Anti-hsp70 protein monoclonal antibody and its preparation method and application

Abstract

The present application relates to a kind of monoclonal antibody that can identify human HSP70 antigen, its preparation method and its use in immunodetection.The present application provides a kind of anti-HSP70 protein rabbit monoclonal antibody, the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1;The amino acid sequence of the variable region of the light chain of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.2.The antibody has high specificity, sensitivity, and can specifically recognize the cell expressing HSP70 protein, is suitable for immunological detection, especially immunohistochemical detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, and particularly relates to an anti-HSP70 protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] Heat shock proteins (HSP) exist widely in organisms and have high conservation. When cells are stimulated by adverse factors such as high temperature, hypoxia, infection and inflammatory factors, the production of HSP can be induced. Therefore, HSP is also called stress protein. HSP helps maintain cellular homeostasis by promoting protein folding and maintaining the natural structure and function of various types of proteins under stress. According to the relative molecular mass size, mammalian HSP includes seven family members of HSP27, HSP40, HSP60, HSP70, HSP90, HSP110 and glucose regulated protein 170 (GRP170). HSP70 has the highest content in cells and is the most important family in HSP. HSP70 plays an important role in guiding protein folding, maintaining protein homeostasis and promoting cell survival under various stress conditions (Fernandez-Fernandez MR1 and Valpuesta JM1, 2018). Studies have found that HSP70 is highly expressed in malignant tumors such as liver cancer. In patients with hepatocellular carcinoma (HCC), overexpression of HSP70 is associated with increased carcinogenicity and decreased survival rate, which is of great significance for the diagnosis and treatment of hepatocellular carcinoma.

[0003] Studies have shown that HSP70 is not expressed or weakly expressed in liver precancerous lesions and proliferative nodules, but is significantly increased in liver cancer tissues. The expression level is related to the degree of malignancy of cancer tissues. With the progression of the disease, the cancer focus increases, and the expression level of HSP70 gradually increases. Accordingly, the content of HSP70 secreted and released into the blood circulation by tumor cells also increases. Therefore, it is considered that the detection of HSP70 in tissues or serum is a sensitive indicator for the early diagnosis and differential diagnosis of hepatocellular carcinoma patients. Hu Peizhen (2001) used immunohistochemical technology to detect the expression of HSP70 in primary liver cancer tissues and found that the expression level of HSP70 in liver cancer tissues was significantly increased. In view of the high expression of HSP70 in liver cancer tissues, the American Association for the Study of Liver Diseases (AASLD) hepatocellular carcinoma diagnosis and treatment guidelines recommend (Wang Liling et al., 2011) that for tissue specimens that are uncertain to be liver cancer tissues, biomarkers such as HSP70 can be used for immunohistochemical detection, so as to enhance the accuracy of diagnosis. SUMMARY

[0004] The present application provides an anti-HSP70 protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 1; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO. 2. (The CDR region is underlined) SEQ ID NO. 1:

[0005] EVQLQESGPELVKPGASMKISCLASGYSFT GYLKF WVKQSHGKNLEWIG LLNPWNCDTLYMQTAKG KATLTLDKSSSTAYMEFLSLTSEDSAVYYCAR DSYDYENWAFY WGQGTLVTVSA

[0006] SEQ ID NO. 2:

[0007] QAVVTQESALTTSPGETVTLTC TSATVAIFTLYNCL WVQEKPDHLFTGLIG MYTNAPR GVPARFSGSLIGDKAALTITGAQTEDEAIYFC SYNWVALWHF FGGGTKLEIK

[0008] Further, the monoclonal antibody specifically recognizes the HSP70 protein.

[0009] Further, the monoclonal antibody is a rabbit monoclonal antibody.

[0010] Further, the clone number of the rabbit monoclonal antibody is 27B15.

[0011] The present application also provides a preparation method of the anti-HSP70 protein monoclonal antibody, wherein the antigen for immunizing the rabbit is a recombinant protein, and the recombinant protein is expressed by E. coli.

[0012] Further, the recombinant protein comprises a GST protein tag, a HSP70 protein fragment and a HIS protein tag.

[0013] Further, the HSP70 protein fragment is a fragment of 116-274 sites, and the amino acid sequence of the fragment is shown in SEQ ID NO. 3.

[0014] SEQ ID NO. 3:

[0015] PEEISSMVLTKMKEIAEAYLGYPVTNAVITVPAYFNDSQRQATKDAGVIAGLNVLRIINEPTAAAI

[0016] AYGLDRTGKGERNVLIFDLGGGTFDVSILTIDDGIFEVKATAGDTHLGGEDFDNRLVNHFVEEFKR

[0017] KHKKDISQNKRAVRRLR

[0018] Further, the plasmid vector selected in the recombinant process is Pet30a-GST.

