CN110054675A - Immunogenic polypeptide and anti-TTC36 antibody CP4-3 and application - Google Patents
Immunogenic polypeptide and anti-TTC36 antibody CP4-3 and application Download PDFInfo
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- CN110054675A CN110054675A CN201910171155.4A CN201910171155A CN110054675A CN 110054675 A CN110054675 A CN 110054675A CN 201910171155 A CN201910171155 A CN 201910171155A CN 110054675 A CN110054675 A CN 110054675A
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- ttc36
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- hybridoma
- albumen
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
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Abstract
The present invention relates to a kind of immunogenic polypeptides, sequence is as shown in SEQ ID NO:1, further relate to a kind of hybridoma, the hybridoma is preserved in China typical culture collection center (CCTCC) on October 17th, 2018, the method of monoclonal antibody and its application and the TTC36 albumen in a kind of non-diagnostic purpose test sample that deposit number CCTCC NO:C2018208 and the hybridoma generate.Truncated immunogenic polypeptide of the invention can effectively induce human or animal to generate the antibody of anti-TTC36 albumen.Monoclonal antibody CP4-3 of the invention can efficiently and specifically combine the TTC36 albumen in sample to have more high specific and affinity compared with polyclonal antibody.For the research of TTC36 protein function, the research of related disease, diagnosing and treating provides better antibody tool.
Description
Technical field
The present invention relates to immune fields, more specifically it relates to which a kind of immunogenic polypeptide and its application, further relate to a kind of miscellaneous
The TTC36 in antibody and its application and a kind of non-diagnostic purpose test sample for handing over oncocyte, the hybridoma to generate
The method of albumen.
Background technique
TTC36 (tetratricopeptide repeat domain 36) albumen contains 3 TRP blocks
(tetratricopeptide), no other structures domain belongs to TPR spline structure albumen.We by phylogenetic analysis find,
The amino acid sequence of TTC36 is highly conserved in mammal, it is seen that it executes important function in vivo.
The researchs such as Jiang, L. discovery TTC36 albumen expresses downward in human liver cancer tissue sample, if in liver cancer cells
Middle overexpression TTC36 gene can promote hepatoma cell apoptosis.James A Nathan researches show that TTC36 may participate in cell
Interior proteins ubiquitin.
At present TTC36 albumen research report and it is few, have some polyclonal antibodies for TTC36 in the market, but
It is that there has been no document reports to study using TTC36 monoclonal antibody, also there are no the monoclonals for being directed to people TTC36 albumen in the market
Antibody is sold.
Due to the high degree of specificity of monoclonal antibody, related mechanism research is carried out using monoclonal antibody, exploitation is examined in vitro
Disconnected property kit, or prepare antibody drug have become increase in pharmacy and biological agent industry it is most fast, make a profit maximum market it
One.Therefore, it is necessary to develop the monoclonal antibody for being directed to TTC36 albumen for associated uses.
Summary of the invention
In order to solve the above problem, the present invention provides a kind of immunogenic polypeptides, and sequence is as shown in SEQ ID NO:1.
The present invention also provides the applications that above-mentioned immunogenic polypeptide is used to prepare anti-TTC36 antibody.
The present invention also provides a kind of methods for preparing TTC36 antibody, including aforementioned polypeptides stimulation test animal is used to exempt from
Epidemic disease system, the step of making the experimental animal generate antibody.
The present invention also provides a kind of hybridoma, the hybridoma is preserved in China on October 17th, 2018
Type Tissue Collection (CCTCC), deposit number CCTCC NO:C2018208.
The present invention also provides a kind of monoclonal antibodies, are generated by above-mentioned hybridoma.
The present invention also provides the applications that said monoclonal antibody is used to detect the TTC36 albumen in non-pathological sample.
The present invention also provides the applications that above-mentioned antibody is used to prepare reagent for disease diagnosis.
In a preferred embodiment, the disease is cancer.
In a preferred embodiment, the disease is liver cancer.
The present invention also provides a kind of methods of the TTC36 albumen in non-diagnostic purpose test sample, comprising the following steps:
S1: make the sample exposure antigen；
S2: the sample is incubated for using said monoclonal antibody；
S3: the monoclonal antibody of display and sample specific binding.
