The Construction & Screening Of CDNA Expression Library Of Oncosphere Of Taenia Solium And Cloning & Expression Of Immunogen Gene

Cysticercosis cellulosae is the zoonosis of parasites, which endanged severely the production of stockbreeding and the health of humanbeing. It consist the three developments of Teania solium, Oncosphere and Cysticercus cellulosae, that the Oncosphere takes an important role in inbreaking body and causing response of immunology. Our strategy was constructing the cDNA expression library of the Oncosphere development stage by keeping away from the genome of Teania solium, and screening, cloning the immunogen genes, as well as expressing and analysis the interesting gene. Results of studies displayed:1. We first constructed successfully cDNA expression library of the Oncosphere development stage of Teania solium. The results showed that the transformation efficacy and recombinant rate of library were 5 X 106 and 100%, respectively. We had been cloned the genes of lOkD, 18kD and A, B, C types of 45W from the cDNA library, which illuminated the library possessed a good quality and high representation.2. We established the techniques of screening the plasmid expression library and cloning the immunogen genes. The library was screened with antiserum, and the genes of TsOl , TsO2, TsO3 were cloned and sequenced. The ORF of TsO1, TsO2, TsO3 are 393bp, 1191bp and 1818bp, respectively. Then entried the TsOl to GenBank and the number of AY327451 was given. It has been proved the TsOl is a new gene by analysis of Blast Web site. All experiment showed, the immunological screening method is a high special, sensitive and fast for screening the plasmid library.3. The interesting gene of TsOl from library was cloned to expression vector of pGEX-4T-l with special primer, and constructed the fusion expression vector of pGEX-GST-TsOl and then transformed into E.coli. of BL21. The fusion expression of TsOl was induced with IPTG, the expression product was analysis by SDS-PAGE and Western-blot. The results showed that the TsOl fusion protein has been expressed successfully in BL21. The fusion protein was about 40kD and 40% of expression level. It can be distinguished by positive serum of Neuro-cysticercosis. The product was a solubility molecular and accord to prediction of protein analysis soft.4. We successfully predicted the structure of TsOl by technique of bioinformatics. The results of prediction displayed that protein of TsOl was a no-globilar, solubility molecular with Zinc finger domain of C2H2 type binding DNA (CLACRLNFTNKSD YQVHRRLDH), Phosphorylation(56 and 68) and O-GlcNAc (2,54,68 and 69)sites.