[0019] The inventor also provides an HSP70 protein immunological detection reagent, which contains the above-mentioned anti-HSP70 protein monoclonal antibody as an effective component.

[0020] Differing from the prior art, the beneficial technical effects of the present application are: the above-mentioned technical solution provides an anti-HSP70 protein rabbit monoclonal antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2. The antibody has high specificity and sensitivity, can specifically recognize cells expressing HSP70 protein, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a gel map of the fusion histidine tag HSP70 recombinant peptide segment purification result of Example 1.

[0022] Figure 2 It is a comparison chart of immunohistochemical staining results of breast cancer; the left is rabbit monoclonal antibody HSP70 (27B15), and the right is commercially available HSP70. DETAILED DESCRIPTION

[0023] In order to explain the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved of the present application in detail, the following will be described in detail in combination with the specific embodiments listed and with the aid of the drawings. The embodiments described in this paper are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0024] In this paper, the term "embodiment" means that the specific features, structures or characteristics described in combination with the embodiment can be included in at least one embodiment of the present application. The term "embodiment" appearing at various positions in the specification does not necessarily refer to the same embodiment, and does not particularly limit the independence or association between other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, each technical feature mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

[0025] Unless otherwise defined, the meanings of technical terms used in the present application are the same as commonly understood by one of ordinary skill in the art to which the present application belongs; the use of related terms in the present application is only for the purpose of describing specific embodiments and is not intended to limit the present application.

[0026] In the description of the present application, the phrase "and / or" is a description of the relationship between objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in the present application generally represents that the associated objects before and after are a "or" logical relationship.

[0027] In the present application, the phrases such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0028] In the present application, the phrases "including", "containing", "having" or other similar expressions used in the sentence are intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of additional elements in the process, method or product including the described elements, so that the process, method or product including a series of elements can not only include those limited elements, but also include other elements not explicitly listed, or also include the elements inherent to such process, method or product.

[0029] As the same understanding as in the "Guidelines for Examination", in the present application, the expressions such as "greater than", "less than", "exceed" are understood as not including the number; the expressions such as "above", "below", "within" are understood as including the number. In addition, in the description of the embodiments of the present application, the meaning of "multiple" is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly limited.

[0030] Preparation of recombinant HSP70 protein fragment of Example 1

[0031] I. Gene optimization and synthesis

[0032] According to the protein sequence with accession number P0DMV8 in Uniprot database, the protein fragment of 116-274 sites is selected, which is the amino acid sequence shown in SEQ ID NO. 3; and the gene fragment is optimized to be suitable for expression in Escherichia coli BL21 (DE3). In the process of PCR, BamH I and Xho I enzyme cutting sites are added at the 5' and 3' ends of the gene, respectively.

[0033] PCR products were recovered after agarose gel electrophoresis, and the recovered fusion protein gene and plasmid vector Pet30a-GST for expression were subjected to BamH I and Xho I enzyme digestion, and then recovered after electrophoresis again, and ligated with T4 DNA ligase. The ligation product was transformed into E. coli competent cells BL21 (DE3), and the colonies on the plate were inoculated and subjected to bacterial liquid PCR identification. The colonies with positive PCR results were selected for sequencing analysis, and the colonies with completely correct sequences were used.

[0034] Different antigens were selected for immunization, and different antibodies with different binding properties were prepared. The presence of multiple variants caused by variable splicing resulted in different recognition abilities and patterns of different antibodies to cells expressing antigens. According to the published sequence, the HSP70 molecule was analyzed, and based on the structure on the cell, antigenicity, hydrophilicity and hydrophobicity of the constituent amino acids, and secondary structure, a suitable soluble expression region with good immunogenicity was selected for recombinant expression. The gene sequence of 116-274 sites of HSP70 was selected for codon optimization, and the molecular weight was about 74 kDa. The recombinant HSP70 peptide segment was obtained by designing the sequence optimization of prokaryotic expression gene sequence. The recombinant immunogen is composed of the HSP70 recombinant peptide segment with antigenicity and the protein tag for purification of the recombinant protein. The protein tags are GST and HIS.

