紫球藻藻红蛋白β亚基基因的克隆及其在毕赤酵母中的表达
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摘要
藻红蛋白潜在的生物活性引起了广泛的重视,如体外的抗氧化活性以及作为光敏剂用于光治疗。藻类养殖及藻红蛋白纯化的高成本已成为藻红蛋白在医药和工业领域广泛应用的重要制约因素。本论文从紫球藻(Porphyridium cruentum)基因组DNA不同提取方法筛选,藻红蛋白β亚基基因的克隆,该亚基基因在巴斯德毕赤酵母中进行胞内和分泌表达等方面开展研究。主要实验结果如下:
采用酚仿法、蛋白酶K法、盐析法、CTAB法和改良SDS法五种方法分别对紫球藻基因组DNA提取,结果显示提取的基因组DNA大小均约23 kb,五种方法所提的DNA纯度及产率有差别,蛋白酶K法提取的DNA得率最大,达3.31μg/μL,CTAB方法次之,但蛋白酶K法提取的DNA含有一些蛋白及RNA等杂质,改良CTAB法提取的DNA样品完整性相对较好,A_(260)/A_(230)和A_(260)/A_(280)分别为2.09和1.85。综合DNA提取纯度、得率、PCR扩增和酶切效果,CTAB法是五种方法中最适合紫球藻基因组DNA提取的方法。
分别提取紫球藻(Porphyridium cruentum)基因组DNA和总RNA,根据藻红蛋白保守序列设计简并引物,采用PCR、RT-PCR获得紫球藻藻红蛋白β亚基、间隔序列及α亚基编码区近5'端cDNA和基因组DNA序列。cDNA序列长987个碱基,包括β亚基起始密码子上游的10个碱基,β亚基编码区的534个碱基(编码177个氨基酸),105个碱基的间隔区以及α亚基编码区近5'端的338个碱基(编码164个氨基酸)。cDNA序列排布顺序为5'UTR-rpeB-间隔区-rpeA;对其基因组结构分析表明,藻红蛋白(Phyeoerythrin,PE)亚基基因编码区无内含子序列。
获得紫球藻藻红蛋白β亚基cDNA片段,插入至巴斯德毕赤酵母表达系统胞内表达载体pPIC3.5K及分泌表达载体pPIC9K中,电转化整合至感受态毕赤酵母GS115中,用MD和MM培养基,筛选重组子,G418筛选出高拷贝整合菌株,甲醇诱导培养96小时后,SDS-PAGE分析表明,pPIC3.5K-PEβ-GS115重组酵母在胞内表达了分子量大小约19kD的重组蛋白,与紫球藻藻红蛋白β亚基分子量大小一致。
Potential biological activities of phycoerythrin such as antioxidant activity in vitro and as photosensitiser for photodynamic therapy(PDT) had gained widely concerning.The cost of mass production of alga and purification of phycoerythrin will seriously constrain the wide range demands of B-phycoerythrin applications in biomedical and food industries.In this paper,we investigated different ways for DNA extraction,Cloned of PEβsubunit gene and then expressed in Pichia pastoris.The results were as follows:
(1) DNA was extracted from Porphyridium cruentum by phenol-chloroform method, CTAB(cetyltrimethyl ammonium bromide) method,proteinase K method,improved SDS method and salting out method respectively.Results showed that there are some differences in purity and yield among those five methods.The size of Porphyridium cruentum genomic DNA is about 23kb.Proteinase K method have better yield up to 3.31μg/μL,while CTAB follows.But DNA extracted by Proteinase K method contains protein and such impurities as RNA.The purity of the DNA by CTAB method is better than that of others.A_(260)/A_(230) and A_(260)/A_(280) of DNA by CTAB method were 2.09 and 1.85,As far as the purist、yield、efficient of PCR amplification and restriction digest concerned,CTAB method is most suitable for extracting genomic DNA from Porphyridium cruentum.
(2) The genomic DNA and total RNA of Porphyridium cruentum were extracted respectively.Degenerate primers were designed based on the conserved PE gene sequences.The DNA and cDNA of 13subunit、5'terminal ofαsubunit and intergenic sequence of PE was amplified using the techniques of PCR and RT-PCR.This sequence contain 987bp,consisting of 10 nucleotides that locate in the upstream of the initiation codon ofβsubunit,534 nucleotides coding forβsubunit(coding 177 amino acids),105 nucleotides for intergenic region and 338 nucleotides that draw near the5'terminal of a subunit(coding 112 amino acids).The structure of this cDNA is 5'UTR-rpeB-intergenic sequence-rpeA;The analysis of genomic sequence showed that Porphyridium cruentum gene ofβsubunit of PE has no introns.
(3) Gene ofβsubunit of PE Were inserted into the expression vector pPIC3.5K and pPIC9K,to produce a recombinant plasmid,which were named pPIC3.5K-PEβand pPIC9K-PEβrespectively.After linearly cleavage,the pPIC3.5K-PEβand pPIC9K-PEβplasmid were used to transform GS 115 yeast by lightning stroke,then used MD and MM mediums to screen the recombinants,further identification was done by clone PCR,applied G418 antibiotic to copy number screening,After pPIC3.5K-PEβ-GS 115 and pPIC9K-PEβ-GS 115 yeasts were induced by methanol for 96 h,SDS-PAGE assays have proved the recombinant yeast pPIC3.5K-PEβ-GS 115 have been induced to express the protein,whose molecular weight was close to 19kDa,which is in accordance with molecular weight ofβsubunit of phycoerythrin.
