125 results about "Cholera toxin" patented technology

Transgenic chloroplast technology could provide a viable solution to the production of Insulin-like Growth Factor I (IGF-I), Human Serum Albumin (HSA), or interferons (IFN) because of hyper-expression capabilities, ability to fold and process eukaryotic proteins with disulfide bridges (thereby eliminating the need for expensive post-purification processing). Tobacco is an ideal choice because of its large biomass, ease of scale-up (million seeds per plant), genetic manipulation and impending need to explore alternate uses for this hazardous crop. Therefore, all three human proteins will be expressed as follows: a) Develop recombinant DNA vectors for enhanced expression via tobacco chloroplast genomes b) generate transgenic plants c) characterize transgenic expression of proteins or fusion proteins using molecular and biochemical methods d) large scale purification of therapeutic proteins from transgenic tobacco and comparison of current purification/processing methods in E. coli or yeast e) Characterization and comparison of therapeutic proteins (yield, purity, functionality) produced in yeast or E. coli with transgenic tobacco f) animal testing and pre-clinical trials for effectiveness of the therapeutic proteins. Mass production of affordable vaccines can be achieved by genetically engineering plants to produce recombinant proteins that are candidate vaccine antigens. The B subunits of Enteroxigenic E. coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are examples of such antigens. When the native LTB gene was expressed via the tobacco nuclear genome, LTB accumulated at levels less than 0.01% of the total soluble leaf protein. Production of effective levels of LTB in plants, required extensive codon modification. Amplification of an unmodified CTB coding sequence in chloroplasts, up to 10,000 copies per cell, resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as oligomers (about 410 fold higher expression levels than that of the unmodified LTB gene). PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that chloroplast synthesized CTB assembled into oligomers and was antigenically identical to purified native CTB. Also, GM1,-ganglioside binding assays confirmed that chloroplast synthesized CTB binds to the intestinal membrane receptor of cholera toxin, indicating correct folding and disulfide bond formation within the chloroplast. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed. The introduced gene was stably inherited in the subsequent generation as confirmed by PCR and Southern blot analyses. Incrased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant based oral vaccines and fusion proteins with CTB needing oral administration a much more practical approach.

The invention belongs to the technical field of medicine, and specifically relates to a primary tumor cell culture medium, culture method and application. The primary tumor cell culture method provided by the invention comprises the steps as follows: preparing a primary cell culture medium which comprises the following components: hydrocortisone, EGF, Insulin, a ROCK inhibitor, but not contains cholera toxin, and is an improvement of the existing primary cell culture medium; culturing tumor tissue epithelial cells on laid-out trophoblast cells by using the primary cell culture medium, and enabling the tumor tissue epithelial cells to proliferate rapidly under the combined action of growth factors secreted by the trophoblast cells and nutrient factors contained in the culture medium; and digesting and sub-culturing the tumor tissue epithelial cells when the tumor tissue epithelial cells grow to a cell density of about 80% to 90%. A convenient primary cell culture method is used to obtain immortalized cells possessing the biological characteristics of a patient's own tumor, and the problem of immortalization of the primary culture of tumor cells is solved, thereby realizing personalized treatment for the patient.

The present invention relates to the field of recombinant toxin protein production in bacterial hosts. In particular, the present invention relates to production processes for obtaining high levels of a recombinant CRM197, Diphtheria Toxin, Pertussis Toxin, Tetanus Toxoid Fragment C, Cholera Toxin B, Cholera holotoxin, and Pseudomonas Exotoxin A, from a bacterial host.

Cultures of keratinocyte cells are provided which are free from nonautologous fibroblasts and organ extracts, and which have a high speed of cell amplification for a minimum seeding density. The cultures can be cryopreserved in a buffered isotonic medium containing serum and a cryoprotectant. The cultures are produced by a process that does not involve the use of a feeder layer and organ extracts. A culture medium which can be used contains Medium 199, serum, epidermal growth factor, cholera toxin and/or hydrocortisone, and optionally insulin. A substance for wound healing and for cosmetic applications is derived from cultured human keratinocytes. A non-viable total keratinocyte lysate for use in promoting wound healing is produced by growing keratinocyte cells on a support, detaching the cells from the support, and lysing the detached cells to obtain the lysate which may be frozen and lyophilized. The cells may be grown without using a support to produce the lysate, or to produce a non-viable keratinocyte cell culture lyophilisate or spray dried non-viable keratinocyte cell composition for use in healing wounds.

