CN116785428A - Compound HPV (human papilloma Virus) biological protein and application thereof - Google Patents
Compound HPV (human papilloma Virus) biological protein and application thereof Download PDFInfo
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Abstract
The invention provides a compound anti-HPV biological protein and application thereof, the compound anti-HPV biological protein takes an anti-HPV single-chain IgY antibody as an active ingredient, wherein, whey protein has immunological activity, can completely enter the near-end small intestine, plays a role in protecting the mucosa of the small intestine, and can effectively assist the immunological effect of the anti-HPV single-chain IgY antibody; the chitosan is used as a drug carrier to stabilize the anti-HPV single-chain IgY antibody, promote the drug absorption, delay or control the dissolution rate of the drug, help the drug to reach target organs, resist acid and ulcer and prevent the drug from stimulating the stomach; poloxamer can increase retention time of anti-HPV single chain IgY antibody in gastrointestinal tract, and increase absorption, so as to improve bioavailability of medicine and promote absorption of medicine. The anti-HPV single-chain IgY antibody aiming at HPV 16E 7 protein for the first time is economical and easy to obtain, has simple preparation and purification processes, lower cost compared with the IgG antibody and higher yield.
Description
Technical Field
The invention relates to the technical field of HPV treatment, in particular to a compound anti-HPV biological protein and application thereof.
Background
Cervical cancer is a common female malignancy worldwide. Persistent infection with high-risk Human Papillomaviruses (HPV) is a clear risk factor for cervical cancer. HPV16 is one of the most common high-risk HPV types. The detection of HPV encoded E6 and E7 early oncoproteins is of great biological significance. However, there is currently no commercialization available for clinical detection of HPV 16E 7 antibodies.
The egg yolk antibody (Immunoglobulin of yolk, igY) refers to a method in which a specific antigen stimulates B lymphocytes of an avian species, and the B lymphocytes differentiate into plasma cells and thereby secrete specific antibodies into the blood circulation, and gradually accumulate in the egg cells as the blood flows through the ovaries, forming the egg yolk antibody. The yolk antibody has the advantages of stable property, strong specificity, low preparation cost, oral administration safety and the like, and has been widely applied to the prevention and treatment of infectious diseases of poultry and livestock.
HPV protein dressing can be used as a novel wound dressing, can be used as a mode for preventing HPV infection, can be used for auxiliary treatment, has no side effect and has huge market space. However, the current HPV protein dressing has slow curative effect and cannot achieve the aim of neutralizing HPV virus very effectively.
Disclosure of Invention
Aiming at the technical problems existing in the prior art, the invention provides a compound anti-HPV biological protein and application thereof.
Specifically, the invention firstly provides a compound anti-HPV biological protein, which comprises the following raw materials in parts by weight: 40-50% of specific IgY antibody against HPV virus, 5-10% of whey protein, 10-20% of chitosan, 5-10% of poloxamer and the balance of distilled water are prepared into 100%.
Preferably, the compound anti-HPV bioprotein consists of the following raw materials in parts by weight: 40% anti-HPV single-chain IgY antibody, 5% whey protein, 10% chitosan and 5% poloxamer and the balance distilled water were formulated to 100%.
Preferably, the compound anti-HPV bioprotein consists of the following raw materials in parts by weight: 45% anti-HPV single-chain IgY antibody, 8% whey protein, 15% chitosan and 8% poloxamer and the balance distilled water were formulated to 100%.
Preferably, the compound anti-HPV bioprotein consists of the following raw materials in parts by weight: 50% anti-HPV single-chain IgY antibody, 10% whey protein, 20% chitosan and 10% poloxamer and the balance distilled water were formulated to 100%.
The invention further provides an anti-HPV single-chain IgY antibody, which is obtained by first separation and purification, wherein the amino acid sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO.1, and further, after codon optimization, the coding sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO. 2.
The invention also aims at providing a preparation method of the compound anti-HPV biological protein, which is characterized by comprising the following steps:
(1) Weighing the relevant raw materials according to the weight ratio;
(2) Firstly, slowly dissolving whey protein, chitosan and poloxamer in distilled water in warm water incubation, and continuously stirring at low temperature to obtain a solution A;
(3) Adding the anti-HPV single-chain IgY antibody into the solution A in the step (2), and stirring and mixing uniformly at a low temperature to obtain a solution B;
(4) And (3) performing vacuum freeze drying on the solution B to obtain the compound anti-HPV biological protein.
Preferably, the compound anti-HPV biological protein can be compounded with common pharmaceutical excipients, and the dosage form is not limited to paste, gel or suppository.
