The 16746-neucleotide (nt) sequence of mitochondrial DNA (mtDNA) of Chinese alligator, Alligator sinensis, was determined using the Long-PCR and primer walking methods. As is typical in vertebrates, the mtDNA encodes ...The 16746-neucleotide (nt) sequence of mitochondrial DNA (mtDNA) of Chinese alligator, Alligator sinensis, was determined using the Long-PCR and primer walking methods. As is typical in vertebrates, the mtDNA encodes 13 proteins, 2 rRNA, 22 tRNA genes, and a noncoding control region. The composition of bases is respectively 29.43% A, 24.59% T, 14.86% G, 31.12% C. The gene arrangement differs from the common vertebrate gene arrangement, but is similar to that of other crocodiles. DNA sequence data from 12S rRNA, 16S rRNA, protein-coding genes and combined sequence data were used to reconstruct the phylogeny of reptiles with the MP and ML methods. With this large data set and an appropriate range of outgroup taxa, the authors demonstrate that Chinese alligator is most closely related to American alligator among three crocodilian species, which suppors the traditional viewpoint. According to the branch lengths of ML tree from the combined data set, the primary divergence between Alligator and Caiman genus was dated at about 74.9 Ma, the split between Chinese alligator and American alligator was dated at 50.9 Ma.展开更多
The recombinant adeno-associated viral vector with human coagulation Factor Ⅸ minigene which wasregulated by CMV promoter was constructed. Largequantity of recombinant adeno-associated viral particles (rAAV/ hFⅨ) wa...The recombinant adeno-associated viral vector with human coagulation Factor Ⅸ minigene which wasregulated by CMV promoter was constructed. Largequantity of recombinant adeno-associated viral particles (rAAV/ hFⅨ) was prepared by the HSV/AAV hybrid helper virus method. Southern dot blot assay and QC-PCR indicated that the titer of the virus was 3.6×1012 v.g./mL. It demonstrated that this method can effectively overcome the hurdles of mass production of AAV vector. Followed by anintramuscular injection of viral vectors (7.5×1011 v.g./mouse) in the quadriceps femoris, an elevation of human Factor Ⅸexpression in the plasma of hemophilia B mice was detected (387 ng/mL) and persisted more than 12 weeks. The level of anti-virus antibody in plasma aligned with the Factor Ⅸexpression curve. The QC-PCR method is easier and moreaccurate than traditional dot hybridization fordetermination of the titer of recombinant adeno-associated virus. Moreover, there are no HSV particles existing inproduced AAV assayed by RT-PCR. AAV is the only virus that has been amplified from AAV-injected muscle by PCR.展开更多
基金This work was supported by the Ministry of Education of China(Grant No.GG-180-2100-2403-1740)SRF for ROCS,from the Ministry of Education of China.
文摘The 16746-neucleotide (nt) sequence of mitochondrial DNA (mtDNA) of Chinese alligator, Alligator sinensis, was determined using the Long-PCR and primer walking methods. As is typical in vertebrates, the mtDNA encodes 13 proteins, 2 rRNA, 22 tRNA genes, and a noncoding control region. The composition of bases is respectively 29.43% A, 24.59% T, 14.86% G, 31.12% C. The gene arrangement differs from the common vertebrate gene arrangement, but is similar to that of other crocodiles. DNA sequence data from 12S rRNA, 16S rRNA, protein-coding genes and combined sequence data were used to reconstruct the phylogeny of reptiles with the MP and ML methods. With this large data set and an appropriate range of outgroup taxa, the authors demonstrate that Chinese alligator is most closely related to American alligator among three crocodilian species, which suppors the traditional viewpoint. According to the branch lengths of ML tree from the combined data set, the primary divergence between Alligator and Caiman genus was dated at about 74.9 Ma, the split between Chinese alligator and American alligator was dated at 50.9 Ma.
基金supported by the National Natural Science Foundation of China(Grant No.30100102).
文摘The recombinant adeno-associated viral vector with human coagulation Factor Ⅸ minigene which wasregulated by CMV promoter was constructed. Largequantity of recombinant adeno-associated viral particles (rAAV/ hFⅨ) was prepared by the HSV/AAV hybrid helper virus method. Southern dot blot assay and QC-PCR indicated that the titer of the virus was 3.6×1012 v.g./mL. It demonstrated that this method can effectively overcome the hurdles of mass production of AAV vector. Followed by anintramuscular injection of viral vectors (7.5×1011 v.g./mouse) in the quadriceps femoris, an elevation of human Factor Ⅸexpression in the plasma of hemophilia B mice was detected (387 ng/mL) and persisted more than 12 weeks. The level of anti-virus antibody in plasma aligned with the Factor Ⅸexpression curve. The QC-PCR method is easier and moreaccurate than traditional dot hybridization fordetermination of the titer of recombinant adeno-associated virus. Moreover, there are no HSV particles existing inproduced AAV assayed by RT-PCR. AAV is the only virus that has been amplified from AAV-injected muscle by PCR.
