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Q type non-viral vector and pharmaceutical composition containing the same

A composition and drug technology, applied in the direction of drug combination, the use of carriers to introduce foreign genetic material, and non-effective ingredients of polymer compounds, etc., can solve the problems of toxic side effects of killing effect, immunogenicity problems, neutralization and failure of toxin killing activity, etc.

Inactive Publication Date: 2008-03-05
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] It is known that there are thousands of human heterologous proteins with anti-tumor or anti-viral activity, but there are two problems in clinical application: one is non-specific (or targeted) killing effect on all cells The serious toxic and side effects caused by it; the second is the serious immunogenicity problem when it is introduced into human blood circulation as a human heterologous protein
Moreover, the toxin protein is a heterologous protein, and the appearance of such a protein in the blood circulation will lead to the formation of its antibody, thereby neutralizing the killing activity of the toxin

Method used

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  • Q type non-viral vector and pharmaceutical composition containing the same
  • Q type non-viral vector and pharmaceutical composition containing the same
  • Q type non-viral vector and pharmaceutical composition containing the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Construction of fusion gene recombinant qmIL2-SON2 and wtIL2SON1

[0056] 1.1 Construction of qmIL2-SON2 sequence:

[0057] The IL2 fragment was amplified using the CW120 containing IL2 sequence of Dr. Chen Weijing in our laboratory, and after sequencing, it was found that compared with the wild type, there are two mutations in the IL2 template sequence that affect amino acid coding. It is necessary to design mutation primers for these mutation sites , Perform back mutation. Finally, the 58 and 125 Cys coding codons of the IL2 sequence were mutated into Ser coding codons, and the remaining sequences were the same as the wild-type sequence.

[0058] While mutating the IL2 sequence, use the upstream and downstream full-length primers FF and RF to introduce the restriction sites EcoRI and Nde I required for cloning at the 5'end of the IL2 sequence, respectively; at the 3'end of the IL2 sequence Introduce the thrombin cleavage site as the Linker sequence inside the fu...

Embodiment 2

[0065] Example 2 Induced expression of fusion gene qmIL2-SON2 or wtIL2SON1, extraction and detection of inclusion bodies

[0066] 2.1 Temperature induction and extraction of inclusion bodies

[0067] 2.1.1 Large volume culture

[0068] The constructed expression recombinant was transformed into Escherichia coli DH5α, and the transformed product was spread on an LB plate containing ampicillin and cultured overnight at 32°C. Take out several 2-3mm single colonies from the plate of the recipient strain, transfer them to a test tube containing 5ml of LB (containing appropriate antibiotics) culture solution, and culture them with vigorous shaking at 37°C overnight until they become turbid. On the second day, the overnight bacteria were also transferred to a medium bottle (150-200ml containing appropriate antibiotics) LB culture medium at a ratio of 1-2%, and cultured with vigorous shaking at 30-32°C overnight until turbid. On the third day, transfer the overnight bacteria to 6 large bo...

Embodiment 3

[0086] Example 3 Purification of protein qmIL2-SON2 or wtIL2SON1 from E. coli inclusion bodies

[0087] 1. Inclusion bodies are in the proportion of 20ml per gram. The inclusion bodies were dissolved with inclusion body dissolving solution (50mM Tris-HCl, 2mM EDTA, 100mM NaCL, 10mM DTT, 7M guanidine hydrochloride, pH9.0), and passed through a 0.45μm pore filter membrane.

[0088] 2. Reverse phase column: use AKTA medium pressure liquid chromatography system, chromatography column Waters AP1 column packing SOURCE 15RPC, flow rate 3ml / min. Buffer: starting buffer A solution: 10% acetonitrile, 0.05% TFA, starting buffer B solution: 90% acetonitrile, 0.05% TFA.

[0089] Gradient: 100% solution A maintains 3 column volumes to elute unbound proteins and impurities; the concentration of solution B rises to 100% within 15 minutes and maintains 2 column volumes. Collect the eluted protein in 8ml.

[0090] After qmIL2-SON2 was purified by reversed-phase column, the purity of the protein was...

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Abstract

The present invention relates to gene therapy medicine. The present invention provides one kind of tumor-targeting gene therapy medicine composition qmIL2-SON2 / NfBS-MiniCMV-PEIIImut comprising non-viral protein carrier qmIL2-SON2 and specific tumor tissue killing gene eukaryon expressing plasmid NfBS-MiniCMV-PEIIImut. The present invention also provides one kind of specific tumor tissue regulating sequence NfkB-MiniCMV promoter containing the coding sequence of Nf-kB protein combining site and shortened miniCMB promoter. The present invention provides all the relevant research results, including the use of composition qmIL2-SON2 / NfBS-MiniCMV-PEIIImut assembled with the non-viral protein carrier and the killing gene recombinant in killing IL2R enriching tumor cell specifically without killing normal cells.

Description

Technical field [0001] The invention belongs to the field of gene therapy drugs. Specifically, the invention provides a targeted tumor gene therapy drug composition qmIL2-SON2 / NfBS-MiniCMV-PEIIImut. Specifically, the composition is composed of a protein non-viral vector qmIL2-SON2 and a tumor tissue-specific killing (toxin) gene eukaryotic expression plasmid NfBS-MiniCMV-PEIIImut. The protein non-viral vector is composed of the cell-binding peptide qmIL2 and the DNA-binding polypeptide SON2, and is a targeted fusion protein non-viral vector qmIL2-SON2 through DNA recombination and expression. The DNA binding polypeptide SON2 in the fusion protein is rich in positive amino acids and is positively charged, so it can bind negatively charged DNA; in the present invention, SON2 is used in combination with the killer gene expression recombinant plasmid NfBS-MiniCMV-PEIIImut to form a compound drug. The qmIL2 in the fusion protein can bind to tumor cells enriched with IL2 receptors on th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K47/42C12N15/63A61P35/00
Inventor 卢圣栋王湛陈伟京李涛杜延平路金芝
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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