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[Prokaryotic Expression and Immunoreactivity Analysis of α-8 Giardin in Giardia lamblia]
Objective:
To clone and express α8-giardin gene of Giardia lamblia, and analyze its immunoreactivity.
Methods:
The open reading frame (ORF) of α8-giardin gene was amplified by PCR. The PCR product was cloned into prokaryotic expression vector pET-30a (+) with restriction enzymes EcoR I and Xho I . The recombinant vector pET30a (+)-α8-giardin was transformed into E. coli BL21 (DE3), and the positive clones were then selected. The constructed pET30 a (+)-α8-giardin was induced with IPTG for expression, and purified through Ni-affinity chromatography. The recombinant protein was examined by SDS-PAGE and Western blotting.
Results:
The length of α8-giardin gene was 930 bp. PCR and restriction enzyme digestion analysis confirmed the construction of recombinant plasmid pET30a (+)-α8-giardin. SDS-PAGE and Western blotting analysis showed that the recombinant protein rGiardin (about M, 36,000) was expressed in E. coli as inclusion body protein, and reacted positively with anti-His tag antibody and rabbit anti-G. lamblia serum.
Conclusion:
The recombinant plasmid pET30a (+)-α8-giardin is constructed, and the purified rGiardin protein shows immunoreactivity.
