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Recombinant bacillus subtilis CoxA16-VP1 expression vector as well as preparation method and application thereof

A Bacillus subtilis, coxa16-vp1 technology, applied in the field of genetic engineering, can solve the problems of no inactivated vaccine and live attenuated vaccine

Inactive Publication Date: 2012-12-19
曹银光 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CoxA16 and poliovirus belong to the genus Enterovirus of the picornaviridae family, but compared with poliovirus, no effective inactivated vaccine or live attenuated vaccine has been applied

Method used

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  • Recombinant bacillus subtilis CoxA16-VP1 expression vector as well as preparation method and application thereof
  • Recombinant bacillus subtilis CoxA16-VP1 expression vector as well as preparation method and application thereof
  • Recombinant bacillus subtilis CoxA16-VP1 expression vector as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Construction of recombinant Bacillus subtilis CoxA16-VP1 expression vector, and verification

[0081] Methods as below:

[0082] (1) Transferring the carrier protein gene:

[0083] (a) PCR method to amplify the CotB gene regulatory sequence and coding sequence of Bacillus subtilis (BGSC NO.1A771 strain):

[0084] Upstream: 5-AGCGCCGgaattcACGGATTAGGCCGTTTGTCC-3 (position: -263 / -244, with EcoRI restriction site)

[0085] Downstream: 5-CAATCATCCaagcttGGATGATTGATCATCTGAAG-3 (position: +806 / +825, containing HindIII restriction site)

[0086] Amplification conditions are: 95°C for 4min, 30 cycles of amplification at 95°C for 30Sec, 53°C for 30Sec, and 75°C for 1min;

[0087] (b) Connect the above PCR amplification product to the pMD18T vector (Dalian Bao Biological Kit), the reaction system is: 5 μL connection buffer + 0.5 μL pMD18T vector + 4.5 μL target fragment; transform Escherichia coli DH5α, and send the positive PCR amplification strain sequencing

[008...

Embodiment 2

[0122] (1) The promoter and partial coding sequence (position -263~825nt) of the CotB gene (BSU36050cotB) of Bacillus subtilis were used as the carrier protein and the partial coding sequence of CoxA16VP1 was connected to the middle part of the amyE gene of the integrated plasmid pDG1662 (BGSCAccession: ECE113) .

[0123] Specific steps are as follows:

[0124] 1. Using the BGSC 1A771 strain genome as a template, pfu DNA polymerase was used to amplify the partial coding sequence of the carrier protein and its promoter by PCR.

[0125] The primer sequences are as follows:

[0126] Upstream: 5-AGCGCCGgaattcACGGATTAGGCCGTTTGTCC-3 (position: -263 / -244, with EcoRI restriction site)

[0127] Downstream: 5-CAATCATCCaagcttGGATGATTGATCATCTGAAG-3 (position: +806 / +825, containing HindIII restriction site)

[0128] PCR amplification conditions: 95°C for 4min, (95°C for 30sec, 53°C for 30sec, 72°C for 1min) × 30 cycles.

[0129] Results: A 1088bp DNA fragment containing the CotB promot...

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Abstract

The invention discloses a recombinant bacillus subtilis CoxA16-VP1 expression vector which is escherichia coli-bacillus subtilis shuttle expression vector that is loaded with encoding gene of capsid protein VP1 of CoxA16 virus. A construction method of the expression vector comprises the steps of: (1) amplifying a CoTB gene regulation sequence and a coding sequence of the bacillus subtilis, and connecting the amplified sequences to pDG1662 to obtain plasmid pDG1662-CoTB; and (2) amplifying a VP1 antigen sequence of the capsid protein VP1 of the CoxA16, connecting the amplified sequence to the plasmid pDG1662-CoTB to obtain plasmid pDG1662-CoTB-VP1 which is the recombinant bacillus subtilis CoxA16-VP1 expression vector. After being introduced into a host cell for cultivation, the recombinant vector can be used for preparing an oral vaccine for controlling or treating diseases caused by the CoxA16.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis CoxA16-VP1 expression vector, a preparation method thereof, an expression host and an application as an oral vaccine, belonging to the field of genetic engineering. Background technique [0002] Coxsackievirus A16 (Coxsackievirus A16, CoxA16) is a member of the genus Enterovirus in the family Picornaviridae. The genome of the CoxA16 virus is a single positive-strand RNA containing about 7400 nucleotides. The virus particles are roughly spherical in shape, with icosahedral stereosymmetry, no envelope and protrusions, and the virus capsid is composed of 60 subunits connected to each other. Each subunit is composed of capsid proteins VP1, VP2, VP3, and VP4 encoded by the viral genome. The three proteins VP1, VP2, and VP3 are exposed on the surface of the viral capsid, while VP4 is embedded in the inner side of the viral capsid and the virus. The core is tightly connected. According to the nucleoti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/66C12N1/21A61K48/00A61K39/39A61P31/14C12R1/125
Inventor 曹银光李志会
Owner 曹银光
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