近江牡蛎热休克蛋白70基因的原核表达研究

Prokaryotic expression of heat shock protein 70 gene in Crassostrea hongkongensis

近江牡蛎(Crassostrea hongkongensis)热休克蛋白70(HSP70)家族成员是重要的抗逆和潜在污染预警分子。本研究将近江牡蛎HSP70基因cDNA连接到原核表达载体pQE30中,构建近江牡蛎HSP70的重组表达质粒pQE30/HSP70。该重组质粒经酶切和测序鉴定后,转入表达宿主大肠杆菌M15进行诱导表达,经SDS-PAGE电泳和Westernblot分析表明,确实获得了重组蛋白的表达。分别在25℃、30℃和37℃下,经0.2mmol/LIPTG诱导融合蛋白表达,其中,在25℃下,诱导的融合蛋白表达量最高,表明该温度为恰当诱导温度;在25℃下,分别诱导重组蛋白表达2h、4h、6h、8h、12h和16h,其中诱导12h的蛋白表达量最高;大部分融合蛋白以可溶形式存在于大肠杆菌M15中;表达的重组融合蛋白分子量约69kD,并能与鼠源抗6伊His的单克隆抗体特异性结合,表明通过原核表达获得了近江牡蛎HSP70蛋白。实验表明,在25℃下,经0.2mmol/LIPTG诱导表达12h,可获得大量可溶近江牡蛎HSP70蛋白。

Heat shock protein 70s（ HSP70s） play an important role in folding,assembling,transfering proteins and degradating,regulating polypeptides of living things,and contribute to the protection of cellular proteins by functioning as molecular chaperones. Moreover,they take part in immune activity against pathogens,sensitively respond to various pollutants and so on. Therefore,HSP70s are key molecules of cells. The oyster,Crassostrea hongkongensis,is one of the most important economic mollusc and inhabits alongshore in South China Sea. It has become an important reared edible species and a monitoring animal for early warning of marine environment pollution in shore. Because oyster’s HSP70 is involved in controlling diseases and monitoring polluting status of marine environment,it is necessary to get the HSP70 protein’s monoclonal antibody for further research of the HSP70’s function. Then enough HSP70 protein should be prepared. The present study aims to synthesize HSP70 with prokaryotic expression method. The recombinant expression plasmid pQE30/HSP70 was constructed by means of linking the oyster’s HSP70 cDNA with prokaryotic expression vector pQE30. It was identified by endonuclease digestion and DNA sequencing of the recombinant plasmid. Then,the recombinant plasmid pQE30/HSP70 was transformed into Escherichia coli M15 and induced to express the fusion protein with 0.2 mmol/L IPTG. The fusion protein was identified by SDS-PAGE and Western blot. The expressed protein quantity induced at 25℃ is the highest among those induced at 25 ℃,30 ℃ and 37 ℃ . Thus,25 ℃ is the fittest inducing temperature. Meanwhile,the expression level induced for 12 h at 25℃ is up to the highest,so the fittest induction time was 12 h. In the fittest condition,HSP70,a 69 kD protein,could be highly expressed in E. coli M15 as a soluble protein,and was identified by means of western blot with the monoclonal antibody against 6×His. The prokaryotic expression vector pQE30/HSP70 was constructed for the fusion protein expression of the oyster HSP70. The recombinant protein was highly expressed as a soluble protein in E. coli M15,which would promote further study of the HSP70 function.