肠炎沙门菌avrA蛋白的原核表达及其生物信息学分析

doi:10.16431/j.cnki.1671-7236.2022.11.007

摘要/Abstract

摘要： 【目的】表达具有生物学活性的肠炎沙门菌（Salmonella Enteritidis） avrA蛋白，并对其进行生物信息学分析，为研究肠炎沙门菌avrA蛋白对猪肠道细胞免疫功能的影响提供参考。【方法】根据GenBank中肠炎沙门菌avrA蛋白全基因序列（基因ID：1254388）设计引物扩增avrA基因，回收的目的片段与表达载体pGEX-4T-1进行重组、克隆，用限制性内切酶和基因测序验证重组表达质粒的正确性。将验证后的重组表达质粒转化大肠杆菌Rosetta 2（DE3）感受态细胞，并在不同条件下对重组表达菌进行诱导表达。应用SDS-PAGE和Western blotting检测avrA蛋白的表达情况。将酶切测序后目的基因序列进行BLAST比对分析，并用ExPASy-Translate Tool软件预测avrA蛋白的氨基酸组成。应用生物信息学软件预测avrA蛋白的跨膜结构域、二级结构、三级结构及抗原性。【结果】肠炎沙门菌avrA基因CDS序列全长为909 bp，编码302个氨基酸。限制性内切酶及基因测序分析结果表明，重组表达质粒pGEX-4T-1-avrA构建成功。SDS-PAGE和Western-blotting结果表明，重组菌所表达的avrA蛋白为可溶性蛋白。重组菌在15 ℃诱导16 h表达的蛋白量为10 mg/L，蛋白溶解度为40%。生物信息学分析表明，avrA蛋白为膜外蛋白，该蛋白的16―301位氨基酸具有来自超家族YopJ丝氨酸/苏氨酸乙酰转移酶的保守结构域；avrA蛋白二级结构中无规则卷曲、α-螺旋、延伸链分别占43.71%、39.40%和16.89%；avrA蛋白三级结构预测结果与二级结构一致；avrA蛋白有9个与B细胞结合的抗原表位，分别在第5―40、51―68、80―92、94、101―109、146―157、160―168、199―231和236―298位氨基酸处。【结论】试验成功克隆出肠炎沙门菌avrA基因，并成功构建重组表达质粒和表达菌，诱导表达的avrA蛋白为可溶性表达；avrA蛋白为膜外蛋白，有9个能与B细胞结合的抗原表位。本研究结果为进一步制备检测肠炎沙门菌感染的抗体提供参考。

关键词: 肠炎沙门菌; avrA蛋白; 原核表达; 生物信息学分析

Abstract: 【Objective】 The aim of this study was to express avrA protein of Salmonella Enteritidis which had biological activity and analyze its bioinformatics, so as to provide references for studying the influence of avrA protein of Salmonella Enteritidis on the immune function of porcine intestinal cells.【Method】 Primers were designed according to the complete gene sequence of avrA protein of Salmonella Enterititis in GenBank (gene ID:1254388), and avrA gene was amplified by PCR.After recombination and cloning of the recovered target fragment and expression vector PGEX-4T-1, the correctness of the recombinant expression plasmid was verified by restriction enzyme and gene sequencing.The verified recombinant expression plasmids were transformed into Escherichia coli Rosetta 2(DE3) competent cells, and the recombinant expression strains were induced to express under different conditions.The expression of avrA protein was detected by SDS-PAGE and Western blotting.The target gene sequences were analyzed by BLAST, and the amino acid composition of avrA protein was predicted by ExPASy-Translate Tool software.The transmembrane domain, secondary structure, tertiary structure and antigenicity of the target protein were predicted by bioinformatics softwares.【Result】 The total length of CDS region of avrA gene was 909 bp, encoding 302 animo acids.The recombinant expression plasmid PGEX-4T-1-avrA was successfully constructed by restriction enzyme and gene sequencing analysis.The results of SDS-PAGE and Western-blotting showed that avrA protein expressed by recombinant bacteria was soluble protein.The protein content of recombinant bacteria induced under 15 ℃ for 16 h was 10 mg/L and the protein solubility was 40%.Bioinformatics analysis showed that avrA protein was an extracellular protein, and amino acids 16-301 of avrA protein had a conserved domain from the superfamily YopJ serine/threonine acetyltransferase.In the secondary structure of avrA protein, random coil, alpha helix and extended chain were accounted for 43.71%, 39.40% and 16.89%, respectively.The predicted tertiary structure of avrA protein was consistent with the predicted secondary structure. AvrA protein had 9 B cell-binding epitopes at amino acid positions 5-40, 51-68, 80-92, 94, 101-109, 146-157, 160-168, 199-231, 236-298, respectively.【Conclusion】 avrA gene of Salmonella Enteritidis was successfully cloned, and the recombinant expression plasmid and expression bacteria were successfully constructed.The induced expressed avrA protein was soluble.AvrA protein was an extramembrane protein with 9 epitopes that could bind to B cells.The results of this study could provide references for the preparation of antibodies to detect Salmonella Enteritidis infection.

