Construction Of Three Vectors For Investigation Of Giardia Lamblia Gene Function

Giardia lamblia (syn. Giardia),a unicellular parasitic protozoa lead to Giardiasis, is one of the earliest differentiation eukaryotes and takes possession of special place in the biological evolution process. With the accomplishment of Giardia genome sequencing, more and more researches focus on the gene functions of Giardia. The in vivo expression technology of foreign gene and recombinant gene is a powerful tool for investigating gene function. But common expression vectors useful for molecular biology study could not be applied to the Giardia gene function research basically. On the basis of above requirements, a rapid tagging recombinant vector with HA tag and a stable expression vector for Giardia will be constructed, then the expression vector with SNAP or CLIP protein tag will be further constructed due to the application of novel protein tags.To obtain the recombinant vector pGL gdh-Neo with the Neo selection marker, the Neo gene was put under the control of gdh promoter and inserted into pGEM-5zf(+). A DNA fragment containing multiple cloning sites(MCS) followed by triple hemagglutinin(3HA) coding sequences was synthesized and cloned into the pGL gdh-Neo to construct pGL MCS-3HA-gdh-Neo, a rapid tagging recombinant vector with multiple cloning sites and triple hemagglutinin(3HA), with a length of4260bp. Taking the H2A as a tagging gene to validate the availability of the recombinant vector pGL MCS-3HA-gdh-Neo. The H2A recombinant vector was transfected into Giardia trophozoites and recombinant strain was analyzed by PCR, Western blot and Immunofluorescence. Recombinant vector integrate into the Giardia genome at the correct locus and the HA-tagged H2A protein was expressed.To obtain the recombinant vector pGL tim-gdh-Neo for integrating into the Giardia genome, the tim gene and its flanking sequence was inserted into the front of gdh5’UTR of pGL gdh-Neo constructed previously. A DNA fragment, containing a2-Tubulin5’UTR, multiple cloning sites(MCS) and3’UTR, was synthesized and cloned into the pGL tim-gdh-Neo by Apa I to construct stable expression recombinant vector with multiple cloning sites and the Neo selection marker, pGL tim-Tub-MCS-gdh-Neo, with a length of5395bp. To validate the effectivity of the recombinant vector, the Luc coding sequence was inserted into MCS of pGL tim-Tub-MCS-gdh-Neo and transfected into Giardia trophozoites. Luciferase activity of the recombinant strain was tested and it was considerably higher than control. Then the SNAP or CLIP sequence was cloned into MCS of pGL tim-Tub-MCS-gdh-Neo to construct stable expression recombinant vector with SNAP or CLIP protein tag.A rapid tagging recombinant vector of genes in Giardia, pGL MCS-SHA-gdh-Neo, a stable expression recombinant vector of Giardia, pGL tim-Tub-MCS-gdh-Neo, and stable expression recombinant vector with SNAP or CLIP protein tag were constructed successfully, which could be used for in situ gene labeling, subcellular localization, gene overexpression or knockout, protein-protein interactions, the labeling of fusion protein in living cells of Giardia. It will provide a powerful platform for research of Giardia gene function.