[Construction and sequencing of recombinant plasmid pcDNA3/GRA1 from Toxoplasma gondii].
OBJECTIVE
To construct a mammalian expression plasmid pcDNA3/GRA1 to express dense granules antigen-1 (GRA1) of Toxoplasma gondii, and to lay a foundation for further studying the protective immunity of pcDNA3/GRA1 as a DNA vaccine.


METHODS
The GRA1 opening reading frame (ORF) was amplified with two specific primers. The ORF and plasmid pcDNA3 were digested with EcoR I and Xho I respectively and the ORF was ligated into the pcDNA3 at polylinker. The recombinant vector pcDNA3/GRA1 was characterized by PCR, restriction enzyme digestion, and sequencing analysis.


RESULTS
The expected ORF, 573 bp long, was amplified by PCR, and inserted into plasmid pcDNA3. PCR, restriction enzyme digestion and sequencing analysis showed that pcDNA3/GRA1 contained GRA1 ORF with the right orientation.


CONCLUSIONS
The mammalian expression vector pcDNA3/GRA1 is successfully constructed.
Cai
Li-ting
LT
Department of Parasitology, Xiangya School of Medicine, Central South University, Changsha 410078, China.
Shu
Heng-ping
HP
Jiang
Li-ping
LP
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D014122:Toxoplasma / Q000235:genetics*
D000953:Antigens, Protozoan; D004274:DNA, Recombinant; C412913:GRA1 antigen; D011993:Recombinant Fusion Proteins