CN114195886A - anti-HPV 39L 1 protein monoclonal antibody, and preparation and application thereof - Google Patents

Abstract

The invention discloses an anti-HPV 39L 1 protein monoclonal antibody, and preparation and application thereof, and aims to solve the problem that an HPV39 subtype is difficult to specifically recognize. The heavy chain variable region of the antibody contains a DNA sequence shown as SEQ ID NO.1 and an amino acid sequence shown as SEQ ID NO.2, and the light chain variable region contains a DNA sequence shown as SEQ ID NO.3 and an amino acid sequence shown as SEQ ID NO. 4; the antibody can be applied to immunological detection or vaccine preparation, and also can be applied to antigen or antibody detection kits. The monoclonal antibody for resisting HPV 39L 1 protein is prepared by immunizing BALB/c mice by an immunological method, can specifically recognize HPV39 high-risk (HR) subtype, has extremely high sensitivity, has good reactivity with HPV39 pseudovirus and HPV 39L 1 protein, and does not have cross reaction with L1 protein of other subtypes. The invention lays a good foundation for further modifying the antibody variable region sequence to prepare the genetic engineering antibody with different combination forms and carrying out clinical detection research on different HPV subtype pathogens.

Description

anti-HPV 39L 1 protein monoclonal antibody, and preparation and application thereof

Technical Field

The invention relates to the technical field of biological immunity, in particular to an anti-HPV 39L 1 protein monoclonal antibody and preparation and application thereof.

Background

Human papilloma virus (H)Human PapillomavirusHPV) belongs to the family of papillomaviruses, is a double-stranded closed circular DNA virus with a 20-hedral structure, has strict species specificity and mainly infects human skin andmucosal tissue. HPV can be transmitted not only by sexual contact but also through the skin and oral cavity, and persistent infection can cause epithelial hyperplasia and progressively cause benign and malignant lesions of various mucosal epithelial cells.

The HPV viral capsid consists of a major capsid protein (L1) and a minor capsid protein (L2); wherein, the L2 protein is a minor capsid protein, is a highly variable nucleoprotein and reflects the polymorphism of HPV antigens; the L1 protein is a main capsid protein of the HPV, accounts for about 80% of the total amount of the virus protein, is a highly conserved glycoprotein, has the molecular weight of 55-60 kDa, has high immunogenicity, and is also the basis of HPV typing.

Studies have shown that HPV L1 protein expressed in vitro can self-assemble into VLPs structures. The VLPs structure assembled in vitro is not different from the natural HPV virus structure with the size of 55 nm, and the VLPs structure can induce an organism to generate type-specific neutralizing antibodies after being immunized, so that the organism can be effectively protected from the infection of homotype viruses. Thus, such vaccines based on the structure of VLPs are commonly used to prevent HPV viral infection. Therefore, the HPV L1 protein is the key for researching HPV VLPs vaccine, and the rapid, specific and sensitive L1 protein detection method has important significance for researching HPV VLPs vaccine.

The current methods for detecting HPV mainly include immunohistochemistry, molecular hybridization, PCR and the like. Since the HPV gene exists in vivo in an episome or in an integrated form integrated into a host chromosome, and none or only a small amount of coat protein exists in the above forms, it is difficult to isolate natural HPV virus in a diseased tissue; based on this, detection of HPV viral antigen proteins is difficult to be clinically applied. Moreover, the related research of the HPV L1 protein detection technology is relatively lagged. At present, monoclonal antibodies resisting HPV 39L 1 protein and a method for specifically detecting the high-risk subtype are not reported.

Therefore, there is a need to prepare a monoclonal antibody capable of specifically recognizing the HPV39 subtype L1 protein by ELISA, Western Blot and other detection methods, and further confirm the heavy chain variable region sequence and the light chain variable region sequence thereof to modify the antibody variable region sequences to prepare genetically engineered antibodies in different combinations, so as to further improve the specificity and affinity of the antibodies.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.

