RU2463343C1 - Ecoli BL21 (DE3)pLysS CELL STRAIN, pTT9/ASFVp30 CLONE CONTAINING RECOMBINANT PLASMID WITH INTEGRATION OF PART OF CP204L GENE OF AFRICAN SWINE FEVER VIRUS, ENCODING CONFORMATIONAL EPITOPE OF PROTEIN p30, FOR PRODUCING DIAGNOSTIC PREPARATIONS - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: disclosed is an E.coli strain which produces a recombinant protein p30 of the African swine fever virus. The strain is homogeneous, stable during passage and culturing in liquid and solid culture media and is resistant to chloramphenicol.

EFFECT: invention can be used to produce a recombinant protein p30 of African swine fever virus for diagnostic purposes.

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Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the manufacture of diagnostic tools for the virus of African swine fever (ASF).

African swine fever (ASF) is a viral disease characterized by high mortality and contagiousness, over-acute, subacute, acute and chronic course. In 2000, the pathogen was assigned to the Asfarviridae family [1].

African swine fever (ASF) has been known for almost 100 years as a natural focal viral infection of wild African and domestic pigs of local and European breeds. It is transmitted from sick animals and virus carriers by contact and alimentary route. In addition, ASF virus can also be transmitted transplacentally, causing abortions in pregnant sows [2].

The economic damage caused by ASF consists of direct losses due to the radical elimination of the disease, restrictions on international trade and is measured in tens of millions of dollars [3].

To date, vaccines against ASF have not been developed, so early and highly effective diagnosis of the disease is a key point to limit its spread and irradiation.

A known E. coli M15 cell strain (Qiagen), in which the accumulation level of recombinant ASF virus p30 protein during IPTG induction was 1 μg with 1 ml of bacterial culture, the expression time was 2.5 hours at a temperature of 24 ± 0.5 ° C [4 ]. However, the low level of accumulation in the indicated cell system did not allow to obtain recombinant antigen in production quantities from small volumes of bacterial culture.

The known E. coli BL21 (DE3) pLysS cell strain is used for highly efficient gene expression under the control of the T7 promoter and allows one to achieve a high level of expression of the target gene upon induction with IPTG [5, 6]. The cells of this strain have the property in the presence of an inducer (IPTG) to predominantly express recombinant proteins and in a minimal amount of their own proteins.

The proposed strain is a strain of E. coli cells BL21 (DE3) pLysS, clone pTT9 / ASFVp30 (clone 11) containing a recombinant plasmid with the insertion of a portion of the gene for African swine fever virus CP204L (ASF) encoding the conformational epitope of protein p30.

The aim of the present invention is to obtain a new strain of E. coli cells BL21 (DE3) pLysS, clone pTT9 / ASFVp30, containing a recombinant plasmid with the insertion of a portion of the gene for African swine fever virus (ASF), coding for the conformational epitope of the p30 protein, a hyperproducer of the recombinant the size of 101-amino acid with a molecular weight of 14,000 Da, which is effectively purified by chromatography on cellulose and suitable for use in diagnostic test systems for detecting antibodies to ASF virus in pork serum and receiving hyperimmune sera to detect antigen ASFV by immunofluorescence.

The technical result from the use of the present invention is to expand the arsenal of sources of recombinant ASFV antigens with high biological antigenic and immunogenic activity, retaining its basic immunobiological properties after chromatographic purification and suitable for producing hyperimmune monospecific serums in laboratory animals in order to study the pathogenesis and reproduction of ASFV virus in vitro and in vivo. The invention involves the use of recombinant p30 produced by E. coli cells of strain BL21 (DE3) pLysS, clone pTT9 / ASFVp30 (clone 11), immunoglobulins G (IgG) for diagnostic purposes (in the indirect immunofluorescence reaction to detect ASF virus antigen in smears fingerprints of samples of pathological material, diagnostic immunoblotting).

This technical result was achieved by obtaining clone pTT9 / ASFVp30 (clone 11) in E. coli cells of strain BL21 (DE3) pLysS, which is a hyperproducer of the recombinant protein. The obtained recombinant transformants were screened for insertion by PCR and the cultural and biological properties of the clones were studied when SOB (pH 7.5-8.0) or LB containing 100 μg / ml ampicillin, 50 μg / ml kanamycin was accumulated in a liquid optimal temperature 37.0 ± 0.5 ° C. The homogeneity of the clones was studied by reseeding on a solid nutrient medium.

It was established that the recombinant clone is resistant to chloramphenicol. Clone cells are able to stably produce recombinant ASF virus p30 in the presence of 1 mM IPTG. The dynamics of the accumulation of the target antigen in different temperature cultivation conditions are presented in table 1. In indirect TF ELISA and SDS-PAAG electrophoresis according to Laemmli U.K. The stability of expression and the polypeptide composition of the expressed proteins are shown.

