A recombinant protein, a monoclonal antibody of the recombinant protein, and a preparation method and application thereof

Abstract

The present invention discloses an AKAP4 recombinant protein, wherein the AKAP4 recombinant protein includes AKAP4 recombinant protein 1, AKAP4 recombinant protein 2, AKAP4 recombinant protein 3 and AKAP4 recombinant protein 4. The present invention also discloses a monoclonal antibody prepared using the AKAP4 recombinant protein, wherein the monoclonal antibody includes monoclonal antibody 1, monoclonal antibody 2, monoclonal antibody 3 and monoclonal antibody 4. The AKAP4 recombinant protein disclosed in the present invention has good immunogenicity and can be used to prepare a detection reagent for the AKAP4 protein. The four monoclonal antibodies prepared in the present invention have good sensitivity and specificity and are suitable for preparing a detection reagent for the AKAP4 protein.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a recombinant protein, a monoclonal antibody of the recombinant protein, and a preparation method and application thereof. Background Art

[0002] Non-small cell lung cancer (NSCLC) includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Compared with small cell carcinoma, its cancer cells grow and divide more slowly, and spread and metastasize relatively late. NSCLC accounts for about 80% of all lung cancers. About 75% of patients are already in the middle and late stages when they are discovered, and the 5-year survival rate is very low. Lung cancer has the fastest growing incidence and mortality rate. Early diagnosis of lung cancer depends on sputum exfoliated cells and tissue biopsy under bronchoscope.

[0003] A-kinase anchor protein 4 (AKAP4) is a member of the cancer testis antigen family and is not expressed in the human body except in testis and placenta tissue. In a new study, researchers from the United States and China identified the protein AKAP4 as a potential biomarker to effectively distinguish people with non-small cell lung cancer (NSCLC) from those without NSCLC. (Kiranmai Gumireddy et al. AKAP4 is a circulating biomarker for non-small cell lung cancer, Oncotarget, 13 May 2015). However, there are no diagnostic reagents or raw materials related to AKAP4. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, one of the objects of the present invention is to provide an AKAP4 recombinant protein and a preparation method and application thereof; a second object of the present invention is to provide a monoclonal antibody against AKAP4 and a preparation method and application thereof.

[0005] Therefore, on one hand, the present invention provides an AKAP4 recombinant protein, which includes AKAP4 recombinant protein 1, AKAP4 recombinant protein 2, AKAP4 recombinant protein 3 and AKAP4 recombinant protein 4; the amino acid sequence of the AKAP4 recombinant protein 1 is shown in SEQ ID NO.2, the amino acid sequence of the AKAP4 recombinant protein 2 is shown in SEQ ID NO.3; the amino acid sequence of the AKAP4 recombinant protein 3 is shown in SEQ ID NO.4; and the amino acid sequence of the AKAP4 recombinant protein 4 is shown in SEQ ID NO.5.

[0006] Preferably, the position of the AKAP4 recombinant protein 1 in SEQ ID NO.1 is aa1-aa180; the position of the AKAP4 recombinant protein 2 in SEQ ID NO.1 is aa320-aa430; the position of the AKAP4 recombinant protein 3 in SEQ ID NO.1 is aa490-aa590; and the position of the AKAP4 recombinant protein 4 in SEQ ID NO.1 is aa690-aa854.

[0007] On the other hand, the present invention also provides a monoclonal antibody prepared using the AKAP4 recombinant protein, the monoclonal antibodies include monoclonal antibody 1, monoclonal antibody 2, monoclonal antibody 3, and monoclonal antibody 4; the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1 are shown in SEQ ID NO.6 and SEQ ID NO.7; the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 2 are shown in SEQ ID NO.8 and SEQ ID NO.9; the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 3 are shown in SEQ ID NO.10 and SEQ ID NO.11; the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 4 are shown in SEQ ID NO.12 and SEQ ID NO.13.

[0008] Preferably, the monoclonal antibody 1 described in the present invention is prepared from AKAP4 recombinant protein 1, the monoclonal antibody 2 is prepared from AKAP4 recombinant protein 2, the monoclonal antibody 3 is prepared from AKAP4 recombinant protein 3, and the monoclonal antibody 4 is prepared from AKAP4 recombinant protein 4.

