Preparation And Research The Biology Of Monoclonal Antibodies Against The Truncated E2 Protein Of CSFV

Swine Fever (SF) is an extensive popular and high contagious disease,serious caused very great economic losses and attacked the swine industry.Swine Fever is still one of the first contagious diseases.Bacterin is the important means of controling SF.With the application of Chinese lapinized vaccine of CSFV, the immuned and infected swinery were difficult to differentiate.DNA bacterin could induce the body fluid and cell immunoreaction, which had been used in the study of CSFV viccine widely because it did not exist the latency risk.In order to establish a diagnostic method of CSFV, empolder new type of Swine Fever bacterin and research the function of E2 protein,in this thesis a prokaryotic expression recombinant plasmid to express protein of E2 were successfully construct.The polyclonal antibody and mcAb against CSFV E2 were obtained. A ELISA technique was primarily established to filtrate hybridize cell, and the methods of identify monoclonal antibodies have been established too, which hoped to make a important use in defending and control Swine Fever.At first, based on the reported genomic sequence of CSFV in the GeneBank and multi-clone sites of expressive plasmid pGEX-4T-1,a couple of primers were designed, which were used to prokaryotically express antigenic A/D region of E2 protein. Using PCR technology, E(A)gene fragment about 290 bp was amplified from a recombinant plasmid PGEX-4T-1-TE2 which was conserved in our experiment.E(A)gene has been cloned into prokaryotic expression vector pGEX-4T-1 to construct the recombinant vector by recombinant DNA technique and the recombinant plasmid has been transformed into E.coli BL21 in order to obtain E(A)protein acting as immune antigen to make monoclonal antibody. Recombinant plasmid pGEX-4T-E(A) is obtained by bacterial culture PCR/plasmid PCR/double restriction endonuclease.The recombinant plasmid pGEX-4T-E(A)was induced to express large-scale fused protein GST-E(A)by IPTG, the induced recombinant bacteria were lysed by freeze-thaw and sonication. At last, we obtained the GST-E(A) inclusion body protein, which could be solubilized by sonication after the detergent lauroylsarcosine was added. The MW of the fusion protein was about 35 000 as analyzed by SDS-PAGE.The concentration of fusion protein GST-E(A) was about 1.2mg/ml by folin-hydroxybenzene, which were enough to fulfill the continuous experiment.Secondly, using the purified GST-E(A) fusion protein as antigen, monoclonal antibodies to CSFV E2 was derived from the 6-8 week-old female BALB/c mice. After were immunized three times the spleen cells of the immunized mouse and SP2/0 cells were fused in proportion of 10:1.The supernatants secreted by the fusing cells were detected 10 to 15 days later. In this research, cell fusion was made twice. The cell fusion rates were about 80% and 70% respectively, and the positive rates were 10% and 5% respectively. Two hybridoma cell lines which produced monoclonal antibodies steadily, named C3 and A1, were obtained by twice subcloning. With repetitious generation, both of the hybridoma cell lines still could secrete high titer antibodies. The titers of cell supernatant and ascites for C3 and A1 were about 1:2000,1:2000and 1:256000,1:128000 respectively. Western- blot indicated C3 and A1 react withCSFVspecially.These results demonstrated that the McAbs were specific reagent for CSFV,and recognized a linear conserved epitope on the E2 protein.Finally, In order to study antigenic property of CSFV E2, we had produced neutralizing mAb C3, which recognizes a linear epitope in CSFV. Using DNAStar software to analyse the epitopes of E(A)gene, and then expressed GST-E(A1)and GST-E(A2)protein.The results of Western-blot showed that the sequence of TAVSPTTLRT were recognised by mAb C3.This result need to validate farther by phage-displayed libraries with four rounds of biopanning.In conclusion, we have successfully expressed CSFV E2 gene, two mAbs against CSFV E2 were produced and the methods have been established to identify monoclonal antibodies and filtrate their epitope,which is helpful to develop novel vaccine and diagnosis reagent for control of CSFV, and these provide some experimental foundations for the future studies on the structure,function,epitope of CSFV E2 protein.