Table 1 Comparative analysis of the effect of induction time of the promoter on the yield of recombinant protein p30. n = 3 Time watch The output of the recombinant protein of the clone rTT9r30-2 M15 (prototype), μg / ml The output of the recombinant protein of the clone pTT9 / ASFVp30 BL21 pLysS, μg / ml 0.5 0.1 0.1-0.2 1,0 0.3 0.5-0.6 1,5 0.6 1.0-1.2 2.0 0.8 1.4-1.6 2.5 1,0 2.0-2.5 3.0 n.i. 2.5-3.0 3,5 n.i. 3.0-3.5 4.0 n.i. 3.5-4.0

The resulting recombinant clone was deposited in the "Collection of pathogenic microorganisms, viruses and bacteria that cause especially dangerous, including zooanthroponoses", All-Russian Research Institute of Veterinary Virology and Microbiology (VNIIVViM) on 09.09.2011 under the registration code 2966.

The strain E. coli BL21 (DE3) pLysS clone pTT9 / ASFVp30 (clone 11) differs from the prototypes by a higher level of expression of the recombinant antigen, 2-4 times higher than the accumulation of the target antigen by the primary producer in E. coli cells of strain M15, which was achieved using competent E. coli cells of strain BL21 (DE3) pLysS. Compared with the producer of recombinant protein p30 in the baculovirus system, the proposed strain differs from the prototype cheap cultivation hundreds of times.

The possibility of using E. coli strain BL21 (DE3) pLysS of clone pTT9 / ASFVp30 (clone 11) for the manufacture of diagnostic tools for ASFV virus was confirmed experimentally.

The strain E. coli BL21 (DE3) pLysS clone pTT9 / ASFVp30 (clone 11) is characterized by the following features and properties: the clone is homogeneous, stable upon passage and cultivation in liquid and solid nutrient media, resistant to chloramphenicol, cultivated in the presence of ampicillin and kanamycin, capable of stably express recombinant p30 ASF virus. The produced protein has a high biological antigenic and immunogenic activity, retains its basic immunobiological properties after chromatographic purification. The yield of purified recombinant antigen with 1 l of bacterial culture is 2-4 mg.

The main marker characteristics of the E. coli strain BL21 (DE3) pLysS clone pTT9 / ASFVp30 (clone 11) are shown in the table.

Marker characteristics of E. coli strain BL21 (DE3) pLysS clone pTT9 / ASFVp30

No. p / p Basic properties Feature Description one 2 3 one. Molecular genetic properties The E. coli strain BL21 (DE3) pLysS clone pTT9 / ASFVp30 (clone 11) contains the recombinant plasmid pTT9p30-9 carrying the 212th to 584th base pair of the ASF virus gene, coding for the sequence of the soluble epitope of the p30 protein of the AC virus 90th to 190th amino acid having a molecular weight of 14,000 Da. The structure of the recombinant plasmid contains the sequence of the cellulose-binding domain (CBD), due to which it is possible to purify the target antigen on a cellulose sorbent. The strain of E. coli cells BL21 (DE3) pLysS, which is used for highly efficient gene expression under the control of the T7 promoter. BL21 (DE3) pLysS cells contain the plasmid pLysS carrying the gene encoding T7 lysozyme. Lysozyme T7 reduces the level of expression under the control of the T7 promoter, but allows to achieve a high level of expression of the target gene upon induction with IPTG. The genotype of E. coli cells BL21 (DE3) pLysS: F-, ompT, hsdS _B (r _B -, m _B -), dcm, g a l, λDE3, pLysS, Cm ^r . 2. Growth properties The clone is reproduced in SOB containing 2% bacto-tryptone (Difco), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl and 20 mM Mg ²⁺ , pH 7.5-8.0 or in LB medium containing 1% bacto tryptone (Difco), 0.5% yeast extract (Difco), 170 mM NaCl, 10 mM MgSO ₄ , pH 7.0-7.4, in the presence of 100 μg / ml ampicillin 50 μg / ml kanamycin at an optimal cultivation temperature of 37.0 ± 0.5 ° C. Before induction of expression of the target antigen, the procedure of growing a refreshed culture (night culture) on a freshly prepared medium is carried out at an optimum temperature of 37.0 ± 0.5 ° C for 3-4 hours to an optical density of the growth medium of 0.6-0.8 units. at a wavelength of 600 nm. The maximum expression level is achieved in the presence of 1 mM IPTG at an optimum temperature of 28.0 ± 0.5 ° C after 4-5 hours of cultivation. 3. Pathogenic properties The strain is apatogenous for laboratory and susceptible animals. four. The properties of the beneficial substance produced by the strain The strain produces a soluble protein representing the conformational epitope p30 of the ASF virus (amino acids 90 to 190), molecular weight 14,000 Da. The structure is a phosphoprotein. Recombinant p30 protein when used in ELISA is 3 times more active compared to cytoplasmic virus-specific antigen. The advantages of using recombinant p30 for ELISA are due to the number of linear and conformational epitopes contained in this protein. It was shown that recombinant p30 contains mainly conformational epitopes; due to this, some sera react better with recombinant proteins than with virus-induced ones. 5. Thermal Resistance (T) The strain E. coli BL21 (DE3) pLysS clone pTT9 / ASFVp30 (clone 11) is inactivated at temperatures above 42.0 ± 0.5 ° C. 6. Inhibitor sensitivity Under the influence of chloroform, ether and other solvents, as well as under the influence of solutions of formaldehyde, copper sulfate, theotropin, the viability of E. coli BL21 (DE3) pLysS cells of clone pTT9 / ASFVp30 (clone 11) is completely lost. 7. Stability of genetic and immunogenic The stability of the genetic and immunogenic characteristics of the strain was maintained when it was passaged in a liquid nutrient medium in

