CN117209613B - Preparation of novel HPV broad-spectrum antigen and application of polyclonal egg yolk antibody thereof - Google Patents
Preparation of novel HPV broad-spectrum antigen and application of polyclonal egg yolk antibody thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a preparation method of a novel HPV broad-spectrum antigen and application of a polyclonal egg yolk antibody thereof, wherein the L1 antigens of HPV16, HPV18, HPV6 and HPV11 are fused together for the first time to obtain the HPV broad-spectrum antigen, the purified HPV broad-spectrum antigen is used for directly immunizing laying hens, and after the antibody is generated, the antibody is purified through steps of salting out, dialysis, concentration and the like. Proved by verification, the antibody has broad-spectrum anti-HPV activity, can effectively prevent or treat most common Human Papilloma Virus (HPV) infection and can prevent the difficult problem of canceration caused by HPV infection.
Description
Technical Field
The invention relates to the technical field of HPV treatment, in particular to preparation of a novel HPV broad-spectrum antigen and application of a polyclonal egg yolk antibody 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. HPV has been identified to date with more than about 200 subtypes, which are classified into high-risk HPV (HR-HPV) and low-risk HPV (LR-HPV) according to their pathogenicity and prognosis, wherein HR-HPV 15 types, including HR-HPV types 16, 18, 31, 33, 35, 39, 45, 5l, 52, 56, 53, 58, etc., may cause precancerous lesions and cervical cancer after infection.
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 bioactivity of the egg yolk antibody (IgY) obtained by intramuscular injection of DNA nucleic acid antigen for immunization of the laying hens is far superior to that of the egg yolk antibody prepared by the common technology (subcutaneous injection of protein antigen), the antibody titer is higher, and the specificity is stronger. 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. The development of the product also produces good economic and social benefits in ecological agriculture and food safety. Has good application value in diagnosing, preventing and treating various diseases of human beings and animals.
Disclosure of Invention
Aiming at the technical problems existing in the prior art, the invention provides the preparation of a novel HPV broad-spectrum antigen and the application of a polyclonal egg yolk antibody thereof. The research fuses L1 antigens of HPV16, HPV18, HPV6 and HPV11 together to obtain HPV broad-spectrum antigen, and the purified HPV broad-spectrum antigen is used for directly immunizing laying hens, and after the antibodies are produced, the antibodies are purified through steps of salting out, dialysis, concentration and the like. Proved by verification, the polyclonal egg yolk antibody has broad-spectrum anti-HPV activity, can effectively prevent or treat most common Human Papilloma Virus (HPV) infection and can prevent the difficult problem of canceration caused by HPV virus infection.
Specifically, the invention firstly provides a novel HPV broad-spectrum antigen, which is obtained by fusing L1 antigen core sequences of HPV16, HPV18, HPV6 and HPV11 through polypeptide linkers.
Preferably, the majority of polypeptide linkers include (GS) n, (GGGS) n, and the like.
Preferably, the broad-spectrum antigen is a tandem of the L1 antigen core sequences of HPV16, HPV18, HPV6 and HPV11 by a GSGS polypeptide linker.
Preferably, the amino acid sequence of the broad-spectrum antigen is shown in SEQ ID NO. 1-3.
Preferably, the coding sequence of the broad-spectrum antigen is obtained through codon optimization, and the coding sequence further subjected to codon optimization is shown in SEQ ID NO. 4-6.
On the other hand, the invention also provides a preparation method of the novel HPV broad-spectrum antigen, which comprises the following steps:
1) Codon optimization is carried out on L1 antigen core sequences of HPV16, HPV18, HPV6 and HPV11 to obtain matched nucleotide sequences;
2) Coupling the nucleotide sequence in the step 1) with a prokaryotic expression vector through an enzyme digestion connection mode;
3) Introducing the recombinant expression vector of the step 2) into escherichia coli for recombinant expression;
4) Selecting the recombinant escherichia coli in the step 3), and carrying out shake flask culture;
5) And (3) collecting the escherichia coli in the step (4), and extracting and purifying recombinant proteins to obtain the HPV broad-spectrum antigen.
