CN108441482A - The monoclonal antibody of AntiCD3 McAb 0, its hybridoma cell strain, preparation method and application - Google Patents

Abstract

The invention discloses an anti-CD30 monoclonal antibody, its hybridoma cell line, preparation method and application. The hybridoma cell line is obtained by fusion of mouse splenocytes and myeloma cells immunized with prokaryotically expressed CD30 protein. The anti-CD30 monoclonal antibody can specifically recognize CD30 protein and has strong reactivity with tumor cells. It can be applied In the diagnosis or treatment of tumors, it is contained in a preparation for diagnosis or treatment of tumors, the invention also discloses the prokaryotic expression method of CD30 protein, the expression gene sequence used, the hybridoma cell line and the preparation of anti-CD30 monoclonal antibody method. The invention prepares anti-CD30 hybridoma cell line by immunizing CD30 protein, can secrete specific anti-CD30 monoclonal antibody, has strong reactivity with tumor cells, and can be applied to the diagnosis and treatment of tumors.

Description

Anti-CD30 monoclonal antibody, its hybridoma cell line, preparation method and application

technical field

The invention belongs to the technical field of biomedical engineering, and specifically relates to an anti-CD30 monoclonal antibody, a hybridoma cell line, a preparation method and an application thereof.

Background technique

Peripheral T-cell lymphoma (PTCL) is a type of malignant lymphoma derived from T cells at different stages of the thymus, with obvious heterogeneity in biological behavior and clinical manifestations, and its incidence has regional and racial differences. PTCL only accounts for 6%-7% of NHL in western countries, and the incidence of lymph node lymphoma is higher, including PTCL-U, ALCL and angioimmunoblastic lymphoma (AlTL); while PTCL is common in Asian countries, accounting for NHL 15%-30% of the total, extranodal lymphoma is more common. Due to the increasing incidence of PTCL year by year, 2007 National Cancer Network (NCCN) guidelines included PTCL for the first time. This shows that western countries have begun to pay attention to the diagnosis and treatment of PTCL.

A large number of studies have shown that CD30 is one of the main tumor markers of lymphoma. CD30 is a cell surface glycoprotein with a molecular weight of 120KDa, which was reported and described by Stein in the 1980s. In 1992, the cDNA encoding human CD30 was successfully cloned, thus confirming that CD30 is a member of the NGFR/TNFR superfamily. Studies have found that CD30 can participate in cell activation and differentiation, in the conduction of activation signals such as NF-kB, and in the activation of T cell immunity. During the pathogenesis of Hodgkin's lymphoma (HL), the expression of CD30 can be significantly increased, and under the joint action of other factors, it has a strong promotion effect on the activation of CD8+ and CD4+ Th2 cells. CD30 expression was significantly increased in these pathological states. Related studies by Smith et al. found that the expression of CD30 was increased in HL. Watanabe et al. also found that the expression of CD30 was increased in various lymphomas including HL through cell culture and other methods. Many studies have found that CD30 is highly expressed in PTCL. Bossard reported that 50% of PTCL patients expressed detectable CD30 by immunohistochemistry (IHC).

Since the first monoclonal antibody drug Rituximab (Rituximab) was approved by the US Food and Drug Administration (FDA) for tumor treatment in 1997, antibody-based immunotherapy has gradually become one of the important strategies in this field. Antibody research against CD30 began in the early 1990s, and there are currently two drugs targeting CD30, iratumumab (SGN-30) and Brentuximab vedotin (SGN-35). Iratumumab is a therapeutic antibody targeting CD30, which has a certain effect on CD30-positive HL patients. Brentuximab vedotin is an immunoconjugate of microtubule inhibitors composed of CD30 antibody and monomethyl auristatin. FDA has approved Brentuximab vedotin for the treatment of relapsed and refractory CD30-positive HL patients. In a phase II multicenter clinical trial of Brentuximab vedotin in the treatment of PTCL, the overall remission rate was 86%, and the complete remission rate was 53%. In another phase I/II study, the investigators evaluated the safety and efficacy of brentuximab vedotin for subsequent treatment of CHOP or in combination with CHP in first-line treatment of CD30+ PTCL. The results showed that the overall remission rate of the patients was 73%-85%, the complete remission rate was 35%-62%, and 62% of the patients had grade 3/4 adverse reactions, including febrile neutropenia.

CD30 is an important molecular target of malignant hematological tumors. Obtaining high-potency and high-specificity anti-CD30 monoclonal antibodies is a prerequisite for the development of new anti-CD30 antibody-drug conjugates. The successful development of this conjugate will provide my country with A new candidate drug for lymphoma with independent intellectual property rights.

Contents of the invention

The purpose of this section is to outline some aspects of embodiments of the invention and briefly describe some preferred embodiments. Some simplifications or omissions may be made in this section, as well as in the abstract and titles of this application, to avoid obscuring the purpose of this section, the abstract and titles, and such simplifications or omissions should not be used to limit the scope of the invention.

