CN108070036B - Purification method of Yersinia pestis F1Vmut fusion protein - Google Patents

Abstract

The invention discloses a purification method of Yersinia pestis F1Vmut fusion protein, which comprises four steps of thallus cracking, anion exchange chromatography, hydrophobic interaction chromatography and ultrafiltration, wherein the purification process only comprises 2 steps of column chromatography and staged elution, more than 80mg of target antigen can be obtained from each liter of culture, the purity is more than 95%, and the antigen is not easy to aggregate and is beneficial to product quality control; the method has high yield of target protein and good linear repetition, is beneficial to industrial amplification, and finally obtains the recombinant antigen with good immunogenicity and protectiveness. The method can realize the large-scale preparation of the recombinant F1Vmut, and lays a good foundation for the industrialization of the recombinant plague vaccine.

Description

Purification method of Yersinia pestis F1Vmut fusion protein

Technical Field

The present invention relates to a protein purification method, and more particularly, to a purification method of a fusion protein.

Background

Plague, also known as black death disease, is a virulent infectious disease caused by Yersinia pestis (Yersinia pestis, hereinafter abbreviated as plague), the main reservoir and source of infection being rodents. There are three main types of plague infecting bacteria: bubonic plague, pulmonary plague and septicemia plague. Wherein the disease course of the pulmonary plague is acute, the lethality is high, and bacteria-carrying droplets generated by cough of the pulmonary plague patients can be transmitted human-to-human through a respiratory tract route, so that more serious interpersonal plague is caused.

After the 90 s of the twentieth century, the trend that plague epidemic becomes active gradually is more obvious, and plague is determined by WHO to be one of 20 epidemic diseases which are newly epidemic. Plague treatment is currently dominated by antibiotic treatment, although early administration of sufficient antibiotic has a good therapeutic effect, side effects are large, and drug-resistant plague strains have been found. Therefore, the prevention of the plague becomes a key, and the research of the plague vaccine has important significance.

The traditional plague vaccines are inactivated vaccines, such as the plague USP killed vaccine used in the United states, and live attenuated vaccines, such as the EV76 live attenuated vaccine in use in China and other countries. However, the vaccine has a series of problems of large side effect, more times of immunity, short protection time, poor protection effect on the pneumonic plague spread by aerosol and the like. The recombinant protein vaccine overcomes many defects of the traditional vaccine, does not contain infectious components, has no pathogenicity, and has the advantage of no replication in vivo, so the recombinant protein vaccine becomes a hotspot of the current plague vaccine research. Two recombinant plague vaccines currently in clinical research are based primarily on plague F1 capsular antigen and V antigen, one containing both F1 and V antigens and the other containing F1-V fusion protein, which has completed phase ii clinical studies and is qualified by the FDA awarded orphan drug in the united states.

At present, the expression of recombinant F1-V fusion protein in domestic and foreign documents is mainly divided into the following two cases: one is expressed in the form of inclusion body without label in colibacillus, and the later purification needs renaturation; one is soluble expression using plant cells, but the expression level is very low. The above method may require complicated operation means for inclusion body renaturation; or, a long production time is required for cell culture, and the expression level of the target protein is low, which is not favorable for stable amplification of the process, and this causes a series of problems in the production and quality control of the vaccine, and therefore, the method is not suitable for the antigen preparation process for vaccine production.

Patent CN101220086B and patent CN101531710B disclose the extraction or preparation of natural or recombinant F1 antigen, but only a single antigen cannot effectively protect against the occurrence of plague, especially pulmonary plague; the patent CN1155707C discloses a plague vaccine in a recombinant F1+ V form, but the clinical test prospect of the vaccine is great, and because two antigens need to be prepared respectively, the investment in the process flow and the product quality control is multiplied, and the problem of easy aggregation caused by the natural property of F1 cannot be solved; patents CN101094685A and CN104470537B disclose salmonella typhi vaccines using flagellin as a carrier or containing F1-V fusion protein, respectively, which belong to different types of vaccines with the vaccine using aluminum adjuvant of the present invention, and the target antigen is in the form of unmodified fusion protein.

At present, the biggest problem for recombinant plague vaccines is that the natural function of F1 is that it aggregates into oligomeric proteins on the outer membrane of plague bacteria to form a glue-like granular layer, a water-soluble capsular substance, and this natural property causes it to be easy to form non-uniform aggregates. Although both the intact form of the F1 polymer and the F1 depolymerized monomer are immunogenic, this can present problems for quality control in vaccine production; moreover, the fusion protein rF1-V constructed by F1 and V can not avoid the random aggregation of the target protein, so that the molecular weight of the purified rF1-V is distributed from 53kDa to 15000kDa, and particularly when the protein buffer solution is a neutral buffer system suitable for a human body, the aggregation of the protein can be accelerated, and even precipitation is easy to occur, which brings troubles for the application of the protein as a vaccine antigen.