The nucleotide sequence in the step 1) is shown as SEQ ID NO. 4-6;
the prokaryotic expression vector in the step 2) is selected from pET28a, pGEX-4T, pET or pColdII, etc.
The E.coli in step 3) is selected from BL21 (DE 3), rosetta or Origami B, etc.
The shake flask culture conditions in the step 4) are that single colonies of recombinant E.coli are picked and added into 10ml of LB for culture, and the culture is carried out at 37 ℃ and 300rpm overnight.
The recombinant protein extraction and purification step in the step 5) comprises the steps of crushing bacterial suspension by using an ultrasonic instrument, centrifugally collecting supernatant, separating and purifying the supernatant by using affinity column chromatography, eluting and ultrafiltering and concentrating to obtain HPV broad-spectrum antigens recombined by L1 proteins of HPV16, HPV18, HPV6 and HPV 11.
Furthermore, the invention also provides a polyclonal egg yolk antibody based on the novel HPV broad-spectrum antigen, which is characterized in that the polyclonal egg yolk antibody is prepared by immunizing laying hens with the novel HPV broad-spectrum antigen.
Further, the preparation method of the polyclonal egg yolk antibody comprises the following steps:
(1) Obtaining the novel HPV broad-spectrum antigen according to the preparation method of the novel HPV broad-spectrum antigen;
(2) Laying hen immunization program: the primary immunogen is novel HPV broad-spectrum antigen (500 mu g) and equal volume Freund complete adjuvant (Freunds complete adjuvant, FCA) are fully and uniformly mixed into emulsion, and 6 points of distributed intramuscular injection of 1 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 a novel HPV broad-spectrum antigen (250 μg) plus freund's incomplete adjuvant (Freunds incomplete adjuvant, FIA) and was mixed well 2 weeks apart for 2 booster immunizations. After 1 week of booster immunization, eggs are collected and kept for later use at 4 ℃;
(3) Isolation and purification of polyclonal egg yolk antibodies: separating and collecting yolk liquid of eggs, adding 5 times volume of PBS buffer, adding 3.5% (w/v) PEG-6000, standing for 1 hr, 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; the dialyzate is sterilized and filtered to obtain the polyclonal egg yolk antibody based on the novel HPV broad-spectrum antigen; freeze drying, and storing at-80deg.C.
Furthermore, the invention also provides application of the novel HPV broad-spectrum antigen in preparation of preparations for preventing or treating HPV virus infection diseases.
Furthermore, the invention also provides application of the polyclonal egg yolk antibody based on the novel HPV broad-spectrum antigen in preparation of a preparation for preventing or treating HPV virus infection diseases.
Such formulations include, but are not limited to, ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, oil preparations or transdermal patches and the like.
The invention has the following advantages: the invention fuses L1 antigens of HPV16, HPV18, HPV6 and HPV11 together for the first time to obtain HPV broad-spectrum antigen, and the purified HPV broad-spectrum antigen is used for directly immunizing laying hens, and after the antibody is produced, the antibody is purified through steps of salting out, dialysis, concentration and the like. Proved by verification, the antibody has broad-spectrum anti-HPV activity, and can effectively prevent or treat most types of Human Papilloma Virus (HPV) infection and prevent canceration.
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.
Example 1
A novel HPV broad-spectrum antigen is obtained by fusing L1 antigen core sequences of HPV16, HPV18, HPV6 and HPV11 through GSGS polypeptide linker, and the amino acid sequence of the novel HPV broad-spectrum antigen is shown as SEQ ID NO. 1.
Example 2
A novel HPV broad-spectrum antigen is obtained by fusing L1 antigen core sequences of HPV16, HPV18, HPV6 and HPV11 through GSGS polypeptide linker, and the amino acid sequence of the novel HPV broad-spectrum antigen is shown as SEQ ID NO. 2.
