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Influenza virus hemagglutinin fusion peptide-containing targeted recombinant molecule gene, protein encoded thereby and application thereof

An influenza virus and hemagglutinin technology, applied in the field of gene therapy, can solve the problem of reducing the efficacy of antibody fusion proteins, and achieve the effects of small immunogenicity, direct killing effect, and high-efficiency apoptosis-promoting biological activity.

Inactive Publication Date: 2011-02-23
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the place where most effector proteins function is the cytoplasm. Although the internalizable single-chain antibody can mediate the effector protein into the endosome of the target cell through the internalization of the target cell, the effector protein cannot freely penetrate into the endosome. Phytosome membrane structures enter the cytoplasm, and the efficacy of antibody fusion proteins is greatly reduced

Method used

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  • Influenza virus hemagglutinin fusion peptide-containing targeted recombinant molecule gene, protein encoded thereby and application thereof
  • Influenza virus hemagglutinin fusion peptide-containing targeted recombinant molecule gene, protein encoded thereby and application thereof
  • Influenza virus hemagglutinin fusion peptide-containing targeted recombinant molecule gene, protein encoded thereby and application thereof

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Experimental program
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Effect test

Embodiment 1

[0023] 1. Construction of HBsAg targeting pro-apoptotic molecular genes

[0024] (1) Construction of pET32a-scFv-Fu-HAfp-tBid plasmid (Fu is the furin protease recognition site, the same below)

[0025] With the plasmid pET32a-HBV-scFv preserved in the inventor's laboratory as a template (Wen WH, Qin WJ, Gao H, Zhao J, Jia LT, Liao QH, Meng YL, Jin BQ, Yao LB, Chen SY, YangAG.An hepatitis B virus surface antigen specific single chain of variable fragment derived from a natural immune antigen binding fragment phage display library is specifically internalized by HepG2.2.15 cells.J Viral Hepat.2007 Jul; 14(7):512-9.), with HBV -scFv-p5 and HAfp-1st-p3 are used as upstream and downstream primers for the first round of amplification, and the obtained PCR product is then used for the second round of amplification with HBV-scFv-p5 and HAfp-2nd-p3 as upstream and downstream primers , to obtain scFv-HAfp fragments. The scFv-HAfp fragment was double-digested with NcoI and EcoRI, and ...

Embodiment 2

[0044] 2. Construction of HER2 targeting pro-apoptotic molecular genes

[0045] The pET44b-scFv-Fu-HAfp-tBid plasmid and the pET32a-e23sFv-TD-tBid plasmid were digested with NcoI and EcoRI, and the pET44b-e23sFv-Fu-HAfp-tBid plasmid was obtained by fragment recovery and ligation, and the vector construction was confirmed by enzyme digestion correct (attached figure 1 ), the sequencing results show that the recombinant molecule has the sequence of influenza virus hemagglutinin fusion peptide.

[0046] 3. Prokaryotic expression and product purification of HBsAg / HER2 targeting pro-apoptotic molecules

[0047] The successfully constructed pET44b-scFv-Fu-HAFP-tBid plasmid and pET44b-e23sFv-Fu-HAfp-tBid were transformed into Escherichia coli protein expression host strain RosettaBlue (DE3), single clones were picked, and inoculated in a medium containing ampicillin (100 μg / mL), tetracycline (12.5 μg / mL), chloramphenicol (34 μg / mL) in the LB medium, 37 ° C, 220 rpm shaking overnig...

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Abstract

The invention provides an influenza virus hemagglutinin fusion peptide-containing targeted recombinant molecule gene. The influenza virus hemagglutinin fusion peptide-containing targeted recombinant molecule gene is formed by recombining an internalizing single-chain antibody gene, an influenza virus hemagglutinin fusion peptide gene and an apoptotic molecule gene, wherein a gene sequence for encoding a furin protease recognition site is inserted between the internalizing single-chain antibody gene and the influenza virus hemagglutinin fusion peptide gene. A single-chain antibody part of an expression product of the targeted recombinant molecule gene of the kind can identify specificity, combines a target cell expressing a specific antigen and mediates the recombination protein internalization into the endosome of the target cell; the single-chain antibody is separated from the other parts after the recombination protein is cut by the furin protease in the endosome; the hemagglutinin fusion peptide is exposed; and the apoptotic molecule is released to cytoplasm by damaging the stability of an endosome membrane under the action of the hemagglutinin fusion peptide; and finally, the apoptotic molecule effectively induces the target cell into atopsis in the cytoplasm and embodies the biological activity of the hemagglutinin fusion peptide.

Description

technical field [0001] The present invention relates to gene therapy in the field of biotechnology, in particular to genetic engineering methods to construct targeted recombinant molecular genes composed of internalizable single-chain antibodies, influenza virus hemagglutinin fusion peptides and pro-apoptotic molecules, and proteins encoded by the above genes And the application of said gene and protein in the preparation of medicine for tumor treatment. Background technique [0002] Antibody drugs are drugs prepared by antibody engineering technology with cell engineering technology and genetic engineering technology as the main body. Due to its high specificity, uniform properties, and the ability to be prepared for specific targets, antibody drugs have been widely used in the treatment of infections, cardiovascular diseases, autoimmune diseases and other diseases, especially in the treatment of tumors with great potential and Application prospect. Antibody drugs have th...

Claims

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Application Information

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IPC IPC(8): A61P35/00C12N15/62A61K47/48A61K38/17A61K48/00C07K19/00A61K47/42
Inventor 杨安钢王涛闫博温伟红贾林涛王芳白文栋赵晶陈锐赵智凝任君琳孟艳玲
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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