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


METHODS
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.


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 E. coli.


CONCLUSIONS
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.
Yang
Hui-ling
HL
Research Centre of Etiology, Nanhua University, Hengyang 421001, China.
Xiao
Jian-hua
JH
Liang
Yu
Y
Zhang
Yu-kuai
YK
Liu
Chuan-ai
CA
D000818:Animals; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014122:Toxoplasma / Q000235:genetics*
D015815:Cell Adhesion Molecules; D016054:DNA, Protozoan; C105877:MIC1 protein, Toxoplasma gondii; D015800:Protozoan Proteins; D011994:Recombinant Proteins