Herpes simplex virus type 1 carrier system, and preparation method and application thereof

A technology of herpes simplex virus and vector system, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of time-consuming, time-consuming and laborious, cumbersome screening of recombinants, etc., and achieve weak toxicity, High safety effect

Inactive Publication Date: 2014-02-19
XINJIANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even so, the acquisition of recombinant HSV-1 is still a time-consuming and laborious work, and it often takes a year to construct a recombinant virus
However, when the proliferation ability of the wild-type virus is significantly higher than that of the mutant strain, it is difficult to screen out the mutant virus strain; if the mutation is missing an essential gene for virus proliferation, it is necessary to propagate the recombinant virus in a complementary cell line, which in turn makes the screening of the recombinant virus difficult. more complicated

Method used

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  • Herpes simplex virus type 1 carrier system, and preparation method and application thereof
  • Herpes simplex virus type 1 carrier system, and preparation method and application thereof
  • Herpes simplex virus type 1 carrier system, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Preparation of shuttle plasmid pKo5 / BN

[0024] According to the genome structure of HSV-1, the Bam H IB fragment (B) and Bam The H IN fragment (N) is located on both sides of the IR region, respectively, and primers are designed according to the sequence of HSV-1, and respectively added to the 5' ends of the upstream and downstream primers of the B fragment Xba I and Speech Ⅰ Restriction site, added at the 5' end of the upstream and downstream primers of the N fragment respectively Eco R V and cla Ⅰ Restriction site, PCR amplification to obtain a 2024bp B fragment and a 2857bp N fragment, clone the B and N fragments into pBluescriptⅡKS(+) in sequence, and then include the B and N fragments and the multiple cloning site between the two fragments Cloning of the DNA fragment into pKO5 Xba I and Sal The shuttle plasmid pKo5 / BN (attached figure 1 ).

Embodiment 2

[0025] Embodiment 2. prepare HSV-1 bacterial artificial chromosome plasmid pHSV Δ IR-BAC

[0026] A large amount of pKo5 / BN was extracted and purified by alkaline lysis, 2μl (1μg / μl) of purified pKo5 / BN was mixed with 40μl DH10B competent cells and pre-cooled in an ice-water bath, and then added to a 0.1cm electric shock cup for electroporation. The parameters are voltage 2.5 kV, capacitance 30 μF and resistance 400Ω. The competent cells after electrotransformation were added to 1 mL of SOC medium and incubated at 30 °C for 1 h, spread on LB solid medium containing zeocin and chloramphenicol, and cultured at 43 °C overnight; when the colony grew to a diameter of about 1 mm, random Pick 10 single clones, mix them in 1 mL of normal saline, take 40 μL and spread them on LB solid medium plates containing chloramphenicol and 5% (v / v) sucrose, and culture them overnight at 30°C; Randomly pick 10 single clones (total 100 single clones) on each plate and streak on the solid LB me...

Embodiment 3

[0027] Example 3. Preparation of recombinant HSV-1 with deletion of IR region: HSV-1 / Δ IR

[0028] Vero cells were cultured according to conventional methods until the cell density reached 70%-80%, and the purified recombinant plasmid pHSVΔIR-BAC (set in three gradients of 2 μg, 4 μg and 8 μg) was transfected into cells by liposome-mediated method. Observe the pathological changes (CPE) of the transfected cells after 2 days, and plaques appear in the cells after 4 days (the cell state and transfection reagents will affect the transfection effect and cause the appearance time of plaques to lag); Discard the cell culture medium, fix the cells with an equal volume mixture of 2% low-melting point agarose and serum-free 2×199V medium, pick up the cells in the plaque area by conventional methods, and add 200 μL of skim milk powder solution (the mass volume of which has been sterilized by conventional autoclaving) Concentration of 9% skimmed milk powder solution) and 2×199V equa...

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Abstract

The invention belongs to the technical field of biology, and relates to a herpes simplex virus type 1 carrier system, and a preparation method thereof. The carrier system comprises a shuttle plasmid by taking DNA (deoxyribonucleic acid) fragment at two sides of an inverted repeat sequence (IR) inside HSV-1 (herpes simplex virus type 1) as homologous recombinant arms, and a bacterial artificial chromosome plasmid pHSV delta IR-BAC (bacterial artificial chromosome) containing IR zone deleted HSV-1 genome. The method for preparing recombinant HSV-1 by using the carrier system comprises the following steps: firstly, cloning a target DNA fragment into pKo5/BN to obtain a recombinant shuttle plasmid; secondly, electro-transforming escherichia coli containing pHSV delta IR-BAC by using the recombinant shuttle plasmid, and screening to obtain recombinant bacteria; finally, extracting the recombinant DNA from the recombinant bacteria, transfecting Vero cells, and purifying to obtain recombinant HSV-1. The carrier system is high in security, can accommodate 30Kb of exogenous DNA, and can construct optional replication type recombinant HSV-1 capable of expressing a plurality of target genes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and the invention relates to a type 1 herpes simplex virus vector system and a preparation method thereof, and also relates to the herpes simplex virus vector carrying and expressing EGFP reporter gene and its application in anti-tumor. Background technique [0002] Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that is very common in the population. The clinical manifestations are mild symptoms such as localized herpes on the mucous membrane or skin, occasionally accompanied by fever. HSV-1 is a double-stranded DNA virus with a genome of 152kb, consisting of a unique long segment (U L ) and short fragments (U S ) with terminal inverted repeats TR at both ends L and TR S , the junction of the two fragments is an internal inverted repeat sequence (IR region). HSV-1 encodes about 90 proteins, the functions of which are mostly clear, about half of which are necessary for viral replicatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/869C12N15/85A61K48/00A61P35/00
Inventor 马正海毛红艳李永鑫
Owner XINJIANG UNIVERSITY
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