Cloning And Sequence Analyzing of the Eg10 Gene on Echinococcus Granulosus from China
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Objective To obtain the Eg10 gene of echinococcus granulosus and analyze its sequence.Methods Total RNA was extractedfrom protoscoleces of Echinococcus granulosus of humam origin.The specific primers were designed according to published nucleotide sequence in Genebank.The Eg10 gene fragment was amplified By RT-PCR and then cloned it into pGEM-T vector for sequencing and analyzing.Results The Eg10 sequence was successfully amplified,which was 938bp.The sequence and its amino acid sequence showed 100% homology with that published in Genebank previously.The deduced sequence of coding amino acid was 100% homology,too. Conclusion The sequenced Eg10 gene has a completely open coding frame(765bp).It will be significant to study its structure and function,as well as immunizing prevention.Keywords:
Coding region
Homology
Cloning (programming)
Sequence (biology)
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Aim The classical methods of constructing cDNA library for isolating and cloning genes, were complicating, hard work, and difficult to obtain full-length genes which contain 3' and 5'-untranslated regions, especially for those 5'-untranslated regions.To construct a full-length cDNA library of Echinococcus granulosus for screening the genes and studying the structure and function of genes.Method The total RNA was isolated by using the TRI zol reagent (GibcoBRL).Using “SMART” method for constructing a full-length cDNA libraries from Echinococcus granulosus.Result and Conclusion The full-length cDNA librarie of Echinococcus granulosus have been constructed.The packaging efficiency and the recombinant rate were about 1.4×10 7pfu/ml and 99% clones.The average length of the cDNA inserts was about 1.1kb.
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Cloning and Sequence Analyzing of Glutathione S-transferase Gene on Echinococcus Granulosus of China
Objective To clone and analyze sequence of Glutathione S-transferase(GST) gene of Echinococcus granulosus.Methods Total RNA was extracted from protoscoles of cysts from humam origin.The specific primers were designed according to published nucleotide sequence in the Genebank database.The GST gene of Echinococcus granuLosus was amplified by RT-PCR and cloned into pGEM-T vector for sequencing and analyzing.Results A cDNA sequence with an open reading frame of 660bp had been amplified successfully by RT-PCR.Comparision of the DNA and amino acid sequence deduced from cDNA with the published GST gene sequence of Echinococcus granulosus in the Genebank revealed 99% homology.Conclusion The GST gene sequence of Echinococcus granulosus was cloned successfully and its biological study could be performed on the basis.
Glutathione S-transferase
Cloning (programming)
clone (Java method)
Homology
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ObjectiveTo obtain and analyze the mMDH gene sequence,and lay bases for screening candidate antigen of Echinococcus granulosus.MethodsTotal RNA was extracted from protoscoles of cysts from humam origin. The specific primers were designed according to the published nucletid sequence in the Genbank database.The mMDH gene of Echinococcus granulosus was amplified by RT-PCR and cloned into pGEM-T vector for sequencing and analyzing.ResultsA cDNA sequence with an open reading frame of 1017bp has been amplified successfully by RT-PCR.Comparision of the DNA and amino acid sequence was deduced from cDNA with the published mMDH gene sequence of Echinococcus granulosus in the Genbank revealed 99% identity.ConclusionmMDH gene was gained in China from protoscoles can be used as candidate antigene gene to develop vaccine and its biological function study.
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Sequence (biology)
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According to the sequence of GPV B strain published in GenBank, a pair of primers were designed by Oligo4.0.NS2 gene of GVP and amplified by PCR. The gene of interest was cloned into the vector pMD18_T and sequenced. The results showed that NS2 gene consisted of 1356 nucleotides and encoded 451 amino acids. The NS2 gene shared 98.75 % and 98.67 % homology with that of B isolate at nucleotide and amino acid level, respectively.
