Preparation Of Rabbit Hemorrhagic Virus Baculovirus Vector Inactivated Vaccine And Its Immune Effect

Rabbit hemorrhagic hisease（RHD）is an acute,severe,highly contagious and high mortality disease caused by rabbit hemorrhagic virus（RHDV）.The typical pathological changes of the disease are hemorrhage of the respiratory system,liver necrosis,and bleeding.It is characterized by parenchymal organ bleeding,edema,congestion,etc.Rabbits are generally susceptible to RHDV from 5 to 6 weeks,and are fully susceptible at 8 to 9 weeks.The mortality rate of adult rabbits is as high as 90%～100%,which brings huge economic losses to the rabbit breeding industry worldwide.Based on the morphological,biochemical,and molecular biological characteristics of RHDV,the International Committee on Taxonomy of Viruses classified RHDV as a genus of rabbit virus in the family Calicivirus.The virus is a single-stranded sense RNA virus.The genome is composed of 7437 nucleotides and has two open reading frames（ORFs）that encode multiple proteins.The capsid protein is the only structural protein of RHDV,called VP60,and Inducing immune responses against viral infections is directly related.There is no specific treatment for RHD.Vaccination of rabbits is an effective treatment for preventing hemorrhagic disease in rabbits.The currently widely used RHDV vaccines are rabbit hemorrhagic virus inactivated vaccine,rabbit viral hemorrhagic multiple killer pasteurellosis double inactivated vaccine,rabbit hemorrhagic virus pasteurella clostridium triplet inactivated vaccine,etc.Although it has a good immune effect for a long time,it has its own inevitable shortcomings.Since the method of culturing rabbit hemorrhagic virus in vitro tissue cells has not been established,the inactivated vaccine of rabbit hemorrhagic virus baculovirus vector in this study is a study of the use of molecular biology to make a genetic engineering subunit vaccine of rabbit hemorrhagic virus.That is,the recombinant rabbit hemorrhagic virus VP60 baculovirus（BAC-VP60 strain）was constructed through molecular biology and other technologies.The infected Sf9 cells were infected to express the VP60 protein and added an appropriate adjuvant.After inactivation,it was made into genetic engineering.Subunit inactivated vaccine.The vaccine can not only play an immune preventive role for rabbits of all ages,but also can well solve the biological safety problems brought about by tissue-inactivated vaccines.1.Preparation and identification of rabbit haemorrhagic virus baculovirus vector virus species.Use SF-SFM medium（containing 10%fetal bovine serum）to culture Sf9insect adherent cells in a cell flask.When the cells are covered with the cell flask,press 1%of the total cell culture fluid（MOI:0.5）Inoculation,culture at 27-28°C,continuous observation for 5 days,when 85%of the cells have lesions（enlargement,rounding,rupture,refractive enhancement,etc.）harvest,place the cell harvest in 2～Store at 8℃.Identification:The virus content(TCID₅₀)of the virus solution was determined to be≥10^8.0/m L;the Hemagglutination titer（HA）was≧2⁸;the recombinant baculovirus DNA was extracted,and PCR amplification and sequencing were performed using primers that amplified the VP60 gene,and the gene sequence was consistent with the capsid protein VP60Gene sequence;identified by Western blotting,the recombinant VP60 protein was specifically expressed,with a target band of about 60 k Da in size;after safety and immunogenicity tests were met;the test was performed according to the current appendix of the Chinese Veterinary Pharmacopoeia,no Contaminated by bacteria,mold,mycoplasma and foreign pathogens,purity meets requirements2.Preparation process and identification of semi-finished product of rabbit hemorrhagic virus baculovirus vector inactivated vaccine.In this study,7.5L,15L,60L bioreactors were used to systematically study the Sf9 insect cell suspension culture process,and the key technical parameters such as initial cell culture concentration,bioreactor agitation method,agitation speed,DO,and medium supply method were optimized..Optimize virus inoculation dose（MOI）,DO,culture temperature parameters,etc.Sf9 insect cell suspension culture process:initial cell growth concentration is 8*10⁴cells/m L,culture volume is 1/3 of tank volume,speed is 60r/min,dissolved oxygen condition is 30%,temperature is 27℃,medium supply method It is a perfusion method.After 96 hours of culture,the cell peak value reaches 8.0～9.5*10⁶cells/m L;Virus culture process:initial concentration of cell poisoning2.0*10⁶cells/m L,dose of poison:0.5MOI,speed 60r/min,30%dissolved oxygen,dissolved oxygen condition 30%,temperature 27℃,one-time harvest culture After 72 hours to 96 hours of culture,the cells were swollen,rounded,ruptured,and the refractive index increased,and the cell cytopathy（CPE）such as granular substances appeared in the cytoplasm.HA)can reach more than 28,even up to more than 2¹⁰.After inactivating the harvested cell culture with 10%formaldehyde,the final formaldehyde concentration was0.2%.Purity test,in line with the current"Chinese Veterinary Pharmacopoeia"standard;at the same time by inactivation test without cell disease and hemagglutination.3.Preparation and inspection of finished product of rabbit hemorrhagic virus baculovirus vector inactivated vaccine.The qualified semi-finished products and aluminum hydroxide glue shall be thoroughly mixed and shaken according to the ratio of 9:1,quantitatively packed,sealed with stoppers,and labeled.According to the biological product inspection procedures,the quality of the finished product of rabbit haemorrhagic virus baculovirus vector inactivated vaccine is tested for indicators such as traits,loading,sterility,safety,potency,efficacy testing,and formaldehyde residue.fulfil requirements.4.Immune effect of inactivated vaccine against rabbit hemorrhagic virus baculovirus vector.Based on the above research,a total of 11 batches of vaccines were prepared in the production workshop,with a total of about xxxx portions and the safety,immune efficacy,immunization period,and shelf life of vaccine products were studied.The results show that all batches of vaccines are safe for rabbits;the vaccines have good immunization efficacy,and the immunization period（immunization duration）reaches 6months;the product is stored at 2-8°C for at least 24 months.In this study,the rabbit hemorrhagic virus baculovirus vector inactivated vaccine was successfully prepared,which can produce a good immune response to all rabbit species,and protect the experimental rabbits against strong virus attacks,and the immune efficacy reaches100%.It shows that the suspension production process is reasonable,stable and applicable.At the same time,it also laid a solid foundation for subsequent larger-scale production,and promoted the introduction of rabbit hemorrhagic virus baculovirus vector inactivated vaccines into the market,and can gradually replace tissue inactivated vaccines with increasingly difficult raw materials.The industrialization of genetically engineered vaccines for haemorrhagic rabbits has potential application and development value.