Prokaryotic secretion expression carrier and application thereof
A secretory expression and vector technology, applied in the field of prokaryotic secretory expression vectors, can solve the problems of no protein folding and assembly, decreased expression of active proteins, misfolded or aggregated products, etc., and achieves improved efficiency, high water solubility, and easy purification. Effect
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Embodiment 1
[0036] Example 1 Construction of recombinant plasmid pGTG containing double gene III-Trx complex device
[0037] 1. PCR amplification of Escherichia coli Trx gene
[0038] The following pair of primers (SEQ ID NO: 2) were synthesized:
[0039] (1) 5'-CATGCCATGGATATGAGCGATAAAAATTATTCACCTGAC-3'
[0040] (2) 5'-CCCCCGGGGGCCAGGTTAGCGTCGAGGA-3'
[0041] The Trx gene was amplified using the E.coli K12 genome as a template. To facilitate cloning, an NcoI restriction site was introduced at the 5' end, and an XmaI restriction site was introduced at the 3' end.
[0042] 2. PCR amplification of gene III signal peptide
[0043]The following pair of primers (SEQ ID NO: 3) were synthesized:
[0044] (1) 5'-CCCCCCGGGATGAAAAAACTGCTGTTCGCGATTCCGCTGGTG-3'
[0045] (2) 5'-CATGCCATGGTGCTATGGCTATAGAACGGCACCACCAGCGGAAT-3'
[0046] The gene III signal peptide was amplified by PCR technique. For the convenience of cloning, an XmaI restriction site was introduced at the 5' end, and an NcoI res...
Embodiment 2
[0049] Example 2 Construction of recombinant human epidermal growth factor secretion expression vector pBAD-GTG-rhEGF and secretion and expression of rhEGF
[0050] 1. Design four pairs of primers, and obtain recombinant human epidermal growth factor (rhEGF) coding gene through whole gene synthesis, insert rhEGF gene into pGTG polyclonal restriction site, obtain recombinant human epidermal growth factor secretion expression vector pGTG-rhEGF .
[0051] 2. Transform the secreted expression plasmid pGTG-rhEGF into the expression host strain E.coli TOP10, screen for positive clones, inoculate 20ml of Amp-containing culture solution, culture overnight at 37°C with shaking, and inoculate 1L of the overnight cultured bacterial solution at a ratio of 1:100 Add 50 mg / ml of Amp (1 μl / ml) to the LB culture medium, culture with shaking at 37°C for 5-6 hours, until OD600=0.5-0.6. Add 2ml, 0.02ml of inducer fructose (1mg / ml), and incubate at 37°C for 4h. Dissolve the expressed bacterium ...
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