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Virus-like particles for pseudorabies virus and preparation method for same

A rabies virus and virus-like technology, which is applied to the purification and concentration methods and identification fields of the virus-like particles, can solve the problems that there are no research institutions and publish rabies virus-like particles, and achieve simple and rapid construction, convenient and rapid design and manufacture Effect

Active Publication Date: 2012-07-04
WUHAN INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no research institution has published relevant studies on the recombinant expression of rabies virus-like particles in vitro

Method used

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  • Virus-like particles for pseudorabies virus and preparation method for same
  • Virus-like particles for pseudorabies virus and preparation method for same
  • Virus-like particles for pseudorabies virus and preparation method for same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The acquisition and cloning of embodiment 1 recombinant rabies virus M protein and G protein:

[0044] 1. Rabies virus CVS strain RNA extraction

[0045] 1) Pre-prepared reagents:

[0046] a) Preparation of buffer AVE containing carrier RNA: 310 μl of buffer AVE was added to a small tube containing 310 μg of carrier RNA to obtain a solution with a concentration of 1 μg / μl.

[0047] b) Preparation of buffer AW1: 25 ml of ethanol (96-100%) was added to unused buffer AW1 (19 ml) to make its final volume 44 ml.

[0048] c) Preparation of buffer AW2: add 30ml of ethanol (96-100%) to the unused buffer AW2 (13ml) to make its final volume reach 43ml;

[0049] 2) Take a 1.5ml Ep tube, add 554.4μl buffer AVL and 5.6μl carrier RNA-containing buffer AVE, and mix well.

[0050] 3) Add 150 μl of CVS virus solution to the Ep tube, shake and mix for 15 seconds, and let stand at room temperature for 10 minutes.

[0051] 4) Briefly centrifuge to remove water droplets hanging on the w...

Embodiment 2

[0164] Embodiment 2 utilizes recombinant bacmid pFD-MG to prepare recombinant virus rV_MG

[0165] 1. Transform DH10Bac highly competent cells with the recombinant plasmid pFDual_MG

[0166] 1) Equilibrate the LB plate (containing gentamicin, tetracycline, kanamycin, IPTG and X-GAL) and SOC medium to room temperature.

[0167] 2) Take the frozen DH10Bac highly competent cells out of the -70°C refrigerator, place them on ice until they melt, and shake the centrifuge tube gently to mix them evenly. And take two 1.5ml Ep tubes and put them on ice.

[0168] 3) Add 50 μl of highly competent DH10Bac cells to each Ep tube in step 2. Add the recombinant plasmid to one of the tubes, and add the control pUC19DNA to the other tube.

[0169] 4) Gently vortex the Ep tube to mix well, and put it in an ice bath for 30 minutes.

[0170] 5) Heat shock for 60s in a precise 42°C water bath (do not shake).

[0171] 6) Quickly move the tube to an ice bath to cool the cells for 2 min.

[0172...

Embodiment 3

[0213] Example 3 Preparation of rabies virus-like particles and identification of rabies virus-like particles by recombinant baculovirus rV_MG infecting cells with expression products

[0214] 1. Preparation of rabies virus-like particles

[0215] 1) Inoculate 4 flasks of Sf9 cells with a density of about 80% with the recombinant baculovirus rV_MG at MOI=5, place them in an incubator at 28° C., and collect the supernatant for 144 hours to concentrate.

[0216] 2) Place the cell culture supernatant in a 50ml centrifuge tube and centrifuge at 1000g for 10min, take the supernatant in an ultrafiltration centrifuge tube,

[0217] 3) Centrifuge the ultrafiltration centrifuge tube at 4°C at 4,000rpm for 1h, concentrate the supernatant and add sterile PBS to 1ml;

[0218] 4) Take 1ml of sucrose with different densities to prepare a continuous gradient of sucrose from high to low, the sucrose concentrations are 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% and 60% respectively;

[0219] 4) ...

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Abstract

The invention provides virus-like particles for pseudorabies virus. The virus-like particles consist of pseudorabies virus glycoprotein G and pseudorabies virus matrix protein M, and have stable and homogenous spatial structures; and the outer membrane protein of the virus-like particles contains all or partial fragments of the pseudorabies virus glycoprotein G, and can induce an organism to generate protective-level cellular immunity and humoral immunity. The virus-like particles do not comprise any nucleic acid component of chromosome of the pseudorabies virus, and avoid quality risks caused by inactivator addition. The virus-like particles can be conveniently purified by the conventional purification technology, and the quality risks possibly caused by inactivator addition in the traditional inactivated vaccines are avoided on principle. Meanwhile, due to a simple and quick construction and preparation mode, novel vaccines aiming at new strains can be conveniently and quickly designed and prepared. Based on the principle, novel polyvalent vaccines and multi-vaccines are easily formed on the basis of the particles; and the particles can widely replace related critical raw materials in the conventional practical technologies such as related antigen and antibody detection, functional protein vectors and the like.

Description

technical field [0001] This patent relates to one or more rabies virus proteins, which are expressed by in vitro recombination or other methods to form virus-like particles with a certain spatial structure. The particle has the important antigenic characteristics of rabies virus, and can produce enough ability to resist virus attack by inducing T cell immunity and B cell immunity. The safety of the particle and the convenience of strain construction are superior to the whole cell inactivated rabies vaccine currently in use. The particles can be widely used in the development of related multi-linked multivalent vaccines, in vitro detection of rabies virus corresponding antigen antibodies, foreign protein carriers and the like. The patent also relates to a method for purifying and concentrating the virus-like particles and an identification method. Background technique [0002] Virus-like particles (Virus-Like Particles, VLPs) are hollow particles containing one or more stru...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/47C12N15/866C12N7/01A61K39/205A61P31/14
Inventor 杨晓明黄晓媛刘雄施金蓉夏志武邱阿明喻刚胡迪超吴杰段凯
Owner WUHAN INST OF BIOLOGICAL PROD CO LTD
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