| Porcine contagious pleuropneumoniae(PCP) is a infective respiratory system disease caused by Actinobacillus pleuropneumoniae. It can infect every stage of the pigs. The infected pigs breathe hardly.The pathological changes are that the lung has different degree of hyperaemia, haemorrhage and fibroid pneumonia.The incidence and mortality of the disease are usually above 20%.The occurance tendence of porcine contagious pleuropneumoniae in our country is rising now, and it has brought serious economic damages to modern pig industry.The virulence factors of APP include capsular polysaccharide, lipopolysaccharide, outer membrane proteins, transferrin binding proteins, and haemolysins, et al. Study the function of the virulence factors can help us to develop new subunit vaccines.This is important to prevent and control porcine contagious pleuropneumonia.Two parts were included in the studies:Part one : Cloning and expression of hemolytic-toxin from Actinobacillus pleuropneumoniae.Four pair of primers were designed according to the sequence of Apxâ… A gene of Actinobacillus pleuropneumoniae serotype 5 and Apxâ…¡A, Apxâ…¢A, Apxâ…£A gene of Actinobacillus pleuropneumoniae serotype 3 published in GenBank. 1095bp,1017bp,1462bp and 537bp gene segments were amplified by PCR with the template of Actinobacillus pleuropneumoniae serotype 5 and serotype 3 which were isolated from Shandong province.The sequencing results showed that the homogeneity of Apxâ… A,Apxâ…¡A, Apxâ…¢A and Apxâ…£A gene were higher than 99% ,compared with the standard strains.The amplified DNA fragments were cloned into pGEX-5X-3 plasmid respectively. SDS-PAGE and westen-blotting analysis showed that the Apxâ… A,Apxâ…¡A, Apxâ…¢A and Apxâ…£A gene were expressed in BL21 and the expression products had immunogenicity.Part two:The purification of expression products and immunoprotection in Mice. The expression products were purified by (NH4)2S04 fractionation and Sephadex G-200 gel filtration.Subunit vaccines were made by different purified expression products and Freund's adjuvant. Mice were immunized at 30 days and 45 days with the subunit vaccines. Then the mice were challenged with the APP of serotype 1, 3, 5, 7or 10 at 60 days. The result of animal immunoprotection test showed that subunit vaccines (Apxâ… A +Apxâ…£A, Apxâ… A + Apxâ…¢A +Apxâ…£A ,Apxâ… A + Apxâ…¡A + Apxâ…¢A + Apxâ…£A) could offer 58.4%,66.6% and 91.7% protection in mice against the challenge of serotype 1, 5 and 7 APP, respectively. The mice which were immunized by four kind of expression products have slight pathological changes. These results suggested that the expression products had good immunogenicity and the subunit vaccine which contained four kind of expression products could induce better immunoprotection in mice. |