[0035] II. Protein expression and purification

[0036] The single colony culture was transferred to 100 mL LB medium at a ratio of 1:100, and ampicillin was added at a final concentration of 10 μg / mL. The culture was incubated at 37°C with shaking until the OD600 was 0.6-0.8. Then, 1 mmol / L IPTG was added, and the culture was incubated at 16°C with shaking for 4 h. After harvesting the bacteria, ultrasonic disruption was performed. The recombinant protein with a histidine tag was subjected to affinity purification using a nickel column. Elution was performed using 500 mmol / L imidazole, and SDS PAGE separation and detection were performed.

[0037] Figure 1 The gel map of the purified recombinant HSP70 protein with a fusion histidine tag is shown. The final purified protein concentration was 0.5 mg / mL, which can be used for animal immunization and antibody screening and identification requirements.

[0038] Example 2: Single B cell sorting for producing rabbit anti-human HSP70 monoclonal antibodies

[0039] I. Immunization and ELISA detection

[0040] The recombinant HSP70 protein in Example 1 was emulsified with Freund's complete adjuvant, and 6 rabbits were selected for immunization, with a dose of 0.7 mg per rabbit. One-time booster immunization was carried out on the 7th, 19th, 40th, 61st, and 112th days, respectively, and the antigen was emulsified with Freund's incomplete adjuvant, with a dose of 0.35 mg per rabbit. Impact immunization was carried out on the 126th day, and the antigen was mixed with normal saline, with a dose of 0.7 mg per rabbit. After the third, fourth, and fifth immunization, serum ELISA titer detection, endogenous WB detection, and IHC detection were carried out, and according to the results (i.e. using positive control photos corresponding to the antibody for detection, and immunohistochemical staining can be observed on the corresponding detection site in the positive control photos), one of the rabbits (No. E10037) was selected for subsequent monoclonal antibody screening.

[0041] II. Spleen cell separation and B lymphocyte sorting

[0042] Monoclonal antibody preparation was carried out on the selected rabbit. The spleen was taken 3 days after impact immunization, the rabbit spleen was placed in RMPI basic medium containing 100 U / ml penicillin and 100 ug / ml streptomycin, cut into pieces with a surgical blade, and then transferred to a 100 μm cell screen for grinding. The obtained cell suspension was filtered to remove large cell clumps and tissue envelopes, and after centrifugation at 400 g for 5 minutes, the supernatant was removed and the spleen cell clumps were retained. The spleen cell clumps were resuspended with a hypotonic solution and the red blood cells were lysed, and then centrifuged at 400 g for 5 minutes, and the spleen cells were retained. The spleen cells were resuspended with RMPI basic medium containing 100 U / ml penicillin and 100 μg / ml streptomycin, centrifuged at 400 g for 5 minutes, and the obtained spleen cells were resuspended with complete medium (RMPI basic medium containing 10% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin) for standby.

[0043] The specific steps of B lymphocyte sorting are described in Chinese Patent 201910125091.4 "Method for efficiently separating single antigen-specific B lymphocytes from spleen cells".

[0044] About 2000 single B cell clones were sorted and cultured, and positive clones specifically recognizing the recombinant HSP70 protein in Example 1 were preliminarily screened by ELISA. According to the ELISA data, 50 supernatants from high to low were selected for IHC verification.

[0045] III. B cell culture positive clone detection

[0046] A single B cell culture supernatant was verified by IHC using a multi-tumor tissue chip and a normal tissue chip containing HSP70 positive and negative, and the IHC results of 13 clone culture supernatants were better. LEM supernatant was prepared for IHC verification to determine the clone (27B15) with excellent sensitivity and specificity.

[0047] The sensitivity and specificity screening criteria are: using the positive control photo corresponding to the antibody for detection, and the corresponding detection site in the positive control photo can observe immunohistochemical staining, while no immunohistochemical staining is observed at the non-detection site. Compared with the control antibody, the staining intensity reaches or is even higher than that of the control antibody.

[0048] IV. Cloning of the gene encoding the rabbit monoclonal antibody (27B15) and construction of the rabbit monoclonal antibody expression plasmid

[0049] After the cell collection and lysis of the positive clones, RNA is extracted and reverse transcribed into cDNA. Using PCR method, the naturally paired rabbit monoclonal antibody light and heavy chain variable region genes are amplified from the cDNA corresponding to the positive clones. The rabbit monoclonal antibody expression vector plasmid is constructed using the above-mentioned naturally paired rabbit monoclonal antibody light and heavy chain variable region gene sequences respectively.

[0050] Example 2 was completed by Wuhan Aibotek Biotechnology Co., Ltd. commissioned by Fujian Maynew Biotechnology Development Co., Ltd., in which immunohistochemical screening was performed by Fujian Maynew Biotechnology Development Co., Ltd.

