CN110054675B - Immunogenic polypeptide, anti-TTC 36 antibody CP4-3 and application - Google Patents

Abstract

The invention relates to an immunogenic polypeptide, the sequence of which is shown as SEQ ID NO:1, a hybridoma, which is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 10 and 17 months, the preservation number of which is CCTCC NO: C2018208, a monoclonal antibody generated by the hybridoma, application of the monoclonal antibody, and a method for detecting TTC36 protein in a sample for non-diagnosis purposes. The truncated immunogenic polypeptides of the invention are effective in inducing the production of antibodies against TTC36 protein in humans or animals. The monoclonal antibody CP4-3 of the invention can efficiently and specifically bind to TTC36 protein in a sample, and has higher specificity and affinity compared with polyclonal antibodies. Provides better antibody tools for functional research of TTC36 protein, research, diagnosis and treatment of related diseases.

Description

Immunogenic polypeptide, anti-TTC 36 antibody CP4-3 and application

Technical Field

The invention relates to the field of immunization, in particular to an immunogenic polypeptide and application thereof, a hybridoma cell, an antibody produced by the hybridoma cell and application thereof, and a method for detecting TTC36 protein in a sample for non-diagnostic purposes.

Background

The TTC36(tetratricopeptide repeat domain 36) protein contains 3 TRP motifs (tetratricopeptide) and no other domains, and belongs to TPR-like structural protein. We found by phylogenetic tree analysis that the amino acid sequence of TTC36 is highly conserved among mammalian species and it can be seen that it performs important functions in vivo.

Jiang, L. and other researches find that the expression of TTC36 protein is down-regulated in human liver cancer tissue specimens, and if the TTC36 gene is over-expressed in liver cancer cells, the liver cancer cell apoptosis can be promoted. James a Nathan's study showed that TTC36 may be involved in intracellular protein ubiquitination.

At present, the research on TTC36 protein is not well reported, and some polyclonal antibodies aiming at TTC36 are available on the market, but the research on using TTC36 monoclonal antibodies is not reported in the literature, and monoclonal antibodies aiming at human TTC36 protein are not available on the market.

Because of the high specificity of monoclonal antibodies, the development of in vitro diagnostic kits or the preparation of antibody drugs by using monoclonal antibodies to perform relevant mechanism research has become one of the fastest growing and most profitable markets in the pharmaceutical and biological preparation industries. Therefore, monoclonal antibodies against TTC36 protein need to be developed for related uses.

Disclosure of Invention

In order to solve the problems, the invention provides an immunogenic polypeptide, the sequence of which is shown as SEQ ID NO. 1.

The invention also provides application of the immunogenic polypeptide in preparing anti-TTC 36 antibodies.

The invention also provides a method for preparing TTC36 antibody, which comprises the step of stimulating the immune system of experimental animal by using the polypeptide to make the experimental animal produce antibody.

The invention also provides a hybridoma cell which is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 10 and 17 months, and the preservation number is CCTCC NO: C2018208.

The invention also provides a monoclonal antibody produced by the hybridoma cell.

The invention also provides application of the monoclonal antibody in detecting TTC36 protein in a non-pathological sample.

The invention also provides the application of the antibody in preparing a disease diagnosis reagent.

In a preferred embodiment, the disease is cancer.

In a preferred embodiment, the disease is liver cancer.

The present invention also provides a method for detecting TTC36 protein in a sample for non-diagnostic purposes, comprising the steps of:

s1: exposing the sample to an antigen;

s2: incubating the sample with the monoclonal antibody;

s3: displaying the monoclonal antibody specifically bound to the sample.

The truncated immunogenic polypeptide of the invention can effectively induce the human or animal to generate antibodies against TTC36 protein, and the antibody of the invention can efficiently and specifically bind to TTC36 protein in a sample, and has higher specificity and affinity compared with polyclonal antibody. Provides better antibody tools for functional research of TTC36 protein, research, diagnosis and treatment of related diseases.

