BACKGROUND： Neurotrophin-4 （NT-4） can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the...BACKGROUND： Neurotrophin-4 （NT-4） can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the blood-brain barrier, which limits its activity in the central nervous system. Delivering NT-4 into the central nervous system v/a cells or vectors may have therapeutic benefit. OBJECTIVE： To construct a recombinant vector with a human embryonic brain-derived NT-4 gene and pEGFP-NI. DESIGN, TIME AND SETTING： Neural genetic engineering experiment. The study was performed at the Neuroscience Institute of Kunming Medical College between October 2007 and March 2008. MATERIALS： The pEGFP-N1 plasmid vector was provided by Kunming Institute of Zoology, Chinese Academy of Sciences; embryonic brain tissues were provided by the First Affiliated Hospital of Kunming Medical College. TRIzol RNA extraction Kit was purchased from Sigma （USA）, One Step RNA PCR Kit （AMV） etc. were from Takara （Dalian, China）. METHODS： Total RNA was extracted from human embryonic brain tissues using Trizol. The agarose gel electrophoresis showed two bands： 18 S and 28 S, which were essential subunits of total RNA. The human NT-4 DNA was obtained via RT-PCR and inserted into the pEGFP-N1 vector using ligation and transformation reaction. MAIN OUTCOME MEASURES： The sequencing results of the DNA in the recombinant of NT-4- pEGFP-NI. RESULTS： The NT-4-pEGFP-N1 vector was sequence-verified and showed the expected molecular weight. CONCLUSION： The recombinant of NT-4-pEGFP-N1 was constructed successfully in vitro.展开更多
Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant pl...Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ.This recombinant plasmid was transformed into both Escherichia coli DH5α and L.lactis MG1363.The enzyme activity,gene sequencing,SDS-PAGE and hereditary stability were assessed and studied.Results The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence,and SDS-PAGE revealed an evident idio-strap at 116 KDa between L.lactis MG1363/pMG36eusp-lacZ in both supernatant and cell samples.β-Galactosidase activity measured 0.225 U/mL in L.lactis pMG36e-usp-lacZ transformants,and its secretion rate was 10%.The plasmid pMG36e-usp-lacZ appeared more stable in MG1363.Conclusion The authors concluded that these new recombinant bacteria well expressed and secreted β-galactosidase,indicating that the β-galactosidase expression system was successfully constructed,and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.展开更多
基金Supported by:the Scientific Research Foundation of Yunnan Provincial Education Academy,No.07C10388
文摘BACKGROUND： Neurotrophin-4 （NT-4） can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the blood-brain barrier, which limits its activity in the central nervous system. Delivering NT-4 into the central nervous system v/a cells or vectors may have therapeutic benefit. OBJECTIVE： To construct a recombinant vector with a human embryonic brain-derived NT-4 gene and pEGFP-NI. DESIGN, TIME AND SETTING： Neural genetic engineering experiment. The study was performed at the Neuroscience Institute of Kunming Medical College between October 2007 and March 2008. MATERIALS： The pEGFP-N1 plasmid vector was provided by Kunming Institute of Zoology, Chinese Academy of Sciences; embryonic brain tissues were provided by the First Affiliated Hospital of Kunming Medical College. TRIzol RNA extraction Kit was purchased from Sigma （USA）, One Step RNA PCR Kit （AMV） etc. were from Takara （Dalian, China）. METHODS： Total RNA was extracted from human embryonic brain tissues using Trizol. The agarose gel electrophoresis showed two bands： 18 S and 28 S, which were essential subunits of total RNA. The human NT-4 DNA was obtained via RT-PCR and inserted into the pEGFP-N1 vector using ligation and transformation reaction. MAIN OUTCOME MEASURES： The sequencing results of the DNA in the recombinant of NT-4- pEGFP-NI. RESULTS： The NT-4-pEGFP-N1 vector was sequence-verified and showed the expected molecular weight. CONCLUSION： The recombinant of NT-4-pEGFP-N1 was constructed successfully in vitro.
基金supported by the National Science Foundation of China (NO. 30800910)
文摘Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ.This recombinant plasmid was transformed into both Escherichia coli DH5α and L.lactis MG1363.The enzyme activity,gene sequencing,SDS-PAGE and hereditary stability were assessed and studied.Results The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence,and SDS-PAGE revealed an evident idio-strap at 116 KDa between L.lactis MG1363/pMG36eusp-lacZ in both supernatant and cell samples.β-Galactosidase activity measured 0.225 U/mL in L.lactis pMG36e-usp-lacZ transformants,and its secretion rate was 10%.The plasmid pMG36e-usp-lacZ appeared more stable in MG1363.Conclusion The authors concluded that these new recombinant bacteria well expressed and secreted β-galactosidase,indicating that the β-galactosidase expression system was successfully constructed,and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.
