CN116120436B

CN116120436B - anti-HBcAg protein monoclonal antibody and cell strain, preparation method and application thereof

Info

Publication number: CN116120436B
Application number: CN202211471983.8A
Authority: CN
Inventors: 黄林军; 杨清海; 陈惠玲; 王小亚
Original assignee: Fuzhou Maixin Biotech Co ltd
Current assignee: Fuzhou Maixin Biotech Co ltd
Priority date: 2022-11-23
Filing date: 2022-11-23
Publication date: 2023-10-27
Anticipated expiration: 2042-11-23
Also published as: CN116120436A

Abstract

The application relates to a monoclonal antibody capable of recognizing human HBcAg antigen, a secretory cell strain, a preparation method thereof and application thereof in immunodetection. According to the technical scheme, the natural HBcAg protein is selected to immunize a mouse, and the cell fusion, screening and subcloning are carried out to obtain the mouse hybridoma cell strain 4C2 capable of efficiently secreting the anti-HBcAg protein monoclonal antibody and the anti-HBcAg protein monoclonal antibody secreted by the cell strain. The antibody obtained by the scheme has high specificity and sensitivity, can specifically identify cells expressing HBcAg protein, and is suitable for immunological detection, in particular immunohistochemical detection.

Description

anti-HBcAg protein monoclonal antibody and cell strain, preparation method and application thereof

Technical Field

The application relates to the field of biomedical engineering, in particular to an anti-HBcAg protein monoclonal antibody, a cell strain, a preparation method and application thereof.

Background

It is counted that about 3000 thousands of Chronic Hepatitis B (CHB) patients exist in China at present, and about 30 thousands of patients die from hepatitis B related liver cirrhosis, liver cancer and end-stage liver disease every year, so that a heavy economic burden is brought. The long-term follow-up of 101 patients with HBeAg positive CHB is carried out, and the results show that the expression of HBcAg in the liver of the patients with CHB has different expression types and different expression intensities at the antiviral baseline, the serological conversion rate of HBeAg of the patients with serous and nuclear serous expression after nucleoside antiviral treatment is higher, and the serological conversion rate of HBeAg of the patients with III-level HBcAg expression intensity is also higher.

Research shows that HBcAg has strong immunogenicity, is favorable for activating immune cells of an organism, but is mainly expressed in liver cells and seldom secreted into peripheral blood, so that the regulation effect of HBcAg on the immunity is affected. Wherein, the total anti-HBc and IgM anti-HBc in serum of plasma type and nuclear plasma type expression patients and patients with higher expression of HBcAg in liver are obviously increased, so that when liver inflammation is damaged, HBcAg is exposed or released into blood circulation, and immune cells are activated. Thus, the type and level of expression of HBcAg in hepatocytes may be one of the factors affecting immune function and antiviral efficacy in patients.

Past clinical practice has shown that HBcAg is difficult to detect in serum, mainly because it is not exposed due to HBsAg encapsulation, and it is more difficult to directly stimulate immune cells. In the conversion of the intrahepatic HBcAg from cell nucleus to cytoplasm expression, the quantitative conversion of serum total anti-HBc and IgM type anti-HBc has obvious relation, and when HBV infected patients fluctuate, the HBcAg is exposed from the cell nucleus to cytoplasm and even outside the cell membrane after the hepatic cell injury, and the immune reaction of the organism (especially the B cell reaction generates HBV specific antibody) is activated, which explains the immunological mechanism of the quantitative prediction of the HBc to the serological conversion of the HBeAg after the antiviral treatment of CHB patients to a certain extent. In conclusion, the detection of HBcAg in liver cells plays an important role in predicting antiviral efficacy of nucleoside drugs for patients with CHB.

Disclosure of Invention

The inventor provides a monoclonal antibody of anti-HBcAg protein, wherein the amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

Further, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown as SEQ ID NO.2, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown as SEQ ID NO. 3.

