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Fusion protein containing collagen binding structure domain

A technology combining structural domains and fusion proteins, applied in the biological field, can solve the problems of multifunctional fusion protein design, preparation and application difficulties, and achieve the effects of avoiding immune response, reducing toxic side effects, and reducing pain

Active Publication Date: 2017-03-15
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existence of these technical bottlenecks makes multifunctional fusion proteins face many difficulties in the process of design, preparation and application

Method used

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  • Fusion protein containing collagen binding structure domain
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  • Fusion protein containing collagen binding structure domain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Cloning of human CBD gene and cloning of mouse IFN-γ gene

[0064] The gene sequence of CBD was cloned from the existing human MMP2.

[0065] Cloning of the IFN-γ gene:

[0066] (1). Use LA enzyme to Xhol with EcoR1 is the enzyme cutting site, and the primers were synthesized by Wuhan Qingke Biotechnology Co., Ltd., and diluted with pure water to a working solution concentration of 10 μM. The IFN-γ gene sequence on which the primers were designed was derived from NCBI Reference Sequence: NM_008337.3).

[0067] (2). Preparation of template: RNA was extracted from mouse cells, and then reverse-transcribed to obtain cDNA as a template for PCR reaction

[0068] (3).PCR reaction system: 50ul reaction system contains: ddH 2 O 37.5ul; 10×LA buffer 5ul; 10mMdNTP 4ul; Primer (F+R) 2ul; mcDNA 1ul; LA enzyme 0.5ul. PCR reaction parameters: 95°C for 1 min; 94°C for 30 s, 57.9°C for 30 s, 72°C for 30 s, 40 cycles. 72°C for 5 min; 16°C for ∞.

[0069] (4). Af...

Embodiment 2

[0076] Example 2: Insert the cloned human MMP2 CBD and CDS region sigpeptide of the mouse IFN-γ DNA sequence into the expression vector PET28a(+) between EcoR1 and HindIII restriction sites. Using the method of fusion and recombination.

[0077] (1). First design the primers for fusion recombination.

[0078] (2). Fragment PCR, fragment PCR reaction system: 50ul reaction system, ddH 2 O 37.5ul; 10×LA buffer 5ul; 10mMdNTP 4ul; Primer (F+R) 2ul; plasmid 1ul; LA enzyme 0.5ul. PCR reaction parameters: 95°C for 5min; 94°C for 30s, 55°C for 30s, 72°C for 30s, 40 cycles. 72°C for 5 min; 16°C∞. After PCR cloning PCR is completed, take 2ul PCR product + 2ul 10×loading buffer, and carry out 1% agarose gel electrophoresis detection.

[0079] (3). PCR reaction system for vector linearization: 50ul reaction system contains: ddH 2 O 33ul; 10×KODbuff 5ul; 2mMdNTP 5ul; 25Mm MgSO4: 3ul; Primer (F+R) 2ul; plasmid 1ul; KOD enzyme 1ul. PCR reaction parameters: 95°C for 1 min; 95°C...

Embodiment 3

[0087] Implementation Example 3: Insert the sig peptide of the CDS region of the IFN-γ DNA sequence of the cloned mouse into the upstream of the CDS region of the DNA sequence of the CBD of human MMP2 in PET-28(a)+CBD

[0088] The operation method is still fusion and recombination, PET-28(a)+CBD is used as a carrier, PET-28(a)+IFN-γ is used as a fragment, the operation is the same as PET-28(a)+CBD, PET-28(a)+IFN - build of gamma.

[0089] image 3 After cloning the PET-28(a)+ IFN-γ-CBD plasmid, run a 1% DNA gel identification map, where the M band represents the DL5000 marker, and the e band represents the constructed PET- After the 28(a)+ IFN-γ-CBD plasmid was digested by ECORⅠ and HindⅢ, PET-28(a)+ and IFN-γ-CBD were excised respectively.

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Abstract

The invention relates to a fusion protein with a recombinant collagen binding structure domain. The amino acid sequence of the fusion protein is shown in SEQID NO:2. The fusion protein is a solution preparation and can be used for preparing antitumor drugs, and the drugs can be administrated by being locally injected in an neoplasm with enough therapeutic dosage at a time. In the fusion protein, a local slow release system formed by the collagen binding structure domain CBD well avoids a strong immune response caused by diffusion of IFN-gamma to surrounding tissues. At the same time, repeated administration of the drugs is not required; not only the pain, brought by traumas caused by constant repeated administration of the drugs, of patients is reduced and the high therapy cost caused by the constant repeated administration of the drugs is reduced, but also the toxic and side effects caused by the large use amount and frequent use of the drugs are reduced.

Description

technical field [0001] The present invention relates to the field of biotechnology. It is specifically related to the recombinant protein containing the collagen binding domain (CBD) of matrix metalloproteinase (MMP2) and the active domain of gamma interferon (IFN-γ) and its fusion protein, especially the one with multiple activities fusion protein. Background technique [0002] Cancer is one of the leading causes of disease and death worldwide. It is a serious threat to human health, and has also attracted great attention. Over the past 20 years, a large number of studies have focused on the molecular events related to tumorigenesis and signaling pathways involved in tumor progression. Studies have found that the molecular mechanism of complex interactions between tumor cells and the tumor microenvironment plays an important role in tumor progression. At the same time, more than 40 years of research have shown that there is a large body of evidence supporting extrac...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/48A61K38/21A61P35/00A61P35/02
CPCA61K38/00C07K14/57C07K2319/21C12N9/6491C12Y304/24024
Inventor 王琳王征李永奎徐妞妞张剑
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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