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Herpes simplex virus I-type gene recombinant attenuated live vaccine 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: 2012-09-12
郑州金森生物科技工程有限公司
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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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  • Herpes simplex virus I-type gene recombinant attenuated live vaccine and preparation method thereof
  • Herpes simplex virus I-type gene recombinant attenuated live vaccine and preparation method thereof
  • Herpes simplex virus I-type gene recombinant attenuated live vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 Construction of recombinant herpes simplex virus attenuated live vaccine JSH01S

[0024] 1. Construct the homologous recombination shuttle vector pShuttle-01S-GFP.

[0025] (1) Referring to the sequence of the US region US2-US5 of the HSV-1 genome with the accession number NC_001806 on NCBI, design the amplification primers for the left flank sequence of the US2 gene and the right flank sequence of the US5 gene, respectively.

[0026] (2) Primer design for US2 flanking sequences

[0027] A NotI site and three protective bases were added to the 5' end of the upstream primer of the US2 left flank sequence, and a PmeI, AseI site and three protective bases were added to the 5' end of the downstream primer of the US2 left flank sequence. After adding 30 cycles, the target band was recovered.

[0028] (3) Primer design for US5 flanking sequence

[0029] Add PmeI and MluI sites and three protective bases to the 5' end of the upstream primer of the US5 right fla...

Embodiment 2

[0040] Example 2 Replication and infection ability of recombinant herpes simplex virus attenuated live vaccine JSH01S is reduced

[0041] 1. Experimental materials: wild-type HSV-1, JSH01S, Vero cells, six-well plate.

[0042] 2. Experimental method: 5×10 5 Vero cells were cultured for 24 hours. According to the multiplicity of infection (MOI) of 0.1 and 1, four wells of the six-well plate were infected with wild-type HSV-1 and JSH01S respectively, and the remaining two wells were used as controls to observe the cytopathic effect of the virus-infected wells. Condition.

[0043] 3. Results: It is expected that wild-type HSV-1 will completely produce CPE in about 48 hours to 72 hours, while JSH01S can produce complete CPE in about 6 to 7 days, indicating that the recombinant strain JSH01S has the ability to replicate and infect compared with wild-type HSV-1 obviously decased.

Embodiment 3

[0044] The pathogenicity of embodiment 3 recombinant herpes simplex virus attenuated live vaccine JSH01S reduces

[0045] 1. Experimental method is identical with the method used in embodiment 2, gives control group wild-type herpes simplex virus type 1 20 microliters through nasal cavity infusion route, and normal dose group gives 20 microliters of JSH01S, and high dose group gives 100 microliters of JSH01S, observes mouse condition.

[0046] 2. Results: It is expected that the mice in the control group will begin to die on the 5th day after nasal instillation, and all die after the 13th day, and the mortality rate is 100%; the mice in the normal dose group and the high dose group are all alive, and the mortality rate is 0, It shows that compared with the wild type, JSH01S has almost no pathogenicity.

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Abstract

The invention discloses a herpes simplex virus I-type gene recombinant attenuated live vaccine and a preparation method thereof. The attenuated live vaccine is characterized in that: US2, US3, US4 and US5 genes in a genome and other duplicated or infected nonessential gene fragments are jointly knocked out, wherein other duplicated or infected nonessential gene fragments are US9-US12 genes. The method comprises the following steps: separating and identifying wild HSV-1 (herpes simplex virus-1) from blister fluid of a patient with remarkable herpes labialis and herpes progenitalis; propagating and extracting a complete genome of virus; co-transfecting a Vero cell with a shuttle vector containing homologous flanking sequences of 700bp to 2000bp on the left and right sides of a fluorescent expression gene and the HSV gene to be knocked off; and selecting the recombinant virus by using one or combination of multiple fluorescent protein genes serving as a marker under fluorescent microscope, and acquiring the required recombinant attenuated live vaccine after plaque purification.

Description

1. 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. 2. Background technology [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 encephal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/245C12N7/04C12N15/38A61P31/22C12R1/93
Inventor 刘景伟
Owner 郑州金森生物科技工程有限公司
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