Example 3
A novel HPV broad-spectrum antigen is obtained by fusing L1 antigen core sequences of HPV16, HPV18, HPV6 and HPV11 through GSGS polypeptide linker, and the amino acid sequence of the novel HPV broad-spectrum antigen is shown as SEQ ID NO. 3.
Example 4
A method for preparing a novel HPV broad-spectrum antigen, comprising the steps of:
1) Obtaining matched nucleotide sequences by codon optimization based on the amino acid sequences of the broad-spectrum antigens of any of examples 1-3; the nucleotide sequence is shown in any one of SEQ ID NO. 4-6;
2) Coupling the nucleotide sequence in the step 1) with a prokaryotic expression vector through an enzyme digestion connection mode; the prokaryotic expression vector is pET28a;
3) Introducing the recombinant expression vector of the step 2) into escherichia coli for recombinant expression; the escherichia coli is BL21 (DE 3);
4) Selecting the recombinant escherichia coli in the step 3), and carrying out shake flask culture; the shake flask culture condition is that a single colony of recombinant escherichia coli is picked and added with 10ml of LB for culture, and the culture is carried out at 37 ℃ and 250rpm overnight;
5) And (3) collecting the escherichia coli in the step (4), extracting and purifying recombinant proteins, crushing bacterial suspension by using an ultrasonic instrument, centrifugally collecting supernatant, separating and purifying the supernatant by using affinity column chromatography, eluting and concentrating by ultrafiltration to obtain the HPV broad-spectrum antigen recombined by L1 proteins of HPV16, HPV18, HPV6 and HPV 11.
Example 5
The invention discloses a polyclonal egg yolk antibody based on a novel HPV broad-spectrum antigen, which is characterized in that the polyclonal egg yolk antibody is prepared by immunizing laying hens with the novel HPV broad-spectrum antigen in any one of embodiments 1-3.
Further, the preparation method of the polyclonal egg yolk antibody comprises the following steps:
(1) Obtaining the novel HPV broad-spectrum antigen of any one of examples 1-3 according to the method of preparing the novel HPV broad-spectrum antigen of example 4;
(2) Laying hen immunization program: the primary immunogen is novel HPV broad-spectrum antigen (500 mu g) and equal volume Freund complete adjuvant (Freunds complete adjuvant, FCA) are fully and uniformly mixed into emulsion, and 6 points of distributed intramuscular injection of 1 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 a novel HPV broad-spectrum antigen (250 μg) plus freund's incomplete adjuvant (Freunds incomplete adjuvant, FIA) and was mixed well 2 weeks apart for 2 booster immunizations. After 1 week of booster immunization, eggs are collected and kept for later use at 4 ℃;
(3) Isolation and purification of polyclonal egg yolk antibodies: separating and collecting yolk liquid of eggs, adding 5 times volume of PBS buffer, adding 3.5% (w/v) PEG-6000, standing for 1 hr, 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; the dialyzate is sterilized and filtered to obtain the polyclonal egg yolk antibody based on the novel HPV broad-spectrum antigen; freeze drying, and storing at-80deg.C.
Example 6
ELISA method is used for determining the binding titers of polyclonal egg yolk antibodies based on novel HPV broad-spectrum antigens to various HPV subtypes. And respectively selecting 10 mug/mL HPV16, HPV18, HPV6, HPV11, HPV31, HPV33, HPV52, HPV58 and HPV81 as detection antigens, coating a 96-well microplate, finally adding the pure egg yolk antibodies with different dilution factors, incubating at 37 ℃ for 1h, washing the plate, adding horseradish peroxidase-labeled rabbit anti-chicken IgG antibodies with dilution of 1:5000, incubating at 37 ℃ for 1h, washing the plate, and adding a substrate for color development. And measuring the absorption value of 450nm wavelength by using an enzyme-labeled instrument, and taking the non-immune egg yolk liquid as a control.
Binding assays of polyclonal egg yolk antibodies to multiple HPV subtypes described in Table 1
From the statistical results of table 1, the polyclonal egg yolk antibody based on the novel HPV broad-spectrum antigen has certain binding activity on various HPV subtypes, especially on HPV16, HPV18, HPV6 and HPV11 subtypes, and the binding activity is slightly higher than that of HPV31, HPV33, HPV52, HPV58 and HPV81. But in the whole, the polyclonal egg yolk antibody has certain binding affinity to a broad spectrum of HPV viruses.
