Sea anemone neurotoxin gene Sr4 and application thereof
A technology of neurotoxin and sea anemone, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of less neurotoxin in sea anemone, and achieve the effect of prolonging the effective refractory period and enhancing atrial contraction
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Embodiment 1
[0028] Example 1: Extraction and RT-PCR amplification of total RNA from tentacles of rose red and green sea anemone
[0029]Extraction of total RNA and synthesis of cDNA: Isolate the tentacles of the rose red and green sea anemone, extract the total RNA of the tentacles by the guanidine isothiocyanate method, and remove the protein by phenol / chloroform extraction. Obtain 100 μg of the total RNA of the venomous gland, and its A 260 / A 280 = 2.03, two clear bands of 18s and 28s were detected by 1% formaldehyde denaturing gel electrophoresis, and the ratio was >1 (see Figure 2), indicating that the integrity of the total RNA was good. Take 1ug of total RNA with SMART III olignuclotide (5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3') and CDSIII / 3'PCR primers (5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30 N -1 N-3′) was reverse transcribed to synthesize the first strand to obtain 10 μl of cDNA first strand product.
[0030] Primer design and RT-PCR amplification: According to the amino ...
Embodiment 2
[0035] Example 2: Determination and analysis of recombinant sea anemone neurotoxin gene sequence
[0036] The recovered electrophoresis product was connected to the T vector, transformed into DH5α Escherichia coli, and the recombinant clones were selected for sequencing. A total of 25 clones were determined, and Blast homology analysis showed that these 25 clones were all neurotoxin gene sequences, and the gene length was 141bp. Each encodes 47 amino acid toxin proteins, one of which is numbered Sr-p4, which is not completely identical to the amino acid sequence of the reported sea anemone neurotoxin protein, and is a new toxin protein.
[0037] 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...
Embodiment 3
[0040] Example 3: Construction of recombinant sea anemone neurotoxin expression plasmid
[0041] A pair of primers were synthesized according to the sequences at both ends of the Sr4 gene, the upstream primers contained Kpn I and Prescission Protease cleavage sites, and the downstream primers contained BamH I cleavage sites.
[0042] Upstream primer (R1): 5'CGG GGT ACC CTG GAA GTT CTG TTC CAG GGG CCC GGG GTT
[0043] Kpn I Precision Protease site
[0044] CCC TGT TTG TGT GTG 3';
[0045] Downstream primer (B2): 5'CGC GGA TCC TTA TTA CTT CTT GCA GCA CCA GCC AAT G 3';
[0046] Bam H I
[0047] The pGEM-T Easy plasmid (purchased from Promega) containing the Sr4 gene was used as a template, and B1 and B2 were used as primers for PCR amplification to obtain a specifically amplified single band with a product size of about 200 bp. The PCR amplification product was first cloned into the BSK plasmid with the combi...
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