[Expression of adenylate kinase of Schistosoma japonicum and evaluation on the immunoreactivity of the recombinant protein].
OBJECTIVE
To express and purify the Schistosoma japonicum adenylate kinase (AK) protein of which the cDNA sequence was subcloned into the pET32a(+) soluble expression plasmid, and to evaluate its immunoreactivity.


METHODS
The recombinant plasmid was transformed into E. coli BL21, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analyzed by SDS-PAGE after boiling and centrifugation. The target protein was purified with the Ni-NTA agarose. The proteins on the gel were transferred to the PVDF film and then the immunoreactivity was tested by Western blotting using anti-6-His antibody, sera from rabbits 6 weeks after infected with cercariae or UV-attenuated cercariae. The purified protein was coated for ELISA to test the cercariae infected rabbit serum and the normal rabbit serum.


RESULTS
The molecular weight of the target fusion protein was Mr 40 000 after being induced with IPTG. The fused protein showed a single band when reacted with anti-6-His antibody, the cercariae infected rabbit serum and attenuated cercariae infected rabbit serum.


CONCLUSIONS
The AK protein is expressed as a fused protein with thioredoxin and its molecular weight is about Mr 40000. This protein has a positive immunoreactivity with sera of rabbits infected with cercariae and UV-attenuated cercariae.
Department of Parasitology, First Military Medical University, Guangzhou 510515, China.
Hong-juan
HJ
Peng
Xiao-guang
XG
Chen
Hua
H
Li
Chun-mei
CM
Wang
D000263:Adenylate Kinase / Q000096:biosynthesis* / Q000276:immunology; D000818:Animals; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D012549:Schistosoma japonicum / Q000201:enzymology* / Q000276:immunology
D011993:Recombinant Fusion Proteins; D000263:Adenylate Kinase