鸡肠炎沙门菌毒力蛋白SipD生物信息学分析及原核表达

doi:10.16431/j.cnki.1671-7236.2024.07.025

Abstract

Abstract: 【Objective】 This study was aimed to predict the potential biological functions of Salmonella Enteritidis (SE) SipD protein,and express and purify SipD protein,so as to provide a theoretical basis for exploring SipD protein as a candidate antigen for anti-Salmonella nanobodies.【Method】 DNAStar software was used to perform similarity alignment on the amino acid sequences of SipD protein among different serotypes of Salmonella strains published in GenBank.Subsequently,the bioinformatics analysis of SipD protein was performed using online software.Primers were designed based on SipD gene sequence in GenBank,using the standard strain of Salmonella Enteritidis (ATCC 13076) as a template.PCR was applied to amplify SipD gene and construct a pET-32a(+)-SipD recombinant plasmid,which was then transformed into Escherichia coli BL21 (DE3) competent cells.IPTG was applied to induce the expression of recombinant SipD protein and Ni²⁺ affinity chromatography was used to purify.SDS-PAGE and Western blotting was applied to detect the expression of SipD protein.【Result】 SipD protein was highly conserved among different serotypes of Salmonella strains. SipD protein,with the molecular formula C₁₆₁₉H₂₅₇₃N_441O540S₈,was an extracellular protein without transmembrane domain and signal peptide.The amino acids 1 to 343 of SipD protein had conserved structural domains from the superfamily of invasive plasmid antigen IpaD.The secondary structure prediction of SipD protein showed that alpha helix,extended chain,beta turn and random coil accounted for 59.18%,6.41%,3.21% and 31.20%,respectively.There were 12 antigenic epitopes that bound SipD protein to B cells.The size of SipD gene was 1 029 bp,which was expressed as a soluble protein in Escherichia coli BL21 (DE3) competent cells.After purification,dialysis and concentration,the protein concentration of SipD protein was 8.6 mg/mL.SDS-PAGE and Western blotting analysis revealed that SipD protein with high purity could be used to immunize alpacas for the preparation of nanobody.【Conclusion】 SipD protein was an extramembrane protein with many antigen binding sites.The recombinant protein of SipD with high purity was obtained by expression and purification,which provided materials for further preparation of nanobody targeting SipD protein of Salmonella Enteritidis in chickens.

Key words: chicken; Salmonella Enteritidis; SipD protein; bioinformatics; prokaryotic expression

CLC Number:

S852.61⁺7

YUAN Liang, HAN Xiaoxi, GONG Yingchao, FAN Xianan, HAO Beili, HAO Shuangshuang, LI Changwen, CHANG Yicong, LI Rui, LIU Fangping. Bioinformatics Analysis and Prokaryotic Expression of SipD Protein of Salmonella Enteritidis in Chickens[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(7): 2998-3007.

