RU2493249C1 - Recombinant strain of yeast hansenula polymorpha - producent of capsid protein of hepatitis e virus - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain of yeast Hansenula polymorpha is proposed - a producent of a recombinant capsid protein of hepatitis E virus E (HEV) of genotype 3. The strain was produced by integration of a DNA sequence into a yeast cell genome, and this sequence codes the fragment of the amino acid sequence from 86 to 607 position of the capsid protein of the hepatitis E virus of genotype 3 (rtHEV-ORF2) under control of the MOX gene promotor. The strain provides for high yield of the recombinant protein rtHEV-ORF2 with simplified methods for production of high-density cultures.

EFFECT: invention may be used for microbiological production of a recombinant protein rtHEV-ORF2, having immunogenic properties of a natural protein.

5 ex

Description

The invention relates to the field of biotechnology, namely genetic engineering, and can be used to create a microbiological yeast producer of the hepatitis E virus capsid protein (HEV).

Hepatitis E is a viral infection belonging to the conditional group of fecal-oral hepatitis, characterized by liver damage, acute cyclic course, often with high fever, arthralgia, as well as severe manifestation in pregnant women. The reservoir and source of infection is a person, patient, or carrier. The viral etiology of hepatitis E was demonstrated on volunteers by infection with fecal isolates. The virus is found in the feces of patients. The transmission mechanism of hepatitis E virus, also called non-A, non-B (NANB), is fecal-oral, the transmission route is aqueous, however, there have been cases of transmission of the virus from person to person. Hepatitis E virus is endemic in an area with extremely poor water supply to the population, and poor water quality in an epidemic sense. Hepatitis E is common in Asia, Africa, Europe, Mexico, India in both epidemic and sporadic forms. It is generally accepted that about 1 million people are sick with hepatitis E each year, and in Asian countries it accounts for more than half of cases of acute hepatitis. The epidemiological feature of this disease is associated with high mortality in pregnant women, which, according to numerous studies, can reach 10-20%. In recent years, there has been a steady increase in the number of individuals with antibodies to HEV in temperate regions and economically developed countries. In these cases, the infection was often caused by the common genotype 3 virus in Europe and the USA, which was also found in wild and domestic pigs, which makes it possible to consider the zoonotic reservoir as a source of infection for most people. A few studies conducted in the Russian Federation also demonstrated the presence of antibodies to the hepatitis E virus in people who do not travel to endemic areas, which indicates the contact of the population with the virus.

Hepatitis E virus belongs to the Hepeviridae family of the genus Hepevirus. Virus particles are spherical formations with a diameter of 27-34 to them, built from identical structural elements, and deprived of the outer shell. Viral particles react with serum antibodies from infected individuals from geographically different regions, suggesting the widespread prevalence of HEV. Serologically and molecularly, genetic HEV is different from other types of hepatitis viruses. The sequence of the HEV genome was first determined in 1991 (Tam A.W. et.al., 1991 Virology, 185, 120-131). The genome of hepatitis E virus is represented by single-stranded RNA of positive polarity. Analysis of the genome sequence showed that its size is 7.2 kb, and it contains 3 open reading frames (ORFs), each of which encodes the synthesis of a specific protein or group of proteins. ORF1 encodes the non-structural proteins of the virus responsible for virus replication, ORF2 encodes the structural capsid protein of the virus (ORF2 protein), ORF3 encodes an auxiliary protein, the function of which has not yet been determined. Using mapping, it was found that linear antigenic epitopes are localized in the polypeptides encoded by ORF2 and ORF3 (Khydyakov Y.E. et al., Virology 1993, 194, 89-96). The recombinant protein ORF2 is capable of self-assembly into virus-like particles (VLP), similar in their antigenic properties to the capsid protein of an infectious virus.