Further, the monoclonal antibody specifically recognizes HBcAg protein.

Further, the monoclonal antibody is mouse IgG ^2b Kappa subtype monoclonal antibodies.

Further, the monoclonal antibody is produced by a hybridoma cell strain with a preservation number of CGMCC NO 21974.

The inventor also provides a preparation method of the anti-HBcAg protein monoclonal antibody, wherein an antigen for immunizing mice is HBcAg protein, and the amino acid sequence of the antigen is shown as SEQ ID NO. 1.

The inventor also provides a hybridoma cell strain secreting anti-HBcAg protein molecules, wherein the cell strain is a mouse hybridoma cell strain 4C2, and the cell strain is preserved in China general microbiological culture collection center (CGMCC) NO 21974 at 2021, 3 months and 24 days, and is provided at the microbiological research institute of national academy of sciences of China, 1 st China, 3 rd, beijing, the region of towards the sun.

The inventor also provides an HBcAg protein immunodetection reagent, which contains an amino acid sequence of a heavy chain variable region, wherein the amino acid sequence is shown as SEQ ID NO. 4; the anti-HBcAg protein monoclonal antibody with the amino acid sequence of the light chain variable region shown in SEQ ID NO.5 is taken as an active ingredient.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

The inventor also provides an HBcAg protein histochemical immunoassay reagent which contains an amino acid sequence of a heavy chain variable region shown in SEQ ID NO. 4; the anti-HBcAg protein monoclonal antibody with the amino acid sequence of the light chain variable region shown in SEQ ID NO.5 is taken as an active ingredient.

Compared with the prior art, the application has the following beneficial technical effects: according to the technical scheme, natural HBcAg protein is selected as antigen peptide to immunize a mouse, and the cell fusion, screening and subcloning are carried out to obtain a mouse hybridoma cell strain 4C2 capable of efficiently secreting anti-HBcAg protein monoclonal antibodies and the anti-HBcAg protein monoclonal antibodies secreted by the cell strain. The antibody obtained by the scheme has high specificity and sensitivity, can specifically identify cells expressing HBcAg protein, and is suitable for immunological detection, in particular immunohistochemical detection.

Drawings

FIG. 1 is a comparative graph of immunohistochemical staining results of paracancerous liver tissue; wherein the left is HBcAg secreted by 4C2, and the right is commercial HBcAg.

Detailed Description

In order to describe the possible application scenarios, technical principles, practical embodiments, and the like of the present application in detail, the following description is made with reference to the specific embodiments and the accompanying drawings. The embodiments described herein are only for more clearly illustrating the technical aspects of the present application, and thus are only exemplary and not intended to limit the scope of the present application.

Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the application. The appearances of the phrase "in various places in the specification are not necessarily all referring to the same embodiment, nor are they particularly limited to independence or relevance from other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the technical features mentioned in each embodiment may be combined in any manner to form a corresponding implementable technical solution.

Unless defined otherwise, technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains; the use of related terms herein is for the purpose of describing particular embodiments only and is not intended to limit the application.

In the description of the present application, the term "and/or" is a representation for describing a logical relationship between objects, which means that three relationships may exist, for example a and/or B, representing: there are three cases, a, B, and both a and B. In addition, the character "/" herein generally indicates that the front-to-back associated object is an "or" logical relationship.

In the present application, terms such as "first" and "second" are used merely to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual number, order, or sequence of such entities or operations.

Without further limitation, the use of the terms "comprising," "including," "having," or other like terms in this specification is intended to cover a non-exclusive inclusion, such that a process, method, or article of manufacture that comprises a list of elements does not include additional elements but may include other elements not expressly listed or inherent to such process, method, or article of manufacture.

