鼠伤寒沙门菌毒力基因SptP的原核表达及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2022.12.023

摘要/Abstract

摘要： 【目的】试验旨在获得高效表达的鼠伤寒沙门菌SptP蛋白并进行生物信息学分析，为其功能研究和互作蛋白的筛选提供理论依据。【方法】通过PCR技术扩增SptP基因，并将该序列连接至pET-32a （+）载体，构建鼠伤寒沙门菌SptP基因原核表达载体pET32a-SptP。通过热激法将重组质粒导入大肠杆菌BL21（DE3）感受态细胞后经IPTG诱导表达、纯化重组蛋白，并经过SDS-PAGE和Western blotting验证；应用在线软件对SptP蛋白进行生物信息学分析。【结果】PCR成功扩增出大小为1 632 bp的SptP基因。SptP重组蛋白在大肠杆菌BL21（DE3）感受态细胞中成功诱导表达、纯化，得到分子质量为79.7 ku的蛋白。SptP蛋白分子式为C₂₆₂₅H₄₂₅₇N₇₄₅O₈₁₂S₂₅，分子质量为60 047.68 u，无跨膜结构，无信号肽存在，理论等电点为8.75，有57个潜在的磷酸化位点，主要定位于细胞核、细胞质、高尔基体、细胞骨架、分泌系统的囊泡、质膜，占比分别为43.5%、34.8%、8.7%、4.3%、4.3%和4.3%。SptP蛋白二级结构由α-螺旋、延伸链、β-转角及无规则卷曲组成，占比分别为43.65%、14.92%、4.42%和37.02%。【结论】本研究构建了表达SptP蛋白的重组质粒pET32a-SptP，获得分子质量为79.7 ku的SptP重组蛋白，阐明了SptP蛋白的基本理化性质和生物学功能，为后续SptP蛋白与宿主细胞互作的作用机制及鼠伤寒沙门菌新疫苗的制备提供理论基础和试验依据。

关键词: 鼠伤寒沙门菌; SptP; 原核表达; 生物信息学分析

Abstract: 【Objective】 The purpose of the experiment was to obtain the highly expressed SptP protein of Salmonella Typhimurium and carry out bioinformatics analysis, so as to provide a theoretical basis for its function research and screening of interacting proteins.【Method】 The SptP gene was amplified by PCR technology, and the sequence was ligated to pET-32a(+) vector to construct the prokaryotic expression vector pET32a-SptP of Salmonella Typhimurium SptP gene.The recombinant plasmid was introduced into Escherichia coli BL21(DE3) competent cells by heat shock method, the recombinant protein was induced to express by IPTG, purified, and verified by SDS-PAGE and Western blotting.Then the SptP protein was analyzed by bioinformatics using online softwares.【Result】 The SptP gene was successfully amplified by PCR with a size of 1 632 bp.The recombinant SptP protein was successfully induced, expressed and purified in E.coli BL21(DE3) competent cells to obtain a protein with a molecular mass of 79.7 ku.The molecular formula of the SptP protein was C₂₆₂₅H₄₂₅₇N₇₄₅O₈₁₂S₂₅, no transmembrane structure, no signal peptide, and a molecular mass of 60 047.68 u, the theoretical isoelectric point was 8.75, and there were 57 possible phosphorylation sites.The SptP protein was mainly located in the nucleus, cytoplasm, Golgi apparatus, cytoskeleton, secretion system vesicles, and plasma membrane, accounting for 43.5%, 34.8%, 8.7%, 4.3%, 4.3% and 4.3%, respectively.SptP protein was mainly composed of alpha helix, extended strand, beta turn and random coil, accounting for 43.65%, 14.92%, 4.42% and 37.02%, respectively.【Conclusion】 In this study, a recombinant plasmid pET32a-SptP expressing SptP protein was constructed, and a SptP recombinant protein with a molecular mass of 79.7 ku was obtained.The basic physical and chemical properties and biological functions of SptP protein were clarified, which provided theoretical and experimental basis for the interaction mechanism between SptP protein and host cells and the preparation of new Salmonella Typhimurium vaccine.

Key words: Salmonella Typhimurium; SptP; prokaryotic expression; bioinformatics analysis

中图分类号:

S852.61^＋2

周南龙, 丁永辉, 李天森. 鼠伤寒沙门菌毒力基因SptP的原核表达及生物信息学分析[J]. 中国畜牧兽医, 2022, 49(12): 4734-4744.

ZHOU Nanlong, DING Yonghui, LI Tiansen. Prokaryotic Expression and Bioinformatics Analysis of Salmonella Typhimurium Virulence Gene SptP[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4734-4744.

