Lactococcus lactis recombinant bacteria for expressing hypoallergenic peanut allergen, and application thereof
A peanut allergen and Lactococcus lactis technology, applied in the field of bioengineering, can solve the problems that hinder the large-scale promotion and application of the Escherichia coli expression system, and achieve the effect of reducing side effects and avoiding allergic reactions
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Embodiment 1
[0019] Embodiment 1 The acquisition of hypoallergenic gene mArah2
[0020] (1) Optimization of natural peanut allergen gene Arah2:
[0021] The signal peptide sequence was deleted in the amino acid sequence of Arah2 (genbank: AAN77576.1), and the codon optimization of the Arah2 gene sequence was carried out in combination with the codon preference of Lactococcus lactis, and subcloned into pUC57 to obtain a natural allergen optimization Plasmid pUC57-nArah2 of the gene nArah2 (synthesized by Shanghai Sangon, where pUC57 is a commercialized plasmid).
[0022] (2) Modified mutation of natural allergen optimization gene nArah2:
[0023] The aa47-56 antigenic epitope is disconnected and rearranged according to the amino acid sequence of 50-151-1-49 to obtain the peanut allergen gene mArah2 with low allergenicity. The relevant sequence is as follows:
[0024] Native gene sequence before optimization (genbank: AY158467.1), SEQ ID No.1.
[0025] The optimized gene sequence (mArah2...
Embodiment 2
[0028] Example 2 Construction of recombinant expression vector containing hypoallergenic gene mArah2
[0029] Rearrangement and splicing of target fragments aa1-49 and aa50-151: Using the plasmid pUC57-nArah2 (synthesized by Shanghai Sangon, where pUC57 is a commercialized plasmid) as a template, use primers P1F: CAGGTGGTCGTGATCGTTA TCGTCAACAATGGGAATTAC / P1R: CCCTCGAGATCTTGTGATGGTGAATATG and primers P2F :CGCCATAT GCCATATTTCTCTTCACAAG / P2R:GTAATTCCCATTGTTGACG ATAACGATCACGACCACCTG Amplifies aa1-49 and aa50-151 fragment genes. The PCR products of the two gene fragments were detected and purified by 1% agarose gel electrophoresis. The target fragments aa1-49 and aa50-151 were spliced by overlapping region amplification gene splicing method. The amplification system is as follows (total volume: 50 μL): 10×KOD plus buffer: 5 μL; dNTP: 5 μL; MgSO4: 2 μL; template 1-49: 2 μL; template 50-151: 2 μL; KOD enzyme: 0.5 μL; μL. After 5 cycles, add 1 μL each of the upstream and downstream...
Embodiment 3
[0030] The preparation of embodiment 3 recombinant Lactococcus lactis
[0031] The ligation reaction product above was transformed into L. lactis NZ9000 (Kuipers et al. Quorum sensing-controlled gene expression in lactic acid bacteria. Journal of Biotechnology. 1998, 64(1): 15-21) competent cells by electroporation. The transformants were picked for sequencing verification, and the recombinant plasmids and transformants with correct sequencing were named pNZ8148-mh2 (self-constructed) and L.lactis NZMH respectively. L.lactis NZ9000 and L.lactis NZ9000 strains recombinantly expressing nArah2 protein Named L. lactis NZ and L. lactis NZNH.
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