Cloning and bioinformatics analysis of IFN-αgene of wild cat
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One pair of primer was designed and synthesized according to the feline interferon alpha gene(IFN-α) nucleic acid sequence.Wild cat IFN-α was amplified by PCR,then cloned and sequenced.The nucleotide sequence of the amplified IFN-α gene and the amino acid sequence deduced from the gene were then analyzed by the DNAstar software and on-line tools,and the similarity and evolution of IFN-α gene were analyzed among wild cat,human and other animals.The results showed the full length sequence of wild cat IFN-α gene was 631 bp,which includes one open-reading frame,encoding 194 amino acid residues,one potential N-glycosalation site and seven O-glcnac sites in deduced amino acid sequence.The results of comparative sequence analysis and phyloge-netic tree analysis indicated there was difference of genus in IFN-α gene,the nearer their relationship,the higher the homology.This study paved the way for future study of biological function and application of feline IFN-α.Keywords:
Homology
Primer (cosmetics)
Cloning (programming)
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According to feline interleukin-18 (IL-18) gene sequence reported in GenBank, feline IL-18 gene was amplified by RT-PCR from the feline peripheral blood mononuclear cells (PBMCf) stimulated by concanavalin (ConA). The PCR product was purified and ligatured with pMD18-T. The recombinant plasmid was used for sequencing. The full length of feline IL-18 gene was 579 bp, which encoded 192 amino acids(Genbank accession No.DQ100372). There were no signal peptide and N-glycosylation sites, while had 4 conservative Cys amino acid in the deduced amino acid sequence. Compared with IL-18 genes of other species, the nucleotide sequence of feline IL-18 gene shares 89.8%、88.6%、88.4% and 88.1% identities with IL-18 genes of canine, ovine, bovine and porcine respectively, but displays significant differences with mouse and chicken IL-18 gene sequence which published in GenBank. Meanwhile, the feline IL-18 gene was subcloned into the prokaryotic expressing vector pET28a and recombinant vector was further transformed into Escherichia coli strain BL21(DE3) for expression under the induction of IPTG. The results of SDS-PAGE revealed that it had a molecular weight of 27.5 kD, which amounts to 13.6% in the total protein of the induced bacteria by the assaying of gel scanning.
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One pair of primers was designed and synthesized according to the canine interferon alpha gene(IFN-α) nucleic acid sequence(AB102731) published in the GenBank.Canine IFN-α gene was amplified by PCR from genome DNA extracted directionally from liver of Zangao canine,then cloned and sequenced.The result showed the full length sequence of Zangao canine IFN-α gene was consisted of 564 bp,which includes one open-reading frame,encoding 187 amino acid residues,three potential N-glycosalation sites and ten cysteines in deduced amino acid sequence.The results of comparative sequence analysis and phylogenetic tree analysis showed there was difference of genus in IFN-α gene,the nearer relationship,the higher homology.This study set a basis the way for future study of biological function and application of canine IFN-α.
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One pair of primers was designed and synthesized according to the canine interferon-α(IFN-α) gene nucleic acid sequence published in the GenBank.The IFN-α gene was amplified from genome DNA of poodle liver by PCR.The purified PCR products were linked to pGEM-T Easy vector and then transformed into competent cells of DH5α.By identification of blue-white spot screening,plasmid PCR and enzyme digestion,positive strain was sent to TaKaRa for sequencing.The sequencing results showed that poodle IFN-α gene was obtained.The cloned poodle IFN-α gene span is 564 bp,which includes one open-reading frame encoding 187 amino acid residues.And there are three potential N-glycosalation sites and ten cysteines in the deduced amino acid sequence.Homology of nucleotide and amino aids of IFN-α gene between poodle and humans and other animals and the phylogenetic tree indicated that IFN-α gene varied in species,and the closer the relationship,the higher the homology.
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According to a forecast sequence of chicken interleukin-5(ChIL-5) gene published in GenBank,a pair of primers was designed and used for cloning ChIL-5 gene by RT-PCR from chicken spleen lymphocytes stimulated by ConA for the first time.The complete nucleotide sequence of the cloned gene contained a 381bp open reading frame encoding 126 amino acids,including the sequence encoding 22-amino-acid signal peptide.The protein encoded by the gene was about 14.63ku in molecular weight.The amino acid sequence deduced from the gene had less than 25% similarity to that of other mammals.However,the predicted tertiary structure of ChIL-5 protein was similar to that of the human,and the structure of the protein mainly consisted of four α-helix.This gene was cloned into pET-28a(+) vector to construct recombinant expression plasmid pET-28a-IL-5,then the recombinant plasmid was transformed into Escherichia coli Rosetta(DE3) and expressed under induction of IPTG.The prokaryotic expression of ChIL-5 gene provided foundation for further studies of the structure and biological function of avian IL-5.