The invention relates to a culture medium for a normal epithelial cell of a human or mammal, primary separation culture, subculture, 3D gas-liquid culture and 3D matrigel culture methods, the normal epithelial cell generated by using the culture medium and the culture methods and application of the normal epithelial cell to a toxicological evaluation system. The culture medium is prepared by mixing DMEM and Ham's F-12NUTRIENT MIX according to the volume ratio of 3:1 and also adding 4-6% of fetal calf serum, 1-3nM triiodothyronine, 0.4-0.65% of insulin-transferrin-selenium reagent, 4-6mu g/ml transferrin, 9-11ng/mL epidermal growth factors, 0.3-0.5mu g/mL hydrocortisone, 0.5-1.5nM cholera toxin, 0.4-0.6mu g/mL amphoterrible B, 35-45mu g/mL gentamicin, 45-55nM calpeptin, 35-45ng/ml recombinant human IL-1RA and 3mu g/ml recombinant human R-Spondin-1. The culture medium disclosed by the invention can be used for carrying out separation culture or subculture on the normal epithelial cell of the human or the mammal and any other various tissue source, rapidly proliferating the normal epithelial cell in vitro and establishing a cell line; and the normal epithelial cell is a normal diploid cell and is applied to the toxicological evaluation system of the human or the mammal.

The present invention discloses the use of the non-toxic cell-binding B subunit of CT (CTB), and holotoxin CT that is devoid of ADP-ribosylating activity, as adjuvants for enhancing transcutaneous immune response to a co-administered protein allergen. It was found that topical administration of CTB to mice induced serum antibody response against itself comparable to those evoked by CT, but was inefficient at promoting systemic antibody responses against an admixed prototype protein allergen. To the contrary co-administration of either CT or CTB with allergen led to vigorous antigen-specific T cell proliferative responses in lymph nodes draining the cutaneous site of administration and at distant systemic sites. Consistent with these observations, it was found that CTB selectively potentiated Th1-driven responses without affecting Th2-dependent responses. Cutaneously applied CT enhanced serum IgE responses to a co-administered allergen, while CTB partially suppressed epicutaneously induced IgE responses to the same allergen.

The present invention provides improved methods and compositions for treating and preventing autoimmune and allergic diseases. More specifically, the invention relates to new immunomodulating complexes that are fusion proteins comprising a mutant subunit of the A1-subunit of the cholera toxin (CTA1), a peptide capable of binding to a specific cellular receptor, and one or more epitopes associated with an autoimmune or allergic disease. In the mutant CTA1 subunit, the amino acids corresponding to the amino acid 7, arginine, and amino acid 187, cysteine, in the native CTA1 have been replaced.

The invention relates to a cholera toxin virulence gene detection kit and a detection method thereof. The kit of the invention contains three pairs of primers which are designed by using vibrio cholera ctxA gene as target gene on the basis of the loop-mediated isothermal amplification technology, namely inner primers FIP/BIP, outer primers F3/B3 and ring primers LF/LB and also contains Bst DNA polymerases, reaction solution, sample pretreatment solution, coloring liquid, stabilizing solution and positive control. The method for detecting the cholera toxin virulence gene comprises the following steps: extracting bacterial DNA, performing the loop-mediated isothermal amplification of the cholera toxin virulence gene and coloring for detection. The kit of the invention has high amplificationefficiency, specificity and sensitivity, low omission ratio and obvious coloring effect and is suitable for the rapid detection of toxigenic vibrio cholera.

