Broad spectrum vaccine, preparing method and application thereof

Abstract

The use of an HLA-G molecule and its antigenic fragments prepared a broad spectrum vaccine which could be used to prevent the invasion of various tumors and various viruses or unknown virus on human and animal body.

Claims

1. A broad spectrum vaccine for preventing various kinds of tumors and various kinds of viral diseases, wherein the broad spectrum vaccine is prepared with an antigenic fragment; the antigenic fragment consists of at least one of amino acid sequences in SEQ ID NO: 1-4; the tumors and the viral diseases comprise the tumors and virus that express HLA-G; wherein, SEQ ID NO:1 is YW EEE TRN TKA HA; SEQ ID NO:2 is RGY YNQ SEA SSH TL; SEQ ID NO:3 is PPK THV THH PVFD; SEQ ID NO:4 is PLM LRW KQS SL; the vaccine works by producing HLA-G antibodies which bind to HLA-G; where in the broad spectrum vaccine is prepared by combining the antigen fragment with a carrier protein into a complex and then purifying the complex.

2. A method of preparing the broad spectrum vaccine according to the claim 1 to prevent various kinds of tumors and various kinds of viral diseases, wherein the broad spectrum vaccine is prepared with an antigenic fragment; the antigenic fragment consists of at least one of amino acid sequences in SEQ ID NO: 1-4; wherein, SEQ ID NO:1 is YW EEE TRN TKA HA; SEQ ID NO:2 is RGY YNQ SEA SSH TL; SEQ ID NO:3 is PPK THV THH PVFD; SEQ ID NO:4 is PLM LRW KQS SL; wherein the broad spectrum vaccine is prepared by combining the antigen fragment with a carrier protein into a complex and then purifying the complex.

3. The method of preparing of the broad spectrum vaccine according to the claim 2, wherein the tumors and the viral diseases express HLA-G, the tumors comprise: (1) squamous call carcinoma of the epithelial tissue, basal cell carcinoma, adenocarcinoma, papillary adnocarcinoma, cystic adnocarcinoma, malignant pleomorphic adnoma, and transitional epithelial carcinoma which root in the epithelial tissue; (2) fibrosarcoma, malignant fibrous tissue cell carcinoma, liposarcoma, leionyosacoma and rhabdomyosarcoma, angiosarcoma, lymphaticsarcoma, osteosarcoma, chondrosarcoma, synovial sarcoma, and malignant mesothelioma; (3) lymphoma and leukemia from lymphopoietic tissue;(4) neurofibrosarcoma, malignant schwannoma, malignant glioblastoma, medulloblastoma, malignant meningioma, neuroblastoma; (5) esophageal squamous cell tumor of the digestive tract, esophageal adenocarcinoma, gastric cancer adenoma, gastric squamous cell carcinoma, hepatic cell tumor, rectal cancer, colon cancer and pancreatic cancer; (6) non-small cell lung cancer from respiratory tract; (7) and melanoma, chorionic carcinoma, ovarian cancer cell tumor, laryngeal cancer, breast cancer, seminoma, prostate cancer, cervical cancer, dysgerminoma, embryonal carcinoma and malignant teratoma, the viruses of the viral diseases comprise: (1) pox virus division in double-stranded DNA virus, iridescent virus, virus, herpes virus, adenovirus, HPV, polyoma virus families, DNA virus, African swine fever virus; (2) single stranded DNA viruses in the combined virus family, parvovirus; (3), retroviruses, pseudoviridae; (4) double-stranded RNA viruses in the, double-stranded RNA virus family, double-stranded RNA globular fungal virus; (5) the paramyxoviruses in the negative single-stranded RNA viruses, the filovirus family, the bornavirus family, the orthonaviridae, the bunya virus family; (6) arteritis virus, coronavirus, and small RNA viruses in the single-stranded RNA virus.

