Cloning, sequencing and expressing of microneme protein 1 partial gene in toxoplasma gondii ZS2 isolate / 中华预防医学杂志

<p><b>OBJECTIVE</b>To construct a recombinant prokaryotic expression vector (plasmid) containing microneme protein 1 (MIC1) partial gene in toxoplasma gondii (T. gondii) ZS2 isolate. The gene was expressed in varied Escherichia coli (E. coli) after sequencing.</p><p><b>METHODS</b>The gene fragment coding MIC 1 from the genomic DNA of T. gondii ZS2 isolate was amplified by polymerase chain reaction (PCR). The gene was inserted to a prokaryotic expression vector pWR450-1 by digesting with restriction enzymes and linking reaction. The positive clone was screened on LB plates containing ampicillin and identified by restrictive enzyme digestion, PCR amplification and sequence analysis. The recombinant plasmid was transferred into E. coli TG1, JM109 (DE3) and DH5 alpha, and was expressed under the induction of IPTG. The expression products were identified by SDS-PAGE. The MIC1 gene structure was analyzed and compared in homology with the gene sequence of RH isolate using computer software.</p><p><b>RESULTS</b>The recombinant plasmid pWR450-1/MIC1, after cloning from acquired 471 bp MIC1 gene fragment and amplified from the genome gene ZS2, was complete homologous to the sequence of RH isolate, reflecting its highly conservative. The gene could be expressed as fusion protein with 70,000 in varied E. coli.</p><p><b>CONCLUSION</b>Recombinant plasmid pWR450-MIC1 was successfully constructed and could be expressed in different strains of E. coli, laying a foundation for research on its structure and function.</p>

Acquiring and analyzing of ornithine aminotransferase gene of Schistosoma japonicum / 中国血吸虫病防治杂志

Objective To screen and identify the novel genes from the adult worm cDNA library of Schistosoma japonicum by the expressed sequence tags (EST) strategy.Methods The cDNA clones were selected randomly and the ESTs were obtained.The novel gene was searched for homologue identity with NCBI blast programmer.The homologue of the two sequences with a high identity was compared at amino acid and nucleotide level with the BLAST programmer,and the analysis of protein was carried out with the pcgene software. Results There was a novel gene of Schistosoma japonicum which included 1 677 bp coding for 424 amino acid residues, and it was given the accession number of sequence in Genbank(AY336497).The cDNA sequence was homologous to the known ornithine aminotransferase gene of Xenopus laevis. The identity of amino acid sequence was 66%. The academic pI was 8.52, and the antigen determinant was probably from 795 to 846 on the cDNA sequence. Conclusion EST strategy is an effective measure to discover new genes of Schistosoma japonicum. The novel gene is homologous to the ornithine aminotransferase gene of Xenopus laevis.This study is helpful to further sequential and functional search of the gene.[