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[Objectives] In order to clarify the difference in activation of the immune system by live or inactivated viruses after entering the body, a high-efficiency animal model was established for the evaluation of the efficacy of immune enhancers to better improve the immune efficacy of inactivated vaccines. In this experiment, the Pseudorabies virus (PRV) Bartha-K61 strain was used to establish a PRV artificial infection mouse model, and to screen the immune-related difference factor after inoculation with live or inactivated antigens. [Methods]PRV Bartha-K61 was inoculated to experimental mice in different doses and methods, and the survival rate was calculated to screen the safe dose. Mice were vaccinated with live virus or inactivated virus at a safe dose, and ELISA kits were used to detect the level of serum PRV-specific gB antibodies and the level of serum TNF-α, IL-1β, IFN-β and IL-6 of mice 7 days after immunization; inactivated PRV was used as a specific antigen to perform lymphocyte proliferation experiments and calculate the lymphocyte proliferation index(stimulation index, SI)of each group to screen the best vaccination route. [Results] 5×104 TCID50 was a safe challenge dose for 6-8 weeks mouse; subcutaneous injection was the best way of infection. Compared with the live virus and the inactivated virus group after 7 days of infection, the serum levels of TNF-α, IL-1β and IFN-β significantly increased (P<0.001, P<0.01), the levels of PRV gB IgG antibody and the lymphocyte SI in the mouse serum also significantly increased (P<0.05, P<0.001); There is no significant difference in cytokines and SI between different inactivation methods (P>0.05). [Conclusions]The experiment successfully established an artificial pseudorabies virus infection model in mice, and clarified that after live or inactivated pseudorabies virus immunized mice, there were differential immune-related factors in the early stage of the body, providing a research platform for the evaluation and screening of subsequent immunopotentiator. [ABSTRACT FROM AUTHOR]

[Objectives] This study aimed at deciphering the mechanisms of CVC1302 in promoting germinal center(GC) responses. [Methods]Pattern antigen 4-hydroxy-3-nitrophenylacetyl(NP)-ovalbumin(OVA)were mixed with CVC1302 followed by emulsified with ISA206-named as NP-CVC1302. Six-week-old BALB/ c femal mice were immunized with NP-CVC1302-NP or ISA206-and the sera were collected to detect the NP-specific antibody titers and isotype antibody titers. The percentages of follicular helper T cells (TFH )-NP + GC B cells and NP + plasma-blasts in inguinal lymph nodes and the numbers of GC were measured at 14 days post immunization(dpi). Furthermore-the percentage of NP + long-lived plasma cells(LLPC)in bone marrow was also detected at 42 dpi by using flow cytometry. [Results] Through the detection-we found that NP-specific antibody titers were significantly higher in NP-CVC1302 group compared with NP group(P< 0.01)-as well as the NP-specific IgG1 and IgG2a antibody titers. Significantly higher percentages of TFH cells-NP + GC B cells and NP + plasma-blasts in inguinal lymph nodes were induced in NP-CVC1302 group compared with NP group(P<0.01 or P<0.001). Moreover-significantly higher percentage of LLPC was transferred into the bone marrow of mice that received NP-CVC1302 compared with those that received NP(P< 0.01). [Conclusions] Results of the study revealed that CVC1302 improved the differentiation of TFH cells-then TFH cells selected the high-affinity GC B cells to differentiate into plasma-blasts-and finally became LLPC locating in the bone marrow-continuing to secrete antibody. This study provided the blueprint for designing new generations of immunopotentiators. [ABSTRACT FROM AUTHOR]

