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Preparation method of shiga-like toxin Stx1B oral vaccine and product of shiga-like toxin Stx1B oral vaccine

A Shiga-like toxin and oral vaccine technology, applied in the preparation of Shiga-like toxin Stx1B oral vaccine, in the field of Stx1B oral vaccine, can solve the problems of low antigen content and inability to effectively induce intestinal immune response, so as to reduce the incidence rate, The effect of reliable immune effect and simple method

Inactive Publication Date: 2012-07-18
CHONGQING UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0003] Escherichia coli O157: H7 mainly invades the digestive tract, so vaccines injected into the immune system often cannot effectively induce specific immune responses in the intestinal tract, but they have not been used without special When dealing with oral vaccine antigens and direct oral immunization, the amount of antigens that can effectively reach the intestinal tract and stimulate the specific response of the intestinal lymphatic system is too low, and cannot effectively induce immune responses in the intestinal tract

Method used

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  • Preparation method of shiga-like toxin Stx1B oral vaccine and product of shiga-like toxin Stx1B oral vaccine
  • Preparation method of shiga-like toxin Stx1B oral vaccine and product of shiga-like toxin Stx1B oral vaccine
  • Preparation method of shiga-like toxin Stx1B oral vaccine and product of shiga-like toxin Stx1B oral vaccine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, cloning of Shiga-like toxin Stx1B gene

[0028] According to the reported Escherichia coli O157:H7 Stx1B gene sequence (GenBank: NC_002655.2), design Stx1B gene-specific primers, the upstream primer is Stx1B-F: 5'- cgagctcg atgaaaaaaacat-3' (SEQ ID No.1), the underline is the SacⅠ restriction site, and the downstream primer is Stx1B-R:5'- cgcggatccgcg tcaacgaaaaataac -3' (SEQ ID No.2), the underline is the BamHI restriction site; at the same time, the genomic DNA of Escherichia coli O157:H7 was extracted, and the extracted genomic DNA of Escherichia coli O157:H7 was used as a template for PCR amplification, PCR program As follows: pre-denaturation at 95°C for 10 minutes cycle once; further denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 50 seconds, 35 cycles, and finally extension at 72°C for 8 minutes to obtain the Stx1B gene. The PCR reaction products were electrophoresed on agarose gel with a mass volume...

Embodiment 2

[0029] Example 2, Construction of Shiga-like toxin Stx1B recombinant expression vector

[0030] The resulting pMD19-Stx1B vector was digested with restriction enzymes BamH I and SacI, and the Stx1B gene was recovered (here, the Stx1B gene obtained by PCR amplification in Example 1 can also be directly digested with BamH I and SacI) ; Simultaneously digest the improved pCAMBA1301 vector with BamH I and Sac I, recover the large fragment of the improved pCAMBA1301 vector, connect the recovered Stx1B gene with the large fragment of the improved pCAMBA1301 vector, and connect overnight at 16°C under the action of T4 DNA ligase to obtain improved pCAMBA1301-Stx1B carrier.

[0031] The resulting improved pCAMBA1301-Stx1B vector was transformed into Agrobacterium tumefaciens EHA105 competent cells by freeze-thaw method to obtain Agrobacterium tumefaciens (EHA105) containing the improved pCAMBA1301-Stx1B vector, which was named Stx1B-EHA105.

Embodiment 3

[0032] Embodiment 3, the preparation of transgenic tobacco

[0033]Transform tobacco with the obtained Stx1B-EHA105 Agrobacterium using the leaf disc method, induce regenerated tobacco on MS solid medium containing 1.0mg / L 6-BA, 50mg / L kanamycin and 500mg / L carbenicillin, and Transfer the regenerated tobacco that grows 2-3cm high to 1 / 2 MS medium containing 50mg / L kanamycin and 500mg / L carbenicillin for rooting culture. During the cultivation period, take a few tobacco leaves and extract the regenerated tobacco genome by CTAB method DNA, and use Stx1B gene-specific primers Stx1B-F (SEQ ID No.1) and Stx1B-R (SEQ ID No.2) for PCR detection to screen Stx1B gene transgenic tobacco, and transfer the rooted transgenic tobacco to soil for growth . Take about 2 g of transgenic tobacco leaves, add pre-cooled 1ml Tris-HCl buffer solution (25mmol / L, pH8. L thiourea, 0.4% CHAPS, 10 mmol / L DTT), grind until homogenized, transfer to a centrifuge tube at 4°C, 12 000 rpm, centrifuge for 3...

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Abstract

The invention discloses a preparation method of a shiga-like toxin Stx1B oral vaccine, which concretely comprises the steps of: cloning a shiga-like toxin Stx1B gene, building a recombinant expression vector containing the Stx1B gene, then preparing a transgenic plant, and finally preparing the shiga-like toxin Stx1B oral vaccine. The prepared shiga-like toxin Stx1B oral vaccine can be directly and orally taken to immunize and can effectively prevent the escherichia coli O157:H7 infection, moreover, the orbidity of edema disease of a pig is reduced, and the death efficiency is also reduced at the same time.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a preparation method of a Shiga-like toxin Stx1B oral vaccine and the prepared Stx1B oral vaccine. Background technique [0002] Escherichia coli ( Escherichia coli ) O157: H7 is a serotype of enterohaemorrhagic Escherichia coli. E. coli O157: H7 infection can produce toxins to kill vero cells. The toxin produced is Shiga-like toxin (Stx), which mainly acts on pigs or humans. Colon and ileum cells, the main symptom after infection of pigs is porcine edema disease. Stx contains 1 A subunit and 5 B subunits, forming a complex toxin of A-5B structure. The A subunit is the virulence unit of Stx, which has the activity of RNA-N-terminal glycosidase, which can interfere with the synthesis of intracellular proteins and cause cell damage and death. The B subunit is non-toxic, but it can bind to cells with a specific receptor (Gb3), produce specific antibodies, and then combin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/108A61P31/04C12N15/31C12N15/63
Inventor 王贵学臧广超黄俊丽张筱娟文文乙豪
Owner CHONGQING UNIV
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