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Published on: April 29, 2020
[Expression and antigenicity analysis of NTPase gene of Toxoplasma gondii]
Dan Sha1, Feng Tan, Chang-Wang Pan
1Wenzhou Medical College, Wenzhou 325035, China.
Objective:
To clone and express prokaryotic recombinant plasmid of nucleoside triphosphate hydrolase (NTPase) gene of Toxoplasma gondii, and analyze its antigenicity.
Method:
NTPase gene was amplified by PCR from RH strain of T. gondii and cloned into pGEM-T Easy vector. Positive clones were screened and identified by BglII, HindIII digestion and sequenced. The target gene was then subcloned into prokaryotic expression vector pBAD-HisB and transformed into E. coli BL21 (DE3). The expressed recombinant protein was purified with Ni-NTA agarose and further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.
Results:
NTPase-II gene was specifically amplified, and the homology of DNA sequence was 100% to that in the GenBank. SDS-PAGE showed that the recombinant NTPase protein with correct molecular weight was expressed highly in E.coli BL21 (DE3). Western blotting testified that the purified recombinant protein could be specifically recognized by mouse serum immunized with T. gondii and mouse anti-recombinant protein serum.
Conclusion:
The NTPase-II gene has been cloned and expressed in E.coli BL21 (DE3), and the purified protein of NTPase-II gene displays a specific antigenicity.
