Biotinylated anti-ttc36 antibodies and uses thereof

Abstract

The present application relates to an anti-TTC36 antibody, the sequences of CDR1-3 of the heavy chain variable region are respectively shown as SEQ ID NO: 5-7; the sequences of CDR1-3 of the light chain variable region are respectively shown as SEQ ID NO: 8-10.The anti-TTC36 antibody of the present application can specifically recognize and combine TTC36 protein, thereby detecting the presence and content of TTC36 protein in a sample, providing convenience for studying and understanding the function of TTC36 protein in life activities, and also can be used as a liver cancer detection reagent.The anti-TTC36 antibody of the present application can also be biotinylated, thereby being easy to use for detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of protein detection, and more particularly, to an anti-TTC36 antibody and application thereof. BACKGROUND

[0002] TTC36 (tetratricopeptide repeat domain 36) protein contains 3 TRP motifs (tetratricopeptide), no other domain, belongs to TPR-like structure protein, and currently there are few reports on its research. It is reported that the protein is related to tumor and ubiquitination mechanism.

[0003] In terms of tumor, it has been found that the TRP domain of TTC36 interacts with the C-terminal EEVD motif of heat shock protein-70 (Hsp70), which may be involved in inhibiting tumor proliferation and metastasis. In addition, the expression of TTC36 protein is down-regulated in human hepatocarcinoma tissue specimens, and further research finds that it is mainly because of the loss of heterozygosity or promoter methylation of TTC36 gene in hepatocarcinoma cells. In addition, if the TTC36 gene is overexpressed in hepatocarcinoma cells, it can inhibit the interaction of HSP70 and Bax, increase the transport of Bax from cytoplasm to mitochondria, and then increase the release of cytochrome C from mitochondria to cytoplasm, thereby promoting the apoptosis of hepatocarcinoma cells.

[0004] In terms of ubiquitination, among the proteins combined with K-48 polyubiquitin chains, there is TTC36 protein, and the function of K-48 polyubiquitin chains is to mediate the transport of target proteins to proteasome for degradation, which suggests that TTC36 may be involved in intracellular protein ubiquitination.

[0005] In addition, we found in the research that the Ttc36 gene full knockout mouse will appear tyrosinemia, and the exogenous TTC36 tyrosinemia is partially recovered by supplement. The results of immunofluorescence and immunoblotting experiments show that TTC36 is mainly expressed in the liver and kidney proximal tubules of mice, and the expression position is the same as that of tyrosine metabolic enzyme HPD. In human body, TTC36 is mainly expressed in liver parenchymal cells and kidney proximal tubules.

[0006] Therefore, TTC36 protein has multiple biological functions in mammals and human body, and its research has important significance. Anti-TTC36 antibody can be used to develop in vitro diagnostic kits or prepare antibody drugs. SUMMARY

[0007] To solve the above problems, the application provides an anti-TTC36 antibody, wherein the sequences of CDR1-3 of the heavy chain variable region are respectively shown as SEQ ID NO: 5-7; and the sequences of CDR1-3 of the light chain variable region are respectively shown as SEQ ID NO: 8-10.

[0008] In one specific embodiment, the sequence of the heavy chain variable region is shown as SEQ ID NO: 2.

[0009] In one specific embodiment, the sequence of the light chain variable region is shown as SEQ ID NO: 4.

[0010] In one specific embodiment, the antibody is a single-chain antibody, which is connected by a connecting polypeptide between the heavy chain variable region and the light chain variable region; and the sequence of the connecting polypeptide is shown as SEQ ID NO: 11.

[0011] In one specific embodiment, the sequence of the antibody is shown as SEQ ID NO: 12.

[0012] In one specific embodiment, the antibody is further coupled with Avi, so as to be biotinylated.

[0013] The application further provides a non-diagnostic application of the above anti-TTC36 antibody in detecting TTC36 protein.

[0014] The application further provides an application of the above anti-TTC36 antibody in preparing a liver cancer diagnostic reagent.

