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Herpes simplex virus I gene recombinant attenuated live vaccine and preparation method thereof

A technology of herpes simplex virus and live attenuated vaccine, which is applied in the field of biomedicine, can solve the problems of complex preparation method of live attenuated vaccine, difficult PCR amplification, low success probability, etc., and achieves improved homologous recombination efficiency and high immunity. Original, targeted effects

Inactive Publication Date: 2016-06-08
JINAN UNIVERSITY
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Problems solved by technology

The attenuated live vaccine knocks out non-essential gene fragments for replication or infection in its genome, can induce the body's immune response system, remove infected viruses in time, and inhibit the further development of infection, but its preparation method has many shortcomings: (1) Select the homologous flanking sequence of 700bp to 2000bp upstream and downstream of the gene sequence to be knocked out and the fluorescent protein gene to form a homologous recombination frame, because the target fragment is too long to make PCR amplification difficult; (2) Select the pShuttle-CMV vector, and

Method used

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  • Herpes simplex virus I gene recombinant attenuated live vaccine and preparation method thereof
  • Herpes simplex virus I gene recombinant attenuated live vaccine and preparation method thereof
  • Herpes simplex virus I gene recombinant attenuated live vaccine and preparation method thereof

Examples

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[0039] Example 1: Preliminary screening of HSV-1 attenuating genes

[0040] Using SiRNA interference technology, the effects of UL5, UL39, UL40, UL41, US6, US8, US12, UL18, UL39, ICP8, ICP34.51 and ICP34.52 genes of HSV-1 on virus proliferation and infectivity were studied. The attenuated genes that were knocked out were screened initially, and the results are shown in Table 1. The PCR primer sequences for the knockout genes UL5, US8, UL41 and UL18 are shown in SEQ ID NO. 1 to SEQ ID NO. 16, respectively, and the primer sequences for other knockout genes are not listed here. The enzyme used for the two PCR amplifications was PrimeSTARMaxDNAPolymerase (TAKARA, R045A), and the reaction system was as follows: PrimeSTARMaxPremix (2×) 25μL, upstream primer (10μM) 1.5μL, downstream primer (10μM) 1.5μL, template 2 0.25μL, add deionized water to 50μL. The reaction conditions are: 98°C for 10s, 56°C for 5s, 72°C for 5s / Kb, a total of 30 cycles, and then 72°C for 5 minutes and 4°C heat pr...

Example Embodiment

[0043] Example 2 Construction of HSV-1UL18 gene knockout virus strain

[0044] The HSV-1UL18 gene knockout virus strain was successfully constructed using the gene knockout system of the present invention. The preparation method includes:

[0045] Construction of S1, BAC-HSV-1 host strain

[0046] S1.1 Expanded culture of HSV-1 virus: culture vero cells, wash the cells with PBS, add HSV-1 virus (purchased from the American Collection of Types of Microorganisms) into the cell culture flask to ensure that the virus solution completely covers the cell surface , Put the cell culture flask with virus liquid at 37℃, 5% CO 2 Incubate in the incubator for 1.5 hours, shaking every 15 minutes during the incubation; after incubation, add DMEM maintenance solution to the cell culture flask, and put it in the cell culture incubator to continue culturing to observe the cytopathic condition. When the cytopathic rate reaches 70-80%, the virus can be collected; the amplified virus is placed at -80℃...

Example Embodiment

[0057] Example 3 Construction of HSV-1US8 gene knockout virus strain

[0058] Similar to Example 2, the HSV-1US8 gene knockout virus strain was constructed using the BAC gene knockout system.

[0059] Use primers (shown in SEQIDNO.5 to SEQIDNO.8) to PCR amplify the homologous recombination frame of the knockout gene US8. The homologous recombination frame includes the 50bp homologous sequence upstream and downstream of the target gene to be knocked out, kanamycine gene and SacB Gene, so that the kanamycine gene and SacB gene are located between the homologous flanking sequences. The electropherogram of the PCR amplified △US8 gene knockout homologous recombination frame is as follows Figure 7 Shown, where, Figure 7 -A refers to the verification electropherogram of the kanamycine+SacB recombinant fragment, Figure 7 -B refers to the verification electropherogram of the homologous fragment of the downstream homologous sequence of the kanamycine+SacB+ knockout gene US8, Figure 7 -C ...

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Abstract

The invention provides a herpes simplex virus I gene recombinant attenuated live vaccine, and belongs to the technical field of biomedicines. The herpes simplex virus I gene recombinant attenuated live vaccine includes a herpes simplex virus I gene knockout virus strain, and the gene knockout virus strain knocks out at least one of UL5, UL18, UL40, UL41, US8, ICP8, ICP34.51 and ICP34.52 genes of a herpes simplex virus I genome. The attenuated live vaccine is prepared through knocking out a specific gene. The attenuated live vaccine has the advantages of safety, effectiveness, difficult toxicity recovery, and effective prevention and treatment of HSV-1 infection.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a herpes simplex virus type I gene recombinant attenuated live vaccine and a preparation method thereof. Background technique [0002] Herpes simplex virus (HSV) is a double-stranded enveloped DNA virus with human beings as its only natural host, and its serology is divided into HSV-I and HSV-2 types. HSV-I mainly infects the skin and mucous membranes of the mouth, eyes, and lips, the central nervous system, and external genitalia, causing diseases such as gingivostomatitis, cold sores, keratoconjunctivitis, herpetic encephalitis, meningitis, and genital herpes; HSV-2 mainly Causes genital herpes; herpes virus is a serious threat to human health. At present, antidrugs such as herpesnet, vidarabine, acyclovir, and famloxivir are often used clinically for treatment. Although they can relieve clinical symptoms, they cannot completely remove the virus dormant in the ...

Claims

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Application Information

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IPC IPC(8): A61K39/245A61P31/20C12N15/63C12N7/04
CPCA61K39/12A61K2039/5254A61K2039/53C12N7/00C12N2710/16634C12N2710/16662
Inventor 王一飞任哲罗帆王巧利廖晓凤
Owner JINAN UNIVERSITY
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