Truncated immunogenic polypeptide of the invention can effectively induce human or animal to generate the antibody of anti-TTC36 albumen, this
The antibody of invention can efficiently and specifically combine the TTC36 albumen in sample to have more Gao Teyi compared with polyclonal antibody
Property and affinity.For the research of TTC36 protein function, the research of related disease, diagnosing and treating provides better antibody tool.
Biomaterial preservation
The monoclonal antibody that the present invention designs is generated by hybridoma, and hybridoma is protected on October 17th, 2018
It is hidden in the China typical culture collection center (CCTCC) of Wuhan University, Wuhan City, Hubei China province, deposit number CCTCC NO:
C2018208 is named as hybridoma cell strain CP4-3.
Detailed description of the invention
Fig. 1 is synthesis polypeptide (GQLRRPRDSR) chromatography qualification figure；
Fig. 2 is immunizing antigen GST-TTC36 purified product electrophoretogram, wherein swimming lane M is Marker, and swimming lane 1 is 0.5mg/
Ml BSA, swimming lane 2 are the GST-TTC36 for diluting 2 times；
Fig. 3 is ELISA method detection Hybridoma Cell Culture supernatant binding force and specific outcome；
Fig. 4 is Western blot detection Hybridoma Cell Culture object supernatant binding force as a result, swimming lane M is Marker, swimming lane
1-3 is respectively the culture supernatant coloration result of hybridoma CP4-3, CP4-6 and CP4-7；
Fig. 5 is ELISA method detection CP4-3 antibody titer result after purification；
Fig. 6 is liver cancer and cancer beside organism's paraffin section immune detection as a result, the left side is the photo of liver cancer tissue slice, the right
For the photo of cancer beside organism's slice；
Fig. 7 is liver cancer and cancer beside organism's immune-blotting method as a result, swimming lane 1 is cancer beside organism, and swimming lane 2 is liver cancer tissue.
Specific embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
1. prepared by antigen
The preparation of 1.1 immunizing antigens
According to the protein sequence of TTC36, multistage polypeptide is synthesized, one of them is 180-189 amino acids, and sequence is
GQLRRPRDSR(SEQ ID NO:1).The polypeptide of synthesis carries out chromatography identification, as a result as shown in Figure 1, polypeptide sequence is correct, and
And purity is 90.356%.The polypeptide is coupled KLH, forms coupling protein TTC36 (180-189aa)-KLH.It is coupled to ox blood
Pure albumen (BSA) forms " polypeptide-BSA " coupling protein, then quantitative 2.0mg/ml, name are as follows: TTC361 (180-
189aa)-BSA。
The prokaryotic expression of 1.2 positive antigen TTC36 albumen
1.2.1 the synthesis of genetic fragment and clone
Codon optimization is carried out to the encoding gene whole process of TTC36, and connects the encoding gene of GST label in C-terminal with benefit
In purifying, obtained sequence is as shown in SEQ ID NO:2.EcoR I and Xho I site is added respectively at the both ends of the sequence, with
Convenient for clone.
Above-mentioned sequence is synthesized, and is cloned into pUC57 plasmid vector in a manner of TA, choosing sequencing, correctly clone carries out down
The experiment of one step.
1.2.2 construction of expression vector and expression bacterial strain
Segment digestion: taking 43 μ l TTC36-pUC57 recombinant plasmids, 1 μ l EcoR I, 1 μ lXho I, and 5 μ l 10 ×
Buffer, 37 DEG C of reaction overnights (using Ago-Gel DNA QIAquick Gel Extraction Kit, BPI)；Carrier digestion: 43 μ l carriers are taken
(pGEX-4T-1) plasmid, 1 μ l EcoR I, 1 μ l Xho I, 5 10 × Buffer of μ l, 37 DEG C of reaction overnight (Ago-Gels
DNA QIAquick Gel Extraction Kit, BPI).
Connection conversion: 1 μ l carrier (pGEX-4T-1), 3 μ l segments, 1 μ l ligase (BPI), 52 × Rapid of μ l are taken
Buffer is mixed, and is reacted at room temperature 30min, is obtained TTC36 protein expression vector.
