Construction Prokaryotic Vector,Optimization Expression Conditions and Polyclonal Antibody Preparation of Vibrio alginolyticus Accessory Colonization Factor ACFA

doi:10.7668/hbnxb.2015.01.007

Abstract

Abstract: To construct prokaryotic expression vector of aquatic pathogenic Vibrio alginolyticus accessory colonization factor A (ACFA), optimize the expression condition of ACFA and prepare the polyclonal antibodies of mouse anti-ACFA.The ACFA expression strain was obtained by molecular clone.ACFA was purified by the way of the gel slices, immunized mice and prepared the polyclonal antibody.The antibody titer was 1:3 200 by ELISA, and Western-Blotting proved that the antiserum had good specificity.By the method of orthogonal experiment, the optimal inducing expressing condition of ACFA protein:strain OD₆₀₀ value, IPTG final concentration, inducing time and temperature, which were 0.5, 0.3 mmol/L, 8 h, 32 ℃, respectively;the optimal culture condition:glucose concentration, rotation rate, and medium volume, which were 0, 230 r/min and 50 mL, respectively.

Key words: Vibrio alginolyticus, ACFA protein, Orthogonal experiment, Polyclonal antibody

CLC Number:

Q816

LIU Xiang. Construction Prokaryotic Vector,Optimization Expression Conditions and Polyclonal Antibody Preparation of Vibrio alginolyticus Accessory Colonization Factor ACFA[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(1): 35-41. doi: 10.7668/hbnxb.2015.01.007.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2015.01.007

http://www.hbnxb.net/EN/Y2015/V30/I1/35

References

[1] 魏霜,冼钰茵,赵晖,等.多重 PCR 检测四种食源性病原弧菌[J].中国农业科学,2013,46(8):1682-1686.
[2] Takekawa N,Kojima S,Homma M.Contribution of many charged residues at the stator-rotor interface of the Na⁺-driven flagellar motor to torque Generation in Vibrio alginolyticus[J].Journal of Bacteriology,2014,196(7):1377-1385.
[3] 熊盼,彭喜春,魏霜,等.溶藻弧菌的毒力相关基因及其对小鼠的致病力[J].微生物学报,2014,54(1):80-88.
[4] Yang Y,Bao C,Liu A,et al.Immune responses of prophenoloxidase in the mud crab Scylla paramamosain against Vibrio alginolyticus infection: in vivo and in vitro gene silencing evidence[J].Fish & Shellfish Immunology,2014,39(2):237-244.
[5] Sun Y,Liu C S,Sun L.A multivalent killed whole-cell vaccine induces effective protection against Edwardsiella tarda and Vibrio anguillarum[J].Fish & Shellfish Immunology,2011,31(4):595-599.
[6] Alcaide E,Blasco M D,Esteve C.Occurrence of drug-resistant bacteria in two European eel farms[J].Applied and Environmental Microbiology,2005,71(6):3348-3350.
[7] Olivier V,Queen J,Satchell K J.Successful small intestine colonization of adult mice by Vibrio cholerae requires ketamine anesthesia and accessory toxins[J].PLOS One,2009,4(10):e7352.
[8] Withey J H,Dirita V J.Activation of both acfA and acfD transcription by Vibrio cholerae ToxT requires binding to two centrally located DNA sites in an inverted repeat conformation[J].Molecular Microbiology,2005,56(4):1062-1077.
[9] Chaparro A P,Ali S K,Klose K E.The ToxT-dependent methyl-accepting chemoreceptors AcfB and TcpI contribute to Vibrio cholerae intestinal colonization[J].FEMS Microbiology Letters,2010,302(2):99-105.
[10] Withey J H,Dirita V J.Vibrio cholerae ToxT independently activates the divergently transcribed aldA and tagA genes[J].Journal of Bacteriology,2005,187(23):7890-7900.
[11] 程海燕,庞欢瑛,鲁义善,等.溶藻弧菌acfA基因克隆与生物信息学分析[J].广东海洋大学学报,2014,34(1):9-14.
[12] Queen J,Satchell K J.Promotion of colonization and virulence by cholera toxin is dependent on neutrophils[J].Infection and Immunity,2013,81(9):3338-3345.
[13] Peterson K M,Mekalanos J J.Characterization of the vibrio cholerae ToxR regulon:identification of novel genes involved in intestinal colonization[J].Infection and Immunity,1988,56(11):2822-2829.
[14] Cai S H,Huang Y C,Lu Y S,et al.Expression and immunogenicity analysis of accessory colonization factor A from Vibrio alginolyticus strain HY9901[J].Fish & Shellfish Immunology,2013,34(2):454-462.
[15] Sharma M K,Jani D,Thungapathra M,et al.Expression of accessory colonization factor subunit A (ACFA) of Vibrio cholerae and ACFA fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum) [J].Journal of Biotechnology,2008,135(1):22-27.
[16] Marrero K,Sánchez A,Rodríguez-Ulloa A,et al.Anaerobic growth promotes synthesis of colonization factors encoded at the Vibrio pathogenicity island in Vibrio cholerae El Tor[J].Research in Microbiology,2009,160(1):48-56.
[17] Liu X,She X T,Zhu Q F,et al.Heterogeneous interactome between Litopenaeus vannamei plasma proteins and Vibrio parahaemolyticus outer membrane proteins[J].Fish & Shellfish Immunology,2013,34(1):192-198.
[18] 李传香,薛淑霞,刘逸尘,等.凡纳滨对虾泛素交联酶E2基因的克隆及表达分析[J].中国水产科学,2014,21(4):661-668.
[19] 刘祥,李惠.大肠埃希菌外膜蛋白OmpA表达质粒构建和诱导条件优化 [J].生物技术,2013,23(4):32-36.
[20] Wang Y D,Peng K C,Wu J L,et al.Transgenic expression of salmon delta-5 and delta-6 desaturase in zebrafish muscle inhibits the growth of Vibrio alginolyticus and affects fish immunomodulatory activity[J].Fish & Shellfish Immunology,2014,39(2):223-230.
[21] Lazo L,Izquierdo A,Suzarte E,et al.Evaluation in mice of the immunogenicity and protective efficacy of a tetravalent subunit vaccine candidate against dengue virus[J].Microbiology and Immunology,2014,58(4):219-226.
[22] Mire C E,Geisbert J B,Agans K N,et al.A recombinant Hendra virus G glycoprotein subunit vaccine protects nonhuman primates against Hendra virus challenge[J].Journal of Virology,2014,88(9):4624-4631.
[23] Xu M M,Sun Q,Su J,et al.Microbial transformation of geniposide in Gardenia jasminoides Ellis into genipin by Penicillium nigricans[J].Enzyme and Microbial Technology,2008,42(5):440-444.
[24] Corrales-Garcia L,Ortiz E,Castañeda-Delgado J,et al.Bacterial expression and antibiotic activities of recombinant variants of human β-defensins on pathogenic bacteria and M.tuberculosis[J].Protein Expression and Purification,2013,89(1):33-43.
[25] Studier F W.Protein production by auto-induction in high density shaking cultures[J].Protein Expression and Purification,2005,41(1):207-234.

Please choose a citation manager

Content to export