KR20070078560A - Chimeric protein consisting of ctb and hpv16 e6 protein and the use thereof - Google Patents

Abstract

A chimeric protein consisting of nontoxic CTB(cholera toxin B subunit) of Vibrio cholerae and HPV16 E6(human papilloma virus type 16 E6) protein and use thereof are provided to produce vaccine effectively absorbed to a mucosa for preventing and treating cervical cancer by using cervical cancer treating effects of HPV16 E6 protein and mucosa-penetrating ability of CTB. The chimeric protein consists of nontoxic CTB(cholera toxin B subunit) of Vibrio cholerae wherein the signal peptide region is deleted to remove toxicity, and HPV16 E6 protein, has the amino acid sequence of SEQ ID NO:6, is encoded by a gene having the nucleotide sequence of SEQ ID NO:5, and is produced by preparing a vector E6-CTB/pET29a containing the chimeric protein gene, transforming Escherichia coli with the vector to produce Escherichia coli BL21:E6-CTB/pET29a(KCTC 10892BP), culturing Escherichia coli BL21:E6-CTB/pET29a(KCTC 10892BP), separating the chimeric protein from the cultured transformed microorganism and removing endotoxin from the separated chimeric protein.

Description

Non-toxic cholera toxin Ｂ protein fused with subunit and protein of human papillomavirus type E6 {Chimeric protein consisting of CTB and HPV16 E6 protein and the use}

1 is a schematic diagram of E6-CTB which is a recombinant gene of HPV16 E6 and CTB of the present invention,

2 is a schematic diagram showing the position of subcloning E6-CTB, a recombinant gene of HPV16 E6 and CTB, to pET29a, an E. coli expression vector,

3 is a photograph showing the results of electrophoresis by applying 1% agarose gel after digestion with the restriction enzymes ECoRI and XhoI to confirm whether the E6-CTB recombinant gene was subcloned into the pET29a vector, which is an E. coli expression vector.

Figure 4 is a schematic diagram showing the process of subcloning the E6-CTB recombinant gene into the E. coli expression vector pET29a to create the plasmid E6-CTB / pET29a,

Figure 5 shows the results of electrophoresis by applying the cell lysate of E. coli BL21: E6-CTB / pET29a transformed with plasmid E6-CTB / pET29a of the present invention to a 12% SDS poleacrylamide gel (SDS poleacrylamide gel) It is a photograph.

The present invention relates to a non-toxic cholera toxin B subunit (CTB) of Vibrio cholerae and a chimeric protein of human papillomavirus (HPV) type 16 E6 and its use.

Papilloma virus is a double-helix DNA virus of about 45-55 nm in length, 8 kb, found in rabbit papilloma. At first, papillomavirus was only known to be involved in the development of malignant tumors as well as benign tumors. Since then, it has been found to be universally present in most animals, and has been treated as one of the animal-based tumor viruses. Until the early 1970s, papillomavirus was well known as a virus that infects humans and causes skin diseases. By the 1970s, however, papillomavirus became known in cancer tissues. In particular, the discovery of papillomavirus of a new species has been found to be closely related to the development of cervical cancer, many experiments have been carried out regarding cervical cancer. As a result, the relationship between papillomavirus and various types of diseases gradually became known and closely related to various types of tumors including cervical cancer, prostate cancer, oral cancer, laryngeal cancer, lung cancer, esophageal cancer, genital benign tumors and skin cancer. I found out. There are currently more than 100 different genotype papillomaviruses known and classified into three categories: high risk, medium risk and low risk. The high-risk group includes type 16 and 18, which account for about 70% of the uterine cancers, and the medium-risk group includes 31, 32, 35, 45, and 52, and the low-risk group of 6 and 11 papillomaviruses. have.