Biological material preservation

The monoclonal antibody designed by the invention is produced by hybridoma cells which are preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university in Wuhan city, Hubei province in China within 10 and 17 months in 2018, the preservation number is CCTCC NO: C2018208, and the monoclonal antibody is named as a hybridoma cell strain CP 4-3.

Drawings

FIG. 1 is a chromatogram identification of a synthetic polypeptide (GQLRRPRDSR);

FIG. 2 is an electrophoretogram of purified products of the immunoantigen GST-TTC36, wherein lane M is Marker, lane 1 is 0.5mg/ml BSA, and lane 2 is GST-TTC36 diluted 2-fold;

FIG. 3 shows the results of ELISA method for detecting the binding force and specificity of hybridoma cell culture supernatant;

FIG. 4 shows the results of detecting the binding force of the culture supernatant of hybridoma cells by immunoblotting, wherein lane M is Marker, and lanes 1-3 are the results of staining the culture supernatants of hybridoma cells CP4-3, CP4-6 and CP4-7, respectively;

FIG. 5 shows the result of the antibody titer of CP4-3 after purification by ELISA;

FIG. 6 shows the results of the immunodetection of paraffin sections of liver cancer and paracarcinoma tissues, the left side is a photograph of a liver cancer tissue section, and the right side is a photograph of a paracarcinoma tissue section;

FIG. 7 shows the result of immunoblotting detection of liver cancer and tissue beside cancer, wherein Lane 1 is tissue beside cancer, and Lane 2 is tissue of liver cancer.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

1. Antigen preparation

1.1 preparation of Immunoantigens

Based on the protein sequence of TTC36, a plurality of polypeptides were synthesized, one of which is amino acid 180-189 and has the sequence GQLRRPRDSR (SEQ ID NO: 1). The synthesized polypeptide was identified by chromatography, and the sequence of the polypeptide was correct and the purity was 90.356% as shown in FIG. 1. The polypeptide is conjugated to KLH to form the conjugated protein TTC36(180-189aa) -KLH. Conjugation to Bovine Serum Albumin (BSA) to form a "polypeptide-BSA" conjugate, followed by quantitation at 2.0mg/ml, designated: TTC361 (180) 189aa) -BSA.

1.2 prokaryotic expression of Positive antigen TTC36 protein

1.2.1 Synthesis and cloning of Gene fragments

The coding gene of TTC36 is codon optimized in the whole course, and GST-labeled coding gene is connected to the C end to facilitate purification, and the obtained sequence is shown in SEQ ID NO. 2. EcoR I and Xho I sites were added to each end of the sequence to facilitate cloning.

The above sequences were synthesized and cloned into pUC57 plasmid vector in TA mode, and correctly sequenced clones were selected for further experiments.

1.2.2 construction of expression vectors and expression strains

Fragment enzyme digestion: take 43. mu.l TTC36-pUC57 recombinant plasmid, 1. mu.l EcoR I, 1. mu.l Xho I, 5. mu.l 10 XBuffer, react overnight at 37 ℃ (using agarose gel DNA recovery kit, BPI); and (3) carrying out enzyme digestion on the vector: mu.l of the vector (pGEX-4T-1) plasmid, 1. mu.l of EcoR I, 1. mu.l of Xho I, 5. mu.l of 10 XBuffer, were taken for overnight reaction at 37 ℃ (agarose gel DNA recovery kit, BPI).

Connection transformation: and (3) mixing 1. mu.l of vector (pGEX-4T-1), 3. mu.l of fragment, 1. mu.l of ligase (BPI) and 5. mu.l of 2 × Rapid Buffer, and reacting at room temperature for 30min to obtain the TTC36 protein expression vector.