[0009] Preferably, the monoclonal antibody 1 and the monoclonal antibody 4 described in the present invention are best paired, wherein the monoclonal antibody is a coating antibody and the monoclonal antibody 4 is a detection antibody.

[0010] In another aspect, the present invention also provides a use of the recombinant AKAP4 protein in preparing monoclonal antibodies against the AKAP4 protein.

[0011] In another aspect, the present invention also provides the use of the monoclonal antibody in preparing an AKAP4 protein detection reagent.

[0012] The AKAP4 recombinant proteins (AKAP4 recombinant protein 1 to AKAP4 recombinant protein 4) disclosed in the present invention have good immunogenicity and can be used to prepare detection reagents for AKAP4 proteins (such as calibrators or immunogens of monoclonal antibodies, etc.). Although the present invention does not provide examples of calibrators, those skilled in the art should know that calibrators of different concentrations can be prepared using the AKAP4 recombinant proteins (AKAP4 recombinant protein 1 to AKAP4 recombinant protein 4) prepared by the present invention in combination with a suitable diluent (such as PBST buffer containing 1% BSA) for the detection of AKAP4 proteins.

[0013] Based on the prepared AKAP4 recombinant proteins (AKAP4 recombinant protein 1 to AKAP4 recombinant protein 4), the present invention screened 4 monoclonal antibodies that specifically bind to the AKAP4 recombinant proteins (AKAP4 recombinant protein 1 to AKAP4 recombinant protein 4). The 4 monoclonal antibodies all have good sensitivity and specificity and are suitable for preparing detection reagents for AKAP4 proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Analysis results of disordered regions of AKAP4 protein. The higher the score, the greater the probability that the position is a disordered region, and 0.5 is used as the dividing line for whether it is a disordered region.

[0015] Figure 2 SDS-PAGE diagram of AKAP4 recombinant proteins 1 to 4, wherein 1 to 4 are AKAP4 recombinant protein 1 to AKAP4 recombinant protein 4.

[0016] Figure 3 Western blot detection results of AKAP4 protein with four monoclonal antibodies. DETAILED DESCRIPTION

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0018] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0019] Example 1: Design and preparation of AKAP4 recombinant protein

[0020] An AKAP4 protein (amino acid sequence as shown in SEQ ID NO.1) was selected from NCBI (NP_003877.2) as the research object, and the disordered region of the protein was analyzed (such as Figure 1 As shown), the AKAP4 protein was truncated to prepare 4 recombinant proteins (a 6His tag was added to the C-terminus of each recombinant protein to facilitate purification), as shown below:

[0021] AKAP4 recombinant protein 1: The position in SEQ ID NO.1 is aa1 to aa180, as shown in SEQ ID NO.2,

[0022] AKAP4 recombinant protein 2: The position in SEQ ID NO.1 is aa320 to aa430, as shown in SEQ ID NO.3,

[0023] AKAP4 recombinant protein 3: The position in SEQ ID NO.1 is aa490 to aa590, as shown in SEQ ID NO.4,

[0024] AKAP4 recombinant protein 4: The position in SEQ ID NO.1 is aa690 to aa854, as specifically shown in SEQ ID NO.5.

[0025] The codon-optimized AKAP4 recombinant protein 1-4 nucleotide sequences were cloned into the pET-30a(+) vector. The codon-optimized AKAP4 recombinant protein 1-4 nucleotide sequences were used as templates and connected using the NdeI (CATATG)-XhoI (CTCGAG) sites to construct recombinant expression plasmids.

[0026] The recombinant expression plasmids were transformed into competent cells respectively, and after inverted culture at 37°C for 12 to 16 hours, the colony morphology was smooth, scattered individually, with neat edges, shiny, moist, and grayish white surfaces, showing the typical Escherichia coli colony morphology. One single colony was selected for PCR identification and sequencing identification. The PCR identification results were consistent with the expected size. The sequence alignment results showed that the sequence homology with SEQ ID NO.2 to 4 was 100%. The screened positive strains were named BL-21-AKAP4 recombinant protein 1 to BL-21-AKAP4 recombinant protein 4. The recombinant Escherichia coli BL-21-AKAP4 recombinant protein 1 to BL-21-AKAP4 recombinant protein 4 were tested for purity, and the test results were all pure, without the growth of foreign bacteria or mold.

