[Gene cloning, optimized expression and immunogenicity evaluation of tetanus toxin fragment C].
OBJECTIVE
To obtain highly purified tetanus toxin fragment C (TTC) with good immunogenicity.


METHODS
The gene fragment encoding TTC was amplified from Clostridium tetani plasmid DNA by PCR, inserted into the vector pET43.1a (+) and expressed in E. coli BL21(DE3)plysS. After purification using Ni2+-chelate affinity chromatography, the expressed fusion protein was digested by thrombin and the resultant TTC protein was purified with Ni2+-chelate affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purifed TTC protein was then used to immunize mice to test its immunogenecity.


RESULTS
The 1373-bp gene fragment encoding TTC was obtained, and the constructed recombinant expression vector pET43.1a (+)-TTC was successfully expressed in E. coli BL21(DE3)plysS. SDS-PAGE identified a recombinant fusion protein with relative molecular mass (Mr) of 117 000, which accounted for 22% of the total bacterial protein. The TTC protein with Mr of 50 000 was obtained after purification of the thrombin digestion products of the fusion protein, with a purity reaching 95.5%. Both the fusion protein and TTC protein could be recognized by anti-tetanus toxin antibody as shown by Western blotting. The titer of the anti-serum from mice immunized with the TTC protein was 1:25 600, and the anti-serum could specifically bind to tetanus toxin.


CONCLUSIONS
Highly purified and immunogenetic TTC protein has been successfully obtained, which provides a good model antigen for studying antigen presentation and immune responses in vivo.
Wang
Ming-yong
MY
Department of Immunology, Southern Medical University, Guangzhou 510515, China. cxaini118@163.com
Zhang
Ya-ni
YN
Lei
Ming
M
Zuo
Da-ming
DM
Zhang
Li-yun
LY
Chen
Zheng-liang
ZL
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D013744:Tetanus Toxin / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D013745:Tetanus Toxoid / Q000276:immunology*
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; D013745:Tetanus Toxoid; C040635:tetanus toxin fragment C