[0051] Example 3 HSP70 Rabbit Monoclonal Antibody Expression

[0052] I. Plasmid amplification and extraction

[0053] Take one tube (100 μl) of competent bacteria (DH5a), insert into ice, ice bath for 5-10 min; add 5 μl of plasmid, gently shake and place on ice for 30 min; gently shake and place in a 42°C water bath for 90 s for heat shock, then quickly place back on ice, stand for 5 min; add 800 μl of LB medium (note: without antibiotics) to the above-mentioned mixture in the clean bench, mix gently, and fix on a shaker at 37°C for 1 h; take 50-100 μl of the above-mentioned transformation mixture in the clean bench, add to the labeled solid LB plate containing Amp, and evenly spread with a glass spreader (sterilized); first stand in a 37°C incubator for 30 min, allowing the surface bacteria to completely penetrate the medium, then invert and place in a 37°C incubator for overnight culture. Use a gun head to pick a single colony into 4 ml of LB medium (containing 2 uL of 200 mg / ml Amp), and culture at 37°C for 16 h at 220 rpm.

[0054] Add 1 ml of bacteria to 100 ml of LB medium (containing 50 μl of 200 mg / ml Amp), and culture at 37°C for 16 h on a shaker; use SanPrep endotoxin-free DNA small amount extraction kit (Shenguo), and follow the instructions for plasmid extraction.

[0055] II. Transfection

[0056] Adjust the concentration of 293F cells to 2.5-3 x 10 6 viable cells / ml with Expi293F™ Expression Medium and incubate overnight.

[0057] 1) Count the viable cells with a hemocytometer, the viable cell concentration is about 4.5-5.5 x 10 6 viable cells / ml, and the viable cell number should meet the requirements of the expression system;

[0058] 2) Dilute the cell concentration to 3 x 10 6 viable cells / ml with Expi293F™ Expression Medium;

[0059] 3) Add the plasmid DNA to Opti-MEM™ I Reduced Serum Medium, gently blow, and mix well by inverting;

[0060] 4) Gently invert ExpiFectamine TM 293 Reagent 4-5 times, mix ExpiFectamine TM 293 Reagent and Opti-MEM™ I Reduced Serum Medium, gently blow and invert 2-3 times, and place at room temperature for 5 min;

[0061] 5) Mix the solutions of steps 3) and 4), gently blow and invert 2-3 times, and mix well;

[0062] 6) Place the solution of step 5) at room temperature for 10-20 min;

[0063] 7) Slowly suck the mixture into the cell culture solution, and gently shake the flask;

[0064] 8) Incubate at 37℃ with 8% CO2 for 5-7 days;

[0065] 9) After 18-22 hours, add ExpiFectamine TM 293 Transfection Enhancer1 and ExpiFectamine TM 293 Transfection Enhancer2 (Note: mix in advance before use), and gently shake to mix well, and continue to culture.

[0066] III. Purification of Monoclonal Antibodies

[0067] The antibodies were purified from the supernatant by HiTrap rHSP70otein A FF affinity chromatography according to the manufacturer's instructions. The purity was determined by SDS-PAGE and the concentration was determined by the Bradford method. The purified antibodies were stored at -20°C.

[0068] Example 4. Immunohistochemical tissue microarray staining and identification

[0069] I. Preparation of the microarray

[0070] Each sample was first stained by HE section to determine the tumor site. The tumor target site was circled and prepared for punching. When making the blank receptor wax block, a plastic frame was placed on the mold, and the melted wax (melting point at 55-58°C) was poured into the mold. After cooling to room temperature, the mold was placed in a -20°C refrigerator for 6 min, and the wax block was taken out of the mold. A 1 mm diameter sample needle was selected on the tissue sample machine to punch a hole in the receptor wax block, with a hole depth of 3-4 mm. Another 1 mm diameter punching needle was used to punch a hole in the marked part of the wax block to collect the tissue core, with a length of about 0.1 mm shallower than the hole depth of the receptor wax block. The collected tissue core was directly inserted or carefully clamped with tweezers into the empty hole of the receptor wax block. This was repeated until all sample points were prepared. Finally, a glass slide was used to flatten all the tissue cores, making the tissue chip wax block flat and smooth. The prepared tissue chip wax block was placed in the wax block making mold and placed in a 60°C oven for 15 min to fuse the tissue core and the wax of the receptor wax block into one. Then the mold was gently taken out of the oven, and the semi-melted wax was cooled at room temperature for about 30 min. Then the tissue chip wax block was taken out of the mold after being frozen in a -20°C refrigerator for 6 min, and was sectioned or stored in a 4°C refrigerator for future use. After trimming, continuous sections were cut with a thickness of 3 μm. The continuous sections were floated in cold water to naturally expand, and then the separated sections were transferred to warm water at 45°C for 30 seconds. The sections were mounted on a glass slide treated with polylysine, and the prepared tissue chip was placed in a 65°C oven for 2 hours for sectioning. After cooling at room temperature, the tissue chip was stored in a -4°C refrigerator.