Therefore, as one aspect of the present invention, the present invention overcomes the deficiencies in the prior art and provides a hybridoma cell line.

In order to solve the above technical problems, the present invention provides the following technical solutions: a hybridoma cell line, whose preservation number in the China Type Culture Collection Center of Wuhan University is CCTCC NO: C201861.

As a preferred solution of the hybridoma cell line of the present invention: the hybridoma cell line is an anti-CD30 hybridoma cell line, and the CD30 protein is a prokaryotic expressed recombinant protein, named as prokaryotic expressed CD30 protein.

As a preferred solution of the hybridoma cell line of the present invention: the hybridoma cell line is obtained by fusion of mouse splenocytes immunized with prokaryotically expressed CD30 protein and myeloma cells.

As another aspect of the present invention, the present invention overcomes the deficiencies in the prior art and provides an anti-CD30 monoclonal antibody.

In order to solve the above technical problems, the present invention provides the following technical solution: an anti-CD30 monoclonal antibody secreted by the hybridoma cells described in claim 1 .

As a preferred solution of the anti-CD30 monoclonal antibody of the present invention: the subclass of the anti-CD30 monoclonal antibody belongs to IgG1, and the anti-CD30 monoclonal antibody can specifically recognize the CD30 protein, and bind to tumor cells With strong reactivity, the anti-CD30 monoclonal antibody at a concentration of 1 mg/ml has an antibody titer of 1:640,000 when the CD30 dosage is 2.5 ng.

As another aspect of the present invention, the present invention overcomes the deficiencies in the prior art and provides the application of the anti-CD30 monoclonal antibody described in claim 4 or 5 in the preparation of pharmaceutical preparations for diagnosing or treating tumors.

As another aspect of the present invention, the present invention overcomes the deficiencies in the prior art and provides a pharmaceutical preparation for diagnosing or treating tumors, which includes the anti-CD30 monoclonal antibody according to claim 4 or 5.

As another aspect of the present invention, the present invention overcomes the deficiencies in the prior art and provides a method for preparing the anti-CD30 monoclonal antibody described in claim 4 or 5.

In order to solve the above-mentioned technical problems, the present invention provides the following technical scheme: the preparation method of the anti-CD30 monoclonal antibody described in claim 4 or 5, which comprises,

Prokaryotic expression and purification of CD30 protein:

Design primers, construct the pET-28a-CD30 prokaryotic expression plasmid, identify the correct plasmid and transform it into BL21, and induce protein expression with IPTG; IPTG-induced bacterial liquid is ultrasonically lysed, and the centrifuged supernatant is combined with Ni filler to elute the protein , to obtain purified CD30 protein, the protein molecular weight is about 40KDa;

Preparation of hybridoma cell lines: emulsify the purified CD30 protein, and inject it into Balb/c mice at multiple points and intraperitoneally. 2 weeks, 4 days after the second multipoint injection plus intraperitoneal injection, the amount of CD30 protein injected into the tail vein was 50 μg/mouse; the splenocytes of the immunized mice were fused with Sp2/0 myeloma cells to obtain hybridoma cells; screening Positive hybridoma cell lines were expanded and cultured.

As the preparation method of the anti-CD30 monoclonal antibody of the present invention, it also includes,

Preparation of anti-CD30 monoclonal antibody: inject the positive hybridoma cell line obtained in claim 8 into the abdominal cavity of Balb/c mouse to make the mouse produce ascites, and collect the ascites;

Purification of anti-CD30 monoclonal antibody: use protein-G column affinity purification to obtain anti-CD30 monoclonal antibody.

Beneficial effects of the present invention: the present invention prepares anti-CD30 hybridoma cell lines by immunizing CD30 prokaryotic expression protein, can secrete corresponding monoclonal antibodies, can specifically recognize CD30 protein, and has strong reactivity with tumor cells, Can be applied to the diagnosis and treatment of tumors, the CD30 monoclonal antibody prepared by the present invention, when the CD30 monoclonal antibody with a concentration of 1mg/ml is 2.5ng/well, the antibody titer reaches 1:640000, and the purified monoclonal antibody The antibody was identified by SDS-PAGE, with clear bands and no impurity bands. There were clear bands at 50KDa and 25KDa, respectively representing the heavy chain and light chain of the antibody.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the accompanying drawings that need to be used in the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For Those of ordinary skill in the art can also obtain other drawings based on these drawings without any creative effort. in:

Fig. 1 is the identification (SDS-PAGE identification) diagram of CD30 prokaryotic expression protein.

Fig. 2 is a diagram of the identification (mass spectrometry identification) of CD30 prokaryotic expression protein.

Fig. 3 is a graph showing the reactivity (indirect immunofluorescence method) between anti-CD30 monoclonal antibody and tumor cells.

Figure 4 is a diagram of the reactivity (immunoblotting) of anti-CD30 monoclonal antibody with CD30 recombinant protein.

