ABSTRACT
To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.
Subject(s)
Animals , Chick Embryo , Chickens , Chromosomes, Artificial, Bacterial , DNA, Recombinant , Herpesvirus 2, Gallid/genetics , Marek DiseaseABSTRACT
Rapid detection and classification of bacteria colonies ( Escherichia coli, Listeria monocytogens and Staphylococcus aureus) were investigated by using hyperspectral imaging. The hyperspectral reflectance images (390-1040 nm ) of bacterial colonies on agar plates were collected. Bacterial spectra were extracted automatically based on the masks produced by segmenting a band difference image using the OTSU method. Full wavelength and simplified PLS-DA models were established for classification of bacterial colonies. For the full wavelength model, the overall correct classification rate ( OCCR) and confident OCCR for the prediction set were 100% and 95. 9%, respectively. Besides, competitive adaptive reweighted sampling ( CARS), genetic algorithm ( GA ) and least angle regression-least absolute shrinkage and selection operator ( LARS-Lasso) were used to select feature wavelengths for the development of simplified models. Among them, the CARS-model outperformed the other two in terms of precision, stability and classification accuracy with OCCR and confident OCCR of 100% and 98. 0% for the prediction set, respectively. It was demonstrated that hyperspectral imaging was an effective technology for nondestructive detection of bacterial colonies with high accuracy and high speed. The allocated feature wavelengths by CARS could lay theoretical basis for developing low cost multispectral imaging systems for bacterial colony detection.
ABSTRACT
Objective:To investigate the Migration ability toward human pancreatic carcinoma cell line and human colon carcinoma cell line with difference HSP 70 plasma membrane expression .Methods: CD3-CD56+NK cells were obtained from human peripheral blood mononuclear(PBMC)in stem cell growth medium SCGM,2μg/ml TKD was added to the medium on 10th day,the ac-tivating receptor CD94/NKG2C expression levels on NK cells was detected with FAC after 4 days.The human pancreatic carcinoma cell line Colo357 and the human colon carcinoma cell line CW 2 were separated into Colo+and CW2+with high HSP70 expression and Colo-and CW2-with low HSP70 expression;Migration assays of NK to the four difference cell lines were performed in a Transwell cell culture system.The cytolytic activity of TKD-activated NK cells against the four subline with HSP 70 expression on their cell surface was analyzed by MTT assay.Results:Flow cytometry analysis showed that CD 3-CD56+NK cells could expanded after 2 weeks in SCGM medium,and the largest percentage of NK cell was (92.50 ±1.25 )%.CD94 expression levels on NK cells increased obviously after TKD inducement the cell surface HSP 70 expression of Colo+, Colo-were ( 78.2 ±2.2 )% and ( 27.3 ±1.2 )% separately , the cell surface HSP70 expression of CW2+,CW2-were (91.1±2.5)%and (18.2±1.0)%separately after FACS;the Migration of NK cells toward Colo+was (68.6±2.8)%,higher than the migration toward Colo-with (22.8±1.5)%;the Migration of NK cells toward CW2+was(73.5±2.7)%,higher than the migration toward CW2-with (18.2±1.3)%;the cytolytic activity of NK against Colo +was(61.2± 3.0)%compared to (24.5 ±1.5)%against Colo-when the ratio of effector cells and target cell was 20 ∶1,the cytolytic activity of NK against CW2+was (63.8±3.2)%compared to (22.4±1.8)% against CW2-when the ratio of effector cells and target cell was 20∶1.Conclusion:TKD-activated NK cells are highly efficient cytolytic effector cells which have stronger significant migration toward HSP70-positive tumor target cells on their cell surface in vitro .
ABSTRACT
BACKGROUND: Both electroacupuncture and salviae act on prevention and treatment ofis chemia-reperfusion injury, but, whether such action is achieved or not by coordination of both? OBJECTIVE: To probe into the effects of electroacupuncture and salviae on expression of myocardial cellular heat-shock protein 70mRNA that acts on protecting myocardium and on dopamine level that is the mark for myocardial injury after ischemia-repeifusion as well as the interaction between electroacupuncture and salviae. DESIGN: Factorial design of two factors. SETTLNG: Department of Anesthesia of Renji Hospital Affiliated to Shanghai Second Medical Scientific University. PARTICIPANTS: The experiment was performed in Animal Experimental Room of Renji Hospital from September 2001 to December 2002, in which 24 healthy New Zealand white rabbits were employed and randomized into 4 groups, 6 rabbits in each, named ① ischemia-reperfusion group (IR group), ② electroacupuncture group (EA group), ③ salviae group (SA group), ④ electroacupuncture + salviae group (ES group). METHODS: ① IR group: ischemia-reperfusion model was prepared by clipped the anterior descend branch of cardiac coronal artery for 30 minutes and released for 2 hours. ② EA group: modeling was done as the previous method. Twenty minutes before ischemia, electroacupuncture was done on Neiguan (PC 6), Yunmen (LU 2) and Lieque (LU 7), 0.8 V in voltage, 3.0-4.0 Hz in frequency. ③ SA group: modeling was done as the previous method. Before ischemia, salviae was injected intravenously once at 1.5 mg/kg, and was injected before and after reperfusion once respectively at 1.0 mg/kg. ④ ES group: modeling was done as the previous method. Both electroacupuncture and medication were applied in the group. The content of dopamine was determined in blood before ischemia,30 minutes after ischemia and 2 hours after reperfusion successively in rabbits of every group. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine myocardial heat-shock protein 70mRNA expression in ischemic and non-ischemic areas. RESULTS: After supplemented, 24 rabbits entered result analysis. ① Expression of myocardial heat-shock protein 70mRNA: that after ischemia and reperfusion was increased significantly compared that before ischemia in every group (P ＜ 0.01). That in the rest 3 groups was all higher than IR group,and that in ES group was higher than EA and SA groups (F=4.48, P ＜0.05). CONCLUSION: ① Both electroacupuncture and salivae stimulate expression of heat-shock protein 70 mRNA after ischemia and reperfusion, enhance protein stability of heat-shock protein 70 and alleviate myocardial damage. ② Both electroacupuncture and salivae inhibit increased dopamine content in the body after ischemia and reperfusion, reduce dopamine mediated injury and protect myocardium. ③ Coordination is present between electroacupuncture and salivae.