Recombinant fusion protein of IL3 and Lidamycin, preparation method and application thereof

A technology of fusion protein and protein, which is applied in the field of proteomics, biopharmaceuticals, and oncology. It can solve the problems of high molecular weight, difficult internalization, immunogenicity, poor stability, etc., and achieve the goal of overcoming lethal side effects, not easy to degrade, and good The effect of applying the foreground

Inactive Publication Date: 2013-05-15
中国医学科学院血液病医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, DT388 itself has a relatively large molecular weight and is difficult to internalize and has certain immunogenicity. The purification process in vitro experiments also showed that its stability is slightly poor, and there are still many shortcomings in practical applications.

Method used

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  • Recombinant fusion protein of IL3 and Lidamycin, preparation method and application thereof
  • Recombinant fusion protein of IL3 and Lidamycin, preparation method and application thereof
  • Recombinant fusion protein of IL3 and Lidamycin, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Construction of recombinant expression plasmids pET28a IL3-LDP and pET28a mIL3-LDP

[0071] PCR amplification of IL3:

[0072] Take about 10ml of peripheral venous blood from normal people, add lymphocyte separation solution after anticoagulant treatment, absorb buffy coat phase, wash twice with 1×PBS, culture with RPMI1640 medium containing 10% fetal bovine serum, and add IL -2 and CD3 / p-gp bifunctional antibodies stimulate cell proliferation. The total RNA of normal human peripheral blood buffy coat cells was extracted, and the corresponding cDNA was obtained by reverse transcription PCR (RT-PCR) as a template to obtain IL3 that was completely consistent with the cDNA sequence of the human IL-3 core coding region retrieved from Genbank. The full length of the gene sequence. The PCR primers were synthesized by Shanghai Yingjun Company, and the corresponding enzyme cutting sites were respectively introduced.

[0073] Specifically, the cDNA of human peripher...

Embodiment 2

[0107] Example 2 Expression and verification of IL3-LDP and mIL3-LDP

[0108] 2.1 Expression of IL3-LDP and mIL3-LDP

[0109] The single bacterium colony of Escherichia coli BL21 containing plasmid pET28a IL3-LDP and pET28a mIL3-LDP obtained in Example 1 was inoculated in 5ml of 2×YT medium containing kanapenicillin (Kan) 50 μg / ml, at a constant temperature In a shaker box at 37°C, 200rpm, shake culture overnight; transfer 500ml of 2×YT medium containing Kan 100μg / ml (1L of 2×YT medium contains 1.6% tryptone, 1.0% yeast extract, 0.5% Sodium chloride, pH 7.4), 37°C, 200rpm, shaking culture for 8h, then centrifuge at 6000rpm, 4°C for 10 minutes on a low-temperature high-speed vacuum centrifuge to collect the bacteria, resuspend the bacteria in 1000ml containing Kan 100μg / ml and 1mM IPTG 2×YT medium, 30°C, 200rpm, shaking culture for 4h; 8,000rpm, 4°C centrifugation for 10 minutes to collect the bacteria, frozen in -20°C refrigerator for later use.

[0110] Thaw the frozen bact...

Embodiment 3

[0113] Example 3 Immunological activity of IL3-LDP and mIL3-LDP

[0114] The in vitro immunofluorescence binding activity of IL3-LDP and mIL3-LDP was determined by flow cytometry. Will 1×10 6 TF-1 cells per ml were resuspended in 100 μL PBS solution containing different concentrations of FITC-labeled anti-CD123-perCP5.5 antibody or IL3-LDP and mIL3-LDP obtained in Example 2, placed at 4°C for 1 h, centrifuged at 2000 g After 10 minutes, the supernatant was discarded, washed three times with PBS, and the positive rate of anti-CD123 binding to TF-1 cells was determined by FACS. Prove that the same concentration of anti-CD123 antibody and IL3-LDP and mIL3-LDP have basically the same binding activity to TF-1 cells, IL3-LDP and mIL3-LDP fusion protein retains the ability to specifically bind to the target antigen ( Figure 4 ).

[0115] After the flow cytometry sample is ready, centrifuge at 2000g for 10 minutes, discard the supernatant; add 4% paraformaldehyde solution for fixa...

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Abstract

The invention relates to a strengthened fusion protein of an anticancer drug lidamycin, i.e. mIL3-LDM from mutated I131L and F132L, and a coding gene thereof. The invention also relates to a genetic engineering construction method of the fusion protein and application of the strengthened fusion protein.

Description

technical field [0001] The invention relates to the fields of oncology, proteomics and biopharmaceuticals, and provides a fusion protein that can produce a targeted tumor killing effect, a preparation method and an application in tumor targeted therapy drugs. Background technique [0002] Leukemia is a malignant clonal disease of hematopoietic stem cells, and its mortality rate ranks first among malignant tumors that cause death in children and adults under the age of 35. Conventional radiotherapy and chemotherapy can remove most proliferating cells and genetically unstable cells in tumor tissue, but cannot remove tumor stem cells, resulting in tumor recurrence and metastasis. At the same time, the lack of targeted conventional radiotherapy and chemotherapy may also damage certain types of normal cells Cells have obvious toxic and side effects. Cancer stem cells have self-protection characteristics similar to normal stem cells, such as effective DNA repair, high expression ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/15C12N1/19C12N1/21A61K38/20A61K47/48A61P35/00A61P35/02
Inventor 杨纯正甄永苏邵荣光熊冬生张砚君苗庆芳刘荣张秀丽彭红薇胡蕴惠李双静林阳李真真颜次慧姜琳琳
Owner 中国医学科学院血液病医院
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