Construction Of GLV Transfer Vector And GFP Expressed In Giardia Iamblia

Giardia lamblia, an early diverging eukaryote that infects several species including humans and a major agent of waterborne diarrhea throughout the world, can be infected with a double-stranded RNA virus, giardiavirus (GLV). Chimera of GLV cDNA and green fluorescent protein (GFP) were constructed and their in vitro transcripts were electroporated into GLV-infected G. lamblia trophozoites, the cis-acting signals of the GLV genome required for expression of foreign gene were determined according to the expression level of GFP in the transfected parasites. The Giardia glutamate dehydrogenase (GDH) promoter was cloned and its transcriptional activity was identified. The stable DNA transfer vector was constructed and the reporter gene was expressed stable under G418 selection. The GLV transcription cassette was generated from locating the cis-acting signals of the GLV genome required for expression of foreign gene from the downstream of the GDH promoter. The GLV vector was constructed by combining the neomycin resistance cassette in which the neomycin phosophotransferase gene was flanked by uncoding regions of GDH and the transcription cassette on a single plasmid. This plasmid was electroporated into G. lamblia, and the transfectants persistently expressed GFP under G418 selection. This stable transfection system should provide a valuable tool for genetic study of G. lamblia.Expression of foreign gene mediated by GLV Chimera pGLV631/GFP/GLV71 6, pGLV631/GFP/GLV2171 and pGLV631/GFP of GLV cDNA and green fluorescentprotein (GFP) were constructed according to the transcriptional start site, replication start site and packaging site of the GLV genome, transcript of the constructs were synthesized in vitro by T7 RNA polymerase and used to transfect G. lamblia BJ trophozoites already infected with GLV. The results indicated that chimera pGLV631/GFP/GLV716 and pGLV631/GFP/GLV2171 appeared fluorescent signal under fluorescence microscope while chimera pGLV631/GFP did not. The expression level of GFP was determined by ELISA with rabbit anti-GFP serum as the primary antibody and goat anti-rabbit IgG-HRP as the second antibody, and it proved that there was not marked difference between pGLV631 /GFP/GLV716 and pGLV631 /GFP/GLV2171, but the expresson level of former was more stable than the latter, so chimera pGLV631/GFP/GLV716 was selected to be used in posterio experiments.Cloning and transcriptional activity evaluation of glutamate dehydrogenase (GDH) gene promoter from Giardia lamblia The promoter GDH5 and poly(A) signal GDH3 of GDH gene from G. lamblia were amplified by PCR and inserted into the pMD18-T vector, resulting in recombinant plasmid pMD/GDH5 and pMD/GDH3. The promoter GDH5 and poly(A) signal GDH3 were released by HindlWISall and BamHl/EcoRl , they were used to construct GFP expression cassette pGDH5/GFP/GDH3. The plasmid was electroporated into G. lamblia trophozoite and the transfectants appeared fluorescent signal under fluorescence microscope at 6 hours post-transfection, indicating that the promoter we had cloned had transcriptional activity.Establishment and identification G418 resistant G. lamblia strain At first NEO resistant cassette pGDH5/NEO/GDH3 was constructed in which its upstream was the promoter GDH5 of GDH gene from G. lamblia and its downstream was poly(A) signal GDH3 . The minimal lethal dose (MLF) of G. lamblia to G418 was determined to be600 u g/mL. Linearized pGDH5/NEO/GDH3 was electroporated into G. lamblia trophozoite. The transfected cells would survive in further culture medium containing G418 as the NEO gene could express G418 resistant products and the G418 resistant cell line was established successfully under G418 selection. The identification of the G418 resistant cell line was conducted by polymerase chain reaction with the specific primers of NEO gene, indicating that linear transfected DNA was integrated into the cell genome. Establishment G418 resistant cell line should greatly enhance our ability to study stable DNA transfection of G. lamblia.Establishment of stable DNA transfection of G. lamblia Recombinant plasmid pNEO/GFP was constructed by combining the GFP expression cassette and the NEO resistant cassette, this plasmid was linearized and electroporated into G. lamblia, and the transfectants were selected by G418. The G418 resistant cell line was established successfully. The NEO and GFP gene were detected by PCR, demonstrating that they were integrated into G. lamblia genome and their transcriptions were all synthesized persistently under the instruction of GDH promoter. The stable DNA transfection system should provide a valuable tool for GLV vector construction.Construction of GLV vector and expression of foreign gene GLV transcription cassette pGDH5/ GLV631/GFP/GLV716 was firstly constructed in which GDH promoter was located upstream from chimera pGLV631/GFP/GLV716, the GLV vector pGLV was genearated by combining GLV transcription cassette and NEO resistant cassette on a single plasmid. This plasmid was electroporated into G. lamblia, and the transfectants persistently expressed GFP under G418 selection. The NEO gene was detected by PCR, demonstrating that the transfected DNA was integrated into G. lamblia genome, while chimera GLV and GFP was replicated as double-strand RNA and packaged into virus-like particle. The stable transfectants had...