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
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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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