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Mono-cloning antibody of anti-human cancer protein iASPP and use thereof

A monoclonal antibody, oncoprotein technology, applied in anti-animal/human immunoglobulin, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problem of lack of antibodies, and achieve the effect of promoting the pathogenesis

Inactive Publication Date: 2006-11-01
INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, further in-depth research on iASPP, such as whether the protein level of iASPP in cancerous tissues of solid tumors and bone marrow cells and peripheral blood cells of hematological tumors has changed in various solid tumors and hematological tumors, is due to lack of corresponding studies. antibodies and cannot perform

Method used

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  • Mono-cloning antibody of anti-human cancer protein iASPP and use thereof
  • Mono-cloning antibody of anti-human cancer protein iASPP and use thereof
  • Mono-cloning antibody of anti-human cancer protein iASPP and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Cloning of embodiment 1 human iASPP gene and construction of eukaryotic and prokaryotic expression vectors

[0028] 1.1 Cloning of human iASPP gene

[0029] Collect U-937 cells in the logarithmic growth phase by centrifugation (1-5)×10 6 , TRIzol extracted total RNA, and measured A with a spectrophotometer 260 / A 280 , Calculate the RNA content, and confirm the integrity of the RNA by 12g / L agarose gel electrophoresis. 20μl reverse transcription reaction system containing 2μg total RNA, 4μl RT buffer, 50pmol / L oligo(dT) 16 , 1 μmol / L dNTPs, 0.1 μmol / L DTT, 15 U RNase inhibitor (Takara), 200 U MLV (Invitrogen), in 65 ° C water bath for 5 min, ice bath for 5 min, 37 ° C water bath for 60 min, 70 ° C for 15 min to stop the reaction, the obtained cDNA Store at -20°C for later use. According to the iASPP CDS sequence (NM_006663), use the gene runner software to design primers, add EcoR I to the 5' end of the antisense strand and add Xho I restriction site to the 5' end ...

Embodiment 2

[0034] Example 2 Expression and purification of screening antigens

[0035] 2.1 The PIAF and PIAS plasmids obtained in Example 1.2 were respectively transformed into Rosetta (DE3) prokaryotic expression strains, and positive clones were screened on kanamycin and chloramphenicol double-resistant LB medium agar plates. Pick a single clone and inoculate it in LB medium containing kanamycin (50mg / L) and chloramphenicol (34mg / L), and culture it with shaking at 37°C until OD 600 Reach 0.6-0.8. Inoculate in LB medium containing the same concentration of antibiotics at a ratio of 1:100, culture with shaking at 37°C until OD 600 After reaching 0.6-0.8, add IPTG to a final concentration of 1 mmol / L to induce expression. After 4 hours, the cells were collected by centrifugation at 4°C. Resuspend in an appropriate amount of 1×SDS loading buffer, place in a boiling water bath for 3-5 minutes, and take 50 μl for SDS-PAGE electrophoresis analysis. The molecular weights of the target frag...

Embodiment 3

[0040] The preparation of embodiment 3 monoclonal antibody

[0041] 3.1 Animal immunity

[0042] The CIAF plasmid obtained in Example 1.3 was extracted using a large extraction plasmid kit, and the plasmid was diluted with PBS to a concentration of 100 μg / 100 μl and injected into the spleen of mice, 100 μl per mouse.

[0043] 3.2 Preparation of feeder cells

[0044] The feeder cells used in the present invention are mouse peritoneal macrophages. The feeder cells were prepared the day before fusion, and the preparation process was as follows:

[0045] Use the same strain of Balb / c mice as the immunized mice, aged 6-8 weeks →

[0046] Kill the mice by cervical dislocation, soak them in 75% alcohol, and disinfect them for 3-5 minutes →

[0047] Mount the mouse on a dissecting board, cut the skin with sterile scissors, and expose the peritoneum→

[0048] Inject 6-8ml of culture solution into the abdominal cavity with a sterile syringe→

[0049] Repeatedly flush the peritonea...

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Abstract

A monoclone antibody of antihuman cancer protein iASPP and its use are disclosed. It includes antigen carrier construction, protein expression and purification to screen iASPP monoclone antibody. It can be used to diagnose and treat various tumors.

Description

technical field [0001] The invention relates to a monoclonal antibody, in particular to a monoclonal antibody against human oncoprotein iASPP and its application. Background technique [0002] At present, malignant tumors have become one of the most serious diseases threatening human health. According to the data of the Ministry of Health in 2001, the mortality rate of malignant tumors in urban areas of my country is 135.59 / 100,000, ranking first among the mortality rates of various diseases. With the development of social economy and the acceleration of industrialization and urbanization, the incidence of lung cancer, liver cancer, gastric cancer, intestinal cancer and other tumors in both urban and rural areas are on the rise. Therefore, the prevention, pathogenesis, diagnosis and treatment of various tumors The research related to the prognosis and prognosis has become an urgent problem to be solved. The main pathophysiological basis of the pathogenesis of malignant tum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/79G01N33/577
Inventor 王建祥王敏刁世勇饶青张新伟廖晓龙
Owner INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
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