4. The method of preparing the broad spectrum vaccine according to the claim 2, wherein the complex is obtained by using carbodiimide, glutaraldehyde, and diisocyanic acid compound or obtained by building fusion gene expression by organisms.

5. The method of preparing the broad spectrum vaccine according to the claim 4, wherein the carrier protein is a hemocyanin, and coupling reagent is aglutaraldehyde.

6. The method of preparing the broad spectrum vaccine according to the claim 2, wherein the tumors comprise: laryngeal cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, colon cancer, prostate cancer, lung cancer, breast cancer, ovarian cancer and cervical cancer; the viruses of the viral diseases comprise: HBsAg, HCAg, HIV, SARS, ebola, zika, dengue virus, and avian influenza virus.

Description

DESCRIPTION OF THE EMBODIMENTS

(1) In addition to the definition, the meaning of technical terms used in the following implemental example was identical with the understanding of technical personnel in the field of the invention. The test reagents used in the following implemental example, such as no special instructions, are conventional biochemical reagents; the experimental methods, if no special instructions, are conventional methods.

(2) The following described the creativity of the invention with the implemental example. In the following implemental example, the immunogen preparing the vaccine coming from market or obtained by through commercial synthesis.

Implemental Example 1

(3) The HLA-G 1, HLA-G 5 and HLA-G 7 were used as immunogen to prepare broad spectrum vaccine. The HLA-G 1, HLA-G 5 and HLA-G 7 as three isomers of the

(4) HLA-G molecule had the same amino acid sequence (SEQ ID NO: 5), the process of preparation of vaccine was same with HLA-G 1. As an example of implemental example, the process of preparing vaccine was as follows:

(5) 33 mg HLA-G 1 was dissolved in 50 ml pH 7.4, 0.01M/L PBS solution. The concentration of HLA-G 1 was 660 mg/L. Placed the solution on an electromagnetic agitator. Placed a stirrer in the solution, and turned on the electromagnetic agitator to stir the solution of HLA-G 1. Then added 3 ml of 1 mMol/L KAL(SO.sub.4).sub.2-12H.sub.2O solution, stirred constantly and drip added 1 ml of 1 mMoL/L NaOH solution. Continue to stir for 10 minutes, stopped stirring, 0.1 Mol/L HCL was used for adjust the pH of the solution to 7.4, after aseptic filtration, the filtrated solution placed at 4° C. for later use.

Implemental Example 2

(6) A broad spectrum vaccine was prepared by HLA-G 2 and HLA-G 6 respectively. HLA-G 2 and HLA-G 6 were two isomers of HLA-G molecules with the same amino acid sequence (SEQ ID NO: 6). The process of preparing the vaccine was same, HLA-G 2 as an example, the process of preparing the vaccine was as follows:

(7) 24 mg HLA-G 2 was dissolved in 50 ml pH 7.4, 0.01 M/L of PBS solution. The concentration of HLA-G 2 was 480 mg/L. Placed the solution on an electromagnetic agitator, placed a stirrer in the solution, turned on the electromagnetic stirrer to stir the HLA-G 2 solution slowly. Then slowly added 2.1 ml of 1 mMol/L KAL(SO.sub.4).sub.2-12H.sub.2O solution, stirred constantly and drip added 0.72 ml of 1 mMol/L NaOH solution, continue to stir for 10 minutes, stopped stirring, 0.1 Mol/L of HCL was used to adjust the pH of the solution to 7.4, after aseptic filtration, the filtrated solution was placed at 4° C. for later use.

Implemental Example 3

(8) HLA-G 3 (SEQ ID NO: 7) was used as immunogen to prepare broad spectrum vaccine: 14 mg of HLA-G 3 was dissolved in 50 ml pH 7.4, 0.01 M/L PBS solution. The concentration of HLA-G 3 was 279 mg/L. Placed the solution on an electromagnetic agitator, and placed a stirrer in the solution, and turned on the electromagnetic agitator to gently stir the HLA-G 3 solution. Then slowly added 1.3 ml of 1 mMol/L KAL(SO.sub.4).sub.2-12H.sub.2O solution, stirred constantly and drip added 0.42 ml of 1 mMol/L NaOH solution. Continue to stir for 10 minutes, stopped stirring, 0.1 Mol/L of HCL solution was used to adjust the pH of the solution to 7.4, after aseptic filtration and filtrated solution placed at 4° C. for later use.

