坛紫菜别藻蓝蛋白α亚基基因的原核表达与鉴定
PROKARYOTIC EXPRESSION AND IDENTIFICATION OF ALLOPHYCOCYANINα SUBUNIT GENE FROM PORPHYRA HAITANENSIS
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摘要: 通过PCR的方法从重组质粒pMD-apcAB中扩增坛紫菜别藻蓝蛋白α亚基基因(apcA),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),IPTG诱导表达,并对表达产物进行Western-blot和质谱鉴定。结果显示:apcA全长486bp,表达的α亚基(apcA)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19.7KD。0.5mmol/L的IPTG在37℃诱导6h时,apcA的表达量达到最大,达菌体总蛋白50%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α亚基。
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关键词:
- 坛紫菜 /
- 别藻蓝蛋白 /
- 原核表达 /
- Western-blot
Abstract: Allophycocyanins (APC) consist ofαandβsubunits, was one of phycobiliproteins, act 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. Although theapcA andapcB genes encoding allophycocyaninαandβsubunits ofCyanophora paradoxa,Anabaena variabilis, Cyanidium caldarium, Synechocystis6714 and Aglaothamnion neglectum(Rhodophyta) were also cloned, the research related toapc genes in macro-alga has not been reported. In this study, theapcA gene ofPorphyra haitanensis was PCR amplified from pMD-apcAB recombinant plasmid and cloned intoE. coli fusion expression pTO-T7 vector which allows the overexpression of a target protein. The recombinat plasmid pTO-T7-apcA was transformed into E. coli BL21(DE3) and induced in 0.5mmol/ L IPTGin 37℃, the induce product was identified by western blotting and mass spectrum.The results showed that the 486bp sequence ofapcA was successfully inserted into pTO-T7 plasmid. SDS-PAGE analysis of the inclusion ofE. coli carrying pTO-T7-apcA showed a band with molecular mass of 19.7KD in agreement with a fusion APCα protein with the first 12 Nterminaminol amino acids of T7g10, which was identical to what had been anticipated. And the recombinant fusion protein accounted for more than 50% of the totalE. 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 allophycocyaninαsubunit ofPorphyra haitanensis.-
Keywords:
- Porphyra haitanensis /
- Allophycocyanin /
- Prokaryotic expression /
- Western－blot
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