Cloning and Expression
of Tetanus Toxin Fragment C in E. coli
HE Hua-Jun1,2, HE Zhi-Yong2, SHI
Hui-Juan2, ZHU Wei3, YANG Guan-Zhen2, YUAN
Qin-Sheng1, WU Xiang-Fu2*
( 1Institute of Biochemistry, East China University of Science
and Technology, Shanghai 200237, China； 2Shanghai Institute of Biochemistry, the
Chinese Academy of Sciences, Shanghai 200031, China； 3Shanghai Institute of Biological
Products, Ministry of Public Health, Shanghai 200052, China )
Abstract The
fragment C of tetanus toxin was amplified from Clostridium tetani DNA
by PCR. This fragment was cloned into expression vector pET-28a(+),under the
control of the T7 promoter. Expression of this plasmid in E.coli
resulted in the production of a protein consisting of 6×His of the vector fused
to the N-terminal 451 amino acids of tetanus toxin. After induction with 1
mmol/L IPTG, TTC was expressed in E.coli BL21(DE3). The
protein product accounted for 8.2% of the bacteria total protein in soluble
form, SDS-PAGE and Western blot analysis of TTC recombinant protein confirmed
this result. The expression products were also purified by Ni2+-IDA-Sephrose
6B column. Immunization of mice with rTTC resulted in the production of
antibodies that were able to protect mice against a challenge with tetanus
toxin；
furthermore, rTTC in vivo appeared to be able to undergo retrograde
axonal transport.
Key words tetanus toxin fragment C； gene cloning； immunization and challenge； retrograde axonal transport
*Corresponding author： Tel, 86-21-64374430-292； Fax, 86-21-64338357； e-mail, [email protected]