参考文献

[1] 徐家芳, 谢永登.沙门氏菌研究进展[J].广西畜牧兽医, 2020, 36(2):92-94. XU J F, XIE Y D.Research progress of Salmonella[J].Guangxi Journal of Animal Husbandry Veterinary Medicine, 2020, 36(2):92-94.(in Chinese)
[2] 李星, 王雪敏, 邵玉新, 等.鸽沙门氏菌病及其抗生素替代品防控措施研究进展[J].中国畜牧兽医, 2021, 48(7):2584-2593. LI X, WANG X M, SHAO Y X, et al.Research progress on pigeon salmonellosis and control measures of antibiotic substitutes[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(7):2584-2593.(in Chinese)
[3] 李正田, 豆腾飞, 李琦华, 等.中草药复方防治雏鸡沙门氏菌病的研究[J].中国畜牧兽医, 2020, 47(3):911-921. LI Z T, DOU T F, LI Q H, et al.Study on the prevention and treatment of salmonellosis in chicks with Chinese herbal medicine compound[J].China Animal Husbandry & Veterinary Medicine, 2020, 47(3):911-921.(in Chinese)
[4] RIVERA-CHÁVEZ F, ZHANG L F, FABER F, et al.Depletion of butyrate-producing Clostridia from the gut microbiota drives an aerobic luminal expansion of Salmonella[J].Cell Host & Microbe, 2016, 19(4):443-454.
[5] ROGERS A W L, TSOLIS R M, BÄUMLER A J.Salmonella versus the Microbiome[J].Microbiology and Molecular Biology Reviews, 2020, 85(1):e00027-19.
[6] POPA G L, PAPA M I.Salmonella spp.infection-A continuous threat worldwide[J].Germs, 2021, 11(1):88-96.
[7] 郑林, 祝令伟, 郭学军, 等.沙门氏菌主要流行血清型耐药性的研究进展[J].江苏农业科学, 2020, 48(6):8-12. ZHENG L, ZHU L W, GUO X J, et al.Research progress of antimicrobial resistance of major epidemic serotypes of Salmonella[J].Jiangsu Agricultural Science, 2020, 48(6):8-12.(in Chinese)
[8] 董晓璐, 秦晓杰, 刘阳泰, 等.食源性沙门氏菌在人体胃肠道中耐受及致病机制研究进展[J].食品与发酵工业, 2021, 47(24):286-292. DONG X L, QIN X J, LIU Y T, et al.Research progress on tolerance and pathogenic mechanism of foodborne Salmonella spp.in human gastrointestinal tract[J].Food and Fermentation Industries, 2021, 47(24):286-292.(in Chinese)
[9] FEASEY N A, HADFIELD J, KEDDY K H, et al.Distinct Salmonella Enteritidis lineages associated with enterocolitis in high-income settings and invasive disease in low-income settings[J]. Nature Genetics, 2016, 48(10):1211-1217.
[10] FISKIN E, BIONDA T, DIKIC I, et al.Global analysis of host and bacterial ubiquitinome in response to Salmonella Typhimurium infection[J].Molecular Cell, 2016, 62(6):967-981.
[11] KIYUNA T, MURAKAMI T, TOME Y, et al.High efficacy of tumor-targeting Salmonella Typhimurium A1-R on a doxorubicin- and dactolisib-resistant follicular dendritic-cell sarcoma in a patient-derived orthotopic xenograft PDOX nude mouse model[J].Oncotarget, 2016, 7(22):33046-33054.
[12] LV Q, LV Y, DOU X, et al.Myricetin inhibits the type Ⅲ secretion system of Salmonella enterica serovar Typhimurium by downregulating the Salmonella pathogenic island Ⅰ gene regulatory pathway[J].Microbial Pathogenesis, 2021, 150:104695.
[13] AL-TAWEEL F B, DOUGLAS C W I, WHAWELL S A.The periodontal pathogen Porphyromonas gingivalis preferentially interacts with oral epithelial cells in S phase of the cell cycle[J].Infection and Immunity, 2016, 84(7):1966-1974.
[14] KNUFF-JANZEN K, TUPIN A, YURIST-DOUTSCH S, et al.Multiple Salmonella-pathogenicity island 2 effectors are required to facilitate bacterial establishment of its intracellular niche and virulence[J].PLoS One, 2020, 15(6):e0235020.
[15] LAWRENCE A E, ABUAITA B H, BERGER R P, et al.Salmonella enterica serovar Typhimurium SPI-1 and SPI-2 shape the global transcriptional landscape in a human intestinal organoid model system[J].mBio, 2021, 12(3):399-401.
[16] DE SOUZA SANTOS M, ORTH K.The role of the type Ⅲ secretion system in the intracellular lifestyle of enteric pathogens[J].Microbiology Spectrum, 2019, 7(3):BAI-0008-2019.
[17] HEMN M, WEI D, RACHEL O, et al.Host-derived Spi6 provides granzyme B-independent protection of intestinal epithelial cells in murine GVHD[J]. Biology of Blood and Marrow Transplantation, 2018, 24(12):2397-2408.
