RESUMO
To understand the HA1 genetic variation characterization of influenza H3N2 virus isolates in Zhu-hai during 2008-2009, we selected 20 of H3N2 Influenza strains cultured in MDCK cell. Viral RNAs were extracted and amplified by using RT-PCR. The amplified products were purified after identified by gel electrophoresis and then the nucleotide sequences of the amplicons were determined. The results were analyzed by the software ClustalX and MEGA4. 1. When compared with the amino acid sequences of the epitopes of HA1 district of H3N2 influenza vaccine recommended by WHO in 2008, changes were found in those of H3N2 influenza strains in Zhuhai in 2008: K140I in all of H3N2 influenza strains, L157S in 08-0343 and 08-0677, K158R in 08-0466, 08-0620 and 08-0667, K173E in 08-0466 and 08-0620, K173N in 08-0667, and I192T in 08-0667. The epitopes of HA1 district of H3N2 influenza strains in Zhuhai in 2009 are different from that of H3N2 influenza vaccine during the same time: K173Q and P194L occur in all of H3N2 influenza strains, N144K, K158N, and N189K occur in the strains except the strain 09-0056. HA1 domain of H3N2 influenza strains in 2009 has lost a glycosylation site at amino acid position 144 while the glycosylation sites of HA1 domain of H3N2 influenza stains isolated in 2008 remained. This study suggested that H3N2 influenza virus in Zhuhai in 2008 was not evolved a novel variant and H3N2 influenza variant in 2009 was attributed to antigenic drift in HA1 district.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Animais , Antígenos Virais/imunologia , Linhagem Celular , China , Cães , Epitopos/imunologia , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H3N2/classificação , Vírus da Influenza A Subtipo H3N2/imunologia , Mutação , Filogenia , Análise de Sequência de DNARESUMO
The effective delivery of exogenous genes into eukaryotic cells is important for fundamental and biotechnological research. Protein-based gene delivery including histone proteins has recently emerged as a powerful technique for non-viral DNA transfer. Histones are DNA-binding proteins that function in DNA packaging and protection. In particular, histone H1 is largely responsible for the stabilization of higher-order chromatin structures. Several studies have examined the use of full-length histone H1-mediated gene transfer, and a few studies have investigated the use of C-terminal histone H1 fragments as gene-transfer materials. Previously, we cloned a novel histone H1 cDNA from the goldfish Carassius auratus and found that a recombinant histone H1 C-terminal short peptide (H1C) of 61 amino acids has comparable DNA binding and protection functions as full-length histone H1. In the present work, we successfully expressed and purified soluble recombinant H1C in an Escherichia coli expression system using a hexahistidine tag fusion strategy and providing tRNAs for rare codons. We confirmed its DNA-binding ability and found that this H1C peptide had similar or higher transfection efficiency in mammalian cells (human 293T and mouse NIH/3T3) than the widely used agent lipofectamine. Therefore, we suggest that this novel goldfish-derived recombinant histone H1 C-terminal short peptide could be used as a peptide-based gene-transfer mediator.
Assuntos
Carpa Dourada/metabolismo , Histonas/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Transfecção/métodos , Animais , Western Blotting , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Carpa Dourada/genética , Histonas/química , Histonas/genética , Humanos , Camundongos , Células NIH 3T3 , Fragmentos de Peptídeos/genética , Ligação Proteica , Proteínas Recombinantes/genéticaRESUMO
OBJECTIVE: To screen cDNA library from Clonorchis sinensis for gene identification, and to clone and construct library of secreted recombinant proteins. METHODS: cDNA library from Clonorchis sinensis was screened for identifying new genes by Blastn protocol, the sequence of which was further analyzed by Motifscan and NCBI Conserved Domain Search protocol. RESULTS: The secreted proteins (Pcs004f03) were identified, with a 689 bp DNA sequence (187aa), and a theoretical molecular weight of Mr 21100. It is predicted that the gene CsCrSP contains one N-glycosylation site, three N-myristoylation sites, three casein kinase II phosphorylation sites, one cAMP-and cGMP-dependent protein kinase phosphorylation site and protein kinase C phosphorylation site, a signal peptide at N-terminals and SCP-like extracellular protein domain. CONCLUSION: The CsCrSP gene is a secretory cysteine-rich protein with a SCP-like extracellular protein domain.
