Prokaryotic Expression,Purification,Identification,and Polyclonal Antibody Preparation of Vibrio cholerae Hemolysin HlyA

doi:10.13560/j.cnki.biotech.bull.1985.2021-1299

Abstract

Abstract:

It is aimed to express and purify hemolysin HlyA protein of Vibrio cholerae in prokaryotic cells,and to prepare and identify the polyclonal antibody of mouse anti-HlyA. The hlyAgene was amplified from the genome Vibrio cholerae via PCR and cloned into the expression plasmid pET28a,pET32a and pCold TF to construct the recombinant plasmid pET28a-hlyA,pET32a-hlyA and pCold TF-hlyA, and they were transformed into E. coli BL21（DE3）. The conditions used to induce expression were optimized and the expressive forms were identified. The soluble form of hlyA protein was purified by Ni-NTA column. The purified HlyA protein was used as antigen for immunization of BALB/c mice to prepare polyclonal antibody. The antibody titer was determined to test the immunogenicity by indirect ELISA. The antibody specific recognition of HlyA protein in Vibrio cholerae was analyzed by Western blot and verified by mass spectrometry. The hemolytic activity of the purified hlyA protein and the neutralizing activity of the antibody were analyzed. The HlyA protein was expressed as inclusion bodies in the pET28a-hlyA and pET32a-hlyA vectors,and as soluble form in the pCold TF-hlyA vector. HlyA was purified by affinity chromatography on an Ni-NTA agarose column to get higher purity. The recombinant HlyA protein could not lyse rabbit red blood cells,but immunized mice can obtain polyclonal antibody with higher titer. The results of Western blot and mass spectrometry showed that the HlyA polyclonal antibody can specifically recognize the HlyA protein in Vibrio cholerae,and the antibody can effectively inhibit the hemolytic activity of Vibrio cholerae. The soluble form of HlyA protein and the high titer anti-HlyA polyclonal antibody were successfully obtained,which laid the foundation for the follow-up study of the role of HlyA protein in the pathogenic process of V. cholerae.

Key words: Vibrio cholerae, HlyA protein, prokaryotic expression, polyclonal antibody

WANG Guang-li, FAN Chan, WANG Hui, LU Hui-fang, XIA Ling-yin, HUANG Jian, MIN Xun. Prokaryotic Expression,Purification,Identification,and Polyclonal Antibody Preparation of Vibrio cholerae Hemolysin HlyA[J]. Biotechnology Bulletin, 2022, 38(7): 269-277.

Figures/Tables 9

Fig.1 Construction of recombinant plasmids pET28a-hlyA,pET32 a-hlyA and pCold TF-hlyA. M：DNA marker DL5000 bp;A：electrophoresis results of hlyA gene PCR amplification（1：hlyA fragment）. B-D：Electrophoresis results of pET28a-hlyA,pET32a-hlyA and pCold TF-hlyArecombinant plasmid bacterial liquid PCR,respectively（1：positive control;2-4：positive cloning bacteria）

Fig.2 SDS-PAGE analysis of pET28a-hlyA expression pro-ducts in E. coli BL21（DE3）induced with IPTG M：180 kD protein marker;A：1：uninduced E. coli BL21（DE3）with recombinant plasmid pET28a-hlyA;2-3：induced E. coli BL21（DE3）with recombinant plasmid pET28a-hlyA at 10℃,120 r/min by 0.05 mmol/L and 0.1 mmol/L IPTG for 16 h;4：induced E. coli BL21（DE3）with recombinant plasmid pET28a-hlyA at 15℃,100 r/min by 0.05 mmol/L IPTG for 12 h;5 and 7：lysate pellet of induced bacteria;6 and 8：lysate supernatant of induced bacteria. B：1：uninduced E. coli BL21（DE3）with recombinant plasmid pET28a-hlyA;2：lysate pellet of induced bacteria at 23℃ by 0.05 mmol/L IPTG for 10 h;3：lysate supernatant of induced bacteria at 23℃

