猪β_2-肾上腺素能受体的克隆、表达及初步分离纯化
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摘要
本研究的目的是为我们建立的用于药物有效成分筛选的受体色谱模型提供高纯度的β_2-肾上腺素能受体(β_2-AR)。为此,我们采用基因工程技术,从猪肝脏组织中克隆出猪β_2-AR基因全长序列,将其在大肠杆菌中进行融合表达,并对表达产物进行了初步的分离和纯化,为该受体的大规模制备奠定了基础。
(1)根据GenBank中猪β_2-AR全长cDNA序列设计一对特异性引物,分别以猪肝脏组织总RNA为模板进行RT-PCR反应和以基因组DNA为模板进行PCR反应,同时扩增目的基因。然后将其与pUC18载体进行体外连接,转化感受态大肠杆菌E.Coil DH5α并筛选阳性克隆。PCR、双酶切和测序鉴定结果表明,二者均扩增出长1257 bp的目的基因片段,可编码418个氨基酸。测序结果与GenBank中收录的猪β_2-AR序列比对,前者同源性为99.52%,编码的氨基酸有99.04%相同;后者同源性为99.44%,编码的氨基酸有98.80%相同。再将本研究中两种扩增方法的测序结果进行比对,同源性为99.84%,编码的氨基酸有99.52%相同;
(2)设计引物时,在上游引物中引入了EcoRⅠ酶切位点,在下游引物中引入了SalⅠ酶切位点,利用EcoRⅠ/SalⅠ将克隆的β_2-AR全长片段从构建的克隆载体pUC18-β_2-AR中切下,将其与被EcoRⅠ/SalⅠ双酶切后的pET32a载体体外连接并转化感受态大肠杆菌E.Coil BL21(DE3),然后以IPTG诱导使其在大肠杆菌中表达。诱导前后全菌体蛋白经SDS-PAGE检测,诱导后较诱导前在66.5 kD处有一明显条带,表明该受体以融合蛋白形式在大肠杆菌中得以表达;对重组菌在不同IPTG浓度和不同培养时间下进行诱导,经SDS-PAGE和薄层扫描分析,结果显示IPTG浓度约为2.0 mmol/L、诱导时间约为4.0h时融合蛋白表达量最高,约为17.1%;
(3)由于所设计的融合β_2-AR受体具有6×His-tag,His标签可与固定化的金属离子Ni~(2+)高度选择,并强力亲和,我们在Ni~(2+)-Sepharose Fast-Flow亲和层析上对其进行了初步分离和纯化。用不同咪唑浓度的磷酸盐溶液进行梯度洗脱,对洗脱峰进行SDS-PAGE分析,结果显示峰2在66.5 kD处有一明显条带,为目标蛋白峰,其纯度约为50%。
This study was aimed to provide high purityβ_2-adrenergic receptor (β_2-AR) for receptor chromatography model which we established to screen the effective ingredients in drugs. Therefore, we used genetic engineering technology to clone the full-lengthβ_2-AR gene sequences in porcine liver tissues and express these gene sequences in E. coli in form of fusion proteins, which were further separated and purified preliminarily. That has laid foundation for the large-scale preparation ofβ_2-AR.
(1) According to the full-length cDNA sequences of porcineβ_2-AR published in GenBank, a pair of specific primers were designed respectively. RT-PCR reaction and PCR reactions were carried out separately in order to amplify target genes simultaneously with pig liver total RNA and genomic DNA as template respectively. And then the target genes were linked to the pUC18 vector in vitro, which was further transformed into competent E. Coil DH5αto screen positive clones. PCR, double digestion and sequencing results showed that both of them had amplified1257 bp target gene fragment encoding 418 amino acids. GenBank sequence of results included in the sequence of porcineβ_2-AR than for the former to 99.52% homology, amino acid are the same as 99.04%; the latter to 99.44% homology, amino acid are the same as 98.80%. Both this study and then the sequencing results for the ratio of its 99.84% homology, amino acid are the same as 99.52%.
(2) In the process of designing primers, restriction site EcoRⅠwas added to the upstream primer and SalⅠrestriction site was added to the downstream primer. The full-lengthβ_2-AR fragment was cut off from the cloning vector pUC18-β_2-AR by using EcoRⅠ/ SalⅠand was linked to the pET32a vector after double enzyme digestion in vitro, which was transformed into competent E. Coil BL21(DE3) for expression in E.coli induced by EPTG. SDS-PAGE detection of the whole cell protein was performed before and after the IPTG induction. Compared with before induction, there is a clear 66.5 kD band after induction, indicating that the receptor proteins had been expressed in form of fusion proteins. Recombinant strains of were induced at different IPTG concentrations and under different culture time. The results of SDS-PAGE and TLC analysis showed that the concentration of IPTG was about 2.0 mmol/L, about 4.0 h induction time when the highest amount of fusion protein expression, about 17.1%;
(3) Because of the integration ofβ_2-AR receptor with 6×His-tag, His tag can combine with Ni~(2+)selectly and strongly. We carried out the initial separation and purification of proteins in Ni~(2+)-Sepharose Fast Flow affinity chromatography, eluted peaks though SDS-PAGE analysis. The results showed that a clear 66.5 kD goaled band appeared on peak 2, purity was about 50%.
