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Compsn of anti-blood generating factor contg adeno-associated virus mediation and its application

An anti-angiogenesis and composition technology, applied in the direction of medical preparations containing active ingredients, viruses, microorganisms, etc., can solve the problems of unsatisfactory effect and short action time, and achieve enhanced anti-tumor effect, long action time, stable effect

Inactive Publication Date: 2007-05-09
林李家宓
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] In order to prevent or treat cancer more effectively, and to overcome the defects of short action time and unsatisfactory effect of tumor angiogenesis inhibitors in the prior art, the present invention provides a drug that can not only inhibit the growth of tumor angiogenesis but also inhibit the growth and metastasis of cancer cells. recombinant construct

Method used

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  • Compsn of anti-blood generating factor contg adeno-associated virus mediation and its application
  • Compsn of anti-blood generating factor contg adeno-associated virus mediation and its application
  • Compsn of anti-blood generating factor contg adeno-associated virus mediation and its application

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0074] 7. Preparation of HGFK1 protein antibody

[0075] Using standard techniques of preparation (Harlow, E., and Lane, D. (1988) Antiodies: a laboratory manual (antibodies: a laboratory manual). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory) or methods known in the art, can be The construct of the present invention or the protein expressed thereof is used to prepare antibodies against the protein of the present invention.

[0076]For example, the constructs of the invention can be purified to the extent necessary to immunize animals such as rabbits. To prevent potential problems with low affinity or low specificity of antisera, 2 or 3 constructs for anti-angiogenic factors can be prepared and each construct injected into at least 2 animals. Antiserum can be generated by a series of injections, preferably comprising at least three booster injections. The primary immunization can be performed with complete Freund's adjuvant, followed by booster immunization with inco...

Embodiment 1

[0084] The construct of the AAV expression vector of embodiment 1 coding human HGFK1 polypeptide

[0085] The cDNA of human HGFK1 (SEQ ID NO: 1 nucleotide sequence) is inserted into the AAV packaging vector of pAM / CAG / EGR-1-pL-WPRE-BGH-polyA that has been digested with the same endonuclease in advance to form AAV - HGFK1 recombinant vector. Then use the three-vector helper virus defect packaging system (AAV, H22 and pFD6) for packaging, that is, use the calcium carbonate method to co-transfect HEK-293 cells with the recombinant vector AAV-HGFK1 and helper plasmids H22 and pFD6, and transfect for 60-72 hours Afterwards, the cells were harvested, the isolated recombinant virus particles were purified by HiTrap Heparin affinity chromatography, and the final titer was determined by real-time PCR.

[0086] After infecting mouse microvascular endothelial cells and rat hepatocellular carcinoma cells with AAV-HGFK1 in vitro, the infected cells were stained by immunohistochemistry. T...

Embodiment 2

[0088] Embodiment 2 encodes p53, the construct pAd-X of the adenoviral vector (Adv) of hTERTC27, IL-2 or HGFK1 gene

[0089] The present invention provides the preparation and packaging method of the construct of recombinant defective adenovirus expression vector encoding human p53, hTERTC27, IL-2 or HGFK1 gene, comprising the following steps: human p53 gene, hTERTC27 gene, IL-2 gene or HGFK1 Genes were ligated into the shuttle plasmid pENTR TM In 2B, the recombinant shuttle plasmids pENTR-p53, pENTR-hTERTC27, pENTR-IL-2 or pENTR-HGFK 1 and pAd / CMV / V5-DEST were obtained by homologous recombination into expression vectors pAd-p53, pAd-hTERTC27, pAd-IL-2 or pAD-HGFK1, the resulting expression vector was treated with Lipofectamine TM The 293A cells were transfected with 2000 Reagent (Invitrogen), and the cells were lysed, concentrated and purified through a cesium chloride column. The purified recombinant adenovirus (Adv-p53, Adv-hTERTC27, Adv-IL-2 or Adv-HGFK1) carrying human...

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Abstract

An adeno-associated virus carrier, the adeno-associated virus carrier mediated configurators AAV-HGFK1 and Adv-HGFK1 expressing the anti-angiogenesis factor, a composition containing configurator AAV-HGFK1 and the adeno-associated virus carrier mediated configurator expressing anticancer gene or cancer suppressing gene or cell factor, and the application of said configurators or composition in preparing the medicines for preventing or treating cancer are disclosed.

Description

field of invention [0001] The present invention relates to a construct or composition for preventing or treating cancer, in particular to an adeno-associated virus vector or an adenoviral vector-mediated construct AAV-HGFK1 or Adv-HGFK1 expressing an anti-angiogenic factor HGFK1, and a construct containing Compositions of AAV-HGFK1 and adenoviral vectors or constructs of adenoviral vectors expressing anti-oncogenes, tumor suppressor genes or cytokine genes. technical background [0002] In the growth, invasion and metastasis of solid tumors, continuous angiogenesis is one of the key factors affecting the process, because new blood vessels provide nutrients, oxygen and various growth factors necessary for tumor cell proliferation. [0003] Accordingly, Folkman first proposed in 1971 that inhibiting tumor angiogenesis could be used as a means of treating tumors (Folkman J. et al., J Exp Med, 1971, 133: 275-88.). Preclinical studies have shown that systemic application of puri...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/395A61P35/00
CPCC12N2750/14143A61K38/1833C12N2710/10343A61K48/005C12N15/86A61P35/00
Inventor 林李家宓沈赞孔祥复
Owner 林李家宓
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