Chimaera for HK97 bacteriophage virus-like particle and application of chimaera

A virus-like, bacteriophage technology, applied in the field of molecular biology genetic engineering, can solve the problems of unreported use of biological immune targeted therapy vectors and rare chimera research.

Inactive Publication Date: 2013-06-05
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

So far, although the research on HK97 phage virus-like particles has been reported in the literature, the research on its chimera is rare, and its use as a carrier for biological immune targeting therapy has not been reported

Method used

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  • Chimaera for HK97 bacteriophage virus-like particle and application of chimaera
  • Chimaera for HK97 bacteriophage virus-like particle and application of chimaera
  • Chimaera for HK97 bacteriophage virus-like particle and application of chimaera

Examples

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Effect test

Embodiment example 1

[0030] Implementation Case 1 Construction of Chimera Expression Plasmid pETDuet-HK97-P

[0031] Using primers 5'-CCATGGCTGAAATCGTAAAAACGCTGT-3'; 5'-CCGGAATTCTTATTTACTAAGTTAGAAGGG-3', the protein GP4 gene encoding the head end of HK97 bacteriophage virus-like particles was amplified by PCR, with a size of 678bp. Using primers 5'-CCATATGTCTGAACTCGCTCTCATTC-3'; 5'-CTCGAGTCAACGAGATTCGTAGTGG-3', the protein GP5 gene encoding the head end of HK97 bacteriophage virus-like particles was amplified by PCR, with a size of 1158bp.

[0032] The GP5 gene fragment and GP4 gene fragment obtained above were inserted into the two multiple cloning sites of the pETDuet-1 plasmid (purchased from Novagen) to construct the chimera expression plasmid pETDuet-HK97-P, and its molecular weight was identified by enzyme digestion ( figure 1 ).

Embodiment example 2H

[0033] Implementation Case 2H Maturation and purification of II-HK97-P virus-like particles

[0034] (1) Transform the plasmid pETDuet-HK97-P into BL21(DE3) competent bacteria, pick a single colony and amplify in 20ml liquid medium at 37°C for 16 hours, take 1ml of the bacterial solution and add it to 1L liquid medium, 37 Amplify for several hours at ℃ to the OD of the medium 600nm At about 0.6, IPTG (20ul, 0.2g / ml) was added to induce the expression of virus-like particle chimera precursor P II-HK97-P.

[0035] (2) After 4 hours, the lysed bacteria were centrifuged and the supernatant was collected at 6% PEG80004°C to settle the virus-like particle chimera precursor P II-HK97-P overnight, and the P II-HK97-P was subjected to UnoQ strong anion exchange chromatography (20mM Tris- HCl, 1M NaCl).

[0036] (3) PII-HK97-P is induced to mature after anion exchange purification: Dilute 1.5ml of 30mg / ml PII-HK97-P virus-like particle chimera precursor into 150ml acidified buffer (pH...

Embodiment example 3H

[0038] Implementation Case 3H Identification of II-HK97-P VLPs

[0039] Get HK97-P virus-like particle chimera precursor, intermediate and mature body each 20ug to carry out 6% SDS-PAGE electrophoresis ( figure 2 ), and take HK97-P virus-like particle chimera precursor P II-HK97-P and mature body H II-HK97-P to carry out the morphology and size under the transmission electron microscope (nickel mesh, 4% uranyl acetate negative staining) observation ( image 3 ). Electron microscope observation revealed that the HK97-P virus-like particle chimera precursor P II-HK97-P is a self-assembled prism-shaped virus-like particle structure with a diameter of about 54nm, while the mature body HII-HK97-P is more morphological and structural than the former The body has a thinner shell with a slightly enlarged diameter of about 66 nm.

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Abstract

The invention provides a chimaera for an HK97 bacteriophage virus-like particle and application of the chimaera, and application of the chimaera to bio-immune targeted therapy. A base sequence for expressing AFHYESQ short peptide is added at the C end of a GP5 gene of the HK97 bacteriophage virus-like particle, the GP5 gene and a GP4 gene for coding protein at the head end of the HK97 bacteriophage virus-like particle are inserted into pETDuet-1 plasmid, a chimaera expression plasmid of the HK97 bacteriophage virus-like particle is constructed, and a precursor after the expression plasmid is subjected to induction expression is purified and acidified to be induced into a mature chimaera for the HK97 bacteriophage virus-like particle. The chimaera for the HK97 bacteriophage virus-like particle serves as a carrier for the bio-immune targeted therapy and is prepared into a vaccine to achieve the bio-immune targeted therapeutic effect.

Description

field of invention [0001] The invention belongs to the field of molecular biology genetic engineering, and specifically relates to the construction and purification of a chimera of HK97 phage virus-like particle protein, and its role in biological immune targeting therapy. Background of the invention [0002] Therapeutic vaccines against chronic diseases are currently one of the hotspots in vaccine research, including chronic viral infections, allergic diseases, tumors, diabetes, hypertension, and Alzheimer's disease. The difference from ordinary preventive vaccines is that therapeutic vaccines are aimed at self-antigens or foreign immune tolerance antigens. Therefore, in order to play a therapeutic role, therapeutic vaccines must first break immune tolerance and produce autoantibodies or self-reactions against specific antigens. T cells. Since the target antigens of therapeutic vaccines are generally short peptides with weak antigenicity, they must be combined with a suita...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/04C12N7/02C12N15/11A61K48/00
Inventor 陈霄邱志华杨仕俊陈芬廖玉华周子华王敏
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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