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: 2011-10-12
ZHENGZHOU VIRIGE BIOTECH
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  • Summary
  • 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 package

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

Example Embodiment

[0061] Example 1. Construction of BAC-HSV-1 HF strain

[0062] Such as figure 1 The construction mode of the BAC-HSV-1 HF strain is shown.

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

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

Example Embodiment

[0066] Example 2. Construction of HSV-1 amplicon plasmid vector

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

[0068] 1. Obtaining the "pac" sequence

[0069] BAC-HSV-1HF was digested with MluI to obtain BAC-TR containing terminal repeat region, and BAC-TR was digested with SacI to obtain a 4Kb fragment containing HSV-1 terminal repeat region "pac" sequence; HphI digested 4Kb fragment A 1.3Kb fragment containing the terminal repeat region "pac" sequence was obtained. In addition, the 1.3Kb fragment is digested by BsrBI to obtain a 188bp Ub-DR1-Uc structure (the packaging signal of the smallest packaging unit). EcoRI digested pGEMT linear T vector (purchased from Dalian Bao Biological Company), Klenow enzyme (purchased from Dalian Bao biological company) filled in the pGEMT linear T vector after EcoRI digestion, and added the 188bp fragment to pGEMT with blunt-ended ends. T7 universal primers were used for sequencing (sequencing was don...

Example Embodiment

[0079] Example 3. Construction of recombinant adenovirus Adv-loxP-OPD-loxP and Adv-loxP-D-loxP and virus packaging

[0080] Such as Figure 7 The construction mode of Adv-loxP-OPD-loxP is shown.

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

[0082] pENTR-MCS was digested with SalI and BamHI, and the 2.6 kb vector pENTR-MCS was recovered from the gel. The vector was filled in and dephosphorized; BAC-LRarm contains two loxP sites in the same direction, which can be digested with PvuI and ScaI. Cut out two fragments of loxP in the same direction and the middle LR homology arm and GFP reporter gene, and fill in the fragments. The vector and the fragment were ligated, transformed, and the pENTR-loxP-LRarm-loxP vector was identified and identified by AvaII restriction enzyme digestion. Theoretically, the sizes should be: 3.1Kb, 1.4Kb, 1.2Kb, 440bp, 282bp, and 153bp. The electrophoresis result is shown by the arrow in Figure 8-1A. It can be seen that 3.1Kb, 1.4Kb, 1.2Kb, 440b...

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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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IPC IPC(8): C12N15/869A61K48/00A61P35/00A61P31/22
Inventor 韩志强孙项东
Owner ZHENGZHOU VIRIGE BIOTECH
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