Fig. 5 is a picture of purified CD30 monoclonal antibody 6B6 (identified by SDS-PAGE).

Detailed ways

In order to make the above objects, features and advantages of the present invention more comprehensible, the specific implementation of the present invention will be described in detail below in conjunction with specific examples.

In the following description, a lot of specific details are set forth in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described here, and those skilled in the art can do it without departing from the meaning of the present invention. By analogy, the present invention is therefore not limited to the specific examples disclosed below.

Second, "one embodiment" or "an embodiment" referred to herein refers to a specific feature, structure or characteristic that may be included in at least one implementation of the present invention. "In one embodiment" appearing in different places in this specification does not all refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

The hybridoma cell line of the present invention was preserved in the Chinese Type Culture Collection Center of Wuhan University on March 20, 2018. Address: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province; Collection Number: CCTCC NO: C201861.

The preparation method of the anti-CD30 monoclonal antibody is characterized in that: comprising the following two parts:

Prokaryotic expression and purification of CD30 protein:

1. Primer design (CD30 protein 141-379aa)

PET28a-CD30-F:CGC GGATCCGGCACGGCGCAGAAGAACAC

PET28a-CD30-R:CCC AAGCTTTTACTTCCCCGTGGAGGAGAG

2. Operation steps

(1) RNA extraction

1 CD30 positive peripheral blood T lymphoma cells, centrifuge at 1000rpm for 8min, discard the supernatant, add 1ml Trizol, and repeatedly pipette with a 1ml pipette until there is no obvious precipitation in the lysate, and let stand at room temperature for 5 minutes;

2 Add 200 μL of chloroform to the lysate obtained in step 1, close the cap of the centrifuge tube tightly, shake it up and down by hand for 15 seconds, centrifuge at 12000 rpm, 4 °C for 10 minutes;

3 Take out the centrifuge tube, the sample is divided into three layers, the transparent supernatant water phase, the middle white layer and the red lower layer, carefully draw about 400 μL of the transparent supernatant water phase into another centrifuge tube;

4. Add 400 μL equal volume of isopropanol to the supernatant aqueous phase obtained in step 3, mix gently, let stand at room temperature for 10 minutes, and centrifuge at 12000 rpm for 10 minutes at 4°C;

5 Carefully remove the supernatant, slowly add 1ml of 75% ethanol (made with DEPC-treated water) along the tube wall, centrifuge at 12000rpm, 4°C for 10min;

6 Carefully suck up the supernatant, dry the pellet at room temperature for 5 minutes, and add 30 μL of RNase-free water to dissolve the RNA pellet;

7 Measure the OD value, and the RNA concentration is 155ng/μL. OD260/OD280 = 1.84

(2) Reverse transcription system and procedures

5xPrimeScript RT Master Mix

(reverse transcriptase 200U/μL; 125mM MgCl ₂ ; 12.5mM dNTPs; olige dT primer) 4 μL

RNase Free dH ₂ O 1 μL

RNA (concentration: 155ng/μL) 2μL

37℃ 15min 85℃ 5min 4℃∞

(3) PCR system (high-fidelity enzyme amplification) and program

Procedure: pre-denaturation at 95°C for 4 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 45 s, and extension at 72°C for 50 s; extension at 72°C for 10 min. Take 5 μL of the PCR product for electrophoresis and perform electrophoresis.

(4) Glue recycling

1. Cut off the agarose gel containing the target DNA under the ultraviolet light, add 800 μL Buffer DE-A, mix well, heat in a water bath at 75°C, and mix once every 2-3 minutes until the gel block is completely melted (about 8 -10min);

2. Add 400μL Buffer DE-B and mix well;

3. Take the mixed solution in step 2, transfer it to the DNA preparation tube, centrifuge at 12000rpm for 1min, discard the filtrate, repeat this step until the mixed solution is centrifuged;

4. Put the preparation tube back into the centrifuge tube, add 500μL Buffer W1, centrifuge at 12000rpm for 30s, discard the filtrate;

5. Put the preparation tube back into the centrifuge tube, add 700μL Buffer W2, centrifuge at 12000rpm for 30s, discard the filtrate; wash once again with 700μL Buffer W2 in the same way, and centrifuge at 12000rpm for 1min;

6. Put the preparation tube back into the centrifuge tube and centrifuge at 12000rpm for 1min;

7. Place the preparation tube in a clean 1.5ml centrifuge tube, add 30 μL Eluent to the center of the preparation membrane, let it stand at room temperature for 3 minutes, and centrifuge at 12,000 rpm for 1 minute to elute the DNA;

8. Measure the concentration, the fragment is 224ng/μL.

(5) Digestion

(6) Cleaning and recycling

1. Add 3 times the volume of Buffer PCR-A to the digested product, mix well and transfer to the preparation tube, place the preparation tube in a 2ml centrifuge tube, centrifuge at 12000rpm for 1min, and discard the waste liquid;

2. Put the preparation tube back into a 2ml centrifuge tube, add 700 μL Buffer W2, centrifuge at 12000 rpm for 1 min, discard the waste liquid, then add 400 μL Buffer W2, and centrifuge at 12000 rpm for 1 min;

3. 12000rpm air separation for 1min;

4. Place the preparation tube in a clean 1.5ml centrifuge tube, add 20 μL Eluent to the center of the preparation membrane, let it stand at room temperature for 3 minutes, and centrifuge at 12,000 rpm for 1 minute to elute the DNA;

5. Measure the concentration, the fragment after digestion is 103ng/μL, and the fragment of pet28a plasmid is 79ng/μL after digestion.