Pan Tao et al disclose a F1mut-V fusion protein (PLOS Pathologens, 2013,9: 1-15), which is prepared by transferring amino acids 1-14 of the N-terminal of F1 protein to the C-terminal, and then fusing the protein with V protein, thereby solving the problem that the protein is easy to irregularly aggregate from the primary structure of the protein, but the protein has a His tag and is one of T4 phage display antigens, and a purification method thereof is not discussed, and a small amount of target protein carrying the tag is obtained only by affinity chromatography. Although the prior art solves the problem of irregular aggregation of the F1 protein from the primary structure, there are still many technical problems that limit the scale-up preparation of F1Vmut fusion proteins. Although escherichia coli has the advantages of clear genetic background, fast growth, economic culture, high expression level, more plasmids to be selected and hosts and the like, the escherichia coli becomes a preferred expression system in the technical field of genetic engineering, exogenous proteins are often expressed at high level and easily form inclusion bodies; or soluble expression is obtained in cells, but after bacteria are broken, a large amount of host protein, enzyme, nucleic acid and other impurities are released together when the host cells release the target protein, so that difficulty is increased for purification of the target protein, problems of low protein purification yield, partial degradation, overproof host DNA and protein residues and the like are caused, and challenges are brought to quality control of vaccine products, so that selection of an efficient and rapid purification process is particularly important. Moreover, the mutation of the F1 protein, the fusion with the V antigen and other reconstruction behaviors bring great changes to the overall physicochemical properties (isoelectric point, hydrophobicity and the like) of the fusion protein, so that the technical problem is brought to the purification of the fusion protein, the three dimensions of time efficiency, purity and yield are difficult to be considered, and the scale preparation process of the fusion protein suitable for being used as a vaccine component is difficult to form.

Therefore, the invention aims to provide a novel purification method, thereby fundamentally solving the problem of large-scale preparation of the F1Vmut fusion protein which can be used as a recombinant plague vaccine component.

Disclosure of Invention

Based on the above object, the present invention provides a method for purifying yersinia pestis F1Vmut fusion protein, comprising the steps of:

(1) cracking host bacteria thallus expressing the fusion protein, and collecting supernatant;

(2) filtering the supernatant obtained in the step (1) and then carrying out anion exchange chromatography;

(3) collecting the liquid obtained in the step (2), filtering and then carrying out hydrophobic interaction chromatography;

(4) and (4) collecting the liquid obtained in the step (3) for standby after ultrafiltration.

The fusion protein F1mutV described in step (1) is structurally designed to: the amino acids at 1-14 sites of the N end of the F1 protein are moved to the C end, and then the protein is fused with the V protein, thereby solving the problem that the protein is easy to generate irregular aggregation from the primary structure of the protein, finally forming a stable and uniform fusion protein monomer in a non-label form, and enabling the fusion protein monomer to be efficiently expressed in escherichia coli.

In a preferred embodiment, in the anion exchange chromatography in step (2), the filtered supernatant is equilibrated with an equilibration buffer, and then loaded onto an anion exchange chromatography column, and the fusion protein is eluted with an elution buffer.

More preferably, the equilibrium Buffer solution is Buffer A [20mmol/L Tris-HCl, 0.5mmol/L EDTA, 5% glycerol, pH 8.0], and the elution is carried out by firstly eluting the hybrid protein to a baseline with an elution Buffer solution Buffer B [20mmol/L Tris-HCl, 50mmol/L NaCl, pH 8.0] 5-10 CV, then eluting with an elution Buffer solution Buffer C [20mmol/L Tris-HCl, 50mmol/L NaCl, pH 8.0] 3-5 CV and collecting the eluent.

In another preferred embodiment, the anion exchange chromatography column is selected from any one of Q Sepharose XL, POROS 50HQ, Q Sepharose Fast Flow, Capto Q, DEAE Sepharose FF and Capto DEAE. More preferred is Q Sepharose Fast Flow.