Fig.3 SDS-PAGE analysis of pET32a-hlyA expression pr-oducts in E. coli BL21（DE3）induced with IPTG M：250 kD protein marker. A：1：uninduced E. coli BL21（DE3）with recombinant plasmid pET32a-hlyA;2-3：induced E. coli BL21（DE3）with recombinant plasmid pET32a-hlyA at 15℃,100 r/min by 0.05 and 0.1 mmol/L IPTG for 16 h;4 and 5：induced E. coli BL21（DE3）with recombinant plasmid pET32a-hlyA at 25℃,120 r/min by 0.05 and 0.1 mmol/L IPTG for 10 h;6-9：lysate supernatant of induced bacteria;10-13：lysate pellet of induced bacteria. B：1：uninduced E. coli BL21（DE3）with recombinant plasmid pET32a-hlyA;2 and 5：induced E. coliBL21（DE3）with recombinant plasmid pET32a-hlyA at 20℃,100 r/min by 0.05 and 0.1 mmol/L IPTG for 12 h;3 and 6：lysate pellet of induced bacteria;4 and 7：lysate supernatant of induced bacteria

Fig.4 SDS-PAGE analysis of pCold TF-hlyA expression products in E. coli BL21 induced with IPTG and the purified HlyA protein M：250 kD protein marker;1：uninduced E. coli BL21 with recombinant plasmid pCold TF -hlyA;2：induced E. coli BL21 with recombinant plasmid pCold TF -hlyA;3：lysate supernatant of induced bacteria;4-9：1×Washing Buffer contain of 10,20,30,40,300,500 mmol/L imidazole through Ni-chelating affinity chromatography

Fig.5 Hemolytic activity determination of recombinant expressive protein HlyA 1：Positive control（1% Trion-X）;2：negative control（PBS）;3：0 μg HlyA protein incubateed with 2% rabbit red blood cells;4：4 μg HlyA protein incubateed with 2% rabbit red blood cells;5：8 μg HlyA protein co-incubated with 2% rabbit red blood cells

Fig.6 Analysis of serum antibody titer in mice before and after immunization with HlyA protein

Fig.7 Analysis specificity of anti-HlyA polyclonal antibody by Western blot 1：supernatant of HN375 bacterial;2：supernatant of HN375△hlyA bacterial

Fig.8 Mass spectrometric profiles of HlyA protein A mass spectrum of the peptide sequence SASFTVDWDHPVFTGGRPVNLQLAS-FNNR that matches the amino acid sequence of the HlyA protein is shown

Fig. 9 Detection of Anti-HlyA polyclonal antibody neutrali-zation activity A：Analysis of the inhibitory effect of anti-HlyA polyclonal antibody on the hemolytic activity of HN375△tagH strain;B：Analysis of the inhibitory effect of anti-HlyA polyclonal antibody on the hemolytic activity of HN375 strain and two clinical strains. *** indicates that there is a significant difference in the data between two groups（P < 0.001）