signs for at least 20 passages (observation period). 8. Contamination with bacteria, fungi, mycoplasmas and extraneous viruses The strain is not contaminated by bacteria, fungi, mycoplasmas. 9. Cultural properties and standard growing conditions The strain is resistant to chloramphenicol. The clone is reproduced in SOB medium or in LB medium in the presence of 100 μg / ml ampicillin and 50 μg / ml kanamycin at an optimal cultivation temperature of 37.0 ± 0.5 ° C, capable of stably expressing recombinant ASF virus p30. 10. Persistence and storage conditions The strain is stabilized in glycerol to a concentration of 50% and stored in a frozen state at a temperature of at least minus 60 ° C. It is freshened by culturing it in a liquid nutrient medium SOB or in LB medium in the presence of 100 μg / ml ampicillin and 50 μg / ml kanamycin at an optimal cultivation temperature of 37.0 ± 0.5 ° C for 18 hours with moderate shaking of 110-120 rpm min once every 3 years. eleven. Originality and patentability The E. coli strain BL21 (DE3) pLysS clone pTT9 / ASFVp30 (clone 11) in terms of antigenicity and immunogenicity of the produced target antigen is an original, new, previously unknown variant of the producer of recombinant ASF virus p30 protein suitable for obtaining hyperimmune serum for immunofluores.

The essence of the invention is illustrated by examples of its implementation and use.

EXAMPLE 1. Cultivation and purification of a recombinant protein produced by a strain.

To build the E. coli strain BL21 (DE3) pLysS clone pTT9 / ASFVp30 (clone 11) and expression of the recombinant p30 protein by bacterial culture, cultivation was performed in SOB medium, pH 7.5-8.0 with the addition of 100 μg / ml ampicillin, 50 μg / ml kanamycin, 20 mM MgCl ₂ and 1.0 mM IPTG at an optimum temperature of 28.0 ± 0.5 ° C for 4 hours. Previously, before induction of the expression of the target antigen, a procedure was performed for growing a night culture that was seeded on freshly prepared medium at an optimum temperature of 37.0 ± 0.5 ° C for 3-4 hours until the optical density of the bacterial mass, measured at a wavelength of 600 nm, reached a value of 0, 6-0.8 units

Next, cell lysates were prepared. For this, the bacterial mass was besieged by centrifugation at 3000 rpm for 40 min at a temperature of 4 ° C. Cell destruction was carried out in an ultrasonic disintegrator with constant cooling of the mixture. The scoring procedure consisted of 3 cycles of 2 minutes at a frequency of 18 kHz / s with a break between cycles of 15 minutes. The scoring process was microscopically monitored by staining aliquots of the lysate with fuchsin Pfeifer. The cell lysate containing recombinant p30 was resuspended in TE buffer (pH 7.2-7.4) per 100 mg of bacterial mass 1 ml of buffer [15].

Since proteolytic degradation of secreted recombinant protein is a significant problem, a protease inhibitor, Pefablock SC, was added to the buffers to a working concentration of 0.25 mg / ml. Both suspensions were clarified by centrifugation at 15,000 rpm for 20 min and filtered through membrane filters with a pore size of 0.45 μm [15].

The total protein content in the lysate was determined by the Lowry method [8].

The purification efficiency of recombinant p30 protein was evaluated in 12% and 10% SDS-PAGE electrophoresis according to Laemmli U.K. [7, 13].

To isolate recombinant p30 protein from the prepared lysate, adsorption under static conditions was used — a fast method that allows the isolation of substances that are in small quantities in crude mixtures [15].

