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Codon-optimized severe fever with thrombocytopenia syndrome virus nucleoprotein gene and its nucleic acid vaccine

A codon-optimized, severe fever technology, applied in gene therapy, antiviral agents, genetic engineering, etc., can solve the problem of inefficient expression of foreign genes

Inactive Publication Date: 2017-11-21
JIANGSU PROVINCE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 with thrombocytopenia syndrome virus nucleoprotein gene and its nucleic acid vaccine
  • Codon-optimized severe fever with thrombocytopenia syndrome virus nucleoprotein gene and its nucleic acid vaccine
  • Codon-optimized severe fever with thrombocytopenia syndrome virus nucleoprotein gene and its nucleic acid vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Design and synthesis of the SFTSV nucleoprotein gene sequence of embodiment 1 codon optimization

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

[0044] In order to clearly show the site for codon optimization, the nucleic ac...

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) Obtaining 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. And the vector plasmid pJW4303 was digested with Hind Ⅲ and BamH Ⅰ. Enzyme digestion reaction system: 10×Buffer Tango TM 4 μl, plasmid pJW4303 or PCR product 10 μl, HindⅢ 1.5 μl, BamH I or Bgl II 1.5 μl, rehydrate to 40 μl, 37°C, 2h.

[0052] 2) Obtaining the N-opt fragment and the large linear fragment of plasmid pJW4303: the recombinant vector pUC57-NP-opt and the vector plasmid pJW4303 provided by Nanjing GenScript Biotechnology Co., Ltd. provided by Nanjing GenScript Biotechnology Co., Ltd. were digested with Hind Ⅲ and BamH Ⅰ respectively , the enzyme digestion reaction system is the same as 1).

...

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 transform Escherichia coli HB101 competent cells

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

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

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

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

[0062] 3.2 Screening positive clones

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

[0064] 3.3 Small extraction of recombinant plasmids pJW4303-N, pJW4303-N-opt, pJW4303-tPA-N-opt (Plasmid ...

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Abstract

The invention belongs to the technical field of biomedicine and relates to codon-optimized severe fever with thrombocytopenia syndrome virus nucleoprotein gene and nucleic acid vaccine thereof. The codon-optimized gene sequence takes into account the codon usage preference of mammalian cells, and the sequence is shown in SEQ ID NO.2. The involved severe fever with thrombocytopenia syndrome virus nucleoprotein nucleic acid vaccine is composed of codon-optimized severe fever with thrombocytopenia syndrome virus nucleoprotein gene sequence and eukaryotic expression vector pJW4303, whose 5' end is connected with tPA signal peptide sequence. The nucleic acid vaccine effectively stimulates the immune system after immunizing the mammal, and produces a better humoral immune response.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to codon optimization, severe fever with thrombocytopenia syndrome virus nucleoprotein gene sequence and nucleic acid vaccine carrying tPA signal peptide. Background technique [0002] Severe fever with thrombocytopenia syndrome virus (Severe fever with thrombocytopenia syndrome virus, SFTSV) is a new virus belonging to the genus Phlebovirus first discovered in my country recently, which can cause severe fever with thrombocytopenia syndrome clinically. Severe fever with thrombocytopenia syndrome (SFTS). So far, this 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, which are not easy to distinguish from other fever and hemorrhagic diseases, and the case fatality rate can be as high as 30%. Literature reports o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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