Mutated e. coli heat-labile enterotoxin
A heat-resistant enterotoxin and Escherichia coli technology, applied in the direction of viruses, antibacterial drugs, antiviral agents, etc., can solve the problems of high toxicity and limited clinical application
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Embodiment 1
[0019] Example 1: Construction of encoding wild-type LT A and LT mutant LT A gene
[0020] A 1.8-kb DNA fragment of the LT gene, including subunit A and subunit B, was isolated from human enterotoxigenic E. coli H10407 and cloned into pBluescript IIKS(-) vector (pBluescript-LThWT). The nucleotide sequence (SEQ ID NO: 6) of the LT gene (encoding subunits A and B) and the amino acid sequence (SEQ ID NO: 5) of the subunit A of the LT are as follows:
[0021] Nucleotide sequence of LT (SEQ ID NO: 6) (subunit A: 1-777; subunit B: 774-1148):
[0022] atgaaaaata taactttcat tttttttatt ttattagcat cgccattata tgcaaatggc 60
[0023] gacaaattat accgtgctga ctctagaccc ccagatgaaa taaaacgttc cggaggtctt
[0024] 120
[0025] atgcccagag ggcataatga gtacttcgat agaggaactc aaatgaatat taatctttat
[0026] 180
[0027] gatcacgcga gaggaacaca aaccggcttt gtcagatatg atgacggata tgtttccact
[0028] 240
[0029] tctcttagtt tgagaagtgc tcacttagca ggacagtcta tattatcagg atattccact
[0030] 300
[0031]...
Embodiment 2
[0206] Example 2: Preparation of wild type LT and mutant LT A LT
[0207]E. coli HB101 was transformed with the pBluescript II KS(-) vector containing native or mutant LT genes (including subunit A and subunit B genes). Native and mutant LTs were purified from cultures grown overnight in 3-liter shake flasks containing L-broth supplemented with 100 μg / ml ampicillin. Cells were collected by centrifugation, resuspended in TEAN buffer (0.2M NaCl, 50 mM Tris, 1 mM EDTA and 3 mM NaN3, pH 7.4), and lysed with a microfluidizer (Microfluidics Corporation, USA). After clarification of the lysate by centrifugation, LT was fractionated by addition of solid ammonium sulfate to 65% saturation. The preparation was then suspended in TEAN buffer, extensively dialyzed against the same buffer, and used as crude LT. Crude LT was chromatographed at 4°C on an immobilized D-galactose (Pierce, Rockford, IL) column equilibrated with TEAN buffer (Uesaka et al., 1994, Microbial Pathogenesis 16:71-76...
Embodiment 3
[0213] Example 3: Determination of wild-type LT and mutant-containing LT A Effect of LT on intracellular cAMP levels
[0214] Caco-2 cells (ATCC HTB-37) at 5×10 per well 4 Concentrations of cells were maintained in 24-well plates in MEM-α medium supplemented with 20% FBS, grown to near confluence, and incubated in 5% CO 2 Incubate for 30 min in MEM-α containing 1% FBS and 1 mM 3-isobutyl-1-methylxanthine (IBMX), then add toxin (Grant et al., 1994, Infection and Immunity 62:4270-4278) . Native or mutant LT was added to each well and incubated for 4 hours. Cells were then washed twice with cold PBS. Intracellular cAMP was extracted by adding 200 [mu]l 0.1 N HCl to each well and incubating for 15 minutes at room temperature. Supernatants of cell lysates were collected after addition of 0.1 N NaOH to each well (Cheng et al., 2000, Vaccine 18:38-49; Park et al., 1999, Experimental and Molecular Medicine 31:101-107). cAMP was measured with a cAMP enzyme immunoassay kit (Assay ...
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