The competent cell (BL21) for taking out -80 DEG C of preservations is placed on ice slowly defrosting.Competent cell is added and is connected
It is mixed in product, places 30min on ice.42 DEG C of heat shock 90s.After ice bath 2min, the LB culture medium of 800 μ l non-resistants is added.37
DEG C culture 45min.5000rpm is centrifuged 3min, abandons most of supernatant, stays about 100-150 μ l, and thallus is resuspended, and selection has corresponding anti-
The LB plate of property, coated plate.It dries, is inverted overnight incubation in 37 DEG C of incubators.Picking positive colony obtains after inspection is errorless
TTC36 protein expression strain.
1.2.3 the expression and purification of TTC36
1.2.3.1 protein expression
(1) bacterium is connect into corresponding resistant LB liquid medium according to 1:50 ratio, 37 DEG C of cultures, 200rpm.
(2) bacterium solution of culture is transferred in 1000ml corresponding resistant LB liquid medium according to 1:50 ratio, 37 DEG C,
200rpm, culture to OD=0.6-0.8,37 DEG C of induction 2h of IPTG (0.5mM).
(3) bacterium: 8000rpm is received, 6min is centrifuged.Abandon supernatant.
(4) carrying out ultrasonic bacteria breaking: thallus is dispelled with 20-30ml 10mM Tris-HCl (pH 8.0) solution, ultrasonic disruption
(500W 180 times, each 5s, is spaced 5s).
(5) electrophoresis determines expression-form: the bacteria suspension after taking 100 μ l ultrasounds, 12000rpm are centrifuged 10min, take on 50 μ l
Clear to manage to another EP, precipitating is dispelled with 50 μ l 10mM Tris-HCl (pH 8.0) solution after supernatant removal is clean.Electrophoresis detection.
1.2.3.2 protein purification
(1) ratio of 1ml Glutathione Sepharose 4B bead is needed to inhale according to every 8mg gst fusion protein
Column material is taken, and 5ml PBS (pH 7.4) is added thereto, is resuspended, 2000rpm is centrifuged 2min, repeats 3-5 times.
(2) thallus of every 500ml bacterium solution is added 20ml PBS (pH 7.4) and is resuspended, ultrasonic disruption, 12000rpm centrifugation
10min。
(3) it takes supernatant to mix with treated bead to shake up, 4 DEG C of overturning shaken over night combine.
(4) 2000rpm is centrifuged 2min, and supernatant is gone in another pipe, and the electrophoresis that keeps sample (for prick post), bead is transferred to 10ml EP
Pipe.
(5) it is washed 3 times (2000rpm is centrifuged 2min, is resuspended after liquid feeding) with 5ml lysate.
(6) it is resuspended with 5ml eluent (10mM Reduced Glutathione, 50mM Tris-HCl, pH 8.0)
Bead, 2-3h is shaken in overturning under the conditions of 4 DEG C, and 2000rpm is centrifuged 2min, and supernatant is gone in another pipe.It is repeated twice.
(7) it is resuspended with 5ml eluent (60mM Reduced Glutathione, 50mM Tris-HCl, pH 8.0)
Bead overturns shaken over night under the conditions of 4 DEG C, and 2000rpm is centrifuged 10min, and supernatant is gone in another pipe.It is repeated twice.
(8) 50 μ l, electrophoresis detection protein purification effect are sampled.
(9) 200 times of sample volume of dialysis Buffer (1 × PBS), 4 DEG C of dialysis are used.Each 3h dialyses 3 times.
(10) 12000rpm is centrifuged 10min, takes supernatant, electrophoresis detection.
As a result as shown in Fig. 2, albumen size is consistent with theoretical value (46kDa), purity 87.25% can be used for being immunized and sieving
Choosing.
2. immune mouse
With " polypeptide-K LH " coupling protein, by 60 μ g albumen/mouse amount, 4 SPF grades of BALB/c of subcutaneous initial immunity
Female mice, number are as follows: 1#, 2#, 3#, 4#.At interval of 2 weeks, be immunized second third time respectively, the amount of being immunized be 30 μ g albumen/
Only.7th day after third time is immune, eye socket takes blood, surveys serum titer, as the result is shown 1# Mouse titers highest.After a week with immune
1# mouse is impacted in 50 μ g of original, abdominal cavity.