As in the understanding of "review guidelines," the expressions "greater than", "less than", "exceeding" and the like are understood to exclude this number in the present application; the expressions "above", "below", "within" and the like are understood to include this number. Furthermore, in the description of embodiments of the present application, the meaning of "a plurality of" is two or more (including two), and similarly, the expression "a plurality of" is also to be understood as such, for example, "a plurality of" and the like, unless specifically defined otherwise.

The HBcAg protein used in this example was purchased from the biotechnology company, tokyo, south kyo, and was extracted from viruses, the accession number in Uniprot database was P03146, and the sequence was the amino acid sequence shown in SEQ ID No. 1.

EXAMPLE 1 establishment of hybridoma cell lines

1. Immunization

HBcAg protein (0.01 mg/mL) from Nanjing Jingda Biotechnology Co., ltd was emulsified with Freund's complete adjuvant (Sigma Co., F5881) and immunized with 4-6 week old female ICR mice (from Beijing Venethol Lihua laboratory animal technologies Co., ltd.) at a dose of 20 μg/mouse by abdominal subcutaneous injection at 6 points. Once every 14 days, the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20 μg/dose. The polyclonal antibody titer of the anti-immunogen in the serum of the mice was detected by indirect ELISA (wavelength 450 nm) 7 days after the 3 rd booster immunization, the mice with the highest titer were immunized by tail vein injection, and the antigens were uniformly mixed with physiological saline at a dose of 20 mug/mouse.

2. Cell fusion

Sterile preparation of immune-up to-standard mouse spleen cell suspension, mixing with mouse myeloma cell sp2/0 (ATCC NumberCRL-8287) at a ratio of 5:1, centrifuging at 1500rpm for 5min. The supernatant was discarded, the tube was placed in a 37℃water bath, 1ml of PEG1500 (Roche Co.) was slowly added over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10ml of serum-free IMDM (Sigma Co.) was added, mixed well, centrifuged at 1000rpm for 5min. After the supernatant was discarded, 10ml of serum (PAA company) was added and the cells were carefully blown up, and 5ml of thymocytes mixed with 10xHAT (Sigma company) were added and mixed. 25ml of semisolid medium containing 2.1% nitrocellulose (Sigma Co.) was added and mixed well, and then poured into 20 cell culture dishes uniformly. Placing the cell culture dish into a wet box, placing 5% CO at 37deg.C ₂ Culturing in an incubator.

3. Cloning and ELISA screening positive hybridoma cells

The size and density of the cloned cell mass are moderate 7 days after fusion, round, solid and large clone mass is sucked into a 96-hole culture plate which is prepared with culture medium in advance under a dissecting lens, and is put into 5% CO at 37 DEG C ₂ Culturing in an incubator. After 3 days, the cell mass was approximately 2/3 of the floor space, and 100. Mu.L of the supernatant was screened by ELISA using the immunogen and the synthetic polypeptide, respectively. Positive clones were completely changed and 200. Mu.L of complete medium containing feeder cells and 1% HT (Sigma Co.) was added. Two days later, a second ELISA screening was performed and positive clones were transferred to 24-well plates in which medium (containing feeder cells and HT) was prepared in advance. Five days later, 100 μl of the supernatant was subjected to a third ELISA screening, and positive clones were successively transferred into 6-well plates and cell culture flasks for expansion culture and frozen storage. EXAMPLE 2 preparation of monoclonal antibodies by ascites Induction

1. Ascites preparation

Cells in logarithmic growth phase were washed and suspended in serum-free medium and counted approximately5×10 ⁵ 1ml. Suspended cells were intraperitoneally injected into mice previously sensitized with paraffin oil. Ascites collection was started after 7 days. The ascites removed was centrifuged at 4000rpm at 4℃for 10min. Carefully aspirate the intermediate ascites and collect in centrifuge tubes and store at 4℃or-20 ℃.

2. Purification of monoclonal antibodies

Antibodies were purified from ascites fluid using HiTrap rProtein A FF (GE company) affinity chromatography as described. SDS-PAGE gel was used to identify purity and concentration was determined by the Bradford method. Purified antibodies were stored at-20 ℃.