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Objective: To study the structure specificity of full length Echinococcus granulosus 95 (Eg95) cDNA in Xinjiang, China and construction of Eg95 DNA vaccine. Methods: According to the sequence of Eg95 antigen cDNA, the primers of Eg95 were designed. The full length Eg95 antigen cDNA was amplified by PCR from cDNA library of E.granulosus in Xinjiang, and was cloned into eukaryotic expression plasmid pcDNA3 to construct the full length DNA vaccine pCDNA3 Eg95. The recombinant plasimid pcDNA3 Eg95 was transformed into E.coli DH5α and the positive clones were screened by restriction endonuclease analysis, and then sequenced. The sequences were analyzed by DNAman and GeneBank/Blast biosoftware. Results: DNA sequence analysis of Eg95 Xinjiang strain (Eg95 XJ) cDNA fragment indicated that the full length of Eg95 XJ was 471bp and code 156aa. The pcDNA3 Eg95 positive clone was the exact recombinant plasmid and could be used as a DNA vaccine. By homology comparison, there were no difference of full length Eg95 cDNA between Eg95 XJ and other Eg95 members of New Zealand strain. Conclusion: pcDNA3 Eg95 DNA vaccine was constructed successfully.
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Objective To clone the egG1Y162 gene and analyze the protein sequence. Methods mRNA was extracted from protoscolex of Echinococcus granulosus and used for RT-PCR. The primers of egG1Y162 were designed according to the sequence of emy162 antigen genes. Using cDNA as a template, egG1Y162 gene was amplified by PCR; and recombinant plasmid PUCm-T/egG1Y162 was constructed. Then recombinant plasmid was identified by PCR, enzyme digestion and sequencing. Results The length of the egG1Y162 cDNA was 459 bp, which coded 153 aa. Homology comparison showed that the egG1Y162 cDNA sequence shared 95% homology with emY162 gene, while shared 30.61% homology with eg95. Conclusion egG1Y162 antigen gene is a new gene and has been cloned successfully. Comparative analysis showes that amino acid sequences of egG1Y162 protein is similar to that of egY162 and significant differences existed in the amino acid sequences of egG1Y162 protein as compared with others antigen proteins.
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Protoscoleces were recovered from the liver of sheep naturally infected with Echinococcus granulosus(Eg),and total RNA and genomic DNA were extracted from the protoscoleces,respectively.One pair of specific primers based on published thioredoxin peroxidase gene of Eg(EgTPx) sequence in GenBank were designed and used to amplify cDNA sequence of EgTPx by RT-PCR from the total RNA and to obtain genomic sequence of EgTPx by PCR from the genomic DNA.The amplified products were purified and cloned into pMD18-T vector and sequenced.Sequence analysis indicated that the EgTPx gene of E.granulosus isolated from Qinghai,China,was successfully cloned with an ORF of 582bp encoding 193 amino acids which shared 99% identity with the published EgTPx mRNA sequence(AF478688) and EgTPx(AAL84333),with three bases changed from T to C respectively and one amino acid changed from Ala82 to Val82.EgTPx presented two highly conserved motifs(FVCP and VCPA) around the catalytic sites Cys48 and Cys169 for the typical 2-Cys peroxiredoxin.Comparison of cDNA and genomic sequences of EgTPx gene revealed the existence of one intron of 69bp and 2 exons.
genomic DNA
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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.
Homology
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Sequence homology
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SO_7' gene of E.tenella isolated from chickens in HeBei was cloned and sequenced. E.tenella RNA was extracted from sporulated oocysts and used as a template for cDNA synthesis. SO_7' cDNA was amplified by RT-PCR. The PCR products were cloned into pMD18-T vector successfully. The recombinant plasmids were analysed with restriction endonucleases and sequenced. The results indicated that SO_7'-HB gene was 651bp in length and included opening read frame which encoded a polypeptide of 216 amino acid. Comparing SO_7'-HB strain gene with the published sequence of SO_7'-LS18,the nucleotide sequence homology was 99.2%.And the homology of its deduced amino acid sequence was 98.6%.
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Aim:To clone the tumor related gene MAGE-1 and analyze its sequence homology in comparison with the known sequence in GenBank.Methods:The total RNA was extracted from hepatic cell carcinoma (HCC) tissue, then RT-PCR was used for cDNA cloning of MAGE-1 gene. The sequence data were compared with the the known sequence in GenBank.Results: The cloned gene had 4 different bases and 99.7% homology with MAGE-1 sequence (GenBank M77481); furthermore, only one amino acid changed.Conclusion: The MAGE-1 gene was cloned and sequenced successfully.
Cloning (programming)
Homology
clone (Java method)
Sequence (biology)
Sequence homology
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