In a preferred embodiment, in the hydrophobic interaction chromatography described in step (3), the mother solution is used to adjust the conductance and the conductance is consistent with the equilibrium Buffer D, after filtration, the equilibrium Buffer D [20mmol/L Tris, 1mol/L (NH) is used₄)₂SO₄，pH 8.0]After balancing, loading the hydrophobic interaction chromatographic column, and continuously leaching to a baseline by using Buffer D; first, 75% Buffer D + 25% Buffer E [20mmol/L Tris-HCl, pH 8.0] was eluted in stages]Eluting the hybrid protein by 5-10 CV, then eluting by 50% Buffer D + 50% Buffer E and 5-10 CV, and collecting the target protein.

Preferably, the hydrophobic interaction chromatography column is selected from any one of Phenyl Sepharose 6Fast Flow, Butyl High Performance, Phenyl High Performance or Butyl Sepharose 4Fast Flow, more preferably Butyl High Performance.

More preferably, the mother liquor is selected from Na₂SO₄、(NH₄)₂S0₄、Na₂HPO₄Or NaCl.

Particularly preferably, the mother liquor is (NH) with the concentration of 0.6-2 mol/L₄)₂SO₄。

In a preferred embodiment, in the ultrafiltration in step (4), Buffer F [20mmol/L PB, 0.15mol/L NaCl, pH 7.2] is used as a displacement Buffer, the obtained fusion protein is concentrated by using a tangential Flow ultrafiltration system, and the collected liquid is filtered by a Rapid Flow Flutter Unit (0.2 μm, Nalgene) for later use.

In another preferred embodiment, the filtration in steps (2) and (3) is performed using a 0.45 μm filter.

The purification method of the recombinant plague F1Vmut fusion protein provided by the invention is simple, efficient and rapid, the non-label recombinant antigen meeting the requirement of vaccine preparation can be obtained only by two-step-stage elution column chromatography and stage elution, the excellent purification effect can be obtained only in 48 hours, the high timeliness is realized, and the conventional Escherichia coli expression system usually adopts at least three-to-four-step chromatographic columns and a complex gradient elution mode. Through the purification process, the molecular weight of the specific protein band is correct (53.8kDa), the purity is more than or equal to 95 percent by SEC-HPLC verification, the loss of target protein is reduced, and foreign DNA residue, host protein residue, bacterial endotoxin inspection and sterile inspection all meet the related requirements of 2015 pharmacopoeia of the people's republic of China on vaccine stock solution. Finally, more than 80mg of target antigen can be obtained per liter of fermentation liquor for preparing the vaccine, and the yield is obviously higher than 2-40mg of antigen/fermentation liquor reported in the prior art. According to the calculation of the dosage (80 mu g) for 1 time of human use, more than 20,000 vaccine finished products can be prepared by 30L fermentation scale, the consistency of scale-up production is easy to realize, the requirement of large-scale industrial production is met, the recombinant plague vaccine stock solution can be used as a stock solution production process of the recombinant plague vaccine, the chromatography step is simple and convenient, and the production time and the input cost are obviously reduced.

The recombinant antigen obtained by the invention has high yield, good purity, stable protein in a monomer form, and good immunogenicity and protectiveness.

Drawings

FIG. 1 is a diagram showing the construction of plasmid pET-F1 Vmut;

FIG. 2 is a PCR identification chart of colony transformed by pET-F1Vmut to Top 10;

FIG. 3 is a SDS-PAGE electrophoretic analysis of the induced expression of the target protein;

FIG. 4 is a pilot fermentation curve;

FIG. 5 is an electrophoretic analysis of SDS-PAGE for purification of F1 Vmut;

FIG. 6 SDS-PAGE electrophoretic analysis of recombinant antigens;

FIG. 7 is a graph showing the results of antibody titers against F1 and against V in sera after immunization of mice;

FIG. 8 is a graph showing the survival of mice after immunization.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

Example 1 construction and expression of recombinant expression plasmid pET-F1Vmut

1. Sequence optimization of target genes

According to F1(AF542378.1) and V (AF074612.1) gene sequences published by GenBank, a base sequence corresponding to 1-14 amino acids at the N end of F1 protein is removed, an NdeI enzyme cutting site is introduced at the 5 'end of the nucleic acid sequence, a base sequence corresponding to 1-21 amino acids at the N end of an F1 molecule and a BamHI enzyme cutting site are introduced at the 3' end of the nucleic acid sequence, the 273 th base of Cys coded by V protein is mutated into a base coded by Ser, double enzyme cutting sites of BamHI and Not I are introduced at two ends, and the optimized nucleotide sequence is shown as SEQ ID NO.1 (the corresponding amino acid sequence is shown as SEQ ID NO. 2). And (4) carrying out gene synthesis according to the optimized nucleotide sequence.