Phylogenetic analysis, based on the full length of the genome sequence of some HEV isolates, indicates the existence of 4 different genotypes, each of which dominates in a particular geographical area. Genotype 1 includes strains from Asia and Africa, genotype 2 - a Mexican strain and several variants from Africa, genotypes 3 and 4 include human and pig HEV strains from industrial countries and Asia. Genotypes 1 and 2 were found only in humans, while genotypes 3 and 4 in humans, as well as pigs and other animals. Genotype 3 is ubiquitous; genotype 4 is more common in China and Japan. The discovery of viruses similar to HEV in pigs (pork HEV) and chickens (avian HEV) suggests that the virus belongs to zoonosis. Despite the existence of different genotypes, one HEV serotype has been identified. All virus subtypes have major cross-reactive epitopes that allow the development of a capsid protein-based recombinant vaccine that is effective against many strains of HEV.

Currently, the development of recombinant vaccines, along with monitoring and identification of HEV carriers, is a promising area in the fight against infection caused by HEV. The difficulties hindering the development of effective vaccines against hepatitis E virus are primarily associated with the development of optimal expression systems for the production of recombinant proteins with immunogenic properties of natural proteins.

Examples of use for the production of recombinant HEV proteins of the bacterial expression system based on E. coli are presented in the scientific and patent literature. Thus, it is known to obtain a recombinant chimeric protein trpE-C2 containing 439 C-terminal amino acids of the HEV genoform ORF2 protein of genotype 1 fused to the N-terminal region of tryptophan synthetase in a bacterial expression system in E. coli (Kamili S., Virus Research 2011, 161 (1): 93-100). The protein expressed in E. coli has high immunoreactivity and contains cross-reactive epitopes recognized by antibodies to HEV. As a result of vaccination of monkeys with trpE-C2 protein adsorbed on aluminum hydroxide, the induction of a humoral immune response was detected.

The preparation of an immunoreactive pE2 polypeptide originating from the carboxyterminal region of a protein encoded by the ORF2 genome of the Chinese strain D11092 is described. This polypeptide, due to the presence of a conformational antigenic determinant, is capable of forming active homodimers from monomers (US 7,204,989, 04.17.2007). The pE2 polypeptide was expressed in E. coli cells using the pGEX vector. When cloning in this vector, the sequence encoding pE2 was linked in frame with the glutathione S-transferase (GST) gene. The result was a hybrid gene encoding a polypeptide with GST at the N-terminal end and pE2 at the C-terminal end. Such a composite protein obtained by expression of a hybrid gene in E. coli cells was soluble and could be isolated from the cell lysate by binding to glutathione agarose. The pE2 polypeptide was separated from GST using a specific protease. The pE2 polypeptide expressed in bacterial systems is considered as a potential candidate for HEV vaccines. The proposed vaccine for immunization against HEV contains an immunologically effective amount of pE2, a pharmacologically acceptable carrier and an adjuvant. The vaccine can be administered orally or parenterally.

Despite the fact that the bacterial expression system is used to obtain recombinant proteins quite widely, there are drawbacks to its use associated with the solubility of proteins produced in E. coli, as well as with the difficulties encountered during serological immunoassays. In addition, recombinant fragments of the HEV capsid protein expressed in bacterial cells have a significantly shortened amino acid sequence, which can negatively affect their antigenic properties. In this regard, it is more preferable to use a eukaryotic expression system to obtain recombinant HEV proteins, in particular baculovirus or yeast.

It is known to use a 549 amino acid residue of the ORF2 protein of the Mexican and Burmese strains as the HEV antigen. This fragment was designated as "antigen 62K" (US 6,291,641, 09/18/2001). The antigen was produced in a baculovirus expression system. For this, a recombinant baculovirus was obtained, which transfected insect cells with SF9. Transfected cells were cultured either in suspension or in a monolayer. Recombinant antigen 62K was obtained mainly in soluble form. The isolated antigen showed immunoreactivity with the serum of individuals infected with HEV, and was able to form VLP according to electron microscopic data.

