[Prokaryotic expression of human cTnI and preparation of rabbit anti-hcTnI antibody].
OBJECTIVE
To construct the prokaryotic expression vector pET-21a(+)-hcTnI and prepare the rabbit anti-hcTnI antibody using hcTnI expressed in E.coli as immunogen.


METHODS
The full-length gene encoding human cardiac troponin I (hcTnI) was synthesized chemically and inserted into expression plasmid pET-21a(+) to construct recombinant plasmid pET-21a(+)-hcTnI. The recombinant plasmid was transformed into E.coli BL21 (DE3) plysS which then expressed hcTnI under IPTG induction. The immunological activity of the expressed hcTnI was analyzed by Western blot. A rabbit was immunized with purified hcTnI to prepare anti-hcTnI antibody and the Ab's properties were identified.


RESULTS
Human cTnI gene was synthesized and confirmed by DNA sequencing. Positive recombinant clones were identified by restriction enzyme digestion analysis and DNA sequencing. After induction with IPTG, hcTnI with M(r) being 24 000 was expressed in E.coli BL21 (DE3) plysS, and the expressed hcTnI accounted for 28% of total bacterial protein. Western blot analysis showed that the hcTnI protein could be recognized by an anti-hcTnI antibody. Rabbit polyclonal antibody with a good specificity was obtained. The titer of the polyclonal antibody was 3x10(-4).


CONCLUSIONS
The recombinant expression plasmid of hcTnI was constructed successfully and expressed in E.coli. The prepared rabbit anti-hcTnI antibody had a high titer and specificity.
Department of Medical Laboratory Sciences, Guangzhou Medical College, Guangzhou 510182, China. xuxia503@yahoo.com.cn
Xia
X
Xu
Neng
N
Yang
Hong-bin
HB
Tu
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification*; D000918:Antibody Specificity; D015153:Blotting, Western; D003429:Cross Reactions; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008297:Male; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011817:Rabbits; D019210:Troponin I / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*
D000906:Antibodies; D019210:Troponin I; D004262:DNA Restriction Enzymes