2. Construction of expression vectors

The synthesized gene is respectively cut by Nde I, BamH I and BamH I, Not I, the fragments are recovered and then connected with pET-32a carrier for transformation (figure 1), the single colony is selected for colony PCR identification (figure 2, M: DL2000DNA molecular weight marker; 1-10: pET-F1Vmut transforms the colony PCR amplification product of Top 10). The positive clones were sent to Beijing Biotechnology engineering (Shanghai) GmbH for DNA sequencing analysis. And (3) converting the positive pET-F1Vmut plasmid with correct sequencing into the engineering bacteria. The engineering bacteria can be Escherichia coli, yeast, mammalian cells, insect cells or Bacillus subtilis, etc., preferably Escherichia coli. The Escherichia coli can be E.coli DH5 alpha and E.coli BL₂₁(DE₃)、E.coli BL₂₁(DE₃) pLysS or e.coli Top10, and the like. The present example uses Escherichia coli BL₂₁(DE₃) A competent cell.

Inducible expression of F1Vmut

Selecting single colony of engineered bacteria from the plate, inoculating in 5mL LB culture solution containing 100mg/mL Amp, culturing at 37 deg.C and 220rpm for 12 hr, transferring into LB culture solution containing Amp at a ratio of 1:100 the next day, culturing at 37 deg.C and 220rpm to OD in middle logarithmic growth phase_600nm0.6-1.0, preferably 0.8, adding IPTG with the final concentration of 20-100 mu mol/L, preferably 50 mu mol/L, and inducing temperature: the continuous culture time is 4 at 25 DEG C8 hours, preferably 5 hours. The expression induction was identified by 12% SDS-PAGE as shown in FIG. 3. In FIG. 3, 1, induction of supernatant non-reduction, 2, induction of supernatant reduction, 3, induction of precipitate non-reduction, 4, induction of precipitate reduction, 5, non-induction of non-reduction, and 6, non-induction of reduction. As can be seen from FIG. 3, the target protein is soluble and expressed, and the size is in accordance with the expectation (52kDa), and the target protein accounts for about 40% of the total protein of the broken strain supernatant by thin-layer scanning analysis, which indicates that the F1Vmut protein is highly soluble and expressed in Escherichia coli.

Example 2 Scale fermentation and purification of F1Vmut

1. Optimization of fermentation conditions

Firstly, optimizing key fermentation parameters in a 5L shake flask, carrying out heavy suspension on collected thalli in proportion, carrying out ultrasonic treatment, analyzing supernatant and precipitate by adopting SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and finally determining the fermentation conditions of a small test as follows: induction of OD_600nmThe value: 0.8, induction temperature: the IPTG concentration was 50. mu. mol/L and the induction time was 5 hours at 25 ℃.

2. Scale up of fermentation

On the basis of the above conditions, pilot-scale fermentation was investigated, and the finally determined pilot-scale fermentation process was as follows: (1) seed activation: one working seed is unfrozen, and is inoculated into a 50mL first-stage culture medium (Amp) prepared in advance according to the inoculum size of 1:50, and is cultured for 8-12 hours at 37 ℃ and 220 r/min. Taking out 20mL of the first-order seeds, inoculating the first-order seeds into 1L of culture medium according to the same inoculation amount, culturing for 14-15 hours in the same way, and then performing OD_600nmShould be 4-6. (2) Culturing in a fermentation tank: inoculating 1L of the secondary seed solution into 29L of sterilized LB culture medium at the rotation speed of 200-400 r/min, controlling the pH to be 7.0 by automatic feedback flow of an acid-base flow adding pump and adding 1mol/L NaOH or 30% phosphoric acid, controlling the dissolved oxygen value to be more than 50% by ventilation and stirring rotation speed, and performing amplification culture at 37 ℃ for 1.2-1.8 hours to obtain OD_600nmThe induction was started at a value of about 0.8, IPTG concentration of 50. mu. mol/L and induction time of 5 hours. Fermentation parameters of the whole fermentation process are monitored and recorded in real time through Sadouris MFCS type intelligent fermentation control software. The real-time fermentation monitoring curve is shown in FIG. 4. In fig. 4, a: ventilation (slpm), B: pH and C: dissolved oxygen%, D: stirring speed (rpm)And E: temperature (. degree. C.). The condition of the whole fermentation process is stable, and various parameters are not changed violently, so that the growth of bacteria is facilitated; and the majority of the target protein is in the bacteria-breaking supernatant, so that the high-efficiency soluble expression of the target protein is realized, and the development of a subsequent continuous process is facilitated.