Key words: Salmonella Enteritidis; avrA protein; prokaryotic expression; bioinformatics analysis

中图分类号:

S852.61

徐云明, 卜永谦, 卞蓉蓉, 杨剑波, 孙智远, 陈益, 柳增善, 任洪林. 肠炎沙门菌avrA蛋白的原核表达及其生物信息学分析[J]. 中国畜牧兽医, 2022, 49(11): 4168-4177.

XU Yunming, BU Yongqian, BIAN Rongrong, YANG Jianbo, SUN Zhiyuan, CHEN Yi, LIU Zengshan, REN Honglin. Prokaryotic Expression and Bioinformatics Analysis of avrA Protein of Salmonella Enteritidis[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4168-4177.

参考文献

[1] 孟霞, 何梦萍, 韦骅栩, 等.肠炎沙门菌SipA多克隆抗体的制备及非编码小RNA RyhB-1/2对其表达调控的分析[J].中国畜牧兽医, 2021, 48(11):4162-4169. MENG X, HE M P, WEI H X, et al.Preparation of polyclonal antibody of SipA in Salmonella Enteritidis and analysis of its expression regulation by non-coding small RNA RyhB-1/2[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(11):4162-4169.(in Chinese)
[2] 崔一, 王振华, 赵素娟, 等.禽源肠炎沙门菌aroA基因缺失株的构建及其生物学特性研究[J].中国畜牧兽医, 2021, 48(6):2247-2254. CUI Y, WANG Z H, ZHAO S J, et al.Construction and biological characteristics of aroA gene deletion strain of Salmonella enteritis from poultry[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(6):2247-2254.(in Chinese)
[3] 程相朝, 田文静, 张梦珂, 等.一株肠炎沙门氏菌鞭毛基因FliC的克隆及生物信息学分析[J].中国畜牧兽医, 2018, 45(12):3379-3386. CHENG X C, TIAN W J, ZHANG M K, et al.Cloning and bioinformatics analysis of a flagellar FliC gene in Salmonella Enteritidis[J].China Animal Husbandry & Veterinary Medicine, 2018, 45(12):3379-3386.(in Chinese)
[4] 刘聪, 黄世猛, 计成, 等.植物乳酸杆菌对感染肠炎沙门氏菌蛋鸡生产性能、蛋品质及血浆生化指标的影响[J].中国畜牧兽医, 2018, 45(5):1196-1202. LIU C, HUANG S M, JI C, et al.Effect of dietary supplement with Lactobacillus plantarum on production performance, egg quality, plasma biochemical parameters in laying hens infected with Salmonella Enteritidis[J].China Animal Husbandry & Veterinary Medicine, 2018, 45(5):1196-1202.(in Chinese)
[5] 杨亚晋, 郭爱伟, 张颖, 等.植物乳杆菌对致病性肠炎沙门氏菌感染蛋鸡的生产性能及其蛋、粪含菌量的影响[J].中国畜牧兽医, 2017, 44(2):482-487. YANG Y J, GUO A W, ZHANG Y, et al.Effects of Lactobacillus plantarum on production performance, egg and feces bacteria content of Salmonella enteric-infected hens[J].China Animal Husbandry & Veterinary Medicine, 2017, 44(2):482-487.(in Chinese)
[6] DHAWIA A, ELAZOMI A, JONES M A, et al.Adaptation to the chicken intestine in Salmonella Enteritidis PT4 studied by transcriptional analysis[J].Veterinary Microbiology, 2011, 153(1-2):198-204.
[7] WISNER A L, POTTER A A, KOSTER W.Effect of the Salmonella pathogensicity island 2 type Ⅲ secretion system on Salmonella survival in activated chicken macrophage-like HD11 cells[J].PLoS One, 2011, 6(12):e29787.
[8] 蔺志杰.肠炎沙门氏菌效应蛋白avrA参与宿主炎性反应机制[D].扬州:扬州大学, 2017. LIN Z J.Impact of Salmonella Enteritidis effector protein avrA on the host inflammatory response[D].Yangzhou:Yangzhou University, 2017.(in Chinese)
[9] VOETSCH A C, VAN GILDER T J, ANGULO F J, et al.FoodNet estimate of the burden of ilness caused by nontyphoidal Salmonella infections in the United States[J].Clinical Infectious Diseases, 2004, 38(Suppl 3):S127-S134.