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 (3)
1. The HPV broad-spectrum antigen is characterized in that the coding sequence of the broad-spectrum antigen is obtained by codon optimization, and the coding sequence after codon optimization is shown as SEQ ID NO. 2.
2. A method for preparing polyclonal egg yolk antibodies based on HPV broad-spectrum antigens according to claim 1, characterized in that said method comprises the steps of:
(1) Obtaining the HPV broad-spectrum antigen of claim 1;
(2) Laying hen immunization program: the primary immunogen is 500 mug HPV broad-spectrum antigen and equal volume Freund's complete adjuvant (Freunds complete adjuvant, FCA) are fully and uniformly mixed to form emulsion, 6 points under the chest and wings of the laying hen are sterilized by conventional iodine and alcohol, and 1 mL emulsion is injected in a distributed intramuscular manner for immunization; the antigen for boosting is 250 mug HPV broad-spectrum antigen and Freund's incomplete adjuvant (Freunds incomplete adjuvant, FIA) are fully mixed, and boosting is carried out 2 times at intervals of 2 weeks; after 1 week of booster immunization, eggs are collected and kept for later use at 4 ℃;
(3) Isolation and purification of polyclonal egg yolk antibodies: separating and collecting yolk liquid of eggs, adding 5 times volume of PBS buffer, adding 3.5% (w/v) PEG-6000, standing for 1 hr, and 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 of 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; the dialyzate is sterilized and filtered to obtain the polyclonal egg yolk antibody based on HPV broad-spectrum antigen; freeze drying, and storing at-80deg.C.
3. Use of the HPV broad-spectrum antigen of claim 1 or the polyclonal egg yolk antibody of claim 2 in the preparation of a reagent for detecting HPV viral infection diseases.
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| CN101481407A (en) * | 2008-01-07 | 2009-07-15 | 马润林 | Modification sequence of recombinant human mammilla tumor virus L1 capsid protein |
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| CN116554280A (en) * | 2023-05-18 | 2023-08-08 | 上海博满生物科技有限公司 | 18-valent HPV composite yolk neutralizing antibody for preventing and treating cervical HPV infection, and preparation method and application thereof |
| CN116606371A (en) * | 2023-05-15 | 2023-08-18 | 上海博满生物科技有限公司 | IgY (IgY) for preventing and treating condyloma acuminatum 16-valent HPV (human papilloma Virus) composite yolk neutralizing antibody as well as preparation and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BR102019025802A2 (en) * | 2019-12-05 | 2022-01-18 | Instituto Butantan | PROCESS OF PRODUCTION OF A PROPHYLATIC AND THERAPEUTIC DNA IMMUNOLOGICAL COMPOSITION AGAINST HPV AND VIRUS-ASSOCIATED CANCER, HYBRID PROTEIN, EXPRESSION VECTOR, IMMUNOLOGICAL COMPOSITION AND THEIR USES |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101481407A (en) * | 2008-01-07 | 2009-07-15 | 马润林 | Modification sequence of recombinant human mammilla tumor virus L1 capsid protein |
| CN112010950A (en) * | 2020-07-28 | 2020-12-01 | 尤丽康（江苏）生物医药有限公司 | Antigen and yolk antibody for inhibiting multiple HPV viruses and preparation method and application thereof |
| CN116606371A (en) * | 2023-05-15 | 2023-08-18 | 上海博满生物科技有限公司 | IgY (IgY) for preventing and treating condyloma acuminatum 16-valent HPV (human papilloma Virus) composite yolk neutralizing antibody as well as preparation and application thereof |
| CN116554280A (en) * | 2023-05-18 | 2023-08-08 | 上海博满生物科技有限公司 | 18-valent HPV composite yolk neutralizing antibody for preventing and treating cervical HPV infection, and preparation method and application thereof |
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