RU2546240C1 - RECOMBINANT STRAIN OF YEAST Hansenula polymorpha - PRODUCER OF MAJOR CAPSID PROTEIN L1 OF HUMAN PAPILLOMAVIRUS OF TYPE 56 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: yeast strain Hansenula polymorpha - producer of recombinant protein L1 of human papillomavirus (HPV) of type 56 is proposed. The strain was obtained by introducing into the genome of yeast cell of one copy of the DNA sequence encoding the capsid protein L1 HPV of type 56, under control of AIBN promoter and one copy of DNA sequence encoding the capsid protein L1 HPV of type 56, under control of the MOX promoter. The invention can be used for microbiological production of the recombinant protein L1 HPV of type 56.

EFFECT: strain provides high yield of recombinant protein L1 HPV having antigenic and immunogenic properties of natural antigen.

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Description

The invention relates to the field of biotechnology, namely genetic engineering, and can be used to create a microbiological yeast producer of capsid protein L1 of human papillomavirus type 56.

The human papilloma virus (HPV - Human Papilloma Virus), affecting epithelial cells, causes various diseases in humans. Depending on the type of infectious virus, which is classified into more than 70 types on the basis of DNA sequence homology, papillomavirus infection manifests itself in the form of benign warts, non-cancerous warts or papillomas on the mucous membranes of the genital organs or respiratory tract. Human papillomavirus type 16 and 18 causes epithelial dysplasia of the genital mucosa, it is these types of papilloma virus that are associated with most pre-invasive and invasive carcinomas of the anogenital sphere (cervical, vaginal, vulvar and anal cancers), of which cervical cancer is one of the most common and dangerous types of tumors in women. In a study conducted on the territory of the Russian Federation, it was shown that in women with a high degree of squamous intraepithelial lesion and cervical cancer, along with HPV type 16, HPV type 56 is found, and in the case of multi-infection, this type of virus prevails (VII All-Russian Scientific and Practical Conference with International participation “Molecular Diagnostics - 2010”, Sat. Proceedings, ed., Academician of the Russian Academy of Medical Sciences V.I. Pokrovsky, vol. 3, pp. 373-409). According to the WHO, annually about 500 thousand women worldwide become ill with cervical cancer, more than half of these cases are fatal. Clinical manifestations of cervical cancer are not detected for a long time, while early diagnosis of human papillomavirus infection is rather complicated. That is why, in the fight against the spread of such a serious disease, preference is given to the prevention of human papillomavirus infection, in particular, by inducing an immune response in humans with vaccination.

The papilloma virus belongs to the family of papoviruses (Papoviridae), has a diameter of 40-50 nm. The virus capsid is formed from 72 protein capsomeres consisting of the main protein L1 and minor protein L2. The genome of the virus is presented in the form of circular double-stranded DNA, which contains 8 early genes (E1-E7) and two late genes encoding the capsid proteins L1 and L2. The main L1 protein (55-60 kDa) is able to assemble itself into virus-like particles (VLP - virus-like particle), similar in structure to virions, but not containing viral DNA. Protein L1 has a high immunogenicity and induces the formation of antibodies that neutralize the infectious virus.

Currently, recombinant HPV LI proteins are promising immunogenic components for the development of prophylactic vaccines against dangerous types of papillomavirus. The difficulties hindering the development of effective human papillomavirus vaccines are primarily associated with the development of optimal expression systems for the production of recombinant HPV proteins with the immunogenic properties of natural HPV L1 proteins.

