坛紫菜别藻蓝蛋白β亚基的表达及鉴定

Expression and Identification of Allophycocyanin β Subunit Gene from Porphyra haitanensis

根据GenBank(AY372218)中的相关序列设计引物,通过PCR的方法从质粒pMD-apcAB中扩增坛紫菜别藻蓝蛋白β亚基基因(apcB),并将其克隆到高效表达外源基因的原核表达质粒pTO-T7.将构建好的质粒导入表达型大肠杆菌BL21(DE3),异丙基-β-D硫代半乳糖苷(IPTG)诱导表达,并对表达产物进行Western-blot和质谱鉴定.结果显示:apcB全长486 bp,表达的坛紫菜别藻蓝蛋白β亚基(APCβ)为带有原核表达载体T7g10的12个起始氨基酸的融合蛋白,其分子量约为18.0 ku.扫描分析的结果表明,0.5 mmol/L的IPTG在37℃诱导6 h时,APCβ的表达量达到最大,达菌体总蛋白40%以上.Western-blot和质谱鉴定的结果表明获得的融合蛋白为重组坛紫菜别藻蓝蛋白β亚基.

Allophycocyanin（APC） consist of α and β subunits, was one of phycobiliproteins;acting as substances of light-havesting and transferring energy to photosystem reaction centers in algal photosynthesis. It also as a kind of bioactive substances applied perspective in quite a lot of fields. In this study,the apcB gene of Porphyra haitanensis was PCR amplified from pMD-apcAB recombinant plasmid and cloned into E. coli fusion expression pTO-T7 vector which allows the overexpression of a target protein. The recombinant plasmid pTO-T7-apcB was transformed into E. coli BL21 （DE3） and induced in 0.5mmol/L IPTG in 37℃, and the induced product was identified by Western blotting and mass spectrum. The results showed that the 486 bp sequence of apcB was successfully inserted into pTO-T7 plasmid. SDS-PAGE analysis of the inclusion of E. coli carrying pTO-T7-apcB showed a β band with molecular mass of 18.0 ku in agreement with a fusion APC protein with the first 12 N-terminaminol amino acids of T7g10, which was identical to what had been anticipated. And the recombinant fusion protein accounted for more than 40% of the total E. coli protein after 6 hours inducing. The result of western blotting confirmed that the recombinant fusion protein could specifically react with antibody against native APC proteins. And the mass spectrum results proved that the target protein was allophycocyanin βsubunit of P. haitanensis.