Construction of TK Gene-deleted PRV SH Strain Containing a Single LoxP Site

China Biotechnology

研究简报

Construction of TK Gene-deleted PRV SH Strain Containing a Single LoxP Site

Pu-Yan Chen

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Abstract Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and Economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. In this study, we constructed a recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID50 assay indicated that rPRV2 grows well on RK-13 cells. The LD50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.

Key words： Cre LoxP GFP recombinant virus PRV TK gene

Received: 20 March 2006 Published: 25 October 2006

Corresponding Authors: Pu-Yan Chen

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Pu-Yan Chen . Construction of TK Gene-deleted PRV SH Strain Containing a Single LoxP Site. China Biotechnology, 2006, 26(10): 35-39.

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https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2006/V26/I10/35

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