CN100402554C - Preparation and application of testis-specific protein 50 human antibody - Google Patents

Abstract

The invention provides a method for preparing a human single-chain antibody of testis-specific protein 50, using phage antibody display technology, taking human testis-specific protein 50 as a target antigen, and screening the variable region of recombinant phage human antibody by immunoaffinity screening method Gene library screening. The TSP50-specific single-chain antibody genes AI, AII and C8 were isolated and cloned. Then, the single-chain antibody genes AI, AII and C8 were respectively inserted into the prokaryotic expression vector PelB containing histidine tails, transformed into Rosetta bacteria, induced to express foreign proteins by IPTG, and the cells were lysed and then used for affinity with Ni-NTA column Chromatography to obtain a purified single-chain antibody, which can be specifically combined with TSP50 whole protein and 50 peptide by ELISA; and the human single-chain antibody is expressed in large quantities by means of genetic engineering. The invention also discloses the use of the testis-specific protein 50 human single-chain antibody in the diagnosis and treatment of TSP50-related diseases such as female breast cancer.

Description

Preparation and application of testis-specific protein 50 human antibody

technical field

The invention belongs to the field of humanized genetic engineering antibodies, and relates to the preparation and application of a human single-chain antibody of testis-specific protein 50 closely related to breast cancer and other tumors. This antibody can be used for the diagnosis and treatment of TSP50-related diseases and belongs to The technical field of biogenetic engineering.

Background technique

Breast cancer is a common malignant tumor in women, and its incidence rate is increasing year by year. From the perspective of epidemiological development trend, breast cancer is more and more concentrated in big cities, ranking first among female malignant tumors. At present, the diagnosis of breast cancer is mainly through imaging and biopsy, and there is no effective early diagnosis method. The treatment of breast cancer is still based on surgery, supplemented by radiotherapy and chemotherapy. With the development of society, radical mastectomy will be replaced by breast-conserving surgery, which is more prone to postoperative recurrence and metastasis, and the side effects and pain caused by radiotherapy and chemotherapy are obvious to all. Therefore, biological treatment will become one of the more promising treatment methods, and proto-oncogene antibody therapy is one of the main methods, but most of the current proto-oncogenes are expressed in a small amount in normal tissues and play an important role. Physiological function, which brings great inconvenience to the application of its antibody.

The testes specific protein 50 (hereinafter referred to as: TSP50) involved in the present invention is a recently newly discovered proto-oncogene, which encodes a protein of 385 amino acids, and this protein has similarities with various serine proteases. structure, but its contact reaction residue Ser is replaced by Thr, so it may be a special type of threonine protease. Under normal circumstances, it is only expressed in human testis tissue, and only exists in spermatogonia, not in sperm, suggesting that it plays an important role in the process of sperm production. In other normal tissues other than testis, this gene is not expressed (Yuan, L., Shan, J., De Risi, D., Broome, J., Levecchio, J., Gal, D., Vinciguerra, V. , and Xu HP. Isolation of a novel gene, TSP50, by a hypomethylated DNA fragment in human breast cancer. Cancer Res. 1999, 59, 3215-3221). However, recent studies have shown that abnormally high expression of TSP50 is found in more than 90% of breast cancer tissues, and this expression is limited to malignant epithelial cells (Shan, J., Yuan, L, Xiao, Q., Chiorazzi, N., Budman, D., Teichberg, S., and Xu, HP, TSP50, A possible protease in human testis, is activated in breast cancer epithelial cells. Cancer Res. 2002, 62, 290-294). Therefore, we speculate that TSP50 is a new tumor-testis antigen (cancer testes antigen, CTA), which plays an important role as a threonine protease in the process of breast cancer and sperm formation, and it will be the basis for the diagnosis and treatment of breast cancer. important target.

In order to produce antibodies that can target specific antigens, the method of Kohler and Milstein is generally used. This method generally involves immunizing mice with antigens, then fusing the spleen cells of the mice with mouse myeloma cells, and selecting from the resulting hybridomas Hybridomas that secrete monoclonal antibodies against specific antigens.

