1977年3月,在巴塞尔生物:中心(那次 John 作了一个关于质粒克隆载体发展的学术报告)首次相见时,我们俩就确信,寻找一个能高效克隆大型 DNA 片段(】10kb)的全新的方法,是基因技术能否广泛应用的重要一环。当时,John 需要(这个需要就是发...1977年3月,在巴塞尔生物:中心(那次 John 作了一个关于质粒克隆载体发展的学术报告)首次相见时,我们俩就确信,寻找一个能高效克隆大型 DNA 片段(】10kb)的全新的方法,是基因技术能否广泛应用的重要一环。当时,John 需要(这个需要就是发明的原动力)从细菌克隆出青霉素酰基转移酶基因。这用传统的载体是难以办到的(那时象 EcoRI、SmaI、HindⅢ和 Sau3A 这些限制性酶只能由自己制备)。John展开更多
A genomic DNA library was constructed to the elite rice cultivar “Minghui 63” using the cosmid SuperCos1 as the vector. The library consisted of 45000 clones with average insert size about 40 kb. It was estimated ...A genomic DNA library was constructed to the elite rice cultivar “Minghui 63” using the cosmid SuperCos1 as the vector. The library consisted of 45000 clones with average insert size about 40 kb. It was estimated that this library had a capacity of 4.2 times equivalent of the haploid genome of rice.展开更多
AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expres...AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expression vectorcosmid pTM3.Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with therecombinant plasmid pTM3-Q534 by lipofectin.RESULTS From the transfected bacteriaTop10F’,2 pTM3-Q534 clones containing therecombinant plasmid were identified fromrandomly selected 10 ampicillin-resistantcolonies.By reverse transcription PCR andindirect immunofluorescence technique,HCVRNA and core protein was identified in HepG2cells transfected with the recombinant plasmid.CONCLUSION The construction of arecombinant plasmid and the expression of coregene cDNA of HCV in HepG2 was successful.展开更多
A comparative study on the phenotypic and genetic characteristics among Acidithiobacillus ferrooxidans （AF2）, a typic strain ATCC23270 and a previously isolated strain AF3 was performed. AF2 can use ferrous ion （F...A comparative study on the phenotypic and genetic characteristics among Acidithiobacillus ferrooxidans （AF2）, a typic strain ATCC23270 and a previously isolated strain AF3 was performed. AF2 can use ferrous ion （Fe^2＋） or elemental sulfur （S^0） as sole energy source, but oxidizes So more effectively than Fe^2＋, which is different from ATCC23270 and AF3. The G＋C content of AF2 is 51.8% （molar fraction）, however, ATCC23270 and AF3 strains have G＋C content of 63.7% and 64.8% （molar fraction）, respectively. The DNA-DNA hybridization results show that AF2 has 41.53% and 52.38% genome similarity to ATCC 23270 and AF3, respectively, but AF3 has a high genome similarity of 89.86% to ATCC 23270 strain. Rusticyanin （rus） and subunit III of aa3-type cytochrome oxidase （coxC） genes are not detected in AF2, but Fe^2＋ oxidase （iro） gene can be detected. To understand the genomic organization of iro gene, a cosmid library of AF2 genome was constructed and iro gene-containing clone was screened. The sequencing result shows that although the nucleotide sequence of iro gene in AF2 is completely identical to that of ATCC 23270 strain, its genomic organization is different from that of ATCC 23270. In AF2, iro is located at downstream ofpurA gene, while it is located at downstream ofpetC-2 gene in ATCC 23270 strain. These results indicate that AF2 is a novel strain ofA. ferrooxidans, and that phenotypic differences among the strains ofA. ferrooxidans are closely correlated with their genetic polymorphisms.展开更多
Objective: To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector pTM3 and to express HCV core antigen in HepG2 cells. Methods: Core gene cDNA of HCV was introduced into eukaryoti...Objective: To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector pTM3 and to express HCV core antigen in HepG2 cells. Methods: Core gene cDNA of HCV was introduced into eukaryotic expression vector pTM3. Using vaccinia virus/bacteriophage T7 hybrid expression system, HepG2cells were trans feeted with the recombinant plasmid pTM3-Q534 by lipofectin. Results: From the transfected bacteria Top10F, 2pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10ampicillin-resistant colonies. By indirect immunofluorescence technique HCV core protein was identified in HepG2cells trans feeted with the recombinant plasmid. Conclusion: The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 were successful.展开更多
Using plant mini-Ti cosmid pEND4K, the cosmid genomic library of Sesbania rostratawas constructed. Sizes of plant DNA inserts in clones were 25- 33 kb. The restrictionmapping showed that different recombinant involved...Using plant mini-Ti cosmid pEND4K, the cosmid genomic library of Sesbania rostratawas constructed. Sizes of plant DNA inserts in clones were 25- 33 kb. The restrictionmapping showed that different recombinant involved various types of plant DNA insert. 4clones containing leghemoglobin gene sequence of S. rostrata were obtained by in situ hy-bridization of colonies. The cloning of leghemoglobin gene sequence has been confirmedby plasmid DNA dot hybridization and Southern blot hybridization.展开更多
文摘1977年3月,在巴塞尔生物:中心(那次 John 作了一个关于质粒克隆载体发展的学术报告)首次相见时,我们俩就确信,寻找一个能高效克隆大型 DNA 片段(】10kb)的全新的方法,是基因技术能否广泛应用的重要一环。当时,John 需要(这个需要就是发明的原动力)从细菌克隆出青霉素酰基转移酶基因。这用传统的载体是难以办到的(那时象 EcoRI、SmaI、HindⅢ和 Sau3A 这些限制性酶只能由自己制备)。John
文摘A genomic DNA library was constructed to the elite rice cultivar “Minghui 63” using the cosmid SuperCos1 as the vector. The library consisted of 45000 clones with average insert size about 40 kb. It was estimated that this library had a capacity of 4.2 times equivalent of the haploid genome of rice.
