[Cloning, sequencing and expressing of microneme protein 1 partial gene in XXXX ZS2 isolate].

@entity869 @entity214 @entity526 @entity3204
OBJECTIVE: To construct a recombinant prokaryotic expression vector (plasmid) containing microneme protein 1 (MIC1) partial gene in @entity3204 ( @entity3204 ) ZS2 isolate. The gene was expressed in varied @entity526 (E. coli) after sequencing. METHODS: The gene fragment coding MIC 1 from the genomic DNA of @entity3204 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 @entity869 and identified by restrictive enzyme digestion, PCR amplification and sequence analysis. The recombinant plasmid was transferred into @entity526 TG1, JM109 (DE3) and DH5 alpha, and was expressed under the induction of IPTG. The expression products were identified by @entity214 -PAGE. The MIC1 gene structure was analyzed and compared in homology with the gene sequence of RH isolate using computer software. RESULTS: 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 @entity526 . CONCLUSION: Recombinant plasmid pWR450-MIC1 was successfully constructed and could be expressed in different strains of @entity526 , laying a foundation for research on its structure and function.