[0027] LB liquid culture medium containing 100 μg / ml kanamycin was inoculated at a ratio of 1%, and cultured at 37°C at 200r / min for 2 to 4 hours. When the bacterial solution OD600nm was 0.6 to 0.8, IPTG was added at a final concentration of 1.0mM to continue induction for 4 hours. The induced recombinant bacteria were centrifuged at 5000g for 10 minutes and the supernatant was discarded. The precipitate was dissolved with PBS for 10 minutes and then crushed with an ultrasonic cell disruptor. After crushing, the supernatant was discarded by centrifugation at 4°C (12000r / min) for 30 minutes, the precipitated inclusion body was lysed with lysis buffer for 10 minutes, and the supernatant was taken for nickel column purification at 12000r / min for 30 minutes. The column was washed with 10 times column volume of lysis buffer; the supernatant was added to the nickel column, and the protein solution was slowly loaded; 10 times column volume of lysis buffer was washed to remove impurities; 5 times column volume of elution buffer was eluted to collect the purified AKAP4 recombinant protein 1 to AKAP4 recombinant protein 4, and stored at -80°C.

[0028] AKAP4 recombinant protein 1 to AKAP4 recombinant protein 4 were tested by SDS-PAGE protein gel electrophoresis. The results were as follows Figure 2 As shown, the SDS-PAGE purity was ≥90%.

[0029] Example 2: Preparation of monoclonal antibodies against AKAP4 recombinant protein

[0030] The AKAP4 recombinant proteins 1 to AKAP4 recombinant proteins 4 prepared in Example 1 were used to prepare AKAP4 protein monoclonal antibodies respectively. The specific method is conventional mouse hybridoma cell technology, which is briefly described as follows:

[0031] BALB / c mice aged 6 to 8 weeks were selected for immunization. For the first immunization, the recombinant protein was emulsified with an equal volume of Freund's complete adjuvant and inoculated intraperitoneally into mice at a rate of 100 μg protein per mouse. Two weeks later, the recombinant protein was emulsified with an equal volume of Freund's incomplete adjuvant and inoculated intraperitoneally into mice at a rate of 100 μg protein per mouse. Two weeks later, the mice were directly immunized with the recombinant protein for the third time via intraperitoneal route at a rate of 100 μg per mouse. On the third day after immunization, mouse spleen cells were taken out and fused with mouse myeloma cells SP2 / 0 and cultured in HAT selective medium. Seven to ten days later, the corresponding recombinant protein was used as the coating antigen, and the cell supernatant was detected by indirect ELISA to screen positive hybridoma cells.

[0032] Balb / c mice aged 6 to 8 weeks were intraperitoneally injected with 0.5 ml of pristane per mouse. After 7 to 10 days, 1×10 hybridoma cells were injected into each mouse. 6 ~2×10 6 After 7 to 10 days, the mouse ascites was extracted and centrifuged at 1000 r / min for 10 minutes at 2 to 8°C to collect the supernatant. The monoclonal antibody was purified using a Protein G affinity chromatography column.

[0033] After the above steps, AKAP4 recombinant protein 1 to AKAP4 recombinant protein 4 of Example 1 were used respectively, and one good hybridoma cell was screened for each recombinant protein, and on this basis, corresponding monoclonal antibodies were prepared, namely monoclonal antibody 1 to monoclonal antibody 4 (corresponding to AKAP4 recombinant protein 1 to AKAP4 recombinant protein 4, respectively, that is, monoclonal antibody 1 was prepared from AKAP4 recombinant protein 1, monoclonal antibody 2 was prepared from AKAP4 recombinant protein 2, monoclonal antibody 3 was prepared from AKAP4 recombinant protein 3, and monoclonal antibody 4 was prepared from AKAP4 recombinant protein 4).

