CN102584995B

CN102584995B - Monoclonal antibody resistant to human phosphodiesterase 4D interaction protein variant 5 and application thereof

Info

Publication number: CN102584995B
Application number: CN 201110297243
Authority: CN
Inventors: 张霖; 牛瑞芳; 罗艺; 杨毅; 魏熙胤; 齐众
Original assignee: TIANJIN TUMOUR HOSPITAL
Current assignee: TIANJIN TUMOUR HOSPITAL
Priority date: 2011-09-29
Filing date: 2011-09-29
Publication date: 2013-07-17
Anticipated expiration: 2031-09-29
Also published as: CN102584995A

Abstract

The invention discloses a monoclonal antibody resistant to a human phosphodiesterase 4D interaction protein variant 5 and an application of the monoclonal antibody, belonging to the preparation of antibody-containing medical preparations or monoclonal antibodies. The antibody specifically identifies 1103rd to 1116th amino acids of the human phosphodiesterase 4D interaction protein variant 5; the antibody is produced by a hybrid tumor cell strain with a collection number of CGMCC (China General Microbiological Culture Collection Center) No.5078, and has an amino acid sequence of GELESVRIHHKHAY. The monoclonal antibody has specific binding capacity with the phosphodiesterase 4D interaction protein variant 5, is used for detecting the expression and location of the phosphodiesterase 4D interaction protein variant 5 in human tissues and cells, and is used as an immunohistochemical detection reagent of the human phosphodiesterase 4D interaction protein variant 5.

Description

Monoclonal antibody and the application thereof of anti-human phosphodiester enzyme 4D interaction protein variant 5

Technical field

The present invention relates to contain the pharmaceutical product of antibody, or MONOCLONAL ANTIBODIES SPECIFIC FOR, specifically be a kind of monoclonal antibody and application thereof of anti-human phosphodiester enzyme 4D interaction protein variant 5.

Background technology

Myomegalin(MMG/MMGL/PDE4DIP, phosphodiesterase 4D interacting protein, phosphodiesterase 4 D interaction protein) is a kind of brand-new albumen, it is distributed in the endochylema, and assemble at golgi body and centrosome place, point out the major function of this molecule to relate to the cAMP signal transduction of these two organoids.The Myomegalin gene is very long, about 150,000 base pairs, but its each exon is very little, approximately has only 30-40 amino acid, so the variant of Myomegalin is a lot.It is not immediately clear that the variant what Myomegalin are specifically arranged exists.

Contain α spiral and coiled-coil structure in the Myomegalin protein structure, and microtubule-associated protein is in conjunction with territory and leucine zipper pattern.Myomegalin is also called PDE4DIP, this be since this albumen can in conjunction with the circulation in the PDE4D(nucleotide phosphodiesterase).In neoplastic disease, can see the unusual performance of Myomegalin.Can detect the PDE4DIP-PDGFRB fusion gene among the patient of myeloproliferative disorder.In esophageal neoplasm, can see the high expression level of Myomegalin variant 5 albumen.Variant 1 albumen then lacks in most tumour cell.This shows that the relation of Myomegalin variant and tumour is not illustrated as yet fully, be worth further research.

1975, Kohler and Milstein found murine myeloma cell and sheep red blood cell (SRBC) mice immunized splenocyte are merged, and the hybrid cell of formation both can produce antibody, but infinite multiplication again, thus founded the monoclonal antibody hybridoma technology.This technical breakthrough has not only been started new era for the Med Biol fundamental research, and also diagnosis, prevention, the treatment for clinical disease provides new instrument.Monoclonal antibody is one of main tool of research protein expression and distribution, monoclonal antibody at Myomegalin can detect cell and tissue, especially the expression level of Myomegalin and location situation in the tumor tissues help to further investigate the cytology function of Myomegalin and the biological action in tumour.

Summary of the invention

The present invention is in order to solve the effect of Myomegalin in the cell biological function, and the relation between Myomegalin and the tumour, be applied to basic medical research and clinical detection problem, and a kind of monoclonal antibody and application thereof of anti-human phosphodiester enzyme 4D interaction protein variant 5 albumen are provided.

The present invention realizes by following technical scheme.

A kind of monoclonal antibody of anti-human phosphodiester enzyme 4D interaction protein variant 5 albumen, characteristics are this antibodies specific identification human phosphodiester enzyme 4D interaction protein variant 5 protein 11 03-1116 site amino acid; This antibody is the hybridoma cell strain generation of CGMCC No.5078 by preserving number, and aminoacid sequence is the aminoacid sequence shown in the sequence table № 1.

A kind of hybridoma, this hybridoma cell strain produce anti-human phosphodiester enzyme 4D interaction protein variant 5 protein monoclonal antibodies, and this hybridoma cell strain preserving number is: CGMCC No.5078.

