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| Prokaryotic Expression, Purification and Polyclonal Antibody Preparation of Crassostrea hongkongensis Metallothionein (MT) |
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Abstract The metallothionein (MT) of bivalves has been taken as a valid biomarker to indicate heavy metal pollution of aquatic environment. The present study was aimed to prepare the polyclonal antibodies against MT of Crassostrea hongkongensis which is an important oyster living largely at nearshore area and estuary of the South China Sea. The sufficient MT was required to immune a New Zealand rabbit (Oryctolagus cuniculus) to generate polyclonal anti-MT antibodies. Therefore, the plasmid pQE30/MT containing C. hongkongensis MT coding sequence and the prokaryotic expression vector pGEX-4T-1 were digested by both BamHⅠand SalⅠ, and the digested pGEX-4T-1 and MT DNA sequence were ligated together into an expression plasmid pGEX-4-1/MT. The constructed plasmid was transformed into the expression host Escherichia coli M15, and the soluble recombinant protein (glutathione S-transferase (GST)-tagged MT, GST-MT) was induced by 0.2 mmol/L IPTG for 16 h at 20 ℃. The GST-MT was purified by affinity chromatography column filled with Glutathione Sepharose 4B, subsequently digested in situ by thrombin, finally the recombinant MT with a molecular weight of 9 kD was purified and collected, and the GST-tag was removed from glutathione sepharose 4B. The purified MT was employed to immunize a New Zealand rabbit for 4 times at 3-week interval. The antiserum was collected from the immunized rabbit heart, and its antibody titer was 1∶128 000 through detection of indirect enzyme-linked immunosorbent assay (ELISA). The anti-MT antibodies were specific against the MT in tissues of C. hongkongensis, and will play an important role in research of heavy metal biomarker characteristics.
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Received: 23 January 2015
Published: 01 June 2015
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