Two Expression Vectors, Designated as pGEX-CDON and pC89S4-CDON, for Producing GST-CDON and pVIII-CDON Fusion Proteins

Abstract:

Deoxynivalenol (DON) mimotope, designated as CDON, is an epitope (CMRPWLQ) immunoscreened from a phage-displayed random peptide library. In order to replace the conjugated toxin with non-toxic recombinant proteins in ELISA, two novel expression vectors, which were designated as plasmid pGEX-CDON and phagemid pC89S4-CDON for producing GST-CDON and pVIII-CDON fusion proteins in E.coli were constructed. After purification, both GST-CDON and pVIII-CDON fusion proteins show good reactogenicity with an anti-DON antibody in a competitive inhibition ELISA test. When GST-CDON was used as coating antigen, the linear range of the competitive inhibition ELISA is from 62ng/ml to 410ng/ml, the linear equation is Y= 186.6-23.87Ln (X), IC₅₀ is 194ng/ml. For pVIII-CDON as coating protein, the linear range of the competitive inhibition ELISA is from 20ng/ml to 470ng/ml, the linear equation is Y = 161.3-25.49Ln (X), R2=0.9962, IC₅₀ is 94ng/ml. ELISA analysis and comparison show the reactogenicity and specificity of pVIII-CDON binding to anti-DON antibody are better than GST-CDON fusion protein. The pVIII-CDON is promising in establishing an ELISA without the use of the toxic mycotoxin conjugate.

• Periodical: Advanced Materials Research (Volumes 726-731)
• Pages: 505-510
• DOI: https://doi.org/10.4028/www.scientific.net/AMR.726-731.505
• Online since: August 2013
• Authors: Fu Yong Xu, Ren Rong Liu*, Ling Xu, Xue Mei Qiu, Li Xin Zhu
• Keywords: Deoxynivalenol, ELISA, Fusion Protein, Glutathione-S-Transferase, Mimotopes, pVIII

References

[1] Foroud NA, Eudes F. Int J Mol Sci. Vol. 10(2009), p.147.

[2] Starkey DE, Ward TJ, Aoki T, Gale LR, Kistler HC, Geiser DM, Suga H, To´th B, Varga J, O'Donnell K. Fung Genet Biol. Vol. 44(2007), p: 1191.

[3] Bae HK, Pestka JJ. Toxicol Sci. Vol. 105(2008), pp.59-66.

[4] Pestka JJ, Smolinski AT. J Toxicol Environ Health. Vol. 8(2005), pp.39-69.

[5] Jestoi, M., Ritieni, A., Rizzo, A. J. Agric. Food. Chem. Vol. 52(2004), p.1464.

[6] Klotzel, M., Gutsche, B., Lauber, U., Humpf, H.U. J. Agric. Food. Chem. Vol. 53(2005), p.8904.

[7] Fang Ji, Hua Li, Jianhong Xu, Jianrong Shi. Toxins. Vol. 3(2011), p.968.

[8] Pestka, J.J., Islam, Z. Amuzie, C.J. Toxicol. Lett. Vol. 178(2008), p.83.

[9] Förster-Waldl E, Riemer AB, Dehof AK, Neumann D, Brämswig K, Boltz-Nitulescu G, et al. Mol Immunol. Vol. 42(2005), p.319.

[10] Jyoti Pande, Magdalena M. Szewczyk, Ashok K. Grover. Biotechnology Advances. Vol. 28 (2010), p.849.

[11] DENG Shunz hou, YU Zhou, YOU Shuz hu, et al. Chin J Public Health. Vol. 22(2006), p.1205 (in chinese).

[12] Smith DB, Johnson KS. Gene . Vol. 67(1988), p.31.

[13] Smith, G.P. Science. Vol. 228(1985), p.1315.

[14] Ying Wan, Yuzhang Wu, Jiang Bian, et al. Vaccine. Vol. 19(2001), p.2918.

[15] Kristensen P, Winter G. Fold Des. Vol. 3(1998), p.321.

[16] Litman GW, Rast JP, Shamblott MJ, Haire RN, Hulst M, et al. Mol Biol Evol. Vol. 10(1993), p.60.