Based on a previously used plasmid pHC11, a new plasmid pHC11R was con-structed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R’ was co-transformed with a PCR amplifi...Based on a previously used plasmid pHC11, a new plasmid pHC11R was con-structed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R’ was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R- IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.展开更多
通过测定重组水蛭素Ⅲ的质粒中目的基因序列、重组水蛭素Ⅲ分子质量、C末端序列以及菌体蛋白和DNA残留量,对重组水蛭素Ⅲ的质量进行研究。结果:重组质粒经NheⅠ、HindⅢ双酶切和基因序列测定,表明工程菌所携带的质粒上含有完整的信号肽...通过测定重组水蛭素Ⅲ的质粒中目的基因序列、重组水蛭素Ⅲ分子质量、C末端序列以及菌体蛋白和DNA残留量,对重组水蛭素Ⅲ的质量进行研究。结果:重组质粒经NheⅠ、HindⅢ双酶切和基因序列测定,表明工程菌所携带的质粒上含有完整的信号肽基因、NheⅠ酶切位点、水蛭素HV3基因和HindⅢ酶切位点;还原性SDS PAGE结果表明在14 000 u处有单一条带,为重组水蛭素Ⅲ二聚体;测定样品分子C端序列为E D YA D E P I P E F,与重组水蛭素Ⅲ的理论值一致;重组水蛭素Ⅲ样品中的残留菌体蛋白和DNA含量分别是0.02%和每剂量样品中残留DNA含量低于2.8 ng。结论:从基因水平、蛋白水平和杂质的角度证明重组水蛭素Ⅲ产品符合质量控制标准。展开更多
This paper deals with a method of identifying, with transposon, the fertility inhibition feature of plasmids. Using the method of labelling the F′lac pro plasmid with transposon Tn5, we identified the fi feature of t...This paper deals with a method of identifying, with transposon, the fertility inhibition feature of plasmids. Using the method of labelling the F′lac pro plasmid with transposon Tn5, we identified the fi feature of the drug resistant plasmids pFD10 and pFD11 with R144 as a control. The results obtained by this method are identical with that by the recombination method. However, the former is simple and more convenient. When the pFD10 plasmid coexisted with the F′lac pro plasmid, the fre-展开更多
文摘Based on a previously used plasmid pHC11, a new plasmid pHC11R was con-structed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R’ was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R- IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.
文摘通过测定重组水蛭素Ⅲ的质粒中目的基因序列、重组水蛭素Ⅲ分子质量、C末端序列以及菌体蛋白和DNA残留量,对重组水蛭素Ⅲ的质量进行研究。结果:重组质粒经NheⅠ、HindⅢ双酶切和基因序列测定,表明工程菌所携带的质粒上含有完整的信号肽基因、NheⅠ酶切位点、水蛭素HV3基因和HindⅢ酶切位点;还原性SDS PAGE结果表明在14 000 u处有单一条带,为重组水蛭素Ⅲ二聚体;测定样品分子C端序列为E D YA D E P I P E F,与重组水蛭素Ⅲ的理论值一致;重组水蛭素Ⅲ样品中的残留菌体蛋白和DNA含量分别是0.02%和每剂量样品中残留DNA含量低于2.8 ng。结论:从基因水平、蛋白水平和杂质的角度证明重组水蛭素Ⅲ产品符合质量控制标准。
文摘This paper deals with a method of identifying, with transposon, the fertility inhibition feature of plasmids. Using the method of labelling the F′lac pro plasmid with transposon Tn5, we identified the fi feature of the drug resistant plasmids pFD10 and pFD11 with R144 as a control. The results obtained by this method are identical with that by the recombination method. However, the former is simple and more convenient. When the pFD10 plasmid coexisted with the F′lac pro plasmid, the fre-
