Research And Development Of Combined Genetic Engineering Inactivated Vaccine Against Porcine Circovirus Disease (Type 2) And Mycoplasma Hyopneumoniae

Porcine circovirus type2(PCV2)is is the main pathogen of Postweaning multisystemic exhaustion syndrome(PMWS)and Porcine circovirus associated diseases(PCVAD).Cap protein is the main immunogenic protein of PCV2,and it is also the main target of genetic engineering vaccine development.Mycoplasmal Pneumonia of Swine(MPS)is a chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae(Mhp).MPS is often co-infected with other pathogens in clinical practice.The co-infection of Mhp and PCV2 was most common.Porcine Mycoplasma pneumoniae membrane protein P46 is a major immunodominant protein on the surface of MHP.It is highly species-specific and is often used as a target protein in the detection of porcine Mycoplasma pneumoniae.At present,the main means of prevention and control of PCV2 and Mhp infection is vaccine immunization.The quality of the existing commercial vaccines is uneven,and the immunization procedure is complicated,which is not conducive to the basic immunization work.With the development of genetic engineering technology,the combination of genetic engineering vaccine with antigen protein of many diseases can not only achieve the use effect of combined vaccine,but also solve the problem of antigen component compatibility of conventional vaccine,and realize multi-protection with one injection.Therefore,it is of great application value to develop a genetically engineered double vaccine that can prevent both PCV2 and Mhp.The objective of this study was to obtain the good immunogenicity of PCV2 Cap protein and Mhp P46 protein through Escherichia coli expression system,prepare the genetic engineering dual vaccine to immunize experimental animals,and preliminarily analyze its immune effect on piglets.1.PCV2 Cap protein expression and immune protection.In order to improve the soluble expression of PCV2 Cap protein in Escherichia coli,the PCV2 ORF2 gene was optimized to make the soluble expression rate of target protein reach more than 90%.The expression products were analyzed and identified by SDS-PAGE and Western-Blot,and the recombinant protein was prepared to immunize healthy susceptible piglets.The serum PCV2 antibody titer in the immunized group was not less than 1:1600.The results of animal challenge experiment showed that the immune group was 5/5 protected.2.Expression of Mhp P46 protein and immune protection.Three TGA codons of Mhp P46 gene were mutated into TGG by point mutation kit,and the recombinant expression plasmid PET-32A-P46 was constructed,which was introduced into competent Escherichia coli cells and induced by IPTG to successfully express Mhp P46 protein.P46 protein immunized susceptible piglets,the immune group were normal in spirit,diet and body temperature,did not show the typical clinical symptoms of mycoplasma pneumonia,necropsy examination showed no lung tissue lesions.The control group showed typical mycoplasma pneumonia symptoms and lesions.P46 protein can effectively protect immunized pigs against Mhp infection.3.Combined Genetic Engineering Inactivated Vaccine against Porcine circovirus disease(Type 2)and Mycoplasma hyopneumoniaeOn this basis,the vaccine preparation process was optimized,the antigen inactivation method and antigen protein concentration of Porcine circovirus disease(Type 2)and Mycoplasma suis pneumonia gene engineering double inactivated vaccine were determined,and 15 AVG adjuvant was selected.The water phase and oil phase were mixed in 85:15 ratio,and the oil in water(O/W)vaccine was emulsified at 4500r/min and 25℃ for 40 minutes.All the tests of the trial vaccine were in accordance with the Quality Standard for the Genetic engineering two-combination inactivated vaccine of porcine circovirus type 2 and Mycoplasma suis Pneumonia,which indicated that the strain constructed was stable and immunogenic,the production process was reasonable and scientific,the product quality was stable,and the vaccine could be large-scale production.The safety test of the genetic engineering dual vaccine was carried out.The results showed that the vaccine was safe and had no side effects,little stress and no other side effects on pigs.The efficacy test of immunizing healthy susceptible piglets with the vaccine: the serum PCV2 antibody titer of the immune group was not less than 1:1600,4/5 of the immune group and 0/5 of the non-immune challenge group were protected by the PCV2 challenge test.After challenge with Mycoplasma pneumoniae virulent strain,the average reduction rate of pneumonia lesions in immunized group was 81.6%.The results indicated that the vaccine had good immune protection to the experimental pigs.The results of immunization duration test showed that the titer of PCV2 antibody in serum of the experimental pigs remained above 1:1600 at 120 and 150 days after immunization.The reduction rate of pneumonia lesions in the challenge immunization group was 77.9% and 72.6% at 120 and 150 days after immunization.Therefore,the duration of immunity of the inactivated Porcine circovirus disease(Type 2)and Mycoplasma porcine pneumonia genetic engineering vaccine was set as 4 months.On the market at present no genetic engineering of type 2 porcine circovirus virus and mycoplasma pneumoniae for swine,by E.coli expression system,this study obtained good immunogenicity of porcine circovirus virus type 2 Cap protein,mycoplasma pneumoniae P46 protein,prepared genetic engineering vaccine for the prevention of PCV2 and Mhp duplex of of experimental animals,security,immune protection is good,and the duration of immunization is 4 months,and the new Veterinary medicine certificate has been obtained from the Ministry of Agriculture,which will provide quality vaccine protection to the pig industry.