Cervical cancer is the second largest cause of cancer death among women worldwide. According to the World Health Organization's estimate, about 510,000 women are diagnosed with cervical cancer each year, or about 56% of them, which is 280,000. A thousand people die. Cervical cancer has the highest incidence in the United States or developed countries, whereas Korea and other developed countries have a high mortality rate, with breast cancer rising to one and two annually. The incidence of cervical cancer has been shown to be associated with low age and age of onset of sexual intercourse, or having an unspecified number of sexual intercourse partners. The incidence increased steadily from 15 to 25 years of age, but declined after that period (Scheurer ME et al., Int . J. Gynecol . Cancer , 15 (5), pp727-746, 2005) . In addition, sexually transmitted factors such as syphilis and chlamydia have been pointed out as a major cause of cervical cancer. The cervix, which is the site of cervical cancer, is more likely to develop because it is prone to chronic inflammation. Skin warts, a common infectious disease of papillomavirus, do not show an inflammatory response, and the host's strong immune response to the virus often causes the lesion to spontaneously degenerate. In this case, the presence of neutralizing antibodies in the host Is found. In women including cervical cancer patients with neutralizing antibodies, 18.1% of the antibodies against papillomavirus type 16 E6 protein and 3.9% of the E7 protein were detected. Among the three women, 40.7% were able to confirm the presence of antibodies of type 16 E6 protein. Papillomaviruses, which cause cervical cancer, are mostly high-risk type 16 or 18. Papillomaviruses of type 16 and 18 have been shown to be induced by the action of early-expressing genes such as E6 and E7 and later-like genes such as L1 and L2 (Sanclemente G and Gill DK, J. Eur . Acad . Dermatol . Venereol ., 16 (3), pp231-240. 2002). Therefore, the research and development of vaccines for papilloma virus mainly focused on the use of E6 and E7 proteins of type 16 and 18. By inducing an immune response against the E6 or E7 protein antigen, the antibody is produced and eliminated, while an immune response occurs to destroy and remove cells infected with the virus, thereby resulting in an effect that is cured from the virus infection.

The development of cervical cancer vaccines has been studied in two ways: preventive and therapeutic vaccines. Prophylactic vaccines focus on the production of neutralizing antibodies by the L1 and L2 proteins of HPV to prevent the host from infecting HPV and, if infected, to further progress the disease. Therapeutic vaccines, on the other hand, use the E6 and E7 genes of HPV to induce immunity, thus killing tumors or cancers. The vaccine mainly developed to date is a type of live recombinant vaccine or peptide produced by the HPV gene using bacteria, yeast and animal cells.

The main antigens that cause disease in our body are various microorganisms, which mainly penetrate through the mucous membrane of our body's respiratory or digestive system through air or food media. The mucous membrane of our body is an important part of the infection and at the same time induce an initial immune response. These mucosal tissues can be divided into two major parts. Nasal Associated Lymphoid Tissue (NALT), consisting mainly of nasal and nasal tissues that react to gaseous antigens inhaled by the nose and mouth, and the small intestine through which food or liquid antigens infiltrate. Gut Associated Lymphoid Tissue (GALT), which consists of the surface of the large intestine. NALT and GALT, the main mucosal tissues, are also called multiple Associsted Lymphoid Tissue (MALT). The GALT is composed of Payer's patches (PP), mesenteric lymph nodes (MLN), and numerous lymphoid cells distributed throughout the mucosa propria and small intestine epithelium. Fayer patches are sites of antigenic stimulation, with dome-shaped areas covered with follicular synthesizing epithelial cells (M cells). Intrapithelial pockets of M cells provide endocytosis and transport pathways for macromolecules, microorganisms, viruses, and the like. The transported molecule is processed by the crude and lymphoid cells bearing antigens to stimulate a local immune response in the host, which depends on the transport of the antigen through M cells as an immune response in the mucosa (Wolf JL and Bye WA, Ann. Rev. Med. , 35, pp 95-112, 1984).

