Expression And Screening B-cell Linear Epitopes Of PPRV Hemagglutinin Protein

Peste des petits ruminants(PPR)is an acute,contagious disease caused by peste des petits ruminants virus(PPRV),which infects mainly homebred and wild ruminants,such as goats and sheep.PPR has been reported in 20 provinces in China since it appearanced in Tibet in 2007.Developing prophylactic measures conbined rapid diagnosis with prevention will have a great significance for coping with the unexpected emergence of PPR in time.In this study,Western blot was employed for screening of linear B-cell epitopes on H protein of PPRV.The GST fusion expressed 16 mers peptides of H Protein were first separated by SDS-PAGE,then the anti-H antiserums prepared by immunizing the rabbits were used as the primary antibody for screening of the positive GST fusion expressed 16 mers peptides.The main researches of this thesis are as follows:1.Construction of prokaryotic expression plasmidThe H gene was amplified by using codon optimized H gene of Peste des petits ruminants virus China/Tibet/Geg/07-30(FJ905304)as template.Then the PCR product was inserted into cloning vector pJET1.2/blunt and the plasmid pJET1.2/blunt-PPRV-H was obtained.Then Constructed prokaryotic expression vectors pET28a-PPRV-H?pET30a-PPRV-H?pET32a-PPRV-H.2.Expression and purification of His-H proteinThe expression conditions of H protein were optimized and the optimal expression conditions were as follows,IPTG concentration 1 mmol / L,and induction time 5 h.The expressed H protein existed mainly as inclusion bodies in E.coli cells.The bacteria were broken by sonication;the precipitate was collected by centrifugation and washed several times with gradient concentration urea solution in order to improve the purity of the inclusion bodies.The recombinant expressed H protein was further purified and concentration,and the high concentration is 2mg/mL.3.Preparation of anti-H antiserumThe New Zealand White Rabbits were immunized with 0.5 mg concentrated H protein,and booster at 14 d,28 d and 42 d after the the first dose.The animals were uthanized at 56 d and the antiserums were collected.The titer of serum from the first rabbit was 1?25600,and that from the 2nd was 1?12800,and the 3rd rabbits was 1?6400.The antiserums could be recognized by the recombinant expressed H protein.4.Screening B cell linear epitope of H proteinThe DNA fragments encoding the serial overlapping 16 peptides were chemically synthesized and inserted in the expressing vector pXXGST and the plasmid pXXGST-3-P76-(1F~75F)were obtained.The sequencing confirmed plasmids were transformed into E.coli BL21.The above mentioned anti-H serum was used for screening the epitope of PPRV H by Western blot method.Totally,30 out of the 75 GST fusion expressed 16 mers peptides were identified as positive epitope candidates.