姜晓杰,高金亮,祁美荣,徐萌杰,王文杰,李炜
JIANG Xiao-jie,GAO Jin-liang,QI Mei-rong,XU Meng-jie,WANG Wen-jie,LI Wei

• 1:鄂尔多斯市疾病预防控制中心

摘要(Abstract)：

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 platensis,construct the prokaryotic expression vectors and express E. coli. Methods The apc gene was amplified by PCR from genomic DNA extracted from S. platensis and cloned into p GEM-T-easy vector,and the inserted fragment was sequenced. The apc A and apc B genes encoding the α and β subunits of APC respectively were amplified by PCR using the cloned apc gene as a template,identified by sequencing and cloned into expression vector p GEX-4T-1 respectively. The constructed recombinant plasmids p GEX-4T-apc A and p GEX-4T-apc B were transformed to E. coli BL21(DE3)plys S and induced with IPTG. The expressed α-APC-GST and β-APC-GST fusion proteins were purified with Glutathione SepharoseTM4 Fast Flow beads and determined for concentration by Bradford protein assay. Results Both apc A and apc B genes were successfully cloned,and the constructed recombinant plasmids were constructed correctly as proved by sequencing. Both the relative molecu-lar 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. 1% of total somatic protein,and reached a purity of 85% and a concen-tration of 0. 3 mg / ml after purification. However,β-APC-GST mainly existed in a form of inclusion body,contained31. 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.

关键词(KeyWords)： 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

Abstract:

Keywords:

基金项目(Foundation): 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

作者(Author): 姜晓杰,高金亮,祁美荣,徐萌杰,王文杰,李炜
JIANG Xiao-jie,GAO Jin-liang,QI Mei-rong,XU Meng-jie,WANG Wen-jie,LI Wei

DOI: 10.13200/j.cnki.cjb.000846

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