Competent cells (BL21) stored at-80 ℃ were removed and slowly thawed on ice. Competent cells were added to the ligation product and mixed well and left on ice for 30 min. Heat shock at 42 ℃ for 90 s. After ice-bath for 2min, 800. mu.l of non-resistant LB medium was added. Culturing at 37 deg.C for 45 min. Centrifuging at 5000rpm for 3min, discarding most of the supernatant, leaving about 100-. Air-dried and cultured in an incubator at 37 ℃ for overnight in an inverted state. Selecting positive clones, and checking to obtain TTC36 protein expression strain.

1.2.3 expression and purification of TTC36

1.2.3.1 protein expression

(1) According to the following steps of 1:50 proportions of the strains were inoculated into the corresponding resistant LB liquid medium and cultured at 37 ℃ and 200 rpm.

(2) The cultured bacterial liquid is transferred into 1000ml of corresponding resistant LB liquid culture medium according to the proportion of 1:50, cultured at 37 ℃ and 200rpm until OD is 0.6-0.8, and induced for 2h at 37 ℃ by IPTG (0.5 mM).

(3) Collecting bacteria: 8000rpm, and 6 min. The supernatant was discarded.

(4) Ultrasonic bacteria breaking: the cells were blown up with 20-30ml of 10mM Tris-HCl (pH 8.0) solution and sonicated (500W,180 times 5s each time, 5s apart).

(5) Electrophoresis to determine the expression pattern: mu.l of the sonicated bacterial suspension was centrifuged at 12000rpm for 10min, 50. mu.l of the supernatant was transferred to another EP tube, and after the supernatant was removed, the pellet was blown off with 50. mu.l of 10mM Tris-HCl (pH 8.0). And (4) carrying out electrophoresis detection.

1.2.3.2 protein purification

(1) The column was pipetted at a rate of 1ml glutaminone Sepharose 4B bead per 8mg GST fusion protein, and 5ml PBS (pH 7.4) was added thereto, resuspended, centrifuged at 2000rpm for 2min, and repeated 3-5 times.

(2) 20ml of PBS (pH 7.4) was added to the cells of 500ml of the cell suspension, resuspended, sonicated, and centrifuged at 12000rpm for 10 min.

(3) The supernatant was mixed with the treated beads and shaken well, combined by shaking overnight at 4 ℃.

(4) Centrifuging at 2000rpm for 2min, transferring the supernatant to another tube, performing sample electrophoresis (for column penetration), and transferring the bead to a 10ml EP tube.

(5) Washed 3 times with 5ml of lysate (centrifuged 2min at 2000rpm, resuspended after addition).

(6) Resuspend the bead with 5ml of eluent (10mM Reduced glutaminone, 50mM Tris-HCl, pH 8.0), shake at 4 ℃ for 2-3h with tumbling, centrifuge at 2000rpm for 2min, and transfer the supernatant to another tube. This was repeated twice.

(7) The beads were resuspended in 5ml of eluent (60mM Reduced glutaminone, 50mM Tris-HCl, pH 8.0), shaken overnight at 4 ℃ with tumbling, centrifuged at 2000rpm for 10min, and the supernatant transferred to another tube. This was repeated twice.

(8) Sampling 50 μ l, and detecting the protein purification effect by electrophoresis.

(9) Dialyzed against 200 times the sample volume Buffer (1 XPBS) at 4 ℃. Dialyzing for 3 times each for 3 h.

(10) Centrifuging at 12000rpm for 10min, collecting supernatant, and performing electrophoresis detection.

The results are shown in FIG. 2, the protein size is consistent with the theoretical value (46kDa), the purity is 87.25%, and the protein can be used for immunization and screening.

2. Immunization of mice

4 SPF-grade BALB/c female mice were initially immunized subcutaneously with a "polypeptide-KLH" conjugate protein in an amount of 60 μ g protein/mouse, numbered: 1#, 2#, 3#, 4 #. Every 2 weeks, the immunization was carried out a second and third time, respectively, at an amount of 30. mu.g protein/mouse. On day 7 after the third immunization, blood was collected from the orbit and the serum titer was measured, showing that the titer was highest in the # 1 mouse. One week later, mice # 1 were challenged intraperitoneally with 50 μ g of immunogen.

