Construction And Immunogenicity Of Recombinant Adenovirus Vector Vaccine Of M. Hyopneumoniae And Porcine Circovirus Type 2

Mycoplasma hyopneumoniae is the main pathogens causing mycoplasma pneumonia of swine（MPS）.Porcine circovirus type 2（PCV2）is the main pathogen of post-weaning multisystemic wasting syndrome（PMWS）.Mhp and PCV2 not only cause primary infections in pigs,but more importantly cause damage to the immune system of infected pigs,resulting in Mhp and PCV2 often being co-infected or secondary to other pathogens,making it more difficult to prevent and control respiratory infections in pigs.The main means of preventing and controlling Mhp and PCV2infections in China is vaccination.In order to reduce the number of vaccinations and to reduce the impact of factors such as stress caused by vaccination on pig production,combined vaccination has become a trend in the development of the animal vaccine industry.Currently,combined vaccines against both Mhp and PCV2 pathogens have been approved for marketing,but most are prepared by mixing whole inactivated Mhp and baculovirus-expressed PCV2 Cap proteins in a certain ratio,while diphasic vaccines for Mhp and PCV2 genetically engineered subunits and live vectors are still not available.Currently,adenoviral vector systems derived from human adenovirus have been widely used in the development of new generation genetically engineered vaccines due to their advantages of good safety,high titer and strong induction of humoral,mucosal and cellular immune responses.Based on this,the Mhp P97R1,P46,P42 proteins and PCV2 Cap proteins,which were confirmed to be immunogenic by prokaryotic and baculovirus expression systems in the laboratory,were cloned into a replication-deficient human type 5 adenovirus vector,and the recombinant adenovirus r Adv-P97R1,r Adv-P46,r Adv-P42 and r Adv-Cap were packaged in the same ratio and then mixed as a live vector vaccine.The recombinant adenoviruses r Adv-P97R1,r Adv-P46,r Adv-P42 and r Adv-Cap were mixed in proportion to each other and used as live vector vaccine to investigate their effect on inducing an immune response in BALB/c mice and to lay the foundation for the development of a diphasic adenovirus vector-based vaccine against Mhp and PCV2.Firstly,to facilitate the identification and detection of the protein in subsequent experiments,the Cap protein was successfully purified and polyclonal antibody against Cap with high specificity and high antibody potency was prepared.Secondly,recombinant adenovirus backbone vectors Adv-P97R1,Adv-P46,Adv-P42,Adv-Cap and Adv-EGFP carrying the P97R1,P46 and P42 genes of Mhp and the Cap gene of PCV2 and carrying the fluorescent tag of EGFP were constructed and transfected with HEK293 cells respectively for direct fluorescence observation.The expression of P97R1,P46,P42 and Cap proteins was confirmed after identification of recombinant adenovirus-infected HEK293 cells by RT-PCR and Western Blot.On this basis,recombinant adenoviruses r Adv-P97R1,r Adv-P46,r Adv-P42 and r Adv-Cap were obtained,which were amplified and purified in large quantities.An equal volume of5×10⁷ TCID₅₀ of r Adv-P97R1,r Adv-P46,r Adv-P42 was mixed with 5×10⁷ TCID₅₀ of r Adv-Cap to immunize BALB/c mice,which were tested by indirect ELISA show that,compared with PBS,wild-type adenovirus r Adv-wild and commercial the recombinant adenovirus stimulated specific Ig G antibodies（p<0.0001）.Meanwhile,compared with the control group,the Ig G1 of anti-P46 and anti-P42 showed an overall decreasing trend,while the Ig G1 of anti-P971 and anti-Cap showed a stable and unchanging trend.The Ig G2a of anti-P97R1 of r Adv-Mhp and r Adv-PCV2 showed an increase and then a decrease and maintained a stable value until the end of the immunity period.The Ig G2a of anti-P97R1 and anti-P46 of the rest of the experimental groups maintained a basically unchanged level until the end of the immunity period.The Ig G2a of anti-P42 showed a transient decrease at the day 21 and day 35,but showed a rising trend later.Spleen lymphocyte proliferation showed a significant proliferative effect（p<0.0001）after stimulation with the corresponding stimulating agent.These results suggest that this recombinant adenovirus can trigger specific humoral and cellular immune effects in mice after immunisation.This study provides the basis for the development of an adenoviral vector-based combined genetically engineered subunit vaccine against Mhp and PCV2.