References 30

[1]	Saka HA, Bidinost C, Sola C, et al. Vibrio cholerae cytolysin is essential for high enterotoxicity and apoptosis induction produced by a cholera toxin gene-negative V. cholerae non-O1, non-O139 strain[J]. Microb Pathog, 2008. 44(2):118-28. doi: 10.1016/j.micpath.2007.08.013 URL
[2]	Ruenchit P, Reamtong O, Siripanichgon K, Chaicumpa W, Diraphat P. New facet of non-O1/non-O139 Vibrio cholerae hemolysin A:a competitive factor in the ecological niche[J]. FEMS Microbiol Ecol, 2017. 93(12).
[3]	Zmeter C, Tabaja H, Sharara AI, Kanj SS. Non-O1, non-O139 Vibrio cholerae septicemia at a tertiary care center in Beirut, Lebanon;a case report and review[J]. J Infect Public Health, 2018. 11(5):601-604. doi: 10.1016/j.jiph.2018.01.001 URL
[4]	Jiang F, Bi R, Deng L, Kang H, Gu B, Ma P. Virulence-associated genes and molecular characteristics of non-O1/non-O139 Vibrio cholerae isolated from hepatitis B cirrhosis patients in China[J]. Int J Infect Dis, 2018. 74:117-122. doi: S1201-9712(18)34456-4 pmid: 29969728
[5]	Dobrović K, Rudman F, Ottaviani D, Šestan Crnek S, Leoni F, Škrlin J. A rare case of necrotizing fasciitis caused by Vibrio cholerae O8 in an immunocompetent patient[J]. Wien Klin Wochenschr, 2016. 128(19-20):728-730. doi: 10.1007/s00508-016-1060-3 URL
[6]	Hao Y, Wang Y, Bi Z, et al. A case of non-O1/non-O139 Vibrio cholerae septicemia and meningitis in a neonate[J]. Int J Infect Dis, 2015. 35:117-9. doi: 10.1016/j.ijid.2015.05.004 URL
[7]	Marinello S, Marini G, Parisi G, et al. Vibrio cholerae non-O1, non-O139 bacteraemia associated with pneumonia[J]. Italy 2016. Infection, 2017. 45(2):237-240.
[8]	Fan Y, Li Z, Li Z, et al. Nonhemolysis of epidemic El Tor biotype strains of Vibrio cholerae is related to multiple functional deficiencies of hemolysin A[J]. Gut Pathog, 2019. 11:38. doi: 10.1186/s13099-019-0316-7 URL
[9]	Chen YT, Tang HJ, Chao CM, Lai CC. Clinical manifestations of non-O1 Vibrio cholerae infections[J]. PLoS One,2015. 10(1):e0116904.
[10]	Chen J, Huang J, Huang M, et al. Two cases of septic shock with different outcomes caused by non-O1/non-O139 Vibrio cholerae isolates[J]. J Int Med Res,2020. 48(6):300060520933459.
[11]	Chatterjee S, Ghosh K, Raychoudhuri A, et al. Incidence, virulence factors, and clonality among clinical strains of non-O1, non-O139 Vibrio cholerae isolates from hospitalized diarrheal patients in Kolkata[J]. India. J Clin Microbiol, 2009. 47(4):1087-95.
[12]	Mukherjee A, Ganguly S, Chatterjee NS, Banerjee KK. Vibrio cholerae hemolysin:The β-trefoil domain is required for folding to the native conformation[J]. Biochem Biophys Rep, 2016. 8:242-248. doi: 10.1016/j.bbrep.2016.09.009 pmid: 28955962
[13]	Ganguly S, Mukherjee A, Mazumdar B, Ghosh AN, Banerjee KK. The β-prism lectin domain of Vibrio cholerae hemolysin promotes self-assembly of the β-pore-forming toxin by a carbohydrate-independent mechanism[J]. J Biol Chem,2014. 289(7):4001-8. doi: 10.1074/jbc.M113.522284 URL
[14]	Diep TT, Nguyen NT, Nguyen TN, et al. Isolation of New Delhi metallo-β-lactamase 1-producing Vibrio cholerae non-O1, non-O139 strain carrying ctxA, st and hly genes in southern Vietnam[J]. Microbiol Immunol, 2015. 59(5):262-7. doi: 10.1111/1348-0421.12248 URL
[15]	Saha N, Banerjee KK. Carbohydrate-mediated regulation of interaction of Vibrio cholerae hemolysin with erythrocyte and phospholipid vesicle[J]. J Biol Chem, 1997. 272(1):162-7. doi: 10.1074/jbc.272.1.162 URL
