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Preparation and use of testes specificity protein 50 human source antibody

A humanized antibody and protein technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, anti-tumor drugs, etc., to achieve the effect of small immunogenicity, low cost, and reduction of harmful reactions

Active Publication Date: 2006-08-30
李玉新 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, recent studies have shown that abnormally high expression of TSP50 is found in more than 90% of breast cancer tissues, and this expression is limited to malignant epithelial cells (Shan, J., Yuan, L, Xiao, Q., Chiorazzi, N., Budman, D., Teichberg, S., and Xu, HP, TSP50, A possible protease in human testis, is activated in breast cancer epithelial cells. Cancer Res. 2002, 62, 290-294)

Method used

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  • Preparation and use of testes specificity protein 50 human source antibody
  • Preparation and use of testes specificity protein 50 human source antibody
  • Preparation and use of testes specificity protein 50 human source antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Screening of phage antibody library with 50 peptide and full-length protein of TSP50

[0043] Phage antibody library: The human antibody library constructed in the pCOMB3 plasmid was donated by the Scripps Research Institute of the United States.

[0044] 1. The first round of screening: Coat polystyrene culture dishes with 50 peptides and full-length proteins (50 μg / ml) of TSP50, and antibody libraries (5.6×10 12 pfu) into the culture dish, incubate for 2 hours, suck out the supernatant, wash the dish 10 times with PBST, dry the dish on sterile filter paper, wash the dish with 1ml of freshly prepared triethylamine; take 2 sterile EP tubes , add 300 μl Tris-HCl respectively, and then add 500 μl triethylamine eluent respectively, and use it to infect TG1 after neutralization.

[0045] 2. Amplification of the antibody library after the first round of screening: Infect 14ml of fresh Escherichia coli TG1 for every 0.8ml of the eluted product after neutralization, and infec...

Embodiment 2

[0048] Preparation and activity detection of monoclonal phage antibody

[0049] The product obtained after three rounds of elutriation and enrichment was spread on 2YT solid medium and grown overnight at 37°C. On the next day, 100 single spots were randomly picked and put into a sterilized test tube containing 2ml 2YT / AG, and cultured at 37°C overnight. The collected phages were added to the wells of the microtiter plate coated with TSP50 protein and incubated for 2 hours. Screened by conventional ELISA method, strong positive clones were obtained.

Embodiment 3

[0051] TSP50 Human Antibody Sequence Determination

[0052] The DNA of ELISA strongly positive clones was extracted, entrusted to Shanghai United Gene Company for sequence analysis, and its amino acid sequence was deduced, the results are shown in Figures 2, 3, and 4.

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Abstract

The present invention provides a preparation method of testicular specific protein 50 humanized single-chain antibody. Said invention utilizes phage antibody presentation technique and adopts the following steps: using human testicular specific protein 50 as target antigen, utilizing immunoaffinity screening process to screen the recombinant phage human antibody variable region gene library, separating and cloning TSP 50 specific single-chain antibody gene AI,AII and C8, then respectively inserting single-chain antibody gene AI, AII and C8 into prokaryotic expression vector PelB, transforming Rosetta microphyte, utilizing IPTG to induce and express exogenous protein, after the thallus is cracked, ulitizing Ni-NTA column affinity chromatography to obtain purified single-chain antibody, utilizing ELISA method to judge that it can be specifically combined with TSP 50 holoprotein and 50 peptide, and using gene engineering measure to massively express said hamonized single-chain antibody. Said invention also discloses application of the testicular specific protein 50 humanized single-chain antibody for diagnosing and curing the diseases of female mammary carcinoma, etc. related to TSP 50.

Description

technical field [0001] The invention belongs to the field of humanized genetic engineering antibodies, and relates to the preparation and application of a human single-chain antibody of testis-specific protein 50 closely related to breast cancer and other tumors. This antibody can be used for the diagnosis and treatment of TSP50-related diseases and belongs to The technical field of biogenetic engineering. Background technique [0002] Breast cancer is a common malignant tumor in women, and its incidence rate is increasing year by year. From the perspective of epidemiological development trend, breast cancer is more and more concentrated in big cities, ranking first among female malignant tumors. At present, the diagnosis of breast cancer is mainly through imaging and biopsy, and there is no effective early diagnosis method. The treatment of breast cancer is still based on surgery, supplemented by radiotherapy and chemotherapy. With the development of society, radical mast...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18A61K39/395A61P35/00G01N33/577
Inventor 李玉新鲍永利徐浩鹏刘洋魏景艳李惟乌垠孟祥颖张靖单继东易静雯
Owner 李玉新
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