[18] PRADHAN D, NEGI V D.Repeated in-vitro and in-vivo exposure leads to genetic alteration, adaptations, and hypervirulence in Salmonella[J].Microbial Pathogenesis 2019, 136:103654.
[19] FU Y, GALÁN J E.The Salmonella Typhimurium tyrosine phosphatase SptP is translocated into host cells and disrupts the actin cytoskeleton[J].Molecular Microbiology, 1998, 27(2):359-368.
[20] MURLI S, WATSON R O, GALÁN J E.Role of tyrosine kinases and the tyrosine phosphatase SptP in the interaction of Salmonella with host cells[J].Cellular Microbiology, 2001, 3(12):795-810.
[21] HARAGA A, MILLER S I.A Salmonella enterica serovar Typhimurium translocated leucine-rich repeat effector protein inhibits NF-kappa B-dependent gene expression[J].Infection and Immunity, 2003, 71(7):4052-4058.
[22] LIN S L, LE T X, COWEN D S.SptP, a Salmonella Typhimurium type Ⅲ-secreted protein, inhibits the mitogen-activated protein kinase pathway by inhibiting Raf activation[J].Cellular Microbiology, 2003, 5(4):267-275.
[23] FU Y, GALÁN J E.A Salmonella protein antagonizes Rac-1 and Cdc42 to mediate host-cell recovery after bacterial invasion[J].Nature, 1999, 401(6750):293-297.
[24] HUMPHREYS D, HUME P J, KORONAKIS V.The Salmonella effector SptP dephosphorylates host AAA+ ATPase VCP to promote development of its intracellular replicative niche[J].Cell Host Microbe, 2009, 5(3):225-233.
[25] JIA S, MCWHORTER A R, ANDREWS D M, et al.Challenges in vaccinating layer hens against Salmonella Typhimurium[J].Vaccines, 2020, 8(4):696-708.
[26] XIA S, HENDRIKSEN R S, XIE Z, et al.Molecular characterization and antimicrobial susceptibility of Salmonella isolates from infections in humans in Henan province, China[J]. Journal of Clinical Microbiology, 2009, 47(2):401-409.
[27] KE B, SUN J, HE D, et al.Serovar distribution, antimicrobial resistance profiles, and PFGE typing of Salmonella enterica strains isolated from 2007-2012 in Guangdong, China[J]. BMC Infectious Diseases, 2014, 14(1):338.
[28] JOHNSON R, MYLONA E, FRANKEL G.Typhoidal Salmonella:Distinctive virulence factors and pathogenesis[J].Cellular Microbiology, 2018, 20(9):e12939.
[29] DENG Q, BARBIERI J T.Modulation of host cell endocytosis by the type Ⅲ cytotoxin, Pseudomonas ExoS[J].Traffic, 2008, 9(11):1948-1957.
[30] CHOI H W, BROOKING-DIXON R, NEUPANE S, et al.Salmonella Typhimurium impedes innate immunity with a mast-cell-suppressing protein tyrosine phosphatase, SptP[J]. Immunity, 2013, 39(6):1108-1120.
[31] LIN Z, TANG P, JIAO Y, et al.Immunogenicity and protective efficacy of a Salmonella Enteritidis SptP mutant as a live attenuated vaccine candidate[J].BMC Veterinary Research, 2017, 13(1):194.
[32] LEBLANC M A, FINK M R, PERKINS T T, et al.Type Ⅲ secretion system effector proteins are mechanically labile[J].Proceedings of the National Academy of Sciences of the United States of America, 2021, 118(12):e2019566118.
[33] HAYASHI F, KAWASHIMA Y, TAKEUCHI S, et al.SptP_106-136 plays a role in the complex formation with SptP-specific chaperone SicP[J].Bioscience, Biotechnology & Biochemistry, 2014, 78(9):1560-1563.
[34] MILETIC S, FAHRENKAMP D, GOESSWEINER-MOHR N, et al.Substrate-engaged type Ⅲ secretion system structures reveal gating mechanism for unfolded protein translocation[J].Nature Communications, 2021, 12(1):1546.
[35] 杨江流, 乔军伟, 宋青山, 等.布鲁氏菌S19毒力基因BvfA的原核表达及生物信息学分析[J].中国畜牧兽医, 2021, 48(8):2746-2753. YANG J L, QIAO J W, SONG Q S, et al.Prokaryotic expression and bioinformatics analysis of virulence gene BvfA of Brucella S19[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(8):2746-2753.(in Chinese)