RU2445357C1 - Pichia angusta recombinant yeast strain - producer of capsid protein l1 of human papillomavirus type 16 - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: strain is obtained by introducing into yeast cells a DNA sequence which codes the capsid protein L1 of HPV type 16 under control of a DAK gene promoter. The present invention provides high output of the recombinant protein L1 of HPV type 16 using simple methods of obtaining high-density cultures and can be used in microbiological synthesis of recombinant protein L1 of HPV type 16, having immunogenic properties of natural protein.

EFFECT: simple method.

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 capsid protein L1 of human papillomavirus type 16.

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. 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 immunologically, by inducing an immune response in humans.

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 protein L1 (55-60 kDa) is able to assemble itself into virus-like particles (VLP - Virus-like particles), 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 L1 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 cells of mammals infected with a recombinant vaccinia virus vector [Zhou J. et al. Virology, v. 185, p. 251-257, 1991]. Known expression of papillomavirus protein L1, capable of aggregating into virus-like particles (VLP) and having immunogenicity, in a eukaryotic system based on cultured insect cells, baculovirus system [Kirnbauer 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, 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 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 HPV16-L1 [Bazan S.B. et al. Arch. Virol. 2009, 154 (10), 1609-17]. A gene encoding an HPV type 16 L1 protein (HPV16-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 a virus-like particle assembly (VLP) step. The formation of biologically active VLPs was confirmed by immunoelectron microscopy and hemagglutination. The use of other types of methylotrophic yeast except P. pastoris as a producer of HPV type 16 L1 protein is not established in the prior art.

The closest analogue is patent RU 2206608, 2003, which describes the creation of a yeast producer of HPV L1 or HPV L2 protein of various types, including HPV 16 and 18, using Saccharomyces cerevisiae as an example. 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.

The preparation of the HPV L1 capsid protein having the antigenic properties of a natural protein using a recombinant producer in accordance with the closest analogue has certain disadvantages compared to previously known recombinant producers. These include the instability of recombinant cells associated with a relatively high frequency of autonomous plasmid losses 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 an effective microbiological yeast producer of HPV protein type L1 type 16. Under the effectiveness refers to 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 strain of the capsid protein L1 HPV16 expression plasmid containing a DNA fragment with a recombinant gene encoding HPV16-L1, under the control of the promoter of the gene DAK (dehydroxyacetone kinase) Hansenula polymorpha (selective marker H. polymorpha LEU2) integrate into the genome of the H. polymorpha recipient strain DL1-L. As a result of the selection of clones capable of synthesizing the HPV16-L1 protein, the transformant with the highest productivity was identified, designated KBT-09 / pHV-161. 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. In accordance with the changes made to the classification of yeast, H. polymorpha is synonymous with the taxonomic name Pichia angusta [Hansenula Polymorpha. Biology and Applications. Edited by G. Gellissen, 2002, pp. 3-4].

The resulting recombinant P. angusta strain allows one to achieve higher levels of synthesis of the recombinant HPV16-L1 protein with simpler methods for producing high density cultures.

The strain was deposited under VKM number Y-2988D, 04/07/2010 at the Institute of Biochemistry and Physiology of Microorganisms RAS named after G.K.Skryabin. 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.

Moreover, all routine genetic engineering operations are carried out in accordance with standard methods and instructions of companies producing enzymes and kits for manipulating DNA in vitro. In all cases, Pfu polymerase (Fermentas) was used for PCR. The synthesis of oligonucleotides and a DNA fragment encoding the HPV16-L1 polypeptide, as well as the determination of the nucleotide sequence, were performed by Evrogen CJSC in Moscow. The nucleotide sequences of the synthesized primers and the double-stranded DNA fragment encoding the HPV16-L1 polypeptide are shown in the List of nucleotide sequences.

Example 1. Obtaining a recombinant gene containing a DAK gene promoter and a sequence encoding an HPV16-L1 polypeptide.

A DNA fragment containing the promoter of the H. polymorpha DAK gene was obtained by polymerase chain reaction (PCR). For this, a reaction mixture was prepared containing a single reaction buffer for Pfu, dATP, dTTP, dGTP, dCTP at a concentration of 200 μM each, primers DAK1 (SEQ ID NO: 1) and DAK2 (SEQ ID NO: 2) at a concentration of 0.5 μM each, DNA from a strain of H. polymorpha DL1-L [Agaphonov et al., 1994, Yeast v10, pp. 509-513; a collection of strains of the laboratory of molecular genetics RKNPK Ministry of Health of the Russian Federation] at a concentration of 10 μg / ml and Pfu polymerase at a concentration of 20 units / ml. Prior to PCR, DAK2 primer was phosphorylated using T4 polynucleotide kinase. This mixture was incubated for 30 cycles of temperature change, each of which included incubations at 95 ° C for 15 s, at 50 ° C for 30 s, at 72 ° C for 60 s. After that, the obtained reaction products were separated on an agarose gel and a fragment of 1095 bp was isolated. This fragment was mixed with a fragment encoding the HPV16-L1 polypeptide (SEQ ID NO: 5) at a concentration of 100 ng / μl each and incubated with T4 DNA ligase in a single ligase buffer at 20 ° C for two hours. After that, 1 μl of the ligase mixture was added to 100 μl of the PCR mixture containing a single reaction buffer for Pfu, dATP, dTTP, dGTP, dCTP at a concentration of 200 μM each, primers DAK1 (SEQ ID NO: 1) and HPV16L1 / 3'a (SEQ ID NO: 3) at a concentration of 0.5 μM each, and Pfu polymerase at a concentration of 20 u / ml. This mixture was incubated for 15 cycles of temperature change, each of which included incubations at 95 ° C for 15 s, at 50 ° C for 30 s, at 72 ° C for 150 s. After this, the obtained reaction products were separated on an agarose gel and a 2600 bp fragment was isolated. This fragment contained a recombinant gene including the promoter of the H. polymorpha DAK gene and a sequence encoding HPV16-L1.