Contents of the invention

The invention provides a preparation method of testis-specific protein 50 human single-chain antibody, the purpose of which is to use TSP50 to screen the human single-chain antibody library, and to express the human single-chain antibody in large quantities by means of genetic engineering.

The invention also discloses the use of the testis-specific protein 50 human single-chain antibody in the diagnosis and treatment of TSP50-related disease-breast cancer.

The technique of the present invention will be described in detail below. The present invention utilizes a phage antibody surface display technology system. The system is to insert the single-chain antibody variable region gene into the phage coat protein gene, so that the single-chain antibody is expressed on the surface of the phage in the form of fusion protein, and then through repeated immune screening-infection-amplification-rescreening process, to obtain Antigen-specific recombinant phage antibodies.

The invention utilizes phage antibody display technology, takes human testis specific protein 50 as target antigen, and screens recombinant phage human antibody variable region gene library by immunoaffinity screening method. The TSP50-specific single-chain antibody genes AI, AII and C8 were isolated and cloned. Then, the single-chain antibody genes AI, AII and C8 were respectively inserted into the prokaryotic expression vector PelB containing histidine tails, transformed into Rosetta bacteria, induced to express foreign proteins by IPTG, and the cells were lysed and then used for affinity with Ni-NTA column The purified single-chain antibody was obtained by chromatography, and it was identified by ELISA that it could specifically bind to TSP50 whole protein and 50 peptide. Further studies have proved that it can inhibit human breast cancer cell lines cultured in vitro.

The concrete technical solution of preparation method of the present invention is as follows:

1. Preparation of TSP50 Human Antibody

1. According to the amino acid sequence of human TSP50 (see accompanying drawing 1) and its homology comparison with other serine proteases, select the 156-206th amino acid in the amino acid hydrophilic region in the contact reaction functional region of TSP50 to synthesize, these 50 The small peptide of amino acids is a TSP50-specific peptide, which is named pep-50. At the same time, according to the cDNA sequence of human TSP50, TSP50-specific primers were designed, and the TSP50 cDNA fragment was fished from human testis tissue by RT-PCR method, and connected into the prokaryotic expression vector pGEX-4T-3 for expression.

2. Use pep-50 and prokaryotic expressed TSP50 protein to screen the human antibody library. The specific method is: first amplify the library, incubate in a petri dish coated with the antigen TSP50, the human antibody presented on the surface of the phage binds firmly to the antigen, washes away the unbound phage, and washes it out with the antigen The bound phages are amplified, and the amplified phages are put into the next round of screening, and this is repeated 3-4 times, and positive clones are obtained by amplifying, pottering, and screening the library.

3. Coat the bacterial styrene microtiter plate with the screened phage, then add the rabbit anti-TSP50 polyclonal antibody, then add the enzyme-labeled goat (or donkey) anti-rabbit antibody, and finally add the substrate for color development, so that the obtained Positive clones were further screened.

4. Extract the phage DNA from the positive phages further screened by the ELISA method, sequence them, and deduce their amino acid sequences. (See Figure 2, Figure 3, Figure 4).

5. Digest the DNA fragment expressing the human antibody in the positive phage with restriction enzymes NcoI and NotI, and connect it into the prokaryotic expression vector PelB, then transform Escherichia coli, and use IPTG to induce the expression of TSP50 human antibody . SDS-PAGE electrophoresis showed that the molecular weight of the fusion protein was 31KD (see Figure 5). in the picture

1: A1 was not induced with IPTG 2: A1 was induced with IPTG

3: Empty plasmid not induced with IPTG 4: Empty plasmid induced with IPTG

5: Marker 6: C8 was not induced by IPTG

7: C8 induced with IPTG 8: A11 induced with IPTG

9: A11 induced with IPTG

6. The prokaryotic expressed human antibody was purified by metal chelate column affinity chromatography.

2. Use of the purified human antibody of the present invention

A typical application of the human antibody of the present invention is to treat TSP50-related diseases, such as breast cancer and the like.