It is known to obtain HPV type 16 L1 and L2 proteins by culturing CV-1 mammalian cells infected with a recombinant vaccinia virus vector [Zhou J et al., Virology v.185, p. 251-257, 1991]. Known is the expression of papillomavirus protein L1, capable of spontaneously collecting into virus-like particles and having immunogenicity, in a eukaryotic system based on cultured insect cells (baculovirus system) [Kimbauer R. et al., Proc. Natl. Acad. Sci USA, v. 89, p. 12180-12184, 1992]. The scientific and patent literature presents examples of the use of the Saccharomyces cerevisiae yeast expression system for the production of recombinant HPV proteins, which are preferred due to the internationally recognized yeast safety, the ability to produce large amounts of protein in the native conformation. So, in patent RU 2206608, 2003, C12N 7/00 describes the creation of a yeast producer of HPV or L2 HPV protein L1 of various types, including HPV 16 and 18, for example, Saccharomyces cerevisiae. Cloning of HPV 16 L1 and L2 genes was performed using genomic DNA extracted from Caski cells (ATCC No. CRL 1550). An expression plasmid yeast vector containing the DNA sequence encoding the corresponding polypeptide was constructed under the control of the GAL10 promoter and transformed S. cerevisiae cells with it. The ability of recombinant S. cerevisiae cells to express HPV L1 or L2 proteins was analyzed by immunoblotting.

Petrov P. M. et al. [WO 2006/065166 A1, 2006] obtained capsid proteins L1 of viral serotypes 16, 18, 31 HPV, causing the most aggressive forms of diseases in humans, from recombinant yeast strains. These strains were obtained by transforming S. cerevisiae yeast cells with plasmids with genes encoding the L1 proteins of these serotypes. DNA fragments encoding these proteins were isolated from clinical material. Created yeast producers during cultivation were able to express L1 proteins in the form of virus-like particles. L1 proteins were used for the preparation of prophylactic and therapeutic vaccines.

The use of methylotrophic yeast Pichia pastoris is known to produce the main capsid protein HPV [Bazan S.B. et al., Arch. Virol. 2009, 154 (10), 1609-17]. A gene encoding HPV-L1 was introduced into the P. pastoris genome under the control of a methanol-regulated promoter. This gene had an optimized codon composition. Purification of the L1 protein was performed using heparin-sepharose chromatography followed by the assembly of virus-like particles. The formation of biologically active VLPs was confirmed by immunoelectron microscopy and hemagglutination.

Patent RU 2445357, 2012 discloses a recombinant producer strain of the HPV type 16 L1 capsid protein Pichia angusta (synonym for Hansenula polymorpha) BKM Y-2988D, obtained by integration into the genome of an expression plasmid containing a DNA fragment with a recombinant gene encoding HPV type 16 L1 encoding under the control of the promoter DAK (dihydroxyacetone kinase gene) H. polymorpha and a selective marker (LEU2 gene H. polymorpha). The described recombinant producer strain, having high productivity, however, does not provide an antigen having optimal immunogenicity for use in the vaccine. To increase the productivity of the producer strain, the recombinant protein expression cassette is introduced into the genome of the recipient strain in a large number of copies. However, in some copies of the expression cassette during the preparation and cultivation of the strain, mutations may occur that alter the properties of the target protein. In this case, control over the occurrence of mutations in the case of high copy number of the gene is almost impossible. Moreover, if a certain level of synthesis is exceeded, the folding process of the target protein may be disrupted, which leads to a decrease in its yield, a deterioration in biological activity, and complicates the purification process.

The patent literature provides information on the production of HPV type 56 L1 capsid protein by the recombinant route and its use in the vaccine composition for the prevention of human papillomavirus infection. Thus, US Pat. No. 6,165,471,26,12,2000 describes L1 capsomeres of various types of HPV, including type 56, which were obtained in the baculovirus expression system using Trichoplusia ni cells infected with recombinant baculovirus. It is known to use a baculovirus expression system to create a multivalent vaccine comprising HPV type L1 16, 18, 31, 45 and optionally type 56 (US 7,416,846, 08/26/2008).

The closest analogue is US patent 7,709,010, 05/04/2010, which describes the creation of a multivalent vaccine against human papillomavirus infection, including VLP L1 of various types of HPV, including type 56. VLP obtained by expression of the capsid protein L1 in recombinant S. cerevisiae yeast cells. The isolated and purified VLPs were adsorbed on an aluminum adjuvant and an ISCOM-type adjuvant. An advantage of the S. cerevisiae yeast expression system is its relatively low cost and easy adaptation to large-scale growth in fermenters.