The application of the monoclonal antibody of described anti-human phosphodiester enzyme 4D interaction protein variant 5 albumen in preparation immunohistochemical methods detection reagent, described detection reagent are used for expression and the location of phosphodiesterase 4 D interaction protein variant 5 in human body tissue and the cell.

With this prokaryotic expression polypeptide immune BALB/c mouse, get mouse spleen and murine myeloma cell (SP2/0) cytogamy, screen with above-mentioned prokaryotic expression polypeptide, obtain positive colony, hypotype is identified and mouse peritoneal injection preparation ascites, ascites antibody carries out the immunohistochemical methods of cell and tissue, observes expression and the detection and localization of Myomegalin variant 5 albumen.

She Ji the present invention like this, its antibody and Myomegalin variant 5 albumen have specific binding capacity, the immunohistochemical methods detection reagent of preparation people Myomegalin variant 5 albumen is used for expression and the location that human body organizes Myomegalin variant 5 in the tumor tissue cell particularly.

Hybridoma cell strain is by China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation;

Address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City;

Preserving number is: CGMCC No.5078, preservation date: on July 22nd, 2011.

Description of drawings

Fig. 1 is the Myomegalin variant 5 protein immunization fluorescent dye figure of Hela cell;

Fig. 2 shows Myomegalin variant 5 the albumen expression in the cancer and location in breast duct;

Fig. 3 shows the expression of Myomegalin variant 5 albumen in normal breast.

Embodiment

Below in conjunction with drawings and Examples the present invention is described in detail.

One, MONOCLONAL ANTIBODIES SPECIFIC FOR Myomegalin variant 5(phosphodiesterase 4 D interaction protein variant 5)

1. experiment material is originated

BALB/c mouse is provided by Tianjin tumour hospital, available from Beijing Vital River Experimental Animals Technology Co., Ltd.; Hela cell, murine myeloma cell (SP2/0) are provided by Tianjin tumour hospital, grind institute available from Chinese Academy of Sciences's blood.Fu Shi Freund's complete adjuvant and freund 's incomplete adjuvant, HAT, HT conditioned medium composition are available from Invitrigen company.DMEM substratum, polyoxyethylene glycol (PEG), RPMI 1640 substratum are available from Hyclone company.The anti-mouse Ig of horseradish peroxidase (HRP) labelled goat, O-Phenylene Diamine (OPD), diaminobenzidine (DAB), 8-azaguanine (8-AG) are available from Sigma company.

2. animal immune

Get 7～10 age in week 4 of BALB/c female mices, with lO0ug Myomegalin variant 5 protein polypeptides (1103-1116 amino acid, have the GST label) (professor Qi Zhong of Hong Kong University of Science and Thchnology provides) be dissolved in 0.2ml 0.1M PBS(pH7.2), with the abundant mixing of equal-volume Freund's complete adjuvant (Sigma), subcutaneous abdomen multi-point injection.The 1st immunity back l5 and 29 days are used fully booster immunization behind the mixing of 100ug polypeptide/0.2ml PBS and equal-volume Freund's incomplete adjuvant (Sigma) respectively, and 3d injects 5Oug polypeptide/0.2ml PBS 1 time by tail vein reinforced immunological before merging.

3. cytogamy

Learn from else's experience 1 of the BALB/c mouse of booster immunization after the sacrificed by exsanguination, is got spleen under the aseptic condition, the preparation splenocyte suspension.Get 2 * 10 ⁷Individual myeloma cell (SP2/0) adds 50%PEG(MW 4000 in 37 ℃ of water-baths) 1 ml fusion, fused cell is inoculated in 96 well culture plates, adopt HAT and HT substratum to carry out selectivity respectively and cultivate.When treating that hybridoma grows to culture hole bottom surface 1/3, make envelope antigen with Myomegalin variant 5 protein polypeptides, with ELISA method screening antibody, select the positive hole of antibody-secreting, adopt limiting dilution assay, the hybridoma dilution is 5-10/ml, be inoculated in 96 well culture plates, select the culture supernatant in individual cells colony hole to do antibody test, positive person uses with quadrat method and clones again, and it is 100% that the antibody-secreting positive rate of hybridoma growth is arranged behind the clone.

4. positive hybridoma ascites MONOCLONAL ANTIBODIES SPECIFIC FOR

BALB/c mouse abdominal injection Pristane0.5ml/, 1 all pneumoretroperitoneums are injected well-grown hybridoma 10 ⁷, about 10 d, when mouse web portion swells to its vigor extreme difference, put to death mouse, extract its ascites, the centrifugal precipitation of going is collected supernatant mensuration and is tired.

5. indirect elisa method detects antibody titer

With Myomegalin variant 5 protein polypeptide coated elisa plates, establish the blank group, 37 ℃ of bags are spent the night.With 37 ℃ of sealings of 0.25%BSA, 1 h.Add ascites antibody, make serial gradient dilution with confining liquid, hatch 1h for 37 ℃, PBS-T washes 5 times.Add the anti-mouse Ig of horseradish peroxidase (HRP) labelled goat, hatch 1h for 37 ℃, PBS-T washes 5 times.OPD substrate Color Appearance System colour developing 5-10 min measures the OD492 value.