The purified TSP50 human antibody was added to the human breast cancer cell line MCF7 cultured in vitro, and after 72 hours, the viability of the MCF7 cells was detected by the MTT method. The results showed that the TSP50 single-chain antibody could significantly inhibit cell proliferation (see Figure 6).

The term "treatment" in the present invention includes "prevention", "suppression" or "treatment" of diseases. "Prophylaxis" is the use of a protective composition prior to disease induction. "Suppression" is the use of the composition after disease induction but before clinical manifestation. "Treatment" is the administration of a protective composition after the disease manifests clinical symptoms.

The human antibody of the present invention can be used in conjunction with other antibodies, especially antibodies that can react with other markers on human cells that are not related to this, such as the marker c-erbB-2 of breast cancer (Bacus SS, Gudkov AV, Esteva FJ, Yarden Y. Expression of erbB Receptors and their Ligands in Breast Cancer: Implications to Biological Behavior and Therapeutic Response. Breast Dis. 1999; 11:63-75.)

Usually, the human antibody of the present invention can be used in a purified form together with a pharmaceutically appropriate carrier. Typically, these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically acceptable adjuvants may be selected from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates, if necessary to maintain the complex in suspension.

Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers.

The human antibodies of the present invention can be used as compositions administered alone or in combination with other drugs, including various immunotherapeutic drugs such as cyclosporine, methotrexate, doxorubicin or cischlor Aminoplatin and immunotoxins. Pharmaceutical compositions may include a "cocktail" of various cytotoxic or other agents in combination with the human antibodies of the invention.

The route of administration of the pharmaceutical composition according to the present invention can be any one generally known by those of ordinary skill in the art.

For therapeutic methods including but not limited to immunotherapy, the human antibodies of the invention can be administered to any patient according to standard techniques. Administration may be by any suitable means, including parenteral, intravenous, intramuscular, intraperitoneal, or also by direct infusion through a catheter. Dosage and frequency of administration depend on age, sex, patient's condition, concomitant use of other drugs, adverse reactions and other factors considered by the clinician.

In addition to therapeutic uses, this construct may also be used for research purposes. The TSP50 human antibody of the present invention can be used in the study of the structure and function of TSP50, and these antibodies can be used as a tool for conformational research to clarify the natural configuration of different parts of TSP50. In addition, TSP50 human antibodies can be used as diagnostic probes. Human antibodies to TSP50 can be labeled according to techniques known in the art. This construct is also suitable for other in vivo purposes. For example, they can be used for positioning or targeted therapy of TSP50-related tumors, such as diagnosing whether breast cancer occurs, and bringing radioactive substances to the local tumor through TSP50 human antibodies.

The positive effects of the present invention are: (1) Since TSP50 is not expressed in normal tissues of women, the application of this antibody will not affect normal physiological functions like other proto-oncogene antibodies. (2) Since the single-chain antibody is formed by linking the variable region of the heavy chain and the variable region of the light chain of the antibody through a connecting chain of a small molecule, its molecular weight is about one-sixth of the molecular weight of immunoglobulin IgG. Therefore, its permeability is better, which is beneficial to the in vivo diagnosis and treatment of solid tumors. (3) The cost of producing antibodies by genetic engineering is low and suitable for large-scale production. (4) Since the antibody is of human origin and does not contain a stable region of the antibody, its immunogenicity is extremely small, and it can significantly reduce harmful reactions to patients when used for treatment.

Description of drawings

Figure 1 shows the amino acid sequence of human TSP50, the underlined part is the synthetic 50 peptide site

Figure 2 shows the sequence of the human antibody AI screened from the phage antibody library.

Figure 3 shows the sequence of the human antibody AII screened from the phage antibody library.

Figure 4 shows the sequence of the human antibody C8 screened from the phage antibody library.

Figure 5 shows the results of electrophoresis analysis of the prokaryotic expressed single chain antibody.