基金the National Natural Science Foundation of China,No.39500129
文摘AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expression vectorcosmid pTM3.Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with therecombinant plasmid pTM3-Q534 by lipofectin.RESULTS From the transfected bacteriaTop10F’,2 pTM3-Q534 clones containing therecombinant plasmid were identified fromrandomly selected 10 ampicillin-resistantcolonies.By reverse transcription PCR andindirect immunofluorescence technique,HCVRNA and core protein was identified in HepG2cells transfected with the recombinant plasmid.CONCLUSION The construction of arecombinant plasmid and the expression of coregene cDNA of HCV in HepG2 was successful.
基金Project(200805032) supported by the Scientific Research Program of Marine Public Welfare Industry of China
文摘A comparative study on the phenotypic and genetic characteristics among Acidithiobacillus ferrooxidans （AF2）, a typic strain ATCC23270 and a previously isolated strain AF3 was performed. AF2 can use ferrous ion （Fe^2＋） or elemental sulfur （S^0） as sole energy source, but oxidizes So more effectively than Fe^2＋, which is different from ATCC23270 and AF3. The G＋C content of AF2 is 51.8% （molar fraction）, however, ATCC23270 and AF3 strains have G＋C content of 63.7% and 64.8% （molar fraction）, respectively. The DNA-DNA hybridization results show that AF2 has 41.53% and 52.38% genome similarity to ATCC 23270 and AF3, respectively, but AF3 has a high genome similarity of 89.86% to ATCC 23270 strain. Rusticyanin （rus） and subunit III of aa3-type cytochrome oxidase （coxC） genes are not detected in AF2, but Fe^2＋ oxidase （iro） gene can be detected. To understand the genomic organization of iro gene, a cosmid library of AF2 genome was constructed and iro gene-containing clone was screened. The sequencing result shows that although the nucleotide sequence of iro gene in AF2 is completely identical to that of ATCC 23270 strain, its genomic organization is different from that of ATCC 23270. In AF2, iro is located at downstream ofpurA gene, while it is located at downstream ofpetC-2 gene in ATCC 23270 strain. These results indicate that AF2 is a novel strain ofA. ferrooxidans, and that phenotypic differences among the strains ofA. ferrooxidans are closely correlated with their genetic polymorphisms.
文摘Objective: To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector pTM3 and to express HCV core antigen in HepG2 cells. Methods: Core gene cDNA of HCV was introduced into eukaryotic expression vector pTM3. Using vaccinia virus/bacteriophage T7 hybrid expression system, HepG2cells were trans feeted with the recombinant plasmid pTM3-Q534 by lipofectin. Results: From the transfected bacteria Top10F, 2pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10ampicillin-resistant colonies. By indirect immunofluorescence technique HCV core protein was identified in HepG2cells trans feeted with the recombinant plasmid. Conclusion: The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 were successful.
基金Project supported by the National Natural Science Foundation of China.
文摘Using plant mini-Ti cosmid pEND4K, the cosmid genomic library of Sesbania rostratawas constructed. Sizes of plant DNA inserts in clones were 25- 33 kb. The restrictionmapping showed that different recombinant involved various types of plant DNA insert. 4clones containing leghemoglobin gene sequence of S. rostrata were obtained by in situ hy-bridization of colonies. The cloning of leghemoglobin gene sequence has been confirmedby plasmid DNA dot hybridization and Southern blot hybridization.