[16]	Yamamoto K, Al-Omani M, Honda T, Takeda Y, Miwatani T. Non-O1 Vibrio cholerae hemolysin:purification, partial characterization, and immunological relatedness to El Tor hemolysin[J]. Infect Immun,1984. 45(1):192-6. doi: 10.1128/iai.45.1.192-196.1984 URL
[17]	Coelho A, Andrade JR, Vicente AC, Dirita VJ. Cytotoxic cell vacuolating activity from Vibrio cholerae hemolysin[J]. Infect Immun,2000. 68(3):1700-5. doi: 10.1128/IAI.68.3.1700-1705.2000 URL
[18]	Figueroa-Arredondo P, Heuser JE, Akopyants NS, et al. Cell vacuolation caused by Vibrio cholerae hemolysin[J]. Infect Immun, 2001. 69(3):1613-24. doi: 10.1128/IAI.69.3.1613-1624.2001 URL
[19]	Chakraborty DC, Mukherjee G, Banerjee P, Banerjee KK, Biswas T. Hemolysin induces Toll-like receptor(TLR)-independent apoptosis and multiple TLR-associated parallel activation of macrophages[J]. J Biol Chem,2011. 286(40):34542-51. doi: 10.1074/jbc.M111.241851 URL
[20]	Debellis L, Diana A, Arcidiacono D, et al. The Vibrio cholerae cytolysin promotes chloride secretion from intact human intestinal mucosa[J]. PLoS One,2009. 4(3):e5074.
[21]	Alm RA, Mayrhofer G, Kotlarski I, Manning PA. Amino-terminal domain of the El Tor haemolysin of Vibrio cholerae O1 is expressed in classical strains and is cytotoxic[J]. Vaccine,1991. 9(8):588-94. doi: 10.1016/0264-410X(91)90247-4 URL
[22]	Tsou AM, Zhu J. Quorum sensing negatively regulates hemolysin transcriptionally and posttranslationally in Vibrio cholerae[J]. Infect Immun,2010. 78(1):461-7. doi: 10.1128/IAI.00590-09 URL
[23]	Tsou AM, Cai T, Liu Z, Zhu J, Kulkarni RV. Regulatory targets of quorum sensing in Vibrio cholerae:evidence for two distinct HapR-binding motifs[J]. Nucleic Acids Res,2009. 37(8):2747-56. doi: 10.1093/nar/gkp121 URL
[24]	Nguyen AN, Disconzi E, Charrière GM, et al. csrB gene duplication drives the evolution of redundant regulatory pathways controlling expression of the major toxic secreted metalloproteases in vibrio tasmaniensis LGP32[J]. mSphere,2018. 3(6).
[25]	McCardell BA, Kothary MH, Madden JM. Two-step purification and partial characterization of a variant of the Vibrio cholerae non-O1 hemolysin[J]. FEMS Microbiol Lett,1999. 180(2):177-82. doi: 10.1111/j.1574-6968.1999.tb08793.x URL
[26]	Dutta S, Mazumdar B, Banerjee KK, Ghosh AN. Three-dimensional structure of different functional forms of the Vibrio cholerae hemolysin oligomer:a cryo-electron microscopic study[J]. J Bacteriol, 2010, 192(1):169-78. doi: 10.1128/JB.00930-09 URL
[27]	尹鑫, 刘澜澜, 贾莹, 等. 鸡氨肽酶N的高效可溶性表达及生物学功能分析[J]. 生物工程学报, 2010, 26(4):470-475.
	Yin X, Liu L, Jia Y, et al. Expression and biological function analysis of chicken aminopeptidase N[J]. Chinese Journal of Biotechnology,2010. 26(4):470-5.
[28]	De S, Olson R. Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins[J]. Proc Natl Acad Sci USA, 2011, 108(18):7385-90. doi: 10.1073/pnas.1017442108 URL
[29]	Nagamune K, Yamamoto K, Naka A, Matsuyama J, Miwatani T, Honda T. In vitro proteolytic processing and activation of the recombinant precursor of El Tor cytolysin/hemolysin(pro-HlyA)of Vibrio cholerae by soluble hemagglutinin/protease of V. cholerae, trypsin, and other proteases[J]. Infect Immun, 1996. 64(11):4655-8. doi: 10.1128/iai.64.11.4655-4658.1996 URL
[30]	Nagamune K, Yamamoto K, Honda T. Intramolecular chaperone activity of the pro-region of Vibrio cholerae El Tor cytolysin[J]. J Biol Chem,1997. 272(2):1338-43. doi: 10.1074/jbc.272.2.1338 URL