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| Prokaryotic Expression and Antibody Preparation of Troponin CⅢ(TnCⅢ) from Bombyx mori |
| 2, 2, 3, 2, 2 |
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Abstract Troponin CⅢ(TnCⅢ) is one subtype of TnC. In this research, TnCⅢ fusion protein was expressed in Escherichia coli and polyclonal antibody was prepared for further research. A subtype cDNA sequence coding TnCⅢ was obtained from our silkworm(Bombyx mori) pupal cDNA library, named BmTnCⅢ(Bombyx mori TnCⅢ). This cDNA contained a full length of open reading frame(ORF) of BmTnCⅢ coded 153 amino acid residues. According to the cDNA sequence, the primers bearing restriction enzyme sites of BamHⅠand HindⅢ were designed for amplifying the full length of ORF. The intact ORF of BmTnCⅢ(GenBank accession No.DQ889211) was amplified from total RNA of silkworm pupa by RT-PCR, and cloned into a prokaryotic expression vector pET-28a. Following the recombinant plasmid pET-28a-BmTnCⅢ was transformed into E. coli Rosetta (DE3), the fusion protein was observed at approximately 21 kD with the induction of 1 mmol/L IPTG. The interest protein His-Tag BmTnCⅢ was purified by affinity chromatography and used as an antigen to immunize a male New Zealand rabbit(Oryctolagus cuniculus). Then, the polyclonal antibody of the fusion protein was harvested and used in Western blotting analysis. The result indicated that this polyclonal antibody had fairly high specificity and can be used to carry out the researches on tissue distribution and localization of BmTnCⅢ.
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Received: 22 July 2009
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