[10] RANDOW F, MUNZ C.Autophagy in the regulation of pathogen replication and adaptive immunity[J]. Trends in Immunology, 2012, 33(10):475-487.
[11] LI S, WANDEL M P, LI F, et al.Sterical hindrance promotes selectivity of the autophagy cargo receptor NDP52 for the danger receptor galectin-8 in antibacterial autophagy[J].Science Signaling, 2013, 6(261):ra9.
[12] GOMES L C, DIKIC I.Autophagy in antimicrobial immunity[J].Molecular Cell, 2014, 54(2):224-233.
[13] MIZUSHIMA N, YOSHIMORI T, OHSUMI Y.The role of Atg proteins in autophagosome formation[J].Annual Review of Cell and Developmental Biology, 2011, 27:107-132.
[14] KANG R, ZCHH J, LOUZE M T, et al.The beclin 1 network regulates autophagy and apoptosis[J].Cell Death and Differentiation, 2011, 18(4):571-580.
[15] LIANG X H, JACKSON S, SEAMAN M, et al.Induction of autophagy and inhibition of tumorigenesis by beclin 1[J]. Nature, 1999, 402(6762):672-676.
[16] STEELE-MORTIMER O.The Salmonella-containing vacuole:Moving with the times[J].Current Opinion in Microbiology, 2008, 11(1):38-45.
[17] BUCHMEIER N A, HEFFRON F.Inhibition of macrophage phagosome lysosome fusion by Salmonella Typhimurium[J]. Infection and Immunity, 1991, 59(7):2232-2238.
[18] KNODLER L A, FINLAY B B.Salmonella and apoptosis:To live or let die?[J].Microbes and Infection, 2001, 3(14-15):1321-1326.
[19] JONES R M, WU H, WENTWORTH C, et al.Salmonella avrA coordinates suppression of host immune and apoptotic defenses via JNK pathway blockade[J].Cell Host & Microbe, 2008, 3(4):233-244.
[20] OWEN K A, MEYER C B, BOUTON A H, et al.Activation of focal adhesion kinase by Salmonella suppresses autophagy via an Akt/mTOR signaling pathway and promotes bacterial survival in macrophages[J].PLoS Pathogens, 2014, 10(6):e1004159.
[21] GANESAN R, HOS N J, GUTIERREZ S, et al.Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy[J].PLoS Pathogens, 2017, 13(2):e1006227.
[22] 焦扬.肠炎沙门菌rfbG、rfbH、avrA基因的功能及其分子机制研究[D].扬州:扬州大学, 2019. JIAO Y.Role of Salmonella Enteritidis genes rfbG, rfbH and avrA and their molecular mechanisms[D].Yangzhou:Yangzhou University, 2019.(in Chinese)
[23] DALBEY R E, KUHN A.Protein traffic in Gram-negative bacteria:How exported and secreted proteins find their way[J].FEMS Microbiology Reviews, 2012, 36(6):1023-1045.
[24] ELLERMEIER J R, SLAUCH J M.Adaptation to the host environment:Regulation of the SPI1 type Ⅲ secretion system in Salmonella enterica serovar Typhimurium[J].Current Opinion in Microbiology, 2007, 10(1):24-29.
[25] SABBAGH S C, FOREST C G, LEPAGE C, et al.So similar, yet so different:Uncovering distinctive features in the genomes of Salmonella enterica serovars Typhimurium and Typhi[J].FEMS Microbiology Letters, 2010, 305(1):1-13.
[26] YIN C, LIU Z, XIAN H, et al.avrA exerts inhibition of NF-κB pathway in its naïve Salmonella serotype through suppression of p-JNK and beclin-1 molecules[J]. International Journal of Molecular Sciences, 2020, 21(17):6063.