(7) Connection

16°C metal bath connection overnight.

(8) Transformation and connection products

Add the ligation product to competent DH5a, incubate on ice for 30 minutes, heat stress in a water bath at 42°C for 90s, then place on ice for 3-5 minutes, add 1ml of liquid LB, shake at 37°C, 100rpm for 1h, centrifuge at 5000rpm for 5min, discard The supernatant and bacterial suspension were spread on LB plates containing Kan resistance, and cultured overnight at 37°C.

(9) Identification

Pick a single colony and place it in about 3ml of LB liquid medium containing Kan resistance, shake it at 37°C at 200rpm and culture overnight for PCR identification and extract the plasmid for sequencing.

(10) Transformation plasmid

The obtained fragment was digested and ligated with the prokaryotic expression vector pET28a, the constructed plasmid was sent for sequencing, and the sequenced correct plasmid was transformed into BL21, the method was the same as (8).

(11) Induced expression of protein

Pick a single colony and place it in about 3ml of LB liquid medium containing Kan resistance, and culture overnight at 37°C with 200rpm shaking, and the next day, take 40μL of the bacterial liquid into 4ml of fresh LB liquid medium containing Kan resistance, shake After 2 hours, take out 1ml as a control, add 3μL of 1M IPTG to the remaining bacterial solution, induce for 4 hours at 37°C, take out the control and induced bacterial solution and centrifuge at 12000rpm for 1min to collect the bacterial solution, wash twice with PBS, and add to the bacterial solution Add 20 μL 4x protein loading buffer to 60 μL PBS, and add 20 μL 4x protein sample buffer, boil for 15 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, take 10 μL samples for spotting, 80 v for 2 hours, stain with Coomassie brilliant blue for 30 minutes, and then decolorize. The protein size expressed by CD30 protein 141-379aa is about 40KDa.

Development of CD30 monoclonal antibody:

1. Development steps and process

1. Animal immunity

Take 500 μL of purified CD30 protein (1 mg/ml), add 200 μL of ddH ₂ O, add an equal volume of Freund’s complete adjuvant (300 μL) to emulsify, and immunize Balb/c mice about 6 weeks old by multi-point subcutaneous injection; 2 One week later, the same dose was taken for the second immunization, and an additional immunization was given two weeks later. The antibody titer was measured four days later, and 50ug protein was injected into the tail vein and intraperitoneal cavity at the same time. Spleen cells ground into single cells were subjected to cell fusion with myeloma cells Sp2/0.

2. Fusion

The feeder cells can be prepared one day before fusion, take 2 ICR mice aged 6-8 weeks, let them die by cervical dislocation, sterilize them in 75% alcohol for 5-10min, fix them on the plate, and cut open the abdomen in an ultra-clean table aseptically skin. Use a sterile syringe to draw 20ml of the HAT selection culture solution and inject it into the abdominal cavity of the mouse, gently rub the abdomen with an alcohol cotton ball, and withdraw the culture medium. Add 10 ml of HAT culture solution, spread into 10 96-well cell culture plates, 100 μL/well, culture in 37°C, 5% CO2 cell culture incubator.

Sp2/0 cells were revived one week before fusion and subcultured in a 37°C, 5% CO ₂ incubator. Collect the cells in the logarithmic growth phase into a centrifuge tube, count the cells, and dilute the cells to 10 ⁶ cells/ml for later use.

Take the Balb/c mice that have been boosted for 3 days, collect blood from the heart to prepare positive serum, kill them by dismounting the cervical spine, disinfect with 75% alcohol for 5 minutes, take out the spleen aseptically in an ultra-clean workbench, wash it several times in a sterile plate, and peel off the connective tissue organize. Place the spleen on a microporous copper grid, add fresh DMEM, and gently grind the spleen with a grinding rod until it becomes a single cell. Transfer the splenocyte suspension in the plate to a 10ml centrifuge tube, centrifuge at 1000r/min for 10min, and collect the splenocytes.

