Prokaryotic Expression And Immunogenicity Analysis Of Truncated Capsid Protein Form PCV2

More than two decades after its emergence,Porcine C ircovirus type 2?PCV2?,an immunosuppressive virus,was the primary cause of Postweaning Multisystemic Wasting Syndrome?PMWS?and its related diseases,which has caused huge economic losses in the pig industry worldwide.So far,vaccination is still the main strategy to prevent PCV2.The structural protein?Cap?of PCV2,as an important immunogenic protein,has the power to self assembling into virus-like particles in vitro,and becomes ideal antigen for PCV2subunit vaccine.Commercial Cap-based subunit vaccine existed at home and abroad.But the high preparation cost encodes its large-scale promotion.Therefore,it's urgent to find a new technology strategy to develop an efficient,safe and cheap subunit vaccine.In this study,we successfully expressed the soluble dCap protein by E.coli p ET28a,a Prokaryotic expression system,and then evaluate its immunogenicity by immunizing pigs.Whatmore,a preliminary research on high density fermentation c ulture of pET28a-dCap are made.The main contents are as follows:1.Prokaryotic expression of truncated capsid protein from PC V2A truncated cap gene?dcap?with a deleted N-41 amino acid gene was optimized and synthesized of PCV2 119-GD01 in our laboratory.Effective secreted expression was achieved in E.coli p ET28a-dCap by IPTG induction.SDS-PAGE and Western-blot show that,a protein,27 kDa,was mainly existed in soluble form,accounts for 40%in total soluble proteins.Furthermore,nice reaction activity was demonstrated with the monoclonal antibody of PCV2/Cap.Therefore secreted expression of dCap protein made a good foundation for the subsequent development of PCV2 subunit vaccine.2.Dose screening of PCV2 subunit vaccine in mice and immunogenicity test in pigsThe dCap protein was purified by N i-NTA spin columns.Different amount of antigen?80?g,60?g,40?g,20?g?mixed with the aluminum hydroxide adjuvant in proportion to immunized SPF Balb/c mice.Specific antibodies were detected by indirect ELISA K it from1 to 7 week.Serum antibody test demonstrated that a positive relation between the antibody level with immune dose and the best immune dose was 80?g.To evaluate the immune effect of vaccine in piglets,two groups of the experimental vaccine are prepared with 0.8 mg dCap protein mixed 201 and alhydrogel adjuvant separately,positive control group,those 3 group are vaccined with 3 week old piglets,individually.At the same time,virus-attack group and negative control group are also established.Blood collection after immunization in every week,and then injection PCV2119-GD01 virus after 28 days and all pigs were sla ughtered 28 days later.The immune efficacy of the vaccine was evaluated by antibody detection,clinical manifestation,body temperature change,average daily weight gain and the visceral virus load.Whatmore,to explored the effect of maternal antibody to the vaccine,the vacinne group was divided into high maternal antibody?H-MDA,S/P>0.3?and low maternal antibody?L-MDA,S/P<0.16?.The results showed:?1?O n the whole,the viral load in immune group was lower than that of negative control group,there are significant difference between 201adjuvant-assisting dCap group and positive control group with virus-attack group in the case of H-MDA;?2?From the perspective of H-MDA and L-MDA:compared with H-MDA,piglets immunized with L-MDA produce higher antibody levels and ADGW.The results indicated that H-MDA could interfere with the immune effect to some extent,but it could be ignored in most farms;?3?From the perspective of different adjuvants:compared with the group of Alhydrogel adjuvant-assisting dCap group,201 adjuvant-assisting dCap group produce higher antibody levels,higher RDGW and significant lower viral load?P<0.05?.Therefore,the immune effect of 201 adjuvant was superior to Alhydrogel adjuvant,which stimulate a rapidly and long-lasting immune response.3.The conditions'optimization of high density fermentation for recombinant E.coli pET28a-dCapThe induction occasion,temperature and time were optimized in 5 L bioreactor.In words,the optimal expression condition follows:using DO-stat?30%⁵0%?to control the stirring speed??800 rpm?,ventilation?8 L?and the rate of feeding flow,10%transfer,37?.When OD₆₀₀ being 50⁵5,the recombinant E.coil was cultured in 30?for 6 hours after added 0.5 mmol/L IPTG.The final O D₆₀₀ reached 60⁶5.The dCap protein accounts for30%in total soluble proteins,and about 2.6 g/L.Mice experiment showed that the experimental group induced a significant higher increase of specific PCV2-Cap antibody than positive control group and negative control group.Therefore,the immune effect of recombinant dCap protein obtained by fermentation was preliminarily verified.Whatmore,this strategy might provide potential uses in developing Cap-based subunit vaccine in large-scale industrial production.