" polypeptide-BSA " coupling protein, 2 μ g/ml, with 2 μ g/ are diluted with the coating buffer of 0.1M carbonate buffer solution (pH9.6)
The concentration of ml is coated with 96 hole elisa plates, and 100 μ l are added in every hole, and 4 DEG C overnight；PBST (0.05% Tween-20, PBS) is washed 5 times,
Then each 3min is closed, 37 DEG C of incubation 2h with the PBS buffer solution containing 3% skimmed milk power；PBST is washed 5 times, each 3min,
It is spare.
Serum 2 times of gradient dilutions since 200 times with PBS, blank control (blank) are PBS, negative control
It (negative) is negative serum (non-immune serum) 200 times of dilutions.Every hole adds 100 μ l, 37 DEG C of incubation 2h, PBST washings
5 times, each 3min, sheep anti-mouse igg antibody (1:5000 dilution) is marked in the HRP that 100 μ l are added in every hole, 37 DEG C of incubation 1h；
PBST is washed 5 times, each 3min, 100 hole μ l/ of TMB developing solution, after room temperature is protected from light incubation 15 minutes, in the 2M sulfuric acid with 50 μ l
It only reacts, detection uses dual wavelength 450nm/630nm.Testing result is as shown in table 1,1# Mouse titers highest.
1 mice serum bioactivity result of table
3. the preparation and screening of hybridoma
(1) fused cell is prepared
1# mouse boosting cell and SP2/0 cell are taken, is merged using PEG method.Cell semisolid culturemedium is merged
(containing HAT) carries out screening and culturing.10 plate × 93 cell monoclonals are chosen, are incubated at 96 porocyte culture plates (in advance with mouse chest
Gland cell bed board, 100 holes μ l/).
(2) filtering hybridoma
ELISA method: " polypeptide-BSA " coupling protein is diluted with the coating buffer of 0.1M carbonate buffer solution (pH9.6), with 1 μ
The concentration of g/ml is coated with 96 hole elisa plates, and 100 μ l are added in every hole, and 4 DEG C overnight；PBST is washed 5 times, each 3min, with containing 3%
The PBS buffer solution of BSA is closed, 37 DEG C of incubation 2h；PBST is washed 5 times, each 3min；It is thin that 100 μ l hybridomas are added in each hole
Born of the same parents' culture supernatant, 37 DEG C of 2h, PBST are washed 5 times, each 3min, then mark sheep anti-mouse igg antibody with the diluted HRP of 1:5000
100 holes μ l/, 37 DEG C of incubation 1h；PBST is washed 5 times, each 3min, and 2M is used after 15 minutes at room temperature in 100 hole μ l/ of TMB developing solution
Sulfuric acid stopped reaction, detection use dual wavelength 450nm/630nm.
The screening strategy of the hybridoma cell strain of stably excreting antibody: ELISA method and Western blot are used, with " more
Peptide-BSA " coupling protein wrapper sheet does and screens for the first time, obtains 12 plants of positive hybridoma cell strain.Positive cell is carried out sub- gram
It is grand, then done programmed screening with " polypeptide-BSA " coupling protein wrapper sheet with ELISA method, obtain the hybridization of stably excreting antibody
5 plants of tumor cell strain.Finally screened again with Western blot and " GST-TTC36 " fusion protein.
Experimental result is as shown in Figure 3: the hybridoma cell strain culture supernatant of number CP4-3, CP4-6 and CP4-7 have
" GQLRRPRDSR " polypeptide binding specificity, CP4-3 binding force highest.
Western blot: preparation 4-12% acrylamide gel；GST-TTC36 albumen is handled with 5 × loading buffer
Afterwards, each 10 μ l/50ng of hole, electrophoretic parameters are constant pressure 60-120V, 60min, are then transferred to 0.45 μm of pvdf membrane, transfer ginseng
Number is constant current 200mA, then 60min. uses cell conditioned medium to be detected as primary antibody (1:4 dilution).