Splenocytes and Sp2/0 cells of the immunized mice were mixed according to the number of cells at 4:1, added to a 50ml fusion tube, centrifuged at 1000r/min for 10min, discarded the supernatant, and rubbed gently in the palm of the hand to fully mix the two cells; then in 37 In a water bath at ℃, add 1ml of preheated PEG into the fusion tube within 60 seconds, slowly at first and then quickly, stirring gently while adding. Immediately, 30ml of anti-blood-free DMEM was added slowly and then quickly within 90s to terminate the reaction. Bath in water at 37°C for 10min, then centrifuge at 1000r/min for 10min, discard the supernatant, suspend the precipitate with HAT, mix evenly into 30ml of HAT selection culture medium containing 20% calf serum preheated at 37°C, spread it into the Feed cells in a 96-well cell plate, 100 μL/well, and place the culture plate in a 37°C, 5% CO ₂ incubator for culture. After 5 days, the cell plate will be half-changed with fresh HAT medium, and after 10 days, the medium will be fully changed with HT medium.

The cells in the 96-well plate that were positive for two consecutive tests were expanded and cultured, and the limiting dilution method was used for subcloning: first, the feeder cells were prepared according to the method described, and dropped into the 96-well plate, leaving the first column empty. Blow up the positive hybridoma cells with HT medium, take 100 μL for counting, and make a doubling dilution in the first column, so that there are about 100 cells in each 10ml medium; gently blow evenly and add feeder cells In the culture plate, 100 μL/well was cultured in a 37° C., 5% CO ₂ cell incubator. After about 7 days, count the number of clones in the cell well, mark and replace with a new medium, and detect when the cells cover 1/3 to 1/2 of the bottom of the well. After 2-3 times of cloning, when all the cell wells of the 96-well plate are positive, the expansion culture can be carried out, and the fixed strains can be frozen.

The hybridoma cells that were positively detected and determined to be strained were expanded and cultured and frozen. The specific process is as follows: the vigorously growing hybridoma cells in good condition were gently blown off the cell bottle with anti-blood and blood-free DMEM, centrifuged at 1000 r/min for 5 min, and the supernatant was discarded. Add serum-free freezing solution, blow the cells apart and distribute them into cell freezing tubes. Put the cryopreservation tube into the freezer box and place it in a -70°C refrigerator. After one day, transfer the cryopreservation tube into liquid nitrogen and make a record.

3. Preparation of antibody ascites

In vivo ascites induction method: take 10-week-old multiparous Balb/c female mice, intraperitoneally inject 0.5 ml of sterilized liquid paraffin; ⁶ cells/piece. Pay attention to observation every day. After the abdomen of the mice has obvious bulge, the skin of the lower abdomen is disinfected with 75% alcohol cotton balls, and the abdominal cavity is punctured with a 16-gauge needle to collect ascites. After ascites regenerates and accumulates, collect again. The collected ascites was centrifuged at 3000 r/min for 10 min, the supernatant was taken, filtered with glass wool, aliquoted, and stored at -70°C.

4. Purification of monoclonal antibodies

Dilute the ascitic fluid 1:1 with Binding Buffer (pH8.0): NaCl 0.15M+Na2HPO4 20mM; add 1ml Binding Buffer to the column in advance, transfer 1ml protein-G resin to the column, drain, and equilibrate with 5ml BindingBuffer; The sample can be added to the column 10ml at a time, and after 10 minutes of action, the column will start to pass through the column (1ml/min flow rate through the column); 30ml Binding Buffer washing (10ml×3 times); 10-15ml Elution Buffer (pH2.5-3.0): Citric acid 0.1 M Wash the column for elution (in order to prevent protein denaturation due to the overacidity of the Elution Buffer pH, adjust the pH to 7.4 with pH 8.5 1M Tris-HCl); regenerate (wash with 10ml Elution Buffer); equilibrate twice with 5ml BindingBuffer.

5. Identification of monoclonal antibody subclasses

According to the instructions of the SIGMA kit, the subclass identification of monoclonal antibody was carried out by the method of capture ELISA, as follows: After diluting the subclass identification reagent of monoclonal antibody at 1:1000, add it to the enzyme-labeled well, 100 μL/well, 2 wells/sample, Incubate at 37°C for 1 hour; wash with PBST three times and pat dry; dilute ascites appropriately and add to the well, 100 μL/well; wash with PBST three times and pat dry; HRP enzyme-labeled goat anti-mouse IgG secondary antibody is diluted at 1:600 and then added , 100 μL/well, incubate at room temperature for 30 minutes; develop color for 10-20 minutes. The subtype of the monoclonal antibody was defined as the OD ₄₅₀ reading value that was significantly higher than that of the subtype reagents added to other wells.

Screening results of some hybridomas obtained:

1. The mouse serum titer was determined as follows:

2. Post-fusion screening (ELISA screening) is as follows:

The present invention will be further described below in conjunction with the accompanying drawings.

The main operation steps of the invention are: constructing CD30 prokaryotic expression plasmid, transforming the plasmid into competent BL21, inducing protein expression with IPTG, and purifying protein with Ni column. CD30 protein was used to immunize Balb/c mice, multiple times of cell fusion by hybridoma technology, using Elisa to screen out hybridoma cell lines that can stably secrete antibodies, the ascites was purified by protein-G, and the obtained monoclonal antibodies were tested for titer and Subclass identification. A monoclonal cell line capable of reacting with tumor cells obtained through screening was named 6B6 (subclass IgG1).