Figure 6 shows that the artificially expressed human antibody can significantly inhibit the proliferation activity of the human breast cancer cell line MCF7.

Detailed ways:

The following examples will describe the present invention more specifically, and should not be considered as limiting the present invention

Example 1:

Screening of phage antibody library with 50 peptide and full-length protein of TSP50

Phage antibody library: The human antibody library constructed in the pCOMB3 plasmid was donated by the Scripps Research Institute of the United States.

1. The first round of screening: Coat the polystyrene culture dish with 50 peptides and full-length protein (50 μg/ml) of TSP50, add the antibody library (5.6×10 ¹² pfu) to the culture dish, incubate for 2 hours, and aspirate the supernatant , washed the plate 10 times with PBST, buckled the plate dry on sterile filter paper, washed the bottle dish with 1ml of freshly prepared triethylamine; took 2 sterile EP tubes, added 300μl Tris-HCl, and then added 500μl triethylamine Amine eluent, after neutralization, was used to infect TG1.

2. Amplification of the antibody library after the first round of screening: Infect 14ml of fresh Escherichia coli TG1 for every 0.8ml of the eluted product after neutralization, and infect in a 37°C incubator for 30 minutes; shake culture at 37°C for 1 hour, and take out 150μl Inoculate into 100ml 2YT/AG1, culture with shaking at 37°C for 1 hour, add helper phage M ₁₃ K ₀₇ , infect at 37°C for 30 minutes, add ampicillin to a final concentration of 50 μg/ml, shake overnight at 37°C. The next day, collect the supernatant of the culture medium by centrifugation, add 1/5 volume of PEG8000/NaCl, mix well, place at 4°C for more than 2 hours, centrifuge to precipitate the phage, dissolve the precipitate in 1ml PBS, take 1μl to measure the titer, and the remaining 4 Store at ℃ for later use.

3. Repeat the screening process and reduce the amount of antigen coating by one time, and repeat this three times. It is the enrichment of specific clones.

Example 2:

Preparation and activity detection of monoclonal phage antibody

The product obtained after three rounds of elutriation and enrichment was spread on 2YT solid medium and grown overnight at 37°C. On the next day, 100 single spots were randomly picked and put into a sterilized test tube containing 2ml 2YT/AG, and cultured at 37°C Overnight. Collect the phages and add them to the wells of the microtiter plate coated with TSP50 protein and incubate for 2 hours. Screened by conventional ELISA method, strong positive clones were obtained.

Example 3:

TSP50 Human Antibody Sequence Determination

The DNA of ELISA strongly positive clones was extracted, entrusted to Shanghai United Gene Company for sequence analysis, and its amino acid sequence was deduced, the results are shown in Figures 2, 3, and 4.

Example 4:

Prokaryotic expression of human antibodies

1. Construction of the recombinant vector: The DNA of the phage antibody-positive clone and the prokaryotic expression vector PelB were digested with NcoI and NotI respectively, and then ligated with T4 ligase. Transformed into Rosetta competent bacteria, picked a single colony the next day and amplified it. Plasmids were extracted, and recombinant plasmids were identified by PCR and enzyme digestion.

2. Induced expression of human antibody: Pick a single positive colony, inoculate it into 12ml 2YT, and culture it with shaking at 37°C until the OD600 is about 0.5. Take out 8ml and put it into a 50ml Erlenmeyer flask, continue shaking culture at 37°C until When OD600 is 0.8-1.2, divide into two parts, add 5 μl of 800 mmol/LIPTG to one part for induction, the final concentration is 1 mmol/L, overnight at 30°C and 140 rpm.

3. Electrophoresis analysis of recombinant protein: centrifuge the bacteria induced by IPTG for 4 hours and 6 hours, freeze the precipitate (obtained by centrifugation of 100ml bacteria) at -20°C, take it out after 1 hour, add PBS12ml, add PMSF (100mM) 8μl, 37 Thaw at ℃, ultrasonically crush, centrifuge at 12,000 rpm at 4°C for 30 minutes, collect the supernatant, store at 4°C, take a little SDS-PAGE gel with 12% separating gel and 5% stacking gel for denaturing electrophoresis, and the results are shown in Figure 5.