[0015] The anti-TTC36 antibody of the application can specifically recognize and combine with TTC36 protein, so as to detect the presence and content of TTC36 protein in a sample, provide convenience for studying and understanding the function of TTC36 protein in life activities, and can also be used as a liver cancer detection reagent. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a gel electrophoresis result diagram of hybridoma cell RNA extraction product.

[0017] Figure 2 It is a gel electrophoresis result diagram of antibody TTC36 antibody heavy chain and light chain variable region PCR amplification product, wherein lanes 1 and 2 are VH, and lanes 3 and 4 are VL.

[0018] Figure 3 It is an SDS-PAGE result diagram of prokaryotic expression product, wherein M is protein marker, 1 is whole bacteria before induction, 2 is whole bacteria after induction, 3 is supernatant after crushing after induction, and 4 is precipitate after crushing after induction.

[0019] Figure 4 It is a protein purification result diagram.

[0020] Figure 5 Figure 1 is a result chart of ELISA detection. Wherein, 1, blank control; 2, Avi control (empty vector expression supernatant); 3, scFv-Avi.

[0021] Figure 6 Figure 2 is a result chart of liver cancer tissue immunoblotting detection. Wherein, 1, paracancerous liver tissue; 2, liver cancer tissue. DETAILED DESCRIPTION

[0022] The principles and features of the present application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the present application, and are not used to limit the scope of the present application.

[0023] 1. Preparation of hybridoma cells

[0024] According to the protein sequence of TTC36, a plurality of polypeptides are synthesized to prepare an immunogen for immunizing mice. The mouse spleen cells are taken and fused with SP2 / 0 cells by PEG method. A hybridoma cell with good specificity and affinity is screened for the next step experiment.

[0025] 2. Extraction of total RNA of hybridoma cells and synthesis of antibody cDNA

[0026] The method is as follows:

[0027] (1) The hybridoma cells are cultured in DMEM 10% FBS medium to 5×10 6 cells are collected at 4°C, 4000 rpm, 5 min, 1 ml Trizol is added, and shaken vigorously for 30 s, and then placed on ice for 10 min, and then centrifuged at 4°C, 12000 rpm for 10 min, and the supernatant is taken into a new 1.5 ml centrifuge tube.

[0028] (2) 200 μl of chloroform is added, shaken for 15 s, and then centrifuged at 4°C, 12000 rpm for 5 min, and the supernatant is transferred to a new centrifuge tube.

[0029] (3) 200 μl of chloroform and 200 μl of water-saturated phenol are added, shaken for 15 s, and then centrifuged at 4°C, 12000 rpm for 5 min, and the supernatant is transferred to a new centrifuge tube.

[0030] (4) 400 μl of chloroform is added, shaken for 15 s, and then centrifuged at 4°C, 12000 rpm for 5 min, and the supernatant is transferred to a new centrifuge tube.

[0031] (5) An equal volume of isopropanol is added, mixed thoroughly, and precipitated at -20°C for 30 min, and then centrifuged at 4°C, 12000 rpm for 15 min, and the supernatant is discarded and the precipitate is retained.

[0032] (6) Use pre-cooled 70% ethanol to wash for 15s, centrifuge at 4℃, 5000rpm for 3min, and then dry in the clean bench.

[0033] (7) The RNA dry matter is dissolved in 30μl DEPC water, and 1% agarose electrophoresis is detected. The results are shown in Figure 1 The total RNA of the hybridoma cells is extracted. The total RNA is sub-packaged and labeled, and then stored at -80℃.

[0034] The total RNA is reverse transcribed to obtain cDNA.

[0035] 3. Construction of pETDuet-P17-scFv-Avi-BirA expression plasmid

[0036] The sequences of the amplified heavy chain variable region (VH) and light chain variable region (VL) of TTC36 from the cDNA are shown in SEQ ID NO: 1 and 3, respectively, and the translated amino acid sequences are shown in SEQ ID NO: 2 and 4, respectively. The CDR1-3 sequences of VH are shown in SEQ ID NO: 5-7, respectively, and the CDR1-3 sequences of VL are shown in SEQ ID NO: 8-10, respectively. The electropherogram of the VH and VL gene fragments is shown in Figure 2 .