The disadvantages of using an expression system based on Saccharomyces cerevisiae include the instability of recombinant cells associated with the relatively high frequency of loss of an autonomous plasmid during cell division. When the strain is cultured under nonselective conditions for the plasmid, this leads to the accumulation of cells that do not contain plasmids and are unable to synthesize the target protein. This significantly complicates the cultivation of the recombinant strain and increases the cost of the target product. To induce the expression of recombinant proteins, a rather expensive reagent, galactose, is used, which increases the cost of the target product.

The objective of the invention is the creation of a recombinant strain of N. polymorpha yeast, a stable effective microbiological yeast producer of the main capsid protein of human papilloma virus type 56, which has the necessary biological properties (proper conformation, immunogenicity, purity) and low cost. The resulting yeast producer should provide the ability to control the absence of mutations in the expressed foreign gene, have a controlled expression level of the recombinant gene, to ensure high efficiency of folding of the synthesized polypeptide with the formation of the natural conformation of the resulting product, and have a sufficiently high yield of the target protein, ensuring the economic feasibility of using the producer.

The problem is solved in that according to the invention, to obtain a recombinant producer strain of the main capsid protein L1 HPV56, two expression cassettes containing one copy of the desired gene are sequentially integrated into the genome of the recipient strain DLT2 H. polymorpha. The first cassette contains a DNA fragment with a recombinant gene encoding HPV56-L1, under the control of the promoter of the MOX gene (methanol oxidase) and a selective marker (H. polymorpha TRP3 gene). The second expression cassette contains a similar HPV56-L1 gene, but under the control of the DAK gene promoter (the gene encodes the dihydroxyacetone kinase) of H. polymorpha yeast and a selective marker (S. cerevisiae LEU2 gene). The transformation produces a recombinant strain containing one copy of the HPV56-L1 gene under the control of the DAK promoter and one copy of the HPV56-L1 gene under the control of the MOX promoter. Moreover, the artificial genes are integrated directly into the genome of the producer strain, i.e. are part of the chromosomes of the yeast 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. In addition, the presence of only one copy of the recombinant gene in each expression cassette with a unique promoter and terminal region makes it possible to control the absence of mutations of this gene during its introduction into the genome and during cultivation of the strain. The absence of mutations is controlled by analyzing the nucleotide sequence of the products of the polymerase chain reaction using primers unique to specific sequences in the coding region of each expression cassette.

An advantage of the proposed invention is the possibility of obtaining a stably high level of synthesis of the target protein without compromising the efficiency of its folding. The use of expression cassettes with two different promoters (MOX and DAK), the maximum activity of which is achieved under different cultivation conditions, provides a more uniform synthesis of the product during the cultivation of the strain. At the same time, the level of protein synthesis is not exceeded, which can lead to a possible violation of the laying efficiency and, as a result, a decrease in the strain productivity.

As a result of the selection of clones capable of synthesizing the HPV56-L1 protein, a transformant that best meets the required properties was isolated. The resulting recombinant strain allows one to achieve higher levels of recombinant protein synthesis with simpler methods for producing high density cultures, as well as to isolate HPV56-L1 protein as VLP with the correct conformation, possessing the necessary antigenic and immunogenic properties, with a sufficiently high yield. VLP formation was confirmed using gel filtration chromatography.

The strain was deposited 04/21/2011 in the collection of NPK Kombioteh CJSC under the number KBT-11 / rRU-562. 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 examples cited, all genetic engineering operations were performed according to standard procedures and instructions from manufacturers of enzymes and in vitro DNA manipulation kits. Transformation of Escherichia coli and Hansenula polymorpha cells was carried out according to the previously described methods (Inoue et al, 1990, Gene, 96: 23-28. And Bogdanova et al., 1995, Yeast 11: 343-353, respectively). The synthesis of DNA fragments, as well as the determination of the nucleotide sequence, were carried out by ZAO Evrogen, Moscow. Synthetic DNA fragments were used, the nucleotide sequences of which are shown in the List of sequences.

