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Construction and purification of ANK gene deleted recombinant goatpox virus.

ABSTRACT: Sheeppox virus (SPPV) and goatpox virus (GTPV) are two pathogens of host specificity. Previous studies have hypothesized that ankyrin (ANK) family may play an important role in determining host range of SPPV and GTPV. In order to verify the function of ANK proteins, it is critical to generate and purify the ANK gene deleted GTPV. In this study, the GFP gene as a reporter gene was connected with two homologous arms of ANK gene by fusion PCR. The ANK gene deleted transfer vectors were generated by inserting the PCR products into PET42b, and were transfected into testicular primary cells which were infected by GTPV. The rGTPV were identified as green fluorescence positive and properly purified. The results showed that GFP gene and two homologous arms of ANK gene were connected. The sequence was inserted in PET42b to form ANK deleted transfer vector. ANK deleted rGTPV was generated successfully by transferring vector and GTPV in cells. The ANK deleted rGTPV was purified and identified in this study. The study successfully generated the ANK deleted rGTPV. It overcomes the technical barrier for future studies about the function of ANK genes.

SUBMITTER: Zhang X

PROVIDER: S-EPMC7749001 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The ank gene of the agent of human granulocytic ehrlichiosis (HGE) codes for a protein with a predicted molecular size of 131.2 kDa that is recognized by serum from both dogs and humans infected with granulocytic ehrlichiae. As part of an effort to assess the phylogenetic relatedness of granulocytic ehrlichiae from different geographic regions and in different host species, the ank gene was PCR amplified and sequenced from a variety of sources. These included 10 blood specimens from patients with confirmed human granulocytic ehrlichiosis (three from New York, four from Wisconsin, two from Slovenia, and one from Sweden). Also examined was a canine granulocytic ehrlichia sample obtained from Minnesota, Ehrlichia equi from California, Ehrlichia phagocytophila from Sweden, and the granulocytic ehrlichia isolate USG3. The sequences showed a high level of homology (>95.5% identity), with the lowest homology occurring between a New York HGE agent and the Swedish E. phagocytophila. Several 3-bp deletions and a variable number of 51- and 81-bp direct repeats were noted. Although the North American HGE sequences showed the highest conservation (>98.1% identity), phylogenetic analyses indicated that these samples represent two separate clades, one including the three New York HGE samples and the USG3 strain and another with the Wisconsin HGE and Minnesota canine sequences. Two of the New York samples and the USG3 strain showed 100% identity over the entire 3,696-bp product. Likewise, three of the Wisconsin human samples and the Minnesota dog sample were identical (3,693 bp). Whereas phylogenetic analysis showed that the E. equi sequence was most closely related to the Upper Midwest samples, analysis of the repeat structures showed it to be more similar to the European samples. Overall, the genetic analysis based on the ank gene showed that the granulocytic ehrlichiae are closely related, appear to infect multiple species, and can be grouped into at least three different clades, two North American and one European.

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Construction and purification of ANK gene deleted recombinant goatpox virus.

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