EXAMPLE 3 characterization of monoclonal antibodies

1. Subclass identification

The coated sheep anti-mouse IgG (Abies sinensis Biotechnology Co., ltd., beijing) was diluted to 0.5. Mu.g/ml with 100mM PBS (pH 7.4), 100. Mu.l/well was added at 4℃overnight. The liquid was emptied and washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. Mu.l of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well and incubated for 1h at 37 ℃. The liquid was emptied and washed 3 times with PBS-T. 0.1ml of hybridoma supernatant was added to each well and incubated at 37℃for 1h. The liquid was emptied and washed 3 times with PBS-T. With blocking liquid 1: HRP-labeled goat anti-mouse (κ, λ) antibody or 1:2000 dilution of HRP-labeled goat anti-mouse (IgM, igG1, igG2a, igG2b, igG3, igA) antibodies (Southern Biotech) 0.1ml were added to the appropriate wells, respectively, and incubated at 37℃for 1h. The liquid was emptied and washed 3 times with PBS-T. Mu.l of a mixture containing 0.15% ABTS (Southern Biotech Co.) and 0.03% H was added to each well ₂ O ₂ The reaction was developed with a citric acid buffer (pH 4.0) and the OD at 405nm was measured in 10-20 min.

The results show that the monoclonal antibody of the application is IgG ₂ ^b Kappa-type murine monoclonal antibodies.

2. Affinity constant determination

The purchased HBcAg protein was coated at a concentration of 2. Mu.g/ml, 100. Mu.l/well, coated overnight at 4℃and washed 3 times with PBS-T. Mu.l of blocking solution was added to each well and blocked at 37℃for 2h, and PBS-T was washed 3 times. The monoclonal antibodies purified in example 3, from 1:200 beginning 2-fold gradient dilution, leaving 1 well blank for control, incubating for 1h at 37℃and washing 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1:diluted 20000, incubated at 37℃for 1h in 100. Mu.l/well and washed 3 times with PBS-T. Mu.l of 0.1% TMB (Sigma Co.) and 0.03% H were added to each well ₂ O ₂ The reaction was stopped by adding 50. Mu.l of 0.5M sulfuric acid solution after 10min of color development in the citric acid-phosphoric acid buffer. The absorbance at a wavelength of 450nm was measured with a microplate reader. Drawing a curve of OD value corresponding to dilution factor of antibody, finding out dilution factor A corresponding to half of maximum binding OD value, and calculating affinity constant of the antibody to 1.92×10 by using the following formula ⁹ 。

Monoclonal antibody reaction specificity and application effect

The recognition specificity of the monoclonal antibody of the present application was detected by immunoblotting using the HBcAg recombinant protein prepared in example 1, and 12% polyacrylamide gel electrophoresis was performed. Gel protein bands were transferred onto PVDF membranes (Millipore Corp.) in a Bio-Rad electrotransfer system according to conventional methods. The membranes were placed in TBS-T blocking solution containing 5% nonfat milk powder overnight at 4 ℃. Monoclonal antibodies (1:1000 dilution) to the antibodies secreted by the 41B1 hybridoma were added and incubated overnight at 4 ℃. After washing the membrane with TBS-T, 1: sheep anti-mouse secondary antibody (fir gold bridge biotechnology limited in beijing) diluted at 5000 was incubated for 1 hour at room temperature. The membrane was again washed with TBST, ECL hypersensitivity developing solution (Beijing pride Gene technologies Co., ltd.) was added, and chemiluminescent image data was collected using a ChemiDocMP multicolor fluorescence imaging system (Bio-Rad).

