Study On Immune Effect Of Chimeric Pcv1-2m Carrying V5 Tag Infective Cloned Plasmid In Mice

Porcine circovirus(PCV)is a member of the circovirus family of the Circoviridae family,is an envelopeless virus,and its genome is a single-stranded circular DNA.At present,four serotypes were founded,namely PCV1,PCV2,PCV3 and PCV4.Among them,PCV2 can cause postweaning multisystemic weaning syndrome and congenital tremor in piglets,miscarriage in pregnant sows,dermatitis nephrotic syndrome in adult pigs,and respiratory disease syndrome,which have caused serious harm to the world's pig industry.At present,vaccine immunization is the main means of preventing and controlling PCV2.The existing commercial PCV2 vaccines are all inactivated vaccines or subunit vaccines,the induced antibodies cannot be distinguished from the antibodies produced by wild-type infections,it is difficult to completely remove infected PCV2 from pigs.Therefore,it is of great significance to research and develop a new vaccine that can not only stimulate the body to produce humoral immunity and cellular immunity,but also distinguish between vaccine immune antibodies and wild virus infection antibodies to prevent and control PCV2-related diseases.In this study,the newly established Taq Man fluorescence quantitative PCR detection method was used to immunize mice PCV1 after immunizing mice constructed with infectious clone plasmid pc DNA3.1(+)-PCV1-2m-V5(PCV1-2m iDNA)constructed with genetic markers in the laboratory.-2m viremia was tested to determine the optimal immunization dose,and then the immune effect of immunizing Kunming mice with PCV1-2m iDNA candidate labeled vaccine was studied in order to evaluate the PCV1-2m iDNA candidate labeled vaccine in the body animal(pig)The foundation of the immune effect.The specific research contents are as follows:1.PCV1-2m rescue virus establishment and preliminary application of Taq Man fluorescence quantitative PCR methodIn order to understand the relationship between PCV1-2m rescued viremia load and induced antibodies after immunizing mice with PCV1-2m iDNA candidate marker vaccine,the optimal immunization dose and the second immunity time were determined.In this study,a pair of specific primers and probes were designed based on the PCV2-ORF2 and V5 tag sequences of pc DNA3.1(+)-PCV1-2m-v5 plasmid.The plasmid was used as the positive standard,after a series of conditions were optimized,Taq Man fluorescence quantitative PCR method was established for quantitative detection of PCV1-2m viremia load.Then,the method was used to detect viremia in 6 groups of mice immunized with different plasmid concentrations at 1 to 9 weeks after immunization;at the same time,the PCV2 antibody titer was detected by ELISA method.The results showed that the fluorescent quantitative PCR method established in the experiment had good sensitivity,specificity and stability,and the detection range can reach 1.29×10~1?1.29×10~9copies/?L;The viremia lasted until the 5th week,and the antibody could last until the 8th week.By analyzing the relationship between the change of antibody titer and the change of viral load,the optimal immune dose of mice was determined as 200?g.After the first immunization,the second immunization was carried out at 1-5w respectively.According to the production of specific antibody and the change of viral load,the 4W after the first immunization was determined as the best second immunization time.2.Study on PCV1-2m iDNA candidate marker vaccine to induce immune effect in miceIn order to understand the immune status of mice induced by PCV1-2m iDNA candidate marker vaccine,six to eight week old SPF Kunming mice were immunized in groups with the best immune dose and second immune time.One to eight weeks after the first immunization,blood was collected from the neck of mice and spleen was collected aseptically for preparation;Flow cytometry was used to determine the content of T and B lymphocyte subsets in peripheral blood cells of immunized mice;ELISA kits were used to detect Th1 and Th2 cytokines;MTT method was used to detect ConA and LPS stimulation of splenic lymphocyte proliferation in mice.The results showed that:compared with the PBS and pc DNA3.1(+)groups,the experimental group pc DNA3.1(+)-PCV1-2m-V5 plasmid immunization,CD4~+,CD19~+and CD4~+/CD8~+ratio in T and B lymphocyte subsets and Th1and Th2 cytokines were significantly increased(P<0.01);ConA and LPS stimulated splenic T lymphocytes and B lymphocytes showed that ConA induced T lymphocytes had a better effect than LPS stimulated B cells.The results showed that the candidate vaccine can induce cellular immunity and humoral immunity simultaneously,and the cellular immunity effect is better.In order to find out whether the immune effect of the candidate vaccine is different from that of different vaccines at home and abroad,as well as the transmission mode and safety of PCV1-2m rescue virus,this study compared the antibody titer of four different vaccines at home and abroad,the transmission mode of the virus after the same group feeding and isolation feeding,as well as the residual situation of the injection plasmid in the viscera and injection site after a period of immunity.The results showed that the candidate vaccines in this study had no significant difference in antibody levels after immunization(P>0.05),but were significantly different from pc DNA3.1(+)and PBS groups(P<0.01);The rescue virus can spread through horizontal contact with peers,but cannot spread horizontally through air;the target fragment has not been detected to be integrated into the mouse,it indicated that the candidate marker vaccine in this experiment is safe for mice in terms of gene integration.