High Level Expression Of The Major Antigenic Domains On Envelope Glycoprotein E2 Of Classical Swine Fever Virus In E.coli And Purification Of Recombinant Products

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major protective antigen and the major inducement that induces neutralizing antibodies against CSFV. Antigenic properties of E2 protein have been elucidated. E2 expressed in insect cell expression system was used to establish indirect ELISA for differentiating specific antibodies against CSFV. In China, application of the indirect ELISA for determination of the antibodies against CSFV by using purified whole classical swine fever lapinized virus (CSFLV) as coating antigen was limited by the availability of the antigen. The glycol chains of E2 protein were unnecessary for the combination of the antibodies against CSFV to E2 protein. It is worthy trying to express the major antigenic domains of E2 protein in engineered E.coli and to establish novel indirect ELISA using recombinant protein as coating antigen. The fused E2 protein was highly expressed in E.coli prokaryotic expression system in the experiments and is to be used as determination reagents for establishing indirect ELISA. 1: In order to establish indirect ELISA, purified CSFLV and antiserum against CSFLV were prepared. The supernatant of the calf testicular cell culture infected with CSFLV was concentrated by polyethylene glycol and separated by gel-filtration chromatography using Sepherose-4B agarose bead as chromatographic material, and the virus angtigenic feature of the collected samples was characterized. The results showed that 811,. 911 samples were high concentration of CSFLV. Typical temperature reacting patterns of the rabbits i.v injected with lml sample 811 .. 911 confirmed the above conclusion. Indirect ELISA method using purified whole CSFLV as coating antigen was established. Optimized concentration of coating antigen 811 . 911 was 17.44ugIml 15.75ug/mI, respectively. The time and temperature for antigen coating~ serum antibodies absorbing and HRP-SPA secondary antibodies absorbing were standardized as I hr. 37 C . Conditions for visualization were: 37C, 15mm. Four rabbits were immunized with the oil-coating-water vaccine consisting of 811 antigen and Freund抯 adjunctive (complete and incomplete) three times every one month. The dose of CSFLV antigen for primary immunization, secondary 3 immunization and third immunization was 625ug. .l000ug. l400ug per rabbit, respectively. Titers of the antibodies against CSFLV in immunized rabbits serum were above 1 :20000(indirect ELISA titration). 2: Based on E2 gene sequence data, a fragment 360bp in E2 gene cloned in plasznid pcDNA3 was amplified by polymerase chain reaction (PCR). The result of PCR showed that the designed fragment was amplified with expected molecular weight and named as e2. The fragment contained a major antigenic unit which included conserved and neutralizing antigenic domain A, D on E2 protein surface and a amino acid sequence with high antigenic index calculated by software DNAstar. Two enzyme sites Sal I Pst I were introduced at 5 terminal. 3 - terminal of the amplified fragment. The e2 fragment was cloned into Sal I Pst I enzyme sites of plasmid pThioHisB. The resulting recombinant plasmid was named after pThioHisB-e2 and its host E.coli. TOPIO was named as TOPe2. Inducing production experiment showed that the fused Thio-e2 protein was expressed in TOPe2 with expected molecular weight 29.7Kd. The result of Western-blotting showed that the expressed fusing protein Thio-e2 had the ant...