US Pat. No. 6,458,562 (10/01/2002), considered as the closest analogue, describes the preparation of various truncated variants of ORF2 protein from the Pakistan HEV line (SAR-55) in the eukaryotic expression system. The obtained proteins at the N-terminus begin with the 112th amino acid, and at the C-terminus end at the amino acids 578-607 or 660 of the wild-type ORF2 protein (as in US Pat. No. 6,787,145, September 7, 2004) and are capable of forming VLP. Viral RNA was isolated from a biological sample collected from primates infected with SAR-55. To obtain the ORF2 protein, a baculovirus vector was created by cloning the nucleotide sequence encoding the ORF2 protein into pBlueBac and transfected with it SF9 cells. After culturing the transfected cells under suitable conditions, the recombinant ORF2 protein was isolated and purified. Using the ELISA method, it was shown that the ORF2 protein expressed in the baculovirus system binds to antibodies produced in response to infection with various strains of HEV and does not bind to antibodies produced in response to hepatitis A, B, C, D. viruses. ORF2 protein used as an antigen in immunoassays, as well as an active antigen in a vaccine. The effective dose of the recombinant ORF2 protein is 10-50 μg. The hepatitis E vaccine also contains a pharmaceutically acceptable carrier, adjuvant, and may contain a stabilizer (polyethylene glycol, sucrose, etc.).

Currently, two candidate HEV vaccines have reached clinical trials. One of them, designated rHEV (GlaxoSmithKline Biological,) contains, as an active principle, a recombinant HEV antigen with a molecular mass of 56 kDa obtained in a baculovirus expression system (Kamili S., Virus Research 2011, 161 (1): 93-100). The antigen used in the HEV vaccine has an amino acid sequence corresponding to amino acid residues 112-607 of the wild-type ORF2 protein. The vaccine study involved 2,000 anti-HEV-negative volunteers from the Nepal Army who were divided into an experimental (20 μg rHEV) and control group (20 μg placebo). Vaccination was carried out according to the scheme of 0, 1 and 6 months. Those receiving all 3 doses of the vaccine had a clinically pronounced acute hepatitis score compared with those receiving placebo. Despite the demonstrated effectiveness of the vaccine, many issues related to the use of the vaccine remain unclear. Thus, the safety and efficacy of using the vaccine in women, the elderly, pregnant women and patients with chronic liver diseases have not been studied, and the efficacy of this vaccine containing the antigen of genotype 1 origin against infections caused by HEV strains of other genotypes has not been investigated.

Another candidate vaccine (HEV239, China) contains a more shortened HEV antigen (amino acid residues 376-606 of the ORF2 protein) of genotype 1 obtained by expression in E. coli (Kamili S., Virus Research 2011, 161 (1): 93-100). The vaccine provided protection in primates against hepatitis E caused by genotypes 1 and 4. Clinical studies of a bacterial recombinant vaccine conducted in South China, an area endemic for genotype 1 and 4, showed the safety, immunogenicity and effectiveness of the vaccine in the general population. In addition, it was found that a vaccine based on genotype 1 provides cross-immunity against genotype 4 in humans. However, clinical trials cannot be considered complete, since many issues remained unclear. So, the duration of the protective effect of the vaccine, the safety of vaccination in pregnant women and people with chronic liver diseases have not been studied.

Significant disadvantages of these vaccines include the fact that the vaccines protect against a clinically pronounced form of hepatitis, but not against HEV infection. Such vaccines cannot have a significant effect on the epidemiology of HEV infection in endemic areas.

Thus, the development of productive recombinant strains expressing active HEV antigens with immunogenic properties of natural proteins and the development of effective vaccines against HEV based on them remain relevant.

The objective of the invention is the creation of an effective microbiological yeast producer of the immunogenic fragment of the capsid protein HEV genotype 3. Efficiency is the possibility of obtaining the target product with the necessary biological properties and low cost.

