Actinomycete signal peptide for expressing intracellular protein to extracellular position and application thereof
An intracellular protein and signal peptide technology, applied in the field of genetic engineering, can solve the problems of low versatility of high-efficiency secretion and expression, no report on recombinant secretion and expression of Escherichia coli, etc. active effect
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[0028] Example 1: Construction of chemical recombinant strains
[0029] Preparation of competent cells: chemical method (Ca Cb) method
[0030] 1% of the inoculum was added to the BL21 overnight culture in 50ml LB; OD600nm was measured every 15-20min, and the cells were collected when they grew to 0.3-0.4; the culture was placed on ice for 10min, and then poured into a 50ml centrifuge tube , 4°C, 4500rpm, centrifuge for 10min (50ml centrifuge tube also needs to be pre-cooled, adjust the centrifuge to 4°C in advance); discard the supernatant, use 30ml of pre-cooled 0.1M MgCl for 50ml of the initial culture medium 2 -CaCl 2 Resuspend the cell pellet in the solution; centrifuge at 4500rpm for 10min at 4°C; discard the supernatant and use 2ml of pre-cooled 0.1M CaCl for 50ml of the initial culture medium 2 Solution (15% glycerol) to resuspend the cell pellet; aliquot 50 μl / tube or 100 μl / tube and store at -70°C.
[0031] Ligation system: vector 4μl, target fragment 5.5μl, T 4 ...
Example Embodiment
[0032] Example 2: Construction of FSA and SFSA recombinant strains
[0033] The full length of the FSA amylase gene is 1359bp. Primers were designed according to the signal peptide sequence, and the signal peptide sequence was recombined with the gene sequence shown in KC441955.1 to obtain the fusion gene fragment SFSA. Among them: 1 round of PCR amplifies the upper and lower fragments respectively, the primers are SFSA-F1: GAGGATCCGATGAGCAGACGAGC, SFSA-R1: GCGAGCGCCGATGACAATAATAATTATAC; SFSA-F2: GTATAATTATTATTGTCATCGGCGCTCGC, SFSA-R2: GACTCGACTTACTCTCCCGAAACCGACCATATG. The two sets of PCR products were respectively recovered by gel cutting, the concentration of the recovered products was determined, and water was added to adjust the concentration of the upstream and downstream homology arms to be basically the same; the 2-round PCR used the 1-round PCR product as the primer and template, and the two amplified products were connected. The PCR products were recovered; 3 rounds...
Example Embodiment
[0037] Example 3: Construction of BLA and SBLA recombinant strains
[0038] The full length of BLA amylase gene is 1455bp. Primers were designed according to the signal peptide sequence, and the signal peptide sequence was recombined with the gene sequence shown in AAU39594 to obtain the fusion gene fragment SBLA. The upper and lower fragments were amplified by one round of PCR. The primers were SBLA-F1: GAGGATCCGATGAGCAGACGAGC, SBLA- R1: CTTTAAGATTTGCCGCGGCGCTCG; SBLA-F2: GCGAGCGCCGCGGCAAATCTTAAAG, SBLA-R2: CCCAAGCTTGGGCTATCTTTGAACATAAATTG. The two sets of PCR products were respectively recovered by gel cutting, the concentration of the recovered products was determined, and water was added to adjust the concentration of the upstream and downstream homology arms to be basically the same; the 2-round PCR used the 1-round PCR product as the primer and template, and the two amplified products were connected. The PCR products were recovered; 3 rounds of PCR used 2 rounds of PCR ...
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