3. Crushing of thallus

After the induction expression in the fermenter is finished, the thalli are collected centrifugally at 4 ℃ for 10min at 8000 g and weighed. And (2) resuspending the strain in 10-50 mmol/L Tris-HCl, 0.5-2 mmol/L EDTA, 0-15% glycerol, pH 7.5-8, preferably Buffer A [20mmol/L Tris-HCl, 0.5mmol/L EDTA, 5% glycerol, pH 8.0] according to the wet weight (g) of the strain and the volume (mL) of a Buffer solution of 1:10-20, preferably 1: 10. After high pressure homogenization (working pressure 900bar), the supernatant was collected by centrifugation at 12000 r/min for 30min at 4 ℃.

4. Anion exchange chromatography

Filtering the supernatant by a 0.45 mu m filter membrane, loading the supernatant on a Q Sepharose Fast Flow anion exchange column (XK 50/380 mL of CV in the volume of 30 columns), continuously washing the supernatant to a baseline by using Buffer A, then washing the hybrid protein by using Buffer B [20mmol/L Tris-HCl, 50mmol/L NaCl, pH 8.0] 5-10 CV to a baseline, and eluting by using Buffer C [20mmol/L Tris-HCl, 50mmol/L NaCl, pH 8.0] 3-5 CV and collecting the target protein.

5. Hydrophobic interaction chromatography

The collected liquid is used first (NH)₄)₂SO₄Mother liquor (20mmol/L Tris-HCl, 3mol/L (NH)₄)₂SO₄pH 8.0) and filtered through a 0.45 μm filter membrane for later use. A Butyl High Performance hydrophobic exchange column (XK 50/20CV 240mL) was used with equilibration Buffer D [20mmol/L Tris-HCl, 1mol/L (NH)₄)₂S0₄，pH 8.0]After equilibration, the sample was loaded and elution with Buffer D was continued to baseline. Eluting the hybrid protein by 25% of B5-10 CV in stages, wherein a B pump is Buffer E [20mmol/L Tris-HCl, pH 8.0]And eluting with 50% B5-10 CV and collecting the target protein.

When anion exchange chromatography such as Source 30Q filler is used, the method has the advantages of uniform filler particles, high resolution, high flow rate and the like, is a technical means suitable for fine purification, and is widely applied to the fine purification process in the prior art. However, there are many technical problems in purifying the F1V fusion protein of the present invention: firstly, the dynamic loading capacity is limited, which can greatly increase the production cost and prolong the production time; secondly, for a high-salt buffer system caused by coarse purification, it is difficult to ensure sufficient desalting effect, and an additional ultrafiltration liquid exchange step is required, thereby increasing the operation time and the loss of target protein. Based on the technical problems, the invention designs and uses hydrophobic interaction chromatography high-resolution fillers such as Phenyl HP, Octyl HP, Butyl HP and the like. The technical scheme is matched with the step of capturing coarse and pure, so that samples can be loaded by directly adjusting the conductance without desalting and ultrafiltration liquid change. Butyl HP is superior to the former two in terms of target protein loading and separation effect, and is a more preferable technical scheme. On the basis of the scheme, the buffer salt concentration, the loading amount, the elution mode and the like of Butyl HP are further optimized to determine relevant parameters of the fine purification stage.

6. Ultrafiltering to change liquid and sterilizing

Using a tangential flow ultrafiltration System (Sartorius PESU 0.1 m)²) Concentrating the obtained target protein by 3-5 times, and replacing Buffer solution with an ultrafiltration system to Buffer F [20mmol/L PB, 0.15mol/L NaCl, pH 7.2]. The collected liquid was filtered through a Rapid Flow Flutter Unit (0.2 μm, Nalgene) and dispensed for use. It can be seen from the protein electrophoretogram of each purification step (fig. 5, 1, homogenate centrifugal precipitation, 2, homogenate centrifugal supernatant, 3, after QFF column, 4, before Butyl HP column, 5, after Butyl HP column, 6, before ultrafiltration liquid change, 7, after ultrafiltration liquid change) that the target antigen is soluble expression, and the expression level is high, about 40% of total protein, the target antigen is concentrated and purified by two-step column chromatography of QFF and Butyl HP, and the loss of ultrafiltration liquid change to the target protein is small, the final process is stable, and can be linearly amplified.

