Actinia neurotoxin gene and its application
A neurotoxin and sea anemone technology, applied in the field of genetics, can solve the problem of less neurotoxins in sea anemones
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Embodiment 1
[0026] Example One The extraction and RT-PCR amplification of the tentacle total RNA
[0027]Extraction of total RNA and synthesis of cDNA; Isolate the tentacles of a sea anemone lateralis, extract the total RNA of the tentacles by guanidine isothiocyanate method, remove the protein by phenol / chloroform extraction, and obtain 100 μg total RNA of venomous glands, the A 260 / A 280 =2.03, two clear bands of 18s and 28s can be seen by 1% formaldehyde denaturing gel electrophoresis, the ratio>1, and several specific RNA bands (see Figure 3), indicating that the integrity of the total RNA is good. 5 μg of RNA was reverse-transcribed with Oligo-dT to synthesize the first-strand cDNA, and 20 μl of the first-strand cDNA product was obtained.
[0028] Primer design and RT-PCR amplification were based on; according to the amino acid sequence at both ends of Ap-B, oligo primer design software was used to assist analysis, and two degenerate primers were designed.
[0029] Amino acid sequ...
Embodiment 2
[0033] Example 2 Determination and Analysis of Recombinant Sea Anemone Neurotoxin Gene Sequence
[0034] The recovered electrophoresis product was connected to a T vector, transformed into DH5α Escherichia coli, and the recombinant clone sequence was selected. A total of 16 clones were determined, and Blast homology analysis showed that 6 of them were neurotoxin gene sequences, and the length of these 7 toxin genes was 141bp, encoding a toxin protein with a length of 47 amino acids. The toxin protein number was As6, its amino acid sequence is not identical with the reported sea anemone neurotoxin protein, it is a new toxin protein.
[0035] At present, there are more than 20 kinds of sea anemone neurotoxins reported, the number of amino acids is between 46-49 (in addition, there are several toxins with amino acid residues between 27-31, called short-chain neurotoxins), of which 12 amino acids are conserved . According to their amino acid sequences, they can be divided into t...
Embodiment 3
[0045] Example 3 Construction of Recombinant Sea Anemone Neurotoxin Expression Plasmid
[0046] A pair of primers were synthesized according to the sequences at both ends of the As6 gene, the upstream primers contained Kpn I and PrescissionProtease cleavage sites, and the downstream primers contained BamH I cleavage sites.
[0047] Upstream primer (B1): 5'CGG GGT ACC CTG GAA GTT CTG TTC CAG GGG CCC GGG GTT
[0048] Kpn I Precision Protease site
[0049] CCG TGT TTG TGT GAC3'
[0050] Downstream primer (B2): 5'CGC GGA TCC TTA TTA CTT CTT GCA GCA CCA GCC AAT G3’
[0051] Bam H I
[0052] Using the pGEM-T Easy plasmid containing the As6 gene as a template, and using B1 and B2 as primers for PCR amplification, a specifically amplified single band was obtained, and the product size was about 200 bp. The PCR amplified product was first cloned into the BSK plasmid with the combination of Kpn I / BamHI to construct BSK-A...
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