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Construction of pseudorabies virus infectious clone. After amplification and purification of the highly pathogenic PRV isolate DCD-1, intact viral DNA was isolated. The intermediate plasmid pBeloBAC11-cm-PRV-pBR322-amp-ccdB, which contained the 80 bp end sequences of the PRV genome, was constructed. BamHI digestion of the intermediate plasmid released the linear vector pBeloBAC11-cm and exposed homology arms. The viral DNA and the linear vector were co-electroporated into a RecETexpressing E. coli strain for direct cloning. The infectious clone was validated by restriction enzyme analysis, a rescue experiment and integrity check. Knockout of the virulence gene (represented by the red box in the genome) and knockin of the immune factor genes and antigen genes were achieved by Redab-mediated deletion or insertion followed by site-specific recombination to remove the selectable marker. The recombinant virus was rescued for in vitro and in vivo characterization.
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Introduction
The herpesviridae are DNA viruses with large and complicated genomes. The herpesvirus bacterial artificial chromosomes (BACs) have been useful for generating recombinant viruses to study the biology and pathogenesis. However, the conventional method using homologous recombination is not only time consuming but also prone to accumulate...
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Context 1
... approach for constructing the PRV DCD-1 infectious clones and mutants is illustrated in Fig. 1. To clone the entire PRV DCD-1 genome, we employed the ExoCET method to ensure successful cloning of a double-stranded stretch of DNA larger than 100 kb into a BAC vector [21]. The intermediate plasmid pBeloBAC11-cm-PRVpBR322-amp-ccdB was constructed by RecET-mediated linear plus linear homologous recombination, incorporating 80 bp ...
Citations
... These plasmids carry the gene for IL-18-γ and PH20. More detailed information for constructing plasmids was described in a previous study [18]. ...
... The final recombinant plasmids pBeloBAC11-DCD1-70#-∆TK-PH20-∆gEgI-FRT (rPRV-PH20) and pBeloBAC11-DCD1-70#-∆gG-IL-18-γ-∆TK-PH20-∆gEgI-FRT (rPRV-IL-18-γ-PH20) were obtained. Recombinant viruses rPRV-gG − -TK − -gEgI − and rPRV-IL-18-γ were originally constructed and preserved for our laboratory [18]. The modification schemes of the four recombinant viruses rPRV-gG − -TK − -gEgI − , rPRV-IL-18-γ, rPRV-PH20 and rPRV-IL-18-γ-PH20 are shown in Figure 1. ...
... Presently, research regarding oncolytic viruses employing PRV as a vector is relatively scarce, with studies primarily conducting in vitro and in vivo tests using a virulencegene-knockout PRV [5,7,18]. In this study, we used PRV as an oncolytic virus vector and carried out genetic engineering to construct PRV recombinant viruses carrying antitumor genes for the first time and verified their therapeutic effect on the mouse pancreatic cancer tumor model. ...
Pseudorabies virus (PRV) is considered to be a promising oncolytic virus that has potential as a cancer gene therapy drug. In this study, PRV-DCD-1-70 was used as a vector to carry exogenous genes IL-18, IFN-γ and PH20 to construct novel recombinant PRV, rPRV-PH20 and rPRV-IL-18-γ-PH20, and their tumorolytic effects were evaluated in vitro and in vivo. Our study showed that recombinant PRV lysed all four tumor cell lines, Pan02, EMT-6, CT26 and H446, and rPRV-IL-18-γ-PH20 showed the best tumor lysis effect. Further studies in mice bearing Pan02 tumors showed that recombinant PRV, especially rPRV-IL-18-γ-PH20, were able to inhibit tumor growth. Moreover, an immunohistochemical analysis indicated that the recombinant PRV effectively increased the infiltration of CD4+T and CD8+T cells and enhanced the anti-tumor immune response of the organism in vivo. Overall, PRV carrying PH20 and IL-18-γ exogenous genes demonstrated anti-tumor effects, providing a foundation for the further development and application of PRV as a novel tumor oncolytic virus vector.
In 2021, a highly virulent strain of duck enteritis virus (DEV), designated as DEV XJ, was isolated from Zhejiang, China, and its complete genome, spanning 162,234 bp with 78 predicted open reading frames (ORFs), was sequenced. While showing relative homology to the DEV CV strain, DEV XJ exhibited distinctions in 38 ORFs, including various immunogenic and virulence-related genes. Amino acid variation analysis, focusing on UL6 and LORF3, indicated a high degree of homology between DEV XJ and the 2085 strain from Europe, as well as the DEV DP-AS-Km-19 strain from India. Subsequently, a full-length infectious bacterial artificial chromosome clone (BAC) of DEV XJ was successfully constructed to delve into the pathogenic mechanisms of this virulent strain. XJ BAC demonstrated substantial similarity to the parental DEV XJ in both in vitro growth properties and the induction of typical pathogenic symptoms in sheldrakes. Furthermore, the US3, LORF3, UL21, and UL36 genes were individually deleted using a two-step RED recombination approach based on the infectious BAC clone. Our findings revealed that the UL21 and UL36 genes play crucial roles in viral proliferation. Although the US3 and LORF3 genes were dispensable for viral replication and cell-to-cell transmission in vitro, they attenuated the replication and transmission efficiency of DEV compared to the WT. In summary, this study accomplished the whole-genome sequencing of a clinically virulent DEV strain and the successful construction of an infectious DEV XJ clone. Moreover, the functional roles of the above-mentioned mutant genes were preliminarily explored through the analysis of their in vitro biological characteristics.
Recombineering is a valuable technique for generating recombinant DNA in vivo, primarily in bacterial cells, and is based on homologous recombination using phage-encoded homologous recombinases, such as Redαβγ from the lambda phage and RecET from the Rac prophage. The recombineering technique can efficiently mediate homologous recombination using short homologous arms (∼50 bp) and is unlimited by the size of the DNA molecules or positions of restriction sites. In this review, we summarize characteristics of recombinases, mechanism of recombineering, and advances in recombineering for DNA manipulation in Escherichia coli and other bacteria. Furthermore, the broad applications of recombineering for mining new bioactive microbial natural products, and for viral mutagenesis, phage genome engineering, and understanding bacterial metabolism are also reviewed.
