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Recombinant HSV (Herpes Simplex Virus) amplicon vector and application thereof

A technology of amplicon and carrier, which is applied in the field of construction of HSV amplicon carrier, can solve the problems of cumbersome operation, low concentration of small circle amplicon DNA, and high cost, and achieve simple construction and preparation, avoid silencing effect, The effect of avoiding pollution

Active Publication Date: 2014-04-09
ZHENGZHOU VIRIGE BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is contamination of auxiliary plasmids in the preparation process, and the prepared MC amplicon needs further purification to remove the mixed pBADφC31RHB and pBADφC31 plasmids. It is expensive and cannot be produced on a large scale. At the same time, the produced small circle amplicon DNA needs to be transfected into eukaryotic cells with a non-viral vector before it can be packaged into an amplicon virus vector, so it has no practical application value;
[0012] 3. Neither the traditional plasmid-type HSV amplicon vector nor the MC small circle HSV amplicon vector can achieve large-scale production of HSV for in vivo anti-HSV virus infection and related disease treatment without helper virus contamination in terms of preparation technology Amplicon Gene Therapy Vectors

Method used

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  • Recombinant HSV (Herpes Simplex Virus) amplicon vector and application thereof
  • Recombinant HSV (Herpes Simplex Virus) amplicon vector and application thereof
  • Recombinant HSV (Herpes Simplex Virus) amplicon vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The construction of embodiment 1, BAC-HSV-1 HF strain

[0062] Such as figure 1 Construction patterns of the indicated BAC-HSV-1 HF strains.

[0063] Using the genome of the HSV-1 HF strain as a template, primers SEQ ID NO: 4 and SEQ ID NO: 5: amplify the upstream homology arm homologous to the UL43 gene of HSV-1, and the two ends of the primers are designed with SacI and MluI enzyme cut linker;

[0064] With primers SEQ ID NO: 6 and SEQ ID NO: 7, amplify the downstream homology arms homologous to the UL47 gene sequence of HSV-1, the enzyme-cut joints of NotI and MluI are designed respectively at the two ends of the primers, and the enzyme-cut ligation method will The homology arm was cloned into the SacI and NotI restriction sites of the BAC plasmid, and the BAC-LR arm was constructed. The BAC containing the HSV-1 homology arm was cut into linear with MluI enzyme, and the linear BAC-HSV-1 homology The source arm and HSV-1 genome were transfected into Vero cells b...

Embodiment 2

[0066] The construction of embodiment 2, HSV-1 amplicon plasmid vector

[0067] Such as image 3 HSV-1 amplicon plasmid vector construction model shown.

[0068] 1. Acquisition of "pac" sequence

[0069] Digest BAC-HSV-1HF with MluI enzyme to obtain BAC-TR containing the terminal repeat region; digest BAC-TR with SacI to obtain a 4Kb fragment containing the "pac" sequence of the HSV-1 terminal repeat region; after digesting the 4Kb fragment with HphI A 1.3Kb fragment containing the "pac" sequence of the terminal repeat region was obtained. In addition, the 1.3Kb fragment can be digested with BsrBI to obtain the 188bp Ub-DR1-Uc structure (the packaging signal of the smallest packaging unit). EcoRI digested pGEMT linear T vector (purchased from Dalian Bao Biological Co., Ltd.), Klenow enzyme (purchased from Dalian Bao Biological Co., Ltd.) filled the pGEMT linear T vector after EcoRI digestion, and added the 188bp fragment into pGEMT with a blunt-ended end to T7 univers...

Embodiment 3

[0079] Embodiment 3, construction of recombinant adenovirus Adv-loxP-OPD-loxP and Adv-loxP-D-loxP and virus packaging

[0080] Such as Figure 7 Construction mode of Adv-loxP-OPD-loxP shown.

[0081] 1. Construction of pENTR-loxP-LRarm-loxP vector

[0082] pENTR-MCS was digested with SalI and BamHI, and the 2.6kb vector pENTR-MCS was recovered from the gel. Cut out the two loxPs in the same direction, the LR homology arm and the fragment of the GFP reporter gene in the middle, and fill in the fragments. The vector and the fragment were connected, transformed, identified as the pENTR-loxP-LRarm-loxP vector, and identified by AvaII digestion. Theoretically, the sizes should be: 3.1Kb, 1.4Kb, 1.2Kb, 440bp, 282bp, and 153bp. The electrophoresis results are shown by the arrows in Figure 8-1A. It can be seen that 3.1Kb, 1.4Kb, 1.2Kb, 440bp, 282bp, and 153bp band. The results showed that the pENTR-loxP-LRarm-loxP vector was constructed successfully.

[0083] 2. Construction o...

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Abstract

The invention relates to a recombinant HSV (Herpes Simplex Virus) amplicon vector and application thereof. By using two replication deficient adenoviruses respectively carrying Cre and an operative linked component loxP-HSVoriS-pac-transgenic expression box-loxP, a novel HSV amplicon vector is recombined in a coinfection cell. Being different from the traditional HSV amplicon vector taking bacterial plasmids as a skeleton, the amplicon vector does not contain a bacteria copying sequence (colEorigin) and a resistance gene component and only contains oriS of HSV, a pac sequence and a transgenic expression box. The recombinant HSV amplicon vector disclosed by the invention is used for preparing a novel HSV amplicon vector, which does not contain the bacterial gene component and is used for various types of transgenic researches and tumour gene treatments, and preparing an adenovirus treatment preparation for specific anti-HSV virus and related diseases. The HSV amplicon vector recombined by the replication deficient adenovirus of the preparation in cells replicates itself by using infected wild HSV virus and competitively inhibits or permanently expresses the antiviral genes so as to inhibit replication of wild HSV virus. Therefore, the recombinant HSV amplicon vector can be used for resisting HSV infection and treating related diseases thereof.

Description

technical field [0001] The invention belongs to the field of biotechnology, more specifically, the invention relates to the construction and application of a recombinant HSV amplicon vector. Background technique [0002] Herpes simplex virus (HSV) is a DNA virus belonging to the subfamily Aviridae of the family Herpesviridae and is divided into two serotypes, HSV-1 and HSV-2. The gene structure of herpes simplex virus (HSV-1) is a double-stranded linear DNA of 152kb, which forms two segments connected by a long segment UL and a short segment US. The HSV genome contains 89 coding genes expressed sequentially in the early (IE), early (E) and late (L) sequences, 3 cis-acting elements (1 oriL and 2 oriS) related to DNA replication, and Packaging signal (pac) sequence associated with viral packaging. On the outside of the herpes virus genome, there are three layers of structure in turn, which are the nucleocapsid (capsid) with a 20-hedron structure, the tegument composed of pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/869
Inventor 韩志强孙项东
Owner ZHENGZHOU VIRIGE BIOTECH
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