Washed cellulose was added to a suspension treated with ultrasound (6 times for 1 min at 18 mg) and clarified lysate at the rate of 50-60 μg of protein per 1 mg of sorbent. The sorbent was incubated at 4 ° C. overnight with constant stirring. Then the sorbent was washed with a 10-fold volume of a 1% solution of X-100 triton. The washing procedure was repeated three times, intensively mixing the sorbent. Recombinant p30 protein was eluted with 100% formamide. The collected fractions were dialyzed overnight at 4 ° C against 0.01 M Tris-HCl (pH 8.5-9.0) containing 0.15 M NaCl [15].

Because the plasmid rTT9p30-9 contains the CBD gene, which encodes a cellulose-binding domain in the structure of the recombinant protein, with the help of which the recombinant p30 protein is able to bind to cellulose matrices, the protein was purified using Avicel® microcrystalline cellulose (Merck), which was previously activated in solution 0.5 M NaOH for 30 minutes, followed by washing to pH 8.0-8.5. Such preliminary preparation of cellulose and carrying out adsorption “in bulk” (adsorption under static conditions) made it possible to increase its sorption capacity to 60 μg of protein per 1 mg of sorbent.

As a result of the experiments, 8 analytical series of purified recombinant p30 were obtained. The yield was approximately 2 to 4 mg of protein per batch obtained from 1 L of the bacterial culture; the protein concentration in the sample reached 0.4-0.5 mg / ml. One analytical series is enough to sensitize about 100 pieces of 96-well panels to detect antibodies to ASF virus in indirect TF ELISA.

The degree of protein purity was confirmed by electrophoresis in SDS page. Analysis of electrophoregrams of purified recombinant protein showed that it was represented by a clear single band in the region of 14 kDa.

EXAMPLE 2. Antigenicity of the recombinant protein produced by the strain, and the safety of its use.

To obtain a monospecific hyperimmune blood serum to the recombinant p30 of the ASF virus, a “priming” procedure was performed - animals aged no more than 6 months, weighing 2.5-3.0 kg were injected intramuscularly or subcutaneously in the thigh area in the immediate vicinity of regional lymph nodes a solution of plasmid DNA at the rate of 100 μg / kg live weight, and after 10 days. the animals were injected with the preparation of purified recombinant ASF virus p30 protein at the rate of 100 μg / kg live weight 3 times with an interval of 10-14 days in the same area. 14 days after the last immunization, animals were bled according to the “Guidelines for Bleeding Laboratory Animals”.

The specificity and activity of the obtained sera was evaluated in the indirect variant of TF ELISA and the reaction of indirect immunofluorescence with FITC conjugate of protein A.

The obtained hyperimmune monospecific rabbit blood serum to recombinant p30 in the indirect variant of TF ELISA specifically reacted with a specific ASF viral antigen and chromatographically purified recombinant ASF virus p30 protein and did not interact with negative antigens - with an intact cell culture lysate of CV-1 cells and a lysate of cell culture CV-1 and lysate coli strain BL21 (DE3) pLysS. The optical density of the wells sensitized with specific antigens in which hyperimmune monospecific rabbit blood serum was incubated exceeded that in 2.1 or more times in the wells in which rabbit negative serum was incubated.

The titer of the obtained serum with recombinant chromatographically purified p30 1: 1600-1: 3200, and with a specific ASF virus antigen 1: 400-1: 800.

EXAMPLE 3. Detection of ASF virus antigens in the culture of infected cells and smears of fingerprints using hyperimmune monospecific serum to recombinant p30.

ASF virus antigens were detected in the culture of infected cells and smears of fingerprints using specific pig serum to ASF virus and the obtained hyperimmune monospecific serum to recombinant ASF virus p30 in the immunofluorescence reaction (RNIF) is an indirect option.

In order to determine the activity of the test rabbit blood serum and its specificity, it was titrated in RNIF.

The resulting hyperimmune monospecific rabbit blood serum to recombinant p30 specifically reacted only with specific antigens of the ASF virus of infected culture test preparations and smears of fingerprints of infected organs and did not interact with intact cells of culture test preparations of transplantable CV-1 cell culture. The titer of the obtained serum in RNIF 1: 128-1: 256.

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Claims (1)

The E. coli BL21 (DE3) pLysS cell strain, clone pTT9 / ASFVp30, containing a recombinant plasmid with an insertion of a region of the African swine fever virus CP204L gene encoding the conformational epitope of p30 protein, inv. No. 2966, deposited in the Collection of microorganisms of the GNU of the All-Russian Research Institute of Veterinary Virology and Microbiology of the Russian Agricultural Academy, for the manufacture of diagnostic preparations.