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

doi:10.16431/j.cnki.1671-7236.2021.11.026

Abstract

Abstract: The purpose of this study was to express Salmonella virulence protein SipA through pET prokaryotic expression system and prepare polyclonal antibodies against SipA protein, so as to analyze the regulatory effect of noncoding small RNAs RyhB-1 and RyhB-2 of Salmonella Enteritidis SE50336 on SipA. pET28a-sipA recombinant plasmid was constructed by pET prokaryotic expression system and transformed into E. coli BL21(DE3) competent cells. After optimizing and obtaining the best protein induction conditions, the protein was induced and expressed. The expression of recombinant protein was analyzed by 10% SDS-PAGE. The recombinant protein was purified by nickel affinity chromatography. BALB/c mice were immunized with purified SipA protein and Freund adjuvant to obtain specific polyclonal antibody against SipA protein, and the specificity of the prepared serum was analyzed. Western blotting technique was used to detect wild strain SE50336 and RyhB single deletion strains SE50336ΔryhB-1, SE50336ΔryhB-2 and RyhB double deletion strain SE50336ΔryhB-1ΔryhB-2 so as to analyze the regulatory effects of RyhB-1 and RyhB-2 on SipA protein. The results showed that the recombinant plasmid pET28a-SipA was successfully constructed and transformed into E. coli BL21(DE3) competent cells. SDS-PAGE showed that the recombinant protein was mainly expressed in the form of inclusion body, with a size of 75 ku. After purification by affinity chromatography, the purity of SipA protein was high. The results of Western blotting showed that the polyclonal antibody could specifically detect the SipA recombinant protein of Salmonella Enteritidis. Compared with wild strain, the expression of SipA protein in RyhB single deletion strain SE50336ΔryhB-1 and SE50336ΔryhB-2 decreased, especially in double deletion strains, indicating that RyhB-1 and RyhB-2 could upregulate the expression of SipA protein. The purification of SipA recombinant protein and the preparation of SipA polyclonal antibody laid a foundation for further study on the regulatory mechanism of RyhB-1 and RyhB-2 on SipA protein.

Key words: Salmonella Enteritidis; SipA; RyhB; prokaryotic expression; polyclonal antibodies

CLC Number:

S852.61⁺2

MENG Xia, HE Mengping, WEI Huaxu, ZHANG Ke, WANG Heng, ZHU Guoqiang. 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 and Veterinary Medicine, 2021, 48(11): 4162-4169.