采用酚仿法、蛋白酶K法、盐析法、CTAB法和改良SDS法五种方法分别对紫球藻基因组DNA提取,结果显示提取的基因组DNA大小均约23 kb,五种方法所提的DNA纯度及产率有差别,蛋白酶K法提取的DNA得率最大,达3.31μg/μL,CTAB方法次之,但蛋白酶K法提取的DNA含有一些蛋白及RNA等杂质,改良CTAB法提取的DNA样品完整性相对较好,A_(260)/A_(230)和A_(260)/A_(280)分别为2.09和1.85。综合DNA提取纯度、得率、PCR扩增和酶切效果,CTAB法是五种方法中最适合紫球藻基因组DNA提取的方法。
分别提取紫球藻(Porphyridium cruentum)基因组DNA和总RNA,根据藻红蛋白保守序列设计简并引物,采用PCR、RT-PCR获得紫球藻藻红蛋白β亚基、间隔序列及α亚基编码区近5'端cDNA和基因组DNA序列。cDNA序列长987个碱基,包括β亚基起始密码子上游的10个碱基,β亚基编码区的534个碱基(编码177个氨基酸),105个碱基的间隔区以及α亚基编码区近5'端的338个碱基(编码164个氨基酸)。cDNA序列排布顺序为5'UTR-rpeB-间隔区-rpeA;对其基因组结构分析表明,藻红蛋白(Phyeoerythrin,PE)亚基基因编码区无内含子序列。
获得紫球藻藻红蛋白β亚基cDNA片段,插入至巴斯德毕赤酵母表达系统胞内表达载体pPIC3.5K及分泌表达载体pPIC9K中,电转化整合至感受态毕赤酵母GS115中,用MD和MM培养基,筛选重组子,G418筛选出高拷贝整合菌株,甲醇诱导培养96小时后,SDS-PAGE分析表明,pPIC3.5K-PEβ-GS115重组酵母在胞内表达了分子量大小约19kD的重组蛋白,与紫球藻藻红蛋白β亚基分子量大小一致。
Potential biological activities of phycoerythrin such as antioxidant activity in vitro and as photosensitiser for photodynamic therapy(PDT) had gained widely concerning.The cost of mass production of alga and purification of phycoerythrin will seriously constrain the wide range demands of B-phycoerythrin applications in biomedical and food industries.In this paper,we investigated different ways for DNA extraction,Cloned of PEβsubunit gene and then expressed in Pichia pastoris.The results were as follows:
(1) DNA was extracted from Porphyridium cruentum by phenol-chloroform method, CTAB(cetyltrimethyl ammonium bromide) method,proteinase K method,improved SDS method and salting out method respectively.Results showed that there are some differences in purity and yield among those five methods.The size of Porphyridium cruentum genomic DNA is about 23kb.Proteinase K method have better yield up to 3.31μg/μL,while CTAB follows.But DNA extracted by Proteinase K method contains protein and such impurities as RNA.The purity of the DNA by CTAB method is better than that of others.A_(260)/A_(230) and A_(260)/A_(280) of DNA by CTAB method were 2.09 and 1.85,As far as the purist、yield、efficient of PCR amplification and restriction digest concerned,CTAB method is most suitable for extracting genomic DNA from Porphyridium cruentum.
(2) The genomic DNA and total RNA of Porphyridium cruentum were extracted respectively.Degenerate primers were designed based on the conserved PE gene sequences.The DNA and cDNA of 13subunit、5'terminal ofαsubunit and intergenic sequence of PE was amplified using the techniques of PCR and RT-PCR.This sequence contain 987bp,consisting of 10 nucleotides that locate in the upstream of the initiation codon ofβsubunit,534 nucleotides coding forβsubunit(coding 177 amino acids),105 nucleotides for intergenic region and 338 nucleotides that draw near the5'terminal of a subunit(coding 112 amino acids).The structure of this cDNA is 5'UTR-rpeB-intergenic sequence-rpeA;The analysis of genomic sequence showed that Porphyridium cruentum gene ofβsubunit of PE has no introns.
(3) Gene ofβsubunit of PE Were inserted into the expression vector pPIC3.5K and pPIC9K,to produce a recombinant plasmid,which were named pPIC3.5K-PEβand pPIC9K-PEβrespectively.After linearly cleavage,the pPIC3.5K-PEβand pPIC9K-PEβplasmid were used to transform GS 115 yeast by lightning stroke,then used MD and MM mediums to screen the recombinants,further identification was done by clone PCR,applied G418 antibiotic to copy number screening,After pPIC3.5K-PEβ-GS 115 and pPIC9K-PEβ-GS 115 yeasts were induced by methanol for 96 h,SDS-PAGE assays have proved the recombinant yeast pPIC3.5K-PEβ-GS 115 have been induced to express the protein,whose molecular weight was close to 19kDa,which is in accordance with molecular weight ofβsubunit of phycoerythrin.
引文
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