The "polypeptide-BSA" coupled protein was diluted with a coating solution of 0.1M carbonate buffer (pH9.6), 2. mu.g/ml, and a 96-well ELISA plate was coated at a concentration of 2. mu.g/ml, 100. mu.l was added to each well, and overnight at 4 ℃; PBST (0.05% Tween-20, PBS) was washed 5 times for 3min each, then blocked with 3% skim milk powder in PBS buffer, and incubated at 37 ℃ for 2 h; PBST was washed 5 times for 3min each time for use.

Serum was diluted with PBS in a 2-fold gradient from 200 fold, blank control (blank) PBS, negative control (negative) 200-fold dilution of negative serum (serum from naive mice). Adding 100 μ l of HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution) to each well, incubating at 37 deg.C for 2h, washing with PBST for 5 times (3 min each time), and incubating at 37 deg.C for 1 h; PBST was washed 5 times, 3min each time, TMB color development solution 100. mu.l/well, incubated 15 min at room temperature in the dark, then quenched with 50. mu.l of 2M sulfuric acid, and detected with dual wavelength 450nm/630 nm. The test results are shown in table 1, and the titer of the 1# mouse is highest.

TABLE 1 mouse serum titer test results

3. Preparation and screening of hybridoma cells

(1) Preparation of fused cells

Taking the splenocytes of the 1# mouse and SP2/0 cells, and fusing by a PEG method. The fused cells were cultured in semi-solid medium (containing HAT) for selection. 10 plates × 93 cell monoclonals were picked and cultured in 96 well cell culture plates (previously plated with mouse thymocytes, 100 μ l/well).

(2) Hybridoma cell selection

ELISA method: the "polypeptide-BSA" coupled protein was diluted with a coating solution of 0.1M carbonate buffer (pH9.6) and coated on a 96-well ELISA plate at a concentration of 1. mu.g/ml, 100. mu.l was added to each well, and overnight at 4 ℃; PBST washing 5 times, each time 3min, with 3% BSA PBS buffer blocking, 37 degrees C were incubated for 2 h; PBST washing 5 times, each time 3 min; adding 100 μ l hybridoma cell culture supernatant into each well, washing with PBST for 5 times (3 min each time) at 37 deg.C for 2h, labeling with HRP (horse radish peroxidase) diluted at 1:5000 for 100 μ l/well, and incubating at 37 deg.C for 1 h; PBST was washed 5 times for 3min each time with 100. mu.l/well of TMB developer, quenched with 2M sulfuric acid 15 min later at room temperature, and detected with dual wavelength 450nm/630 nm.

Screening strategy of hybridoma cell strain capable of stably secreting antibody: and (3) adopting an ELISA method and an immunoblotting method, coating a plate by using a polypeptide-BSA coupled protein, and performing primary screening to obtain 12 positive hybridoma cell strains. And subcloning the positive cells, and performing secondary screening by using an ELISA method and a polypeptide-BSA coupled protein coated plate to obtain a hybridoma cell strain 5 strain capable of stably secreting the antibody. Finally, the screening was performed by immunoblotting and the "GST-TTC 36" fusion protein.

The results of the experiment are shown in FIG. 3: the culture supernatants of hybridoma cell lines numbered CP4-3, CP4-6 and CP4-7 have GQLRRPRDSR polypeptide binding specificity, and CP4-3 has the highest binding force.

Immunoblotting: preparing 4-12% of acrylamide glue; after GST-TTC36 protein was treated with 5 × loading buffer, 10 μ l/50ng per well, electrophoresis parameters were constant voltage 60-120V for 60min, then transferred to 0.45 μm PVDF membrane, constant current 200mA for 60min, and then detected with cell supernatant as primary antibody (1:4 dilution).

