CN110054688B - anti-TTC 36 monoclonal antibody and application thereof - Google Patents

Abstract

The invention relates to a hybridoma cell, which is preserved in a China Center for Type Culture Collection (CCTCC) in 2018, 10 and 17 months, and the preservation number is CCTCC NO: C2018196; also relates to a monoclonal antibody produced by the hybridoma cell, application thereof and a method for detecting TTC36 protein in a sample for non-diagnostic purposes. 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 antibodies. Provides better antibody tools for functional research of TTC36 protein, research, diagnosis and treatment of related diseases.

Description

anti-TTC 36 monoclonal antibody and application thereof

Technical Field

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

Background

TTC36(tetratricopeptide repeat domain 36) protein contains 3 TRP modules (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 reported, and some polyclonal antibodies aiming at TTC36 are available on the market, but the research on using TTC36 monoclonal antibody is not reported in the literature, and the monoclonal antibody aiming at human TTC36 protein is not sold 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 a hybridoma, wherein the hybridoma is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 10 and 17 months, and the preservation number is CCTCC NO: C2018196.

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 invention also provides a method for detecting TTC36 protein in a sample for non-diagnostic purposes, which is characterized by comprising the following steps:

s1: exposing the sample to an antigen;

s2: incubating the sample with the monoclonal antibody of claim 2;

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

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 antibodies. 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 days in 2018, the preservation number of the hybridoma cells is CCTCC NO: C2018196, and the hybridoma cells are named as hybridoma cell strains AT 36-38.

Drawings

FIG. 1 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. 2 shows the results of detecting the binding force of the culture supernatant of hybridoma cells by immunoblotting, wherein lane M is Marker, and lanes 1-6 are the culture supernatants of hybridoma cells 1D7, 12A6, 14A3, 22H1, 38D7 and 53B 12;

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

FIG. 4 shows the result of the ELISA method for detecting the titer of the purified 38D7 antibody;

FIG. 5 shows the results of the immunoassay of paraffin sections of normal liver tissues, the left side is a photograph under a 100-fold microscope, and the right side is a photograph under a 400-fold microscope;

FIG. 6 shows the results of the immunoassay of paraffin sections of normal kidney tissues, the left side of the section is a photograph taken under a 100-fold microscope, and the right side of the section is a photograph taken under a 400-fold microscope;

FIG. 7 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. 8 shows the result of immunoblotting detection of liver cancer and a tissue beside cancer, wherein Lane 1 is a tissue beside cancer, and Lane 2 is a 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.

Prokaryotic expression of TTC36 protein

1.1 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. 1. EcoR I and Xho I sites were added to each end of the sequence to facilitate cloning.

The above sequence was synthesized and cloned into pUC57 plasmid vector, and the correctly sequenced clone was selected for further experiments.

1.2 construction of expression vectors and expression strains

1.2.1 enzyme digestion:

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).

1.2.2 ligation transformations

Connecting: 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.

1.3 expression and purification of TTC36

1.3.1 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 by IPTG (0.5mM) at 37 ℃.

(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.

1.3.2 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 ℃.3 times of dialysis for 3 hours each time.

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

The result is shown in figure 1, the protein size is consistent with the theoretical value (46kDa), the purity is higher than 85 percent, and the requirement of the protein for immunization is met.

2. Immunization of mice

4 BALB/c female mice of SPF grade were initially immunized subcutaneously with purified "GST-TTC 36 protein", 60. mu.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 2# mouse. One week later, mice # 2 were challenged intraperitoneally with 50 μ g of immunogen.

Coating with GST-TTC36 protein, 2 μ g/ml, 4 deg.C overnight; 2% of skimmed milk, sealing at 37 ℃ for 2 h; serum was diluted in a 2-fold gradient starting at 200-fold, with a blank control (blank) in PBS and a negative control (negative) at 200-fold dilution in negative serum. The test results are shown in table 1, with the highest titer in the 2# mice.

TABLE 1 mouse serum titer test results

3. Preparation and screening of hybridoma cells

(1) Preparation of fused cells

Taking the mouse spleen cell and SP2/0 cell, fusing by 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. mu.l/well).

(2) Hybridoma cell selection

Immunoblotting: preparing 4-12% acrylamide glue; after GST-TTC36 protein treatment, 10 μ l/50ng of each well was subjected to electrophoresis at a constant pressure of 60-120V for 60min, and then transferred to a 0.45 μm PVDF membrane at a constant current of 200mA for 60min, and then the cell supernatant was used as a primary antibody (1:4 dilution) for detection.

