Barrel-shaped alpha conantokin Bt1.3 and application thereof
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A technology of sequence and DNA molecules, applied in the barrel-shaped α-conus polypeptide Bt1.3 and its application field
Inactive Publication Date: 2011-09-21
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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The latest research on the analgesic target of Vc1.1 shows that Vc1.1 exerts analgesic effect through GABAB-mediated inhibition of N-type calcium channels (Klimis H et al. PAIN 2011, 152: 259-266), but there are also May be related to a currently undiscovered mechanism
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Embodiment 1
[0031] The synthesis of embodiment 1, α-cono polypeptide Bt1.3
[0032] 1. Gene cloning of α-cono polypeptide Bt1.3
[0033] 1. Synthesis of cDNA
[0034] Live barrel-shaped cone snails (Conus betulinus) collected from the Xisha Islands in the South China Sea were used as sample materials. The total RNA of cono venom ducts was extracted by Trizol method. The 3'Full-RACE kit (3'-Full RACE Core Set Ver 2.0) from Dalian TaKaRa Company was used to synthesize cDNA, the specific method is as follows:
[0035] First add the following components in proportion:
[0036]
[0037] Mix well, denature at 70°C for 10 minutes, and quench on ice for 2 minutes. Then, using the reaction solution as a template, carry out reverse transcription according to the following reaction system and conditions:
[0038]
[0039] React at 42°C for 1 hour, and react at 70°C for 15 minutes, and store the reverse transcription product at -20°C.
[0040] 2. 3'-RACE amplification of cDNA
[0041] Desi...
Embodiment 2
[0072] Embodiment 2, the application of Bt1.3-I polypeptide
[0073] 1. Evaluation of analgesic activity
[0074] 1. Construction of rat sciatic nerve hemisection model
[0075] The rat sciatic nerve hemisection model was used as an analgesic model. This model is caused by direct puncture of rat sciatic nerve, which can induce strong mechanical hyperalgesia, autophagy rarely occurs in animals, and can better simulate neurogenic pain. At the same time, the operation process is simple and fast, and it is a good analgesic Model.
[0076] SD rats, male, weighing 200-220 g, were provided by the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army (SCXK-Jun 2007-004). According to the literature (Seltzer et al. Pain,. 1990, 43, 205-218.) method to construct the sciatic nerve hemisection model rats. The specific steps are as follows: Under anesthesia and aseptic operation, the right sciatic nerve is exposed at the high pos...
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Abstract
The invention discloses barrel-shaped alpha conantokin Bt1.3 and application thereof. The amino acid sequence of the barrel-shaped alpha conantokin Bt1.3 is a sequence 2 in a sequence table; and the nucleotide sequence of a coding gene of the conatokin is a sequence 1 in the sequence table. The connection modes of a disulfide bond of the conantokin are Cys1-Cys3 and Cys2-Cys4; and Cys starting from the amino terminal of the conantokin is respectively marked as Cys1, Cys2, Cys3 and Cys4 in sequence. Experiments prove that: Bt1.3 linear peptide (RKCCSNPACNRYNPAIC) is synthesized by an Fmoc-solid-phase peptide synthesis method, and is folded by one step in buffer solution of NH4HCO3 to form the Bi1.3 conantokin; and analgesic experiments prove that the alpha conantokin has high analgesic activity in rat sciatic nerve hemisection models.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a barrel-shaped α-cono polypeptide Bt1.3 and its application. Background technique [0002] Neuropathic pain is a pain syndrome caused by primary lesions or dysfunction of the central or peripheral nervous system. Typical symptoms of neuropathic pain include paresthesia, hyperalgesia, and hyperalgesia. At present, many major diseases (such as cancer, AIDS, traumatic nerve injury, leprosy, sclerosis, herpes zoster) can cause damage to the nervous system function and cause neuropathic pain. The commonly used drugs in clinical practice are mainly anticonvulsant drugs (such as leticitalide, oxcarbazepine), opioids (such as morphine, etc.) The treatment didn't work well. Therefore, it is necessary to develop a new generation of analgesic drugs with high activity, low tolerance and non-addiction. The latest research shows that the neuronal subtypes nAChRs α9α10, α7 and α3β2 are related...
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