Example 5:

Affinity Purification of Recombinant Human Antibodies

Take 300ml of the induced fresh recombinant bacteria solution, centrifuge at 7000rpm/min for 30min, collect the bacteria, add 5ml of buffer A (6M urea, 100mM Na ₂ PO ₄ 2H ₂ O, 10mM Tris-HCl, pH8.0), 5mg lysozyme, mix thoroughly, add PMSF to a final mass concentration of 100mg/L, and stir overnight at 4°C. The lysate solution was centrifuged at 14000r/min (4°C) for 20min, and the supernatant was passed through a Ni2+-NTA metal chelate layer suction column, followed by 20ml of buffer A and buffer B (6M urea, 100mM Na ₂ PO ₄ 2H ₂ O, 10mM Tris-HCl, 1M NaCl, pH6.3), buffer C (pH5.9, its composition is the same as that of buffer B), buffer C+imidazole (100mM) for elution, and collected buffer C+imidazole (100mM) The peaks were eluted and the protein content determined.

Embodiment 6:

Antibody labeled with horseradish peroxidase

Dissolve 8 mg of horseradish peroxidase in 1 ml of distilled water, add 200 μl of 0.1 M sodium metaperiodate solution, and let the mixture stand at room temperature for 30 minutes. The enzyme solution was dialyzed overnight at 4°C against 1 mM acetate buffer (pH 4.5). Add 100 μl of 0.2M carbonate buffer (pH 9.5) to adjust the pH to 9.5.

On the other hand, 8 mg of the purified anti-human TSP50 human antibody was dissolved in 0.1M phosphate buffer (pH 7.4), and dialyzed overnight at 4°C using 0.01M sodium carbonate buffer (pH 9.5). Mix the peroxidase with the antibody, let it stand at room temperature for 2.5 hours, add sodium tetrahydroborate, and let it stand at 4°C for 2 hours. Dialyze the obtained peroxidase-labeled antibody at 4°C overnight with phosphate buffer to obtain the enzyme-labeled antibody conjugate, add an equal volume of glycerol to store at -20°C, and select an appropriate dilution for use.

Use case 1:

Immunohistochemical detection of TSP50 expression in breast cancer

Take paraffin sections of breast cancer and paracancerous tissues, routinely dewax into water, soak in 0.3% H ₂ O ₂ -methanol for 30-40 minutes, wash with PBS three times, then block with 0.1% gel for 30-60 minutes, discard For excess blocking solution, add HRP-labeled TSP50 human antibody, 37°C for 1 hour, wash with PBS three times, each time for 5 minutes, add freshly prepared DAB (0.5mg/ml) + H ₂ O ₂ (1μl/ml), 3-5 minutes, routine dehydration, sealing, microscopic examination.

Example 2

Intracellular Localization of TSP50 Using Human Antibody

Inoculate the well-cultured human breast cancer cell line MCF-7 cells into a 12-well culture plate with coverslips, 5× ¹⁰⁵ cells per well, and culture overnight at 37°C in a CO2 incubator, discard the supernatant, and use Wash twice with PBS, then fix with 4% paraformaldehyde at room temperature for 30 minutes, wash with PBS three times, add ice-bathed methanol for 20 minutes, block with 1% BSA at room temperature for 1 hour, add FITC-labeled human antibody, 4 ℃ for 2 hours, the cover glass was sealed on the glass slide, and the localization of TSP50 in the cells was observed by confocal phase contrast microscope.

Use case 3:

Localized diagnosis of breast cancer by TSP50 human antibody-mediated medicinal radionuclide ¹³¹ I

Female nude mice were taken, and 0.2ml of well-growing human breast cancer passage cell line MCF-7 cell suspension (cell number 8×10 ⁶ ) was injected into the fat pads on both sides of the abdominal mammary glands to prepare tumor-bearing mouse models.

