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Efficient expression method and application of virus recombinant glycoprotein and eukaryotic cells thereof

A glycoprotein and virus technology, applied in the field of genetic engineering, can solve problems affecting the prevention and treatment of rabies, different virus expression hosts, and uneven quality, and achieve good application prospects and huge market prospects

Active Publication Date: 2019-01-01
北京派迪畅科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the eukaryotic expression of recombinant glycoprotein of rabies virus is too low, and the conformation of prokaryotic expressed protein is wrong, so the research on recombinant rabies vaccine has not progressed
The widely used human rabies vaccine is still an inactivated vaccine, which has certain safety risks
[0005] In addition, due to the different virus strains used by different manufacturers, different virus expression hosts, different production processes, and uneven quality, it directly affects the prevention and treatment of rabies.
At present, the experiment of inhibiting virus-infected cells is generally used to evaluate the effectiveness of vaccines, which is time-consuming and labor-intensive.

Method used

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  • Efficient expression method and application of virus recombinant glycoprotein and eukaryotic cells thereof
  • Efficient expression method and application of virus recombinant glycoprotein and eukaryotic cells thereof
  • Efficient expression method and application of virus recombinant glycoprotein and eukaryotic cells thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 The synthesis of rabies virus recombinant glycoprotein and nucleic acid thereof and the construction of expression vector

[0049] The signal peptide of the human IgG protein heavy chain is used to replace the signal peptide of the rabies virus glycoprotein, and the resulting recombinant protein is the rabies virus recombinant glycoprotein, and its amino acid sequence is shown in SEQ ID NO:1. The rabies virus is from Rabies virus strain CTN-1.

[0050] According to the protein sequence shown in SEQ ID NO:1, through codon correspondence and host expression frequency, the nucleic acid encoding the rabies virus recombinant glycoprotein is obtained and optimized by artificial synthesis.

Embodiment 2

[0051] Embodiment 2 utilizes eukaryotic cell expression rabies virus recombinant glycoprotein

[0052] (1) Construction of recombinant plasmids

[0053] The optimized rabies virus recombinant glycoprotein nucleic acid sequence was digested with a restriction endonuclease and inserted into the expression vector pcDNA3( figure 1 ), transform Escherichia coli DH5α competent cells. After verifying that the vector was successfully constructed, recombinant plasmids were prepared. The partial sequence of the constructed recombinant plasmid is shown in SEQ ID NO:2.

[0054] (2) Plasmid extraction (using Tiangen plasmid extraction kit)

[0055] Shake bacteria: Add 4ml of medium containing corresponding antibiotics to 14ml lock cap tube, dip a sterilized toothpick to pick up the gene-synthesized puncture bacteria, put them into LB medium, and cultivate overnight.

[0056] 1. Column balance: add 500 μL of balance solution BL to the adsorption column CP4 (the adsorption column is placed...

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Abstract

The invention provides virus recombinant glycoprotein which is formed by replacing a signal peptide of target viral glycoprotein with a signal peptide of human or animal secretory protein. The invention also provides a method for efficiently expressing the recombinant glycoprotein by using eukaryotic cells. According to the virus recombinant glycoprotein and the method provided by the invention, the world technical problems of low expression and poor secretion of the virus recombinant glycoprotein in the eukaryotic cells are solved, a solid foundation is laid for developing vaccines and virusprotective antibody detection reagents and a good application prospect is realized.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a virus recombinant glycoprotein and its high-efficiency expression method and application in eukaryotic cells. Background technique [0002] Rabies is a Class B infectious disease under the statutory reporting management in my country. It is one of the acute infectious diseases with the highest human mortality rate so far. Its pathogen is Rabies virus, and humans and all warm-blooded animals are susceptible to this virus. The lethal rate of rabies is close to 100%. Although human anti-rabies virus antibody drugs have been greatly developed in recent years, there is still no very effective therapeutic drug developed. Therefore, rabies vaccine is still the most effective way to prevent and treat rabies. s method. [0003] The rabies virus genome is a non-segmented single-stranded negative-strand RNA with a total length of about 12kb, encoding enzyme protein (L), glyco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/08C12N15/47C12N15/40C12N15/85C07K16/10G01N33/569G01N33/532A61K38/16A61K39/205A61P31/14
CPCA61P31/14G01N33/532G01N33/56983C12N15/85C07K14/005C07K16/10C12N2760/20122C12N2760/14122A61K39/00A61K38/00
Inventor 孙乐张翠娟
Owner 北京派迪畅科技发展有限公司
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