Disclosure of Invention

The invention aims to provide an anti-HPV 39L 1 protein monoclonal antibody, which aims to solve the technical problem that an HPV39 subtype is difficult to specifically recognize; the antibody can be applied to immunological detection, and can also provide convenience for developing a vaccine based on HPV L1 protein; simultaneously discloses a simple and convenient method for preparing the monoclonal antibody of the anti-HPV 39L 1 protein.

In order to solve the technical problems, the invention adopts the following technical scheme:

screening to obtain an anti-HPV 39L 1 protein monoclonal antibody, which comprises the following sequences or activity modification (addition, deletion and replacement) sequences thereof:

a heavy chain variable region DNA sequence shown as SEQ ID NO. 1; a heavy chain variable region amino acid sequence as shown in SEQ ID NO. 2; a light chain variable region DNA sequence shown as SEQ ID NO. 3; the variable region amino acid sequence of light chain as shown in SEQ ID No. 4.

Preferably, the light chain type of the monoclonal antibody is Kappa and the subtype is IgG 1.

The ELISA titer of the monoclonal antibody is not less than 1:1.024 multiplied by 10⁶。

The monoclonal antibody has an affinity of not less than 2.56X 10^-10 mol/L。

The monoclonal antibody against HPV 39L 1 protein can be applied to immunological detection (such as ELISA, IFA, Western Blot and the like) or vaccine preparation; the monoclonal antibody for resisting HPV 39L 1 protein can also be applied to an antigen or antibody detection kit.

The preparation method of the anti-HPV 39L 1 protein monoclonal antibody comprises the following steps:

(1) immunizing a mouse by taking the purified HPV 39L 1 protein as an antigen;

(2) fusing the immune spleen cells of the mice and myeloma cells of the mice to obtain hybridoma cells;

(3) adopting a method of multiple ELISA detection combination and subcloning to obtain positive hybridoma cells;

(4) extracting positive monoclonal antibody hybridoma cell strain RNA, performing reverse transcription to obtain cDNA, and performing PCR amplification to obtain a heavy chain variable region sequence and a light chain variable region sequence of the monoclonal antibody;

(5) carrying out multiple cloning culture on the positive clone to obtain an HPV 39L 1 monoclonal antibody hybridoma cell strain;

(6) and injecting the hybridoma cell strain into the abdominal cavity of a mouse to produce the monoclonal antibody.

Compared with the prior art, the invention has the main beneficial technical effects that:

1. the monoclonal antibody resisting HPV 39L 1 protein can rapidly and specifically recognize HPV39 and L1 protein thereof, lays a foundation for solving the HPV 39L 1 protein rapid detection technology, and has wide research application value and commercial application value in HPV39 related immunodetection.

2. The monoclonal antibody resisting the HPV 39L 1 protein has high specificity and high sensitivity, and does not recognize other high-risk subtypes HPV such as HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV51, HPV52, HPV53 and HPV 58.

3. The antibody preparation method of the invention is to use HPV 39L 1 protein obtained from escherichia coli to immunize BALB/c mice, and adopts hybridoma technology to prepare the monoclonal antibody of anti-HPV 39L 1 protein, which can provide convenience for developing vaccines based on HPV L1 protein.

4. On the basis of the heavy chain variable region sequence and the light chain variable region sequence of the monoclonal antibody disclosed by the invention, modification such as addition, deletion, replacement and the like of one or more amino acids can be carried out through conventional genetic engineering and protein engineering to obtain an active fragment or a conservative variant thereof, but the monoclonal antibody can still be specifically combined with HPV 39L 1 protein, so that the basis is laid for preparing genetic engineering antibodies in different combination forms by further modifying the antibody variable region sequence, and the specificity and the affinity of the antibody are further improved.

Drawings

FIG. 1 is a graph showing the results of measuring the serum titer of the immunized mouse by ELISA in the present invention.

FIG. 2 is a diagram showing the results of IFA detection of mAb 4B4 in the examples of the present invention.