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[Objective] The research aimed to clone and analyze a new IFN-τ gene from yellow cattle in Shenyang area.[Method] A pair of specific primers were designed and synthesized according to the sequence(AY665673) of bovine IFN-τ gene published on NCBI website.The genomic DNA extracted from the kidney tissues of yellow cattle was taken as template for PCR amplification.After being reclaimed,the amplified products were connected with vector pUCm-T and transformed into competent cell DH5α of Escherichia coli.After the obtained plasmid was identified by PCR and double-enzyme digestion,it was made for sequencing and sequence analysis.[Result] Through PCR amplification,a specific band with the size of about 600 bp was obtained.Bovine IFN-τ gene was successfully recombined with the vector and the positive clone was obtained.The results of sequence analysis showed that the size of the cloned fragment was 586 bp and stop codon appeared at 376 bp and led to the translation termination.The nucleotide homology of this fragment with that of the sequence of AY665673 gene was 93.3% and their homology on the amino acid was 88.1%.[Conclusion] The cloned fragment was a new bovine IFN-τ gene probably.
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clone (Java method)
genomic DNA
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One pair of primers was designed according to chicken interferon-alpha(IFN-α)gene sequences retrieved from the GenBank.Chicken IFN-α genes were amplified from genome DNA extracted directionally from α livers of Guishi chicken,Hailan chicken and Luoman chicken.The purified PCR products were cloned into pGEM-T Easy vector and sequenced.The result showed that the full length sequence of chicken IFN-α gene was consisted of 582 bp,which includes one open-reading frame(582 bp),encoding 193 amino acid residues.When the nucleotide sequences of three line chicken IFN-α genes were compared,the nucleotide sequence of Hailan chicken IFN-α gene was just the same as Luoman chicken IFN-α gene,the nucleotide sequence of Guishi chicken IFN-α shared 99.3% homology with that of Hailan chicken and Luoman chicken IFN-α,and putative amino acid sequence shared 97.9%.When three line chicken IFN-α genes were compared with seven chicken IFN-α genes published previously in GenBank,there was identity of 98.1%~100%at nucleotide acid level,and that of 96.9%~100% separately at deduced amino acid level.
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According to published GenBank sequences,a primer for the increase of mink β-interferon(IFN-beta) gene was designed and synthesized by DNAstart analysis software,total RNA was extracted from the infected mink spleen,mink β-interferon gene was increased about 561 bp through RT-PCR method.After this gene was cloned to pEasy-T1 carrier,the facts prove that this gene is mink β-interferon according sequence analysis.The result suggests that β-interferon nucleotide sequence homology are 100.0% and amino acid homology are also 100.0% when compared sequence result with published gene sequence(EF581890.1) from GenBank.The nucleotide sequence homology and amino acid homology are 83.0%~100.0% and 69.4%~100.0% respectively when compared with another canine animals.
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One pair of primers was designed and synthesized according to the nucleic acid sequence of porcine toll-like receptor 7(TLR7)gene.The TLR7 gene from Sanyuan pig was amplified by RT-PCR from the total RNA extracted from peripheral blood lymphocytes,and then cloned and sequenced.The result showed that the full length of TLR7 gene sequence from Sanyuan pig was consisted of 3 160 bp,which included one open-reading frame(3 153 bp),encoding 1 051 amino acid residues.There were fifteen potential N-glycosalation sites and four potential phosphorylation sites.The cleavage sites of signal peptide were predicted to be in 29th or 30th of the amino acid sequence,and the transmembrane helices in proteins was in 844-866th amino acid sequence.The results of comparative sequence analysis and phylogenetic tree analysis indicated that the TLR7 gene had difference of genus,and the nearer the genetic relationship,the higher homology.This study paved the way for future study of porcine TLR7 gene expression,biological function,mechanisms of infection with ssRNA viruses,antiviral immunity and breeding.
TLR7
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The chicken interleukin-15(IL-15) was amplified from ConA-activated chicken splenocytes by RT-PCR using a pair of primers which were designed and synthesized according to the published ChIL-15 gene sequence in GenBank. The gene was inserted into pMD18-T vector and sequenced. The result of sequence analysis showed that the open reading frame (ORF) of Sichuan Caoke chicken IL-15 gene was 564 bp,encoding 187 amino acids. The homology of Caoke chicken ChIL-15 gene with others in GenBank were 98.6% to 99.5% in nucleotide and amino acids level. Bioinformatics analysis of this sequence showed that the protein of ChIL-15 was hydrophilic and highly antigenic,includes three potential N-glycosylation site,three potential protein kinase C phosphorylation site,two potential casein kinase Ⅱ phosphorylation site.
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According to the published hyaluronidase 2 gene sequences of sheep(NM_001009754),two specific pairs of primers were designed.The Full-length gene of Hyal2 was amplified by overlapping PCR and then was subcloned into PMD19-T vector and sequenced.The Hyal2 gene were1431bp long and contain an open reading frame(ORF) which encoding 476 amino acids.The comparison of the sequences showed that: the homology of Hyal2 gene compared with the template nucleotide was 99.8%,amino acid homology was 99%.Phylogenetic analysis showed that the relationship between target gene and bovine was closer.Bioinformaticsanalysis showed that the protein of the gene was a hydrophilic protein.
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