Human antibody AI labeled with medical radionuclide ¹³¹ I ( ⁹⁹ Tc ^m , ¹²³ I and ¹¹¹ In can also be used) was injected through the rat tail blood vessel by the known Idogen or chloramine T method. After 24, 48, 60, 72, 84, and 96 hours, the radioimmunoimaging method was used to detect the imaging of ¹³¹ I-labeled antibody in tumor-bearing nude mice. The rate is 86.9%. It accumulates in the cancer area from 36 hours, and it is most clear at 60 hours. The T/NT is 5.2.

Practical example 4

Detection of TSP50 Protein Expression by ELISA

Kit composition:

1. Standard product: 400μl of breast cancer marker molecule TSP50 protein solution, the concentration is 0.1mg/ml

2. Human antibody: mouse anti-TSP50 human antibody 50μl, antibody concentration 0.1mg/ml, ELISA titer 1:1000

3. Polyclonal antibody: 50 μl of rabbit anti-TSP50 polyclonal antibody, the antibody concentration is 1 mg/ml, and the ELISA titer is 1:1000. It is self-made in our laboratory, and its preparation method is a known technology.

4. Enzyme-labeled antibody: 50 μl of goat anti-rabbit IgG antibody (purchased from Promega), the antibody ELISA titer is 1:1000

The kit includes at least the above-mentioned reagents, and at the same time, for convenience, it can also be loaded with ELISA plate, concentrated washing solution, concentrated blocking solution, chromogenic reagent A, chromogenic reagent B, stop solution, semi-logarithmic coordinate paper, etc.

The concentrated washing solution is phosphate buffer, the blocking solution is BSA-phosphate buffer, the chromogenic agent A is 1% phosphophenylenediamine (OPD), the chromogenic agent B is hydrogen peroxide, and the stop solution is 2M sulfuric acid .

Instructions:

1. Dilute various concentrated solutions to the application concentration, specifically, dilute the concentrated cleaning solution 100 times (the concentration of the test is 0.01M); dilute the blocking solution 10 times (the BSA concentration of the test is 1%)

2. Prepare standard products with different dilutions: Dilute the standard products to 100μg/ml, 10μg/ml, 1μg/ml, 100ng/ml, 10ng/ml, 1ng/ml with diluted washing solution.

3. Dilute the purified anti-TSP50 human antibody to 0.4 μg/ml with 0.01M PBS buffer (pH 7.4), coat a 96-well microtiter plate, 100 μl/well, overnight at 4°C.

4. Discard the antibody coating solution and block with 1% BSA at 43°C for 30 minutes.

5. Discard the blocking solution and wash twice with 0.1% Tween20-PBS, 5 minutes each time.

6. Add different dilutions of TSP50 protein and the liquid to be tested at the same time, 100 μl/well, 43°C, 30 minutes.

7. Wash three times with 0.1% Tween20-PBS, 5 minutes each time.

8. Add rabbit anti-TSP50 polyclonal antibody, 100 μl/well, 43°C, 30 minutes.

9. Wash three times with 0.1% Tween20-PBS, 5 minutes each time.

10. Add goat anti-rabbit IgG, 100 μl/well, 43°C, 30 minutes.

11. Wash three times with 0.1% Tween20-PBS, 5 minutes each time.

12. Add OPD chromogenic substrate and react at room temperature for 5 minutes.

13. Add 50 μl 2M H ₂ SO ₄ to each well to stop the reaction, and measure the OD492 value with a microplate reader.

According to the TSP50 standard test results, a standard curve is made, and all the samples to be tested can find their corresponding values from this standard curve according to their OD492 values, and this value is the content of TSP50 in the sample.

