An about 1.40 Kb target gene fragment was yielded by PCR amplification with the plasmid pRB 129,which was identified by restriction enzyme digestion that the PCR product was TUB2 gene.The gene was digested by the rest...An about 1.40 Kb target gene fragment was yielded by PCR amplification with the plasmid pRB 129,which was identified by restriction enzyme digestion that the PCR product was TUB2 gene.The gene was digested by the restriction enzyme and was linked with pTA plasmid to construct pTA TUB2 plasmid.The plasmid was transformed into Chaetomium spp.by PEG method and the transformation rate was 27/(2×10 5) and it is nine times higher than that of pRB 129.The transformants can grow on the PDA containing 1 000 μg·mL -1 carbendazim,which is 1 000 times higher than the original Chaetomium spp.The resistance was stable after 10 times transfer on non selective medium.展开更多
Objective:This study aimed to prepare Golgi protein-73(GP73)-DC vaccine by constructing human lentiviral vector of GP73and transfecting this vector in vitro into a murine dendritic cell line DC 2.4.This will lay the f...Objective:This study aimed to prepare Golgi protein-73(GP73)-DC vaccine by constructing human lentiviral vector of GP73and transfecting this vector in vitro into a murine dendritic cell line DC 2.4.This will lay the foundation for further study on targeted therapy of hepatocellular carcinoma using GP73-DC vaccine.Methods:Human GP73 gene was cloned into lentivirus GV358vector and validated by polymerase chain reaction(PCR)and sequencing.The validated GV358-GP73vector was then cotransfected with pHelper 1.0plasmid into 293Tcells,in order to prepare lentivirus GP73vector.Finally,the protein expression of GP73 was achieved by transfecting the lentivirus GP73vector into DC 2.4cells in vitro and determined by Western blotting.Results:PCR and sequencing confirmed that the GV358-GP73lentivirus vector was constructed correctly,and the titer level reached 2×108TU/mL.After transfection in DC 2.4cells,the GP73protein level was significantly enhanced.Conclusion:The lentivirus GV538-GP73vector was constructed successfully,and could be effectively expressed in DC 2.4cells.展开更多
从临床发病犬采集粪便样品,以F81传代细胞进行病毒分离,经血球凝集实验(HA)和血球凝集抑制实验(HI)初步鉴定为犬细小病毒。为进一步确诊,根据Genbank中已发表的犬细小病毒VP2基因序列设计并合成一对引物,通过聚合酶链反应(PCR)扩增出CPV...从临床发病犬采集粪便样品,以F81传代细胞进行病毒分离,经血球凝集实验(HA)和血球凝集抑制实验(HI)初步鉴定为犬细小病毒。为进一步确诊,根据Genbank中已发表的犬细小病毒VP2基因序列设计并合成一对引物,通过聚合酶链反应(PCR)扩增出CPV VP2基因,酶切,测序加以鉴定。其序列与国际已发表的CPV-d(type 2)、CPV-1(5 type 2a)、CPV-3(9 type 2b)VP2序列同源性分别为98.97%,98.75%,98.69%,氨基酸序列的同源性分别为98.12%,97.60%,97.60%,从而证明此分离株为犬细小病毒。在获得VP2基因的基础上,为实现VP2蛋白的表达,构建了真核表达质粒pMel BacC-VP2。展开更多
基金Heilongjiang Province Natural Science fund( Grant No.C0 2 0 3 )
文摘An about 1.40 Kb target gene fragment was yielded by PCR amplification with the plasmid pRB 129,which was identified by restriction enzyme digestion that the PCR product was TUB2 gene.The gene was digested by the restriction enzyme and was linked with pTA plasmid to construct pTA TUB2 plasmid.The plasmid was transformed into Chaetomium spp.by PEG method and the transformation rate was 27/(2×10 5) and it is nine times higher than that of pRB 129.The transformants can grow on the PDA containing 1 000 μg·mL -1 carbendazim,which is 1 000 times higher than the original Chaetomium spp.The resistance was stable after 10 times transfer on non selective medium.
基金supported by the National Natural Science Foundation of China (No.81360347)the Key University and College Project of Guangxi Provisional Department of Education(No.ZD2014027).
文摘Objective:This study aimed to prepare Golgi protein-73(GP73)-DC vaccine by constructing human lentiviral vector of GP73and transfecting this vector in vitro into a murine dendritic cell line DC 2.4.This will lay the foundation for further study on targeted therapy of hepatocellular carcinoma using GP73-DC vaccine.Methods:Human GP73 gene was cloned into lentivirus GV358vector and validated by polymerase chain reaction(PCR)and sequencing.The validated GV358-GP73vector was then cotransfected with pHelper 1.0plasmid into 293Tcells,in order to prepare lentivirus GP73vector.Finally,the protein expression of GP73 was achieved by transfecting the lentivirus GP73vector into DC 2.4cells in vitro and determined by Western blotting.Results:PCR and sequencing confirmed that the GV358-GP73lentivirus vector was constructed correctly,and the titer level reached 2×108TU/mL.After transfection in DC 2.4cells,the GP73protein level was significantly enhanced.Conclusion:The lentivirus GV538-GP73vector was constructed successfully,and could be effectively expressed in DC 2.4cells.
文摘从临床发病犬采集粪便样品,以F81传代细胞进行病毒分离,经血球凝集实验(HA)和血球凝集抑制实验(HI)初步鉴定为犬细小病毒。为进一步确诊,根据Genbank中已发表的犬细小病毒VP2基因序列设计并合成一对引物,通过聚合酶链反应(PCR)扩增出CPV VP2基因,酶切,测序加以鉴定。其序列与国际已发表的CPV-d(type 2)、CPV-1(5 type 2a)、CPV-3(9 type 2b)VP2序列同源性分别为98.97%,98.75%,98.69%,氨基酸序列的同源性分别为98.12%,97.60%,97.60%,从而证明此分离株为犬细小病毒。在获得VP2基因的基础上,为实现VP2蛋白的表达,构建了真核表达质粒pMel BacC-VP2。
