Recombined attenuated live vaccine for preventing and treating I-type infection of herpes simplex virus and preparation method thereof

A herpes simplex virus and attenuated live vaccine technology, applied in the field of biomedicine, can solve the problems of difficult to obtain pathogenicity, time-consuming and laborious, etc., and achieve the effects of difficult virulence recovery, reduction of morbidity, and control of recurrence and transmission

Inactive Publication Date: 2013-03-20
樊科伟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditional live attenuated vaccines are mainly obtained through continuous culture and screening, but this method is time-consuming and laborious, and it is difficult to obtain herpes simplex virus strains with reduced pathogenicity by this method

Method used

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  • Recombined attenuated live vaccine for preventing and treating I-type infection of herpes simplex virus and preparation method thereof
  • Recombined attenuated live vaccine for preventing and treating I-type infection of herpes simplex virus and preparation method thereof
  • Recombined attenuated live vaccine for preventing and treating I-type infection of herpes simplex virus and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of recombinant herpes simplex virus attenuated live vaccine JSH01L

[0024] 1. Isolation of wild-type HSV-1

[0025] (1) Take the herpes fluid and secretions of patients with clinical HSV infection, add 1ml of PBS buffer solution and filter with a 0.45 micron filter.

[0026] (2) Take a six-hole plate, pass 2×10 per hole 5 For each Vero cell, 200 microliters of virus filtrate was added to each well of 5 wells, and one well was reserved as a control.

[0027] (3) Put it into a 37°C carbon dioxide incubator and culture it for 48-72 hours, then observe the cytopathic effect (CPE) in the well.

[0028] (4) After the cells in the wells of the six-well plate have complete CPE, put the medium and the cells into the centrifuge tube together.

[0029] (5) Freeze and thaw the centrifuge tube three times at 37°C-minus 80°C, centrifuge at 5000 rpm for 10 minutes, transfer the supernatant into a 2ml EP tube, and put it in a minus 80°C refrigerator for later...

Embodiment 2

[0076] The pathogenicity of embodiment 2 recombinant herpes simplex virus attenuated live vaccine JSH01L reduces

[0077] 1. Experimental materials:

[0078] Wild-type herpes simplex virus type I: the titer is measured after being amplified with Vero cells, and the titer is about 1×10 9 IU / ml;

[0079] Recombinant live attenuated herpes simplex virus vaccine JSH01L: the titer after amplification with Vero cells is about 1×10 9 IU / ml;

[0080] Ordinary level experimental mice: 30, female, weighing 20 ± 2g, each of which is marked on different parts of its body, and the corresponding marks are converted into a total of 30 Arabic numerals from 1 to 30, and divided according to the principle of randomization 10 rats / group were divided into three groups: control group, normal dose group and high dose group.

[0081] 2. Experimental operation:

[0082] The control group was given 20 microliters of wild-type herpes simplex virus type I via nasal infusion, the normal dose group w...

Embodiment 3

[0084] Embodiment 3 Live vaccine JSH01L is to the immune protection experiment of mouse

[0085] 1. Experimental materials:

[0086] Wild-type herpes simplex virus type I: the titer is measured after amplifying HSV-1 with Vero cells, and the titer is about 1×10 9 IU / ml;

[0087] Recombinant live attenuated herpes simplex virus vaccine JSH01L: the titer after amplification with Vero cells is about 1×10 9 IU / ml;

[0088] Normal cell lysate: normal cultured Vero cells in the petri dish were scraped off and then filtered after repeated freezing and thawing at -80°C and 37°C.

[0089] Ordinary level experimental mice: 30, female, weighing 20 ± 2g, each of which is marked on different parts of its body, and the corresponding marks are converted into a total of 30 Arabic numerals from 1 to 30, and divided according to the principle of randomization 15 rats / group were divided into negative group and positive group.

[0090] 2. Immunization and attack plan

[0091] Immunity dose:...

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Abstract

The invention provides a recombined attenuated live vaccine for preventing and treating I-type or II-type infection of herpes simplex virus and preparation method thereof. The recombined attenuated live vaccine is characterized by omitting the UL43, UL44, UL45, UL46 and UL47 genes in the genome thereof; the method comprises the steps of: separating and identifying a wild-type herpes simplex virus; carrying out enlarged cultivation, extracting the complete genome of the virus, and designing a primer to amplify a homologous flanking sequence formulating the omitted gene segment; cloning the homologous flanking sequence and a fluorescent protein gene to a pShuttle-CMV carrier to carry out homologous recombination; selecting the recombined virus by taking the fluorescent protein gene as a mark; and carrying out plaque purification to obtain the gene-recombined attenuated live vaccine of the herpes simplex virus. The attenuated live vaccine prepared by the invention undergoes gene engineering recombination so that toxicity returning is not easy to happen, and the other biological characteristics of the attenuated live vaccine are similar to those of the wild-type herpes simplex virus.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a gene recombinant attenuated live vaccine capable of preventing and treating diseases caused by herpes simplex virus type I infection and a preparation method thereof. Background technique [0002] Herpes simplex virus (HSV) is a virus transmitted through close contact with skin and mucous membranes, including herpes simplex virus type I (abbreviated as HSV-1) and herpes simplex virus type II (abbreviated as HSV-2). Viruses have more than 83% common gene characteristics and more than 50% gene homology. Wherein the infection rate of type I can be as high as more than 80% in the crowd, and long-term dormancy in the sensory ganglion, although most HSV-1 infected persons have no obvious symptoms, but a few infected persons can produce lifelong repeated outbreaks of facial herpes, Highly blinding intraophthalmitis and fatal herpes pneumonia and herpes encephalitis. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/245C12N7/04C12N15/38A61P31/22C12R1/93
Inventor 刘景伟
Owner 樊科伟
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