Infection of infectious diseases begins with the invasion of pathogenic agents through these mucosal surfaces, and the defense of the immune system also involves mechanisms for countering pathogenic agents on mucosal surfaces, so vaccines injected into blood vessels or muscles are associated with mucosal immunity and The disadvantage is that it does not effectively induce a series of related reactions. In order to alleviate these shortcomings, studies on vaccine delivery through mucosal tissue distribution areas such as the digestive system, urogenital organs, or respiratory organs have been actively conducted. As a result, a study on the oral administration method to be absorbed through the intestinal mucosa is performed. In addition to the advantages that the oral administration vaccine is simple in addition to the advantages of simple absorption method, if the delivery efficiency is guaranteed (delivery) is very easy to deliver a large amount of immunity is also useful. However, the results of the study were not as good as expected because the vaccine did not properly absorb the antigen onto the mucosal surface.

Heat-labile intestines, bacterial internal toxins such as E. coli and E. coli and Vibrio Cholera's intestinal invasion genes that inhabit human gut and cause diarrhea while studying to solve vaccine problem that is effectively absorbed by mucous membrane. The use of toxins (Heat labile enterotoxin (LT)) and cholera toxin (Cholera toxin, CT) has been effective to increase the absorption into the mucosa. LT and CT are limited in their use due to their inherent toxicity, so there is no toxicity in their genes and the mucosal penetration ability is the same E. coli heat-labile enterotoxin B subunit ( Esherichia coli The heat-labile enterotoxin B subunit (LTB) and the non-toxic cholera toxin B subunit (CTB) of Vibrio cholera were used. LTB and CTB are similar in size and structure, with similar biochemical characteristics, and various studies have used them as an adjuvant to assist in the absorption of vaccines. LTB and CTB were found to play a role in the transport of mucosal secretory immunoglobulin A (S-IgA) as well as a variety of systemic antibody responses not associated with antigens.

Patents for human papillomavirus type 16 E6 protein that can be usefully used for the treatment of uterine cancer include protein mass production method (Korea Patent Publication No. 10-196416), patent for uterine cancer therapeutic composition (Korea Patent Publication No. 10-207956 ), An anticancer agent (Korean Patent Publication No. 10-380288), and the like, but examples of using the fusion protein of the present invention as an active ingredient of a vaccine for the treatment of cervical cancer have not been taught or described in any of the above documents.

 Accordingly, the present inventors have made a microbial system capable of expressing a fusion protein of a non-toxic cholera toxin B subunit and human papillomavirus type 16 E6, thereby purifying the fusion protein of the present invention obtained by purifying and removing endotoxin as an antigen. The present invention was completed by inducing specific antibodies to human papillomavirus type 16 E6 and at the same time effectively inducing mucosal immunity.

It is an object of the present invention to provide a fusion protein of a nontoxic cholera toxin B subunit and human papillomavirus type 16 E6 and an oral vaccine for the prevention and treatment of cervical cancer containing the same.

In order to achieve the above object, the present invention is a non-toxic cholera toxin B subunit (CTB) gene of Vibrio cholerae and human papillomavirus type 16 E6 (HPV16 E6) recombinant A fusion protein expressed from the gene E6-CTB.

The present invention also provides a recombinant gene encoding the fusion protein.

The present invention also provides a vector comprising the recombinant gene.

The present invention also provides a transformed microorganism characterized in that the vector is introduced.

In addition, the present invention comprises a first step of introducing a recombinant gene encoding a fusion protein into the transforming microorganism; A second step of culturing the transformed microorganism to express the fusion protein in bulk; It provides a method for producing a fusion protein comprising the third step of separating, purifying and removing endotoxin of the fusion protein from the cultured transformed microorganism.

The present invention also provides a vaccine for oral administration containing the fusion protein produced by the above method as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention expresses the gene of the non-toxic cholera toxin B subunit (CTB) of Vibrio cholerae and the gene E6-CTB recombined with the gene of human papillomavirus type 16 E6 (HPV16 E6). To provide a fusion protein.