Materials and sources:

Experimental animals: Balb/c mice and ICR mice were purchased from the Comparative Medicine Center of Yangzhou University.

Protein: CD30 protein was induced, expressed and purified by our laboratory.

Reagents: goat anti-mouse IgG fluorescent secondary antibody and antibody subclass identification kit were purchased from Sigma; protein-G was purchased from Invitrigen.

1. Prokaryotic expression and purification of CD30 protein

1) Prokaryotic expression of CD30 protein

Design prokaryotic expression CD30 protein primers, upstream primers:

CGCGGATCCGGCACGGCGCAGA AGAACAC, the downstream primer CCCAAGCTTTTACTTCCCCGTGGAGGAGAG, the primers were used to capture the CD30 gene extracellular region fragment (141aa-379aa) from the CD30 positive peripheral blood T lymphoma cells by RT-PCR method, and the obtained fragment was digested with the prokaryotic expression vector pET28a Ligation, metal bath at 16°C overnight, the next day, transform the ligation product into competent DH5a, spread the bacterial solution on a solid LB plate containing kan resistance, cultivate overnight at 37°C, pick a single colony on the LB plate to 3ml In the LB liquid medium containing Kan resistance, culture overnight at 37°C, 200rpm shaking, primer PCR identification of the overnight cultured bacterial liquid, transfer the positive bacterial liquid 1:100 to 20ml LB liquid medium containing Kan resistance, Cultivate overnight at 37°C, extract the plasmid from 20ml of the bacterial solution cultured at 37°C overnight, and send the plasmid for sequencing after being correctly identified by the above primers PCR On the LB plate, cultivate overnight at 37°C, pick a single colony on the LB plate into 3ml LB liquid medium containing Kan resistance, and culture overnight at 37°C, 200rpm shaking; the above primers PCR to identify the overnight cultured bacterial liquid, after correct Use an inoculation loop to draw three lines on a solid LB plate containing kan resistance, and culture overnight at 37°C. Pick a single colony into 3ml LB liquid medium containing Kan resistance, 37°C, shake at 200rpm and culture overnight, take 2ml bacterial liquid into 200ml fresh LB liquid medium containing Kan resistance, 37°C , 200rpm shaking culture, when the OD600 value of the bacterial solution reaches about 0.6, take out 1ml as a control, add 100ul 1M IPTG to the remaining bacterial solution, induce 4h at 37°C, and collect the bacterial cells by centrifugation of the induced bacterial solution.

2) CD30 protein purification and identification

The bacterial solution collected by centrifugation was washed twice with PBS, added 30ml lysis buffer to suspend the bacteria, and then sonicated for 20min. After sonication, centrifuged at 12000rpm at 4°C for 20min, took the supernatant, filtered it with a 0.22um or 0.45um disposable filter, and added 5ml to the precipitate Suspend in PBS, mix the Ni filler with the filtrate in 11), combine with shaking at 4°C for 1 hour, transfer the treated mixture to a protein purification column, let it stand for 5 minutes, let the liquid flow down naturally, and then use 10-15ml lysis buffer Wash the miscellaneous protein, wash the miscellaneous protein with wahing buffer until the protein concentration in the eluent is 0mg/ml, add 2ml elution buffer to elute the target protein, and then add 1ml each time until the protein is eluted cleanly, and the eluted protein is marked on the E-1, E-2, E-3, etc., measure the concentration of the eluted protein, store it at -80°C after labeling, do not freeze repeatedly, collect the bacteria by centrifugation of the control bacterial solution, wash twice with PBS, add 60ul PBS to suspend , add protein loading buffer to boil, take 60ul each of the precipitation suspension and protein eluent, add protein loading buffer and boil, take 10ul each of the above samples and run SDS-PAGE, decolorize and take pictures after Coomassie brilliant blue staining, and send the protein after tapping Identification by mass spectrometry. The results are shown in Figures 1 and 2.

2. Preparation of anti-CD30 monoclonal antibody 6B6

1) Preparation of monoclonal antibody

Take 50ul of purified CD30 protein (1mg/ml), add 200ul of ddH ₂ O, add an equal volume of Freund's complete adjuvant (300ul) to emulsify, and immunize Balb/c mice about 6 weeks old by multi-point subcutaneous injection; 2 One week later, the same dose was taken for the second immunization, and an additional immunization was given two weeks later. The antibody titer was measured four days later, and 50ug protein was injected into the tail vein and intraperitoneal cavity at the same time. Splenocytes ground into single cells were fused with myeloma cells Sp2/0, and positive cell clones were screened.