FIG. 3 is a diagram showing the result of the specificity of monoclonal antibody 4B4 detected by Western Blot in the example of the present invention; wherein, the Lane M is a protein standard molecular weight Marker; lane 1 is BL21 negative control; lane 2 is the prokaryotic HPV 39L 1 protein.

FIG. 4 is a graph showing the cross-reactivity of the monoclonal antibody 4B4 with HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV51, HPV52, HPV53 and HPV58 detected by ELISA in the present invention.

FIG. 5 is a diagram showing the result of cross-reactivity of monoclonal antibody 4B4 with HPV16, HPV18, HPV31, HPV45, HPV58 and other subtypes in Western Blot detection in the example of the present invention; wherein, the Lane M is a protein standard molecular weight Marker; lanes 1-6 are L1 proteins of HPV16, HPV18, HPV31, HPV35, HPV45 and HPV58 expressed by pronucleus respectively, and NC is BL21 negative control; PC is HPV 39L 1 protein expressed by pronucleus.

FIG. 6 is a diagram showing the cross-reactivity results of IFA detection mAb 4B4 with HPV16, HPV18, HPV31, HPV35, HPV45, HPV51, HPV52, HPV53, HPV56 and HPV58 of other subtypes in the example of the present invention; wherein 16, 18, 31, 35, 39, 45, 52 and 58 are p16SheLL, p18SheLL, p31SheLL, p35SheLL, p39SheLL, p45SheLL, p52SheLL and p58SheLL respectively to transfect 293T cells; 51. 53 and 56 are pVITRO-HPV 51L 1L2, pVITRO-HPV 53L 1L2 and pVITRO-HPV 56L 1L2 transfected cells respectively; NC is 293T cell negative control.

Detailed Description

The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way.

Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described in this disclosure, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

The first embodiment is as follows: preparation of hybridoma cell strain secreting monoclonal antibody against HPV 39L 1 protein

1. Primary reagents and materials

Freund's complete adjuvant, Freund's incomplete adjuvant, HAT, HT, PEG-1500, RPMl-1640 cell culture medium, fetal bovine serum were purchased from Gibco, HRP-labeled goat anti-mouse IgG was purchased from Sigma, liquid AEC enzyme substrate kit was purchased from China fir Jinqiao, BCA protein concentration determination kit was purchased from Solambio, monoclonal antibody subtype determination kit was purchased from Beijing Yi Qiao Hibiscus biotechnologies, Inc.; commercial anti-HPV 39L 1 monoclonal antibodies were purchased from Abcam (Abcam, uk); plasmids p16SheLL, p18SheLL, p31SheLL, p35SheLL, p39SheLL, p45SheLL, p52SheLL, p58SheLL, pVITRO-HPV 51L 1L2, pVITRO-HPV 53L 1L2, pVITRO-HPV 56L 1L2, supplied by Prof John Schiller (Adne, USA), BALB/c mice were purchased from Zhengzhou university animal center, containing the corresponding HPV L1 and L2 ORF region genes.

2. Preparation of immunogen and coating antigen

Specific preparation methods of the HPV 39L 1 protein immunogen are described in the literature (Chen Y, Liu Y, Wang A, Zhang G, Dong Z, Qi Y, Wang J, Zhao B, Li N, Jiang M. human papillomavir L1 protein expressed in Escherichia coli self-assembly inter Virus-lipid tissue enzyme high hly immunogenic [ J ]. Virus Research 2016, 220: 97-103.).

The coating antigen is SUMO-HPV39-L1 ultrasonic supernatant obtained by performing escherichia coli induced expression on a recombinant prokaryotic expression vector pE-SUMO 39L 1, SUMO tag ultrasonic supernatant obtained by performing escherichia coli induced expression on the prokaryotic expression vector pE-SUMO, HPV39 VLPs and HPV39 pseudovirus prepared by co-transfecting 293FT cells with pseudovirus preparation plasmid pShel39 and reporter gene GFP, and the specific preparation method refers to the above documents.

