Abstract
The S-layer protein CTC surface display system of Bacillus thuringiensis was used to test the possibility of displaying avian influenza virus nucleoprotein (NP) on the cell surface of B. thuringiensis. By fusing np with the anchoring motif of ctc, four recombinant plasmids were constructed. They harbored fusion gene ctc-np, csa-ctc-np (csa representing csaAB operon, very important in anchoring S-layer protein on cell surface), ctc-npp (npp representing the part fragment of np), and csa-ctc-npp, respectively. Five recombinant strains were obtained by transferring recombinant plasmids to B. thuringiensis plasmid-free derivative strain BMB171. The vegetative cells of five strains were used as agglutinogens for slide agglutination assays. The assays showed recombinant NP proteins successfully displayed on the cell surface of five strains. After immunization of chickens with spores by oral route, all five strains elicited a humoral response to NP and exhibited immunogenicity as indicated by enzyme-linked immunosorbent assay (ELISA). ELISA also showed that one of five strains, CN (bearing csa-ctc-npp), exhibited the highest immunogenicity among five strains, which suggested that the best way of constructing ctc fusion gene was the csa-ctc-npp. The strategy developed in this study suggests the possibility of generating a heat-stable and oral veterinary vaccine with B. thuringiensis surface display system.
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References
Bone EJ, Ellar DJ (1989) Transformation of Bacillus thuringiensis by electroporation. FEMS Microbiol. Lett 49(2–3):171–177
Glover T, Mitchell K (2001) An introduction to biostatistics. McGraw-Hill Education (Asia) Co. 201–207
Guo X, Xiong Z, Zhou M, Jia S, Xu Y (1991) The construction of multifunctional shuttle vectors of Bacillus subtilis–Escherichia coli. Chin J Biotechnol 7(3):224–229. (in Chinese with English abstract)
Li L, Yu Z (1999) Transformation and expression properties of a Bacillus thuringiensis plasmid-free derivative strain BMB171. Chinese Journal of Applied and Environment Biology 5(4):395–399. (in Chinese with English abstract)
Li Z, Xu Q, Bi D, Wang G, Cheng F (2001) Diagnosis of avian influenza and isolating and primary identification of avian influenza virus in Hubei province of China. Chinese Journal of Preventive Veterinary Medicine 23(2):146–148. (in Chinese)
Liu M, Hu S, Sun H, Bi D, Sun M (2007) Effect of Bacillus thuringiensis with crystal negative strain on safety of mice and chickens. Chinese Journal of Safety and Environment 7(5):12–15. (in Chinese with English abstract)
Mesnage S, Weber-Levy M, Haustant M, Mock M, Fouet A (1999) Cell surface-exposed tetanus toxin fragment C produced by recombinant Bacillus anthracis protects against tetanus toxin. Infect Immun 67(9):4847–4850
Mesnage S, Fontaine T, Mignot T, Delepierre M, Mock M, Fouet A (2000) Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation. EMBO J 19(17):4473–4484
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory Press
Sára M, Sleytr UB (2000) S-Layer proteins. J Bacteriol 182(4):859–868
Schnepf E, Crickmore N, van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62(3):775–806
Sleytr UB, Beveridge TJ (1999) Bacterial S-layers. Trends in Microbiology 7(6):253–260
Sneath PHA, Mair NS, Sharpe ME, Holt JG (1986) Bergey’s Manual of Systematic Bacteriology, Vol. 2. Williams and Wilkins, Baltimore, pp 1105–1139
Sun M, Zhu C, Yu Z (2001) Cloning of parasporal body protein gene resembling to S-layer protein genes from Bacillus thuringiensis CTC strain. Acta Microbiol Sin 41(2):141–147. (in Chinese with English abstract)
Wang L, Sun M, Yu Z (2004) Capacity of Bacillus thuringiensis S-layer protein displaying polyhistidine peptides on the cell surface. Appl Biochem Biotechnol 119(2):133–143
Wu H, Liu X, Sun M, Yu Z (2004) Cell surface display of α-amylase and metallothionein by using S-layer protein of Bacillus thuringiensis strain CTC. Acta Microbiol Sin 44(5):658–662. (in Chinese with English abstract)
Wu R, Hu S, Xiao Y, Li Z, Shi D, Bi D (2007) Development of indirect enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection and quantification of antibodies against avian influenza virus. Vet Res Commun 31:631–641
Acknowledgements
This work was supported by grants from the National High Technology Research and Development Project (863) of China (2006AA02Z174 and 2006AA10A212), Major Program for Science and Technology Development of Hubei Province of China (2006AA202A05), and Opening Study Program of State Key Laboratory of Agricultural Microbiology of China (AML02004).
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Liu, M., Li, S., Hu, S. et al. Display of avian influenza virus nucleoprotein on Bacillus thuringiensis cell surface using CTC as a fusion partner. Appl Microbiol Biotechnol 78, 669–676 (2008). https://doi.org/10.1007/s00253-008-1345-1
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DOI: https://doi.org/10.1007/s00253-008-1345-1