The invention discloses a limbal stem cell serum-free medium, consisting of the following raw materials (calculated by weight percent): DMEM/F12 60ml-90ml, BSA 0.5g-4g, EGF 1Mug-4Mug, insulin 0.1mg-1mg, hydrogen 20ug-100ug, cholera toxin 1ug-10ug, transferrin 0.1mg-1mg, selenious acid 0.1ug-1ug, GCLCM 10ml-40ml, penicillin 5*103IU-2*104IU and streptomycin 5*103IU-2*104IU. The invention adopts the method that the culture medium is added with fibroblast conditioned medium, and the nutritive factors are supplemented with the hormone and cytokine combination, fibroblast conditioned medium and BSA. As a result, the nutritive needs for long-time in-vitro expansion and cultivation of the limbal stem cell serum-free medium are satisfied, and the medium is serum-free and suitable for commercial production.

Vaccines against prion disease eliciting a humoral immune response when administered mucosally are described. The vaccines comprise a prion protein, a prion protein fragment, or a non-amyloidogenic prion protein homolog and an adjuvant suitable for inducing a humoral immune response after mucosal administration. Suitable adjuvants include cholera toxin subunit B, heat-labile enterotoxin and aluminum hydroxide. Alternatively, the vaccine comprises a vector encoding a prion protein, fragment, or homolog in an attenuated Salmonella host. The vaccines can be used to prevent or treat prion disease in humans and other mammals.

The invention provides a recombination antigen composition, a vaccine and a carrier and a method for preparing the antigen composition. A recombination helicobacter pylori antigen which comprises a helicobacter pylori antigen, the fusion proteins of a cholera toxin CT-A2 subunit and cholera toxin CT-B proteins is optimized. The compatibility of the CT-A2 and the CT-B proteins is used, a chimeric structure of CT-A2-5CT-B is formed, and accordingly an antigen with higher titer is formed through the helicobacter pylori antigen and the fusion proteins of the cholera toxin CT-A2 subunit. Meanwhile, the effect that the CT-B enhances immunization is used, and the effect that the antigen immunogenicity of the recombination antigen composition is enhanced is achieved. In addition, the chimeric protein constituted by the recombination antigen stimulates a mucous membrane to generate secreting type IgA immunization.

The invention discloses a cell culture medium. The raw material formula of the cell culture medium comprises 33.33ml of DMEM/High Glucose (DMEM high-glucose culture medium), 66.67ml of HAM'S/F-12 culture medium, 3-5mg/mL of plant-origin recombinant human serum albumin, 100U/ml of penicillin, 100 micrograms/milliliter of streptomycin, 2.5 micrograms/milliliter of amphotericin B, 0.2-0.5 micrograms/milliliter of hydrocortisone, 3-6 micrograms/milliliter of insulin, 6-10ng/ml of cholera toxin B, 9-12ng/ml of human epidermal growth factor EGF, 22-25 micrograms/milliliter of adenine, and 7-9 micromoles/liter of Y-27632. The cell culture medium can be used for culturing primary human tumor cells or tumor stem cells. The cell culture medium is improved; when the cell culture medium is applied to culturing the primary human tumor cells or the tumor stem cells, the cell culture medium is capable of better accelerating the growth of the primary tumor cells and obtaining the human tumor stem cells of a higher ratio.

The invention provides a human vascular endothelial cell culture solution. The human vascular endothelial cell culture solution is prepared from a basic culture medium, fetal calf serum, human recombined epidermal growth factors, human recombined fibroblast growth factors, vascular endothelial growth factors, insulin-like growth factors, vitamin C, cortisol, cholera toxin, aminoethanol and ethanolamine phosphate. Compared with a traditional vascular endothelial cell culture medium, the human vascular endothelial cell culture solution has the advantages that the culture solution can be suitable for multiple kinds of vascular endothelial cells, cell proliferation is effectively promoted, the division time is effectively shortened, the characteristics of the vascular endothelial cells are maintained, the passage number of the in-vitro human vascular endothelial cells is greatly increased, and the survival time of the in-vitro human vascular endothelial cells is greatly prolonged; meanwhile, all the ingredients of the cell culture solution can be obtained in a general laboratory, preparation is convenient, a special finished product culture medium is not needed, therefore, the cost for culturing the endothelial cells is greatly reduced, and the human vascular endothelial cell culture solution is suitable for application and popularization.