As a result as shown in figure 4, supernatant of the only hybridoma cell strain CP4-3 secretion containing antibody detects 46kDa's or so
Specific band is consistent with theoretical value, and explanation can be with GST-TTC36 albumen specific bond.CP4-3 is used to test in next step.
(3) monoclonal antibody subtype identification
Using Southern Biotech antibody typing kit, antibody that the positive cell strain screened is generated into
Row subtype identification.The results are shown in Table 2, and wherein CP4-3 is IgG1, κ type.
2 antibody subtype testing result of table
4. the preservation of hybridoma
The above experimental result shows that the monoclonal antibody specificity that hybridoma CP4-3 is generated is high, and affinity is best.
The hybridoma cell strain is preserved in the Chinese Typical Representative culture of Wuhan University, Wuhan City, Hubei China province by October 17th, 2018
Collection (CCTCC), deposit number CCTCC NO:C2018208, is named as hybridoma cell strain CP4-3.
5. Antibody preparation and purifying
CP4-3 is cultivated to logarithmic phase, rear overhang is washed with Hanks liquid and plays quantitative, every mouse (BALB/c female mice) abdomen
Chamber injection 5 × 105A cell after having beaten ascites, takes ascites after 7-10 days.
Mouse ascites, 4 DEG C of centrifugation 10min of 12000rpm, then with 0.22 μm of filter membrane are diluted with 1:3 with coupling buffer
Filtering removes fat, cell residue and finely ground particle substance.Contained with the corresponding coupling buffer balance of 5-10 times of column volume
The pillar of protein G agarose micelle (Protein G Agarose).With on the filtered ascites injection pillar of syringe holder
End interface collects efflux in 50ml centrifuge tube.Column is crossed with the coupling buffer of 5 times of column volumes to rinse；Neutralizer is taken to be added
Centrifuge tube；Eluent is added dropwise to 1.0ml, detects pH value after mixing.The amount that neutralizer is adjusted according to result, makes neutralization results pH
7.0.It with 5 times of column volume elution buffer antibody elutions, is collected in centrifuge tube, detects antibody concentration, the antibody of purifying is dense
Degree, which is adjusted, arrives 1mg/ml standard, is used for follow-up function test experience.
6.ELISA detects potency
With 2 μ g/ml " polypeptide-BSA coupling protein ", 4 DEG C of coatings are overnight；2% 37 DEG C of skim milk closing 2h；By purifying
Antibody (1mg/ml) 2 times of gradient dilutions since 200 times, blank control (blank) are PBS, and negative control (negative) is
200 times of culture medium dilutions.
Every hole adds 100 μ l samples, and 37 DEG C of incubation 2h, PBST is washed 5 times, each 3min, then the HRP of 100 μ l is added in every hole
It marks sheep anti-mouse igg antibody (1:5000 dilution), 37 DEG C of incubation 1h；PBST is washed 5 times, each 3min, 100 μ l/ of TMB developing solution
Hole after room temperature is protected from light incubation 15 minutes, with the 2M sulfuric acid stopped reaction of 50 μ l, is detected using dual wavelength 450/630nm.
As a result as shown in figure 5, CP4-3 antibody titer can reach 51200 or more.
7.CP4-3 antibody is used for the immune detection of pathological sample
Following detection method can be not only used for the detection of pathological sample, it can also be used to which the non-diagnostic purpose of sample detects.
(1) the immunohistochemistry detection of liver cancer tissue
Normal liver tissue wax embedding block by human liver cancer and cancer is taken, 5 μm of slices, heating repairs antigen, with 4% formaldehyde
Then the anti-human TTC36 antibody (PBS that dilution is 1%BSA) of 500ng/50 μ l after purification, 25 DEG C of rooms are added dropwise in fixed 10min
Temperature is incubated for 2h, is then washed 5 times with PBST, and HRP is added dropwise and marks sheep anti-mouse igg antibody, is incubated at room temperature 1h, then HE is redyed, Yu Xian
Micro- microscopic observation.
