钝顶螺旋藻藻蓝蛋白β、α亚基基因的克隆及各亚基原核表达载体的构建与表达

Molecular cloning,prokaryotic expression vector construction and expression of C-Phycocyanin beta-and alpha-subunit genes of Spirulina platensis

目的克隆钝顶螺旋藻藻蓝蛋白(CPC)β、α亚基的基因序列,分别构建β和α亚基的原核表达载体,并在大肠杆菌内表达这2个亚基。方法首先利用PCR技术扩增钝顶螺旋藻藻蓝蛋白基因序列,将扩增产物克隆入pGEM-T easy载体内,测序分析插入片段的正确性。然后以克隆的cpc基因为模板分别扩增藻蓝蛋白的β和α亚基的编码基因cpcB和cpcA,经测序分析正确后将此二基因分别克隆入表达载体pGEX-4T-1,获得重组质粒pGEX-4T-cpcB和pGEX-4T-cpcA。将所构建的重组质粒转化入大肠杆菌BL21(DE3)pLysS内,用IPTG诱导重组蛋白表达。利用蛋白纯化树脂Glutathione Sepharose^(TM)4 Fast Flow纯化目的蛋白,并测定其浓度。结果在本次实验中成功克隆了钝顶螺旋藻的藻蓝蛋白β和α亚基的编码基因cpcB和cpcA,构建了它们的原核表达载体,并在大肠杆菌中分别表达了融合蛋白β-CPC-GST和α-CPC-GST,这2个重组蛋白的分子量分别为45 kDa和44 kDa,其中β-CPC-GST主要为包涵体,α-CPC-GST主要为分泌型表达。结论克隆并在大肠杆菌内成功表达了钝顶螺旋藻藻蓝蛋白的β和α亚基。

Objective To clone the C-Phycocyanin(cpc)gene of Spirulina platensis and further construct prokaryotic expression vectors carrying cpcA and cpcB,respectively,for expression of beta-and alpha-subunits of phycocyaninin E.coli.Methods C-Phycocyanin gene was amplified by PCR from genomic DNA extracted from S.platensis.PCR product was cloned into pGEM-T-easy vector and the insert was sequenced.C-Phycocyanin beta-and alpha-subunit genes,cpcB and cpcA,were further obtained by PCR using the cloned C-Phycocyanin as a template.Prokaryotic expression plasmids pGEX-4T-cpcB and pGEX-4T-cpcA were then constructed by cloning the PCR amplified cpcB and cpcA genes into the expression vector pGEX-4T-1.The resultant recombinant pGEX-4T-cpcB andpGEX-4T-cpcA plasmids were transformed into E.coli BL21(DE3)plysS and induced with IPTG for protein expression.Recombinantly preparedβ-CPC-GST andα-CPC-GST fusion proteins were purified with Glutathione Sepharose^(TM)4 Fast Flow beads and their concentrations were determined by Bradford protein assay.Results cpcB and cpcA were cloned and two prokaryotic expression vectors carrying those two genes were successfully constructed.Recombinant fusion proteinsβ-CPC-GST andα-CPC-GST,with the molecular weights of 45 kDa and 44 kDa were expressed in E.coli,respectively.SDS-PAGE analysis showed that recombinantα-CPC-GST existed mainly in soluble protein,while most of theβ-CPC-GST was expressed as inclusion bodies.Conclusion Recombinantα-CPC-GST andβ-CPC-GST fusion proteins of S.platensis were successfully expressed in E.coli.