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Clostridium difficile exotoxin B amino-terminal gene sequence with optimized codon and nucleic vaccine of clostridium difficile exotoxin B

A codon-optimized Clostridium difficile technology, applied in the field of Clostridium difficile exotoxin B amino-terminal gene sequence and its nucleic acid vaccine, can solve the problems of low immunogenicity and ineffective expression of foreign genes, and achieve improved protection 、To overcome the effects of weak animal protection

Inactive Publication Date: 2012-07-04
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 B amino-terminal gene sequence with optimized codon and nucleic vaccine of clostridium difficile exotoxin B
  • Clostridium difficile exotoxin B amino-terminal gene sequence with optimized codon and nucleic vaccine of clostridium difficile exotoxin B
  • Clostridium difficile exotoxin B amino-terminal gene sequence with optimized codon and nucleic vaccine of clostridium difficile exotoxin B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Design and Synthesis of Codon-optimized Clostridium difficile Exotoxin B N-terminal Gene Sequence

[0052] First, use the software MacVector 7.2 to analyze the amino-terminal gene sequence SEQ ID NO.2 encoding Clostridium difficile exotoxin B to find out its codon usage bias and the sites that differ from mammalian and Escherichia coli codon usage bias. For codon sites with different usage preferences, codons preferred by both mammalian cells and Escherichia coli were substituted to design a codon-optimized Clostridium difficile exotoxin B N-terminal gene sequence SEQ ID NO.1. The protein amino acid sequence encoded by the codon-optimized gene sequence is consistent with its original amino acid sequence (SEQ ID NO.3). The designed sequence was synthesized by American GENEART Company, loaded into vector pMK-RQ, and constructed into recombinant plasmid pMK-RQ-TcdB-N. The synthesized sequence was confirmed to be correct by sequencing.

[0053] Compared with the...

Embodiment 2

[0054] Example 2 Construction of eukaryotic expression vector pJW4303-TcdB-N

[0055] 1) Obtaining the TcdB-N fragment and the large linear fragment of the plasmid pJW4303: the plasmids pJW4303 and pMK-RQ-TcdB-N (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-TcdB-N) 10 μl, Pst I 1.5 μl, BamH I 1.5 μl, rehydrate to 40 μl, 37°C, 2h.

[0056] 2) Purification of enzyme-cut products: After the above enzyme-cut products were subjected to 10g / L agarose gel electrophoresis, they were placed under a UV detector, followed by the gel recovery kit (Agarose Gel DNA Purification Kit Ver.2.0, Dalian Bao Biological Co., Ltd.) 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 1mg=1μl. Add 4 times the volume of DR-I Buffer, place in a 75°...

Embodiment 3

[0058] Example 3 Identification of recombinant plasmid pJW4303-TcdB-N

[0059]3.1 pJW4303-TcdB-N transformed Escherichia coli HB101 competent cells (American Standard Biological Collection, USA)

[0060] 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.

[0061] 2) Place the Ep tube in a 42°C water bath for 90s.

[0062] 3) Slowly add 0.5 mL of LB medium to the Ep tube, shake at 37°C, 80 rpm, for 45 min.

[0063] 4) Spread the bacterial solution on an LB plate containing ampicillin (0.1 g / L), and culture overnight at 37°C.

[0064] 3.2 Screening positive clones

[0065] 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.

[0066] 3.3 Small amount extraction of recombinant plasmid pJW4303-TcdB-N (plasmid small amount extraction kit: TaKaRa MiniBES...

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Abstract

The invention belongs to the technical field of biomedicines and relates to a clostridium difficile exotoxin B amino-terminal gene sequence with optimized codon and a nucleic vaccine of the clostridium difficile exotoxin B. The gene sequence with optimized codon gives consideration to the preferences of the codon in the mammalian cell and the escherichia coli. The related vaccine of the clostridium difficile comprises the exotoxin B amino-terminal gene sequence with optimized codon and a eukaryotic expression vector pJW4303. The clostridium difficile exotoxin B amino-terminal gene sequence with optimized codon not only can be used for constructing the nucleic vaccine, effectively simulates the immune system of the host after immunizing the mammals so that the specific humoral immune response is generated and shows that the specific antibodies have good protective effects in the in vivo and in vitro models, but also is suitable for carrying out prokaryotic expression on the protein in the escherichia coli and lays the foundation for obtaining the protein in quantity.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a codon-optimized Clostridium difficile exotoxin B amino terminal gene sequence and a nucleic acid vaccine thereof. Background technique [0002] Clostridium difficile is a spore-forming, obligate anaerobic, Gram-positive Clostridium bacterium whose main virulence factors are exotoxin A and exotoxin B. Since 2000, Clostridium difficile-associated diarrhea has become a pressing medical problem. On the one hand, the morbidity, severity, and mortality of Clostridium difficile infection are getting higher and higher, and the drug resistance rate is also increasing year by year. First of all, Clostridium difficile widely exists in the hospital environment, and the existence of its special living state of spores that can tolerate a variety of commonly used disinfectants in the hospital exacerbates its dissemination in the hospital. One of the most important pathogens of diarrhea. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31A61K48/00A61K39/114A61P1/12A61P31/04C12R1/145
Inventor 王世霞金柯黄祖瑚卢山
Owner 王世霞
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