Example 2. Obtaining a plasmid vector pAM517 containing the recombinant HPV16-L1 gene and a selective marker of the H. polymorpha LEU2 gene.

A DNA fragment with a recombinant gene encoding HPV16-L1, under the control of the promoter of the H. polymorpha DAK gene, was digested with restriction enzyme BglII. The preparation of plasmid AMIpLD1 [Agaphonov et al., 1999, Yeast 15: 541-551] containing the H. polymorpha LEU2 gene as a yeast selective marker was digested with BamHI restriction enzyme and treated with calf thymus alkaline phosphatase. The resulting preparations of the recombinant gene DNA fragment and the linearized plasmid AMIpLD1 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 Escherichia coli strain DH5α. Plasmid DNA was isolated from several grown transformants and used as template for analytical PCR with primers HPV16L1 / 3'a (SEQ ID NO: 3) and HPV16L1 / 5'a (SEQ ID NO: 4). PCR products were analyzed by agarose gel electrophoresis. A plasmid preparation was selected from which a 1500 bp fragment was formed as a result of PCR. The determination of the nucleotide sequence of this plasmid confirmed that it contains a plot encoding HPV16-L1 under the control of the DAK promoter. H.polymorpha. The resulting plasmid was designated pAM517.

Example 3. The introduction of the plasmid pAM517 in the genome of H.polymorpha DL1-L.

The strain H.polymorpha DL1-L, which is an auxotroph of leucine, was grown for 15-18 hours in liquid YPD 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 pAM517, 5 μg of carrier DNA (fragmented and denatured herring DNA) and 60 μl of a 70% solution of polyethylene glycol 4000. 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 and incubated for several days until transformant colonies appeared. The cells of the grown transformants were scattered with a depleting streak on fresh YNB-D plates, and after two days of incubation at 37 ° C, fast-growing colonies were selected.

Selected clones were tested for their ability to synthesize HPV LI protein with cell growth in a medium containing methanol. One of the transformants with the highest productivity was designated KBT-09 / pHR-161.

Example 4. Cultivation and isolation of the recombinant protein HPV16-L1 from H. polymorpha cells.

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% bactopeptone and 4% glycerol. When the raw biomass reached a density of 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.

After fermentation of the producer strain, the HPV16-L1 protein 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 Dyno-Mill mill of type KDL using glass balls with a diameter of 0.5-0.7 mm. To remove the bulk of the non-target proteins and the selection of antigen, the obtained extract was saturated with ammonium sulfate to 45% saturation and the precipitated proteins were separated by centrifugation at 12000g for 10 minutes. The precipitate was diluted in a minimum volume of PBS buffer (pH 7.2) + 0.01% Tween-80 and dialyzed into the same buffer for 24 hours at room temperature to precipitate a portion of the remaining impurity proteins, which were then separated by centrifugation at 12000g for 10 minutes. The resulting supernatant was dialyzed into binding buffer: PBS (pH 7.0) containing 0.33 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 HPV16-L1 protein was eluted with a linear gradient from 0.33 to 1.5 M NaCl. The pooled antigen-containing fractions (by immunoblot and SDS-PAGE) were concentrated by ultrafiltration (500 KDa) and separated by zonal ultracentrifugation (Beckman Coulter, USA) in a glycerol / sucrose gradient. The fractions containing the target protein were combined and then purified by gel filtration on a Toyopearl HW-65 sorbent (ToyoSoda Corp., Japan). The purity of the thus obtained recombinant HPV-L1 protein was determined by electrophoresis. If the protein purity was insufficient (less than 95%), an additional purification was performed using cation exchange chromatography on a Toyopearl SP-650C sorbent. The yield of the target protein was at least 50 mg per 1 liter of culture fluid.

Example 5. Analysis of recombinant HPV16-L1.

1. The purity of recombinant HPV-L1 is determined by polyacrylamide gel electrophoresis under reducing conditions (SDS-PAGE) by silver staining and Coomassie Brilliant Blue R-250. The purity of the isolated recombinant HPV-L1 protein is at least 95%.

2. The immunospecificity of recombinant HPV16-L1 is determined by immunoblotting. Primary monoclonal antibodies to the HPV-L1 protein of serotype 16 (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 method of improved ECL chemiluminescence (Amersham, UK). Recombinant HPV16-L1 antigen is presented as a protein monomer band of 55-60 kDa in size.

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: Phages in yeast not detected. 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 method for determining the activity: in the clarified homogenized cells by immunoblotting or enzyme immunoassay. The strain activity is at least 50 mg / l of culture fluid.

The isolated HPV type 16 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)

Recombinant yeast strain Pichia angusta BKM Y-2988D, transformed with a DNA sequence encoding HPV type 16 L1 polypeptide - producer of recombinant human papillomavirus virus (HPV) type 16 capsid protein (HPV) type 16.