Implemental Example 4

(9) HLA-G 4 (SEQ ID NO: 8) was used as immunogen to prepare broad spectrum vaccine. 23 mg HLA-G 4 was dissolved in 100 ml pH 7.4, 0.01 M/L PBS solution. The concentration of HLA-G 4 was 459 mg/L. Place the solution on an electromagnetic agitator, place a stirrer in the solution, and turn on the electromagnetic agitator to gently stir the HLA-G 4 solution. Then slowly add 2.1 ml of 1 mMol/L KAL(SO.sub.4).sub.2-12H.sub.2O solution, stirred constantly and drip added 0.7 ml of 1 mMOL/L NaOH solution, continue to stir for 10 minutes, stopped stirring, 0.1 Mol/L of HCL is used to adjust the pH of the solution to 7.4, after aseptic filtration place at 4° C. for later use.

Implemental Example 5

(10) when use of two or more than two kinds of HLA-G isomer as joint immunogen, prepared broad spectrum vaccine, the operation use the basis of implemental example 1, in the 33 mg of HLA-G 1, 3 ml, of 1 mMol/L KAL(SO.sub.4).sub.2-12H.sub.2O solution and 1 ml of 1 mMol/L NaOH solution, the 33 mg, 3 ml and 1 ml were multiplied by n, n represents the number of kind of HLA-G isomers. For example, two kinds of isomers as the combined immunogen, n=2, three kinds of isomers as the combined immunogen, n=3. And so on.

Implemental Example 6

(11) The polypeptide (small peptides) of SEQ ID NO: 1 was used as immunogen to prepare broad spectrum vaccine. Dissolved 33 mg hemocyanin in 50 ml pH 7.4, 0.01 mol/L PBS solution, under electromagnetic stirring, again dissolves 3.3 mg small peptide (immunogen) in above PBS solution. Continue to stir 10 minutes, take 5% glutaraldehyde solution was diluted to 1000 times by above PBS solution. Then took the diluted glutaraldehyde solution 0.15 ml with drop wise to add the above solution under the condition of stirring, continue to stir for 30 minutes, put the solution in 4° C. refrigerator overnight.

(12) In the next day, the solution went through the sephadex-G25 chromatography column, collected 280 nanometer absorptive peaks and removed the unbounded glutaraldehyde and immunogen peptides. Then to use an affinity chromatography column of anti-HLA-G, removed hemocyanin which did not combine the small peptide of immunogen, and to use pH 3.5, 0.05 Mol/L glycine-HCL buffer desorbs hemocyanin combined with small peptide, and immediately to use 0.05 Mol/L NaOH solution neutralized the desorbed solution. To concentrates the desorbed solution appropriately. Then to use the method of implemental example 1 obtains a broad spectrum of small peptide vaccine.

Implemental Example 7

(13) The polypeptide (small peptides) of SEQ ID NO: 2 was used as immunogen to prepare broad spectrum vaccine. Dissolved 33 mg hemocyanin in 50 ml pH 7.4, 0.01 mol/L PBS solution, under electromagnetic stirring, again dissolved 3.3 mg small peptide (immunogen), continue to stir 10 minutes, took 5% glutaraldehyde solution was diluted to 1000 times by abortive PBS solution. Then took the diluted glutaraldehyde solution 0.15 ml drop wise to added the above solution under the condition of stirring, continue to stir for 30 minutes. The above solution was put in 4° C. refrigerator overnight.

