枯草芽孢杆菌精氨酸脱羧酶基因speA的表达与蛋白纯化

Expression and purification of arginine decarboxylase gene speA of Bacillus subtilis

根据枯草芽孢杆菌(Bacillus subtilis)BJ3-2的精氨酸脱羧酶(ADC)的编码基因spe A序列设计特异性酶切引物,克隆基因speA序列。测序结果显示,基因speA全长为1473 bp,编码490个氨基酸,分子质量为58 ku。基因spe A克隆至原核表达载体,获得重组菌pET28a-spe A/BL21,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示,1.0 mmol/L的异丙基β-D-硫代半乳糖苷(IPTG)28℃诱导4 h,上清液和菌体均能表达出ADC蛋白,上清液经纯化、透析、冷冻干燥可获得纯度97%的ADC酶,酶联免疫吸附检测(ELISA)ADC酶活为16780 U/mg。为speA基因的表达、纯化及酶学性质研究奠定了理论基础。

Specific enzyme primers were designed according to the arginine decarboxylase （ADC） genesequenceofBaciHussubtilisBJ3-2, thegene speA was cloned and obtained. The results of sequence analysis indicated that the full-length of gene speA was 1 473 bp, which could encode 490 amino acids with deduced molecular mass of 58 ku. The gene speA was cloned into prokaryotic expression vector to obtain recombinant strain pET28a-speA/BL21. The results of SDS-PAGE showed that the target protein was induced with 1.0 mmol/L IPTG at 28 ℃ for 4 h, and the ADC protein could be expressed in supernatant fluid and bacteria. After purification, dialysis and freeze-drying, the ADC with 97% purity was obtained in super- natant fluid. The ADC activity was 16 780 U/mg by ELISA. The study laid a theoretical foundation for expression, purification and enzymatic proper- ties ofgene speA.