A kind of purification method of Yersinia pestis f1vmut fusion protein

Abstract

The invention discloses a yersinia pestis F1Vmut fusion protein purification method. The method includes four steps including bacterial lysis, anion exchange chromatography, hydrophobic interaction chromatography and ultrafiltration. According to a purification process, more than 80mg of antigens in purity higher than 95% can be obtained by consumption of each liter of culture only through two-step column chromatography and stepwise elution, and the antigens are less prone to aggregation and beneficial to product quality control. By adoption of the method, high target protein yield and linearrepeatability are realized, industrial amplification is benefited, and the recombinant antigens excellent in immunogenicity and protectiveness are finally obtained. In addition, by adoption of the method, recombinant F1Vmut large-scale preparation can be realized, and a great foundation is laid for industrialization of recombinant plague vaccines.

Description

technical field [0001] The present invention relates to a protein purification method, more specifically, the present invention relates to a fusion protein purification method. Background technique [0002] Plague, also known as the Black Death, is a severe infectious disease caused by Yersinia pestis (hereinafter referred to as Yersinia pestis). The main storage host and source of infection are rodents. There are three main types of Yersinia pestis: bubonic, pneumonic, and septicemic plagues. Among them, the course of pneumonic plague is acute and the fatality rate is high. The bacteria-carrying droplets produced by the cough of pneumonic plague patients can be transmitted from person to person through the respiratory route, leading to more severe human plague. [0003] After entering the 1990s, the plague epidemic gradually became more active, and plague was identified by the WHO as one of the 20 re-epidemic infectious diseases. At present, the treatment of plague is mai...

AI Technical Summary

Problems solved by technology

The above methods either require complex operation means for inclusion body renaturation; or require a long production time for cell culture, and the expression of the target protein is low, which is not conducive to the stable amplification of the process, which will bring problems to the production and quality control of the vaccine. A series of problems, so it is not suitable as an antigen preparation process for vaccine production

[0006] Patent CN101220086B and patent CN101531710B disclose the extraction or preparation of natural or recombinant F1 antigens, but only a single antigen cannot effectively prevent the occurrence of plague, especially pneumonic plague; patent CN1155707C discloses a plague vaccine in the form of recombinant F1+V, but the vaccine The prospect of clinical trials of F1 is worrisome, and due to the need to prepare two antigens separately, the investment in process flow and product quality control will increase exponentially, and the problem of easy aggregation caused by the natural properties of F1 cannot be solved; patents CN101094685A and CN104470537B respectively disclose The Salmonella typhi vaccine that uses flagellin as a carrier or contains F1-V fusion protein belongs to a different type of vaccine from the vaccine using aluminum adjuvant in the present invention, and its target antigen is in the form of unmodified fusion protein

[0007] Currently, the biggest problem with recombinant plague vaccines is that the natural function of F1 is that it aggregates into oligomeric proteins on the outer membrane of Yersinia pestis to form a colloidal granular layer and a water-soluble capsular substance. This natural property will make it easy to form heterogeneous aggregates

Although the intact form of F1 aggregates and F1 depolymerized monomers are immunogenic, this will cause problems for the quality control of vaccine production; and the fusion protein rF1-V constructed by F1 and V cannot avoid the target protein The random aggregation of rF1-V leads to the molecular weight distribution of the purified rF1-V from 53kDa to 15000kDa, especially when the protein buffer is a neutral buffer system suitable for the human body, it will accelerate the protein aggregation and even easily precipitate. Bringing trouble to the protein's use as a vaccine antigen

[0008] Pan Tao et al. disclosed a F1mut-V fusion protein (PLOS Pathogens, 2013, 9: 1-15), the fusion protein is to move the N-terminal 1-14 amino acids of the F1 protein to the C-terminus, and then combine it with V Protein fusion solves the problem that the protein is prone to irregular aggregation from the primary structure of the protein. However, the protein has a His tag and is one of the antigens presented by T4 phage. and chromatography to obtain a small amount of tagged target protein

Although the existing technology has solved the problem of irregular aggregation of F1 protein from the primary structure, there are still many technical problems that limit the large-scale preparation of F1Vmut fusion protein

Although Escherichia coli has the advantages of clear genetic background, fast growth, economical culture, high expression level, and many plasmids and hosts to be selected, it has become the preferred expression system in the field of genetic engineering technology. , easy to form inclusion bodies; or obtain soluble expression in the cell, but after breaking the bacteria, when the host cell releases the target protein, a large amount of impurities such as host protein, enzyme and nucleic acid are released together, which increases the purification of the target protein. Difficulties, resulting in problems such as low protein purification yield, partial degradation, host DNA and protein residues exceeding the standard, and bring challenges to the quality control of vaccine products, so it is particularly important to choose an efficient and fast purification process

Moreover, the overall physical and chemical properties (isoelectric point, hydrophobicity, etc.) of the fusion protein have been greatly changed due to the mutation of the F1 protein and the fusion with the V antigen, which has brought great changes to the purification of the fusion protein. Technical problems make it difficult to take into account the three dimensions of timeliness, purity, and yield, and it is difficult to form a large-scale preparation process for fusion proteins suitable as vaccine components

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