In the present invention, the non-toxic cholera toxin B subunit (CTB; cholera toxin B subunit) is a non-toxic cholera toxin B subunit having an amino acid sequence of SEQ ID NO: 2, wherein the human papillomavirus type 16 E6 (HPV16 E6) Wherein the non-toxic cholera toxin B subunit comprises the amino acid sequence of SEQ ID NO: 4, wherein the non-toxic cholera toxin B subunit comprises a sequence described as amino acids 23-124 which is the site where the signal peptide is deleted; Do. However, the fusion protein of the present invention is not limited to this sequence, and all possible fusion proteins that fuse a non-toxic cholera toxin B subunit (CTB) with human papillomavirus type 16 E6 (HPV16 E6) It includes.

The fusion protein of the present invention can be obtained as follows.

In order to obtain the fusion protein of the present invention, the gene of the nontoxic cholera toxin B subunit (CTB) of SEQ ID NO: 1, preferably the CTB gene having deleted the signal peptide site and the human papillomavirus 16 of SEQ ID NO: 3 A gene of type E6 (HPV16 E6) can be obtained to construct a recombinant gene E6-CTB of SEQ ID NO: 5 through a ligation reaction. The plasmid of the present invention can be prepared by inserting a recombinant gene E6-CTB prepared by the above method into a vector suitable for expression in Escherichia coli, preferably pET29a (Novagen, USA). The plasmid prepared as described above was transformed by inserting into a suitable host microorganism, preferably E. coli BL21 (DE3) (Accession No. KCTC 10892BP), and the non-toxic cholera toxin B subunit of the present invention of SEQ ID NO: 6 (CTB). LB medium (tryptone per liter) containing a medium suitable for the expression of the fusion protein E6CTB of cholera toxin B subunit) and human papillomavirus type E6 (HPV16 E6), for example kanamycin (70 μg / ml); Isopropyl-beta-di-thiogalactopyranoside (IPTG) was added to the culture to induce the expression of the fusion protein while culturing the transformed strain in 10 g, 5 g of yeast extract and 10 g of sodium chloride, The fusion protein E6CTB of the present invention can be obtained by purification using a nickel bead column to which histidine residues at the C-terminus of the fusion protein E6CTB of the present invention are attached and removing endotoxins.

The present invention provides cDNA and human papillomavirus in which the signal peptide region of a non-toxic cholera toxin B subunit (CTB) of a recombinant gene encoding a fusion protein that can be produced by the above method, preferably Vibrio cholera, is removed. Recombinant gene linking the DNA of type 16 E6 (HPV16 E6), SEQ ID NO: 5 includes a recombinant gene having a nucleotide sequence. However, the genes of the present invention are not limited to the above sequences, and fusion proteins of all non-toxic cholera toxin B subunits (CTB) and human papillomavirus type 16 E6 (HPV16 E6) of all Vibrio cholera Contains the gene that encodes.

The present invention provides a vector comprising a recombinant gene encoding the fusion protein, preferably a vector having a plasmid E6-CTB / pET29a having a cleavage map shown in FIG.

The present invention also provides a transformed microorganism, preferably transformed E. coli, characterized in that the vector is introduced.

The present inventors named the transformed Escherichia coli into which the vector was introduced, and named it "BL21: E6-CTB / pET29a", and deposited it on the Korea Biotechnology Research Institute Gene Bank on January 16, 2006 (Accession No .: KCTC 10892BP). .

In addition, the present invention comprises a first step of introducing a recombinant gene encoding a fusion protein into the transforming microorganism; A second step of culturing the transformed Escherichia coli and expressing the fusion protein in a large amount; It provides a method for producing a fusion protein comprising the third step of separating, purifying and removing endotoxin of the fusion protein from the cultured transformed microorganism.

The present invention provides an oral vaccine for the prevention or treatment of cervical cancer containing the fusion protein E6CTB of the present invention obtained as the active ingredient as an active ingredient.

The vaccine containing the fusion protein of the present invention as an active ingredient may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the vaccines may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

The vaccine comprising the fusion protein according to the present invention is in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be formulated and used. Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the vaccine comprising the fusion protein of the present invention may vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but the amount of 0.1 to 1000 mg / kg 1 week It may be administered several times to several times. The dosage may also be increased or decreased depending on the route of administration, degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

Below, The invention is illustrated in detail by the following examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Reference Example 1. Cultivation of Cells Used

DMEM (Dulbecco's modified Eagle's) with 10% fetal bovine serum (Sigma, USA) added to cultivate CaSki cell line (ATCC CRL-1550) purchased from the American Type Culture Collection (ATCC) medium, Sigma, USA) medium was used, and cultured at 37 ℃ maintaining a carbon dioxide (CO ₂ ) concentration of 6%.

