To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a pro...To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a probe.The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid.The non-GFP labeled MIM-I-BAR encoding plasmid was also constructed as a control. Being successfully transformed into BL21 （DE3 ）cells,the GFP-conjugated MIM-I-BAR （MIM-I-BAR-GFP ） exhibits strong visible fluorescence,and the expression product can be easily detected by visual inspection, a fluorescence microscope, Western blot or ultraviolet and visible spectrophotometer. Moreover, examination of expression efficiency under various culture conditions revealed that the MIM-I-BAR-GFP gene has a high protein yield at 10 ℃,but not at the culture temperature of 37 ℃.This property is much different from that of the non-fluorescent MIM-I-BAR gene. This optimal expression condition is also proved to be feasible for protein production in midi-scale. The fluorescent recombinant MIM-I-BAR-GFP protein can serve as a useful tool in scientific research, biomedical application and pharmaceutical development.展开更多
This paper reports the rationale and methods of DNA extraction and purification from nine species of Compositae and four commercial drugs of corresponding plant Elephantopus scaber. The comparison of three methods: Cs...This paper reports the rationale and methods of DNA extraction and purification from nine species of Compositae and four commercial drugs of corresponding plant Elephantopus scaber. The comparison of three methods: CsCl gradient, CTAB/CsCl gradient and CTAB miniprep extraction by yield, purity and factors affecting PCR was carried out. In conclusion, CTAB miniprep method provides a rapid, effective, economic approach for isolating genomic DNA for Chinese drug identification by genomic fingerprints.展开更多
[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of ...[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of Wuzhishan miniature pig as the template, three pairs of primers were designed by the reported cDNA sequence of porcine GHRH, and cDNA was also amplified by RT-PCR. After being recovered and purified, PCR products were ligated to pMD18-T and then transformed into Escherichia coli DH5a. The transformation products were analyzed by PCR and double enzyme digestion to screen positive clones, and the positive clones were sequenced after identification in LB liquid medium. [ Result] cDNA of Wuzhishan miniature pig GHRH receptor gene was obtained successfully, and its length was 1 577 bp coding 423 amino acids. BLAST analysis showed that there were only 23 nuoleotides in difference between this fragment and pomine GHRH receptor gene, and its homology was 98%. However, both GHRH receptor genes were constituted by 423 amino acids with the sequence homology of 96%. [ Conclusion] This study provides theoretical basis for further studies on the dwarf mechanism of Wuzhishan miniature pig.展开更多
Objective： To establish a method of genomic DNA extraction from whole blood using Fe3O4/Au composite particles as a carrier. Methods： Two crucial conditions （sodium chloride concentration and amount of the magnetic...Objective： To establish a method of genomic DNA extraction from whole blood using Fe3O4/Au composite particles as a carrier. Methods： Two crucial conditions （sodium chloride concentration and amount of the magnetic particles） were optimized and 8 different human whole blood samples were used to purify genomic DNA under the optimal condition. Then agarose gel electrophoresis and polymerase cbain reaction （PCR） were performed. Results： The optimal binding condition was 1.5 mol/L NaC1/10% PEG, and the optimal amount of Fe3O4/Au composite particles was 600μg. The yields of the genomic DNA from 100μl of different whole blood samples were 2-5 μg, and the ratio of A260/A280 was in the range of 1.70-1.90. The size of genomic DNA was about 23 kb and the PCR was valid. Conclusion： The purification system using Fe3O4/Au composite microparticles has advantages in high yield, high purity, ease of operating, time saving and avoiding centrifugation. The purified sample was found to function satisfactorily in PCR amplification.展开更多
Cyanophages are ubiquitous and essential components of the aquatic environment and play an important role in the termination of algal blooms.As such,they have attracted widespread interest.PP was the first isolated cy...Cyanophages are ubiquitous and essential components of the aquatic environment and play an important role in the termination of algal blooms.As such,they have attracted widespread interest.PP was the first isolated cyanophage in China,which infects Plectonema boryanum and Phormidium foveolarum.In this study,this cyanophage was purified three times by a double-agar overlay plaque assay and characterized.Its genome was extracted,totally sequenced and analyzed.Electron microscopy revealed a particle with an icosahedral head connected to a short stubby tail.Bioassays showed that PP was quite virulent.The genome of PP is a 42,480 base pair(bp),linear,double-stranded DNA molecule with 222 bp terminal repeats.It has high similarity with the known Pf-WMP3 sequence.It contains 41 open reading frames(ORFs),17 of which were annotated.Intriguingly,the genome can be divided into two completely different parts,which differ both in orientation and function.展开更多
基金The National Basic Research Program of China(973Program)(No.2011CB933503)the National Natural Science Foundation of China for Key Project of International Cooperation(No.61420106012)China Postdoctoral Science Foundation(No.2013M541592)
文摘To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a probe.The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid.The non-GFP labeled MIM-I-BAR encoding plasmid was also constructed as a control. Being successfully transformed into BL21 （DE3 ）cells,the GFP-conjugated MIM-I-BAR （MIM-I-BAR-GFP ） exhibits strong visible fluorescence,and the expression product can be easily detected by visual inspection, a fluorescence microscope, Western blot or ultraviolet and visible spectrophotometer. Moreover, examination of expression efficiency under various culture conditions revealed that the MIM-I-BAR-GFP gene has a high protein yield at 10 ℃,but not at the culture temperature of 37 ℃.This property is much different from that of the non-fluorescent MIM-I-BAR gene. This optimal expression condition is also proved to be feasible for protein production in midi-scale. The fluorescent recombinant MIM-I-BAR-GFP protein can serve as a useful tool in scientific research, biomedical application and pharmaceutical development.