Example 1. Obtaining a plasmid vector p56HPV3 containing the recombinant gene DAK-HP V56-L1 and a selective marker gene LEU2 S. cerevisiae.

The DNA fragment DAK-HPV56 (SEQ ID NO: 1) and the preparation of the plasmid AMIpSLI (Agaphonov et al., 1999, Yeast 15: 541-551) containing the S. cerevisiae LEU2 gene as a yeast selective marker was digested with BsrGI and HindIII restriction enzymes . The resulting preparations of the recombinant gene DNA fragment and the AMIpSLI linearized plasmid were introduced into the reaction mixture for ligation at a concentration of 30 ng / μl. After incubation with T4 DNA ligase for 2 hours, 2 μl of the reaction mixture was used to transform E. coli strain DH5a. Plasmid DNA was isolated from several grown transformants. Based on restriction analysis, a plasmid containing the DAK-HPV56 fragment was selected. The determination of the nucleotide sequence of this plasmid confirmed that it contains a gene encoding HPV56-L1 under the control of the H. polymorpha DAK promoter. The resulting plasmid was designated p56HPV3.

Example 2. Obtaining a plasmid vector p56HPV2 containing the recombinant gene MOX-HP V56-L1 and the incomplete gene TRP3 H. polymorpha as a selective marker.

The MOX-HPV56 DNA fragment (SEQ ID NO: 2) and the preparation of the plasmid pTZ-MOX (Agaphonov et al., 1995, Yeast, 11: 1241-1247) containing the incomplete H. polymorpha TRP3 gene as a yeast selective marker were hydrolyzed restriction enzymes BglII and Xhol. The product of hydrolysis of the MOX-HPV56 fragment and the product of the hydrolysis of plasmid pTZ-MOX 4.3 kbp were isolated after electrophoretic separation in agarose gel. The obtained DNA preparations were introduced into the reaction mixture for ligation at a concentration of 30 ng / μl. After incubation with T4 DNA ligase for 2 hours, 2 μl of the reaction mixture was used to transform E. coli strain DH5α. Plasmid DNA was isolated from several grown transformants. Based on restriction analysis, a plasmid containing the MOX-HPV56 fragment was selected. The determination of the nucleotide sequence of this plasmid confirmed that it contains a gene encoding HPV56-L1 under the control of the MOX promoter H. polymorpha. The resulting plasmid was designated p56HPV2.

Example 3. Obtaining a strain of N. polymorpha containing the expression cassette HPV56-L1 with the MOX promoter.

Strain H. polymorpha DLT2 [Agaphonov et al., 1994, Yeast v10, pp. 509-513; the collection of strains of the laboratory of molecular genetics RKNPK Ministry of Health of the Russian Federation] was transformed with plasmid pKAM157, hydrolyzed with restriction enzymes Ec1136P and XhoI. Transformants were selected on a medium containing 6.7 g / l of a mixture of salts and vitamins "Yeast Nitrogen Base" (Difco), 20 g / l of D-glucose, 20 g / l of agar and 60 mg / l of leucine. Among the obtained transformants, clones capable of producing HPV56-L1 were selected. One of these transformants was designated DLT2 / p56HP2.

Example 4. Obtaining a strain of N. polymorpha containing two expression cassettes HPV56-L1, one of which is with the MOX promoter, and the other with the DAK promoter.

Strain DLT2 / p56HP2 (Example 3) was transformed with plasmid p56HP3 digested with BsrGI restriction enzyme. Transformants were selected on a medium containing 6.7 g / l of a mixture of salts and vitamins "Yeast Nitrogen Base" (Difco), 20 g / l of D-glucose and 20 g / l of agar. One of the obtained transformants was designated KBT11 / rRU-562. This transformant was confirmed to have the intact DAK-HPV56 gene (see Example 5) and the intact MOX-HPV56 gene (see Example 6).

