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Homologous recombination vector based on HSV-1 as well as target sequence and application of homologous recombination vector

A technology for HSV-1 and HSV-11716, applied in homologous recombination vectors and their target sequences and applications, can solve problems affecting the safety characteristics of vaccine strains, and achieve efficient, fast and accurate screening methods, good expression efficiency, and good safety effect

Active Publication Date: 2020-12-11
昆明医科大学第二附属医院
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Problems solved by technology

However, as more and more functional elements of the LAT locus (maintaining LAP transcriptional activity) are discovered, the position of foreign gene insertion will become more important; on the other hand, the transcription of the downstream region of LAT (containing virally encoded miRNAs) changes May also affect the safety profile of vaccine strains

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  • Homologous recombination vector based on HSV-1 as well as target sequence and application of homologous recombination vector
  • Homologous recombination vector based on HSV-1 as well as target sequence and application of homologous recombination vector
  • Homologous recombination vector based on HSV-1 as well as target sequence and application of homologous recombination vector

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Embodiment Construction

[0023] The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0024] 1. Design of HSV-1 recombinant virus

[0025] Such as figure 1 As shown, two types of recombinant HSV vectors, LMR and LMRx, were designed in this study. Among them, in the LMR series vectors, the exogenous expression cassettes are respectively inserted into the first StyI site of Exon1 downstream of the LAP promoter; while in the LMRx series vectors, the exogenous expression cassettes are respectively inserted into the BstEII site in the 2.0Kb Intron. In each series of vectors, "complete expression cassette" (cBh promoter-reporter gene-bGH poly), "no transcription termination sequence" (cBh promoter-reporter gene) and "coding sequence only" (IRES-reporter gene) are designed In order to compare the effects of the addition of exogenous promoters and transcription termination sequences on the latent ef...

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Abstract

The invention discloses a homologous recombinant vector based on HSV-1 as well as a target sequence and application of the homologous recombinant vector, and provides a set of complete HSV-1 expression vector construction and screening process to optimize an HSV-1 replication-defective central nervous system expression vector, so that efficient insertion and replacement of exogenous genes can be realized. Besides, an optimal latent expression vector construction method is screened out, that is, a complete exogenous gene expression cassette, namely, cBh promoter-repoter Gene-bGH polyA is inserted into a BstEII site in 2.0 Kb Intron at the downstream of an LAP promoter. A recombination virus can realize long-term efficient expression of a reporter gene in a hippocampus of a mouse, and has good safety.

Description

technical field [0001] The invention relates to the construction strategy and expression optimization field of HSV-1 central nervous system latent expression vector, in particular to an HSV-1-based homologous recombination vector and its target sequence and application. Background technique [0002] The HSV 1716 strain replication-deficient vector has a large capacity and can express foreign genes latently for a long time. The natural optic nerve characteristics of HSV-1 enable it to propagate antegradely or retrogradely through synapses in the central nervous system, and can remain latent in neurons for a long time without affecting their normal functions, realizing the efficient and specialized expression of exogenous genes in the central nervous system. 1. Long-term expression. Vaccine-type HSV-1 vectors have relatively high safety. Studies have shown that the modified 1716 strains are due to γ34.5 - / - Mutations that make it lack the ability to replicate in non-cancero...

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Application Information

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IPC IPC(8): C12N15/869C12N15/90C12N15/38C12N15/11
CPCC12N15/86C12N15/907C07K14/005C12N2710/16643C12N2710/16622C12N2310/20
Inventor 曹霞黄新伟李秀清杨丽娟王鹏飞燕晋媛聂祖庆李志伟文婕
Owner 昆明医科大学第二附属医院
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