ABSTRACT
To clone porcine bone marrow stromal antigen-2 (BST-2) gene, construct its recombinant eukaryotic expression plasmid and induce the expression of the fusion antiviral protein, we amplified BST-2 gene by RT-PCR from the total RNA extracted from PK15 cells. The recombinant expression plasmid pcDNA-BST-2 was constructed and then was transfected into HEK293T cells to expresse the BST-2 fusion protein. Western blot and indirect immunofluorescence assay (IFA) were performed, and the biological activity was detected. The results showed that the construction of recombinant plasmid pcDNA-BST-2 was confirmed by restriction enzyme digestion and sequencing. The expressed product had antiviral activity against Vesicular stomatitis virus (VSV), Avian influenza virus (AIV) and Porcine reproductive and respiratory syndrome virus (PRRSV). In conclusion, the research paves the way for further research on bioactivity assayand antiviral medication.
Subject(s)
Animals , Humans , Antigens, CD , Genetics , Allergy and Immunology , Cell Line , Chickens , Cloning, Molecular , Gene Expression , Influenza in Birds , Allergy and Immunology , Virology , Orthomyxoviridae , Physiology , Porcine Reproductive and Respiratory Syndrome , Allergy and Immunology , Virology , Porcine respiratory and reproductive syndrome virus , Physiology , Swine , Vesicular Stomatitis , Allergy and Immunology , Virology , Vesicular stomatitis Indiana virus , Physiology , Virus ReplicationABSTRACT
<p><b>BACKGROUND</b>Multidrug resistance to chemotherapeutic agents is an important clinical problem during the treatment of leukemia. The resistance process is multifactorial. To realize the total factors involved in multidrug resistance, we analyzed the differentially expressed proteins of K562 and K562/ADM cells and we investigated one of the up-regulated proteins (CRKL) using siRNA to determine its role in K562/ADM cells.</p><p><b>METHODS</b>Altered protein expressions between K562/S (K562 ADM-sensitive cell line) and K562/ADM (K562 multidrug resistant cell line induced by adriamycin) were identified by 2D-DIGE coupled with mass spectrometry. Meanwhile, we confirmed the differential expression of CRKL and Stathmin in both K562 and K562/ADM cells by Western blot analysis. Furthermore, we used RNA interference to silence the CRKL gene expression.</p><p><b>RESULTS</b>Among the 9 differentially expressed proteins, 3 were up-regulated in K562/ADM cells, while 6 were down-regulated in the K562/ADM cells compared with its parent cell line. The expression of CRKL was up-regulated significantly in K562/ADM cells, and it can be decreased by recombinant lentivirus. Moreover, the multidrug resistance of K562/ADM cells was efficiently reversed by silence of CRKL gene expression.</p><p><b>CONCLUSIONS</b>The data provided the differentially expressed proteins in K562 and its resistant cell line and highlights the power of 2D-DIGE for the discovery of resistance markers in cancer. We found CRKL may be a new protein involved in the multidrug resistance of leukaemia cells.</p>
Subject(s)
Humans , Adaptor Proteins, Signal Transducing , Genetics , Amino Acid Sequence , Doxorubicin , Pharmacology , Drug Resistance, Multiple , K562 Cells , Chemistry , Molecular Sequence Data , Neoplasm Proteins , Nuclear Proteins , Genetics , Proteomics , StathminABSTRACT
Ribosome inactivating protein(RIP) is a kind of toxin plant pr ot ein in which extensively lives in the body of higher plants and controls ribosom e's function. Beside it can control protein's combination,it has lots of biolog ical reactivity as resisting giving birth and tumor and controlling HIV. At fir st, RIP is isolated from seeds of bitter melon.The result of SDS-PAGE indicate s that there are lots of RIP in the abstraction liquid.Then we study the antivi ral action of RIP through the cell of CEF and SPF chicken embryos.The results s how that RIP can resist NDVF_48E_8,MDVCVI_988 and FPV-SD4 to so me extent.