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Preparation method of recombinant α-bungarotoxin αbtx

A bungarotoxin and expression vector technology, applied in the field of preparation of recombinant α-bungarotoxin αBtx, can solve the problems of no wild αBtx activity, lack of folding partners, unable to obtain expression products and the like

Active Publication Date: 2017-04-05
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the particularity of the synthesis and processing of αBtx, the characteristics of 5 pairs of intramolecular disulfide cross-links, and the difference in the preference of mRNA codons between the prokaryotic expression system and the snake venom gland cells, the lack of folding partners for eukaryotic genes after translation, The prokaryotic expression of αBtx full-sequence cDNA makes it impossible to obtain the expression product, or even if the expression product is obtained, it does not have the activity of wild αBtx

Method used

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  • Preparation method of recombinant α-bungarotoxin αbtx
  • Preparation method of recombinant α-bungarotoxin αbtx
  • Preparation method of recombinant α-bungarotoxin αbtx

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Embodiment 1

[0033] 1. Technical route: such as figure 1 The shown rαBtx (V31) preparation flow chart:

[0034] 1. Codon-optimized synthesis of αBtx(V31) mature peptide coding gene;

[0035] 2. Construction of pET16b-rαBtx recombinant fusion expression plasmid;

[0036] 3. Construction of pET16b-rαBtx recombinant non-fusion expression plasmid;

[0037] 4. Optimization of the mRNA secondary structure of the rαBtx non-fusion expression coding sequence;

[0038] 5. Construction of BL21(DE3)-rαBtx engineering bacteria;

[0039] 6. Screening of rαBtx pre-expression and high-expression clones and separation and purification of rαBtx inclusion bodies;

[0040] 7. Optimization of renaturation conditions of rαBtx protein and mass renaturation preparation.

[0041] 8. Purification and separation of rαBtx monomeric protein and detection of purity and activity.

[0042] 2. Steps:

[0043] 1. Optimized synthesis of rαBtx coding gene and construction of recombinant expression plasmid.

[0044] (...

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Abstract

The invention discloses a preparation method of recombinant alpha-Btx (alpha-Bungarotoxin). The method comprises steps as follows: (1) optimizing the coding sequence of the recombinant alpha-Btx, wherein the optimized coding sequence is represented by SEQ ID NO.15; (2) on the basis of the coding sequence represented by the SEQ ID NO.15, constructing an expression vector of the r-alpha-Btx (recombinant alpha-Btx); (3) converting the expression vector into escherichia coli, constructing alpha-Btx recombinant expression engineering bacteria, and inducing an r-alpha-Btx expression with IPTG (isopropyl-beta-d-thiogalactoside) after r-alpha-Btx recombinant expression engineering bacteria are cultured.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of recombinant α-bungarotoxin αBtx. Background technique [0002] α-Bungarotoxin (α-Βungarotoxin, αBtx) is an important component in the venom of Bungarus Multicinctus. It is a protein composed of 73 amino acids with a molecular weight of 8kDa. αBtx can specifically bind and block the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction in mammals, decoupling the nerve-muscle electrochemical coupling, resulting in the inability of skeletal muscle to contract. αBtx has very high affinity and specificity to nAChR, and is an ideal tool for studying nAChR. The αBtx used in the past was extracted from the venom of the coral snake. The process is complicated and the cost is high. In addition, the venom production of the coral snake is low, so the commercialized αBtx is very expensive. Finding and establishing a method and way to obtain a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/70C07K14/435
Inventor 徐江陈永恒陈主初陈林李柱一徐志凯吴兴安李佳李俊
Owner XIANGYA HOSPITAL CENT SOUTH UNIV