Example 5. Determination of the presence of an intact DAK-HPV56 gene in the genome of H. polymorpha transformants.

Genomic DNA preparations were obtained from the analyzed clones and used as a template for polymerase chain reaction (PCR) with dakFw1 oligonucleotides (SEQ ID NO: 5) and AMI20 (SEQ ID NO: 6) as primers. PCR was performed using Pfu polymerase (Fermentas). In the presence of the DAK-HPV56 gene in the genome, a PCR product of 1819 bp was formed. To confirm the intactness of this gene, the nucleotide sequence of the obtained PCR product was determined.

Example 6. Determination of the presence of an intact gene MOX-HPV56 in the genome of H. polymorpha transformants.

Genomic DNA preparations were obtained from the analyzed clones and used as a template for polymerase chain reaction (PCR) with oligonucleotides MOX20 (SEQ ID NO: 3) and MOX3TRP3 (SEQ ID NO: 4) as primers. PCR was performed using Pfu polymerase (Fermentas). When the MOX-HPV56 gene was present in the genome, a 1797 bp PCR product was formed. To confirm the intactness of this gene, the nucleotide sequence of the obtained PCR product was determined.

Example 7. Cultivation of H. polymorpha strain KBT11 / pPV-562 containing two HPV56-L1 expression cassettes, one with the MOX promoter and the other with the DAK promoter.

The fermentation of the strain producing yeast H. polymorpha was carried out in two stages. At the first stage, in the fed-batch mode at a temperature of 30 ° C, biomass was increased in a culture medium containing 4% yeast extract, 2% bacto-peptone and 4% glycerol. When the density of the raw biomass reached 150 g / l (after about 28-32 hours of fermentation), the inductor was added to a concentration of 0.5-0.8% and maintained at this level for 48-72 hours.

Example 8. Isolation and purification of recombinant protein HPV56-L1 from H. polymorpha cells.

After fermentation of the producer strain, the HPV56-L1 protein (amino acid sequence — SEQ ID NO: 7) was isolated according to the published procedure [Kim H.J. et al., Protein Expression and Purification, v.70, p. 68-74, 2010] with minor changes. Cells from the culture fluid were besieged by centrifugation at 4000 g for 15 minutes at 4 ° C. The precipitated biomass was resuspended to a concentration of 380 g of wet cells per liter of suspension in extraction buffer: PBS (pH 7.2) with 1.7 mM EDTA, 2 mM PMSF, 0.01% Tween-80. Next, the cells were destroyed in a Dyne-Mill mill of type KDL using glass balls with a diameter of 0.5-0.7 mm. To remove the bulk of non-target proteins, the obtained extract was saturated with ammonium sulfate to 45% of saturation, and the precipitated proteins were separated by centrifugation at 12000 g for 30 minutes. The pellet was resuspended in 9 volumes of PBS buffer (pH 7.2) + 0.01% Tween-80 to achieve 5% saturation with ammonium sulfate and incubated for 24 hours at room temperature to completely dissolve the target protein and precipitate a portion of the impurity proteins, which were then separated by centrifugation at 12000 g for 30 minutes. The resulting supernatant was dialyzed into binding buffer: PBS (pH 7.0) containing 0.2 M NaCl and 0.01% Tween-80, followed by adsorption on a Heparin Sepharose CL-6B sorbent (GE Healthcare, USA). After washing the sorbent with the same binding buffer, the HPV56-L1 protein was eluted with a linear gradient from 0.1 M to 1.0 M NaCl. The pooled antigen containing fractions (by immunoblot and SDS-PAGE) were concentrated by tangential flow ultrafiltration on a 500 KDa membrane and separated by zonal ultracentrifugation (Beckman Coulter, USA) in a glycerol / sucrose gradient. The fractions containing the target protein were combined, and then purified from glycerol and sucrose by gel filtration on a sorbent Toyopearl HW-65 (ToyoSoda Corp., Japan). The purity of the HPV56-L1 recombinant protein thus obtained was determined by SDS-PAGE electrophoresis. If the protein purity was insufficient (less than 95%), additional purification was performed using cation exchange chromatography on a Toyopearl SP-650M sorbent.

