Pseudorabies virus(PRV)is the etiological agent of pseudorabies in pigs,which is char-acterized by dyspnea,reproductive disorders,and neurological diseases,and it spreads widely a-round the world.Since 2011,the newly emerged PRV variants have resulted in poor immunity pro-tection of traditional vaccine strains,and the original method of vaccine strain preparation is time-consuming and labor-intensive.Therefore,it is urgently needed to develop an efficient screening method of the vaccine strain at present.Using CRISPR/Cas9 gene editing technology in this study,two single guide RNAs(sgRNA)were designed targeting the virulence gene TK of PRV variant strain CH/JX/2016,and then the enhanced green fluorescent protein the reporter(EGFP)gene was inserted at the TK locus by a homologous repair plasmid.After multiple rounds of plaque puri-fication,the rPRV-ΔTK/EGFP strain was obtained.The results showed the cleavage efficiency of the two sgRNAs was extremely high.The preparation of rPRV-ΔTK/EGFP strain was succeed af-ter only three rounds of purification,and the EGFP expressed normally.The CRISPR/Cas9 system can edit the PRV gene simply,rapidly,and efficiently,and exhibits great potential in the construction of vaccine candidate strains.Meanwhile,the rescued rPRV-ΔTK/EGFP strain not only could be used as a tracer strain in PRV variant infection progresses,but also for subsequent antivi-ral drug screening.

Porcine deltacoronavirus(PDCoV)is one of the important pathogens associated with swine viral diarrhea,which results in enormous economic losses in the pig industry.Among the known encoding proteins,the nucleocapsid(N)protein is relatively conserved and serves as the dominant antigen of coronaviruses,making it an ideal candidate for the early and accurate diagnosis of infection.In this study,the PDCoV N protein was induced and expressed in a prokaryotic system using IPTG treatment at low temperatures.Six-week-old BALB/c mice were immunized with the recombinant PDCoV N protein purified by nickel column.Isolated spleen cells were harvested and fused with SP2/0 cells once the serum titers reached 1∶10 000,as determined by indirect ELISA.The positive monoclonal hybridoma with the highest titer of secretory antibodies were screened af-ter three rounds of limited dilution.Ultimately,indirect immunofluorescent assay(IFA)and West-ern blot were employed to confirm the characterization of the antibodies,and the subtypes of the heavy and light chains of the antibodies were determined.One strain of hybridoma against the PD-CoV N protein,designated as 2G12,was obtained,with a titer approaching 1∶40 960.The IFA as-say showed PDCoV was specifically bound to 2G12.Furthermore,the Western blot assay further showed PDCoV in the supernatant of infected cells or recombinant PDCoV N protein,reacted well with 2G12.The subtype of MAbs was determined as IgG1 with the light chains being κ.The MAbs generated against the PDCoV N protein in this study provide valuable support for the development of a diagnostic kit for PDCoV and play an important role in the functional research of the PDCoV N protein.

Porcine deltacoronavirus(PDCoV)is one of the important pathogens associated with swine viral diarrhea,which results in enormous economic losses in the pig industry.Among the known encoding proteins,the nucleocapsid(N)protein is relatively conserved and serves as the dominant antigen of coronaviruses,making it an ideal candidate for the early and accurate diagnosis of infection.In this study,the PDCoV N protein was induced and expressed in a prokaryotic system using IPTG treatment at low temperatures.Six-week-old BALB/c mice were immunized with the recombinant PDCoV N protein purified by nickel column.Isolated spleen cells were harvested and fused with SP2/0 cells once the serum titers reached 1∶10 000,as determined by indirect ELISA.The positive monoclonal hybridoma with the highest titer of secretory antibodies were screened af-ter three rounds of limited dilution.Ultimately,indirect immunofluorescent assay(IFA)and West-ern blot were employed to confirm the characterization of the antibodies,and the subtypes of the heavy and light chains of the antibodies were determined.One strain of hybridoma against the PD-CoV N protein,designated as 2G12,was obtained,with a titer approaching 1∶40 960.The IFA as-say showed PDCoV was specifically bound to 2G12.Furthermore,the Western blot assay further showed PDCoV in the supernatant of infected cells or recombinant PDCoV N protein,reacted well with 2G12.The subtype of MAbs was determined as IgG1 with the light chains being κ.The MAbs generated against the PDCoV N protein in this study provide valuable support for the development of a diagnostic kit for PDCoV and play an important role in the functional research of the PDCoV N protein.

Objective To establish a tree shrew mode of breast tumor.Method Forty-five 3 to 4 month-old female tree shrews were orally gavaged with 20 mg 7,12-dimethylbenz(a)anthracene (DMBA) or peanut oil per animal for three times.Following that, fifteen DMBA administrated tree shrews were implanted 90 day-release medroxyprogesterone acetate ( MPA) pellets.The tree shrews were palpated once weekly to detect mammary tumors for 45 weeks after first DMBA administration.Results DMBA were able to induce breast tumors (12.5%) in tree shrews, and MPA increased the tumor incidence (50%) while no breast tumors were observed in the control group.Three induced breast tumors were intraductal papillary carcinomas and one was IDC by H&E stain.Conclusion All induced tumors are similar with spontaneous tumors in structure and molecular markers.

With extensive application of interventional radiology technique, skilled and standardized manipulation of DSA is very helpful for interventional operations. It is introduced in this paper how to operate the Siemens dBA which is the first domestic one and settle with the problems met in routine work.

Objective To assess the usefulness of the DPF with Store Fluoro for interventional treatment. Methods Store Fluoro were performed in 187 cases and the dose was compared with that not using it. Results The doctor and the patients′ radiology dose and the volume of the contrast were all reduced. Conclusion The Store Fluoro plays an important role in interventional treatment.