As shown in FIG. 4, only the antibody-containing supernatant secreted by the hybridoma cell line CP4-3 detected a specific band of about 46kDa, which is consistent with the theoretical value, indicating that the antibody specifically binds to GST-TTC36 protein. CP4-3 was used for the next experiment.

(3) Monoclonal antibody subtype identification

The antibody produced by the positive cell line selected was then used for subtype identification using Southern Biotech antibody typing kit. The results are shown in Table 2, where CP4-3 is IgG1, kappa type.

TABLE 2 antibody subtype test results

4. Preservation of hybridoma cells

The results of the above experiments show that the monoclonal antibody produced by the hybridoma CP4-3 has high specificity and the best affinity. The hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university in Wuhan City, Hubei province, China for 10 months and 17 days in 2018, the preservation number is CCTCC NO: C2018208, and the hybridoma cell strain is named as hybridoma cell strain CP 4-3.

5. Antibody preparation and purification

CP4-3 was cultured to log phase, washed with Hanks solution and then suspended to quantify, i.e., 5X 10 injections were administered intraperitoneally to each mouse (BALB/c female mice) ⁵ After the ascites is beaten out, the ascites is taken 7-10 days later.

The ascites of the mice were diluted 1:3 with a coupling buffer, centrifuged at 12000rpm at 4 ℃ for 10min and then filtered through a 0.22 μm filter to remove fat, cell debris and small particulate matter. The column containing Protein G Sepharose (Protein G Agarose) was equilibrated with 5-10 column volumes of the corresponding coupling buffer. The filtered ascites fluid was injected into the upper port of the column using a syringe and the effluent collected in a 50ml centrifuge tube. Washing the column with 5 times of column volume of coupling buffer solution; adding the neutralization solution into a centrifugal tube; the eluent is added dropwise to 1.0ml, and the pH value is detected after the mixture is mixed evenly. The amount of the neutralizing solution was adjusted in accordance with the results so that the pH was 7.0 as a result of the neutralization. Eluting the antibody by using an elution buffer solution with 5 times of column volume, collecting the antibody in a centrifuge tube, detecting the concentration of the antibody, and adjusting the concentration of the purified antibody to 1mg/ml standard for a subsequent function detection experiment.

ELISA assay potency

Coating with 2. mu.g/ml of "polypeptide-BSA coupled protein" overnight at 4 ℃; sealing 2% skimmed milk at 37 deg.C for 2 hr; purified antibody (1mg/ml) was diluted in a 2-fold gradient from 200-fold, blank control (blank) in PBS, and negative control (negative) in medium at 200-fold dilution.

Adding 100 μ l sample into each well, incubating at 37 deg.C for 2h, washing with PBST for 5 times (3 min each time), adding 100 μ l HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution) into each well, and incubating at 37 deg.C for 1 h; PBST was washed 5 times for 3min each time with 100. mu.l/well of TMB color developing solution, incubated for 15 min at room temperature in the dark, then quenched with 50. mu.l of 2M sulfuric acid, and detected with dual wavelength 450/630 nm.

As a result, the CP4-3 antibody titer reached 51200 or more, as shown in FIG. 5.

Use of CP4-3 antibody for immunodetection of pathological samples

The following detection methods can be used for both pathological sample detection and non-diagnostic purpose detection of samples.

(1) Immunohistochemical detection of liver cancer tissue

Taking a paraffin embedded block of human liver cancer and paracancer normal liver tissues, slicing by 5 mu m, repairing antigen by a heating method, fixing for 10min by 4% formaldehyde, then dropwise adding 500ng/50 mu l of purified anti-human TTC36 antibody (the diluent is PBS of 1% BSA), incubating for 2h at 25 ℃, then washing for 5 times by PBST, dropwise adding HRP-labeled goat anti-mouse IgG antibody, incubating for 1h at room temperature, then HE counterstaining, and observing under a microscope.

