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Cloning of Thymidine Kinase Gene of Duck Plague Virus Using degenerate pcr

Agricultural Sciences in China 2005年第8期4卷 634-640页

作者： HAN Xian-jie WANG Jun-wei Department of Veterinary Medicine Northeast Agricultural University Harbin 150030 P.R.China Department of Animal Husbandry and Veterinary Science Laiyang Agricultural College Laiyang 265200 P.R.China

The DNA of duck plague virus （DPV） thymidine kinase （TK） gene was cloned and sequenced from a vaccine virus in the study. degenerate oligonucleotide primers for the consensus site of herpesvirus UL24, TK, and glycoprotein H（gH） gene were used in the polymerase chain reaction （pcr） to amplify DNA product with 3 741-base-pairs （bp） in size. DNA sequence analysis revealed a 1 077-base-pairs （bp） open reading frame （ORF） encoding a 358 amino acid polypeptide homologous to herpesvirus TK proteins. The predicted TK protein shared 31.2, 41.3, 35.7, 37.4, and 28.4% identity with herpes simplex virus typel, equine herpesvirus type 4, Marek＇s disease virus 2, herpesvirus turkey, and infectious laryngotracheitis virus, respectively. Comparison of the amino acid sequences of other herpesvirus TK proteins showed that these proteins were not conserved on the whole, otherwise the portion of the TK proteins corresponding to the nucleotide binding domain and the nucleoside binding site were highly conserved among herpesvirus. Comparison with the amino acid sequences of the conserved nucleotide and nucleoside binding domains of other eleven herpesvirus TK proteins to the predicted DPV peptide confirmed its identity as the DPV TK protein.

关键词： Duck plague virus degenerate pcr Thymidine kinase gene

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Cloning and Sequence of Glycoprotein H Gene of Duck Plague Virus

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Agricultural Sciences in China 2006年第5期5卷 397-402页

作者： HAN Xian-jie WANG Jun-wei MA Bo Faculty of Veterinary Medicine Northeast Agricultural University Harbin 150030 P.R. China Faculty of Animal Science and Veterinary Medicine Laiyang Agricultural College Qingdao 266109 P.R. China

The glycoprotein H （gH） gene homologue of duck plague virus （DPV） was cloned by degenerate polymerase chain reaction （pcr） and sequenced. It was located immediately downstream from the thymidine kinase gene （TK）. In addition, the 3＇-end of the gene homologue to herpesvirus UL21 was located downstream from the gH gene. DPV gH gene open reading frame （ORF） was 2 505 bp in length and its primary translation product was a polypeptide of 834 amino acids long. It possessed several characteristics of membrane glycoproteins, including an N-terminal hydrophobic signal sequence, an external domain containing eight putative N-linked glycosylation sites, a C-terminal transmembrane domain, and a charged cytoplasmic tail. Comparison with other herpesvirus revealed identities of 20.2, 25.1, 23.0, 23.0, 26.5 and 26.0% with the gH counterparts of the human herpesvirus virus 1 （HSV1）, equine herpesvirus 4 （EHV4）, bovine herpesvirus 1 （BHV1）, pseudorabies virus （PRV）, gallid herpesvirus 2 （GHV2） and gallid herpesvirus 3 （GHV3）, respectively.

关键词： duck plague virus glycoprotein H gene degenerate pcr

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Detection and Protein Modelling for Consensus Region of hsp70 Gene Family in Egyptian Arundo donax

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Journal of Life Sciences 2014年第7期8卷 593-602页

作者： Mohamed A. Ezz Mona I. Salah Abdullah I. Ammoura GEBR1 Bioinformatics Department Sadat University P.O. Box 79/22857 Egypt College of Bioteehnology Misr University for Scienceand Technology 6th of October City October 12451 Egypt Molecular Genetics Lab College of Biotechnology Misr University for Scienceand Technology 6th of October City October12451 Egypt

degenerate primers are particularly useful in amplifying homologous genes from different organisms. This paper describes a method for designing degenerate primers for a given multiple alignment of DNA sequences of hsp70 gene family using ClustalW algorithm and detect the consensus region in gene family of hsp70 in a plant species which have not any recorded information about hsp70 gene family in the Genbank of National Centre of Biotechnology Information （NCBI） like Arando donax .The sequenced consensus sequence ofArando donax is considered a gene marker in building the genome map sequence. The BLASTn program is used to find a homology between more than one accession numbers of DNA sequences, （X67711.2） was for Oryza sativa （hsp70）, （AY372071. l） was for Nicotiana tabacum （hsp70） and （L41253.2） was for Lycopersicon esculentum （Hsc70）. In silco pcr module was performed to detect the melting temperatures （Tm） and predicted the pcr product size （783 bp）.The result of designed degenerate primers showed that there was a homology founded among the designed primers and the DNA templates of the recorded sequences （AY372071.1, X67711.2 and L41253.2） with at least 80% identity. The designed degenerate primers were used to isolate a consensus region ofhsp70 gene family ofArando donax at the expected molecular weight （783 bp）. The isolated pcr product, （783 bp） ofArando donax was sequenced and submitted to the Database of Japan （DDBJ） with accession number AB819871. The ORF finder tool translated the accession number AB819871 and gave a selected frame which used to build 3D structure model. In conclusion, this study focused on the importance of designing the degenerate primers to isolate the gene family and predict the 3D structure of gene family depending on the ORF finder tool of Genebank.

