ABSTRACT
OBJECTIVE: To obtain the prokaryotic expression of transketolase genes and analyze its value as a diagnostic antigen for echinococcosis. METHODS: TK gene was amplified by PCR and cloned into prokaryotic vector pMD19-EgTK, and then subcloned into the expression vector pET-28a. The target gene TK prokaryotic expression plasmid pET-28a was constructed and transferred into BL21. The purified protein was identified by SDS-PAGE and Western blotting. The blood samples of patients with cystic echinococcosis (CE group), alveolar echinococcosis (AE group) and healthy people (healthy group) were collected and detected by ELISA with the recombinant EgTK protein as a diagnostic antigen. RESULTS: The recombinant plasmid pET-28a (+)-EgTK was constructed successfully, and there was a band around 70 kDa by using Western blotting. ELISA showed that the difference among the 3 groups of sera reaction A450 was significantly different (F = 44.47, P < 0. 01), and the A450 values ofthe CE group (1.46±0.41) and AE group (1.28±0.29) were higher than that of the healthy group (0.66±0.23), but there was no significant difference between the former two. CONCLUSIONS: The recombinant EgTK protein is better to distinguish the echinococcosis group and healthy group, but it can't do a differential diagnosis between CE and AE cases.
Subject(s)
Echinococcosis, Hepatic/diagnosis , Echinococcosis/diagnosis , Echinococcus granulosus/enzymology , Transketolase/immunology , Animals , Antigens, Helminth/immunology , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Humans , Recombinant Proteins/immunology , Transketolase/geneticsABSTRACT
Objective To obtain the prokaryotic expression of transketolase genes and analyze its value as a diagnostic anti-gen for echinococcosis.Methods TK gene was amplified by PCR and cloned into prokaryotic vector pMD19-EgTK,and then subcloned into the expression vector pET-28a.The target gene TK prokaryotic expression plasmid pET-28a was constructed and transferred into BL21. The purified protein was identified by SDS-PAGE and Western blotting. The blood samples of patients with cystic echinococcosis(CE group),alveolar echinococcosis(AE group)and healthy people(healthy group)were collected and detected by ELISA with the recombinant EgTK protein as a diagnostic antigen.Results The recombinant plasmid pET-28a (+)-EgTK was constructed successfully,and there was a band around 70 kDa by using Western blotting.ELISA showed that the difference among the 3 groups of sera reaction A450was significantly different(F=44.47,P<0.01),and the A450values of the CE group(1.46±0.41)and AE group(1.28±0.29)were higher than that of the healthy group(0.66 ± 0.23),but there was no significant difference between the former two.Conclusion The recombinant EgTK protein is better to distinguish the echinococ-cosis group and healthy group,but it can't do a differential diagnosis between CE and AE cases.