Another object of the present invention is to provide a method for preparing an anti-HPV single-chain IgY antibody, comprising the steps of:
(1) HPV 16E 7 full-length protein acquisition: amplifying full length HPV 16E 7 (NC_ 001526.4) gene sequence from HPV16 whole virus plasmid, connecting the amplified and purified product with carrier to prepare recombinant plasmid, transforming competent cells, inducing expression of HPV 16E 7 protein, separating and purifying to obtain HPV 16E 7 protein, freeze drying and storing at-80 deg.C for use.
(2) Laying hen immunization program: the primary immunogen is HPV 16E 7 protein (500 mu g) and equal volume Freund's complete adjuvant (Freunds complete adjuvant, FCA) are fully and uniformly mixed into emulsion, and 4 points of distributed intramuscular injection of 2 ml emulsion under the chest and wings of the conventional iodine and alcohol sterilized laying hen are used for immunization. The antigen subsequently boosted was HPV 16E 7 protein (250 μg) plus freund's incomplete adjuvant (Freunds incomplete adjuvant, FIA) well mixed 2 times at 2 week intervals. After 1 week of booster immunization, eggs were collected and kept at 4℃for further use.
(3) Isolation and purification of specific IgY antibodies: separating and collecting yolk liquid of eggs, adding 5 times volume of PBS buffer, adding 3.5% (w/v) PEG-6000, standing for 30min, centrifuging at 5000g for 20min; filtering the supernatant by absorbent cotton, adding ammonium sulfate into the supernatant for salting out to ensure that the saturation of the ammonium sulfate reaches 40% -50%, and standing for 12h at 4 ℃; standing, centrifuging, discarding supernatant to obtain precipitate, and dissolving with 5-7ml PBS buffer solution per ml precipitate to obtain solution; loading the solution into a dialysis bag, dialyzing with distilled water to remove ammonium ions, and dialyzing with PBS buffer solution for the last time; sterilizing and filtering the dialyzate to obtain specific IgY antibody extracting solution; freeze drying, and storing at-80deg.C.
(4) And (3) sending the specific IgY antibody prepared in the step (3) to Beijing Anbiqi biological company for sequencing analysis, and coupling the light chain and the heavy chain of the specific IgY antibody through a polypeptide linker to obtain the anti-HPV single-chain IgY antibody.
Preferably, the polypeptide linker comprises (GGGS) n repeat units.
Further preferably, the polypeptide linker comprises GGGSGGGS.
Furthermore, the invention also provides application of the anti-HPV single-chain IgY antibody in preparation of compound anti-HPV biological proteins.
Furthermore, the invention also provides a kit for preventing or treating HPV virus, which is characterized in that the kit comprises compound anti-HPV biological proteins.
Preferably, the kit or reagent further comprises instructions describing the steps of performing the therapeutic method.
Preferably, the type of detecting HPV virus is HPV type 16.
The invention has the following advantages:
(1) The compound anti-HPV biological protein provided by the invention takes an anti-HPV single-chain IgY antibody as an active ingredient, wherein the whey protein has immune activity, can completely enter the proximal small intestine, plays a role in protecting the mucosa of the small intestine, and can effectively assist the immune effect of the anti-HPV single-chain IgY antibody; the chitosan is used as a drug carrier to stabilize the anti-HPV single-chain IgY antibody, promote the drug absorption, delay or control the dissolution rate of the drug, help the drug to reach target organs, resist acid and ulcer and prevent the drug from stimulating the stomach; poloxamer can increase retention time of anti-HPV single chain IgY antibody in gastrointestinal tract, and increase absorption, so as to improve bioavailability of medicine and promote absorption of medicine.
(2) The anti-HPV single-chain IgY antibody aiming at HPV 16E 7 protein for the first time is economical and easy to obtain, has simple preparation and purification processes, lower cost compared with the IgG antibody and higher yield.
Drawings
FIG. 1 shows the neutralization test of HPV16 virus according to examples 1-3 and 5 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
The following examples are given by way of illustration of the invention and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Example 1
The compound anti-HPV biological protein consists of the following raw materials in parts by weight:
40% anti-HPV single-chain IgY antibody, 5% whey protein, 10% chitosan and 5% poloxamer and the balance distilled water were formulated to 100%.
The anti-HPV single-chain IgY antibody is obtained by first separation and purification, the amino acid sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO.1, and further, after codon optimization, the coding sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO. 2.
Example 2
The compound anti-HPV biological protein consists of the following raw materials in parts by weight:
45% anti-HPV single-chain IgY antibody, 8% whey protein, 15% chitosan and 8% poloxamer and the balance distilled water were formulated to 100%.
The anti-HPV single-chain IgY antibody is obtained by first separation and purification, the amino acid sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO.1, and further, after codon optimization, the coding sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO. 2.
