Clostridium difficile exotoxin A carboxy-terminal gene sequence with optimized codon and nucleic acid vaccine thereof

A codon-optimized, Clostridium difficile technology, applied in the field of Clostridium difficile exotoxin A carboxy-terminal gene fragment and its nucleic acid vaccine, can solve the problems of ineffective expression of foreign genes and low immunogenicity of nucleic acid vaccines

Inactive Publication Date: 2011-09-28
王世霞 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the differences in codon usage preferences between prokaryotes and eukaryotes, the foreign genes used to construct nucleic acid vaccines cannot be effectively expressed in mammalian hosts, so they cannot effectively stimulate the host's immune system to produce better immunity. Protective effects
This is the main reason for the low immunogenicity of current nucleic acid vaccines

Method used

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  • Clostridium difficile exotoxin A carboxy-terminal gene sequence with optimized codon and nucleic acid vaccine thereof
  • Clostridium difficile exotoxin A carboxy-terminal gene sequence with optimized codon and nucleic acid vaccine thereof
  • Clostridium difficile exotoxin A carboxy-terminal gene sequence with optimized codon and nucleic acid vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Design and synthesis of codon-optimized Clostridium difficile exotoxin A carboxy-terminal gene sequence

[0041] First, the software MacVector 7.2 was used to analyze the carboxy-terminal gene sequence encoding Clostridium difficile exotoxin A to find out its codon usage bias and the sites that differ from mammalian and Escherichia coli codon usage bias. For the codon sites with different usage preferences, codons preferred by both mammalian cells and Escherichia coli were substituted, and a codon-optimized C-terminal gene sequence of Clostridium difficile exotoxin A was designed. The protein amino acid sequence encoded by the codon-optimized gene sequence is consistent with its original amino acid sequence. The designed sequence was synthesized by American GENEART Company, loaded into vector pMK-RQ, and constructed into recombinant plasmid pMK-RQ-TcdA-C. The synthesized sequence was confirmed to be correct by sequencing.

[0042] In order to clearly show t...

Embodiment 2

[0065] Example 2. Construction of eukaryotic expression vector pJW4303-TcdA-C

[0066] (1) Obtaining the TcdA-C fragment and the large linear fragment of the plasmid pJW4303: the plasmids pJW4303 and pMK-RQ-TcdA-C (synthesized by GENEART, USA) were double-digested with Pst I and BamH I, respectively. The enzyme digestion reaction system is: 10×BufferTango TM 4 μl, plasmid (pJW4303, pMK-RQ-TcdA-C) 10 μl, Pst I 1.5 μl, BamHI 1.5 μl, water up to 40 μl, 37°C, 2h.

[0067] (2) Purification of digested products: The digested products were subjected to 10 g / L agarose gel electrophoresis, placed under a UV detector, and purified according to the gel recovery kit (Agarose Gel DNA Purification Kit Ver. Company) instructions, cut out the gel containing the target fragment, weigh the mass of the gel block with an analytical balance, and calculate the volume of the gel block according to 1 mg=1 μl. Add 4 times the volume of DR-I Buffer, place in a 75°C water bath to heat and melt the ge...

Embodiment 3

[0069] Example 3. Identification of recombinant plasmid pJW4303-TcdA-C

[0070] 3.1 pJW4303-TcdA-C respectively transformed HB101 competent cells

[0071] 1) Add 10 μl of the linker to an Ep tube containing 100 μl of HB101 competent cells, gently tap the tube wall several times, mix well, and place on ice for 30 minutes.

[0072] 2) Place the Ep tube in a 42°C water bath for 90s.

[0073] 3) Slowly add 0.5 mL of LB medium to the Ep tube, shake at 37°C, 80 rpm, for 45 min.

[0074] 4) Spread the bacterial solution on an LB plate containing ampicillin (0.1 g / L), and culture overnight at 37°C.

[0075] 3.2 Screening positive clones

[0076] Pick 5 single colonies at random, inoculate them in 5 culture test tubes (LB medium containing 0.1 g / L ampicillin), shake at 200 rpm at 37°C, and culture overnight.

[0077] 3.3 Small extraction of recombinant plasmid pJW4303-TcdA-C (Plasmid small extraction kit: TaKaRa MiniBEST PlasmidPurification Kit Ver.2.0, TaKaRa Company)

[0078] 1)...

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Abstract

The invention belongs to the technical field of biomedicine, and relates to a Clostridium difficile exotoxin A carboxy-terminal gene fragment with an optimized codon and a nucleic acid vaccine thereof. In the gene sequence with the optimized codon, the codon usage biases of mammalian cells and Escherichia coli are considered; and the Clostridium difficile vaccine consists of the exotoxin A carboxy-terminal gene sequence with the optimized codon and a eukaryotic expression vector pJW4303. The Clostridium difficile exotoxin A carboxy-terminal gene fragment with the optimized codon can be used for constructing the nucleic acid vaccine, effectively stimulates an immune system of a host after a mammal is immunized so as to produce better humoral immune response, is also applicable to the prokaryotic expression of a protein in the Escherichia coli, and lays a foundation for the batch acquisition of the protein.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a codon-optimized Clostridium difficile exotoxin A carboxyl terminal gene fragment and a nucleic acid vaccine thereof. Background technique [0002] Clostridium difficile is a Gram-positive, spore-forming, obligately anaerobic Clostridium bacterium. Currently, the prevention, diagnosis, and treatment of Clostridium difficile infection are facing many difficulties. On the one hand, Clostridium difficile widely exists in the hospital environment, and as its dormant spores can tolerate a variety of disinfectants commonly used in hospitals, it has become the main pathogen of nosocomial infectious diarrhea after Campylobacter; Zazole and vancomycin, as the two main drugs for the treatment of Clostridium difficile infection, their drug resistance is also constantly accumulating and spreading, and the curative effect is decreasing day by day, and for the treatment of relapsed cases, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31A61K48/00A61K39/08A61P31/04C12R1/145
Inventor 王世霞金柯黄祖瑚卢山
Owner 王世霞
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