As a result as shown in fig. 6, CP4-3 antibody test is arrived, TTC36 albumen in liver is predominantly located at the cell of liver cell
In matter, and compared with cancer beside organism, expression of the TTC36 albumen in liver cancer tissue is decreased obviously.
(2) liver cancer tissue immune-blotting method
Liver cancer tissue immunoblotting (Western-bloting) detection: preparation 4-12% acrylamide gel, by liver cancer tissue
It is cracked with Carcinoma side normal tissue, after Tissue lysates are handled with 5 × loading buffer, each glue hole loading 200ng/10
μ l, electrophoretic parameters are set as constant pressure 60-120V, 60min, are then transferred to 0.45 μm of pvdf membrane, and transfer parameter is set as constant current
Then 200mA, 60min use antibody purification as primary antibody (1:2000 dilution), β-Actin is used as internal reference, and HRP marks sheep anti mouse
IgG is secondary antibody (1:2000 dilution), is detected.
As a result as shown in fig. 7, compared with normal liver tissue, the obvious color of TTC36 band of liver cancer tissue is more shallow, explanation
CP4-3 antibody can effectively detect that TTC36 is decreased obviously in liver cancer tissue expression.
By above-mentioned testing result it is found that TTC36 can be used as detection tissue whether the molecular marker of canceration, also, CP4-3 is anti-
Body can effectively and specifically detect TTC36 albumen in pathological tissue.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Sequence table
<110>Shenzhen, the People's Hospital, Longhua District
Liu Yunhong
<120>immunogenic polypeptide and anti-TTC36 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 (10)
1. a kind of immunogenic polypeptide, which is characterized in that sequence is as shown in SEQ I D NO:1.
2. the application that immunogenic polypeptide described in claim 1 is used to prepare anti-TTC36 antibody.
3. a kind of method for preparing TTC36 antibody, which is characterized in that including using polypeptide stimulation test described in claim 1
Animal immune system, the step of making the experimental animal generate antibody.
4. a kind of hybridoma, the hybridoma was preserved in China typical culture collection on October 17th, 2018
The heart (CCTCC), deposit number CCTCC NO:C2018208.
5. a kind of monoclonal antibody, which is characterized in that generated by hybridoma as claimed in claim 4.
6. the application that monoclonal antibody described in claim 5 is used to detect the TTC36 albumen in non-pathological sample.
7. the application that antibody described in claim 5 is used to prepare reagent for disease diagnosis.
8. application according to claim 7, which is characterized in that the disease is cancer.
9. application according to claim 8, which is characterized in that the disease is liver cancer.
10. a kind of method of the TTC36 albumen in non-diagnostic purpose test sample, which comprises the following steps:
S1: make the sample exposure antigen；
S2: the sample is incubated for using the monoclonal antibody described in claim 5；
S3: the monoclonal antibody of display and sample specific binding.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002053704A2 (en) * | 2001-01-04 | 2002-07-11 | Myriad Genetics, Inc. | Protein-protein interactions |
| CN109082471A (en) * | 2018-09-18 | 2018-12-25 | 蚌埠医学院 | A kind of patients with lung adenocarcinoma prognosis prediction peripheral blood mRNA marker and its screening technique and application |
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2019
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002053704A2 (en) * | 2001-01-04 | 2002-07-11 | Myriad Genetics, Inc. | Protein-protein interactions |
| CN109082471A (en) * | 2018-09-18 | 2018-12-25 | 蚌埠医学院 | A kind of patients with lung adenocarcinoma prognosis prediction peripheral blood mRNA marker and its screening technique and application |
Non-Patent Citations (3)
| Title |
|---|
| YURU ZHOU等: ""The expression patterns of Tetratricopeptide repeat domain 36 (Ttc36)"", 《GENE EXPR PATTERNS》 * |
| 刘德康等: "Ppp5c和TTC16基因的TPR结构域与Hsp70、Hsp90蛋白的相互作用及对细胞周期的影响", 《现代生物医学进展》 * |
| 陈曦: ""一种新的HSP90和HSP70伴侣蛋白TTC36在人视网膜色素上皮细胞中的定位和功能研究以及基因LimsA的克隆分析"", 《中国优秀博硕士学位论文全文数据库（硕士）医药卫生科技辑》 * |
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