The hybridoma cells that were positively detected and determined to be strained were expanded and cultured and frozen. The specific process is as follows: the vigorously growing hybridoma cells in good condition were gently blown off the cell bottle with anti-blood and blood-free DMEM, centrifuged at 1000 r/min for 5 min, and the supernatant was discarded. Add serum-free freezing solution, blow the cells apart and distribute them into cell freezing tubes. Put the cryopreservation tube into the freezer box and place it in a -70°C refrigerator. After one day, transfer the cryopreservation tube into liquid nitrogen and make a record.

In vivo ascites induction method: take 10-week-old multiparous Balb/c female mice, intraperitoneally inject 0.5 ml of sterilized liquid paraffin; ⁶ cells/piece. Pay attention to observation every day. After the abdomen of the mice has obvious bulge, the skin of the lower abdomen is disinfected with 75% alcohol cotton balls, and the abdominal cavity is punctured with a 16-gauge needle to collect ascites. After ascites regenerates and accumulates, collect again. The collected ascites was centrifuged at 3000 r/min for 10 min, the supernatant was taken, filtered with glass wool, aliquoted, and stored at -70°C.

2) Purification of monoclonal antibody 6B6

Dilute the ascitic fluid 1:1 with Binding Buffer (pH8.0): NaCl 0.15M+Na2HPO4 20mM; add 1ml Binding Buffer to the column in advance, transfer 1ml protein-G resin to the column, drain, and equilibrate with 5ml BindingBuffer; The sample can be added to the column 10ml at a time, and after 10 minutes of action, the column will start to pass through the column (1ml/min flow rate through the column); 30ml Binding Buffer washing (10ml×3 times); 10-15ml Elution Buffer (pH2.5-3.0): Citric acid 0.1 M Wash the column for elution (in order to prevent protein denaturation due to the overacidity of the Elution Buffer pH, adjust the pH to 7.4 with pH 8.5 1M Tris-HCl); regenerate (wash with 10ml Elution Buffer); equilibrate twice with 5ml BindingBuffer. The purified monoclonal antibody 6B6 was used to measure the antibody titer with the purified prokaryotic expressed CD30 protein. When the concentration of 1mg/ml monoclonal antibody 6B6 was 2.5ng/well, the antibody titer reached 1:640000. The purified monoclonal antibody was identified by SDS-PAGE, with clear bands without impurities, and clear bands at 50KDa and 25KDa (as shown in Figure 5), representing the heavy chain and light chain of the antibody respectively.

Table 1 Antibody titer after purification of monoclonal antibody 6B6

3) Identification of monoclonal antibody 6B6 subclass

According to the instructions of the SIGMA kit, the subtype identification of monoclonal antibody was carried out by the method of capture ELISA, as follows: After diluting the subtype identification reagent of monoclonal antibody 1:1000, add it to the enzyme-labeled well, 100 μl/well, 2 wells/sample, Incubate at 37°C for 1 hour; wash with PBST three times and pat dry; dilute ascites appropriately and add to the well, 100 μl/well; wash with PBST three times and pat dry; HRP enzyme-labeled goat anti-mouse IgG secondary antibody is diluted 1:600 and added. 100μl/well, incubate at room temperature for 30min; develop color for 10-20min. The subtype of the monoclonal antibody was defined as the OD ₄₅₀ reading value that was significantly higher than that of the subtype reagents added to other wells. After identification, the monoclonal antibody 6B6 belongs to IgG1 antibody subclass.

4) Indirect immunofluorescence method to identify the reactivity of monoclonal antibody and tumor cells

Spread the tumor cells into a 96-well plate. After the monolayer is covered, wash three times with PBS, 5min each time, add -20℃ pre-cooled methanol and fix at 4℃ for 15min. After washing three times with PBS, take 50μl and dilute 1000 Add 6 times anti-CD30 monoclonal antibody 6B6 to the wells, incubate at 37°C for 1 hour, wash with PBS three times, add 1:200 diluted fluorescent secondary antibody in the dark, incubate at 37°C for 1 hour, wash with PBS three times, dry, and observe under a fluorescent microscope . The results are shown in Figure 3. CD30 monoclonal antibody 6B6 still had strong reactivity with tumor cells at a concentration of 1*10 ^-4 mg/ml.

5) Identify the reactivity of monoclonal antibody with recombinant CD30 protein by immunoblotting

Prepare the separating gel according to the formula in Table 2. (unit: ml, total amount: 7.5ml/gel)

Table 2 Separating Gel Formula (15ml)

Gel concentration 10% H2O 6ml 1.5M Tris-HCl (pH=8.8) 3.75ml 10% SDS 150μl 10%AP 75μl TEMED 7.5μl 30% Acry/bis 5ml Protein isolate size (KD) 20～80

Slowly add a layer of distilled water (about 1ml) on the separation gel, flatten the separation gel, and place it at 30°C to solidify. After the separation gel was coagulated, the stacking gel was prepared, as shown in Table 3. (unit: ml, total amount: 2.5ml/gel)

Table 3 Stacking Gel Formula (5ml)

Stacking Gel Concentration 4% H2O 3.6ml 1M Tris-HCL (pH=6.8) 600μl 10% SDS 50μl 10%APS 25μl TEMED 5μl 30% Acry/bis 670μl

