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Preventive recombinant VLP (virus-like particle) vaccine for HIV-1 (human immunodeficiency virus) B/C subtype and preparation method thereof

A HIV-1B, preventive technology, applied in the field of medical biology, can solve complex and difficult problems, achieve simple process, ensure safety, and help induce protective immune response

Inactive Publication Date: 2015-09-16
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unlike non-enveloped viruses, HIV-1 is an enveloped virus, and its surface antigen that can induce neutralizing antibodies is a membrane protein, and each structural protein is sheared, packaged into virus-like particles, budding, and secreted outside the cell It is particularly complicated, so it is very difficult to obtain a VLP with an envelope through in vitro expression

Method used

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  • Preventive recombinant VLP (virus-like particle) vaccine for HIV-1 (human immunodeficiency virus) B/C subtype and preparation method thereof
  • Preventive recombinant VLP (virus-like particle) vaccine for HIV-1 (human immunodeficiency virus) B/C subtype and preparation method thereof
  • Preventive recombinant VLP (virus-like particle) vaccine for HIV-1 (human immunodeficiency virus) B/C subtype and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: the establishment of the recombinant MVA virus working seed bank of coding HIV-1B / C subtype Gag / Pol, Env protein

[0023]Using genetic engineering technology, the HIV-1B / C subtype Gag / Pol and Env genes were respectively fused to the internal ribozyme entry site sequence (IRES) gene of encephalomyocarditis virus (ECMV) (Genbank No. .: JB759789) at the 5' and 3' ends to construct the polycistronic gene expression element of HIV-1B / C subtype Gag / Pol-IRES-Env, and then insert this element into the modified Ankara poxvirus (Modified vaccinia virus Ankara, MVA) shuttle vector pSC11 plasmid (see below for references), after homologous recombination and plaque purification, recombinant MVA viruses encoding HIV-1B / C subtype Gag / Pol and Env proteins were obtained (refer to Caroline Staib, Gerd Sutter. Live viral vectors: Vaccinia virus [M] In: Vaccine Protocols, 2nd edition. Edited by Andrew Robinson, Martin Cranage, Michael J. Hudson. Humana Press Inc.; 2003:51-68)...

Embodiment 2

[0024] Embodiment 2: the titration of the recombinant MVA virus working seed bank of encoding HIV-1B / C subtype Gag / Pol, Env protein

[0025] Since MVA is a highly attenuated strain with limited virulence and weak cytopathic effect (CPE), virus titration is usually performed in combination with immunological methods. Taking a 96-well plate as an example, after taking out the virus seed, perform 10-fold serial dilution in MEM complete medium containing 2% FBS, and infect BHK-21 cells that have been cultured for 24 hours and have a cell confluence of 80-90%. From American type culture collection, ATCC, ATCC No.: CCL-10) 100 μl per well, set up duplicate wells and negative controls. After 48 hours of infection, the culture supernatant was discarded, and the fixed solution mixed with acetone and methanol at 1:1 was used to fix the cells at room temperature for 2 minutes, and the vaccinia virus (Vaccinia virus, VV) rabbit polyclonal antibody (purchased from Bio- Rad Company, Cat.No...

Embodiment 3

[0026] Embodiment 3: Expression of HIV-1B / C subtype VLP

[0027] Primary CEF cells were prepared, and after the cells grew to a confluence of more than 90%, they were infected with recombinant MVA viruses encoding HIV-1B / C subtype Gag / Pol and Env proteins at different multiplicity of infection (MOI) Primary CEF cells, comparing different MOI (10 -2 、10 -3 、10 -4 ) and FBS concentrations (0%, 2%) in the culture supernatant of the difference in the expression of VLP. After 48 hours of infection, collect the culture supernatant; replace the fresh medium, continue to cultivate for 48 hours, and collect the culture supernatant; the supernatants collected twice were centrifuged at low speed to remove cell residues; the SW28 rotor (purchased from Beckman, Model: Optima XE90) , 20% sucrose cushion layer, 28000rpm, 4°C ultracentrifugation for 2h, discard the supernatant, and resuspend the ultracentrifugation pellet with 400μl PBS (pH7.2); Antibodies (purchased from Abcam, Cat. No.:...

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Abstract

The invention discloses a preventive recombinant VLP (virus-like particle) vaccine for HIV-1 (human immunodeficiency virus) B / C subtype and a preparation method thereof. The preparation method comprises the following steps: infecting primary chicken embryo fibroblast with a recombinant MVA virus encoding HIV-1 B / C subtype Gag / Pol and Env proteins; expressing Gag, Pol and Env proteins; and packaging and secreting virus-like particles which contain HIV-1 B / C subtype Gag and Env proteins and are similar to a natural HIV-1 virus in structure. According to the vaccine disclosed by the invention, the natural structure of an HIV-1 virus antigen is simulated to the greatest extent; induction of protective immune response is facilitated; the vaccine does not contain an HIV gene and therefore the safety is ensured; the VLP can be obtained in a large quantity by utilizing recombinant MVA infection and therefore the process is simple; and the preventive recombinant VLP vaccine can be applied to prevention of related diseases caused by HIV-1 B / C subtype infection.

Description

technical field [0001] The invention belongs to the field of medical biotechnology. Specifically, the present invention relates to a prophylactic recombinant VLP vaccine for HIV-1B / C subtype and a preparation method. Background technique [0002] AIDS, also known as Acquired Immune Deficiency Syndrome (AIDS), is a global disease caused by HIV (Human Immunodeficiency Virus), with rapid spread and high mortality. Since AIDS was first discovered in 1981, HIV infection has spread rapidly around the world. By the end of 2002, at least 193 countries and regions in the world had HIV-infected deaths, and it was still rapidly expanding at a rate of 15,000 people infected every day, of which more than 95% of HIV-infected people lived in developing countries, which has caused a global cumulative More than 60 million people were infected and more than 20 million people died of AIDS. AIDS is the leading cause of death in Africa and the fourth leading cause of death in the world. The n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/21A61P31/18
Inventor 王涛于晓方庞正袁文肃武晓丽杨英
Owner TIANJIN UNIV