Through the purification process described above, excellent purification effect was obtained only by 48 hours: the molecular weight of the specific protein band is correct (53.8kDa), the purity is more than or equal to 95 percent by SEC-HPLC verification, and foreign DNA residue, host protein residue, bacterial endotoxin inspection and sterility inspection all meet the related requirements of 2015 pharmacopoeia of the people's republic of China on vaccine stock solution. More than 80mg of the target antigen per liter of fermentation broth can be obtained for Vaccine preparation, which is significantly higher than the prior art reported 2-40mg antigen per fermentation broth (Heath D G, et al. [ J ]. Vaccine,1998, 16(11-12):1131.Powell B S et al. [ J ]. Biotechnology Progress,2005, 21(5):1490.Goodin J Let al. [ J ]. Protein Expression & Purification,2007, 53(1): 63-79.). The fermentation scale of 30L can prepare more than 20,000 vaccine finished products according to the calculation of the dose (80 mu g) for 1 time of human use, and the requirement of large-scale industrial production is met.

Example 3 immunogenicity analysis of F1Vmut

1. SDS-PAGE analysis of recombinant antigens

Before mice were immunized, the F1Vmut prepared according to the invention (left at 25 ℃ for 1 month) was subjected to SDS-PAGE analysis together with recombinant F1 antigen, V antigen and unmodified fusion protein F1-V antigen stored at-70 ℃. Through comparative analysis of reduction and non-reduction electrophoresis (FIG. 6, 1: non-reduction F1; 2: non-reduction V; 3: non-reduction F1-V; 4: non-reduction monomer F1 Vmut; 5: protein molecular weight marker; 6: reduction F1; 7: reduction V; 8: reduction F1-V; 9: reduction F1Vmut), compared with F1 and F1-V, a polymer with irregular molecular weight is easily formed, and V finally exists mostly in a dimer form, the F1Vmut constructed by the invention exists in a stable monomer form.

2. Immunogenicity assays

Recombinant F1 and V stored in the room and unmodified recombinant F1-V fusion protein are used as a reference, and the F1Vmut purified by the steps is respectively mixed with an aluminum hydroxide adjuvant in a high dose and a low dose according to a proportion to prepare the vaccine. Female BALB/c (6-8 weeks) mice were randomly divided into 4 groups of 10 mice each, inoculated with recombinant F1+ V (6. mu.g + 14. mu.g), F1-V (20. mu.g), F1Vmut (5. mu.g) and F1Vmut (20. mu.g), intramuscular injection of 0.1 mL/mouse in the hind leg, and booster immunization with the same dose 4 weeks later. Before the first immunization and after the last immunization, tail blood is collected, serum is separated, and specific antibodies in the serum are detected by ELISA. Each well of a 96-well plate is respectively coated with 100 mu L of recombinant F1 and V with the final concentration of 5 mu g/mL, immune serum is used as a primary antibody, goat anti-mouse-IgG marked by HRP is used as a secondary antibody, the antibody titer of the anti-F1 and the antibody titer of the anti-V are respectively measured, the absorbance is measured at 450nm, and 630nm is used as a reference wavelength. The positive judgment standard is that the ratio of the A value of the experimental hole to the A value of the negative control is more than or equal to 2.1.

The anti-F1 and anti-V antibody titers of each group of mice were measured by ELISA and presented as log 10. + -. S.D. of the reciprocal of the endpoint titer, and the comparison of antibody titers among groups was performed by multiple comparisons using GraphPad Prism 5.01 software (one-way ANOVA). As can be seen from FIG. 7, each of the F1+ V, F1-V and F1Vmut groups induced the production of F1 antibody and V antibody with higher antibody titers.

Example 4 analysis of protective Effect of F1Vmut

0.5 ml/mouse was used for subcutaneous challenge with the virulent pestis standard strain 141. The animals are raised in cages and observed for 23 days, the dead animals are autopsied during the period, and the viscera are taken for bacteriological examination, so that the growth of the plague bacillus is positive. After 23 days of observation, the surviving animals were sacrificed and the livers and spleens were taken for bacteriological examination. As shown in FIG. 8, the control group (aluminum adjuvant group) died completely within 5 days, and the experimental group showed a 90% protection rate except for F1+ V (6. mu.g + 14. mu.g), and all groups showed a 100% protection rate, and the mice were protected 100% by F1Vmut immunization of only 5. mu.g.

