[Construction of rapid tagging vector of genes in Giardia lamblia].

OBJECTIVE

To construct a recombinant vector for rapid gene tagging in Giardia lamblia.

METHODS

To obtain the recombinant vector pGL gdh-Neo with the Neo selection marker, the Neo gene was put under the control of gdh promoter by overlap PCR 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 a recombinant vector pGL MCS-3HA-gdh-Neo. Giardia H2A gene was selected as a tagging gene to validate the effectivity of the recombinant vector pGL MCS-3HA-gdh-Neo. The histone H2A coding sequence was amplified by PCR, digested with EcoR I and Spe I, and inserted into MCS of pGL MCS-3HA-gdh-Neo. The resulting plasmid was then linearized and transfected into Giardia trophozoites. The H2A recombinant strain selected by G418 was analyzed by PCR,Western blotting and immunofluorescence.

RESULTS

A rapid tagging recombinant vector with multiple cloning sites and triple hemagglutinin (3HA) was constructed with a length of 4 260 bp. The H2A recombinant vector was transfected into Giardia trophozoites and integrate into the Giardia genome at the correct locus. The HA-tagged H2A protein was expressed with a molecular weight (Mr) of 16 900.

CONCLUSION

A rapid tagging recombinant vector of genes in Giardia lamblia, pGL MCS-3HA-gdh-Neo, has been constructed.