(14) In the next day, the solution went through the sephadex-G25 chromatography column collected 280 nanometer absorptive peaks and removed the unbounded glutaraldehyde and small immunogen peptides. Then to use an affinity chromatography column of anti-HLA-G, removed the hemocyanin which did not combine the small peptide of immunogen. To use pH 3.5, 0.05 Mol/L of glycine-HCL buffer desorbed hemocyanin combined with small peptide of immunogen, and immediately to use 0.05 Mol/L NaOH solution neutralizes the desorbed solution. To concentrates the desorbed solution appropriately. Then to use of the method of implemental example 1 obtained a broad spectrum of small peptide vaccine.

Implemental Example 8

(15) The polypeptide (small peptide) of The SEQ ID NO: 3 was used as immunogen to prepare broad spectrum vaccine. Since SEQ ID NO: 3 with SEQ ID NO: 1 had same number of amino acids, and all of them had 13 amino acids, and the molecular weight of the two was similar, so the specific operation was similar to the implemental example 6.

Implemental Example 9

(16) The polypeptide (small peptide) of SEQ ID NO: 4 was used as immunogen to prepare broad spectrum vaccine. Dissolves the 33 mg hemocyanin in 50 ml pH 7.4, 0.01 Mol/L PBS solution, under electromagnetic stirring condition, again adds 3.3 mg immunogen small peptide, continue to stir 10 minutes, took 5% glutaraldehyde solution was diluted 1000 times by use of the PBS solution. Then took 0.15 ml of the diluted glutaraldehyde solution under the stirring condition drop wise added to the above solution, continue stirs for 30 minutes, put the solution in 4° C. refrigerator overnight.

(17) In the next day, the solution went through the sephadex-G25 chromatography column, collected 280 nanometer absorptive peak solution and removes the unbound glutaraldehyde and immunogen peptides. Then to use an affinity chromatography column of anti-HLA-G to remove the hemocyanin which did not combine the immunogen small peptide, to use pH 3.5, 0.05 Mol/L of glycine-HCL buffer desorbs the complex compound which was hemocyanin combined with the immunogen small peptide, and immediately use of 0.05 Mol/L NaOH solution to neutralize the desorbed solution. After appropriately concentrated the desorbed solution, and again treated by use of the method of implemental example 1, obtained a broad spectrum vaccine prepared by use of immunogen small peptide.

Implemental Example 10

(18) The SEQ ID NO: 9 peptides were used as immunogen to prepare broad spectrum vaccine. 3.3 mg immunogen polypeptide was dissolved in 50 ml pH 7.4, 0.01 M/L PBS solution. The concentration of immunogen polypeptide was 66 mg/L. Placed the solution on an electromagnetic agitator, to place a stirrer in the solution, turned on the electromagnetic stirrer, and gently stir the solution of the immunogen peptides. Then slowly added 0.3 ml, 1 mMol/L KAL(SO.sub.4).sub.2-12H.sub.2O solution, stirred constantly, and drip added 0.1 ml 1 mMol/L of NaOH solution, continue to stir for 10 minutes, stopped stirring, 0.1 Mol/L of HCL was used to adjust the pH of the solution to 7.4, after aseptic filtration, the filtrated solution is placed at 4° C. for storage.

Implemental Example 11

(19) The SEQ ID NO: 10 peptide was used as immunogen to prepare broad spectrum vaccine. 5.3 mg immunogen polypeptide was dissolved in 50 ml pH 7.4, 0.01M/L Of PBS solution. The concentration of immunogen polypeptide was 66 mg/L. Placed the solution on an electromagnetic agitator and to place a stirrer in the solution, turned on the electromagnetic stirrer, and gently to stir the solution of the immunogen peptide. Then slowly to add 0.48 ml, 1 mMol/L of KAL(SO.sub.4).sub.2-12H.sub.2O solution, stirred constantly, and drip added 0.16 ml, 1 mMol/L of NaOH solution, continue to stir for 10 minutes, stopped stirring, 0.1 Mol/L of HCL was used to adjust the pH of the solution to 7.4, after aseptic filtration to place at 4° C. store.

