Expression Of ORF2 Gene Of PCV2 In E. Coli And Comparision Of Immunity Effect With Inactivated Porcine Circovirus 2 Antigen

Porcine circovirus type2(PCV-2) is one of the major pathogens inducing a series of diseases such as the postweaning multisystemic wasting syndrome (PMWS),PDNS and PRDC.The prevalence of Porcine circovirus type 2 (PCV2)-associated disease has caused great economic loss and increasing attention has been paid by the pig farms around the world. Structural protein encoded by PCV-2 ORF2 contains specific antigen which can distinguish PCV-1 with PCV-2 and it is expected to play a role in serological diagnosis.Evidences from home and abroad show that gap protein can induce a certain level of antibody and immune protective effects on the body. It is the detection of recombinant vaccines and gene of choice. This article is intended to discuss the immune effect of the inactivated vaccines which was made by emulsifying freund incomplete adjuvant with different doses of expression protein that was obtained in escherichia coli and PCV2 inactivated antigen.Test one the expression of PCV2 ORF2 gene in escherichia coliAccording to the gene sequences of the PCV-2 ORF2,a pair of primers was designed and the products of the PCR were treated by agarose gel electrophoresis.A 531bp specific fragment was obtained just as the expected result. Sequencing results showed that the sequences of the fragment and the targeted gene were the same. Recombinant expression plasmid was constructed successfully via introducing endonuclease digestion products of PCR into the vector pET-32a.Positive recombinant plasmid pET32a-ORF2 and unexpressionsupporter pET32a screened were detected by PCR and endonuclease digestion.After sequence analysis,both of them were transformed respectively into escherichia coli BL21 and then induced the expression by using 1 mmol/L IPTG at 37℃.A large amount of protein fragments encoded by PCV-2 ORF2 were expressed in terms of inclusion body in escherichia coli.SDS-PAGE was used to analyse the induced products after 1,2,3,4,5,6h inducement.The results showed that expressing protein quantity increased along with the induced time and the quantity of the expression after 5h inducement was the highest.Purified by inclusion bodies lotion,specific bands were found at western blot,indicating that ideal purified effect of the expressing products were obtained.The expressed recombinant proteins can be recognized by PCV-2 monoclonal antibody.Test two the comparison of immune effect between expressed CAP protein and the inactivated antigen PCV2Expressed protein diluted to 2.5mg/ml,1.25 mg/ml and the inactivated antigen PCV2 diluted to 105TCID50/ml were respectively emulsified into vaccines.it is negatively in twenty piglets of PCV2 antibody and antigen at 30 days were randomly divided to 4 groups,5 piglets a group.Each piglet was injected intramuscular vaccines above mentioned and PBS 2ml respectively.3 weeks after the initial immunization booster it and 3 weeks after booster immunization successful drug。The 4th and 7th after the successful drug,each piglet was stimulated by KLH.The 4th,7th,11th,19th after successful drug,each piglet was stimulated by mercaptoacetic acid mediumand isolated and killed 30 days.After the initial immunization,blood of piglets were collected regularly to detect the level of ELISA antibody and neutralizing antibody.The effect of the vaccines was evaluated by the indices such as clinical symptoms and body temperature changes after successful drug,relative weight gain,viremia,Detox situation and pathological changes.The results indicated that the level of ELISA antibody and neutralizing antibody of the first group was significantly higher than the control groups (P<0.01).Relative daily weight gain of the first group was higher than the other groups after successful drug,the number of piglets in the first group which infected in vivo and in vitro detoxification was the smallest,the duration of Carriers of the virus and Detox was the shortest(disappeared at the 11th and the 7th).The results of the pathological sections showed that pathological changes of the first group was the slightest after successful drug。The data demonstrated that the effect of expressed protein vaccine was significantly superior to the other vaccines.