Example 3
The compound anti-HPV biological protein consists of the following raw materials in parts by weight:
50% anti-HPV single-chain IgY antibody, 10% whey protein, 20% chitosan and 10% poloxamer and the balance distilled water were formulated to 100%.
The anti-HPV single-chain IgY antibody is obtained by first separation and purification, the amino acid sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO.1, and further, after codon optimization, the coding sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO. 2.
Example 4
The method for preparing the compound anti-HPV bioprotein according to any one of embodiments 1-3, characterized by comprising the following steps:
(1) Weighing the relevant raw materials according to the weight ratio of any one of the examples 1-3;
(2) Firstly, slowly dissolving whey protein, chitosan and poloxamer in distilled water in warm water incubation, and continuously stirring at low temperature to obtain a solution A;
(3) Adding the anti-HPV single-chain IgY antibody into the solution A in the step (2), and stirring and mixing uniformly at a low temperature to obtain a solution B;
(4) And (3) performing vacuum freeze drying on the solution B to obtain the compound anti-HPV biological protein.
Example 5
A preparation method of an anti-HPV single-chain IgY antibody comprises the following steps:
(1) HPV 16E 7 full-length protein acquisition: amplifying full length HPV 16E 7 (NC_ 001526.4) gene sequence from HPV16 whole virus plasmid, connecting the amplified and purified product with carrier to prepare recombinant plasmid, transforming competent cells, inducing expression of HPV 16E 7 protein, separating and purifying to obtain HPV 16E 7 protein, freeze drying and storing at-80 deg.C for use.
(2) Laying hen immunization program: the primary immunogen is HPV 16E 7 protein (500 mu g) and equal volume Freund's complete adjuvant (Freunds complete adjuvant, FCA) are fully and uniformly mixed into emulsion, and 4 points of distributed intramuscular injection of 2 ml emulsion under the chest and wings of the conventional iodine and alcohol sterilized laying hen are used for immunization. The antigen subsequently boosted was HPV 16E 7 protein (250 μg) plus freund's incomplete adjuvant (Freunds incomplete adjuvant, FIA) well mixed 2 times at 2 week intervals. After 1 week of booster immunization, eggs were collected and kept at 4℃for further use.
(3) Isolation and purification of specific IgY antibodies: separating and collecting yolk liquid of eggs, adding 5 times volume of PBS buffer, adding 3.5% (w/v) PEG-6000, standing for 30min, centrifuging at 5000g for 20min; filtering the supernatant by absorbent cotton, adding ammonium sulfate into the supernatant for salting out to ensure that the saturation of the ammonium sulfate reaches 40% -50%, and standing for 12h at 4 ℃; standing, centrifuging, discarding supernatant to obtain precipitate, and dissolving with 5-7ml PBS buffer solution per ml precipitate to obtain solution; loading the solution into a dialysis bag, dialyzing with distilled water to remove ammonium ions, and dialyzing with PBS buffer solution for the last time; sterilizing and filtering the dialyzate to obtain specific IgY antibody extracting solution; freeze drying, and storing at-80deg.C.
(4) And (3) sending the specific IgY antibody prepared in the step (3) to Beijing Anbiqi biological company for sequencing analysis, and coupling the light chain and the heavy chain of the specific IgY antibody through a polypeptide linker to obtain the anti-HPV single-chain IgY antibody. Wherein the polypeptide linker comprises (GGGS) n Repeating units.
Example 6
The neutralization effect of the compound anti-HPV bioprotein compositions of examples 1-3 and the anti-HPV single-chain IgY antibody of example 5 on HPV16 virus was examined by the Elisa method, and the control group was 100% prepared from 10% whey protein, 20% chitosan and 10% poloxamer, and the balance distilled water. The results are shown in figure 1, and the test results show that the compound anti-HPV bioprotein composition of the examples 1-3 can effectively neutralize human papillomavirus HPV16, and compared with the anti-HPV single-chain IgY antibody in the independent example 5, the compound anti-HPV bioprotein composition of the examples 1-3 shows stronger neutralization effect with HPV16, can effectively inhibit HPV16 virus, and has good clinical application prospect.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The compound anti-HPV biological protein consists of the following raw materials in parts by weight: 40-50% of anti-HPV single-chain IgY antibody, 5-10% of whey protein, 10-20% of chitosan and 5-10% of poloxamer and the balance of distilled water are prepared into 100%;
wherein the amino acid sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO.1, and further, after codon optimization, the coding sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO. 2.