[0034] Example 3: Performance testing of monoclonal antibodies

[0035] 1. Monoclonal antibody subtype identification

[0036] The commercial mouse monoclonal antibody subtype identification (MAI) ELISA kit (Shanghai Kanglang Biotechnology Co., Ltd.) was used to identify the subtypes of the heavy and light chains of the monoclonal antibodies according to the instructions. After testing, the heavy chain subtypes of the four monoclonal antibodies were all IgG1, and the light chains were all Kappa.

[0037] 2. Monoclonal Antibody HRP Labeling and Paired Detection

[0038] Use a commercial kit (HRP coupling kit -Lightning- (ab102890)) were used to label the four monoclonal antibodies with HRP. The pairing of the four monoclonal antibodies was tested using the double antibody sandwich method to verify the pairing effect of the monoclonal antibodies. The results showed (as shown in Table 1) that monoclonal antibody 1 and monoclonal antibody 4 were the best pairing, and the best result was obtained when monoclonal antibody 1 was used for coating and monoclonal antibody 4 was used for detection.

[0039] Table 1 Monoclonal antibody cross-matching results

[0040]

[0041] Four monoclonal antibodies were used to coat the ELISA plate, 100 ng / well, 0.1 ml / well, and coated overnight at 4°C; after washing, the plate was blocked with PBST containing 1% BSA and incubated at 37°C for 2 hours; the prepared AKAP4 recombinant protein (the full-length AKAP4 recombinant protein shown in SEQ ID NO.1 was prepared according to the method of Example 1) was diluted to 100 ng / ml with PBS, and then added to the coated ELISA plate, 0.1 ml / well, and incubated at 37°C for 1 hour; after washing, HRP-labeled monoclonal antibodies diluted 1:20000 were cross-added, 0.1 ml / well, and incubated at 37°C for 30 minutes; after washing 3 times, 0.1 ml of TMB single-component colorimetric solution was added, and incubated at 37°C for 10 minutes; stop solution (2M sulfuric acid) was added, 0.1 ml / well, and the OD450nm value was measured, and the highest OD450nm value was the best.

[0042] 3. Monoclonal Antibody Sequence Detection

[0043] According to the antibody subtype results, the antibody gene sequence was cloned using a method based on the RACE technology route. Hybridoma cells with good growth status were collected, and the total RNA of the hybridoma cells was obtained using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the operating method of Takara's SMARTer RACE manual, and the full-length sequence of the target antibody was amplified. After sequencing and analysis, the sequences of monoclonal antibodies 1 to 4 are shown in Table 2.

[0044] Table 2 Sequences of monoclonal antibodies

[0045] project Heavy chain variable region amino acid sequence Light chain variable region amino acid sequence Monoclonal Antibody 1 As shown in SEQ ID NO.6 As shown in SEQ ID NO.7 Monoclonal antibody 2 As shown in SEQ ID NO.8 As shown in SEQ ID NO.9 Monoclonal antibody 3 As shown in SEQ ID NO.10 As shown in SEQ ID NO.11 Monoclonal antibody 4 As shown in SEQ ID NO.12 As shown in SEQ ID NO.13

[0046] 4. Detection of AKAP4 protein by monoclonal antibody

[0047] The four HRP-labeled monoclonal antibodies were used to perform western blot detection on the total protein of NSCLC tissue specimens (all four monoclonal antibodies were diluted 1:2000 for detection). The results showed that ( Figure 3 ), all four monoclonal antibodies could specifically bind to AKAP4 protein in NSCLC tissue specimens, indicating that all four monoclonal antibodies had good specificity and sensitivity to AKAP4 protein.

[0048] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Monoclonal antibody that specifically binds to AKAP4 protein, It is characterized in that The monoclonal antibody is any one of monoclonal antibody 1, monoclonal antibody 2, monoclonal antibody 3, monoclonal antibody 4 or a pairing combination thereof, wherein: The amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.6 and SEQ ID NO.7; The amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.8 and SEQ ID NO.9; The amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 3 are shown in SEQ ID NO.10 and SEQ ID NO.11; The amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 4 are shown in SEQ ID NO.12 and SEQ ID NO.

13.

2. Use of the monoclonal antibody according to claim 1 in preparing an AKAP4 protein detection reagent.

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