Example 5 determination of variable region sequence of antibodies

Taking fresh hybridoma cells, taking supernatant, performing antigen binding characteristic verification, confirming that cell strain used for cloning can indeed secrete required antibody, and centrifuging and collecting 10 after confirming the result ⁶ The above hybridoma cells. Trizol method is used to extract total RNA of hybridoma cells, 9. Mu.L of total RNA is taken, 2.5. Mu.L of oligo (dT) 12-18primer (10 mM) and 5. Mu.L of dNTPs are added, and the mixture is uniformly mixed, and the mixture is incubated at 70℃for 5 minutes and then placed on ice for 5 minutes, or subjected to denaturation operation in accordance with the reverse transcriptase used. Then 5. Mu.L RT buffer (5X), 2.5mu.L of DTT (0.1M) and 1. Mu.L of reverse transcriptase were reacted at 42℃for 1 hour. The reaction was terminated by incubating at 70℃for 15 minutes, and the obtained cDNA was stored at-20 ℃. The first strand cDNA obtained was subjected to PCR amplification, 25pmol of each primer was added to a 50. Mu.L reaction system, and the sequences of the primers for heavy chain variable region and light chain variable region amplification were designed and synthesized in accordance with the primer sequences of murine monoclonal antibodies in the book of recombinant antibody (scientific Press, 2005) which was mainly compiled by Shen Beifen.

The rest dNTPs and buffer solution are added according to the conventional method, and finally 1 mu L of cDNA template and 1U of hot start Taq DNA polymerase are added. The PCR amplification procedure was set to 94℃for 40 seconds, 52℃for 40 seconds, 72℃for 40 seconds, 20 to 25 cycles were performed, and finally 72℃was extended for 3 minutes, and the product was allowed to stand by at 4℃or directly electrophoresed. And (3) taking 20 mu L of PCR products for electrophoresis analysis, separating on 1.5% agarose gel, wherein the length of a light chain (kappa light chain) is 320-340bp, the length of a heavy chain is 340-370bp, and performing gel cutting recovery when specific products exist in the region, and cloning to a T vector or an expression vector for sequencing.

EXAMPLE 6 immunohistochemical tissue chip staining and identification

Chip preparation process

HE section staining was performed on each sample to determine tumor sites. The tissue chip was fabricated using a full-automatic tissue chip instrument from 3 DHISTECH. Putting the prepared tissue chip wax block into a wax block manufacturing mould, putting the mould into a 68 ℃ oven for 10 minutes to enable the tissue core and the wax of the receptor wax block to be fused into a whole, then slightly taking out the mould from the oven, cooling the paraffin in a semi-fused state for about 30 minutes at room temperature, putting the mould into a-20 ℃ refrigerator for 6 minutes, taking out the tissue chip wax block from the mould, and slicing or putting the mould into a 4 ℃ refrigerator for preservation for later use. After trimming, continuous slicing is carried out, the thickness is set to be 3 mu m, the continuous slicing is floated in 40% alcohol, the continuous slicing is naturally unfolded, then the separated slices are transferred into warm water with the temperature of 50 ℃ for 30 seconds, the slices are pasted on a glass slide which is treated by polylysine, the prepared tissue chips are placed into an oven with the temperature of 68 ℃ for baking the slices for 2 hours, and the tissue chips are taken out, cooled at room temperature and placed into a refrigerator with the temperature of minus 4 ℃ for preservation.

IHC staining and analysis

Conventional xylene dewaxing 3Hydration in 100%, 95%, 85% gradient ethanol for 6min each time, 3 min each time, and finally tap water washing. Antigen retrieval was performed and the sections were then placed in a wet box and rinsed 3 x 3 min in PBS. Dropwise adding 3%H ₂ O ₂ Incubate for 10min, rinse with PBS for 3 x 3 min. The sections were spun dry, incubated for 1 hour at room temperature (25 ℃) with primary antibody diluted in the appropriate ratio (the dilution ratio of the antibody was designed according to the concentration of the antibody for the first time), washed 3X 3 minutes with PBS, incubated for 15-30 minutes with secondary antibody at room temperature, washed 3X 3 minutes with PBS, spun off the PBS, and developed for 3-10 minutes with freshly prepared DAB developing solution. Hematoxylin counterstain for 25 seconds and PBS returns to blue for 30 seconds. Sequentially dehydrating according to an alcohol gradient of 85% (3 minutes) to 95% (3 minutes) to 100% (3 minutes), and finally making xylene transparent for 3 minutes, and sealing the gel.

