摘要
目的克隆钝顶螺旋藻别藻蓝蛋白(allophycocyanin,APC)α、β亚基的基因序列,分别构建该蛋白的α和β亚基的原核表达载体,并在大肠埃希菌中进行表达。方法 PCR扩增钝顶螺旋藻APC基因(apc)序列,克隆至p GEM-T easy载体中,测序分析插入片段的正确性。以克隆的apc基因为模板,分别扩增APC的α和β亚基的编码基因apc A和apc B,测序分析正确后,分别克隆入表达载体p GEX-4T-1中,构建重组质粒p GEX-4T-apc A和p GEX-4T-apc B。将重组质粒转化入大肠埃希菌BL21(DE3)p Lys S感受态细胞中,IPTG诱导表达,表达产物经Glutathione Sepharose TM 4Fast Flow树脂纯化,Bradford法测定纯化蛋白的浓度。结果成功克隆了钝顶螺旋藻的APCα和β亚基的编码基因apc A和apc B,构建的重组质粒经测序证明构建正确,表达的融合蛋白α-APC-GST和β-APC-GST的相对分子质量均为43 000,其中α-APC-GST主要以可溶性形式存在,表达量占菌体总蛋白的32.1%,纯化后的蛋白纯度达85%,蛋白浓度为0.3 mg/ml;β-APC-GST主要以包涵体形式存在,表达量占菌体总蛋白的31.4%,纯化后的蛋白纯度达65.4%,蛋白浓度为0.1 mg/ml。结论已成功克隆并在大肠埃希菌中表达了钝顶螺旋藻APC的α和β亚基。
Objective To clone the allophycocyanin （APC） α and β genes of Spirulina platens is, construct the prokaryotic expression vectors and express E. coli. Methods The apc gene was amplified by PCR ＂from genomie DNA extracted from S. platensis and cloned into pGEM-T-easy vector, and the inserted fragment was sequenced. The apcA and apcB genes encoding the α and β subunits of APC respectively were amplified by PCR using the cloned apc gene as a tem- plate, identified by sequencing and cloned into expression vector pGEX-4T-1 respectively. The constructed recombinant plasmids pGEX-4T-apcA and pGEX-4T-apcB were transformed to E. coli BL21 （DE3） plysS and induced with IPTG. The expressed α-APC-GST and β-APC-GST fusion proteins were purified with Glutathione SepharoseTM 4 Fast Flow beads and determined for concentration by Bradford protein assay. Results Both apcA and apcB genes were successfully cloned, and the constructed recombinant plasmids were constructed correctly as proved by sequencing. Both the relative molecular masses of expressed fusion proteins α-APC-GST and β-APC-GST were 43 000. Recombinant protein α-APC-GST mainly existed in a soluble form, contained 32. l% of total somatic protein, and reached a purity of 85% and a concentration of 0. 3 mg/ ml after purification. However, β-APC-GST mainly existed in a form of inclusion body, contained 31. 4% of total somatic protein, and reached a purity of 65. 4% and a concentration of 0. 1 mg/ ml after purification. Conclusion The α and β subunit genes of S. platensis were successfully cloned and expressed in E. coli.
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第4期356-359,363,共5页
Chinese Journal of Biologicals
基金
内蒙古自然科学基金(2013MS0521)
内蒙古自治区应用技术与研究开发资金计划
鄂尔多斯市科技创新基金
关键词
钝顶螺旋藻
别藻蓝蛋白亚基
基因
原核表达
Spirulina platensis
Allophycocyanin subunit
Gene
Prokaryotic expression