Sequence listing

<110> military medical research institute of military science institute of people's liberation force of China

<120> purification method of Yersinia pestis F1Vmut fusion protein

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1472

<212> DNA

<213> Yersinia pestis

<400> 1

catatggaac cagcccgcat cactcttaca tataaggaag gcgctccaat tacaattatg 60

gacaatggaa acatcgatac agaattactt gttggtacgc ttactcttgg cggctataaa 120

acaggaacca ctagcacatc tgttaacttt acagatgccg cgggtgatcc catgtactta 180

acatttactt ctcaggatgg aaataaccac caattcacta caaaagtgat tggcaaggat 240

tctagagatt ttgatatctc tcctaaggta aacggtgaga accttgtggg ggatgacgtc 300

gtcttggcta cgggcagcca ggatttcttt gttcgctcaa ttggttccaa aggcggtaaa 360

cttgcagcag gtaaatacac tgatgctgta accgtaaccg tatctaacca atccgcggca 420

gatttaactg caagcaccac tgcaacggca actcttgtcg aaccagcccg catcactctt 480

ggatccatta gagcctacga acaaaaccca caacatttta ttgaggatct agaaaaagtt 540

agggtggaac aacttactgg tcatggttct tcagttttag aagaattggt tcagttagtc 600

aaagataaaa atatagatat ttccattaaa tatgatccca gaaaagattc ggaggttttt 660

gccaatagag taattactga tgatatcgaa ttgctcaaga aaatcctagc ttattttcta 720

cccgaggatg ccattcttaa aggcggtcat tatgacaacc aactgcaaaa tggcatcaag 780

cgagtaaaag agttccttga atcatcgccg aatacacaat gggaattgcg ggcgttcatg 840

gcagtaatgc atttctcttt aaccgccgat cgtatcgatg atgatatttt gaaagtgatt 900

gttgattcaa tgaatcatca tggtgatgcc cgtagcaagt tgcgtgaaga attagctgag 960

cttaccgccg aattaaagat ttattcagtt attcaagccg aaattaataa gcatctgtct 1020

agtagtggca ccataaatat ccatgataaa tccattaatc tcatggataa aaatttatat 1080

ggttatacag atgaagagat ttttaaagcc agcgcagagt acaaaattct cgagaaaatg 1140

cctcaaacca ccattcaggt ggatgggagc gagaaaaaaa tagtctcgat aaaggacttt 1200

cttggaagtg agaataaaag aaccggggcg ttgggtaatc tgaaaaactc atactcttat 1260

aataaagata ataatgaatt atctcacttt gccaccacct cctcggataa gtccaggccg 1320

ctcaacgact tggttagcca aaaaacaact cagctgtctg atattacatc acgttttaat 1380

tcagctattg aagcactgaa ccgtttcatt cagaaatatg attcagtgat gcaacgtctg 1440

ctagatgaca cgtctggtaa ataagcggcc gc 1472

<210> 2

<211> 487

<212> PRT

<213> Yersinia pestis

<400> 2

His Met Glu Pro Ala Arg Ile Thr Leu Thr Tyr Lys Glu Gly Ala Pro

1 5 10 15

Ile Thr Ile Met Asp Asn Gly Asn Ile Asp Thr Glu Leu Leu Val Gly

20 25 30

Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val

35 40 45

Asn Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser

50 55 60

Gln Asp Gly Asn Asn His Gln Phe Thr Thr Lys Val Ile Gly Lys Asp

65 70 75 80

Ser Arg Asp Phe Asp Ile Ser Pro Lys Val Asn Gly Glu Asn Leu Val

85 90 95

Gly Asp Asp Val Val Leu Ala Thr Gly Ser Gln Asp Phe Phe Val Arg

100 105 110

Ser Ile Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp

115 120 125

Ala Val Thr Val Thr Val Ser Asn Gln Ser Ala Ala Asp Leu Thr Ala

130 135 140

Ser Thr Thr Ala Thr Ala Thr Leu Val Glu Pro Ala Arg Ile Thr Leu

145 150 155 160

Gly Ser Ile Arg Ala Tyr Glu Gln Asn Pro Gln His Phe Ile Glu Asp

165 170 175

Leu Glu Lys Val Arg Val Glu Gln Leu Thr Gly His Gly Ser Ser Val

180 185 190

Leu Glu Glu Leu Val Gln Leu Val Lys Asp Lys Asn Ile Asp Ile Ser

195 200 205

Ile Lys Tyr Asp Pro Arg Lys Asp Ser Glu Val Phe Ala Asn Arg Val

210 215 220

Ile Thr Asp Asp Ile Glu Leu Leu Lys Lys Ile Leu Ala Tyr Phe Leu

225 230 235 240

Pro Glu Asp Ala Ile Leu Lys Gly Gly His Tyr Asp Asn Gln Leu Gln