The inventive subject matter relates to an immunogenic composition composed of a chimeric molecule including a conformationally stable adhesin from Escherichia coli fused to a bacterial toxin A subunit, such as cholera toxin A subunit or heat-labile Escherichia coli toxin A subunit. The invention also relates to the adhesin-toxin chimera noncovalently associated with a toxin B subunit of the same or different species as the A subunit. The invention also relates to a method of utilizing an adhesin/toxin fusion composition to elicit an immune response.

The invention discloses food grade lactic acid bacteria active carrier Group A rotavirus vaccine and a preparation method thereof. The food grade lactic acid bacteria active carrier Group A rotavirus vaccine is characterized in that VP6 antigen protein from Group A virus, common serotype VP7 antigen protein (P serotype) and VP4 antigen protein (G serotype-expressed separately in the form of VP5* and VP8* protein subunit), vaccine adjuvant escherichia coli thermal unstable toxin B (LTB) and cholera toxin subunit B (CTB) are expressed and secreted by thyA gene deletion lactic acid bacteria cell or shown by the cell wall. Expressions of antigen protein and vaccine adjuvant protein are controlled by inducible or constitutive promoter, protein expression cassette is integrated onto the chromosome of the expression host lactic acid bacteria strain, and external antibiotics resistance gene introduced in gene manipulation is removed. The lactic acid bacteria active carrier rotavirus vaccine has the advantages of having wide serotype covering range, being easy to produce in large scale, and being safe and convenient to use without a refrigerator and a needle tubing.

The invention relates to nutritional and pharmaceutical compositions comprising non-digestible galactooligosaccharides (GOS) and uses thereof. In particular, it relates to the use of GOS species in preventing or treating disease caused by bacterial toxins. Provided is the use of GOS having a polymerization degree of 5 or higher, preferably 6 or higher, for the manufacture of a nutritional or pharmaceutical composition for the treatment or prevention of an acute or chronic disease associated with or caused by the adhesion and/or uptake of a cholera toxin family member. Also provided is a method for providing a GOS fraction capable of inhibiting cholera toxin (Ctx) binding to GM1 and fractions obtainable thereby.

The invention relates to preservation of human local living parts and in particular relates to a culture medium for keeping primary airway epithelial cells in the physiological state in vivo during in vitro culture. The invention is characterized in that the culture medium contains the following components (per liter), 12 g of DMEM/F12 medium, 30 mg of penicillin G sodium salt, 50 mg of streptomycin, 10 mg of insulin, 5 mg of transferring, 25 Mug of epidermal growth factors, 100 Mug of cholera toxin, 108.741 Mug of hydrocortisone, 1 g of bovine pituitary extract, 3 g of bovine serum albumin, 1.5*10<-2> Mug of retinoic acid, 50 mg of gentamycin, 0.5 mg of amphotericin B, 50 mL of fetal calf serum and the balance of water. The primary airway epithelial cells cultured by the culture medium have the ciliary beating function and can maintain the physiological state in vivo.

The invention discloses a method for rapidly separating and culturing human bronchial epithelial cells, and an optimized medium. The optimized medium is prepared from a DMEM culture medium, an F-12 Nutrient mix culture medium, 0.5 mg/ml hydrocortisone, fetal bovine serum, 25mu g/ml epidermal growth factor, 5 mg/ml insulin, 11.7mu M cholera toxin, 10 mM ROCK1 inhibitor, and 10 mM BMP4 antagonist. The method for rapidly culturing the human bronchial epithelial cells by using the optimized medium not only greatly simplifies the steps of culturing epithelial cells which require 3T3 J2 cells as a feeder layer, and only needs to culture the primary human bronchial epithelial cells digested by pronase in the optimized medium, but also can be used for obtaining humane bronchial epithelial cells which are rapidly separated and can be indefinitely subcultured; the subcultured human bronchial epithelial cells are subjected to in vitro expansion and culture; when the human bronchial epithelial cells are subcultured for 20 generations or more, a sufficient amount of seed cells can be obtained so as to be used in a variety of follow-up in vitro and in vivo studies such as in vitro cell interaction model studies.

