| Porcine contagious pleuropneumonia(PCP)is one of the most important bacterial diseases affecting the global pig industry.It is a respiratory disease caused by Actinobacillus pleuropneumoniae(APP)with lung fibrous pleural pneumonia as the main lesion.The disease is currently widespread in the world,caused serious economic losses to the intensive pig industry.C urrently,the prevention of the disease in C hina is inactivated vaccine,but because of the different serotype,complexing virulence factors and other reasons the inactivated vaccine can not provide completely protective effect.Therefo re,development of new and efficient vaccine has become hot spots in disease research.As important virulence factors and immunogen of APP,hemolytic exotoxin and outer membrane proteins play an important role in the pathogenesis and the process of the disease.Based on this,the study takes four kinds of exotoxin and outer membrane protein as a component subunit vaccine to explore its immune effect,as follows:The Apx?A and Apx?A genes were amplified from the commercial inactivated vaccine by PCR and cloned into the expression vector p ET-32 a.The recombinant plasmid p ET-Apx?A and p ET-Apx?A was confirmed by PCR,double digestion,.DN A sequencing.The results showed that the recombinant expression plasmid was successfully constructed.The recombinant plasmid was transformed into E.coli BL21(DE3)competent cells and induced by IPTG.The recombinant protein was detected by SDS-PAGE and Western blot.The results showed that r Apx?A and r Apx?A were about 50 and 45 k D Western blot analysis showed that the recombinant protein had good reactogenicity.Based on the preliminary study of the laboratory,the mice immunized with the oil adjuvant obtained r Apx?A and r Apx?A protein.The immunization group was as follows: Group ? was treated with PBS as blank control group.Group II commercialized trivalent inactivated vaccine was positive control group.Group III was a trivalent inactivated vaccine and white oil adjuvant emulsified r Apx?A,r Apx?A,r Apx?A,r Apx?A and r OMP protein mixed vaccine.The r Apx?A,r Apx?A,r Apx?A,r Apx?A and r OMP proteins emulsified with Freund's adjuvant were used as test group,r Apx?A,r Apx?A,r Apx?A,r Apx?A,and r OMP were tested in group V.Each group was given three immunization,each interval for two weeks,and the mice were given blood after one week of each immunization,i ELISA was used to determine the change of serum specific antibodies in mice.Each group of mice were treated with APP1(4.56 x 107cfu)one week after the third immunization.Results showed that the vaccine ?,? group antibody levels are basically identical which was higher than group ?,and higher than group ?.Group ? and group V had the same protective effect on APP1 challenge(4/10),which was higher than group ?(2/10)and group I(0/10),but lower than group ?(5/10).It showed that the subunit vaccine in this study was safe for mice and had good protective effect.Both the white oil adjuvant and the Freund's adjuvant could stimulate the antibody production.This study provides a reference for PCP's new vaccine and diagnostic methods. |
PDF Full Text Request