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Establishment method of clostridium difficile exotoxin A carboxyl-terminal sequence codon optimization gene segment and expression vector and application of expression protein thereof

A technology of codon optimization and Clostridium difficile, applied in the direction of anti-bacterial immunoglobulin, biochemical equipment and methods, applications, etc., can solve the problems of codon self-preference difference and low expression efficiency of Escherichia coli

Active Publication Date: 2014-06-18
SHANDONG INT BIOTECH PARK DEV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] CN 101870978A discloses a codon-optimized carboxy-terminal sequence of Clostridium difficile toxin A that can be expressed in both Escherichia coli and mammals. Due to its compatibility, there are prokaryotic expression systems and eukaryotic expression systems Differences in codon self-preference, which are not expressed very efficiently in E. coli

Method used

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  • Establishment method of clostridium difficile exotoxin A carboxyl-terminal sequence codon optimization gene segment and expression vector and application of expression protein thereof
  • Establishment method of clostridium difficile exotoxin A carboxyl-terminal sequence codon optimization gene segment and expression vector and application of expression protein thereof
  • Establishment method of clostridium difficile exotoxin A carboxyl-terminal sequence codon optimization gene segment and expression vector and application of expression protein thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1 Codon-optimized Clostridium difficile exotoxin A carboxy-terminal gene sequence design

[0042] Select the 2616-base sequence at the carboxy-terminal of the Clostridium difficile exotoxin A gene (encoding 872 amino acids in total), analyze the coding sequence, and find out the repetitive sequence that affects gene synthesis. The schematic diagram of the repetitive region is shown in figure 1 . Simultaneously analyze the sites with different codon usage preferences in this sequence and E. coli, and replace low-frequency codons with high-frequency codons; in addition, on the basis of taking into account the codon preferences of E. The sequence of the synonymous codon was replaced to obtain a new optimized coding gene sequence.

[0043] The method for codon modification includes the following aspects, that is, based on the homology analysis of the amino acid sequence of the repeat region (see figure 2 ), find out the conserved amino acid sequence (conservative...

Embodiment 2

[0051] Example 2 Codon-optimized Clostridium difficile exotoxin A carboxy-terminal gene sequence synthesis

[0052] According to the optimization principle of Example 1 above, the corresponding nucleotide sequence was designed with reference to the codon preference of Escherichia coli, and the carboxy-terminal gene of the C. difficile exotoxin A gene after codon optimization was sent to gene synthesis in two stages. Restriction sites are designed at both ends of each gene for subsequent molecular cloning.

[0053] The synthetic sequence of the first optimized combined sequence SEQ ID NO.29 Fragment 1 (SEQ ID NO.32) is as follows. Introduce the EcoRI digestion recognition sequence "GAATTC" at the end:

[0054] 5'gatatctcat tattctattt tgatcctata gaatttaact tagtaacagg atggcaaact 60

[0055] atcaacggca agaaatatta cttcgatata aatactggag cagctttaac tagttataag 120

[0056] atttattaatg gtaaacactt ttactttaat aatgatggtg tgatgcagct gggagtattt 180

[0057] aaaggacctg atggatttga atatttt...

Embodiment 3

[0110]Example 3 Construction and identification of the first optimized combination prokaryotic expression vector pET3b(+)-TcdA-C-1

[0111] 1. Acquisition of the target gene fragment: Use EcoRV and EcoRI to double digest the subcloning plasmid pUCE-TcdA-C1 (synthesized by Gene Company, containing the first combined optimized fragment 1 sequence), recover the fragment 1 sequence by gel recovery; use EcoRI and XhoI double Restriction digestion of the subcloning plasmid pUCE-TcdA-C2 (synthesized by Gene Company, containing the sequence of the first optimized combination Fragment 2), gel recovery to obtain the sequence of Fragment 2;

[0112] 2. Acquisition of the prokaryotic vector pET32b(+): design PCR primers to amplify the partial sequence of pET32b(+), and introduce an XhoI restriction site at one end. The PCR product purification kit recovers the product after the PCR reaction, and then releases the vector containing the cohesive end by single enzyme digestion with XhoI. Th...

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Abstract

The invention relates to an establishment method of a codon-transformed clostridium difficile exotoxin A carboxyl-terminal gene segment and an expression vector and high-efficiency expression thereof in escherichia coli. The codon-transformed gene sequence integrates the escherichia coli codon usage preference and reduces the influence of the original gene duplication sequence on gene synthesis and expression. The high-efficiency expression vector involved in the study consists of a codon-transformed clostridium difficile exotoxin A carboxyl-terminal gene and a prokaryotic expression vector pET32b(+); after IPTG induction, the high-efficiency expression vector can express the fusion protein with a His tag with high efficiency. The fusion protein can release about 872 amino acid segments at the clostridium difficile exotoxin A carboxyl terminal after thrombin digestion. In the study, the codon-transformed clostridium difficile exotoxin A carboxyl-terminal gene segment can be subjected to high-efficiency fusion expression in escherichia coli; the purified protein can be used as an antigen for preparing a clostridium difficile exotoxin A antibody and an anti-clostridium difficile infection vaccine.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a codon-modified Clostridium difficile exotoxin A carboxyl terminal gene fragment, an expression vector construction method and a high-efficiency expression method in Escherichia coli. Background technique [0002] Clostridium difficile is a Gram-positive, spore-forming, obligate anaerobic Clostridium bacterium that is currently a major cause of nosocomial infections. C. difficile infection is contact-transmitted and colonizes the colon by the oral-fecal route, through direct contact with patients, contaminated equipment or objects (such as bedpans, baths, and electronic rectal thermometers), through indirect contact with healthcare workers, through Incubation periods ranging from a few days to 8 weeks lead to nosocomial outbreaks of Clostridium difficile infection. The difficulty in the treatment of Clostridium difficile is that the dormant spores can tolerate a variety of di...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/70C12N15/66C07K16/12A61K39/08A61P31/04
Inventor 刘红冯东晓邢平平徐从伦李敏郭桂平于锦丽赵志伟
Owner SHANDONG INT BIOTECH PARK DEV
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