The invention provides for anti-pathogen vaccines produced in recombinant bacteria and/or transgenic plants and administered through standard vaccine introduction methods or oral administration. In one embodiment, the vaccine is administered through the consumption of the edible plant as food, or the bacteria administered orally. The present invention also provides a method of using genetically modified microorganisms, such as lactic acid bacteria, including Lactococcus lactis strains, as oral vaccines. In one embodiment, Lactococcus lactis expressing the avian influenza HA gene can be used as an oral vaccine for protection against H5N1 virus infection. In another embodiment, said Lactococcus lactis is administered as an oral vaccine in conjunction with an adjuvant such as cholera toxin B.

The invention discloses a culture medium that is used in transportation of tissue engineered skin. A basic culture fluid is added with additional hydrocortisone, epidermal growth factors, isoproterenol, insulin, transferrin, triiodothyronine, adenine, vitamin C, cholera toxin, L-serine, L-novain, palmic acid, linoleic acid, arachidonic acid, bovine pituitary extract, bovine serum albumin, penicillin, streptomycin and fungizone B. The obtained culture medium has strong stability and long shelf life of about 12 months, completely meet the demands of large-range transportation of the tissue engineered skin products, can effectively maintain the structure and functions of the tissue engineered skin in the transportation process, becomes solid after being cooled, and consequently has convenient carry in the transportation process.

The invention relates to a loop-mediated isothermal amplification method for rapid detection of cholera toxin vibrio cholera. A reagent comprises a loop-mediated isothermal amplification reaction liquid, Bst DNA polymerase, and a chromogenic reagent, wherein the reaction liquid contains a reaction buffer liquid, dNTP, magnesium sulfate, an upstream inner primer 5-TGAATCCACGGCTCTTCCCT-TGGTTATGGATTGGCAGG-3, a downstream inner primer 5-GGTTGTGGGAATGCTCCAAG-ACTTTGGGTTTTTTCATCGC-3, an upstream outer primer 5- GATATTGCTCCAGCAGCA-3, a downstream outer primer 5-CGTCAAGGAATTTTACACCTAG-3, and betaine. The method for detecting the cholera toxin vibrio cholera comprises the steps of the extraction of bacterial DNA, the loop-mediated isothermal amplification of the cholera toxin vibrio cholera, and chromogenic detection. The method has the advantages of rapidness, strong specificity, high sensitivity, and low cost.

The invention relates to a drug-delivery carrier protein based on a cholera toxin CT structure and an in-vitro construction method of the drug-delivery carrier protein. The drug-delivery carrier protein is a chimeric protein CTB5/EGFP-CTA2-TAT. The construction method comprises the following steps: constructing carriers petduet-CTB and pet22b-EGFP-CTA2-TAT; introducing host bacteria, carrying purification or renaturation, and assembling an AB5-structured chimeric protein in vitro. The drug-delivery carrier protein prepared by virtue of the construction method is high in purity, is relatively stable and is strong in repeatability; furthermore, no burden is produced during cell expression, and the protein raw material can be rapidly obtained; the drug-delivery carrier protein has high combining activity with GM1 as well as relatively efficient transmembrane capacity.

The related test paper to fast detect comma bacillus 01 group, 0139 group and cholera toxin comprises: base on PVC plate carrier, from bottom to top, connecting a sample pad, a colloidal gold conjunct to overlay and label single antibody for former comma bacillus and toxin, the cellulose nitrate covered by other three monoclonal antibody on T1, T2 and T3 areas and anti-rat IG on C area, and an absorption pad. This invention has accurate result, strong specificity, and can play important role in early diagnosis for cholera.