Example  One. CTB  And HPV16 E6 Recombinant Gene Production

1-1. CTB  Gene acquisition

Genomic DNA of Vibrio cholera was purchased from the American Standard Strains (ATCC, American Type Culture Collection, USA) to obtain genes expressing the non-toxic cholera toxin B subunit (CTB) protein of the present invention. Using the cholera genomic DNA purchased as a template, the CTB gene of the present invention was amplified by performing a polymerase chain reaction (PCR) under the following conditions. Forward primer with SEQ ID NO: 7 (5'- CC GGA TCC ACA CCT CAA AAT ATT ACT GAT-3 ') designed to amplify the CTB gene and SEQ ID NO: 8 (5'- G CTC GAG ATT TGC) Reverse primers of CAT ACT AAT TGC GGC -3 ') were added 5 pmole, 10 μM dNTP, 1 μl of Taq polymerase (2 unit / μl), 1 × reaction buffer, and 50 μl of final volume with distilled water. After 45 cycles, the polymerase chain reaction was performed. The reaction time was denaturation (95 ℃, 30 seconds), annealing (45 ℃, 1 minute), elongation (72 ℃, 1 minute). As a result, the amplified CTB gene was applied to 1% agarose gel, followed by electrophoresis to confirm the size (306 bp), and the desired CTB gene was amplified, and used as a material for the following experiment (hereinafter, referred to as CTB). ).

1-2. HPV16 E6  Gene acquisition

In order to obtain the HPV16 E6 gene of the present invention, CaSki cells, which are human cervical cancer cells, were cultured as in the reference example, and the cells were harvested to separate total RNA using Trizol (TRIzol, Invitrogen, USA).

After synthesizing cDNA as a template of the total RNA of the CaSki cells, the synthesized cDNA was used as a template, and a forward primer of SEQ ID NO: 9 (5′-GC GAA TTC ATG CAC CAA AAG AGA ACT-3 ′) And Reverse Transcription-Polymerase chain using a reverse primer of SEQ ID NO: 10 (5'-GC GGA TCC CAG CTG GGT TTC TCT ACG TGT-3 ') to amplify the HPV16 E6 gene reaction, RT-PCR). After completion of PCR, the amplified HPV16 E6 gene was applied to 1% agarose gel, followed by electrophoresis to confirm the size (474 bp), and confirmed that the desired HPV16 E6 gene was amplified, and used as a material for the following experiment (hereinafter, Named HPV16E6).

1-3. E6 - CTB  Recombinant Gene Production

In order to locate HPV16E6 at the front and connect CTB at the back, 10 μl of each of HPV16E6 and CTB obtained above was digested with restriction enzyme BamHI (Roche, USA) at 37 ° C. for 1 hour, and then cut. Two genes were recombined via ligation (ligation, 37 ° C., 1 hour) using 2 μl, 2 μl of DNA ligase (T4 DNA ligase, Roche, USA), 2 μl of 10 × buffer, and 13.5 μl of sterile water. After electrophoresis by applying to agarose gel, the size (780 bp) was confirmed to confirm the preparation of HPV16E6-CTB recombinant gene (hereinafter referred to as E6-CTB), and used as a material for the following experiment (see FIG. 1). ).