Immunohistochemical staining results were divided into: positive and negative. Positive expression must be positive at the cell and tissue specific antigenic sites. Under the conditions of clear tissue staining distribution and accurate cell positioning, the staining results are further divided according to the difference of staining intensity, and the specific steps are as follows:

1. the sample is weak positive; marked as "+";

2. the sample is moderately positive; marked as "++";

3. the sample was highly positive; marked as' ++ ".

4. The sample was negative, labeled "-".

Third, data statistics

1. Tumor tissue chip detection results:

the present antibody HBcAg (4C 2) and commercially available antibody HBcAg (rabbit polyclonal antibody) were synchronously detected in liver tissues beside 20 cases of cancer, and the detection results were compared.

The immunohistochemical results of HBcAg were counted. The whole test process adopts double-blind design, and the statistical result is as follows:

the result shows that the anti-HBcAg protein monoclonal antibody secreted by the 4C2 cell strain has accurate dyeing location, clear dyeing, no nonspecific dyeing and clean background. In immunohistochemical detection, the positive rate was comparable to that of commercially available antibodies, but the positive intensity was higher than that of commercially available antibodies. The HBcAg secreted by the 4C2 cell strain has higher sensitivity, and false negative results are effectively avoided.

FIG. 1 is a graph showing the results of immunohistochemical staining of paracancerous liver tissue (left is 4C2 secreted HBcAg, right is commercially available HBcAg).

2. Normal tissue chip detection results:

the normal tissue chip comprises 30 normal tissue samples, wherein the normal tissue samples are mainly selected from fresh and timely fixed surgical specimens; each tissue included 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsils, skeletal muscle, thymus (young children), skin, bone marrow, peripheral nerves, lung, mesothelial cells.

The antibody (4C 2) and the commercial antibody are synchronously detected on a normal tissue chip, and the detection results of yin and yang are consistent, which shows that the specificity of the antibody in normal tissues is equivalent to that of the commercial antibody.

Finally, it should be noted that, although the embodiments have been described in the text and the drawings, the scope of the application is not limited thereby. The technical scheme generated by replacing or modifying the equivalent structure or equivalent flow by utilizing the content recorded in the text and the drawings of the specification based on the essential idea of the application, and the technical scheme of the embodiment directly or indirectly implemented in other related technical fields are included in the patent protection scope of the application.

Claims

1. A monoclonal antibody of anti-HBcAg protein, which is characterized in that the amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

2. The monoclonal antibody according to claim 1, wherein the DNA sequence encoding the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No.2 and the DNA sequence encoding the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No. 3.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes HBcAg protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG2b kappa subtype monoclonal antibody.

5. A monoclonal antibody of anti-HBcAg protein is produced by a hybridoma cell strain with a preservation number of CGMCC NO 21974.

6. A hybridoma cell strain secreting anti-HBcAg protein molecules, wherein the cell strain is a mouse hybridoma cell strain 4C2, and the cell strain is preserved in the China general microbiological culture collection center with the preservation number of: CGMCC NO 21974.

7. An immunoassay reagent for HBcAg protein, characterized in that it comprises the monoclonal antibody against HBcAg protein according to claim 1 as an active ingredient.

8. The immunoassay reagent of claim 7, wherein said immunoassay comprises immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

9. An immunohistochemical detection reagent for HBcAg protein, characterized in that the reagents for immunohistochemical detection comprise the monoclonal antibody against HBcAg protein according to claim 1 as an active ingredient.