Method for preparing DNA vaccine with herpes simplex virus type I UL5 gene deletion

A herpes simplex virus and DNA vaccine technology, applied in the field of biomedicine, can solve the problems of complex preparation methods of attenuated live vaccines, difficulties in PCR amplification, and low probability of success, etc., and achieve virus titer and infectivity weakened, high Immunogenicity, efficiency-enhancing effect

Active Publication Date: 2016-05-11
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 The efficiency of integrating the source recombination frame into the correct position of the gene to be knocked out is low, and the probability of success is low; (3) The fluorescent protein gene is used to replace the gene of the virus to be knocked out for screening and identification. Although this method is fast and simple, it failed in the end. The fluorescent protein gene is removed, and the constructed attenuated live vaccine still expresses the protein
[0005] Therefore, the prior art has disadvantages such as complex preparation methods of live attenuated vaccines and low success efficiency.

Method used

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  • Method for preparing DNA vaccine with herpes simplex virus type I UL5 gene deletion
  • Method for preparing DNA vaccine with herpes simplex virus type I UL5 gene deletion
  • Method for preparing DNA vaccine with herpes simplex virus type I UL5 gene deletion

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Experimental program
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Embodiment 1

[0040] The construction of the eukaryotic cell line of embodiment 1UL5 gene expression vector

[0041] Using the BAC-17.37 shuttle plasmid as a template, design primers (SEQIDNO.5, SEQIDNO.6) to amplify the UL5 gene (2696bp), and subclone the gene fragment into pCDNA3.1 after digestion with BamHI and BstXI restriction endonucleases (+) Plasmid (purchased from Youbao Biological Company, Cat. No. VT1001) to obtain pCDNA3.1-UL5 plasmid;

[0042] The Cre gene fragment was amplified by reverse transcription of the P1 phage RNA, the pCDNA3.1-UL5 plasmid and the Cre gene fragment were digested with BamHI and ligated with T4 ligase, and a single clone was selected for Cre gene and UL5 gene PCR identification and sequencing, the results are as follows figure 1 and figure 2 As shown, the eukaryotic expression pCDNA3.1-UL5-CRE plasmid was obtained;

[0043] The pCDNA3.1-UL5-CRE plasmid was transfected into Vero cells (purchased from ATCC, USA) using lipo2000 liposomes (purchased from...

Embodiment 2

[0045] The homologous recombination knockout of embodiment 2HSV-1UL5 gene

[0046] 2.1 Construction of pREDI recombinant plasmid

[0047] Design the PCR amplification primers of the promoter PrhaB, and obtain the PrhaB fragment (2.0kb) through PCR amplification; design the PCR amplification primers of I-SceI, and obtain the I-SecI fragment (0.7kb) through PCR amplification; The I-SecI fragment is connected by recombinant PCR to obtain the PrhaB-I-SecI fragment; the PrhaB-I-SecI fragment is connected to the pKD46λ-redrecombinase vector to obtain the pREDI recombinant plasmid. The pREDI recombinant plasmid has λ-redrecombinase induced by L-arabinose and I-SecI endonuclease gene induced by rhamnose.

[0048] 2.2 Construction of recombinant knockout system

[0049] The constructed pREDI recombinant plasmid was transformed into a host bacterium containing the BAC-HSV-1 vector, and the host bacterium was EPI300E.Coli to obtain a recombinant knockout system for homologous recombina...

Embodiment 3B

[0056] The excision of embodiment 3BAC carrier and the acquisition of HSV-1UL5 gene-deficient strain

[0057] The recombinant strain without UL5 gene and foreign gene was expanded and cultivated, and the BAC mass extraction kit was used for plasmid extraction to obtain the BAC-17.37ΔUL5 plasmid, which was electrotransformed into Vero-pCDNA3.1 constructed in Example 1 - UL5-Cre cells were cultured in DMEM medium containing 2% by volume of fetal bovine serum and 1ug / ml of G418. 24 hours before the occurrence of cell lesions, the cell culture medium was removed, and then cultured with 1% agar Sugar and 2% FBS were covered with DMEM medium. When visible pathological effects were formed, phosphate buffer containing 400ug / ml X-gal (purchased from BioSynth AG, Switzerland) was added, and then the white spots were selected by the blue-white spot test, repeatedly frozen and thawed, and centrifuged The supernatant was used to obtain the HSV-1 UL5 gene-deleted DNA vaccine.

[0058] Infe...

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Abstract

The invention provides a method for preparing a DNA vaccine with herpes simplex virus type I (HSV-1) UL5 gene deletion. The method includes the following steps: S1, carrying out homologous recombination knockout of a herpes simplex virus type I UL5 gene, to obtain a recombinant strain containing no UL5 gene and no exogenous gene, wherein a homologous-recombination-knockout recombination knockout system is a host strain containing a BAC-HSV-1 vector and a pREDI recombinant plasmid; and S2, cutting a BAC vector and obtaining the DNA vaccine with HSV-1 UL5 gene deletion. The invention belongs to the technical field of biological medicine; the attenuated live vaccine can be prepared by the method provided by the invention, is safe and effective and less prone to virulence reversion, and can effectively prevent and treat HSV-1 infection.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method of a herpes simplex virus type I UL5 gene-deleted DNA vaccine. Background technique [0002] Herpes simplex virus (HSV) belongs to the Herpesviridae family and the Alphaherpesvirinae family. HSV virus is a double-stranded DNA virus, which contains four viral structures including envelope, cortex, capsid and genome from the outside to the inside. HSV-1 is a kind of herpes simplex virus, which mainly causes infections of the throat, mouth, eyes, face and nervous system, and can cause herpetic encephalitis, cold sores and other diseases. According to survey results, more than 90% of people will be infected with herpes virus in their lifetime, and herpes simplex virus infection has become the fourth largest infectious disease in the world. [0003] The proliferation of HSV virus in the cell is an orderly and complex process. The intracellular pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/245C12N15/63
CPCA61K39/12A61K2039/53C12N15/63C12N2710/16034
Inventor 王一飞任哲程江王巧利廖晓凤
Owner JINAN UNIVERSITY
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