References

[1] ADETUNJI A,CASEY T,FRANCO J,et al.Proteomic analysis of the effect of Salmonella challenge on broiler chicken[J].Molecules,2022,27(21):7277.
[2] 刘东海.肠炎沙门菌greAgreB基因缺失株构建及生物学特性研究[D].邯郸:河北工程大学,2022. LIU D H.Construction and biological characterization of greAgreB gene deletion strain of Salmonella Enteritidis[D].Handan:Hebei University of Engineering,2022.(in Chinese)
[3] 费晓.我国部分地区种鸡场沙门菌流行现状调查、耐药性分析及分子分型[D].扬州:扬州大学,2017. FEI X.Survey on the current status of Salmonella epidemic,drug resistance analysis and molecular typing in breeder farms in some regions of China[D].Yangzhou:Yangzhou University,2017.(in Chinese)
[4] YU X,ZHU H,BO Y,et al.Prevalence and antimicrobial resistance of Salmonella enterica subspecies Enterica serovar Enteritidis isolated from broiler chickens in Shandong province,China,2013-2018[J].Poultry Science,2021,100(2):1016-1023.
[5] EHUWA O,JAISWAL A K,JAISWAL S.Salmonella,food safety and food handling practices[J].Foods,2021,10(5):907.
[6] 孙朋浩,邱荣超,李书宁,等.广州市生鲜鸡中肠炎沙门菌的监测及耐药基因的分子鉴定[J].畜牧兽医学报,2018,49(2):388-395. SUN P H,QIU R C,LI S N,et al.Surveillance of Salmonella Enteritidis in fresh chickens in Guangzhou and molecular identification of drug resistance genes[J].Acta Veterinaria et Zootechnica Sinica,2018,49(2):388-395.(in Chinese)
[7] SHI Y,CHEN X,SHU J,et al.Harmine,an inhibitor of the type Ⅲ secretion system of Salmonella enterica serovar Typhimurium[J].Frontiers in Cellular and Infection Microbiology,2022,12:967149.
[8] GALÁN J E,WOLF-WATZ H.Protein delivery into eukaryotic cells by type Ⅲ secretion machines[J].Nature,2006,444(7119):567-573.
[9] SIEROCKI R,JNEID B,ORSINI DELGADO M L,et al.An antibody targeting type Ⅲ secretion system induces broad protection against Salmonella and Shigella infections[J].PLoS Neglected Tropical Diseases,2021,15(3):e0009231.
[10] HU Y,HUANG H,CHENG X,et al.A global survey of bacterial type Ⅲ secretion systems and their effectors[J].Environmental Microbiology,2017,19(10):3879-3895.
[11] LUNELLI M,HURWITZ R,LAMBERS J,et al.Crystal structure of PrgI-SipD:Insight into a secretion competent state of the type three secretion system needle tip and its interaction with host ligands[J].PLoS Pathogens,2011,7(8):e1002163.
[12] RATHINAVELAN T,LARA-TEJERO M,LEFEBRE M,et al.NMR model of PrgI-SipD interaction and its implications in the needle-tip assembly of the Salmonella type Ⅲ secretion system[J].Journal of Molecular Biology,2014,426(16):2958-2969.
[13] LARA-TEJERO M,GALÁN J E.Salmonella enterica serovar Typhimurium pathogenicity island 1-encoded type Ⅲ secretion system translocases mediate intimate attachment to nonphagocytic cells[J].Infection and Immunity,2009,77(7):2635-2642.
[14] KANIGA K,TROLLINGER D,GALÁN J E.Identification of two targets of the type Ⅲ protein secretion system encoded by the inv and spa loci of Salmonella Typhimurium that have homology to the Shigella IpaD and IpaA proteins[J].Journal of Bacteriology,1995,177(24):7078-7085.
[15] GLASGOW A A,WONG H T,TULLMAN-ERCEK D.A secretion-amplification role for Salmonella enterica translocon protein SipD[J].ACS Synthetic Biology,2017,6(6):1006-1015.
[16] HOTINGER J A,MAY A E.Antibodies inhibiting the type Ⅲ secretion system of Gram-negative pathogenic bacteria[J].Antibodies (Basel),2020,9(3):35.
[17] 史超,张伟,刘素平,等.单增李斯特菌IspD蛋白生物信息学分析及表达纯化[J].中国畜牧兽医,2023,50(9):3799-3810. SHI C,ZHANG W,LIU S P,et al.Bioinformatics analysis,expression and purification of IspD protein of Listeria monocytogenes[J].China Animal Husbandry&Veterinary Medicine,2023,50(9):3799-3810.(in Chinese)
[18] 王路才,撒朗文朱,张焕容.禽源肠炎沙门菌的分离鉴定及耐药性与致病性分析[J].中国畜牧兽医,2023,50(4):1663-1674. WANG L C,SALANG W Z,ZHANG H R.Isolation,identification,drug resistance and pathogenicity of Salmonella Enteritis from Avian origin[J].China Animal Husbandry&Veterinary Medicine,2023,50(4):1663-1674.(in Chinese)
[19] ALTEKRUSE S F,BAUER N,CHANLONGBUTRA A,et al.Salmonella Enteritidis in broiler chickens,United States,2000-2005[J].Emerging Infectious Diseases,2006,12(12):1848-1852.
[20] SCALLAN E,HOEKSTRA R M,ANGULO F J,et al.Foodborne illness acquired in the United States-Major pathogens[J].Emerging Infectious Diseases,2011,17(1):7-15.
[21] HU Z,LIU L,GUO F,et al.Dietary supplemental coated essential oils and organic acids mixture improves growth performance and gut health along with reduces Salmonella load of broiler chickens infected with Salmonella Enteritidis[J].Journal of Animal Science and Biotechnology,2023,14(1):95.
[22] 杨文文,李玉保,路建彪,等.山东省鸡源沙门氏菌的分离鉴定及毒力基因分析[J].中国畜牧兽医,2021,48(8):3069-3078. YANG W W,LI Y B,LU J B,et al.Isolation,identification and virulence gene analysis of Salmonella from chicken in Shandong province[J].China Animal Husbandry&Veterinary Medicine,2021,48(8):3069-3078.(in Chinese)
[23] MASTROENI P,VILLARREAL-RAMOS B,HORMAECHE C E.Adoptive transfer of immunity to oral challenge with virulent Salmonellae in innately susceptible BALB/c mice requires both immune serum and T cells[J].Infection and Immunity,1993,61(9):3981-3984.
[24] JNEID B,MOREAU K,PLAISANCE M,et al.Role of T3SS-1 SipD protein in protecting mice against non-typhoidal Salmonella Typhimurium[J].PLoS Neglected Tropical Diseases,2016,10(12):e0005207.
[25] GALÁN J E.Salmonella interactions with host cells:Type Ⅲ secretion at work[J].Annual Review of Cell and Developmental Biology,2001,17:53-86.
[26] HOTINGER J A,PENDERGRASS H A,MAY A E.Molecular targets and strategies for inhibition of the bacterial type Ⅲ secretion system (T3SS);Inhibitors directly binding to T3SS components[J].Biomolecules,2021,11(2):316.
[27] DESIN T S,MICKAEL C S,LAM P K,et al.Protection of epithelial cells from Salmonella enterica serovar Enteritidis invasion by antibodies against the SPI-1 type Ⅲ secretion system[J].Canadian Journal of Microbiology,2010,56(6):522-526.
[28] BARTA M L,SHEARER J P,ARIZMENDI O,et al.Single-domain antibodies pinpoint potential targets within Shigella invasion plasmid antigen D of the needle tip complex for inhibition of type Ⅲ secretion[J].The Journal of Biological Chemistry,2017,292(40):16677-16687.
[29] PRAEKELT U,REISSBRODT R,KRESSE A,et al.Monoclonal antibodies against all known variants of EspA:Development of a simple diagnostic test for enteropathogenic Escherichia coli based on a key virulence factor[J].Journal of Medical Microbiology,2014,63(Pt12):1595-1607.
[30] KINOSHITA M,KATO H,YASUMOTO H,et al.The prophylactic effects of human IgG derived from sera containing high anti-PcrV titers against pneumonia-causing Pseudomonas aeruginosa[J].Human Vaccines&Immunotherapeutics,2016,12(11):2833-2846.
[31] VELGE P,WIEDEMANN A,ROSSELIN M,et al.Multiplicity of Salmonella entry mechanisms,a new paradigm for Salmonella pathogenesis[J].MicrobiologyOpen,2012,1(3):243-258.

Bioinformatics Analysis and Prokaryotic Expression of SipD Protein of Salmonella Enteritidis in Chickens

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