The problem is solved in that, according to the invention, to obtain a recombinant producer of a shortened capsid protein rtHEV-ORF2 (recombinant truncated HEV-ORF2) expression plasmid containing a DNA fragment with a recombinant gene encoding rtHEV-ORF2, under the control of the promoter of the MOX gene gene promoter ( oxidase) Hansenula polymorpha and a selective marker (LEU2 gene of Saccharomyces cerevisiae) are integrated into the genome of the recipient strain of H. polymorpha DL1-L. As a result of the selection of clones capable of synthesizing the rtHEV-ORF2 protein, the transformant with the highest productivity was identified, designated KBT-11 / pHEV-001. The artificial gene is integrated into the genome, i.e. is part of one of the chromosomes of H. polymorpha. This ensures high mitotic stability of the strain, making it possible to optimize the conditions for its cultivation without taking into account the risk of accumulation of cells that have lost the ability to synthesize a foreign protein

The resulting recombinant H. polymorpha strain allows one to achieve higher levels of synthesis of the rtHEV-ORF2 recombinant protein with simpler methods for producing high density cultures.

The strain was deposited under the number KBT-11 / pHEV-001, 12.24.2011 in the collection of microorganisms of NPK Kombiotekh CJSC. The strain is new and is neither described in the patent nor in the scientific and technical literature.

The invention can be illustrated by the following examples.

In the given examples, all genetic engineering operations were performed according to standard procedures and instructions of companies producing enzymes and sets for manipulating DNA in vitro. The transformation of Escherichia coli and Hansenula polymorpha cells was carried out according to the previously described methods (Inoue H. et.al., Gene, 1990, 96: 23-28. And Bogdanova AI et.al., Yeast. 1995, 11 (4): 343- 53, respectively). The synthesis of DNA fragments, as well as the determination of the nucleotide sequence, were carried out by ZAO Evrogen, Moscow. A synthetic MOX-rtHEV-ORF2 double-stranded DNA fragment was used, containing the M.polymorpha MOX promoter and an open reading frame encoding the rtHEV-ORF2 polypeptide corresponding to the 523 amino acid fragment from position 86 to 607 of the amino acid sequence of the HEV genotype 3 capsid protein (SEQ ID NO :one).

Example 1. Obtaining the expression vector pHEV1 containing the MOX gene promoter and the sequence encoding the rtHEV-ORF2 polypeptide.

To obtain the pHEV1 expression vector, the pAM270 plasmid was used, which contains the gene encoding β-lactamase, and bacterial origin of replication to maintain the plasmid in E. coli cells, as well as S. cerevisiae LEU2 gene as a yeast selective marker and a sequence providing autonomous replication of the plasmid in Hansenula polymorpha cells. The MOX-rtHEV-ORF2 synthetic fragment (SEQ ID NO: 2) and plasmid pAM270 were hydrolyzed with restriction enzymes BsrGI and BglII. Hydrolysis DNA fragments were purified by agarose gel electrophoresis. A reaction mixture was prepared to conduct a ligase reaction with a total volume of 10 μl containing 20 ng of isolated fragments, a single ligase buffer and 1 unit of T4 ligase DNA. The mixture was incubated at room temperature for 2 hours, after which it was used to transform E. coli strain DH5alpha. Transformants were selected on LB medium (1% Tryptone, 0.5% yeast extract, 1% NaCl) containing ampicillin (100 mg / L). Plasmid DNA preparations were obtained from several transformants. A plasmid containing the insert of the MOX-rtHEV-ORF2 fragment was selected based on restriction analysis. To confirm the absence of unwanted mutations in the cloned insert, its nucleotide sequence was determined. The result was a plasmid pHEV1 containing the gene encoding rtHEV-ORF2, under the control of the MOX promoter and the S.cerevisiae LEU2 gene as a selective marker.

Example 2. Obtaining a producer strain of H. polymorpha expressing rtHEV-ORF2.

