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Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa).

Soh HS, Chung HY, Lee HH, Ajjappala H, Jang K, Park JH, Sim JS, Lee GY, Lee HJ, Han YH, Lim JW, Choi I, Chung IS, Hahn BS - Springerplus (2015)

Bottom Line: Binding of recombinant LTB and CTB proteins was confirmed based on GM1-ganglioside binding enzyme-linked immunosorbent assays (GM1-ELISA).LTB and CTB proteins in rice transgenic lines were detected by Western blot analysis.The results of the present study suggest that the combined expression of CTB and LTB proteins can be utilized to produce vaccines against enterotoxigenic strains of Escherichia coli and Vibrio cholera, for the prevention of diarrhea.

View Article: PubMed Central - PubMed

Affiliation: Division of Environmental Agricultural Research, Gyeonggido Agricultural Research & Extension Services, Hwaseong, 445-784 South Korea.

ABSTRACT
We expressed the heat-labile enterotoxin B (LTB) subunit from enterotoxigenic Escherichia coli and the cholera toxin B (CTB) subunit from Vibrio cholerae under the control of the rice (Oryza sativa) globulin (Glb) promoter. Binding of recombinant LTB and CTB proteins was confirmed based on GM1-ganglioside binding enzyme-linked immunosorbent assays (GM1-ELISA). Real-time PCR of three generations (T3, T4, and T5) in homozygous lines (LCI-11) showed single copies of LTB, CTB, bar and Tnos. LTB and CTB proteins in rice transgenic lines were detected by Western blot analysis. Immunogenicity trials of rice-derived CTB and LTB antigens were evaluated through oral and intraperitoneal administration in mice, respectively. The results revealed that LTB- and CTB-specific IgG levels were enhanced in the sera of intraperitoneally immunized mice. Similarly, the toxin-neutralizing activity of CTB and LTB in serum of orally immunized mice was associated with elevated levels of both IgG and IgA. The results of the present study suggest that the combined expression of CTB and LTB proteins can be utilized to produce vaccines against enterotoxigenic strains of Escherichia coli and Vibrio cholera, for the prevention of diarrhea.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of the binary vector containing LTB and CTB. Glb, Glb promoter (0.9 kb); TpinII, potato protease inhibitor II terminator (1.0 kb); P35S, CaMV 35S promoter (0.84 kb); Bar, bar gene (0.55 kb); Tnos, nopaline synthase terminator (0.25 kb); MAR, 5'-matrix attachment region of chicken lysozyme gene (1.3 kb).
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Fig1: Schematic representation of the binary vector containing LTB and CTB. Glb, Glb promoter (0.9 kb); TpinII, potato protease inhibitor II terminator (1.0 kb); P35S, CaMV 35S promoter (0.84 kb); Bar, bar gene (0.55 kb); Tnos, nopaline synthase terminator (0.25 kb); MAR, 5'-matrix attachment region of chicken lysozyme gene (1.3 kb).

Mentions: The pMJ103 vector integrates the genes LTB and CTB in the inverted repeat regions through homologous recombination (Figure 1). The vector containing LTB and CTB genes is driven by the constitutive globulin promoter (Glb), along with potato protease inhibitor II terminator (Pin II). The expression vector contained the LTB and CTB genes, and the vector was labeled pMJ103-LTB-CTB (Figure 1). The LTB and CTB gene insertions were confirmed based on digestion of the vector with NotI. The digestion yielded about 3 kb of between the CTB inserts and Pin II (data not shown).Figure 1


Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa).

Soh HS, Chung HY, Lee HH, Ajjappala H, Jang K, Park JH, Sim JS, Lee GY, Lee HJ, Han YH, Lim JW, Choi I, Chung IS, Hahn BS - Springerplus (2015)

Schematic representation of the binary vector containing LTB and CTB. Glb, Glb promoter (0.9 kb); TpinII, potato protease inhibitor II terminator (1.0 kb); P35S, CaMV 35S promoter (0.84 kb); Bar, bar gene (0.55 kb); Tnos, nopaline synthase terminator (0.25 kb); MAR, 5'-matrix attachment region of chicken lysozyme gene (1.3 kb).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380882&req=5

Fig1: Schematic representation of the binary vector containing LTB and CTB. Glb, Glb promoter (0.9 kb); TpinII, potato protease inhibitor II terminator (1.0 kb); P35S, CaMV 35S promoter (0.84 kb); Bar, bar gene (0.55 kb); Tnos, nopaline synthase terminator (0.25 kb); MAR, 5'-matrix attachment region of chicken lysozyme gene (1.3 kb).
Mentions: The pMJ103 vector integrates the genes LTB and CTB in the inverted repeat regions through homologous recombination (Figure 1). The vector containing LTB and CTB genes is driven by the constitutive globulin promoter (Glb), along with potato protease inhibitor II terminator (Pin II). The expression vector contained the LTB and CTB genes, and the vector was labeled pMJ103-LTB-CTB (Figure 1). The LTB and CTB gene insertions were confirmed based on digestion of the vector with NotI. The digestion yielded about 3 kb of between the CTB inserts and Pin II (data not shown).Figure 1

Bottom Line: Binding of recombinant LTB and CTB proteins was confirmed based on GM1-ganglioside binding enzyme-linked immunosorbent assays (GM1-ELISA).LTB and CTB proteins in rice transgenic lines were detected by Western blot analysis.The results of the present study suggest that the combined expression of CTB and LTB proteins can be utilized to produce vaccines against enterotoxigenic strains of Escherichia coli and Vibrio cholera, for the prevention of diarrhea.

View Article: PubMed Central - PubMed

Affiliation: Division of Environmental Agricultural Research, Gyeonggido Agricultural Research & Extension Services, Hwaseong, 445-784 South Korea.

ABSTRACT
We expressed the heat-labile enterotoxin B (LTB) subunit from enterotoxigenic Escherichia coli and the cholera toxin B (CTB) subunit from Vibrio cholerae under the control of the rice (Oryza sativa) globulin (Glb) promoter. Binding of recombinant LTB and CTB proteins was confirmed based on GM1-ganglioside binding enzyme-linked immunosorbent assays (GM1-ELISA). Real-time PCR of three generations (T3, T4, and T5) in homozygous lines (LCI-11) showed single copies of LTB, CTB, bar and Tnos. LTB and CTB proteins in rice transgenic lines were detected by Western blot analysis. Immunogenicity trials of rice-derived CTB and LTB antigens were evaluated through oral and intraperitoneal administration in mice, respectively. The results revealed that LTB- and CTB-specific IgG levels were enhanced in the sera of intraperitoneally immunized mice. Similarly, the toxin-neutralizing activity of CTB and LTB in serum of orally immunized mice was associated with elevated levels of both IgG and IgA. The results of the present study suggest that the combined expression of CTB and LTB proteins can be utilized to produce vaccines against enterotoxigenic strains of Escherichia coli and Vibrio cholera, for the prevention of diarrhea.

No MeSH data available.


Related in: MedlinePlus