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Recombinant vector capable of improving solubility of viral glycoprotein, preparation method and applications thereofthereof

A technology for recombinant vectors and glycoproteins, which is applied in the fields of biotechnology and biomedicine, and can solve problems such as increasing the difficulty of protein purification

Active Publication Date: 2018-10-16
广州源博医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies have shown that RSVpre F expresses proteins in this system as inclusion bodies, and only a small amount of protein dissolves (about 10%) after its direct cleavage, which increases the difficulty of purification of RSVpre F proteins

Method used

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  • Recombinant vector capable of improving solubility of viral glycoprotein, preparation method and applications thereofthereof
  • Recombinant vector capable of improving solubility of viral glycoprotein, preparation method and applications thereofthereof
  • Recombinant vector capable of improving solubility of viral glycoprotein, preparation method and applications thereofthereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0042] Implementation Case 1: Optimization of M protein and RSV pre F protein gene sequences of PIV3

[0043] The M protein of PIV3 and the RSV pre F protein gene sequence reported in the literature were retrieved from Genebank, and the kozak sequence was added in front of the promoter. Optimized according to the host cell codon preference of the Spodoptera frugiperda (Sf9) cell line, the M protein and RSV pre F protein gene sequences of the optimized PIV3 were obtained: respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2 Show; in order to facilitate the purification of pre F protein with His sequence.

Embodiment example 2

[0044] Implementation case 2: Obtaining the M protein and RSV pre F protein gene sequence shuttle vector with PIV3

[0045] The present invention requires the co-expression of the viral M protein and the RSV preF protein, so it is necessary to construct polycistrons to meet the purpose. The current strategy for constructing polycistronic vectors mainly includes: 1) co-transfection of multiple vectors; 2) construction of multi-promoter expression vectors; 3) construction of polycistronic expression vectors using the IRES system; 4) use of self-cleaving polypeptides Ligation connects the gene of interest. Such as 2A sequence, LP4 sequence, Nia protease recognition sequence, etc. The construction of the co-expression vector of the virus M protein and the RSV pre F protein in the present invention includes but is not limited to the above methods.

[0046] Method 1: After amplifying the M protein and RSV pre F protein gene sequences of PIV3 by designing primers with restriction s...

Embodiment example 3

[0049] Example 3: Packaging of recombinant baculovirus expressing soluble RSV pre F protein

[0050] The recombinant baculovirus vector obtained in Example 2 was transfected into Sf9 cells, and the recombinant baculovirus containing and expressing soluble RSV pre F protein was packaged in the cell line, which were named BV-MRSVpreF and BV-M2ApreF respectively, and PCR It was determined that two recombinant baculoviruses contained two genes ( figure 2 ).

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Abstract

The invention discloses a recombinant vector capable of improving the solubility of viral glycoprotein, and a preparation method thereof, wherein the recombinant vector is formed by co-expressing multi-virus derived M proteins and viral glycoprotein, and comprises a skeleton carrier, a multi-virus derived M protein expression cassette and a viral glycoprotein expression cassette, and the multi-virus derived M protein expression cassette and the viral glycoprotein expression cassette are inserted into the skeleton carrier. According to the present invention, the characteristic that the viral Mprotein can help the transportation of the transmembrane glycoprotein to the cell surface is used so as to prepare the recombinant vector capable of improving the solubility of viral glycoprotein, wherein the expression vector can solve the disadvantages of easy aggregation and difficult dissolution of the viral glycoprotein in the eukaryotic expression cells in the prior art can be solved, and issuitable for the large-scale expression and purification of soluble viral glycoprotein; and with the expression method, the dissolution efficiency of viral glycoprotein is increased, the high immunogenicity of viral glycoprotein is maintained, and the method is suitable for the industrial production and purification of the protein.

Description

technical field [0001] The invention belongs to the fields of biotechnology and biomedicine, and in particular relates to a recombinant carrier for improving the solubility of viral glycoproteins and its preparation method and application Background technique [0002] The viral surface glycoprotein with cystic sugar can mediate a series of functions such as the interaction between the virus and the host cell, membrane fusion, and the release of genetic material, and is the key molecule for its entry into the cell. Therefore, it is also used as a common target for the preparation of vaccines. [0003] Since most glycoproteins have the functions of fusing cell membranes and binding to receptors, etc., a large amount of glycoproteins is likely to be toxic to cells, resulting in a low expression level. In addition, the viral surface glycoprotein is different from the soluble protein. The viral surface glycoprotein has a single or multiple transmembrane structure, and it is easy...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/86C12N15/62C12N15/66C12N5/10A61K39/12A61P31/12
CPCA61K39/12A61K2039/575C07K14/005C12N15/85C12N15/86C12N2710/14043C12N2760/18551C12N2800/22
Inventor 王弋刘昕
Owner 广州源博医药科技有限公司
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