The results are shown in FIG. 2, and all hybridoma cells secrete antibody-containing supernatants with specific bands at about 46kDa, which is consistent with theoretical values, and shows that the antibody-containing supernatants can be specifically combined with GST-TTC36 protein.

ELISA method: coating the plate with GST-TTC36 protein, diluting the protein with a coating solution containing 0.1M carbonate buffer, pH9.6, coating a 96-well ELISA plate at a concentration of 1. mu.g/ml, adding 100. mu.l per well, and standing overnight at 4 ℃; PBST after 5 times of washing, with 3% BSA PBS buffer blocking, 37 degrees C were incubated for 2 h; PBST washing 5 times, each time 3 min; adding 100 mu 1 of anti-human TTC36 chimeric antibody into two holes, washing for 5 times by PBST (PBST) at 37 ℃ for 2h, 3min each time, then using 100 mu 1/hole of HRP (horse radish peroxidase) labeled goat anti-human IgG antibody diluted by 1:5000, and incubating at 37 ℃; PBST was washed 5 times, 3min each time, TMB developing solution 100. mu.l/well, quenched with 2M sulfuric acid 15 min later at room temperature, and detected with dual wavelength 450nm/630 nm.

The selected clone is screened for the first time by using a tag protein GST-TTC36 fusion protein coated plate and screened for the second time by using the tag protein GST protein coated plate by adopting an ELISA method to obtain a positive hybridoma cell strain which stably identifies immunogen (namely, the positive hybridoma cell strain only combines with TTC36 but not GST protein).

The results are shown in fig. 3, and the 1D7, 12a6, 38D7 hybridoma cell lines secrete antibody-containing supernatants with TTC36 binding specificity: both bound to GST-TTC36 protein coated wells and did not bind to GST protein coated wells, with 38D7 binding the highest; the supernatants secreted by the 14A3, 22H1 and 53B12 hybridoma cell lines and containing the antibody have no TTC36 binding specificity, and are combined with GST-TTC36 protein coated holes and GST coated holes.

(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 38D7 is IgG2a, 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 hybridoma 38D7 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: C2018196, and the hybridoma cell strain is named as hybridoma cell strain AT 36-38.

5. Antibody preparation and purification

38D7 was cultured to log phase, washed with Hanks solution and then suspended to quantitate, each mouse (BALB/c female mice) was intraperitoneally injected with 5X 10⁵After 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 A sepharose 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 μ g/ml "GST-TTC 36 protein" at 4 deg.C overnight; 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. And (3) titer judgment: maximum dilution corresponding to a minimum OD reading greater than one-half of the maximum OD.

As a result, as shown in FIG. 4, the 38D7 antibody titer reached 51200 or more.

7.38D 7 antibody for immunodetection of pathological samples

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

(1) Immunohistochemical detection of normal tissue

Taking paraffin embedded blocks of human livers and kidneys, slicing by 5 mu m, repairing antigens 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), taking PBS of 1% BSA as a negative control, incubating for 2h at 25 ℃ and room temperature, then washing for 5 times by PBST, dropwise adding a labeled goat anti-mouse IgG antibody by HRP, incubating for 1h at room temperature, HE counterstaining, and observing under a microscope.

As a result, as shown in fig. 5 and 6, the anti-human TTC36 chimeric antibody was able to bind to TTC36 protein in liver parenchymal cells, the renal proximal tubule, and TTC36 protein was mainly distributed in cytoplasm.

(2) 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.

The results are shown in fig. 7, and the 38D7 antibody detects that the expression of TTC36 protein in liver cancer tissue is obviously reduced compared with that in the para-carcinoma tissue.

(3) 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. 8, compared with the normal liver tissue, the TTC36 band of the liver cancer tissue is obviously lighter in color, which indicates that the 38D7 antibody can effectively detect that the expression of TTC36 in the liver cancer tissue is obviously reduced.

From the above-mentioned test results, TTC36 can be used as a molecular marker for detecting whether the tissue is cancerous, and the 38D7 antibody can effectively 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> anti-TTC 36 monoclonal antibody and application thereof

<160>1

<170>SIPOSequenceListing 1.0

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<211>567

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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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 (5)

1. 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: C2018196.

2. A monoclonal antibody produced by the hybridoma cell of claim 1.

3. Use of the monoclonal antibody of claim 2 for detecting TTC36 protein in a non-pathological sample.

4. The use of the antibody of claim 2 for the preparation of a diagnostic reagent for liver cancer.

5. 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 2;

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

Images