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Methods for analyses of protein-protein interactions include: yeast two hybrid (Y2H), phage dis- play (PD), co-immunoprecipitation (Co-IP), glutathione S-transferase pull-down (GST pull-down), cellular co-localization, far-western blotting, virus overlay protein binding assay (VOPBA), surface plasmon resonance (SPR), and fluorescence resonance energy transfer (FRET). Technologies for the detection of protein-nucleic acid interactions include: yeast one hybrid (Y1H), chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), Southwestern blotting, reporter gene, Co-IP, GST pull-down, and PD. These methods are often used in the study of the human enterovirus A71 (EV-A71) by our research team. Reviews in the Chinese literature in this field are lacking, so we reviewed applications of these methods in the study of EV-A71. This review may impart important knowledge in the research of other viruses with regard to protein-protein and protein-nucleic acid interactions.
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Electrophoretic Mobility Shift Assay , Enterovirus A, Human , Chemistry , Genetics , Metabolism , Fluorescence Resonance Energy Transfer , RNA, Viral , Metabolism , Two-Hybrid System Techniques , Viral Proteins , MetabolismABSTRACT
Objective To describe the genetic characterization of complete genome from a human coxsackievirus A16 (CA16) strain KMM08,isolated in Yunnan,China,in 2008.Methods By using RT-PCR,the seven fragments contained about 1000 nucleotides in the complete genome were sequenced.The sequences were aligned with other enterovirus sequences downloaded from GenBank using Mega 4.1,RDP3 and SimPlot 3.5.1 software.Results As in other human enterovirus,its genome was 7409 nucleotides in length,encoding for 2193 amino acids.KMM08 strain was closely related to other reference strains of B genotype.In the complete genome,the homology of nucleotide and amino acid among the eleven CA16 isolated strains were 79.0％-98.2％ and 94.5％-99.3％,respectively.The rates of homology were 79.1％ and 94.8％ when comparing with that of G10 strains and 78.7％ and 89.0％ comparing with that of BrCr strains,respectively.SZ-HK08-3 strain had high homology when compared to other strains.In different segment of genome,the rates of homology were 97.0％-99.0％ and 98.0％-100.0％ when compared with that of SZ-HK08-3 strains,respectively.The rates of homology were 74.2％-86.9％ and 90.9％-97.0％ when compared with that of G10 strains,respectively and were 65.0％-84.9％ and 71.0％-95.2％ when compared with that of BrCr strains.Data from Phylogenetic analysis showed that KMM08 belong to genotype B.The putative recombinant Tainan-5079-98 was detected positive with RDP3 and SimPlot 3.5.1.Conclusion KMM08 strains isolated in Yunnan in 2008 belonged to B genotype of coxsackivirus A16.The possible occurrence of inter-typic recombination would involve EV71 and CA16.
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The genome of a new SV40 strain(SV-IMB)isolated from a rhesus monkey was completely sequenced and compared with other isolates.The results showed that the whole genome contains 5246bp,and the average identity of SV-IMB was 98.1% as compared to other SV40 isolates.Its regulatory region is composed of a complete enhancer and a defective enhancer.Amino acid changes occurred to some extent in both the large T antigen (T-Ag) and VP1 region.The findings demonstrate that the SV-IMB is a new SV40 isolate.
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The proteins encoded by the Herpesviridae β-gene play a critical role in the replication stage of the virus. In this paper, phylogenetic analyses provided evidence that someβ-gene products, such as UL2 and UL23 from HSV1, have their homologous genes in its family, and also exist in prokaryotic organisms, indicating that these viruses appear to have been assembled over evolutionary time by numerous independent events of horizontal gene transfer.
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As an immediate-early protein of herpes simplex virus, infected-cell polypeptide 0 (ICP0) exhibits complicated interactions with host cells, and its regulatory function on gene expression is of great importance. Since the ICP0 encoding sequence contains many rare codons which are absent in E.coli, and ICP0 is highly unstable in prokaryotic cells, expression of entire ICP0 in prokaryotic cells has never been reported. In order to further investigate the function of ICP0, a recombinant plasmid was constructed by subcloning a cDNA fragment encoding an amino-terminal of 105 residues of the ICP0 protein into pGEX-5x-1 vector. The resulting GST-105 fusion antigen peptide was expressed with high efficiency in E.coli. Antibodies prepared after the immunization of mice with purified fusion protein can recognize not only the denatured ICP0 protein, but also the native ICP0 protein with normal biological conformation.
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Herpes simplex virusⅠ(HSVⅠ) regulating the pathway of transcription and translation modify in host cell is a very systematic and complicate system. A clear understanding of the concrete mechanisms of infection will greatly help to comprehend the virus replication and the interaction with the host cell. By the analysis of 2-DE, the heterogeneous nuclear ribonucleoprotein H2 in human fetal liver cell represent distinction after the HSVⅠinfection.Utilization of Northern blot and Western blot technologies verified the expression of hnRNP H2 in different stage of virus infection is varied.
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Object: To study the proliferation of hCTGF on cells and its biological function on bone injury healing.Methods: The fibroblast with potential differentiation was transfected by eukaryotic gene delivery system and then transferred into the experimental animal model with bone fracture.The data were collected by molecular biological and clinical orthopedic technique detection analysis.Results: The results demonstrated an obvious proliferation of hCTGF on cells,suggesting that hCTGF have the biological activity of repairing bone injury via gene therapy.The results provide a new activity factor and treatment approach for bone injury in clinics.
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<p><b>OBJECTIVE</b>To study the relationship between the structure and functional activity of hTNF alpha.</p><p><b>METHODS</b>Four hTNF alpha mutants were constructed, different binding structures and gene responses related with these mutants were studied by the methods of immunoprecipitation and mRNA differential display.</p><p><b>RESULTS</b>The specific activities and LD50 of the different hTNF alpha mutants indicated their different bioactivities. It was shown that the hTNF alpha mutants had the relative binding affinities to the wild types. The mRNA differential display assay proved that the hTNF alpha mutants stimulated different gene responses.</p><p><b>CONCLUSION</b>These results suggest that the specific anti-tumor activities of hTNF alpha mutants are accomplished by inducing different or same gene response at different quantities after its binding to specific receptor.</p>