As shown in FIG. 6, the CP4-3 antibody detected that TTC36 protein in liver was mainly located in the cytoplasm of liver cells, and the expression of TTC36 protein in liver cancer tissue was significantly reduced compared with that in the para-carcinoma tissue.

(2) Liver cancer tissue immunoblotting detection

Detection of liver cancer tissue immunoblotting (Western-blotting): preparing 4-12% acrylamide gel, cracking liver cancer tissues and paracancer normal tissues, treating a tissue lysate with 5 × loading buffer, loading 200ng/10 μ l of each gel hole, setting an electrophoresis parameter to be constant voltage of 60-120V for 60min, then transferring to a 0.45 μm PVDF membrane, setting a transfer parameter to be constant current of 200mA for 60min, then using a purified antibody as a primary antibody (1:2000 dilution), using beta-Actin as an internal reference, using HRP-labeled goat anti-mouse IgG as a secondary antibody (1:2000 dilution), and detecting.

The result is shown in fig. 7, compared with the normal liver tissue, the TTC36 band of the liver cancer tissue is obviously lighter in color, which indicates that the CP4-3 antibody can effectively detect that the expression of TTC36 in the liver cancer tissue is obviously reduced.

From the above-mentioned results, it was found that TTC36 can be used as a molecular marker for detecting whether tissues are cancerous, and that the CP4-3 antibody can efficiently and specifically detect TTC36 protein in pathological tissues.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Shenzhen Shenzhong Longhua region people hospital

Liu transporting flood

<120> immunogenic polypeptide, anti-TTC 36 antibody CP4-3 and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gly Gln Leu Arg Arg Pro Arg Asp Ser Arg

1 5 10

<210> 2

<211> 567

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgggcaccc cgaatgacca agcggttctg caagcgattt tcaacccgga caccccgttt 60

ggcgatattg tgggcctgga cctgggcgag gaagcggaga aagaggagcg tgaggaagat 120

gaagtgttcc cgcaggcgca actggagcag agcaaagcgc tggaactgca aggtgttatg 180

gcggcggagg cgggcgacct gagcaccgcg ctggaacgtt ttggtcaggc gatctgcctg 240

ctgccggagc gtgcgagcgc gtacaacaac cgtgcgcagg cgcgtcgtct gcaaggtgat 300

gtggcgggtg cgctggagga cctggaacgt gcggttgaac tgagcggtgg ccgtggtcgt 360

gcggcgcgtc agagcttcgt gcaacgtggt ctgctggcgc gtctgcaggg tcgtgacgat 420

gacgcgcgtc gtgatttcga gcgtgcggcg cgtctgggca gcccgtttgc gcgtcgtcaa 480

ctggttctgc tgaacccgta tgcggcgctg tgcaaccgta tgctggcgga tatgatgggt 540

caactgcgtc gcccgcgtga tagccgt 567

Claims (8)

1. An immunogenic polypeptide, which is characterized in that the sequence is shown as SEQ ID NO. 1.

2. Use of the immunogenic polypeptide of claim 1 for the preparation of an anti-TTC 36 antibody.

3. A method of producing TTC36 antibody, comprising the step of stimulating the immune system of a test animal with the polypeptide of claim 1, thereby allowing the test animal to produce antibodies.

4. A hybridoma cell is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 10 and 17 months, and the preservation number is CCTCC NO: C2018208.

5. A monoclonal antibody produced by the hybridoma cell of claim 4.

6. Use of the monoclonal antibody of claim 5 for detecting TTC36 protein in a non-pathological sample.

7. The use of the antibody of claim 5 for the preparation of a diagnostic reagent for liver cancer.

8. A method for detecting TTC36 protein in a sample for non-diagnostic purposes, comprising the steps of:

s1: exposing the sample to an antigen;

s2: incubating the sample with the monoclonal antibody of claim 5;

s3: displaying the monoclonal antibody specifically bound to the sample.

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