Nakamum M,et al.The primary structures and properties of non-stomach lysozymes of sheep and cow,and implications for functional divergence of lysozyme[J].Eur J Biochem,1993,213(2):649-658.
[22] Takeuchi K,Irwin D M,Gallup M,et al.Multiple cDNA sequences of bovine tracheal lysozyme[J].J Biol Chem,1993,268(36):27440-27446.
[23] Irwin D M.Evolution of cow nonstomach lysozyme genes[J].Genome,2004,47(6):1082-1090.
[24] Jiang M,Chen Y,Wang Y,et al.Yak (Bos grunniens) lysozyme:Molecular cloning,expression and its antibacterial activities[J].Anim Biotechnol,2010,21(1):25-35.
[25] Zhang P,Wang Y,Jiang M F,et al.Molecular cloning,recombinant protein expression,tissue distribution and functional analysis of a new c-type lysozyme from Lezhi black goat rumen[J].Can J Anim,2014,94(1):27-34.
[26] 胡宇飞,江明锋,张鹏,等.乐至黑山羊肾溶菌酶基因克隆及原核表达[J].中国畜牧兽医,2013,40(9):46-50.
[27] 李建波,江明锋,王永.藏绵羊乳腺溶菌酶基因的克隆、原核表达及抗菌活性研究[J].中国生物工程杂志,2013,33(8):37-42.
[28] 李建波,江明锋,王永.藏绵羊肾溶菌酶基因的克隆、表达、纯化和酶学特性研究[J].中国畜牧杂志,2013,50(3):14-17.

Cloning, Prokaryotic Expression and Bioinformatic Analysis of Stomach Lysozyme 1 Gene from Tibetan Sheep

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[2]	LI Can, LI Chuchu, ZENG Wei, ZHANG Ning, JI Chunxiao, CHEN Tao. Recombinant Expression and Function Study of RegⅢγ Protein in Porcine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2414-2426.
[3]	SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.
[4]	LIU Jialong, ZHANG Yiwen, ZHANG Yueran, SUN Qijuan, CHEN Jian, HE Lei, JIA Yanyan, DING Ke, YU Zuhua. Cloning,Prokaryotic Expression and Bioinformatics Analysis of ELAVL1 Gene in Chicken [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1807-1817.
[5]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[6]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[7]	WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.
[8]	LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.
[9]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.
[10]	WANG Wu, WANG Bi, BAI Yu, CHEN Qirong. Research Progress on Antibacterial Activity of Modified Lysozyme [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 942-951.
[11]	ZHANG Zhitao, LI Xianglong, KONG Lingli, LUO Yuxin, WANG Dongying. Prokaryotic Expression of Serine/Threonine Protein Phosphatase 2A in Fasciola gigantica and Expression Analysis at Different Stages [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1107-1117.
[12]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[13]	LYU Shuangfei, MA Xun, WANG Jing, KONG Cuilian, KOU Lijun, LIU Caixia, SHI Weidi, KANG Lichao, REN Huijie. Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 674-683.
[14]	ZHANG Kai, ZHANG Chunping, GE Shengqiang, SUN Chunxi, CHENG Jie, FU Chunyu, WANG Gang, NIU Xing, PENG Jun. Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 270-279.
[15]	ZHU Lilin, WANG Houkun, CHEN Xueyang, LIU Jing, LIANG Xiongyan, FANG Chun, YANG Yuying. Effect of Chicken IFIT5 on Replication of J Subgroup Avian Leukemia Virus Within DF-1 Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 310-316.