(1)根据GenBank中猪β_2-AR全长cDNA序列设计一对特异性引物,分别以猪肝脏组织总RNA为模板进行RT-PCR反应和以基因组DNA为模板进行PCR反应,同时扩增目的基因。然后将其与pUC18载体进行体外连接,转化感受态大肠杆菌E.Coil DH5α并筛选阳性克隆。PCR、双酶切和测序鉴定结果表明,二者均扩增出长1257 bp的目的基因片段,可编码418个氨基酸。测序结果与GenBank中收录的猪β_2-AR序列比对,前者同源性为99.52%,编码的氨基酸有99.04%相同;后者同源性为99.44%,编码的氨基酸有98.80%相同。再将本研究中两种扩增方法的测序结果进行比对,同源性为99.84%,编码的氨基酸有99.52%相同;
(2)设计引物时,在上游引物中引入了EcoRⅠ酶切位点,在下游引物中引入了SalⅠ酶切位点,利用EcoRⅠ/SalⅠ将克隆的β_2-AR全长片段从构建的克隆载体pUC18-β_2-AR中切下,将其与被EcoRⅠ/SalⅠ双酶切后的pET32a载体体外连接并转化感受态大肠杆菌E.Coil BL21(DE3),然后以IPTG诱导使其在大肠杆菌中表达。诱导前后全菌体蛋白经SDS-PAGE检测,诱导后较诱导前在66.5 kD处有一明显条带,表明该受体以融合蛋白形式在大肠杆菌中得以表达;对重组菌在不同IPTG浓度和不同培养时间下进行诱导,经SDS-PAGE和薄层扫描分析,结果显示IPTG浓度约为2.0 mmol/L、诱导时间约为4.0h时融合蛋白表达量最高,约为17.1%;
(3)由于所设计的融合β_2-AR受体具有6×His-tag,His标签可与固定化的金属离子Ni~(2+)高度选择,并强力亲和,我们在Ni~(2+)-Sepharose Fast-Flow亲和层析上对其进行了初步分离和纯化。用不同咪唑浓度的磷酸盐溶液进行梯度洗脱,对洗脱峰进行SDS-PAGE分析,结果显示峰2在66.5 kD处有一明显条带,为目标蛋白峰,其纯度约为50%。
This study was aimed to provide high purityβ_2-adrenergic receptor (β_2-AR) for receptor chromatography model which we established to screen the effective ingredients in drugs. Therefore, we used genetic engineering technology to clone the full-lengthβ_2-AR gene sequences in porcine liver tissues and express these gene sequences in E. coli in form of fusion proteins, which were further separated and purified preliminarily. That has laid foundation for the large-scale preparation ofβ_2-AR.
(1) According to the full-length cDNA sequences of porcineβ_2-AR published in GenBank, a pair of specific primers were designed respectively. RT-PCR reaction and PCR reactions were carried out separately in order to amplify target genes simultaneously with pig liver total RNA and genomic DNA as template respectively. And then the target genes were linked to the pUC18 vector in vitro, which was further transformed into competent E. Coil DH5αto screen positive clones. PCR, double digestion and sequencing results showed that both of them had amplified1257 bp target gene fragment encoding 418 amino acids. GenBank sequence of results included in the sequence of porcineβ_2-AR than for the former to 99.52% homology, amino acid are the same as 99.04%; the latter to 99.44% homology, amino acid are the same as 98.80%. Both this study and then the sequencing results for the ratio of its 99.84% homology, amino acid are the same as 99.52%.
(2) In the process of designing primers, restriction site EcoRⅠwas added to the upstream primer and SalⅠrestriction site was added to the downstream primer. The full-lengthβ_2-AR fragment was cut off from the cloning vector pUC18-β_2-AR by using EcoRⅠ/ SalⅠand was linked to the pET32a vector after double enzyme digestion in vitro, which was transformed into competent E. Coil BL21(DE3) for expression in E.coli induced by EPTG. SDS-PAGE detection of the whole cell protein was performed before and after the IPTG induction. Compared with before induction, there is a clear 66.5 kD band after induction, indicating that the receptor proteins had been expressed in form of fusion proteins. Recombinant strains of were induced at different IPTG concentrations and under different culture time. The results of SDS-PAGE and TLC analysis showed that the concentration of IPTG was about 2.0 mmol/L, about 4.0 h induction time when the highest amount of fusion protein expression, about 17.1%;
(3) Because of the integration ofβ_2-AR receptor with 6×His-tag, His tag can combine with Ni~(2+)selectly and strongly. We carried out the initial separation and purification of proteins in Ni~(2+)-Sepharose Fast Flow affinity chromatography, eluted peaks though SDS-PAGE analysis. The results showed that a clear 66.5 kD goaled band appeared on peak 2, purity was about 50%.
引文
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