Example  2. Recombinant Expression Vector E6 - CTB Of pET29a Preparation and Transformation of Strains

E. coli expression vectors pET29a (Novagen, Madison, WI, USA) and E6-CTB of Experimental Examples 1-3 were treated with restriction enzymes EcoRI and XhoI, followed by ligation of the expression vector with recombinant genes. E. coli expression strain BL21 (DE3) (Stratagene, USA) was transformed (transformation), and transformants were cultured on LB plates to which kanamycin (70 μg / ml, Sigma, USA) was added to culture the colonies. Using a kit from cultured colonies (QIAprep miniprep kit, Qiagen, Germany), plasmid DNA was extracted, treated with restriction enzymes EcoRI (Roche, USA) and XhoI (Roche, USA), and electrophoresed to confirm the transformation. It was. An expression vector (5743 bp) into which an E6-CTB recombinant gene having a size of about 780 bp was inserted from the transformant was identified as E6-CTB / pET29a (see FIGS. 3 and 4).

The transformed strain was cultured overnight in LB medium to which kanamycin (70 μg / ml, Sigma, USA) was added, and the selected transformed strain BL21: E6-CTB / pET29a was deposited as a strain in the Gene Bank of the Biotechnology Research Institute. (Accession No. KCTC 10892BP).

Example  3. Fusion Protein E6CTB Expression, purification and Endotoxin removal

3-1. Fusion protein E6CTB Expression of

The deposited transformed strain BL21: E6-CTB / pET29a (Accession No. KCTC 10892BP) was transferred to 1 L of LB medium containing kanamycin (70 μg / ml) (10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride). Inoculum was shaken at 37 ° C and absorbed at 600 nm using a spectrophotometer (Ultrospec1100pro, Amersham Bioscience, USA) at 0.4 nm to 0.6. Isopropyl-beta-di-thiogalactopyranoside (IPTG; isopropyl-) β-D-thiogalactopyranoside, Sigma, USA) was added to a final concentration of 0.5 mM and shaken at 25 ° C. to induce the expression of the fusion protein E6CTB. After 4 hours of incubation, the cultured cells in which the expression of proteins were induced were centrifuged at 3500 rpm and 4 ° C. for 10 minutes using a centrifuge (UNION 55R, Hanil, Korea) to discard the culture medium and obtain cells. After the obtained cells were resuspended by adding Tris buffer solution (Tris-HCl, pH8.0) as a dispersion solution, the cells were washed three times or more by centrifugation under the same conditions as above, and then discarded. After dissolving with 1 × phosphate buffered saline (PBS, Phosphate buffered saline) containing a inhibitor (protease inhibitor, Sigma, USA), the tube was kept cold and crushed with a sonicator (GE, USA). After crushing, the supernatant obtained by centrifugation (5000 rpm, 15 minutes) was stored at 4 ℃, and the precipitate was resuspended by adding and resuspending 8 M urea (urea) and centrifuged at 12000 rpm, 4 ℃ for 15 minutes and then the supernatant was Obtained and stored at 4 ° C. In order to confirm the expression of the fusion protein E6CTB of the present invention, four kinds (transgenic cells not expressed, transformants expressed for 4 hours, supernatants and precipitates obtained by ultrasonic disruption of the expressed transformants were obtained by 8M urea). A sample of the supernatant obtained by resuspension and sonication was prepared. The samples were subjected to electrophoresis on 12% SDS-polyacrylamide gel to confirm their expression. As shown in FIG. 5, the expression of the fusion protein E6CTB of the present invention was confirmed in a supernatant sample obtained by dissolving the pellets in 8M urea and sonicating the pellets.

3-2. Fusion protein E6CTB Expression, purification and Endotoxin removal

To purify the fusion protein E6CTB, the transforming strain BL21: E6-CTB / pET29a was incubated in 100 ml of LB medium containing kanamycin, and the cells obtained by centrifugation (0.6 g) were tris-HCl (pH8.0). After washing, 1X PBS was added and the suspended cells were disrupted using an ultrasonic grinder.