The strain H. polymorpha DL1-L, which is an auxotroph for leucine, was grown for 15-18 hours in YPD liquid medium (2% peptone, 1% yeast extract, 2% glucose) at 37 ° C. The resulting culture was diluted 100 times with fresh YPD medium and incubated for 4 hours under the same conditions. Cells from 200 μl of the resulting culture were pelleted by centrifugation, washed with sterile distilled water and suspended in 30 μl of T buffer (10 mM Tris-HCl, pH 7.4; 100 mM CH ₃ COOLi; 0.5 mM EDTA). To the suspension were added 1 μg of plasmid pHEV1, 5 μg of carrier DNA (fragmented and denatured herring DNA) and 60 μl of a 70% polyethylene glycol 4000 solution. All components were mixed and incubated for 30 min. at 30 ° C and 18 min at 45 ° C. The mixture was diluted in 1 ml of YPD medium and incubated at 37 ° C for 1 hour. Cells were plated on Petri dishes with YNB-D medium (0.67% Yeast Nitrogen Base [Difco], 2% glucose and 2% agar) and incubated for several days until transformant colonies appeared. The cells of the grown transformants were scattered with a draining stroke on fresh YNB-D plates to obtain individual colonies. Among the grown subclones, the fastest growing subclones were selected and were again redistributed with an exhausting stroke into YNB-D plates. The procedure was repeated several times until the colonies growing during sieving became homogeneous in size.

The subclones of transformants thus obtained were tested for their ability to express rtHEV-ORF2 protein. One of the transformants with the highest productivity was designated KBT-11 / pHEV-001.

Example 3. Cultivation of the recombinant strain KBT-11 / pHEV-001.

The fermentation of the strain producing yeast H. polymorpha was carried out in three stages at a temperature of 30 ° C. At the first stage, in the regime of periodic fermentation, biomass was increased in a culture medium containing 4% yeast extract, 2% bacto-peptone and 4% glycerol. After consumption of all glycerin by yeast (determined by a sharp increase in the concentration of dissolved oxygen), they switched to periodic fermentation with fed glycerol (fed-batch). When the raw biomass reached a density of 120 g / l (after about 28-32 hours of fermentation), they went to the induction stage by adding the inductor in portions of 0.5 volume% of the volume of the culture medium every 2 hours. The induction stage lasted from 2 to 5 days, depending on the level of antigen accumulation in yeast cells, determined by immunoblotting.

Example 4. Isolation and purification of the recombinant protein rtHEV-ORF2.

After fermentation, cells from the culture fluid were besieged by centrifugation at 4000 g for 15 minutes at + 6 ° C. The precipitated biomass was washed by resuspension and re-centrifugation in extraction buffer (PBS, pH 7.2, with 1.7 mM EDTA, 2 mM PMSF, 0.5% [v / v] Tween-20). After washing, the biomass was resuspended in an extraction buffer at a concentration of 380 g of wet cells per liter of suspension and cells were destroyed in a Dyno-Mill KDL mill using glass balls with a diameter of 0.5-0.7 mm. Undamaged cells and debris were besieged by centrifugation at 12,000 g, 30 minutes at + 6 ° C.

Polyethylene glycol (PEG-8000) up to 6% [w / v] and NaCl up to 400 mM were added to the clarified homogenate obtained and incubated for 18 hours with constant stirring at + 4 ° C. The precipitated proteins, including rtHEV-ORF2, were separated by centrifugation at 12000 g for 30 minutes. The precipitate was dissolved in 5 volumes of PBS buffer and the pH was adjusted to 8.0 with sodium hydroxide solution. Insoluble proteins and aggregates precipitated for 30 minutes at 12,000 g. Using tangential ultrafiltration on a 300 KDa membrane, low molecular weight proteins were removed from the supernatant and simultaneously transferred to ion exchange chromatography buffer (50 mM Tris-HCl, pH 8.0, 10 mM NaCl), followed by application to a chromatographic column with a strong anion-exchange coarse porous sorbent MacroCap-Q (GE, USA). Unbound proteins were washed with ion exchange chromatography buffer containing 50 mM NaCl. The rtHEV-ORF2 protein was eluted with a linear gradient of 50 to 500 mM NaCl in 10 column volumes at a linear speed of 100 cm / h.