3. Immunization of BALB/c mice

(1) Adding antigen with the component of HPV 39L 1 protein into Freund's complete adjuvant and Freund's incomplete adjuvant respectively, and emulsifying to prepare Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen, wherein the volume ratio of HPV 39L 1 (50 mug/piece) to Freund's complete adjuvant and Freund's incomplete adjuvant is 1: 1;

(2) 4 female BALB/c mice 8 weeks old, approximately 100. mu.l/mouse, were immunized with Freund's complete adjuvant immunogen by back subcutaneous multi-site injection;

(3) BALB/c mice were boosted 14 and 28 days after the first immunization with Freund's incomplete adjuvant immunogen in the same manner and dose, respectively;

(4) after 7 days of the third immunization, blood is collected from the tail, and the serum titer of the mice is measured;

(5) 3-5 days before cell fusion, selecting a mouse with the highest serum titer, and performing super-strong immunity on a BALB/c mouse by using HPV 39L 1 protein without an adjuvant through an intraperitoneal injection method.

4. Determination of serum antibody titer in immunized mice

Measuring the titer by ELISA:

(1) diluting the purified HPV 39L 1 protein to 1 mug/mL by using a coating solution, adding 50 mul of the coating solution into each hole, incubating for 2 hours at 37 ℃, discarding the coating solution, and washing for 3 times by using PBST;

(2) sealing with 200 μ l of sealing solution (5% skimmed milk powder + PBST) overnight at 4 ℃;

(3) adding 50 mul of each serum to be detected diluted by 4 times of dilution buffer solution (PBST) into each well (the initial dilution multiple is 1: 800), incubating for 1h at 37 ℃, discarding supernatant, and washing for 6 times by PBST;

(4) to each well was added a dilution buffer at 1:50 mul of HRP-labeled goat anti-mouse IgG diluted by 5000 is kept at 37 ℃ for 1h, then the supernatant is discarded, and the supernatant is washed by PBST washing liquid for 6 times;

(5) adding 50 mul of DAB color development liquid into the concave hole, and adding 2M H2SO 450 mul of stop solution to terminate the reaction after the light shielding action at room temperature is carried out for 6-8 min;

(6) OD450 values were measured with a microplate reader.

The measurement results are shown in FIG. 1; the ELISA result shows that the ELISA titer of the No.1 mouse is up to 1: 204800 and 1 mouse is selected for cell fusion to prepare monoclonal antibody.

5. Cell fusion

(1) Preparation of splenocytes: and (3) introducing the BALB/c mouse No.1 after 5 days of hyperimmunization to the neck for death, and disinfecting the mouse body surface by using 75% alcohol. Taking out the spleen of the mouse by aseptic operation, washing the spleen with GNK solution preheated at 37 ℃ for 2 times, adding a little HAT culture medium, shearing the spleen on sterile 120-mesh nylon gauze by using small scissors, filtering spleen cells into an aseptic beaker, transferring the aseptic beaker into an aseptic cell centrifuge tube, centrifuging the aseptic beaker for 10 min at 1000 r/min, and washing the cells with GNK solution for 1-2 times for later use.

(2) Cell fusion and culture of fused cells: cell fusion is carried out by adopting a polyethylene glycol method, and spleen cells of an immunized mouse and mouse myeloma cells SP2/0 are mixed according to the cell number of 10: 1, the fused cells were gently suspended in HAT selection medium, the fused cells were dispersed in 96-well cell culture plates at a volume of 220. mu.l/well, and the plates were incubated at 37 ℃ and 5% CO₂Culturing in an incubator, observing small cell masses by a microscope after culturing for 3-4 days, changing a HT culture medium for half amount of liquid change after 7d fusion, sucking 25 mu l of cell culture supernatant at 10 d, and performing primary screening by ELISA.

Example two: identification of hybridoma cell strain secreting anti-HPV 39L 1 protein monoclonal antibody

1. Screening and identification of hybridoma cells and subcloning

(1) ELISA screening

In the first round of ELISA screening, SUMO 1-39L 1 recombinant protein (diluted by CBS 1: 100) is used as a coating antigen, 22 ELISA plates are coated to perform primary screening on culture supernatant of hybridoma cells; and transferring the preliminarily screened 8 hybridoma cell lines to 1 24-hole cell plate for continuous culture.