Implemental Example 12

(20) The SEQ ID NO: 11 peptides were used as immunogen to prepare broad spectrum vaccine. 5.9 mg immunogen polypeptide was dissolved in 50 ml pH 7.4, 0.01M/L of PBS solution. The concentration of immunogen polypeptide was 119 mg/L. Place the solution on an electromagnetic agitator, to place a stirrer in the solution, turn on the electromagnetic stirrer, and gently to stir the solution of the immunogen peptides. Then slowly to add 0.54 ml, 1 mMol/L of KAL(SO.sub.4).sub.2-12H.sub.2O solution, stirred constantly, and drip added 0.18 ml 1 mMol/L of NaOH solution, continue to stir for 10 minutes, stopped stirring, 0.1 Mol/L of HCL was used to adjust the pH of the solution to 7.4, after aseptic filtration, the filtered solution is placed at 4° C. for storage.

Implemental Example 13

(21) The SEQ ID NO: 12 peptides were used as immunogen to prepare broad spectrum vaccine. 11.6 mg immunogen polypeptide was dissolved in 50 ml pH 7.4, 0.01M/L of PBS solution. The concentration of immunogen polypeptide was 231 mg/L. Placed the solution on an electromagnetic agitator, to place a stirrer in the solution, turned on the electromagnetic stirrer, and gently to stir the solution of the immunogen peptides. Then slowly to add 1.1 ml, 1 mMol/L of KAL(SO.sub.4).sub.2-12H.sub.2O solution, stirred constantly, and drip added 0.35 ml, 1 mMol/L of NaOH solution, continue to stir for 10 minutes, stopped stifling, 0.1 Mol/L of HCL was used to adjust the pH of the solution to 7.4, after aseptic filtration, the filtered solution is placed at 4° C. for storage.

Implemental Example 14

(22) The experiment preventing tumor in sensitized rabbits:

(23) The experimental group took 9 healthy adult rabbits, divided into three groups of A, B, and C, 3 rabbits in each group, the control group took the normal adult 9 rabbits, also divided into three groups of A, B, and C, 3 rabbits in each group. The experimental group was immunized by broad spectrum vaccine. The experimental A group was immunized by the vaccine obtained from the example 1, the experimental B group was immunized by the vaccine obtained from the example 6, the experimental C group was immunized by the vaccine obtained from experiment 10.

(24) The dose of immune was 20 micrograms/kg, once of every 10 days subcutaneous injection for 3 times. When the titer of antibody reached high than 1:10 thousand (ELISA), the immunization was stop. The control group was immunized by use of pH 7.4, 0.01 mol/L PBS and 1 ml using for each times, total injection need 3 times.

(25) The experiment of inoculation of tumor cell: the rabbits of experiment group and control group were injected was same number of ovarian tumor cell lines A2780, 0.5 ml of 2000 /ml, 3 dot of subcutaneous injection. After 1-2 months of observation, tumor cell was not found in the experimental group, the protection rate was 100%, while the control group all of the rabbits had tumors. Experimental results show that HLA-G broad spectrum vaccine was effective in prevention of cancer.

Implemental Example 15

(26) To use the identical method with implemental example 14 did preventive experiment for prevention of other tumors. The experimental process and the dosage of reagent could be conventional adjusted according to the needs of different tumor models and it was easily for the technical person in this field. The various kinds of vaccines obtained in the implemental example 1 to 13 in the invention took part in the experiment preventing tumor model at not less than five kinds of animal tumor. After immunization used the vaccines in this invent, the result that the experiment group did not found tumor growth, and the control group all of the rabbits had tumor growth.