文摘This paper reports the rationale and methods of DNA extraction and purification from nine species of Compositae and four commercial drugs of corresponding plant Elephantopus scaber. The comparison of three methods: CsCl gradient, CTAB/CsCl gradient and CTAB miniprep extraction by yield, purity and factors affecting PCR was carried out. In conclusion, CTAB miniprep method provides a rapid, effective, economic approach for isolating genomic DNA for Chinese drug identification by genomic fingerprints.
基金Supported by Natural Science Foundation of Hainan Province(30515)~~
文摘[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of Wuzhishan miniature pig as the template, three pairs of primers were designed by the reported cDNA sequence of porcine GHRH, and cDNA was also amplified by RT-PCR. After being recovered and purified, PCR products were ligated to pMD18-T and then transformed into Escherichia coli DH5a. The transformation products were analyzed by PCR and double enzyme digestion to screen positive clones, and the positive clones were sequenced after identification in LB liquid medium. [ Result] cDNA of Wuzhishan miniature pig GHRH receptor gene was obtained successfully, and its length was 1 577 bp coding 423 amino acids. BLAST analysis showed that there were only 23 nuoleotides in difference between this fragment and pomine GHRH receptor gene, and its homology was 98%. However, both GHRH receptor genes were constituted by 423 amino acids with the sequence homology of 96%. [ Conclusion] This study provides theoretical basis for further studies on the dwarf mechanism of Wuzhishan miniature pig.
基金Supported by the National High Technology Research and Development Program of China (2006AA020705)
文摘Objective： To establish a method of genomic DNA extraction from whole blood using Fe3O4/Au composite particles as a carrier. Methods： Two crucial conditions （sodium chloride concentration and amount of the magnetic particles） were optimized and 8 different human whole blood samples were used to purify genomic DNA under the optimal condition. Then agarose gel electrophoresis and polymerase cbain reaction （PCR） were performed. Results： The optimal binding condition was 1.5 mol/L NaC1/10% PEG, and the optimal amount of Fe3O4/Au composite particles was 600μg. The yields of the genomic DNA from 100μl of different whole blood samples were 2-5 μg, and the ratio of A260/A280 was in the range of 1.70-1.90. The size of genomic DNA was about 23 kb and the PCR was valid. Conclusion： The purification system using Fe3O4/Au composite microparticles has advantages in high yield, high purity, ease of operating, time saving and avoiding centrifugation. The purified sample was found to function satisfactorily in PCR amplification.
文摘Cyanophages are ubiquitous and essential components of the aquatic environment and play an important role in the termination of algal blooms.As such,they have attracted widespread interest.PP was the first isolated cyanophage in China,which infects Plectonema boryanum and Phormidium foveolarum.In this study,this cyanophage was purified three times by a double-agar overlay plaque assay and characterized.Its genome was extracted,totally sequenced and analyzed.Electron microscopy revealed a particle with an icosahedral head connected to a short stubby tail.Bioassays showed that PP was quite virulent.The genome of PP is a 42,480 base pair(bp),linear,double-stranded DNA molecule with 222 bp terminal repeats.It has high similarity with the known Pf-WMP3 sequence.It contains 41 open reading frames(ORFs),17 of which were annotated.Intriguingly,the genome can be divided into two completely different parts,which differ both in orientation and function.
