The Study On Epitope Of Foot-and-mouth Disease Virus Type O And A. Pleuropneumoniae RTX Toxin

Foot-and-mouth disease (FMD) and Porcine contagious pleuropneumonia (PCP) aresevere, contagious diseases of pigs that cause important economic losses in industrializedpigs production. It is important field of preventive veterinary medicine of research aboutdiagnosing, preventing and controling this infectious deseases. In order to designing newvaccine and diagnosing reagent in the future the epitopes of FMD and PCP wereresearched.1. The epitopes of FMDV structural and non-structural protein 3ABCThe secondary structure of capsid protein and non-structural protein 3ABC ofFMDV was predicted by four parameters. The results showed that three FMDV structuralproteins contained many potential epitopes of the B cells.Using purifying FMDV antibody and non-structural protein 3ABC antibodyasantigens the mimotopes were screened from phage display 12-mer random peptide libraryfor 4 rounds. The 13,12,8 sense and specific positive phages clones of FMDV-P1,FMDV-VP1and FMDV-NSP3ABC were obtained. The ssDNA of the positive cloneswere abstracted for sequencing and the sequences of peptides displayed on the positiveclones were analyzed by ExPASy. By 4 rounds of screening, the results of P1 showedthat 13 positive clones had core motifs of VXLPK, the 12 positive clones had core motifsof VFLPK and KPXEP, the 12 positive clones ofthe 3ABC had core motifs of LSFPS. Theseven line epitopes of VP1 were located on 42aa-46aa,136aa-144aa,141aa-150aa,144aa-149aa,148aa-158aa,150aa-158aa,199aa-208aa of VP1. For the core sequence ofKPXEP had high homological to 43aa-47aa sides, we verified this line epitope and thatepitope amino acids of FMDVO/ES/2001 were Lys,Pro and Glu which were differentfrom other viruses. Except from VP1-5 clone, all 7 displayed sequences of this studyincluded Pro which also was included in 208 sides of VP1. So, Pro of 208 sidesconstructed above discussing epitopes. However, the 8 line epitopes of VP2 were locatedon 3aa-7aa,29aa-32aa,72aa-77aa,80aa-84aa,127aa- 129aa,131 aa- 139aa,207aa-212aa,213aa-215aa of VP2; the 3 line epitopes of VP3 were located on 29aa-36aa,33aa-38aa,131aa-138aa of VP3; the lline epitopes of VP4 were located on 6aa-8aa of VP4; the 4line epitopes of 3ABC were located on 1aa-5aa,153aa-156aa,399aa-402aa,420aa-423aa.The 9 groups of positive phage were immunized the 6-week KM mouse as thenegative control group and with FMDV inactivate vaccine. At 2-week the dose is 10¹² pfuphage per mice at first and second immunization or 10¹⁴ pfu phage per mice at thirdimmunization.. After three times bloods were taken and then the specific antibodies were detected with ELISA, and immune response types were decided with IgG1/IgG2a tilters.The specific antibody rate of 8 groups mice were 33.33%, 33.33%, 16.67%, 16.67%,20.00%, 33.33%, 50.00%, 50.00%, 0.00% (control group) after second immunization and83.33%, 83.33%, 66.67%, 83.33%, 100.00%, 83.33%, 83.33%, 100.00%, 0.00% (controlgroup). The result indicated that the mimotopes could induce the specific antibody againstFMDV. And the quantity of IgG1 was much more than the one of IgG2a in mice serum.2. The epitopes of three exotoxins of APPPorcine contagious pleuropneumonia (PCP) caused by Actinobacilluspleuropneumoniae (APP) is a severe, contagious pulmonary disease of pigs. The epitopesof four exotoxins of APP(Apxâ… ,Apxâ…¡,Apxâ…¢and Apxâ…£) were predicted. The resultsshowed that they had a lot of potential epitopes of B cell and the epitopes of Apxâ… ,Apxâ…¡,Apxâ…¢distributed behind 420aa sides. For protein of Apxâ…£consisted of 13 speciesamino acid, it had simple secondary structure and the potential B cell epitopes distributedall region of protein averagely and serried. Using purified IgG of three exotoxins(Apxâ… ,Apxâ…¡,Apxâ…¢), Mimotopes were screened from phage display 12-mer random peptidelibrary. The 5 sensitive and specific positive phages clones of different IgG wereobtained. The ssDNA of the positive clones were abstracted for DNA sequencing and thesequences of peptides displayed on the positive clones were analyzed by ExPASy method.The results showed that positive clones of Apxâ… had core motifs of RVDV, positiveclones of Apxâ…¢had core motifs of DXLXXXR,DML,SXXXRPXA. According to BLASTand analyzing predicted results, the five line epitopes of Apxâ… were located on196aa-199aa,129aa-132aa,694aa-696aa,475aa-478aa,687aa-692aa of Apxâ… ; the fourline epitopes of Apxâ…¡were located on 938aa-943aa,541aa-544aa,819aa-821aa,316aa-319aa of Apxâ…¢; the nine line epitopes of Apxâ…¢were located on 58aa-60aa,110aa-112aa,240aa-243aa,682aa-684aa,777aa-780aa,806aa-808aa,840aa-843aa,901aa-905aa, 959aa-962aa of Apxâ…¢. Common epitopes of Apxâ… and Apxâ…¡weresequences of Thr-Trp-Thr-Val,Arg-Val-Asp and Leu-Thr-Phe; Common epitopes ofApxâ…¡and Apxâ…¢were sequences of Ala-Leu-Ser.KM mices were immunized with positive phage clones and subunit vaccine. Whenmices were booster immunized, they caused differently degree antibody. The resultsshowed that the positive phage clones had immunogenicity. Isoforms of IgG wereidentified by Kits. The results showed IgG1/IgG2a＞4.