Practical example 5

Identification of Antitumor Activity of Prokaryotic Expressed Human Antibody

The subcultured and well-grown human breast cancer cell line MCF7 cells were digested with 0.25% trypsin, adjusted to a cell concentration of 1× ¹⁰⁵ , and then added to a 96-well plate, 100 μl per well, 37°C, 5% CO ₂ Incubator overnight, three kinds of TSP50 human antibodies expressed in prokaryotic were added to each well at different concentrations, and the final concentrations were 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μg/ ml, each concentration was set up in triplicate wells, and _PBS was used as negative control at the same time. Remove the cell supernatant, add 100 μl DMSO to each well, and measure the OD570 value with a microplate reader to judge the viability of the cells after the intracellular particles are completely dissolved. The results showed that the three strains of TSP50 single-chain antibodies could significantly inhibit the proliferation of breast cancer cells (see Figure 6).

Practical example 6

Therapeutic effect of TSP50 human antibody ^131I on breast cancer

Female nude mice were taken, and 0.2ml of well-growing human breast cancer passage cell line MCF-7 cell suspension (cell number 8×10 ⁶ ) was injected into the fat pads on both sides of the abdominal mammary glands to prepare tumor-bearing mouse models.

Human antibody AII labeled with medical radionuclide ¹³¹ I ( ⁹⁹ Tc ^m , ¹²³ I and ¹¹¹ In) was injected through the rat tail blood vessel by the well-known Idogen or chloramine T method. After 1, 3, 5, 7, and 14 days, the calculated value of CT was used to observe the effect of ¹³¹ I-labeled antibody on tumor growth in tumor-bearing nude mice. The results showed that the tumors of 89.3% of tumor-bearing mice were reduced, and 78.1% of The tumors of tumor mice shrunk by more than 50%.

Practical example 7

Epitope structure analysis of TSP50 with TSP50 human antibody

Use the human antibody of TSP50 to screen the phage 15 peptide library (commercial peptide library can be purchased). The specific method is: first amplify the peptide library, and then add it to a petri dish that has been coated with anti-TSP50 human antibody. After incubation, the small peptides that can bind to the antibody presented on the surface of the phage will be firmly bound to the antibody, the unbound phage will be washed away, the phage bound to the antibody will be eluted and amplified, and the amplified phage will be put into the next step. This round of screening was repeated 3-4 times, and positive clones were obtained by amplifying, washing and screening the peptide library. The resulting positive clones were further screened by competitive ELISA to find clones that compete with TSP50 for binding to human antibodies. The specific method is: coat the microtiter plate with human antibodies, add TSP50 protein and the obtained phage after blocking with BSA, and at the same time use a separate Add TSP50 protein wells as the control group, then add horseradish peroxidase-labeled TSP50 polyclonal antibody, OPD color development, measure OD492 value, find phages that can compete with TSP50 to bind to the antibody and extract their DNA for sequencing to find out Its conservative sequence is the epitope of TSP50 inferred theoretically, and the epitope structure of TSP50 is further determined by computer simulation.

Claims (3)

1. three kinds of testes specificity albumen 50 human single chain variable fragments antibodies, it is characterized in that: they all are to utilize phage antibody to present technology, with human testicle specificity protein white 50 is target antigen, the screening human antibody library, and gained antibody gene sequence clone expressed to the prokaryotic expression carrier PelB and prepare, this three-type-person source antibody has following sequence general formula:

MAEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV 50

SAISGSGGSTYYADSVKGRFTISRDN-①-KNTLYLQMNSLREDTAVYYCAR- 100

②-WGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSVALG 150

QTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSS 200

SGNTASLTITGAQAEDEADYYCNSRDSSG-③-VFGGGTKLTVVLGAA 245

Wherein, 1. be S, 2. be RQRLRHYANN, when 3. being F, this sequence is AI;

1. being P, 2. is RQRKSGRK, and when 3. being K, this sequence is AII;

1. being S, 2. is VRHRASRANL, and when 3. being RV, this sequence is C8.

2. testes specificity albumen 50 human single chain variable fragments antibodies of claim 1 are used to prepare the diseases related diagnosis of testes specificity albumen 50 and the purposes of medicine.

3. pharmaceutical composition, it contains any one human antibody and medicinal acceptable vehicle in the with good grounds claim 1.

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