关键词： ClustalW tool degenerate pcr different melting temperatures in silico pcr SWISS-MODEL workspace.

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Molecular Studies on Heat Shock Protein 70 Gene （Hsp70） Using ClustalW Multiple Sequence Alignment Analysis

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Journal of Life Sciences 2010年第6期4卷 1-8页

作者： Mohamed A. Ezz Mohamed H. Soliman Abd Alkader Y. Gamal El-Din Aly Z.E. Abdelsalam Molecular Genetics Lab Biotechnology College Misr University for Science and Technology 6th of October City October 12451 Egypt Department of Genetics Faculty of Agriculture Cairo University Giza 12613 Egypt A in Shams Center for Genetics Engineering and Biotechnology Faculty of Agriculture Ain Shams University Cairo 11241 Egypt

degenerate primers are particularly useful in amplifying homologous genes from different organisms. This paper describes a method for designing degenerate primers for a given multiple alignment of DNA sequences of Hsp70 gene family using ClustalW algorithm. The authors used an in silico approach to find a homology between more than one accession numbers of DNA sequences, X67711.2 was for Oryza sativa Hsp70, AY372071.1 was for Nicotiana tabacum Hsp70 and L41253.2 was for Lycopersicon esculentum Hsc70. The three accession numbers which were retrieved by the BLASTn program depend on their expected value （E-value）. Multiple sequence alignment was performed by ClustalW algorithm to produce a conserved blocks and determine the consensus region which had been used to produce the forward and reverse primer by the primer select module of DNAStar Lasergene V7 and In-Silco pcr module of FASTpcr program ver.4.0.8 was performed to detect the melting temperatures （Tm） and predict the pcr product size, The results of designed degenerate primer showed that there was a homology found between the designed primers and the DNA templates for the three accession numbers with at least 80% identity. The result of degenerate pcr showed that the three bands of the amplified pcr products of the three accession numbers were detected at the same molecular weight of marker （400 bp） with a difference about 15 pb compared to the in silco pcr product （385 pb）. In conclusion, this study focused on the importance of using the clustalW algorithm for designing the degenerate primer.

关键词： ClustalW tool degenerate pcr different melting temperatures In-Silico pcr.

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The effect of osmotic pressure to aquaporin gene Glp-F expressed by ***

The effect of osmotic pressure to aquaporin gene Glp-F expre...

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重庆微生物学会第九届会员代表大会暨学术年会

作者： Changqing Li~(1,3),Zhibang Yang~(1,2),et al (1.Department of pathobiology and Immunology Experimental Teaching Center of Bacic Medicine,2.Institute of Neuroscience,Chongqing university of medical sciences,Chongqing 400016 3. Department of analysis Sixth People’s Hospital Of Chengdu,Chengdu,610051)

Objective:*** growth in Nutrient Agar which have different osmotic conditions, determinate OD600 at 24,48,72,96 *** reverse transcription assay was used to quantify the levels of Glp-F mRNA in different osmolality ***:Basic media made as Nutrient Agar. Media-N were made hypertonic by adding NaCl to increase the concentrations up to 125,250,500 or 750 ***-N were made hypotonic by diluting them with water(50%and 33%).Media-H were made hypertonic by adding NaCl and HgCl

关键词： degenerate pcr RT-pcr Glp-F aquaporin

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Isolation and identification of small species-specific secreted proteins of Marssonina brunnea

Isolation and identification of small species-specific secre...

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2013全国植物生物学大会

作者： CHENG Qiang CAO Youzhi HUANG Minren Jiangsu Key Laboratory for Poplar Germplasm Enhancement and Variety Improvement Nanjing Forestry University

Fungal plant pathogens produce proteinaceous effectors that can increase their pathogenicity. These fungal effectors are frequently small secreted proteins with limited phylogenetic *** a proteomics approach for analyzing the secretome is a promising strategy for the identification of effector proteins of phytopathogenic ***,the primary limitation of this strategy is the low identification rate of spectrometry-based proteomic methods that rely heavily on the accuracy and integrity of the reference sequence database. Marssonina brunnea is an important fungal pathogen of the Populus *** further our understanding of the pathogenesis of ***,we initiated a proteome-level study of the fungal *** de novo peptide sequencing by MS/MS,we obtained peptide sequences for 32 protein *** proteins were identified by sequence homology to conserved proteins in public databases using MS-driven *** identify additional protein spots,we combined a degenerate pcr method,based on the Consensus-degenerate Hybrid Oligonucleotide Primer(CODEHOP) method,and a rapid amplification of cDNA ends method to clone the full-length cDNA fragments encoding the proteins identified in the gel. Using this method,we cloned the full-length cDNA fragments encoding 9 putative Lysm effector *** method provides an efficient approach to identification of species specific effector proteins of non-sequenced fungal organisms.

关键词： effector Consensus-degenerate Hybrid Oligonucleotide Primer degenerate pcr

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