Since the expressed fusion protein E6CTB exists in the zone of the centrifuged precipitate, the lysate was centrifuged, the supernatant was discarded, and the remaining precipitate was dissolved in 8M urea (UREA) to collect a solution containing the fusion protein E6CTB. Then, for purification of the E6CTB protein, 1 ml of a nickel bead column (His-trap chelating HP column, Amersham bioscience, Uppsala, Sweden) using a histidine residue expressed at the C-terminus of the fusion protein E6CTB of the present invention. After adsorption, the adsorbed fusion protein E6CTB was purified using an increased concentration of imidazole to 100 mM. The fusion protein E6CTB obtained by the above process was measured for the concentration of endotoxin using a LAL assay (Limulus amoebocyte lysate assay) method using LAL assay kit (QCL-1000, Camblex bioscience, USA), The purified product was applied to an endotoxin removal column (AffinityPak Detoxi-Gel, Pierce, Rockford, USA) to remove the endotoxin to complete purification of the fusion protein.

The fusion protein of the nontoxic cholera toxin B subunit (CTB) and human papillomavirus type 16 E6 (HPV16 E6) of the present invention induces an antibody specific for human papillomavirus type 16 E6. Simultaneously and effectively induce mucosal immune response, it can be used as an active ingredient in the manufacture of oral vaccines for the prevention or treatment of cervical cancer.

<110> Good Cell Life Co., Ltd. <120> Chimeric protein consisting of CTB and HPV16 E6 protein and the          use <130> DIF / 2005-12-0011 / SY <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 375 <212> DNA <213> Vibrio cholerae <220> <221> sig_peptide (222) (4) .. (66) <400> 1 atgattaaat taaaatttgg tgtttttttt acagttttac tatcttcagc atatgcacat 60 ggaacacctc aaaatattac tgatttgtgt gcagaatacc acaacacaca aatatatacg 120 ctaaatgata agatattttc gtatacagaa tctctagctg gaaaaagaga gatggctatc 180 attactttta agaatggtgc aatttttcaa gtagaagtac caggtagtca acatatagat 240 tcacaaaaaa aagcgattga aaggatgaag gataccctga ggattgcata tcttactgaa 300 gctaaagtcg aaaagttatg tgtatggaat aataaaacgc ctcatgcgat tgccgcaatt 360 agtatggcaa attaa 375 <210> 2 <211> 124 <212> PRT <213> Vibrio cholerae <220> <221> SIGNAL (222) (2) .. (22) <400> 2 Met Ile Lys Leu Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser Ser   1 5 10 15 Ala Tyr Ala His Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu              20 25 30 Tyr His Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr          35 40 45 Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys      50 55 60 Asn Gly Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp  65 70 75 80 Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala                  85 90 95 Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys             100 105 110 Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn         115 120 <210> 3 <211> 477 <212> DNA <213> Human papillomavirus type 16 <400> 3 atgcaccaaa agagaactgc aatgtttcag gacccacagg agcgacccag aaagttacca 60 cagttatgca cagagctgca aacaactata catgatataa tattagaatg tgtgtactgc 120 aagcaacagt tactgcgacg tgaggtatat gactttgctt ttcgggattt atgcatagta 180 tatagagatg ggaatccata tgctgtatgt gataaatgtt taaagtttta ttctaaaatt 240 agtgagtata gacattattg ttatagtttg tatggaacaa cattagaaca gcaatacaac 300 aaaccgttgt gtgatttgtt aattaggtgt attaactgtc aaaagccact gtgtcctgaa 360 gaaaagcaaa gacatctgga caaaaagcaa agattccata atataagggg tcggtggacc 420 ggtcgatgta tgtcttgttg cagatcatca agaacacgta gagaaaccca gctgtaa 477 <210> 4 <211> 158 <212> PRT <213> Human papillomavirus type 16 <400> 4 Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro   1 5 10 15 Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp              20 25 30 Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu          35 40 45 Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly      50 55 60 Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile  65 70 75 80 Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu                  85 90 95 Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn             100 105 110 Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys         115 120 125 Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met     130 135 140 Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu 145 150 155 <210> 5 <211> 801 <212> DNA <213> Artificial Sequence <220> <223> E6-CTB recombinant DNA <220> <221> misc_recomb (222) (1) .. (6) <223> EcoRI cleavage site <220> <221> misc_recomb (222) (481) .. (486) <223> BamHI cleavage site <220> <221> misc_recomb (222) (796) .. (801) <223> XhoI cleavage site <400> 5 gaattcatgc accaaaagag aactgcaatg tttcaggacc cacaggagcg acccagaaag 60 ttaccacagt tatgcacaga gctgcaaaca actatacatg atataatatt agaatgtgtg 120 tactgcaagc aacagttact gcgacgtgag gtatatgact ttgcttttcg ggatttatgc 180 atagtatata gagatgggaa tccatatgct gtatgtgata aatgtttaaa gttttattct 240 aaaattagtg agtatagaca ttattgttat agtttgtatg gaacaacatt agaacagcaa 300 tacaacaaac cgttgtgtga tttgttaatt aggtgtatta actgtcaaaa gccactgtgt 360 cctgaagaaa agcaaagaca tctggacaaa aagcaaagat tccataatat aaggggtcgg 420 tggaccggtc gatgtatgtc ttgttgcaga tcatcaagaa cacgtagaga aacccagctg 480 ggatccacac ctcaaaatat tactgatttg tgtgcagaat accacaacac acaaatatat 540 acgctaaatg ataagatatt ttcgtataca gaatctctag ctggaaaaag agagatggct 600 atcattactt ttaagaatgg tgcaattttt caagtagaag taccaggtag tcaacatata 660 gattcacaaa aaaaagcgat tgaaaggatg aaggataccc tgaggattgc atatcttact 720 gaagctaaag tcgaaaagtt atgtgtatgg aataataaaa cgcctcatgc gattgccgca 780 attagtatgg caaatctcga g 801 <210> 6 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> E6CTB chimeric protien <400> 6 Glu Phe Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu   1 5 10 15 Arg Pro Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile              20 25 30 His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg          35 40 45 Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg      50 55 60 Asp Gly Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser  65 70 75 80 Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr                  85 90 95 Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys             100 105 110 Ile Asn Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu         115 120 125 Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg     130 135 140 Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu 145 150 155 160 Gly Ser Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn                 165 170 175 Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser             180 185 190 Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala         195 200 205 Ile Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys     210 215 220 Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr 225 230 235 240 Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His                 245 250 255 Ala Ile Ala Ala Ile Ser Met Ala Asn Leu Glu             260 265 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> CTB forward primer <400> 7 ccggatccac acctcaaaat attactgat 29 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> CTB reverse primer <400> 8 gctcgagatt tgccatacta attgcggc 28 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> HPV16 E6 forward primer <400> 9 gcgaattcat gcaccaaaag agaact 26 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HPV16 E6 reverse primer <400> 10 gcggatccca gctgggtttc tctacgtgt 29

