| Chlamydia felis?C.felis?is the pathogen of Chlamydia felis'disease.It is the main pathogen causing Feline upper respiratory tract disease?FURTD?,together with Feline Herpeto Virus-1?FHV-1?and Feline Calici Virus?FCV?.Light causes conjunctivitis?rhinitis and pharyngitis,and severe cases can cause pneumonia?arthritis?claudication,and so on.In clinical cases,humans and animals infected with Chlamydia felis are cured by antibacterial treatment?such as Amphotericin?,but are prone to relapse.Because the status of Chlamydia felis infection is widespread and recurrence,the study of safe and effective vaccine is the best way to control the transmission of Chlamydia felis infection,so as to prevent human and animal infection of Chlamydia felis disease.At present,the research on Chlamydia felis vaccine mainly focuses on new vaccines,live attenuated vaccines or inactivated biological vaccines.During the research process,it was found that the use of whole-bacteria-based sterilization vaccine or live attenuated vaccine was not a good choice,because some specific antigenic components stored by the whole bacteria vaccine could lead to certain pathological reactions,while the live attenuated vaccine might be anti-strong,has a potential threat to public health.The new vaccine is safe and effective,does not contain the side effects of the Chlamydia whole-cell vaccine.There is no risk of persistent infection of the body caused by the return of the ancestors,and it can also induce cellular immunity and humoral immunity to protect the body from infection by Chlamydia.However,the choice of protective antigen and antigen presenting vector is the two major problems currently facing in the development of safe and effective new vaccines.The major outer membrane protein?momp?is highly conserved in different chlamydial serotypes.This protein contains both T cell and B cell epitopes and can induce specific anti-Chlamydia felis immune response.Therefore,this experiment selected the main outer membrane protein of Chlamydia felis as a protective antigen.It has been reported in the literature that the recombinant rabies vaccine an immunogen has the characteristics of safety,high efficiency,can stimulate the body to produce high levels of neutralizing antibodies,and can effectively induce the immune response of T cells.The recombinant rabies virus rescued by reverse genetics has been used as a vector for the expression of different proteins due to its high virus yield,high expression level of foreign protein,and ease of mass production.Baculovirus expression vectors have been favored in recent years.Baculovirus expression vector system based on AcMNPV has developed rapidly and has been widely used.Baculovirus expression vector has many advantages:the recombinant protein expressed by baculovirus expression vector has similar biological activity with natural protein;Exogenous gene has a large capacity,which is suitable for the cloning of large genes and multiple genes at the same time.Biological safety is good,baculovirus does not infect mammals,including humans;The recombinant protein is highly expressed under the regulation of double promoters.Rabies virus expression vector and Baculovirus expression vector are candidates for antigen presentation vector by virtue of their own advantages.In this study,Rabies Virus SRV9 strain reverse genetic vector and Baculovirus expression vector were used to construct a recombinant virus expressing momp,and the immunogenicity was evaluated through serological experiments.Rescue of recombinant Rabies Virus?SRV9-MOMP?:a recombinant plasmid pUC57-SRV9-MOMP?P/M?containing the structure of MOMP was constructed by inserting the main outer membrane protein gene?MOMP?of chlamydia felis into the spacing region of the P and M genes based on the reverse genetic operating system of RABV SRV9 strain.After PCR identification and gene sequencing,the correct recombinant rabies virus infectious whole genome plasmid pUC57-SRV9-MOMP?P/M?was transfected with its auxiliary plasmid to BSR-T7 cells to save the recombinant virus SRV9-MOMP.The insertion and expression of exogenous genes were identified from two aspects of genome and protein,and the growth kinetics,genetic stability and pathogenicity of recombinant virus were studied.Immunofluorescence observation showed that recombinant rabies virus was successfully saved.RT-PCR results showed that the MOMP gene had been successfully inserted into the recombinant virus genome and was genetically stable during passage.Western Blot analysis proved the successful expression of momp in SRV9-MOMP.The growth kinetics curve showed that there was no significant difference in the proliferation level of the recombinant virus and the mother virus in BSR-T7 cells.The titer of SRV9-MOMP virus reach 1×108.125TCID50/mL;The intracranial challenge experiment of lactating mice showed that the pathogenicity of recombinant virus was weaker than that of mother virus.This part of the experiment constructed a recombinant rabies virus infectious whole genome plasmid pUC57-SRV9-MOMP?P/M?inserted into the MOMP gene of chlamydia felis,which saved the recombinant SRV9-MOMP virus with high titer and stable exogenous protein MOMP.The recombinant virus can be used as a vaccine candidate for further immune research.Construction of recombinant baculovirus?Ac-momp?:based on the baculovirus expression vector,the chlamydia felis MOMP gene was cloned into the multiple cloning site of the pFastBacI vector to obtain the recombinant plasmid pFastBacI-MOMP?pF-MOMP?.The correct recombinant plasmid identified by PCR was transformed into DH10Bac E.coli receptor state.After blue-white spot screening,the correctly identified recombinant Bacmid-MOMP?Bac-MOMP?was transfected into Sf9 cells to save the recombinant baculovirus AcMNPV-MOMP?Ac-MOMP?expressing MOMP.Observation of cell morphology and fluorescence showed that the recombinant baculovirus was successfully saved.Western Blot analysis showed that momp could be stably inherited in recombinant baculovirus?Ac-momp?.The recombinant virus is safe and easy to be prepared in large quantities,and can be used as a vaccine candidate for further immunological research.In order to investigate the immunoprotective efficacy of the inactivated viruses SRV9-MOMP and Ac-MOMP:the recombinant viruses SRV9-MOMP and Ac-MOMP were inactivated and immunized with the highest dose,immunized by intramuscular injection,immunized 2 times,14 apart.Serum was collected 2 weeks,4 weeks,6 weeks,and 8 weeks after the first immunization.Finally,Chlamydia felis was used for the challenge test.Neutralization test was used to detect the in vivo production of momp neutralizing antibody levels by inactivated recombinant virus SRV9-MOMP and Ac-MOMP.The immunoprotective efficacy of inactivated virus was evaluated.The neutralizing antibody level of RABV in cats immunized with inactivated virus SRV9-MOMP was detected by FAVN.The results of the neutralization test showed that the serum of the immunized virus SRV9-MOMP and Ac-MOMP-immunized cat contained high-efficiency anti-momp neutralizing antibody,which can protect the body from infection by Chlamydia trachomatis.The FAVN assay indicated that the inactivated virus SRV9-MOMP immunized cat serum contained high titers of RABV neutralizing antibodies.The results of this experiment showed that both inactivated virus SRV9-MOMP and Ac-MOMP could induce cats to produce momp neutralizing antibodies against Chlamydia felis.After boosting immunity,antibody level increased significantly.Compared with inactivated recombinant baculovirus Ac-MOMP,Inactivated SRV9-MOMP induces cats to produce higher momp neutralizing antibodies and high titers of RABV neutralizing antibodies.Based on the inactivated SRV9-MOMP recombinant virus,it has good immunogenicity,high titer and high-efficiency expression of foreign proteins,and can be prepared in large quantities.It provides a new idea for the development of a new"low-cost-efficient"Chlamydia felis vaccine. |
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