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Codon optimized severe fever associated thrombocytopenia syndrome virus nucleoprotein gene and nucleic acid vaccine thereof

A codon-optimized, severe fever technology, used in gene therapy, antiviral agents, genetic engineering, etc., can solve the problem that foreign genes cannot be expressed efficiently

Inactive Publication Date: 2015-08-12
JIANGSU PROVINCE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, nucleic acid vaccines also have many shortcomings, the most important of which is that the research and application objects of nucleic acid vaccines are mainly eukaryotes, and the vast majority of target genes come from prokaryotes such as viruses or bacteria. There are obvious differences in codon usage preferences, which leads to the inability of the foreign gene of the nucleic acid vaccine to be efficiently expressed in eukaryotic cells, and cannot effectively stimulate the body's immune system to respond

Method used

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  • Codon optimized severe fever associated thrombocytopenia syndrome virus nucleoprotein gene and nucleic acid vaccine thereof
  • Codon optimized severe fever associated thrombocytopenia syndrome virus nucleoprotein gene and nucleic acid vaccine thereof
  • Codon optimized severe fever associated thrombocytopenia syndrome virus nucleoprotein gene and nucleic acid vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Design and synthesis of codon-optimized SFTSV nuclear protein gene sequence

[0043] First use the software OptimumGene TM Analyze SEQ ID NO.1 of the SFTSV nucleoprotein gene sequence to find out its codon usage preference and the sites that differ from mammalian preference. For codon sites with different preferences, the codons preferred by mammalian cells were replaced, and the codon-optimized SFTSV nucleoprotein gene sequence SEQ ID NO.2 was designed. The amino acid sequence of the protein encoded by the codon-optimized gene sequence is consistent with its original amino acid sequence SEQ ID NO.3. The above-mentioned codon-optimized gene sequence was submitted to Nanjing GenScript Biotechnology Co., Ltd. for synthesis and loaded into the vector pUC57 to construct a recombinant plasmid pUC57-N-opt. The sequence confirmed that the synthesized sequence was correct.

[0044] In order to clearly show the site of codon optimization, the nucleic acid sequence N-opt a...

Embodiment 2

[0049] Example 2 Construction of eukaryotic expression vectors pJW4303-N, pJW4303-N-opt, pJW4303-tPA-N-opt

[0050] (1) Obtaining the target fragment and vector

[0051] 1) Obtainment of N fragment and large linear fragment of plasmid pJW4303: N fragment was obtained by double digestion with Hind III and Bgl II from pUC57-N provided by Nanjing GenScript Biotechnology Co., Ltd. The vector plasmid pJW4303 was digested with Hind Ⅲ and BamH Ⅰ. Enzyme digestion reaction system: 10×Buffer Tango TM 4μl, plasmid pJW4303 or PCR product 10μl, HindIII 1.5μl, BamH I or Bgl II 1.5μl, make up to 40μl, 37℃, 2h.

[0052] 2) Obtain the N-opt fragment and the large linear fragment of plasmid pJW4303: Use Hind Ⅲ and BamH Ⅰ to digest the recombinant vector pUC57-NP-opt and vector plasmid pJW4303 containing the target sequence provided by Nanjing GenScript Biotechnology Co., Ltd. , The digestion reaction system is the same as 1).

[0053] 3) Obtain the tPA-N-opt fragment and the large linear fragment o...

Embodiment 3

[0056] Example 3 Identification of recombinant plasmids pJW4303-N, pJW4303-N-opt, pJW4303-tPA-N-opt

[0057] 3.1 pJW4303-N, pJW4303-N-opt, pJW4303-tPA-N-opt transformed E. coli HB101 competent cells

[0058] 1) Add 10μl of the linker to the Ep tube containing 100μl of HB101 competent cells, gently tap the tube wall several times, mix thoroughly, and ice bath for 30min.

[0059] 2) Place the Ep tube in a 42°C water bath for 90 seconds.

[0060] 3) Slowly add 0.5 mL of LB medium to the Ep tube, 37° C., 80 rpm, and shake for 45 min.

[0061] 4) Spread the bacterial solution on an LB plate containing ampicillin (0.1g / L), and cultivate overnight at 37°C.

[0062] 3.2 Screen positive clones

[0063] A single colony was randomly picked and inoculated in a culture test tube (LB medium containing 0.1g / L ampicillin) at 37° C., shaking at 200 rpm, and cultured overnight.

[0064] 3.3 Extract a small amount of recombinant plasmids pJW4303-N, pJW4303-N-opt, pJW4303-tPA-N-opt (the plasmid small amount e...

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Abstract

The invention belongs to the technical filed of biological medicines, and relates to a codon optimized severe fever associated thrombocytopenia syndrome virus nucleoprotein gene and a nucleic acid vaccine thereof. The sequence of the codon optimized gene considers the codon usage bias of mammal cells, and is represented by SEQ ID NO.2. The severe fever associated thrombocytopenia syndrome virus nucleoprotein nucleic acid vaccine is composed of the codon optimized severe fever associated thrombocytopenia syndrome virus nucleoprotein gene sequence and an eukaryotic expression vector pJW4303, and the 5' end of the sequence is connected with a tPA signal peptide sequence. The nucleic acid vaccine effectively stimulates the immune system after immunizing mammals, and generates good humoral immune response.

Description

Technical field [0001] The invention belongs to the technical field of biomedicine, and relates to codon optimization and severe fever with thrombocytopenia syndrome virus nucleoprotein gene sequence carrying tPA signal peptide and a nucleic acid vaccine thereof. Background technique [0002] Severe fever with thrombocytopenia syndrome virus (SFTSV) is a new virus belonging to Bunyaviridae Sandfly virus that was first discovered in my country recently. It can cause severe fever with thrombocytopenia clinically. Syndrome (Severe fever with thrombocytopenia syndrome, SFTS). So far, the case has been reported in Hubei, Henan, Jiangsu, Liaoning, Shandong, Anhui and other provinces. The clinical manifestations of severe fever with thrombocytopenia syndrome are mainly fever, thrombocytopenia, and leukopenia. It is not easy to distinguish from other fever and bleeding diseases. The fatality rate can be as high as 30%. There have been many articles on human-to-human transmission of SFTSV...

Claims

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Application Information

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IPC IPC(8): C12N15/40A61K48/00A61K39/12A61P31/14
Inventor 李军金柯周宜庆韩亚萍刘源蒋龙凤
Owner JIANGSU PROVINCE HOSPITAL
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