6. antibody subtype is identified

With sheep anti mouse Ig coated elisa plate, 37 ℃ of bags are spent the night.With 37 ℃ of sealings of 0.25%BSA, 1 h.Add the hybridoma culture supernatant, hatch 1h for 37 ℃, PBS-T washes 5 times.Add the anti-mouse IgG1 of horseradish peroxidase (HRP) labelled goat, IgG2a, IgG2b, IgG3, IgM, IgA respectively, hatch 1h for 37 ℃, PBS-T washes 5 times.OPD substrate Color Appearance System colour developing 5-10 min measures the OD492 value.

7. result

After the cytogamy, serve as to detect antigen with reorganization Myomegalin variant 5 protein polypeptides, the indirect ELISA screening obtains a strain monoclonal antibody, and hypotype is accredited as IgG1, and mouse ascites is tired and is 1:8000.

Two, the specific detection of Myomegalin variant 5 monoclonal antibodies

1. experimental technique

With the enzymic digestion of Hela cell tryptase, get an amount of cell point on slide glass, cultivate after 8 hours for 37 ℃, cold methanol is fixed, 2%BSA seals 1h, (the 37 ℃ of 2h of antibody incubation of γ-tubulin) add different fluorescein-labeled two anti-room temperature black outs and hatch 1h after the PBS washing, observe under the fluorescent microscope to add Myomegalin variant 5 monoclonal antibodies, tubulin.See accompanying drawing 1.

2. the result judges

Tubulin (γ-tubulin) as dyeing is all arranged on cytocentrum and golgi body, be strong with the centrosome; Myomegalin variant 5 mainly has obvious fluorescence developing at golgi body, and the centrosome place illustrates that this antibody has tangible specificity for Myomegalin albumen, the 1103-11160 site amino acid of specific recognition people Myomegalin protein variant 5 a little less than omiting.

Three, the application of the monoclonal antibody of Myomegalin variant 5 albumen in preparation immunohistochemical methods detection reagent:

1. mammary cancer section preparation

Tianjin tumour hospital pathology chamber is taken from the mammary cancer section, conventional dewaxing.

2. immunohistochemical methods step

The slide glass taking-up is placed 500ml methyl alcohol+15ml 3% H2O2 solution, soaked 15 minutes, remove endogenous peroxydase.Immerse in the citrate buffer solution (PH=6.0) microwave oven antigen retrieval 12 minutes.1 normal sheep serum of every point, 37 ℃ of temperature were bathed 20 minutes.Use Myomegalin variant 5 monoclonal antibody 1:800 dilution, 4 ℃ are spent the night.PBS rinsing 3 times adds the anti-mouse IgG of horseradish peroxidase (HRP) labelled goat, hatched 15 minutes for 37 ℃, and the DAB colour developing, routine is redyed and mounting.

3. the result judges

Infiltration ductal carcinomas of breast dyeing shows that Myomegalin variant 5 albumen are evenly painted palm fibre/yellow particle in the positive cell endochylema, or (with) intensively being dispersed in distribution, interfibrillar substance cell, lymphocyte etc. are not painted, and background is clear.Normal breast is seldom painted, sees accompanying drawing 2,3.Therefore, the immunohistochemical methods detection reagent of people Myomegalin variant 5 albumen of this Antibody Preparation, described detection reagent are used for expression and the location of Myomegalin variant 5 in human body tissue and the cell.

SEQUENCE LISTING

＜110〉Tianjin Tumour Hospital

＜120〉monoclonal antibody and the application thereof of anti-human phosphodiester enzyme 4D interaction protein variant 5

<130> 1

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 14

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<220>

<221> protein

<222> (1)..(14)

<400> 1

Gly Glu Leu Glu Ser Val Arg Ile His His Lys His Ala Tyr

1 5 10

Claims

1. the monoclonal antibody of an anti-human phosphodiester enzyme 4D interaction protein variant 5 is characterized in that: the 1103-1116 site amino acid of this antibodies specific identification human phosphodiester enzyme 4D interaction protein variant 5; This antibody is the hybridoma cell strain generation of CGMCC No. 5078 by preserving number, and the aminoacid sequence that described monoclonal antibody is identified is the aminoacid sequence shown in the sequence table № 1.

2. hybridoma, it is characterized in that: this hybridoma produces anti-human phosphodiester enzyme 4D interaction protein variant 5 monoclonal antibodies, and this hybridoma cell strain preserving number is: CGMCC No.5078.

3. the application of the monoclonal antibody of the described anti-human phosphodiester enzyme 4D interaction protein variant 5 of claim 1 in preparation immunohistochemical methods detection reagent, described detection reagent are used for expression and the location of phosphodiesterase 4 D interaction protein variant 5 in human body tissue and the cell.