Construction Of Gene Targeting Vector Of Tetrahymena Thermophila And Its Site-Detected Integration And Gene Expressionin In The Tetrahymena

The ciliated protozoan Tetrahymena thermophila has proven to be a valuable biological model for molecular and genetics studies of eukaryotic cells, which have provided insights into the mechanism of ribozymes, self-splicing RNA, telomere structure and function, DNA sequence reorganization and so on. In the paper, a gene targetting vector suitable to T.thermophila was constructed and integrated into genome of T.thermophila. The work has made a substantial foundation for the expresstion of antigen gene in the T. thermophila.1. The plasmid p508.8 with the H4-I gene of T. thermophila was large-scale prepared by the method of alkaline lysis and analyzed by the method of fractional digestion. At first the the plasmid was digested by the two restricted endonuclease of HindIII and EcoRI and H4-I gene fragment was obtained ; then the residue of vector was digested by PstI and the 3400bp and 750bp fragment was obtained; At the third step,the 3400bp fragment was further cut into two small fragment of 2000 bp and 1500bp by PvuII. The fractional digestion DNA was identified by electrophoresis on the agarose gel after purified by gel extraction and alcohol precipitation.2. We constructed a gene targetting vector suitable to Tetrahymena thermophila. The vector has a insert fragment coding neo which was used as the selected marker, as well as the 5' and 3' untranslated region of H4-I gene flanking the neo gene in the insert .The 5' flanking region has the initiation codon ATG, as well the 3' flanking region initiates with the termination codon TGA of H4-I gene. The vector will further be transformed into T. thermophila whose inserts can be released as linear fragment and integrated by precise homologous recombination into the genome, based on the presence of homologous arm flanking the targeted neo gene in the insert.3. The gene targeting vector pSK-H4-I-neo was transformed into the conjungants of T. thermophila and was integrated into the genome of Tetrahymena by homologous recombination. The integration of neo gene had been identified through the...