In the second round of ELISA screening, SUMO 1-39L 1 purified protein (diluted by CBS 1: 100) and SUMO-tag recombinant protein ultrasonication supernatant (diluted by CBS 1: 1000) are respectively selected as coating antigen, and after false positive is eliminated by detection of SUMO-tag recombinant protein, 4 positive hybridoma cell strains are screened out.

And the third round of ELISA screening selects HPV39 PsV prepared in advance as a coating antigen (diluted by CBS 1: 100), and the positive hybridoma cells are subjected to the third round of ELISA screening, so that 3 positive hybridoma cell strains are screened.

(2) Subcloning of positive cells and further screening and identification

Positive hybridoma cells were subcloned by limiting dilution.

Diluting the positive hybridoma cells to about 3cells/ml with 1640/10 complete medium, adding 100 mul per well into a 96-well plate pre-paved with 100 mul feeder cells, and placing at 37 ℃ with 5% CO₂Culturing for 6-8 days in an incubator, screening and identifying ELISA, transferring the positive monoclonal cell strain into a 24-hole cell culture plate for amplification culture, performing subcloning for 2-3 times if necessary until a hybridoma cell strain capable of stably secreting an anti-HPV 39 monoclonal antibody is obtained, namely obtaining the target hybridoma cell 4B4, performing amplification culture on the screened positive monoclonal cell with the cell number of 1-2 multiplied by 10⁶Freezing and storing in a tube.

2. Monoclonal hybridoma cell stability identification

The obtained positive monoclonal hybridoma 4B4 was serially passaged up to 35 times, and the culture supernatants of the respective passages were subjected to stability measurement by ELISA.

The results are shown in Table 1.

TABLE 1 potency of antibody secretion by hybridoma cells of different generations

。

ELISA results show that the cells transmitted to the corresponding 20-generation hybridoma cell line 4B4 can stably secrete specific monoclonal antibodies, and the result shows that the hybridoma cell line has high degree of monoclonality and stable properties, and can be used as seeds for long-term storage and for large-scale preparation of monoclonal antibodies.

Example three: preparation and purification of anti-HPV 39L 1 protein monoclonal antibody ascites

1. Preparation of anti-HPV 39L 1 protein monoclonal antibody ascites

The monoclonal hybridoma cell strain 4B4 in the second example is subjected to amplification culture, the titer of culture supernatant is measured by an ELISA method, the stable plant shape of the monoclonal cell is ensured, and the collected cells are used for preparing a large amount of monoclonal antibodies, and the specific steps are as follows:

(1) female BALB/c mice born were selected and injected intraperitoneally with 500. mu.l of sterile paraffin to stimulate immune cells to promote proliferation of hybridoma cells.

(2) Observing the state of the mice, and after 7-10 days, each mouse is about 1 multiplied by 10⁷Injecting the amount of each cell into the monoclonal positive cells prepared in advance, and observing the state of the mouse in time;

(3) extracting ascites after 10 days, centrifuging at 8000 r/min at 4 deg.C for 20 min to remove oil and cell precipitate, collecting ascites supernatant, and storing at-80 deg.C;

(4) one week later, the obtained monoclonal hybridoma cells were again injected intraperitoneally at an injection rate of 2X 10⁵(ii) individual cells;

(5) one week later, after the abdomen of the mouse expands, ascites is extracted, the supernatant is taken after centrifugation, and the ascites IgG is crudely extracted by using an ammonium caprylate method;

(6) mouse IgG was purified by DE-52 ion exchange column;

(7) ascites titers were determined by ELISA.

