Amplification,Cloning and Bioinformatic Analysis of Porcine TLR7 Gene
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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.Keywords:
TLR7
Homology
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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.
Homology
Cloning (programming)
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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.
Cloning (programming)
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One pair of specific primers to porcine IL-18 protein gene was designed and synthesized (according) to the published sequence in GenBank, and was used to amplify porcine IL-18 cDNA by RT-PCR from total RNA of porcine spleen cells. RT-PCR product was cloned into pMD 18-T vector. The nucleotide acid sequence was determined after being identified by restriction endonucleases digestion. The result show that the full length of porcine IL-18 cDNA is 474 bp, which encodes 157 amino acids.
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[Objective] To clone the porcine interleukin-18(IL-18) cDNA and explore the immunological effectiveness of porcine IL-18 as an adjuvant of genetic vaccine. [Method] The spleen lymphocytes were isolated from Henan three-way cross-breeding pigs. According to the porcine IL-18 gene in GenBank, a pair of specific primers was designed. The full length cDNA of porcine IL-18 was amplified by RT-PCR. Subsequently, porcine IL-18 cDNA was cloned into pGEM-T vector and sequenced and analyzed. [Result] The porcine IL-18 gene demonstrated an open reading frame of 579 bp encoding an inactive precursor protein with 192 amino acids. The precursor protein had no typical hydrophobic signal peptide and cleaved by interleukin-1 beta (IL-1β) converting enzyme (ICE) in caspase-1 splice site; the porcine mature protein had biological activity. After comparing with other porcine IL-18 genes, the nucleotide sequence homology was over 96% and the deduced amino acid homology was more than 98%. [Conclusion] A full length procine IL-18 gene was gained. It lays the foundation for porcine IL-18 as an adjuvant of genetic vaccine.
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In order to clone and analyze the gene Reg3 in intestinal canal tissues of pig,the total RNA was extracted and mRNA sequence of Reg3 gene was amplified by RT-PCR method which used two designed primers.The PCR productswere ligated into the pMD-19T vector,and then transformed into competent cells of E.coli JM109.The sequence was analyzed to identify the recombinant plasmid.The identification analysis showed that the Reg3 nucleotide sequence shared 83.7%,82.3% and 82.2% homology with that of human,sheep and horse respectively,revealing porcine Reg3 gene had been successfully cloned in the present study.The sequence has been submitted to GenBank(Accession FJ531494).
Homology
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clone (Java method)
Sequence (biology)
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The full-length cDNA sequence of a porcine gene, MOSPD2, was amplified using the rapid amplification of cDNA ends method based on a pig expressed sequence tag sequence which was highly homologous to the coding sequence of the human MOSPD2 gene. Sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 491 amino acids that has high homology with the motile sperm domain-containing protein 2 (MOSPD2) of five species: horse (89%), human (90%), chimpanzee (89%), rhesus monkey (89%) and mouse (85%); thus, it could be defined as a porcine MOSPD2 gene. This novel porcine gene was assigned GeneID: 100153601. This gene is structured in 15 exons and 14 introns as revealed by computer-assisted analysis. The phylogenetic analysis revealed that the porcine MOSPD2 gene has a closer genetic relationship with the MOSPD2 gene of horse. Tissue expression analysis indicated that the porcine MOSPD2 gene is generally and differentially expressed in the spleen, muscle, skin, kidney, lung, liver, fat and heart. Our experiment is the first to establish the primary foundation for further research on the porcine MOSPD2 gene.
Homology
Coding region
Pair-rule gene
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Primers were designed according to the known sequences of Sterol Regulatory Element Binding Proteins-1c (SREBP-1c) genes of human, rat and pig. RT-PCR was then used to amplify porcine SREBP-1c gene with total RNA of spleen tissue. A 760 bp segment of cDNA was cloned and sequenced. Homogeneous comparison showed that the sequence of porcine SREBP-1c had 99.9% and 99.1% homogeneity with the two known partial mRNA sequences of porcine SREBP-1c gene. The complete cDNA was obtained mainly based on the known partial sequences, which has 3 830 bp, encoding 1 151 amino acids with a calculated molecular weight of 121 479 Da. It is the first time that we get complete encode sequence of porcine SREBP-1c gene. The complete cDNA sequence has high homogeneity with SREBP-1c gene of other species. A characteristic structure of HLH (Helix-Loop-Helix) and four transmembrane segments were found in the amino acids. The sequence had been submitted to GenBank (Accession No.NM_-214157).
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Toll-like receptors (TLR) were the key component of innate immune system. In the present study,porcine Toll-like receptor 3,7 and 8 (pTLR3,pTLR 7 and pTLR 8) genes were amplified by RT-PCR from porcine alveolar macrophages (PAM). The PCR products were cloned and sequenced. The results showed that the pTLR3,pTLR7 and pTLR8 genes contained ORF of 2,718 bp,3,153 bp,3,087 bp in length that encoded 906 amino acids (aa),1,051 aa and 1,029 aa,respectively. The nucleotide sequences of pTLR3,pTLR7 and pTLR8 shared 99% homology with the published pTLR sequences in GenBank. The structure prediction showed that the pTLR3,pTLR7 and pTLR8 were transmembrane proteins.
Cloning (programming)
Homology
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The coding sequence of porcine Chemerin and its receptor gene were amplified based on the in silico sequence information in the study.Total RNA was extracted from all kinds of organizations and mRNA sequence of gene was amplified by RT-PCR using designed primers.The PCR products were ligated into the pMD-19T vector,and then transformed into competent cells of E.coli JM109.Porcine Chemerin gene cDNA sequence,was 558 bp in length,with an open reading frame of 492 bp encoding 163 amino acids.ChemerinR gene cDNA sequence,was 1 470 bp in length,with an open reading frame of 1 092 bp encoding a 363 amino acids.Homology analysis showed that porcine Chemerin and nucleic acid sequences,bovine,human and rat had the homology of 86.7%,79.1% and 73%,amino acid sequence had the homology of 83.1%,73.4% and 67.7%.Homology analysis showed that porcine ChemerinR and nucleic acid sequences,human,rat and mouse had the homology of 80.4 %,77.6% and 72.6%,amino acid sequence had the homology of 88.2%,80.7% and 79.3%.Semi-quantitative RT-PCR analysis showed that Chemerin mRNA ubiquitously expressed in various tissues tested,but ChemerinR preferentially expressed in the bladder,brain and muscle.
Chemerin
Homology
Coding region
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In order to clone and analyze the gene KISS-1 in various tissues of pig,the total RNA was extracted and mRNA sequence of KISS-1 gene was amplified by RT-PCR method which used two designed primers.The PCR products were ligated into the pMD-19Tvector,and then transformed into competent cells of E.coli DH5α.The sequence was analyzed to identify the recombinant plasmid.The identification analysis showed that the KISS-1 nucleotide sequence shared 81.0%,76.8%,71.4% and 72.4% homology with that of Bos taurus,human,rat and mouse respectively,revealing porcine KISS-1 gene had been successfully cloned in the present study.The sequence has been submitted to GenBank(Accession EU934235).The expression of porcine KISS-1 gene in various tissues was found.
Cloning (programming)
KISS (TNC)
Homology
clone (Java method)
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