从黑曲霉N25(A.niger China strain)中提取出染色体DNA,根据已经测定出的植酸酶phyA基因全序列设计了一对引物,采用高保真度的聚合酶Advangage-HF扩增到了去除信号肽和内含子后约1.4kb片段,对该片段进行了克隆及序列测定。将该序列与植...从黑曲霉N25(A.niger China strain)中提取出染色体DNA,根据已经测定出的植酸酶phyA基因全序列设计了一对引物,采用高保真度的聚合酶Advangage-HF扩增到了去除信号肽和内含子后约1.4kb片段,对该片段进行了克隆及序列测定。将该序列与植酸酶phyA基因全序列进行了比较。以此片段构建成功了pPIC9K-phyA载体(命名为pPNP-1),并转化毕赤巴斯德酵母。经G418抗性筛选、酶活性测定、Southern印迹和Western印迹,获得了高效表达的转化子PP-NP-1(23869.4u/ml)、PP-NP-2(20533.0u/ml)、PP-NP-3(35646.7u/ml),其酶活性分别是出发菌株的酶活(513.4u/ml)的46.46倍、39.99倍和69.46倍,且转化子具有很好的遗传稳定性。展开更多
The yeast Pichia pastoris was transformed by the multi\|copy Pichia expression vector that can express secreted human albumin.The high level expression of cell line was selected after screening.The expression of human...The yeast Pichia pastoris was transformed by the multi\|copy Pichia expression vector that can express secreted human albumin.The high level expression of cell line was selected after screening.The expression of human recombinant albumin in Pichia pastoris induced by different methods were compared.The retio of secreted human albumin is 80% in total secreted proteins and the expression level reaches as high as is 10g/L.展开更多
Human β-defensin 3(hBD-3) is a short polypeptide with a wide range of antimicrobial activity,which was purified from human lesional psoriatic scales in 2001.To obtain high level expression in E.coli of β-defensin 3,...Human β-defensin 3(hBD-3) is a short polypeptide with a wide range of antimicrobial activity,which was purified from human lesional psoriatic scales in 2001.To obtain high level expression in E.coli of β-defensin 3,four pairs of oligonucleotide with cosmic site were synthesised using E.coli biased codons according to the amino acid sequence of β-defensin 3, connected and amplified by PCR. The PCR product encoding human β-defensin 3 was cloned into pET30a vector.The recombinant vector was transformed into E.coli BL21(DE3)PlysS and the expression was induced by IPTG. The recombinant fusion protein was analyzed by SDS-PAGE and purified by affinity column. The mass of the fusion protein consisted of 30.9% in total bacteria proteins. The recombinant fusion protein was digested by enterokinase, resulting in the recombinant hBD-3. Antimicrobial activity analysis showed that both recombinant hBD-3 fusion protein and recombinant hBD-3 had similar potency as the native protein in suppressing growth of both gram positive bacteria S.aureus and gram negative one E.coli in a dose dependent manner.展开更多
文摘从黑曲霉N25(A.niger China strain)中提取出染色体DNA,根据已经测定出的植酸酶phyA基因全序列设计了一对引物,采用高保真度的聚合酶Advangage-HF扩增到了去除信号肽和内含子后约1.4kb片段,对该片段进行了克隆及序列测定。将该序列与植酸酶phyA基因全序列进行了比较。以此片段构建成功了pPIC9K-phyA载体(命名为pPNP-1),并转化毕赤巴斯德酵母。经G418抗性筛选、酶活性测定、Southern印迹和Western印迹,获得了高效表达的转化子PP-NP-1(23869.4u/ml)、PP-NP-2(20533.0u/ml)、PP-NP-3(35646.7u/ml),其酶活性分别是出发菌株的酶活(513.4u/ml)的46.46倍、39.99倍和69.46倍,且转化子具有很好的遗传稳定性。
文摘The yeast Pichia pastoris was transformed by the multi\|copy Pichia expression vector that can express secreted human albumin.The high level expression of cell line was selected after screening.The expression of human recombinant albumin in Pichia pastoris induced by different methods were compared.The retio of secreted human albumin is 80% in total secreted proteins and the expression level reaches as high as is 10g/L.
文摘Human β-defensin 3(hBD-3) is a short polypeptide with a wide range of antimicrobial activity,which was purified from human lesional psoriatic scales in 2001.To obtain high level expression in E.coli of β-defensin 3,four pairs of oligonucleotide with cosmic site were synthesised using E.coli biased codons according to the amino acid sequence of β-defensin 3, connected and amplified by PCR. The PCR product encoding human β-defensin 3 was cloned into pET30a vector.The recombinant vector was transformed into E.coli BL21(DE3)PlysS and the expression was induced by IPTG. The recombinant fusion protein was analyzed by SDS-PAGE and purified by affinity column. The mass of the fusion protein consisted of 30.9% in total bacteria proteins. The recombinant fusion protein was digested by enterokinase, resulting in the recombinant hBD-3. Antimicrobial activity analysis showed that both recombinant hBD-3 fusion protein and recombinant hBD-3 had similar potency as the native protein in suppressing growth of both gram positive bacteria S.aureus and gram negative one E.coli in a dose dependent manner.