2. Monoclonal antibody ascites ELISA potency assay

(1) Diluting HPV 39L 1 into a coating solution with the concentration of 1 mug/mL by CBS liquid to coat an ELISA plate, sealing at 50 mug/hole for overnight at 4 ℃;

(2) diluting the 4B4 monoclonal antibody by 5% of skim milk in a multiple ratio, sequentially adding the diluted monoclonal antibody into an enzyme label plate at 50 mu L/hole, and incubating the monoclonal antibody at 37 ℃ for 30min, wherein the positive control is a commercial anti-HPV 39L 1 monoclonal antibody (Abcam, UK);

(3) discarding the primary antibody, washing the plate by PBST, cleaning and drying;

(4) adding diluted goat anti-mouse IgG (secondary antibody) marked by HRP into reaction holes, and incubating for 30min at 37 ℃ at 50 mu l/hole;

(5) washing with PBST, and patting to dry;

(6) adding 50 mu l of TMB color developing solution prepared on site into each hole, and carrying out a dark room reaction for 6 min;

(7) adding 50 mul 2M H into each hole₂SO₄Terminating the reaction;

(8) the microplate reader reads the OD450 value of each well.

The ELISA detection result shows that the ascites titer of the monoclonal antibody is 1:1.024 multiplied by 10⁶。

Example three: anti-HPV 39L 1 protein monoclonal antibody performance assay

1. Monoclonal antibody subtype identification

The subclasses and types of mAbs were identified according to the instructions for use of the Mouse Monoclonal Antibody Isotyping Kit. The measurement results are shown in Table 2.

TABLE 2 monoclonal antibody subtype identification

Monoclonal antibody type

IgG1

IgG2a

IgG2b

IgG2c

IgG3

IgM

Kappa

Lambda

4B4

+

-

-

-

-

-

+

-

Note: + indicates positive and-negative.

The identification results of the subclasses and subtypes of the monoclonal antibody show that the subtype of the monoclonal antibody 4B4 is IgG1, and the light chain type is Kappa type.

2. Monoclonal antibody affinity assay

Diluting HPV 39L 1 protein into coating solutions with concentrations of 0.5 mug/mL and 1 mug/mL by CBS solution, respectively coating an ELISA plate by the coating solutions, determining the titer of monoclonal antibody ascites by an indirect ELISA method, drawing corresponding 2 indirect ELISA reaction curves by taking the concentration of monoclonal antibody as abscissa and the OD450 value as ordinate, and calculating the corresponding antibody concentration when the OD450 value of the upper flat section of each curve is taken as 100% on the curves.

The affinity constants of the mabs were calculated according to the formula Kaff = (n-1)/2(n [ Ab '] t- [ Ab ] t), where n = [ Ag ] t/[ Ag' ] t, [ Ag '] t are 2 different concentrations of the coating source, and [ Ab ] t, [ Ab' ] t are antibody concentrations corresponding to 50% OD450 values at the concentrations of the coating sources.

The affinity constant K value of the 4B4 monoclonal antibody is calculated to be 2.56 multiplied by 10 according to the result of the affinity measurement^-10mol/L。

3. Specific identification of monoclonal antibody 4B4

ELISA, Western blot and IFA experiments are respectively used for identifying the specificity of the monoclonal antibody 4B4, and the HPV 39L 1 recombinant protein obtained by prokaryotic expression and other irrelevant proteins of escherichia coli are taken as controls for ELISA detection.

The result shows that the monoclonal antibody 4B4 can specifically react with the HPV 39L 1 recombinant protein and does not react with unrelated proteins.

Taking HPV 39L 1 recombinant protein obtained by prokaryotic expression and other irrelevant proteins of escherichia coli as a control, carrying out SDS-PAGE electrophoresis, then transferring the protein onto a nitrocellulose membrane for Western blot detection, taking 4B4 as a primary antibody, taking goat anti-mouse IgG marked by HRP as a secondary antibody (1: 5000), and developing by using an AEC enzyme substrate kit.

As shown in fig. 2: the monoclonal antibody 4B4 can react specifically with recombinant HPV 39L 1 protein obtained by prokaryotic expression, but not react with unrelated protein.

As shown in fig. 3: the plasmid p39SheLL containing HPV L1 and L2 ORF region gene is used to transfect 293T cell for IFA detection, and the result shows that the monoclonal antibody 4B4 can react specifically with HPV39 and does not react with 293T cell contrast.