245 250 255

Asn Gly Ile Lys Arg Val Lys Glu Phe Leu Glu Ser Ser Pro Asn Thr

260 265 270

Gln Trp Glu Leu Arg Ala Phe Met Ala Val Met His Phe Ser Leu Thr

275 280 285

Ala Asp Arg Ile Asp Asp Asp Ile Leu Lys Val Ile Val Asp Ser Met

290 295 300

Asn His His Gly Asp Ala Arg Ser Lys Leu Arg Glu Glu Leu Ala Glu

305 310 315 320

Leu Thr Ala Glu Leu Lys Ile Tyr Ser Val Ile Gln Ala Glu Ile Asn

325 330 335

Lys His Leu Ser Ser Ser Gly Thr Ile Asn Ile His Asp Lys Ser Ile

340 345 350

Asn Leu Met Asp Lys Asn Leu Tyr Gly Tyr Thr Asp Glu Glu Ile Phe

355 360 365

Lys Ala Ser Ala Glu Tyr Lys Ile Leu Glu Lys Met Pro Gln Thr Thr

370 375 380

Ile Gln Val Asp Gly Ser Glu Lys Lys Ile Val Ser Ile Lys Asp Phe

385 390 395 400

Leu Gly Ser Glu Asn Lys Arg Thr Gly Ala Leu Gly Asn Leu Lys Asn

405 410 415

Ser Tyr Ser Tyr Asn Lys Asp Asn Asn Glu Leu Ser His Phe Ala Thr

420 425 430

Thr Ser Ser Asp Lys Ser Arg Pro Leu Asn Asp Leu Val Ser Gln Lys

435 440 445

Thr Thr Gln Leu Ser Asp Ile Thr Ser Arg Phe Asn Ser Ala Ile Glu

450 455 460

Ala Leu Asn Arg Phe Ile Gln Lys Tyr Asp Ser Val Met Gln Arg Leu

465 470 475 480

Leu Asp Asp Thr Ser Gly Lys

485

Claims (2)

1. A method for purifying yersinia pestis F1Vmut fusion protein having an amino acid sequence as shown in SEQ ID No.2, said method comprising the steps of:

(1) cracking host bacteria thallus expressing the fusion protein, and collecting supernatant;

(2) filtering the supernatant obtained in the step (1), balancing the supernatant with an equilibrium Buffer solution, loading the supernatant onto an anion exchange chromatography column Q Sepharose Fast Flow for anion exchange chromatography, and eluting the fusion protein with an elution Buffer solution Buffer A, wherein the elution is to use Buffer B to wash the hybrid protein to a baseline at 5-10 CV, then use the elution Buffer solution to elute and collect the eluent at 3-5 CV, the Buffer A is 20mmol/L Tris-HCl, 0.5mmol/L EDTA, 5% glycerol, the pH is 8.0, the Buffer B is 20mmol/L Tris-HCl, 50mmol/L NaCl, the pH is 8.0;

(3) collecting the liquid obtained in the step (2), filtering, adjusting the conductivity by using mother liquor, enabling the conductivity to be consistent with the conductivity of a balance Buffer solution Buffer D, balancing by using the balance Buffer solution Buffer D after filtering, loading a sample hydrophobic interaction chromatographic column Butyl High Performance, and continuously leaching to a baseline by using the Buffer D; eluting 75% Buffer D + 25% Buffer E and 5-10 CV hybrid protein by stages, eluting 50% Buffer D + 50% Buffer E and 5-10 CV hybrid protein, and collecting target protein, wherein the Buffer D is 20mmol/L Tris-HCl and 1mol/L (NH)₄)₂S0₄pH 8.0, wherein Buffer E is 20mmol/L Tris-HCl, pH 8.0, and the mother liquor is (NH) with the concentration of 0.6-2 mol/L₄)₂SO₄；

(4) And (3) collecting the liquid obtained in the step (3), taking Buffer F as a replacement Buffer solution, concentrating the obtained fusion protein by using a tangential Flow ultrafiltration system, and filtering the collected liquid by a Rapid Flow Flutter Unit for later use, wherein the Buffer F is 20mmol/L PB, 0.15mol/L NaCl and the pH is 7.2.

2. The method of claim 1, wherein the filtration in steps (2) and (3) is performed using a 0.45 μm filter.

Images