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Construction method and application of HSV1-H129-BAC and mutant thereof

A technology of H129-BAC and construction methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, carriers, etc., can solve the inconvenient molecular biology mutation transformation, self-replication, herpes virus genome cannot be preserved for a long time, etc. problem, to achieve the effect of promoting wide application and convenient detection

Active Publication Date: 2016-05-11
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The first object of the present invention is to provide a type I herpes simplex virus infectious clone H129-BAC, which has a bacterial artificial chromosome BAC, which can be used in specific bacteria (such as DH10B, DY380, EL250, DY330 and GS1783, etc.) Maintain low-copy self-replication, which solves the disadvantages that the herpes virus genome cannot be preserved for a long time, cannot self-replicate, and is inconvenient for molecular biology mutation transformation due to the large genome

Method used

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  • Construction method and application of HSV1-H129-BAC and mutant thereof
  • Construction method and application of HSV1-H129-BAC and mutant thereof
  • Construction method and application of HSV1-H129-BAC and mutant thereof

Examples

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Effect test

Embodiment 1I

[0066] Example 1 Construction method of herpes simplex virus type I infectious clone H129-BAC

[0067] 1. Using the wild-type H129 viral genome as a template, amplify its homologous recombination arm sequence and clone it into a BAC vector containing the GFP gene to obtain a plasmid containing both the H129 homologous recombination arm sequence and the BAC sequence.

[0068] (1) Extraction of wild-type H129 virus genome

[0069] Use the wild-type H129-WT virus (Szpara, M.L.Parsons, L.Enquist, L.W.Sequencevariabilityinclinicalandlaboratoryisolatesofherpessimplexvirus1revealsnewmutations.[J].JVirol.84:5303-13) to infect vero cells at a multiplicity of infection of 1. After infection for 12h, scrape Remove the cells, collect the cell pellet by centrifugation, wash it once with solutionI (10MmTris, 10MmEDTA, pH8.0), and then use 0.5ml of solutionI solution (containing 0.25mgofproteinaseK / ml, purchased from Roche, USA; the final concentration is 0.6% Sodium dodecyl sulfate, purchase...

Embodiment 2I

[0130] The construction method of the variant virus of embodiment 2 type I herpes simplex virus H129-BAC

[0131] (1) Cassette construction

[0132] Cloning the GFP gene into the vector pRK-zeo by PCR, digestion, ligation and transformation to construct CassetteCMV-promoter-GFP-zeo R , the GFP gene sequence is shown in SEQ ID NO: 2; the primers used in PCR are shown in SEQ ID NO: 6 and SEQ ID NO: 7.

[0133] same:

[0134] Cloning the mGFP gene into the vector pRK-kan to construct CassetteCMV-promoter-mGFP-kan R , the mGFP gene sequence is shown in SEQIDNO:4; primers used during PCR are shown in SEQIDNO:8 and SEQIDNO:9;

[0135] (2) PCR amplification of each Cassette

[0136] The reaction system of PCR (PrimeStarDNAPolymerase, purchased from Japan TaKaRa company) is:

[0137]

[0138]

[0139] Primers are listed in the table below:

[0140]

[0141] The amplification reaction procedure is:

[0142]

[0143] After the reaction, the PCR product was subjected t...

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Abstract

The invention relates to the biotechnical field, and concretely relates to an HSV1-H129-BAC and a mutant thereof. The above virus and the mutant thereof carry a bacterial artificial chromosome (BAC), can keep low-copy number self-replication in specific bacteria, and solve the disadvantages of unable long-term preservation, unable self-replication and inconvenient molecular biologic mutation reconstruction due to too large herpes virus genomes. The virus and the mutant thereof also carry a GFP reporter gene, and substantially develop the application prospect of the herpes virus as a tool virus. The invention also provides a construction method of the H129-BAC and the mutant thereof. The method allows H129 to be reconstructed in order to obtain H129-BAC and endows the H129-BAC with more mutant types; and the construction method, the H129 and the mutant thereof form a genetic modification platform with high application possibility.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular, to the construction method and application of HSV1-H129-BAC and its variants. Background technique [0002] Herpes simplex virus (HSV) belongs to the alpha herpes virus subfamily, which is divided into two types, HSV type 1 and HSV type 2, namely HSV1 and HSV2. Both HSV1 and HSV2 are DNA viruses. The full length of the HSV1 genome is 152kb, encoding more than 80 different viral proteins, and the size of the virus plasmid is about 180nm. HSV1 has a wide range of hosts for animal infection. Commonly used experimental animals are rabbits, guinea pigs and mice. HSV1 can proliferate in a variety of cells, and African green monkey kidney cells (Vero cells), human embryonic kidney cells, and hamster kidney cells are commonly used to isolate the virus from subcultured cells. [0003] HSV1-H129 is a typical tool virus for anterograde labeling of neural circuits. It has strict anterog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/66C12N15/65G01N33/68C12Q1/70C12Q1/06C12Q1/18C12R1/19
CPCC12N7/00C12N15/65C12N15/66C12N2710/16621C12N2710/16631C12N2710/16643C12N2710/16652C12N2800/107C12Q1/06C12Q1/18G01N33/68
Inventor 罗敏华曾文波江海飞赵非谌章舟徐富强
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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