The pooled fractions containing the rtHEV-ORF2 target protein (by immunoblot and SDS-PAGE) were concentrated by ultrafiltration on a 300 KDa membrane and separated by zonal ultracentrifugation (Beckman Coulter, USA) in a sucrose gradient from 10 to 50% w / w in PBS buffer (pH 7.2). Fractions containing the target protein were purified from aggregates and transferred to a buffer of 10 mM NaH ₂ PO ₄ , pH 7.2, 0.1% Tween-20 by gel filtration on a Toyopearl HW-65F sorbent (ToyoSoda Corp., Japan). The purity of the thus obtained recombinant protein was determined by electrophoresis. The yield of the target protein was at least 50 mg of rtHEV-ORF2 protein per kilogram of crude biomass.

Example 5. Analysis of the recombinant rtHEV-ORF2 antigen.

The purity of the recombinant rtHEV-ORF2 was determined using denaturing polyacrylamide gel electrophoresis (SDS-PAGE) by staining with Coomassie Brilliant Blue R-250. The purity of the isolated recombinant antigen was at least 95%.

The immunospecificity of recombinant rtHEV-ORF2, as well as the level of expression during fermentation, was determined by immunoblotting. Primary monoclonal antibodies to the HEV capsid protein of genotype 3 (ab1011241, AbCam, UK) were stained with an immunoblot followed by visualization with specific antibodies to mouse immunoglobulins conjugated to horseradish peroxidase according to the improved ECL chemiluminescence technique (Amersham, UK). The recombinant rtHEV-ORF2 antigen on the immunoblot is presented as a 56 kDa protein band.

The degree of aggregation and the approximate size of virus-like particles were determined by size exclusion chromatography on a TSK G5000PW analytical column (ToyoSoda Corp., Japan) in 10 mM NaH ₂ PO ₄ buffer, pH 7.2, 0.03% Tween-20. The first sharp peak elutes at ~ 0.4 column volumes and is a protein aggregate. The subsequent gentle peak, eluting at ~ 0.55 column volumes, consists of virus-like particles of rtHEV-ORF2. Peaks corresponding to low molecular weight proteins, including recombinant antigen monomers, elute in the range of 0.7–0.9 column volumes.

Cultural and morphological features of the strain: round-shaped cells, small in size, form large round colonies with a pronounced convex middle on an agarized YPD medium.

Storage - at -70 ° C in the form of a suspension of cells in a sterile 15 or 50% glycerol solution.

Genetic features: the strain is not zoopathogenic or phytopathogenic.

Method, conditions and composition of media for the propagation of the strain: incubation by pumping at 30 ° C in a nutrient medium composition: 2% peptone, 1% yeast extract, 2% glucose.

The method for determining the activity: in the clarified homogenized cells by immunoblotting or enzyme immunoassay. The strain activity is not less than 100 mg / l of culture fluid.

The isolated rtHEV-ORF2 protein can be used in test systems, as well as to create vaccines based on it for the prevention of diseases associated with the hepatitis E virus in humans and some animals (pigs, deer).

Claims (1)

Recombinant yeast strain Hansenula polymorpha KBT-11 / pHEV-001 containing a DNA sequence integrated into the genome, including a recombinant gene encoding the rtHEV-ORF2 polypeptide with the amino acid sequence of SEQ ID NO: 1, under the control of the promoter of the Hansenula polymorpha MOX gene and LEomyces cerechisac as a selective marker, the producer of the recombinant hepatitis E virus genotype 3 capsid protein rtHEV-ORF2.

Images