[Objectives]The purpose of this study is to verify the feasibility of purification of type O foot and mouth disease virus(FMDV) inactivated antigen with Gram-positive bacterial enhancer matrix (GEM) technology. [Methods]A dual functional molecular consisting of anchor protein gene and FMDV specific single domain antibody gene of type O FMDV were expressed in Escherichia coli,and the recombinant protein was named as VPA. The sedimentation named GEM-VPA was obtained with centrifugation following mixing the recombinant protein with GEM particles,and incubated at 37℃ for 30 min;GEM-VPA was mixed with FMDV antigen,and then the sedimentation was collected with low speed centrifugation after incubated at 37℃ for another 1 h,which was the purified antigen named GEM-FMDV. The feasibility for the method was evaluated with SDS-PAGE,Western-blot,protein quantification and 146S detection,and the immunogenicity for purified FMDV antigen was estimated with animal test. [Results]The dual functional protein designed in this study could be partly soluble when expressed in Escherichia coli. Moreever,this recombinant protein could bind with GEM particles and inactivated FMDV antigen at the same time. SDS-PAGE and Western-blot proved that the purified FMDV antigen existed in the sedimentation after centrifugation. Results from 146S determination indicated that the FMDV antigen recovery in this study could be above 99%,and the removal efficiency of the heterologous proteins from FMDV antigen was higher than 90%. More importantly,animals injected with the purified antigen developed more vigorous immune response and lesser side effect. [Conclusions]It is feasible for FMDV antigen concentration and purification with GEM technology. [ABSTRACT FROM AUTHOR]

[Objectives] The objective of this project is to determined whether the recombinant fluorescent antibody expressed in Escherichia coli can be used to detect Classical swine fever virus(CSFV) in direct immune fluorescence assay. [Methods] The gene VHH-EGFP synthesized according to condon usage of E.coli was cloned into pET32a (+) and then transformed into E.coli BL21(DE3). The recombinant cells were cultured in LB (containing 50 μg·mL-1 of ampicillin) and incubated in 37 °C until A600=0.6-0.8, then the target gene was induced with 0.4 mmol·L-1 IPTG for 24 h at 15 °C. The induced bacterial cell pellet was collected and cracked, the interest protein was identified by 120 g·L-1 SDS-PAGE and Western blot, after confirmation, the expressed target protein was purified with Ni-NTA His·Bind Resin and detemined by BCA protein assay kit, and then it was incubated with cells infected with CSFV, PCV2, PRV, PPV to analyze its specificity and sensitivity. [Results] SDS-PAGE and Western-blot indicated that the recombinant fluorescent antibody was successfully expressed in E.coli BL21(DE3). Although it was mainly expressed as inclusion body form, the soluble protein could be purified and concentrated by nickel column. The PK15 cells infected with CSFV showed numerous obvious green fluorescences points after it was incubated with recombinant antibody. However, there was no or only few nonspecific fluorescence when the recombinant protein incubates with cells infected with PCV2, PRV, PPV. Compared with the IFA based on the commercial monoclonal antibody, the DFA constructed in this study had more sensibility and accuracy. [Conclusions] The optimized VHH-EGFP gene could be efficiently expressed in E.coli BL21(DE3) system, and the purified recombinant fluorescent antibody was more effective comparable to the commercial monoclonal antibody, and it can be produced with a cheaper and easier process. [ABSTRACT FROM AUTHOR]

[Objectives]The paper aimed to detect microRNA(miRNA)expression profile in Japanese encephalitis virus(JEV)infected swine testis(ST)cells.[Methods]Total RNA of JEV-infected and uninfected ST cells was extracted individually by Trizol and then analyzed miRNA expression profiles by high-throughput sequencing technology. miRNA target prediction algorithm miRanda was employed to predict potential target genes of differently expressed miRNA. Gene ontology(GO)analysis was performed on the host target genes,and 6 signifacant differences in expression of miRNA were verified by quantitative real time polymerase chain reaction(RT-qPCR).[Results]The results indicated that total 112 miRNA were obviously differently expressed in the JEV-infected cells including 80 up-regulated miRNA and 32 down-regulated miRNA. The six of those miRNA were verified by RT-qPCR,the results were well matched with those of sequencing data. GO analysis of the target genes showed that these miRNA involved in the biological processes of cell metabolism and cell adhesion.[Conclusions]Our findings indicate that miRNA may play critical roles in the pathogenesis of JEV infection. [ABSTRACT FROM AUTHOR]