Preparation Of Monoclonal Antibodies And Expression In Pichia Pastoris For A08 And A Rhoptry Protein Of Eimeria Tenella

Eimeria tenella which cause severe intestines disease and death is an important parasite for chicken, it can inflict large economic losses on the poultry industry. The invasion stage of host cell by parasite is the major pathopoiesis period, this process involve a number of invasion-related factor. In order to develop the novel approaches to control coccidiosis, we will have prepared the monoclonal antibody against protein A08(EtA08) and a rhoptry protein(EtRP) of E. tenella, and constructed the Pichia pastoris expression system of A08 gene.1. Preparation and characterization of monoclonal antibody against EtA08 and EtRPAfter BALB/c mice were immunized four times by EtA08 and EtRP which were expressed in E.coli, cell fusion was conducted according to standard procedure. After screening by indirect ELISA and Western blot, two hybridoma cell lines that secreted specific McAb against EtA08 were obtained and designated as 1G7 and 4F11 respectively, one against EtRP was obtained and designated as 2E3. The subclass of 1G7 and 2E3 both were characterized as IgG2a, and 4F11 was IgM. The chromosome numbers of the three hybridoma cell lines all were 94 to 108. The titres of culture supernatants of 1G7,4F11,2E3 were 1:1.6×10~5,1:64,1:40, respectively, and their titres of mice ascites were 1:1×10~6,1:1.02×10~5,1:2.56×10~4, respectively. The distribution of EtRP on the sporozoites were investigated using the McAb 2E3. The results indicated that the protein stained apical tips of sporozoites, but when sporozoites were incubated in DMEM at 41℃for 1 h, the protein distributed in whole body of sporozoites. The results of invasion inhibition assay showed that, the inhibition rate of McAb 2E3 inhibiting sporozoites invaded DF-1 cells was 63.5%, it demonstrated that EtRP surely played a important role in the process of sporozoites invasion.2. Expression in Pichia pastoris of EtA08 geneAccording to the A08 gene sequence of E.tenella was reported, a pair of specific primers was designed to amplify the A08 gene with the A08 cDNA as template by PCR, and the PCR product was approximately 1083 bp in size. The A08 gene was cloned into the pGEM-T-easy vector. Identification by double-enzymatic digestion and sequencing indicated that the A08 gene cloned successfully. Then the target gene from pGEM-T-A08 which was digested by EcoR I and Not I, was cloned into pPIC9k vector which was digested with the same endonuclease to pGEM-T-A08, and the recombinant plasmid pPIC9k-A08 was constructed. After being identificated by double-enzymatic digestion and linearized with Sal I, the recombinant plasmid was transformed into P.pastoris GS115 by electroporation. The resistant recons were screened by G418 and the expression of protein A08 in recons was induced with methanol. SDS-PAGE analysis of the expressed product showed that the A08 of E.tenella was expressed successfully in P.pastoris. Western-blot preliminary analysis demonstrated that the expressed product had immunoreactivity.The above results of this paper have established a favourable basis for further studying the biologic function of protein A08 and rhoptry protein EtRP of E. tenella, and provided important reference value for developing recombinant vaccines and new drugs against coccidiosis.