4. The cross-reactivity of monoclonal antibody 4B4 with subtypes HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52, and the like.

Respectively identifying the cross-reactivity of the monoclonal antibody 4B4 with subtypes such as HPV16, HPV18, HPV31, HPV35, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58 and the like by ELISA, Western blot and IFA experiments, taking recombinant L1 proteins of the subtypes such as HPV39, HPV16, HPV18, HPV33, HPV45, HPV52, HPV58 and the like obtained by prokaryotic expression and ultrasonic supernatants corresponding to unloaded pE-Sumo and pET32a as negative controls, and carrying out ELISA detection.

The results are shown in FIG. 4: the monoclonal antibody 4B4 can react specifically with HPV 39L 1 recombinant protein and does not react with other 6 subtypes of HPVL1 protein and other unrelated proteins.

Taking L1 recombinant protein ultrasonic supernatant of different subtype HPV obtained by prokaryotic expression to perform SDS-PAGE electrophoresis, then transferring the supernatant to a nitrocellulose membrane to perform Western blot detection, taking 4B4 as a primary antibody (1: 1000), taking HRP-labeled goat anti-mouse IgG as a secondary antibody (1: 2000), and developing with an AEC enzyme substrate kit.

As shown in fig. 5: the monoclonal antibody 4B4 can react specifically with recombinant HPV 39L 1 protein obtained by prokaryotic expression, but not react with other 6 subtypes of HPVL1 protein and other unrelated proteins.

Plasmids p16SheLL, p18SheLL, p31SheLL, p35SheLL, p39SheLL, p45SheLL, p52SheLL, p58SheLL, pVITRO-HPV 51L 1L2, pVITRO-HPV 53L 1L2 and pVITRO-HPV 56L 1L2 containing corresponding HPV L1 and L2 ORF region genes are respectively selected to transfect 293T cells for IFA detection.

As shown in fig. 6: monoclonal antibody 4B4 reacted specifically with HPV39, but reacted weakly or not with other subtypes and 293T controls.

Example four: determination of monoclonal antibody variable region sequence

Extracting positive monoclonal hybridoma cell strain RNA, reverse transcribing into cDNA, and PCR amplifying to obtain heavy chain variable region sequence and light chain variable region sequence of monoclonal antibody.

Designing a heavy chain variable region primer sequence according to the sequence characteristics of the mouse-derived monoclonal antibody:

P1：5’- GGTGCAGATCAGCAGTCTGG -3’；

P2：TGAGGAGACGGTGACCGT-3’。

design of light chain variable region primer sequence:

P3: 5’-ATGGAGTCAGACACACTCCT-3’；

P4: 5’-TTATTTCCAGCTTGGTCCC-3’。

the variable region sequences of the monoclonal antibody 4B4 were prepared by molecular cloning techniques and sequenced by Henan Shang Asia Biotechnology Ltd. The gene sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 4B4 are respectively shown in SEQ ID NO.1 and SEQ ID NO.3, the amino acid sequences of the heavy chain variable region and the light chain variable region of 4B4 deduced from the gene sequences are shown in SEQ ID NO.2 and SEQ ID NO.4, and the sequence structures are shown in tables 3 and 4.

TABLE 3 amino acid arrangement structure of heavy chain variable region

Name (R)	Sequence of
		FR-H1	QSGGGLVQPKGSLRLSCVASGFTFN
CDR-H1	TYAMN
		FR-H2	WVRQAPGKGLEWVA
CDR-H2	RIRTKSNGYATYYADSVKD
		FR-H3	RFTISRDDSQSMVYLQMNNLKTEDTAMYFCVR
CDR-H3	PGGTYFDY
		FR-H4	WGQGTTVTVSS

TABLE 4 amino acid arrangement structure of light chain variable region

Name (R)	Sequence of
		FR-L1	DIVLTQSPASLAVSLGQRATISY
CDR-L1	SASKSVSTSGYSYMH
		FR-L2	WNQQKPGQPPRLLIY
CDR-L2	LVSNLDS
		FR-L3	GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC
CDR-L3	QHIR

While the invention has been described in detail with reference to the drawings and examples, it will be understood by those skilled in the art that various changes, modifications, substitutions, combinations and simplifications can be made therein without departing from the spirit of the invention in which the invention is embodied in the form of an equivalent or equivalent variant.