2. The compound anti-HPV bioprotein of claim 1, wherein the compound anti-HPV bioprotein consists of the following raw materials in weight ratio: 40% anti-HPV single-chain IgY antibody, 5% whey protein, 10% chitosan and 5% poloxamer and the balance distilled water were formulated to 100%.
3. The compound anti-HPV bioprotein of claim 1, wherein the compound anti-HPV bioprotein consists of the following raw materials in weight ratio: 45% anti-HPV single-chain IgY antibody, 8% whey protein, 15% chitosan and 8% poloxamer and the balance distilled water were formulated to 100%.
4. The compound anti-HPV bioprotein of claim 1, characterized in that the compound anti-HPV bioprotein consists of the following raw materials in weight ratio: 50% anti-HPV single-chain IgY antibody, 10% whey protein, 20% chitosan and 10% poloxamer and the balance distilled water were formulated to 100%.
5. The anti-HPV single-chain IgY antibody is obtained by first separation and purification, the amino acid sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO.1, and further, after codon optimization, the coding sequence of the anti-HPV single-chain IgY antibody is shown as SEQ ID NO. 2.
6. The method for preparing the anti-HPV single-chain IgY antibody of claim 5, comprising the steps of:
(1) HPV 16E 7 full-length protein acquisition: amplifying full-length HPV 16E 7 (NC_ 001526.4) gene sequence from HPV16 whole virus plasmid, connecting the amplified and purified product with a vector to prepare a recombinant plasmid, transforming competent cells, inducing expression of HPV 16E 7 protein, separating and purifying to obtain HPV 16E 7 protein, freeze-drying, and storing at-80 ℃ for later use;
(2) Laying hen immunization program: the primary immunogen is HPV 16E 7 protein (500 mu g) and equal volume Freund's complete adjuvant (Freunds complete adjuvant, FCA) are fully and uniformly mixed into emulsion, and 4 points of distributed intramuscular injection of 2 ml emulsion under the chest and wings of the conventional iodine and alcohol sterilized laying hen are used for immunization. The antigen subsequently boosted was HPV 16E 7 protein (250 μg) plus freund's incomplete adjuvant (Freunds incomplete adjuvant, FIA) well mixed 2 times at 2 week intervals. After 1 week of booster immunization, eggs are collected and kept for later use at 4 ℃;
(3) Isolation and purification of specific IgY antibodies: separating and collecting yolk liquid of eggs, adding 5 times volume of PBS buffer, adding 3.5% (w/v) PEG-6000, standing for 30min, centrifuging at 5000g for 20min; filtering the supernatant by absorbent cotton, adding ammonium sulfate into the supernatant for salting out to ensure that the saturation of the ammonium sulfate reaches 40% -50%, and standing for 12h at 4 ℃; standing, centrifuging, discarding supernatant to obtain precipitate, and dissolving with 5-7ml PBS buffer solution per ml precipitate to obtain solution; loading the solution into a dialysis bag, dialyzing with distilled water to remove ammonium ions, and dialyzing with PBS buffer solution for the last time; sterilizing and filtering the dialyzate to obtain specific IgY antibody extracting solution; freeze drying, and storing at-80deg.C;
(4) And (3) sending the specific IgY antibody prepared in the step (3) to Beijing Anbiqi biological company for sequencing analysis, and coupling the light chain and the heavy chain of the specific IgY antibody through a polypeptide linker to obtain the anti-HPV single-chain IgY antibody.
7. The method of claim 6, wherein the polypeptide linker comprises a (GGGS) n repeat unit.
8. The method of claim 7, wherein the polypeptide linker comprises GGGSGGGS.
9. The method for preparing the compound anti-HPV bioprotein of any one of claims 1-4, wherein the method comprises the following steps:
(1) Weighing the relevant raw materials according to the weight ratio;
(2) Firstly, slowly dissolving whey protein, chitosan and poloxamer in distilled water in warm water incubation, and continuously stirring at low temperature to obtain a solution A;
(3) Adding the anti-HPV single-chain IgY antibody into the solution A in the step (2), and stirring and mixing uniformly at a low temperature to obtain a solution B;
(4) And (3) performing vacuum freeze drying on the solution B to obtain the compound anti-HPV biological protein.
10. The method of claim 9, wherein the compound anti-HPV biologic protein is formulated with common pharmaceutical excipients, and the dosage form is not limited to a paste, or gel, or suppository.
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| CN1944462A (en) * | 2006-09-29 | 2007-04-11 | 西安交通大学 | Preparation of anti PV IgY antibody and its use in PV diagnostic reagent and preventing medicine |
| US20070166699A1 (en) * | 2003-09-17 | 2007-07-19 | Amynon Biotech Gmbh | Combination of anti-hpv-16 and 18 antibodies and uses thereof |
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