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Genomic DNA Sequences and Expression, Purification of BmαTX14 Neurotoxin from Scorpion Buthus Martensii Karsch

Author: LvMeng
Tutor: LiWenZuo
School: Wuhan University
Course: Microbiology
Keywords: Scorpion Scorpion toxin BmαTX14 Gene structure Pichia pastoris Eukaryotic expression
CLC: Q516
Type: Master's thesis
Year: 2005
Downloads: 161
Quote: 0
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Abstract


Scorpion toxin is a class of small peptides of 20-90 amino acids . Such small peptides typically contain 3 or 4 pairs of disulfide bonds , can be selectively and animals can be excited on the cell membrane , sodium, potassium , calcium , chloride ion channel binding , affect the state of the ion channels , Be study the physiological role and function of the ion channels the importance of positioning tools. In addition, traditional Chinese medicine in China , Scorpion antispasmodic , Xifeng , tongluo pain, and attack drug relieves congestion has a long history . (447bp) primers were designed according to the cDNA sequence isolated from scorpion venom gland cDNA library , gene bmatx14 the PCR method , from the Scorpion genome amplified to the full-length gene composed by 855bp bmatx14 . Sequence analysis showed that the bmatx14 gene encoding a signal peptide of 16 amino acid residues Gly codon first base (G) after a 408bp intron . The analysis showed that the genomic structure : rich in AT, the content of 73.5% , significantly higher than the exon ( 57.6% ) ; 5 ' and 3 ' splice site sequences were 5' - AG | gtaaga - 3 ' , 5' - tgacag | GA - 3 ' ; at the 3' splice acceptor splice site 74 nucleotides upstream of at sequence element comprising a splice branching point : 5' - TAAT - 3 ' . the bmatx14 gene structure typical alpha - Na - channel toxins features . Scorpion venom composition is complex , difficult to purified monomer content , and thus the study and application of scorpion toxin subject to certain restrictions . In this study, Pichia pastoris eukaryotic expression system to establish the scorpion toxin efficient eukaryotic expression , purification system , the purified recombinant BmαTX14 by a preliminary function study showed alpha - Na ... channel toxins characteristics . Will the scorpion venom bmatx14 mature peptide cDNA amplification products were cloned into the eukaryotic expression vector pPIC9K between the carrier signal peptide and the mature peptide design histidine tag ( His - tag ) . The recombinant expression vector was transformed into Pichia pastoris strain GS115 , multi-copy transformants G418 gradient filter , it is possible to

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CLC: > Biological Sciences > Biochemistry > Protein > Protein primary structure
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