[27] JIAO Y, ZHANG Y G, LIN Z, et al.Salmonella Enteritidis effector avrA suppresses autophagy by reducing beclin-1 protein[J]. Frontiers in Immunology, 2020, 11:686.
[28] 陈静, 吴燕, 李梅, 等.丝状支原体山羊亚种LPPA蛋白原核表达及间接ELISA方法的建立[J].中国畜牧兽医, 2022, 49(1):318-327. CHEN J, WU Y, LI M, et al.Prokaryotic expression of LPPA protein of Mycoplasma mycoides subsp.capri and establishment of indirect ELISA method[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(1):318-327.(in Chinese)
[29] LIN Z, ZHANG Y G, XIA Y, et al.Salmonella Enteritidis effector avrA stabilizes intestinal tight junctions via the JNK pathway[J].Journal of Biological Chemistry, 2016, 291(52):26837-26849.
[30] 王宇琛, 田广原, 周雅坪, 等.BPIV3 NP蛋白的原核表达及其对BPIV3灭活疫苗免疫增强作用的研究[J].中国畜牧兽医, 2022, 49(8):3122-3130. WANG Y C, TIAN G Y, ZHOU Y P, et al.Prokaryotic expression of BPIV3 NP protein and its immunoenhancing effect on the BPIV3 inactivated vaccine[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(8):3122-3130.(in Chinese)
[31] 杜文琪, 夏立叶, 李桂梅, 等.A型塞内卡病毒VP1蛋白的原核表达及纯化[J].中国畜牧兽医, 2022, 49(7):2708-2715. DU W Q, XIA L Y, LI G M, et al.Prokaryotic expression and purification of Seneca virus A VP1 protein[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(7):2708-2715.(in Chinese)
[32] 贺会利, 徐敏, 余娟, 等.鸡传染性支气管炎病毒(IBV)ZZ2004株核衣壳蛋白基因的克隆及原核表达[J].中国畜牧兽医, 2014, 41(9):15-20. HE H L, XU M, YU J, et al.Cloning and prokaryotic expression of nucleoprotein gene of Infectious bronchitis virus (IBV)ZZ2004 strain[J]. China Animal Husbandry & Veterinary Medicine, 2014, 41(9):15-20.(in Chinese)
[33] JING S R, ZENG W K, HUANG F, et al.Immunogenic characteristics of RSV F2 protein expressed in E.coli Rosetta (DE3)[J].Journal of Kunming University of Science and Technology (Natural Science Edition), 2012, 37(2):54-60.
[34] TEGEL H, TOURLE S, OTTOSSON J, et al.Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)[J].Protein Expression and Purification, 2010, 69(2):159-167.
[35] 蔺志杰, 汤佩佩, 陈韵, 等.肠炎沙门氏菌效应蛋白avrA的原核表达及多克隆抗体的制备[J].中国兽医科学, 2017, 47(7):866-870. LIN Z J, TANG P P, CHEN Y, et al.Prokaryotic expression and polyclonal antibody preparation of Salmonella Enteritidis protein avrA[J].Chinese Veterinary Science, 2017, 47(7):866-870.(in Chinese)
[36] AUSTIN E F S, JOHN S, DANIEL M C, et al.Yersinia pseudotuberculosis YopJ limits macrophage response by downregulating COX-2-mediated biosynthesis of PGE2 in a MAPK/ERK-dependent manner[J].Microbiology Spectrum, 2021, 9(1):e0049621.
[37] CHRIS A M, FERNANDO P L, MOHAMMAD A, et al.Heightened virulence of Yersinia is associated with decreased function of the YopJ protein[J].Infection and Immunity, 2021, 89(12):e0043021.
[38] YE C, KAI G, XIANG H, et al.Yersinia YopJ negatively regulates IRF3-mediated antibacterial response through disruption of STING-mediated cytosolic DNA signaling[J].Biochimica et Biophysica Acta, 2016, 1863(12):3148-3159.