As a result of these operations, it is possible to isolate about 15% of the L1 protein present in the clarified cell lysate (table 1). The yield of purified HPV56-L1 was at least 30 mg / kg wet yeast biomass.

Table 1 Balance of isolation and purification of HPV56-L1. (The concentration of total protein in the first stages was determined by the Biuret method. In the remaining stages, by the Lowry method. The content and purity of HPV56-L1 was determined by immunoblotting and SDS-PAGE electrophoresis). HPV56-L1 (mg / ml) HPV56-L1 (mg) Total protein (mg) Purity% Exit % Lysate 0.11 84 33600 0.3 one hundred 5-45% SA extract 0.08 69 12880 0.5 82 Heparin sepharose 0.20 32 136 24 38 Toyopearl HW-65 0.18 13 fourteen 95 fifteen

Example 9. Analysis of recombinant HPV56-L1.

1) The purity of the recombinant target protein is determined by polyacrylamide gel electrophoresis under reducing conditions (SDS-PAGE) by staining with Coomassie Brilliant Blue R-250 and analyzing the intensity of the bands of scanned gels using the NIH Image computer program. The purity of the selected recombinant protein HPV56-L1 is at least 95% (Fig.1.A).

2) Immunospecificity of recombinant HPV56-L1 is determined by immunoblotting. The primary monoclonal antibodies to the HPV-L1 protein (Camvir-1, AbCam, UK) are 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). Recombinant HPV56-L1 antigen is presented as a 55-65 kDa protein monomer band (Fig. 1B).

3) HPV56-L1 recombinant protein VLP formation was confirmed by gel filtration chromatography on a Tosoh Bioscience (Japan) TSK G5000PW polymer column with a diameter of 7.5 mm and a length of 60 cm connected to a PrePW column with a diameter of 7.5 mm and a length of 7.5 cm. The pore size of the sorbent is 1000A, which allows you to separate correctly assembled VLP from the monomers of the L1 protein, as well as aggregates. Separation was carried out in a buffer of 10 mM KH ₂ PO ₄ , (pH 7.2), 0.03% Tween-20 at a flow rate of 0.6 ml / min. The process was monitored by absorbance at 280 nm. The elution time of protein L1 corresponds to the elution time of virus-like particles (Fig. 2).

4) Immunogenicity of recombinant HPV56-L1 was determined in a test on Balb / c mice weighing 12-14 g after a single vaccination with HPV56-L1 protein adsorbed on aluminum hydroxide. The ED / 50 value (ED / 50 is the dose of antigen that causes seroconversion in 50% of mice) is less than 100 ng, which is a good indicator of the immunogenic properties of the recombinant protein.

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 30-50% 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 conditions and composition of the medium for fermentation: pumping at 30 ° C and pH 5.0-5.5 in a nutrient medium containing up to 4% glycerol.

The strain activity was determined in the clarified cell homogenizate by immunoblotting or enzyme immunoassay. The strain activity is at least 15 mg / l of culture fluid.

The isolated HPV type 56 L1 protein can be used in test systems, as well as to create vaccines based on it for the prevention of diseases associated with human papillomavirus.

Claims (1)

A recombinant strain of Hansenula polymorpha yeast KBT1l / pPV-562 containing a DNA fragment integrated into the genome with a recombinant gene encoding an HPV type 56 L1 polypeptide, under the control of the promoter of the DAK N. polymorpha gene (SEQ ID NO: 1) and a DNA fragment with a recombinant gene, encoding HPV type 56 L1 polypeptide, under the control of the promoter of the MOX gene N. polymorpha (SEQ ID NO: 2) - producer of recombinant human papillomavirus L1 capsid protein (HPV) type 56.

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