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Home > Archive>Volume 36, Issue 8, 2017 >877-883. DOI:10.3969/j.issn.1673-1689.2017.08.014
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Prokaryotic Expression，Purification of Myofibril-Bound Serine Proteinase from Crucian Carp（Carassius auratus） and Preparation of Its Polyclonal Antibody
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    Abstract:

    An E. coli expression strain Rosetta（pET-28a-MBSP） of myofibril-bound serine proteinase（MBSP） from crucian carp（Carassius auratus） was constructed. The recombinant MBSP（rMBSP） was expressed and purified in order to prepare its polyclonal antibody，which would lay a foundation for further studies on the MBSP. The MBSP gene was transformed into Rosetta by sub-cloning it into pET-28a vector to construct the strain Rosetta（pET-28a-MBSP）. The recombinant strain was induced to express MBSP by lactose. The recombinant protein was purified by Ni-NTA agarose affinity column chromatography，MALDI-TOF-MS identification，and then use as the antigen to immune the rabbits to prepare polyclonal antibody. Antibody titer was assayed by ELISA and specificity was detected by western blot. SDS-PAGE，western blot and MALDI-TOF-MS analysis showed that the recombinant protein was the recombinant MBSP（rMBSP） with molecular weight of approximately 28×103，which was similar to native MBSP from the skeletal muscle of crucian carp. And the rMBSP was expressed in prokaryotic expression system in mainly inclusion body. The serum with a high titer and specificity was obtained from immunized rabbit. The recombinant MBSP was expressed and purified successfully. The MBSP's polyclonal antibody with a high titer and specificity was obtained by immunization using the purified MBSP.

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LI Ting, LI Huan, CHEN Haiying, DU Cuihong. Prokaryotic Expression，Purification of Myofibril-Bound Serine Proteinase from Crucian Carp（Carassius auratus） and Preparation of Its Polyclonal Antibody[J]. Journal of Food Science and Biotechnology,2017,36(8):877-883.

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