[0037] The above VH and VL are connected by a flexible linker (SEQ ID NO: 11) to obtain a single-chain antibody TTC36-scFv (SEQ ID NO: 12). The gene fragment (SEQ ID NO: 13) encoding the single-chain antibody TTC36-scFv is inserted into an expression plasmid vector containing P17, Avi, and BirA genes to obtain a pETDuet-P17-scFv-Avi-BirA expression plasmid.

[0038] 4. Prokaryotic expression and purification of scFv-Avi fusion protein

[0039] 1) Inducing expression

[0040] The recombinant plasmid pETDuet-P17-scFv-Avi-BirA is transformed into expression bacteria E. coli BL21 (DE3) competent bacteria. The verified transformants are inoculated in LB liquid medium containing ampicillin (Amp) and cultured overnight at 37℃.

[0041] 50μL of the overnight culture is transferred to a new 5mL LB culture solution (containing 100μg / mL Amp) at a ratio of 1:100, and cultured for about 3h until the OD 600When the OD600 reached 0.8-1.0, IPTG was added to a final concentration of 0.5 mM, and the culture was incubated at 25 °C, 180 rpm overnight for scFv-biotin induction expression.

[0042] 2) Detection of induced products

[0043] In order to detect whether TTC36 scFv-biotin can be normally expressed in E. coli BL21 (DE3), SDS-PAGE was used for detection, and the expression position of the target protein and whether it formed inclusion bodies were explored.

[0044] 500 μL of bacterial solution before induction was centrifuged at 5000 rpm for 5 min, 200 μL was resuspended with PBS as a pre-induction control; the remaining 4.5 mL of induced culture was centrifuged at 5000 rpm for 5 min, the supernatant was removed, and the bacterial body was resuspended with 2.5 mL of PBS. 500 μL of bacterial solution was taken and stored at 4 °C, which was the whole protein after induction. The remaining 2 mL was ultrasonically broken in an ice water mixture.

[0045] The ultrasonically broken bacterial solution was centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant and precipitate were collected, and the precipitate was resuspended with 2 mL of PBS.

[0046] The above samples were subjected to SDS-PAGE to detect the induction effect. The results are shown in Figure 3 As shown in the figure, obvious protein bands can be seen at the target position, indicating that the induction is successful.

[0047] 3) Purification of TTC36 scFv fusion protein

[0048] Streptavidin magnetic beads (GenScript) were used to purify the supernatant after ultrasonic breaking. The steps are as follows:

[0049] (1) Add 100 μl of binding / washing buffer to resuspend the magnetic beads.

[0050] (2) Add the biotinylated antibody solution (supernatant after ultrasonic breaking) to the magnetic beads, and invert the test tube gently to mix.

[0051] (3) Incubate the test tube at room temperature (on a shaker or rotator) for one hour.

[0052] (4) Collect the magnetic beads using a magnetic stand, and discard the supernatant.

[0053] (5) Add 1 ml of binding / washing buffer to the tube and mix well, collect the magnetic beads using a magnetic stand and discard the supernatant. Repeat the washing step 3 times.

[0054] (6) Add 100 μl binding / washing buffer (0.1 M phosphate buffer, 0.15 M NaCl, pH 7.2) to resuspend the magnetic beads.

[0055] (7) Add the antigen sample to the tube and mix by gently inverting the tube. Incubate at room temperature for 30 minutes or at 4°C overnight.

[0056] (8) Wash the magnetic beads with 1 ml of binding / washing buffer. Collect the magnetic beads using a magnetic stand and discard the supernatant. Repeat the washing step 3 times.

[0057] (9) Add 100 μl of elution buffer (0.1 M glycine-HCl, pH 2.5-2.8) to the tube. Mix well and incubate at room temperature for 5 minutes.

[0058] (10) Collect the magnetic beads using a magnetic stand and save the supernatant containing the target antigen.

[0059] (11) Adjust the pH by adding neutralization buffer (1 M Tris-HCl, pH 8.5) at 5 μl per 50 μl of eluate.

[0060] The results of the SDS-PAGE gel analysis are shown in Figure 4 The majority of the impurities in the sample were removed and a clear protein band was observed at the desired position, indicating successful purification.