Claims (12)

A fusion protein expressed from the gene of the non-toxic cholera toxin B subunit (CTB) of Vibrio cholerae and the gene of human papillomavirus type 16 E6 (HPV16 E6) from the recombined gene E6-CTB . 2. The fusion protein of claim 1, wherein said non-toxic cholera toxin B subunit has a deletion of a signal peptide site. The fusion protein of claim 1, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 6. 6. Recombinant gene encoding the fusion protein of claim 1. The recombinant gene of claim 4, wherein the recombinant gene has a nucleotide sequence of SEQ ID NO: 5. 6. A vector comprising the recombinant gene of claim 4. 7. The vector according to claim 6, wherein the vector is plasmid E6-CTB / pET29a having a cleavage map shown in FIG. A transformed microorganism characterized in that the vector of claim 6 is introduced. The transformed microorganism according to claim 8, wherein the transformed microorganism is E. coli BL21: E6-CTB / pET29a into which the vector of claim 6 is introduced (Accession No. KCTC 10892BP). Introducing a recombinant gene encoding a fusion protein into the transforming microorganism; A second step of culturing the transformed microorganism to express the fusion protein in bulk; Method for producing a fusion protein comprising the third step of separating, purifying and removing endotoxin from the cultured transformed microorganism. The method of claim 10, wherein the transformed microorganism is the transformed microorganism of claim 8. Oral administration vaccine containing the fusion protein produced by the method of claim 10 as an active ingredient.

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