SEQUENCE LISTING

<110> Zhengzhou university

<120> monoclonal antibody against HPV 39L 1 protein, and preparation and application thereof, Ministry of Henan province reproductive health science and technology research institute (Henan province birth defect intervention engineering research center)

<130> /

<160> 4

<170> PatentIn version 3.2

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Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro

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Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Asp Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys

115 120 125

Claims (10)

1. An anti-HPV 39L 1 protein monoclonal antibody, which is characterized by comprising the following sequences or activity modified sequences thereof:

a heavy chain variable region DNA sequence shown as SEQ ID NO. 1;

a heavy chain variable region amino acid sequence as shown in SEQ ID NO. 2;

a light chain variable region DNA sequence shown as SEQ ID NO. 3;

the variable region amino acid sequence of light chain as shown in SEQ ID No. 4.

2. The anti-HPV 39L 1 protein monoclonal antibody according to claim 1, wherein the heavy chain variable region amino acid arrangement structure is as follows:

name (R) Sequence of FR-H1 QSGGGLVQPKGSLRLSCVASGFTFN CDR-H1 TYAMN FR-H2 WVRQAPGKGLEWVA CDR-H2 RIRTKSNGYATYYADSVKD FR-H3 RFTISRDDSQSMVYLQMNNLKTEDTAMYFCVR CDR-H3 PGGTYFDY FR-H4 WGQGTTVTVSS

。

3. The anti-HPV 39L 1 protein monoclonal antibody according to claim 1, wherein the light chain variable region amino acid arrangement structure is as follows:

name (R) Sequence of FR-L1 DIVLTQSPASLAVSLGQRATISY CDR-L1 SASKSVSTSGYSYMH FR-L2 WNQQKPGQPPRLLIY CDR-L2 LVSNLDS FR-L3 GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC CDR-L3 QHIR

。

4. The anti-HPV 39L 1 protein monoclonal antibody according to claim 1, characterized in that the light chain type of the monoclonal antibody is Kappa and the subtype is IgG 1.

5. The monoclonal antibody against HPV 39L 1 according to claim 1, wherein the ELISA titer of the monoclonal antibody is at least 1:1.024 x 10⁶。

6. The monoclonal antibody against HPV 39L 1 protein according to claim 1, wherein the affinity of the monoclonal antibody is 2.56 x 10^-10mol/L。

7. The use of the monoclonal antibody against HPV 39L 1 protein according to claim 1 in the preparation of an HPV39 detection reagent.

8. The use of the monoclonal antibody against HPV 39L 1 protein according to claim 1 in the preparation of a vaccine.

9. An antigen or antibody detection kit comprising the anti-HPV 39L 1 protein monoclonal antibody of claim 1.

10. The method for preparing the monoclonal antibody against HPV 39L 1 protein of claim 1, comprising the following steps:

(1) immunizing a mouse by taking the purified HPV 39L 1 protein as an antigen;

(2) fusing the immune spleen cells of the mice and myeloma cells of the mice to obtain hybridoma cells;

(3) adopting a method of multiple ELISA detection combination and subcloning to obtain positive hybridoma cells;

(4) extracting positive monoclonal antibody hybridoma cell strain RNA, performing reverse transcription to obtain cDNA, and performing PCR amplification to obtain a heavy chain variable region sequence and a light chain variable region sequence of the monoclonal antibody;

(5) carrying out multiple cloning culture on the positive clone to obtain an HPV 39L 1 monoclonal antibody hybridoma cell strain;

(6) and injecting the hybridoma cell strain into the abdominal cavity of a mouse to produce the monoclonal antibody.

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