Cloning and Analysis of A New Bovine Interferon-τ Gene
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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.Keywords:
Cloning (programming)
Homology
clone (Java method)
genomic DNA
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The total RNA was prepared from ConA stimulated thymocytes of a 5-week-old chicken according to the instructions of the trizol reagent.The CD8 alpha chain cDNA without its singal peptide sequence was amplified by reverse transcription polymerase chain reaction(RT-PCR) using a pair of gene specific primers.The obtained 651 bp fragment was identified by restriction enzyme digestion,the fragment was cloned to the pMD18-T vector and sequenced(the sequence has been deposited in GenBank with accession number of DQ202314).The sequence showed 98% homology with that of published chicken CD8 alpha chain.Subsequently, the verified fragment was digested with EcoRⅠ and SalⅠ and ligated to the pGEX-4T-1 vector using T_4 DNA ligase.The recombinant prokaryotic expression plasmid,namely pGEX-4T-1-CD8α,was transformed into Escherichia coli strain BL21(DE3) and expressed as a GST fusion protein.The products were analyzed by SDS-polyacrylamide gel electrophoresis and a protein about 50 000 was observed.These suggest that the cloned gene encoding chicken CD8 alpha chain without signal peptide was expressed in prokaryotic system.
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Based on the published nucleotide sequence of bovine interferon-gamma(BovIFN-γ)gene,a pair of RT-PCR primers were designed and synthesized.The mature peptide fragments of γ-interferon gene was amplified using reverse transcription polymerase chain reaction(RT-PCR)from total RNA of bovine peripheral blood lymphocytes,and then carried out cloning,sequencing and sequence analysis.The results showed that the mature peptide of BovIFN-γ gene can be successfully amplified by primers designed.Additionally,the homology between the amplified gene fragment sequence and the relevant sequence in GenBank can reach up to 99.77%。
Cloning (programming)
Homology
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A specific pair of primers was designed and synthesized according to thesequence of swine IFN-α gene published NCBI GenBank, and a fragment of 500bp was amplified by PCR from the liver of (3-weeks-old) swine and the fragment was cloned into pMD18-T vector. The recombinant plasmids were (transferred) into the E.coli DH5α competent cells, which were incubated on ampicillin~(+)plates with X-gal and IPTG at 37℃overnight. The white clones were selected and incubated. The restriction endonuclease (analysis), PCR and sequence analysis indicated that the swine IFN-α cDNA was 501bp in full-length and (encodes) 167 amino acids. The homologies of nucleotide sequence between the swine IFN-α gene and M28623 and X57191 from GenBank were 99.4%and 99.2% respectively.
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This experiment was conducted to study molecular evolution,cloning and sequence analysis of Qinghai Semifine-wool sheep interleukin-8(IL-8) gene.A pair of specific primers was designed based on GenBank.The IL-8 gene was amplified by RT-PCR and cloned into pMD18-T vector.The positive recombinant plasmids which was transformed to E.coli DH5α was identified by PCR and restriction enzyme digestion and sequenced analysis.The results showed that sheep peripheral blood lymphocytes was amplified bands consistent with the expected size(370 bp),the homology analysis showed that IL-8 gene was more conservative,which homology was 96.1% to 99.0%.
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Cloning (programming)
Sequence homology
Restriction map
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Objective: To clone the full-length gene of beta 2-adrenergic receptor (β2-AR).Method: Primers for PCR were designed according to the porcine β2-AR cDNA sequence in the GenBank.The β2-AR gene was amplified from the total RNA of porcine liver by RT-PCR technology,which was then cloned into pUC18 vector.The recombinant was transformed to competent cell E.coil DH5α,screening the positive clones.Result: The amplified β2-AR gene has 1257 bases,encoding 418 amino acids.Compared with the porcine β2-AR DNA sequence inscribed in GenBank, the homology is 99.52% with 99.04% amino acids identical.Conclusion: Full-length sequence of β2-AR Gene was successfully obtained,establishing foundation for gene expression and the construction of receptor-drugs screening model.
Cloning (programming)
Homology
clone (Java method)
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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).
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clone (Java method)
Sequence (biology)
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According to the DNA sequence of Orf virus which was published on Genbank,two pairs of primers have been designed to amplify B2L gene of JS04 by PCR.The amplified production was combined with pMD19-T vector,then was transformed into DH5.Positive clones identified correctly were sequenced.The length of B2L gene was 1137bp,and the corresponding amino acid sequence was conducted.Sequence analysis showed that the B2L gene homology among ORFNZ2,Shanhjahnpur82/04 strains was 97.8%～ 99.5%.The amino acid sequence homology was 97.6%～ 99.5%.The analysis result of phylogenetic tree showed that the B2L gene.
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Cloning (programming)
Sequence homology
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Genome RNA was extracted from bone marrow of pig (Duroc╳Landrace╳Tai-hu) and Protegrin-1 (PPG-1) mRNA was amplified using RT-PCR. A DNA fragment about 565 bp in length was obtained and the PCR product was cloned into pGEM-T vector. Protegrin-1 gene was isolated and sequenced from the positive clones screened and the corresponding amino acid sequence was deduced from its nucleotide sequence. Result of sequence analysis showed that this fragment was the partial sequence of PPG-1 gene cDNA. Its homology with PPG-1 from Genebank was up to 98.7%.The deduced sequence for the mature peptide was composed of 18 amino acid residues, which was consistent with that of porcine PPG-1 in Genebank.
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Homology
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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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Based on the published nucleotide sequence of bovine interleukin-2,a pair of RT-PCR primers were designed and synthesized.Total bovine cell RNA,isolated from ConA-stimulated peripheral blood of bovine,was used as template to generate complementary DNA by reverse transcription.The 0.477 kb DNA fragment was amplified by polymerase chain reaction(PCR),and cloned into the pMD18-T vector.DNA sequencing confirmed the fragment was bovine interleukin-2 and the sequence was identical to the sequence of IL-2 publised in GenBank;Sequence analysis showed that the cloned sequence has very high similarity with that of ovine and Bubalus bubalis IL-2,over 95%;Comparative analysis of both amino acid sequence and antigenecity showed that the interleukin-2 of bovine has high similarity to the one of ovine and Bubalus bubalis.
Cloning (programming)
Bubalus
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