[0071] II. IHC staining and analysis

[0072] Routine dewaxing in xylene for 3 times, 6 minutes each time, hydration in 100%, 100%, 95%, 85% gradient ethanol for 3 minutes each time, and finally tap water flushing. Antigen retrieval was performed, and then the sections were placed in a wet box and washed with PBS for 3 x 3 minutes. 3% H2O2 was added dropwise and incubated for 10 minutes, and then washed with PBS for 3 x 3 minutes. The sections were spun dry, and the primary antibody was added dropwise at an appropriate dilution (the dilution ratio of the antibody was designed according to the concentration of the antibody for the first dilution) and incubated at room temperature (25°C) for 1 hour, washed with PBS for 3 x 3 minutes, the secondary antibody was added dropwise and incubated at room temperature for 15-30 minutes, washed with PBS for 3 x 3 minutes, the PBS was spun off, and fresh DAB developing solution was used for color development for 3-10 minutes. Hematoxylin counterstaining was performed for 25 seconds, and PBS was returned to blue for 30 seconds. Sequential dehydration was performed according to an alcohol gradient of 85% (3 minutes)-95% (3 minutes)-100% (3 minutes)-100% (3 minutes), and finally xylene clearing was performed for 3 minutes, and neutral resin was used for mounting.

[0073] The immunohistochemical staining results are divided into positive and negative. Positive expression must be at the site of cell- and tissue-specific antigens to be considered positive. In the case of clear tissue staining distribution and accurate cell localization, the staining results are further divided according to the difference in staining intensity, as follows:

[0074] 1. The sample is weakly positive, marked as "+";

[0075] 2. The sample is moderately positive, marked as "++";

[0076] 3. The sample is highly positive, marked as "+++".

[0077] 4. The sample is negative, marked as "-".

[0078] III. Data Statistics

[0079] 1. Tumor tissue chip detection results:

[0080] The antibody HSP70 (27B15) of the present application and the commercially available antibody HSP70 (rabbit polyclonal antibody) were simultaneously detected in 25 cases of hepatocellular carcinoma and the detection results were compared. The immunohistochemical results of HSP70 were statistically analyzed. The entire test process adopted a double-blind design, and the statistical results are as follows:

[0081]

[0082] The results show that the rabbit monoclonal antibody HSP70 (27B15) has accurate staining localization, clear staining, no non-specific staining, and a clean background. In the immunohistochemical detection, the positive rate is higher than that of the commercially available antibody, and the positive intensity is higher than that of the commercially available antibody. There is 1 case of hepatocellular carcinoma, and the control HSP70 antibody is negative, while the HSP70 antibody of the present application is weakly positive. This indicates that it has higher sensitivity and effectively avoids false negative results.

[0083] Figure 2 Figure 6: Comparison of immunohistochemical staining results for hepatocellular carcinoma (left: rabbit monoclonal antibody HSP70 of the present application, right: commercially available HSP70).

[0084] 2. Normal tissue chip test results:

[0085] The normal tissue chip includes 30 normal tissue samples, which are mainly selected from fresh and timely fixed surgical specimens; each tissue includes 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectum, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infant), skin, bone marrow, peripheral nerve, lung, mesothelial cell.

[0086] The rabbit monoclonal antibody HSP70 (27B15) and the commercially available HSP70 antibody were simultaneously tested on the normal tissue chip, and the positive and negative test results were consistent, indicating that the specificity of the antibody in the normal tissue was equivalent to that of the commercially available antibody.

[0087] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of the present application, the patent protection scope of the present application should not be limited. Any equivalent structure or equivalent flow replacement or modification based on the essential concept of the present application, using the content described in the specification and drawings, and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, etc., are all included in the patent protection scope of the present application.

Claims

1. An anti-HSP70 protein monoclonal antibody, characterized by, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody specifically recognizes HSP70 protein.

3. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is a rabbit monoclonal antibody.

4. An HSP70 protein immunoassay test reagent, characterized by, The immunodetection reagent contains the anti-HSP70 protein monoclonal antibody of claim 1 as an effective component.

5. The immunoassay test reagent of claim 4, wherein the antibody is a monoclonal antibody. The immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

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