Expression Of E2 Gene Of Classical Swine Fever Virus Shimen Strain On Immortalised Swine Vein Endothelial Cells

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious vital disease of pigs. It is harmful to the global pig farmings and is listeded among type-A diseases of Office International des Epizooties(OIE).The genome of CSFV consists of a single-stranded positive RNA of about 12.3 kb, enco- ding three envelope glyprotein, i.e. E2, E0(Erns)and E1. E2 is the major protective antigen of the virion. It can stimulate organism to produce neutralizing antibody to resist CSFV velogenic strain aggression. Because it is impossible to discriminate between immunization with conventional vaccination and natural infection on serodiagnosis, and attenuated vaccines can through placenta to results in fetal death,weak piglet,congenitally infected pigs which can induce immunotolerance. But genetically engineering vaccine is a new method to solve these problems. So, the paper chose E2 as target gene to be expressed on immortality swine vein endothelial cells (SUVEC), to construct initial genetically engineering vaccine. And it has suitable developing potential on application, because the SUVEC is the physiological cell line without oncogenicity. The genetically engineering vaccines have no problem on biosafty. Main research contents include:1. The construction of recombinant retroviral vector pBABE-puro-E2. By a series of molecular biological methods, E2 gene of CSFV Shimen strain was inserted into retroviral vector pBABE-puro named pBABE-puro-E2. And the result of enzyme digestion and PCR identification showed the construction was successful. So, the recombinant plasmid was extracted with alkaline lysis method and purified with PEG 8 000, for the next transfection to procure retrovirus pseudovirion.2. Packing retrovirus pseudovirion. The recombinant plasmid pBABE-puro-E2 and the pVSV-G plasmid were transferred into 293GP cells by calcium phosphate cotransfection, and procured the packing retrovirus pseudovirion.3. Selected the positive cells of expressing E2 protein and rudiment immunity test. Packing retrovirus pseudovirion infected the SUVEC and the positive cells were gotten by optimal concentration of puromycin, then the cells were analyzed by flow cytometry. Identified by indirect immunofluorescence test and RT-PCR, the CSFV shimen strain of E2 proteins were successfully expressed on SUVEC. The positive SUVEC as a gene engineering vaccine were inoculated into the Balb/c mice by intraperitoneal injection. After the third immunization, the mice were killed to collected sera and detected for anti-CSFV antibodies by ELISA. The result indicated that the sera of mice contained anti-CSFV antibodies and the valence of antibody is 1:3 500.