New type mutant of heatlabile enterotoxin from bacteria coli, and preparation method
A heat-resistant enterotoxin and Escherichia coli technology, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of low expression efficiency and unfavorable large-scale production, etc.
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Embodiment 1
[0103] Example 1: Construction of a novel Escherichia coli heat-labile enterotoxin mutant
[0104] 1. Cloning the heat-labile enterotoxin gene of wild Escherichia coli:
[0105] (1) Using an improved genome extraction method to extract the plasmid carrying the LT gene from wild-type toxigenic Escherichia coli [Feng Qiang, Zou Quanming, Cai Shaoxi, etc. New method for the extraction of LT plasmids and the construction and sequence analysis of the non-toxic mutant LTS63K Journal of Immunology, 2002, 18(5): 385-388]
[0106] (2) The following primers were designed according to the Escherichia coli heat-labile enterotoxin gene sequence published by GenBank: P1: GGAATTCCCATATGAAAAATATAAC, P2: TAGGATCCTCCTAGCATTAGACAT.
[0107] (3) The lt gene was amplified from the plasmid encoding the LT gene by PCR method, and the gene was cloned into pMD18-T. 2. The first modification of the heat-labile enterotoxin gene of wild-type Escherichia coli:
[0108] In order to insert the Escherichi...
Embodiment 2
[0113] Implementation Example 2: Expression of novel Escherichia coli heat-labile enterotoxin mutants
[0114] A single colony of E.coli BL21(DE3) newly transformed with the recombinant plasmid was cultured in a test tube containing 4 mL of LB medium for 8 hours. Inoculate 500mL LB medium with 50μL bacterial solution and continue to cultivate in the flask for 8h to A 600nm ≈1.5. At the same time, the bacterial plasmid in the test tube was identified, and 500 mL of the seed bacteria with the correct plasmid was used for inoculation. The seed bacteria were inoculated in 5L of modified M9-CAA medium, and the antibiotic concentration was Amp 100 μg / mL. After fermenting in BIOSTAT 10L self-controlled fermenter for 3 hours, 2L of feed was added, and the ingredients of feed were: 20% glucose, 8% tryptone, and 8% yeast extract. A 600nm At ≈30, add IPTG to a final concentration of 0.5mmol / L for induction. After 5 hours of induction, the bacteria were harvested by centrifugation [F...
Embodiment 3
[0115] Implementation Example 3: Purification and preservation of recombinant LT and recombinant LTDITH
[0116]Resuspend the wet bacteria collected by centrifugation with 10 times the volume of TEAN (pH7.3) buffer, break the bacteria three times with a high-pressure homogenizer at 70 MPa, check the bacteria-breaking effect with an oil microscope; collect the cell disruption solution by centrifugation at 16000g×30min at 4°C The supernatant was centrifuged at 16000g×30min at 4°C to discard the precipitate; the supernatant was passed through a 0.22 μm filter membrane and the filtrate was collected; the Immobilized D(+)-galactose affinity column ( Pierce, USA), Tuning A 280nm baseline. The ultrafiltered supernatant was applied to the column at a flow rate of 1.5 mL / min. After loading 200mL of sample, use TEAN (pH7.3) buffer to wash the impurities to A 280nm Return to baseline; use 0.3mol / L galactose (Galactose) elution buffer to elute the LT or LTDITH bound to the column, coll...
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