中文摘要：

双壳贝类金属硫蛋白（metallothionein,MT）可作为指示重金属污染的生物标志物,为了进一步研究MT作为重金属污染指示分子特点及其他生物学功能,需要抗MT的特异抗体。本研究构建了双壳贝类近江牡蛎（Crassostrea hongkongensis）金属硫蛋白重组表达质粒p GEX-4T-1/MT,将其转化到大肠杆菌（Escherichia coli）菌株M15中诱导表达,优化诱导条件后,在20℃、0.2 mmol/L异丙基β-D-硫代半乳糖苷（isopropylβ-D-1-thiogalactopyranoside,IPTG）诱导16 h后产生大量可溶的谷胱甘肽巯基转移酶（glutathione S-transferase（GST）-tagged MT,GST-MT）融合蛋白。12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulphate-polyacrylamide gel electrophoresis,SDS-PAGE）和Western blot分析结果表明,成功获得了重组表达蛋白GST-MT。采用谷胱甘肽琼脂糖树脂（glutathione sepharose 4B）纯化该融合蛋白,于25℃凝血酶原位酶切16 h获得了重组MT,其分子量为9 k D。用纯化的重组MT免疫新西兰兔（Oryctolagus cuniculus）,获得效价大于1∶128 000的抗近江牡蛎MT的多克隆抗血清,该抗体能与近江牡蛎组织中的MT分子特异结合,实现了近江牡蛎MT的原核高效表达和多抗制备。

英文摘要：

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 BamH I and Sal I, 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 withGlutathione 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.