(27) These tumors include: (1) squamous cell carcinoma of the epithelial tissue, basal cell carcinoma, adenocarcinoma, papillary adenocarcinoma, cystic adenocarcinoma, malignant pleomorphic adenoma, and transitional epithelial carcinoma; (2) is derived from mesenchymal tissue fibrosarcoma, liposarcoma, malignant fibrous tissue cell carcinoma, leiomyosarcoma and rhabdomyosarcoma, angiosarcoma, lymphatic sarcoma, osteosarcoma, chondrosarcoma, synovial sarcoma, and malignant mesothelioma, etc.; (3) lymphoma and leukemia from lymphopoietic tissue; (4) neurofibrosarcoma, malignant schwannoma, malignant glioblastoma, medulloblastoma, malignant meningioma, neuroblastoma, etc.; (5) esophageal squamous cell tumor of the digestive tract, esophageal adenocarcinoma, gastric cancer adenoma, gastric squamous cell carcinoma, hepatic cell tumor, rectal cancer, colon cancer and pancreatic cancer; (6) non-small cell lung cancer from respiratory tract; (7) is derived from the other groups of melanoma, chorionic carcinoma, ovarian cancer cell tumor, laryngeal cancer, breast cancer, seminoma, prostate cancer, cervical cancer, dysgerminoma, embryonal carcinoma and malignant teratoma, etc.

Implemental Example 16: To Prevent Virus Attack in Sensitized Rabbits

(28) The experimental group collected 9 healthy adult rabbits, divided into three groups of A, B and C, 3 rabbits in ach group, and the control group took 9 normal adult rabbits. Experimental group was immunized by the broad spectrum vaccine, the rabbits in group A immunized by the vaccine obtained in implementation example 2, the rabbits in group B immunized by the vaccine obtained in the implementation 7, and group C immunized by use of the vaccine obtained in implementation 11. when immune titer of the antibody reached more than 1:50000 (ELISA), to stop the immunization. The rabbits of control group were immunized by use of pH 7.4, 0.01 mol/L PBS.

(29) The experimental group and the control group inoculated the same dose of avian influenza virus (0.05-1.0 microgram/kg/time). The infection was carried out by drop in eye or nose, once a day, with a total infection of 2 times, and observed for 5-20 days. Statistics of the number of rabbits in the experimental group and the control group was attacked. Experimental results showed that none of the nine rabbits were attacked in the experimental group, and had a 100% protection rate. The all of nine rabbits in the control group were all ill. The experimental results confirmed that this vaccine has the effective function for preventing avian influenza virus.

Implemental Example 17

(30) The same method with experiment 16 was used for the experiment of prevention of other viruses. The experimental process and dosage of reagent could be conventional adjusted according to the needs of different virus infective models, it was easy for the technical person in this field.

(31) All kinds of vaccines obtained in the implemental example of 1-13 of this invent took part in preventive experiments of virus infective model at no less than 5 kinds of virus infective model. The experimental result shown that after immunization used the vaccines obtained from this invention, the experimental rabbits were found no infected with the virus, whereas the control rabbits were infected with viral diseases.

(32) These viruses include: (1) pox virus division in double-stranded DNA virus, iridescent virus, virus, herpes virus, adenovirus, HPV, polyoma virus families, more DNA virus, vesicles, African swine fever virus, etc.; (2) single stranded DNA viruses in the combined virus family, cyclones, parvovirus, etc.; (3) DNA and RNA retroviruses of the liver DNA virus family, retroviruses, pseudoviridae, transposidae, and so on; (4) double-stranded RNA viruses in the virus, double RNA virus family, bicomponent double-stranded RNA globular fungal virus, detoxividae, etc.; (5) exposed RNA viruses in exposed RNA viruses; (6) the paramyxoviruses in the negative single-stranded RNA viruses, the elastoviruses, the filovirus family, the bornavirus family, the orthonaviridae, the bunya virus family, the salavirus family, and so on; (7) the light bacteriophage, arteritis virus, coronavirus, and small RNA viruses in the single-stranded RNA virus.

(33) Described above is only a preferred example of this invention, not to limit creation of the invention. All of within the spirits and principles of the invention, by any modification, equivalent replacement, improvement, etc., shall be contained within the protection scope of the invention.