References

[1] TORO M, RETAMAL P, AYERS S, et al. Whole-genome sequencing analysis of Salmonella enterica serovar Enteritidis isolates in Chile provides insights into possible transmission between gulls, poultry, and humans[J]. Applied and Environmental Microbiology, 2016, 82(20):6223-6232.
[2] GANTOIS I, DUCATELLE R, PASMANS F, et al. Mechanisms of egg contamination by Salmonella Enteritidis[J]. FEMS Microbiology Reviews, 2009, 33(4):718-738.
[3] GAST R K, REGMI P, GURAYA R, et al. Colonization of internal organs by Salmonella Enteritidis in experimentally infected laying hens of four commercial genetic lines in conventional cages and enriched colony housing[J]. Poultry Science, 2019, 98(4):1785-1790.
[4] DOS SANTOS A M P, FERRARI R G, CONTE-JUNIOR C A.Type three secretion system in Salmonella Typhimurium:The key to infection[J]. Genes Genomics, 2020, 42(5):495-506.
[5] D'COSTA V M, COYAUD E, Boddy K C, et al. BioID screen of Salmonella type 3 secreted effectors reveals host factors involved in vacuole positioning and stability during infection[J]. Nature Microbiology, 2019, 4(12):2511-2522.
[6] STEELE-MORTIMER O, BRUMELL J H, KNODLER L A, et al. The invasion-associated type Ⅲ secretion system of Salmonella enterica serovar Typhimurium is necessary for intracellular proliferation and vacuole biogenesis in epithelial cells[J]. Cellular Microbiology, 2010, 4(1):43-54.
[7] LILIC M, GALKIN V E, ORLOVA A, et al. Salmonella SipA polymerizes actin by stapling filaments with nonglobular protein arms[J]. Science, 2003, 301(5641):1918-1921.
[8] MCINTOSH A, MEIKLE L M, ORMSBY M J, et al. SipA activation of caspase-3 is a decisive mediator of host cell survival at early stages of Salmonella enterica serovar Typhimurium infection[J]. Infection & Immunity, 2017, 85(9):e00393-17
[9] PAPENFORT K, VOGEL J.Regulatory RNA in bacterial pathogens[J]. Cell Host & Microbe, 2010, 8(1):116-127.
[10] WATERS L S, STORZ G.Regulatory RNAs in bacteria[J]. Cell, 2009, 136(4):615-628.
[11] MASSE E, GOTTESMAN S.A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli[J]. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(7):4620-4625.
[12] LECLERC J M, DOZOIS C M, DAIGLE F.Role of the Salmonella enterica serovar Typhi Fur regulator and small RNAs RfrA and RfrB in iron homeostasis and interaction with host cells[J]. Microbiology, 2013, 159(Pt 3):591-602.
[13] PADALON-BRAUCH G, HERSHBERG R, ELGRABLY-WEISS M, et al. Small RNAs encoded within genetic islands of Salmonella Typhimurium show host-induced expression and role in virulence[J]. Nucleic Acids Research, 2008, 36(6):1913-1927.
[14] KIM J N.Roles of two RyhB paralogs in the physiology of Salmonella enterica[J]. Microbiological Research, 2016, 186-187:146-152.
[15] CHAREYRE S, MANDIN P.Bacterial iron homeostasis regulation by sRNAs[J]. Microbiology Spectrum, 2018, 6(2).doi:10.1128/microbiolspec.RWR-0010-2017.
[16] PORCHERON G, DOZOIS C M.Interplay between iron homeostasis and virulence:Fur and RyhB as major regulators of bacterial pathogenicity[J]. Veterinary Microbiology, 2015, 179(1-2):2-14.
[17] 陈斌杰.肠炎沙门菌非编码小RNA RyhB调控毒力相关靶基因的筛选、鉴定和功能研究[D].扬州:扬州大学, 2019. CHEN B J.Screening, identification and functional study of virulence-related target genes regulated by S.Enteritidis non-coding small RNA RyhB[D].Yangzhou:Yangzhou University, 2019.(in Chinese)
[18] CHEN B, MENG X, NI J, et al. Positive regulation of type Ⅲ secretion effectors and virulence by RyhB paralogs in Salmonella enterica serovar Enteritidis[J]. Veterinary Research, 2021, 52(1):44.
[19] REHMAN T, YIN L, LATIF M B, et al. Adhesive mechanism of different Salmonella fimbrial adhesins[J]. Microbial Pathogenesis, 2019, 137:103748.
[20] BOHN E, SONNABEND M, KLEIN K, et al. Bacterial adhesion and host cell factors leading to effector protein injection by type Ⅲ secretion system[J]. International Journal of Medical Microbiology, 2019, 309(5):344-350.
[21] 袁婷婷, 秦毅斌, 王若木, 等.隆林黑猪氨肽酶N基因的生物信息学分析和原核表达[J]. 中国畜牧兽医, 2020, 47(6):1648-1658. YUAN T T, QIN Y B, WANG R M, et al. Bioinformatics analysis and prokaryotic expression of porcine aminopeptidase N gene in Longlin Black pigs[J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(6):1648-1658.(in Chinese)
[22] 刘雪婷, 王召阳, 鑫婷, 等.非洲猪瘟病毒B438L蛋白的原核表达及其多克隆抗体的制备与鉴定[J]. 中国畜牧兽医, 2021, 48(3):991-1000. LIU X T, WANG Z Y, XIN T, et al. Prokaryotic expression and polyclonal antibody preparation and identification of African swine fever virus B438L protein[J]. China Animal Husbandry & Veterinary Medicine, 2021, 48(3):991-1000.(in Chinese)
[23] HOE C H, RAABE C A, ROZHDESTVENSKY T S, et al. Bacterial sRNAs:Regulation in stress[J]. International Journal of Medical Microbiology, 2013, 303(5):217-229.
[24] JØRGENSEN M G, PETTERSEN J S, KALLIPOLITIS B H.sRNA-mediated control in bacteria:An increasing diversity of regulatory mechanisms[J]. Biochimica et Biophysica Acta-Gene Regulatory Mechanisms, 2020, 1863(5):194504.
[25] WANG L, CAI X, WU S, et al. InvS coordinates expression of PrgH and FimZ and is required for invasion of epithelial cells by Salmonella enterica serovar Typhimurium[J]. Journal of Bacteriology, 2017, 199(13):e00824-16.

Preparation of Polyclonal Antibody of SipA in Salmonella Enteritidis and Analysis of Its Expression Regulation by Non-coding Small RNA RyhB-1/2

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