Vector system for herpes simplex virus/phiC31 integrase (HSV/phiC31) heterozygosity amplicon and preparation method of vector system

A carrier system and amplicon technology, which is applied in the field of new HSV/ΦC31 heterozygous amplicon carrier system and its preparation, can solve the problem of long-term expression of transgenes, and achieve the effect of avoiding the risk of insertion mutation

Inactive Publication Date: 2011-04-06
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a heterozygous HSV / ΦC31 amplicon carrier system with site-specific integration, to overcome the problem that the transgene mediated by the HSV amplicon carrier cannot be expressed for a long time, and can greatly reduce or even avoid The risk of insertion mutation makes this vector a new type of viral vector system integrating effectiveness, safety and ease of operation

Method used

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  • Vector system for herpes simplex virus/phiC31 integrase (HSV/phiC31) heterozygosity amplicon and preparation method of vector system
  • Vector system for herpes simplex virus/phiC31 integrase (HSV/phiC31) heterozygosity amplicon and preparation method of vector system
  • Vector system for herpes simplex virus/phiC31 integrase (HSV/phiC31) heterozygosity amplicon and preparation method of vector system

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Embodiment 1

[0045] Embodiment 1 Novel heterozygous HSV / ΦC31 amplicon vector preparation method

[0046] The oligonucleotide core sequence attB (44bp) of the ΦC31 integrase recognition site was artificially synthesized, annealed and digested with KpnI / SpeI and cloned into plasmid pCMV-C31 to obtain recombinant plasmid pCMV-attb-C31. Using the recombinant plasmid as a template, design primers for PCR to obtain the PCR product (CMV-attb-C31-pA) composed of CMV regulatory elements, ΦC31 integrase gene and its polyA (pA) element, and clone it into the carrying There is an HSV amplicon vector (pHSV-GFP) encoding the green fluorescent protein gene (GFP), and the clone is sequenced to obtain a heterozygous HSV / C31 amplicon vector pHSV-GFP-CMV-attB-C31.

Embodiment 2

[0047] Embodiment 2 Preparation of novel heterozygous HSV / ΦC31 amplicon vector virus

[0048] The heterozygous HSV / ΦC31 amplicon vector or general HSV amplicon vector was mixed with 5 cosmids COS6Δa, COS14, COS28, COS56, COS48Δa at a mass ratio of 2:1:1:1:1:1. Transfection was performed on VERO 2-2 cells using liposomal LipofectamineTM. After 5 hours of transfection, 2% fetal bovine serum was added, and after culturing for 2-3 days, the cells and supernatant were collected, frozen and thawed three times, centrifuged at 1500 rpm for 5 minutes, and the virus supernatant was harvested.

Embodiment 3

[0049] Example 3 Novel heterozygous HSV / ΦC31 amplicon carrier virus system infection cell analysis

[0050] Infect the 293 cell line on a 96-well plate with 10ul of the heterozygous HSV / ΦC31 amplicon vector virus. GFP expression can be observed 2 days after the infection of the human embryonic kidney 293 cells, and flow cytometry detection shows , 4 days, 6 days and 8 days later, the percentages of positive cells expressing green fluorescent protein were 35%, 36.8%, 36.9%, and 37.6%, respectively, showing that the percentage of positive cells expressing green fluorescent protein did not change as the cells were passaged. After the control pHSV-GFP infected human embryonic kidney 293 cells, the expression of GFP could also be observed, but flow cytometry showed that the proportion of positive cells expressing green fluorescent protein gradually increased after 2 days, 4 days, 6 days and 8 days after infection. reduce. The results suggested that the novel heterozygous HSV / ΦC31 ...

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Abstract

The invention belongs to the field of gene engineering, relating to a vector system for herpes simplex virus / phiC31 integrase (HSV / phiC31) heterozygosity amplicon and a preparation method of the vector system. The vector system comprises heterozygosity HSV / phi C31 amplicon vector and five helper free virus packaging cosmid, namely COS6 delta a, COS14, COS28, COS56 and COS48 delta a; the heterozygosity HSV / phiC31 amplicon vector plasmid is composed of HSV amplicon vector pHSV-GFP, cytomegalovirus (CMV) promoter, attB locus, a phiC31 intergrase gene coding sequence and poly-adenine, wherein theattB locus is positioned between the CMV promoter and the phiC31 integrase gene coding sequence. The vector system of the invention has function of locus-specific conformability, can express exogenous target gene in various host cells for a long time, and can be used for the research on functions of exogenous genes or gene therapy.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a novel HSV / ΦC31 heterozygous amplicon carrier system and its preparation method and application. Background technique [0002] The vectors based on herpes simplex virus type 1 (herpes simplex virus type 1, HSV-1) include two types: recombinant virus type and recombinant plasmid type. The latter are also known as replication-defective (Replication-defective) vectors or amplicon vectors (amplicon vectors). The amplicon vector consists of a plasmid containing the HSV DNA replication origin sequence oris and the packaging signal pac [Spaete RR et al. The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning-amplifying vector. Cell; 1982; 30: 295- 304]. Under the action of the HSV helper virus, it is allowed to be packaged into the HSV virus in the form of concatemers. In the absence of the HSV helper virus, these vectors can be packaged by co-transfection with a s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/869C12N15/64
Inventor 朱焕章孔垂瑾
Owner FUDAN UNIV
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