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Purification method and application of recombinant Sendai virus-like particles

A technology of Sendai virus and purification method, applied in the field of virus biology, can solve the problems of low purification efficiency, difficult to remove impurities, etc., and achieve the effect of simple operation and high recovery rate

Pending Publication Date: 2021-11-12
雪莱(武汉)生物医药技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult to separate the target protein from the miscellaneous protein by using conventional ion exchange chromatography, and the purification efficiency is very low when other purification methods are used. Moreover, in the process of expressing the target gene, some recombinant proteins will also be produced. Proteins similar to Sendai virus-like particles are purified according to existing technologies, and it is difficult to remove these impurities and other problems

Method used

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  • Purification method and application of recombinant Sendai virus-like particles

Examples

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Effect test

Embodiment 1

[0035] Embodiment 1 Purification method of recombinant Sendai virus-like particles

[0036] A method for purifying recombinant Sendai virus-like particles, comprising the steps of:

[0037] S1, cell lysis: collect the cells containing the recombinant Sendai virus-like particles, add the cell lysate, the components of the cell lysate are as follows: 50mM Tris-HCl buffer (pH 7.4), 150mM NaCl, 1% TritonX-100 , 0.5% SDS, 0.5% deoxycholate, 4% β-mercaptoethanol and 1mM EDTA, after crushing, a cell lysate is obtained, and the crushing is carried out by a high-pressure homogeneous method, and the specific conditions are as follows: Break the cells 4-6 times under certain conditions;

[0038] S2. Ion-exchange chromatography: use anion-exchange chromatography column (one of Capto-DEAE anion-exchange column or Capto-Q anion-exchange column) to purify the cell lysate obtained in step S1, the cell lysate The specific method for purification is as follows: Use 50mM Tris-HCl buffer (pH 8....

Embodiment 2

[0051] Example 2 Performance Test of Recombinant Sendai Virus-Like Particles

[0052] The purity and yield of the recombinant Sendai virus-like particles prepared in Example 1 and Comparative Examples 1-5 were tested, and the results are shown in Table 1 below:

[0053] Table 1 Performance test results of recombinant Sendai virus-like particles

[0054] Purity (%) of recombinant Sendai virus-like particles Yield (%) of recombinant Sendai virus-like particles Example 1 95 87 Comparative example 1 80 75 Comparative example 2 65 59 Comparative example 3 84 79 Comparative example 4 85 80 Comparative example 5 83 77

[0055] As can be seen from the performance test results in Table 1, the purity of the recombinant Sendai virus-like particles prepared in Example 1 is 95%, and the yield is 87%. Perform ion-exchange chromatography, then carry out hydroxyapatite chromatography, and finally carry out affinity chromatography, a...

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Abstract

The invention discloses a purification method and application of recombinant Sendai virus-like particles. The purification method comprises the following steps of S1, cell lysis, S2, ion exchange chromatography, S3, hydroxyapatite chromatography, and S4, affinity chromatography. Through specific and sequential purification steps, the ion exchange chromatography is firstly carried out, then the hydroxyapatite chromatography is carried out, and finally the affinity chromatography is carried out, and the purification steps have a synergistic effect, so that impurities can be removed to a great extent, and finally the recombinant Sendai virus-like particles with extremely high purity are obtained; the purification method is simple to operate, high in recovery rate and beneficial to large-scale industrial production; the recombinant Sendai virus-like particles purified by the method can directly infect cells, generate corresponding antigens and induce an immune reaction of a body; and corresponding adjuvants can also be added to prepare vaccines.

Description

technical field [0001] The invention belongs to the technical field of virus biology, and in particular relates to a method for purifying recombinant Sendai virus-like particles and its application. Background technique [0002] Sendai virus (SeV), also known as Hemagglutinating Virus of Japan (HVJ), belongs to Paramyxoviridae (Paramyxoviridae), Paramyxovirinae (Paramyxovirinae) Respirovirus (Respirovirus), first appeared in 1953 Sendai, Japan found that after the isolation of the first strain of the virus, the Fushimi strain, the Sendai virus was extensively studied and reported. The Sendai virus gene consists of 15384 nucleotides, the 3-end is the start sequence, and then 6 structural genes -NP-P-M-F-HN-L- are arranged in series, and the 5-end is the termination sequence. For each gene RNA polymerase Transcription starts from the start sequence (GS) at the 3 end, followed by the untranslated region (UTR), the open reading frame (ORF), and the termination sequence (GE) at ...

Claims

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Application Information

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IPC IPC(8): C07K14/115C07K1/22C07K1/18C07K1/16A61K39/21A61P31/18
CPCC07K14/005A61K39/12A61P31/18C12N2760/18823C12N2760/18851C12N2760/18843
Inventor 萧哲
Owner 雪莱(武汉)生物医药技术有限公司
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