Insert the pre-prepared comb quickly after adding the stacking gel, and wait for it to solidify at 30°C. Mix 5 times of loading buffer with the sample, 100℃ metal bath or water bath for 10min, and centrifuge at 12000r/min for 5min. After the gel is solidified, put it into the electrophoresis tank, add electrophoresis buffer to cover the gel, pull out the comb vertically, load the sample, and perform electrophoresis. Use 60V voltage for the sample in the stacking gel until the sample is electrophoresed to the interface, adjust the voltage to 100V, and continue electrophoresis until the bromophenol blue is about to flow out of the gel surface. After electrophoresis, take out the gel, rinse it in deionized water, and remove the stacking gel; cut the same area of nitrocellulose membrane and 6 layers of filter paper according to the size of the gel, and soak in the pre-cooled transfer buffer for 30 minutes; (negative electrode) sponge-3 layers of filter paper-gel-nitrocellulose membrane-3 layers of filter paper-sponge (positive electrode) in order to install the transfer device, mark it well, there should be no air bubbles in each layer; add transfer buffer, the The electrophoresis tank was placed in an ice bath and transferred at a current of 200mA for 100min. After the transfer, remove the transfer device, rinse the PVDF membrane with deionized water, add blocking solution and shake at room temperature for 2 hours; wash with TBST 5 times, each time for 5 minutes, add the monoclonal antibody diluted with blocking solution, and the membrane Incubate together at 4°C overnight or at room temperature for 1h. Wash 5 times with TBST, 5 min each time. Horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody diluted 1:8000 was added and incubated at room temperature for 1 h. Wash 5 times with TBST, 5 min each time. Transfer the film to a freshly prepared developer solution and place it in the dark for 1-3 minutes, and develop it in a darkroom developer. The results are shown in Figure 4. The monoclonal antibody 6B6 has a strong reactivity with the recombinant CD30 protein at a concentration of 5*10 ^{- 3} mg/ml.

It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation, although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solutions of the present invention shall be covered by the claims of the present invention.

Claims (9)

1. A hybridoma cell line, the preservation number of which is CCTCCNO: C201861 in the China Center for Type Culture Collection of Wuhan University. 2. hybridoma cell strain according to claim 1, is characterized in that: described hybridoma cell strain is the hybridoma cell strain of anti-CD30, and described CD30 albumen is the recombinant protein of prokaryotic expression, named after prokaryotic expression CD30 protein . 3. The hybridoma cell strain according to claim 1 or 2, characterized in that: the hybridoma cell strain is obtained by fusion of splenocytes of mice immunized with prokaryotic expressed CD30 protein and myeloma cells. 4. An anti-CD30 monoclonal antibody, characterized in that it is secreted by the hybridoma cells according to claim 1. 5. The anti-CD30 monoclonal antibody according to claim 4, characterized in that: the subclass of the anti-CD30 monoclonal antibody belongs to IgG1, and the anti-CD30 monoclonal antibody can specifically recognize the CD30 protein, And it has strong reactivity with tumor cells. When the anti-CD30 monoclonal antibody at the concentration of 1 mg/ml is 2.5 ng of CD30, the antibody titer reaches 1:640000. 6. The use of the anti-CD30 monoclonal antibody according to claim 4 or 5 in the preparation of pharmaceutical preparations for diagnosis or treatment of tumors. 7. A pharmaceutical preparation for diagnosing or treating tumors, characterized in that it comprises the anti-CD30 monoclonal antibody according to claim 4 or 5. 8. The preparation method of the anti-CD30 monoclonal antibody according to claim 4 or 5, characterized in that: comprising, Prokaryotic expression and purification of CD30 protein: Design primers, construct pET-28a-CD30 prokaryotic expression plasmid, identify the correct plasmid and transform it into BL21, and induce protein expression with IPTG; IPTG-induced bacterial liquid is ultrasonically lysed, and the supernatant after centrifugation is combined with Ni filler to elute the protein , to obtain purified CD30 protein, the protein molecular weight is about 40KDa; Preparation of hybridoma cell lines: emulsify the purified CD30 protein, and inject it into Balb/c mice at multiple points and intraperitoneally. 2 weeks, 4 days after the second multipoint injection plus intraperitoneal injection, the amount of CD30 protein injected into the tail vein was 50 μg/mouse; splenocytes from immunized mice were fused with Sp2/0 myeloma cells to obtain hybridoma cells; screening Positive hybridoma cell lines were expanded and cultured. 9. preparation method as claimed in claim 8, is characterized in that: also comprises, Preparation of anti-CD30 monoclonal antibody: inject the positive hybridoma cell line obtained in claim 8 into the abdominal cavity of Balb/c mouse to make the mouse produce ascites, and collect the ascites; Purification of anti-CD30 monoclonal antibody: use protein-G column affinity purification to obtain anti-CD30 monoclonal antibody.