[0061] 5. ELISA detection of TTC36-scFv-biotin activity

[0062] The activity of the purified TTC36 scFv was preliminarily identified using direct ELISA. The coating antigen was TTC36-GST and GST, and the blocking solution was 3% bovine serum albumin (BSA). The steps were as follows:

[0063] (1) Coating: Take the GST-TTC36 protein, 0.1 μg / well, coat a 96-well enzyme-labeled plate at 4°C for 12 h.

[0064] (2) Wash the plate: Pour out the coating solution and wash with PBS-T (PBS buffer containing 0.1% Tween 20) 3 times and pat dry.

[0065] (3) Blocking: Add 200 μl of 3% BSA to each well and block at room temperature for 1 h.

[0066] (4) Wash the plate: Pour out the blocking solution and wash with PBS-T 3 times and pat dry.

[0067] (5) Add antibody: after purification, TTC36-scFv-Avi fusion protein and control were diluted with 5% skim milk PBST to different concentrations, 100 μL was added to each enzyme-labeled plate, and incubated at 37°C for 1 h.

[0068] (6) Wash plate: pour off the supernatant, wash the plate 5 times with a plate washer, and pat dry.

[0069] (7) Add streptavidin-labeled horseradish peroxidase (Streptavidin-HRP, Jin Si Biological) diluted 1:5000 with blocking solution, 100 μl was added to each well, and incubated at 37°C for 30 min.

[0070] (8) Wash plate: pour off the supernatant, wash the plate 5 times with a plate washer, and pat dry.

[0071] (9) Color development: add color development substrate TMB solution, 100 μl / well, and color develop at 37°C for 10 min (water-soluble product is blue).

[0072] (10) Termination: add termination solution, 50 μl / well, and read OD values at 450 nm wavelength with an enzyme-labeled instrument.

[0073] The results are shown in Figure 5 , which shows that the GST-TTC36-scFv-biotin fusion protein can specifically bind to TTC36 protein.

[0074] 6. Liver cancer tissue immunoblotting detection

[0075] Liver cancer tissue immunoblotting (Western-blotting) detection: liver cancer tissue and normal tissue adjacent to cancer were lysed, and the tissue lysate was treated with 5x loading buffer, and SurePAGE precast gel (Jin Si Biological) was used for protein electrophoresis, 200 ng / 10 μl was loaded into each gel well, and the electrophoresis parameters were set as constant voltage 160 V for 30 min, then transferred to 0.45 μm PVDF membrane, and the transfer parameters were set as constant current 200 mA for 60 min, then TTC36-scFv (biotinylated) antibody was used as primary antibody (1:2000 dilution), β-Actin was used as internal reference, and streptavidin-labeled HRP (Streptavidin-HRP) was used as "secondary antibody" (1:5000 dilution) for detection.

[0076] The results are shown in Figure 6 , compared with normal liver tissue (normal tissue adjacent to cancer), the TTC36 band of liver cancer tissue was obviously lighter in color, which showed that the TTC36-scFv single-chain antibody of the application can specifically bind to TTC36 in the tissue.

[0077] The above merely describes preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An anti-TTC36 antibody, characterized in that, The sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 5-7, respectively; the sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 8-10, respectively.

2. The anti-TTC36 antibody according to claim 1, characterized in that, The sequence of the heavy chain variable region is shown in SEQ ID NO:

2.

3. The anti-TTC36 antibody of claim 1, wherein, The sequence of the light chain variable region is shown in SEQ ID NO:

4.

4. The anti-TTC36 antibody of claim 1, wherein, It is a single-chain antibody, connected by a connecting polypeptide between the heavy chain variable region and the light chain variable region; the sequence of the connecting polypeptide is shown in SEQ ID NO:

11.

5. An anti-TTC36 antibody, characterized in that, The sequence is shown in SEQ ID NO:

12.

6. The anti-TTC36 antibody of claim 4, wherein, It is also coupled with Avi.

7. Use of the anti-TTC36 antibody of any one of claims 1-6 in the preparation of a liver cancer diagnostic reagent.

Images