Dominant epitope peptide of Ebola virus envelope glycoprotein, coding gene and application thereof
阅读说明：本技术 一种埃博拉病毒包膜糖蛋白的优势表位肽、其编码基因及应用 (Dominant epitope peptide of Ebola virus envelope glycoprotein, coding gene and application thereof ) 是由 杨琨 姜东伯 刘洋 孙报增 孙昊 孙元杰 杨舒雅 张溪洋 潘婧宇 胡尘辰 刘天玥 于 2021-09-06 设计创作，主要内容包括：本发明属于微生物免疫技术领域,具体涉及一种埃博拉病毒包膜糖蛋白的优势表位肽、其编码基因及应用。所述表位肽由SEQ ID NO.1-25所示序列组成。本发明通过ELISpot试验预测并验证了来源于EBOV GP的潜在表位,并对其在世界各地不同分离株中的保护性质进行了多种深入分析,以验证筛选出的优势分离株。我们进一步预测了这些表位与各自MHC I类分子合适位点的结合亲和力及其免疫原性,这将最终有助于理解EBOV GP的免疫生物学,并在未来完善针对该病毒的表位疫苗设计。(The invention belongs to the technical field of microbial immunity, and particularly relates to dominant epitope peptide of Ebola virus envelope glycoprotein, and a coding gene and application thereof. The epitope peptide consists of a sequence shown in SEQ ID NO. 1-25. The invention predicts and verifies potential epitopes derived from EBOV GP through ELISpot test, and carries out a plurality of deep analyses on the protection properties of the potential epitopes in different isolates around the world so as to verify the screened dominant isolate. We further predicted the binding affinity of these epitopes to the appropriate sites of the respective MHC class I molecules and their immunogenicity, which will ultimately help to understand the immunobiology of EBOV GP and to refine epitope vaccine design against this virus in the future.)
1. A dominant epitope peptide of Ebola virus envelope glycoprotein is characterized in that the epitope peptide consists of a sequence shown in SEQ ID NO. 1-25.
2. A gene encoding the dominant epitope peptide of claim 1.
3. Use of the dominant epitope peptide of claim 1 for the preparation of an ebola virus epitope vaccine.
4. Use of the gene of claim 2 for the preparation of an ebola virus epitope vaccine.
5. An Ebola virus epitope vaccine prepared by using the dominant epitope peptide of claim 1, wherein the active ingredient is derived from the dominant epitope peptide of claim 1.
6. Ebola virus epitope vaccine prepared by using the gene of claim 2, wherein the active ingredient is derived from the gene of claim 2.
Technical Field
The invention belongs to the technical field of microbial immunity, and particularly relates to dominant epitope peptide of Ebola virus envelope glycoprotein, and a coding gene and application thereof.
Background
Ebola virus disease caused by ebola virus was first present in the vicinity of zaire and sudan. Ebola virus genus can be further subdivided into 5 species, named respectively: zaire Ebola virus (Zaire ebolavirus, ZEBOV/EBOV), Sudan Ebola virus (Sudan ebolavirus, SuDV), Tayi forest Ebola virus (Zaire ebolavirus, Zebov/EBOV) (B-Zedob)Forest ebolavirus, TAFV), bendbuiebo eboThe viruses of the bradbugyo ebolavirus (BDBV) and the Reston ebolavirus (Reston ebolavirus, RESTV). Human-to-human transmission can be caused by exposure to infectious body fluids and tissues through contact with infected items and mucosal or skin lesions. The presence of ebola virus has been found in a variety of bodily fluids, including saliva, blood, breast milk, feces, and semen. In addition, there are many potential routes of transmission, including direct contact, contamination, droplet transmission, and aerosols, among others. Ebola viruses belong to the family of filoviridae, are non-segmented negative-strand RNA viruses with a genome size of about 19 kb. The genome has 7 genes, including those encoding Nucleoprotein (NP), Viral Protein (VP)35, VP40, VP24, VP30, polymerase (L gene) and Glycoprotein (GP). Glycoprotein GP is widely recognized as the only protein on the surface of the virus, mediating attachment and binding to the cell surface. Thus, GP is considered to be the most immunologically active viral target.
Activation of an anti-viral CD8+ effector T cell response requires the presentation of viral antigens by Antigen Presenting Cells (APCs) through Major Histocompatibility Complex (MHC) class I molecules. Recognition of viral peptides by CD8+ T cells plays an important role in combating viral infection. However, only polypeptides that bind tightly with high affinity to MHC class I molecules are expressed on the surface of APC cells and presented for recognition by CD8+ T cells, thereby eliciting an immune response. Thus, immunogenic epitopes are crucial for elucidating antiviral immunity. At the same time, antigen conservation is also important for pathogen-host interactions and for establishing population immunity. The presence of evolutionarily conserved epitopes is crucial for virus survival, and therefore epitope conservation may provide broad protection against long-term immunity of these viruses, regardless of their strain.
Recently, epitope-based strategies have been used in alternative vaccine design and immunotherapy. The reasonable design of the epitope-based vaccine is very promising, because the safety is improved by reducing virus components causing adverse reactions, and meanwhile, the purpose of finally improving the vaccine efficiency is achieved by combining multiple epitopes to cover a wide range of people. Therefore, screening and identifying the dominant epitope peptide of the EBOV GP is very important for designing the epitope vaccine of the Ebola virus.
Disclosure of Invention
In view of the above technical problems, it is an object of the present invention to provide a dominant epitope peptide of Ebola virus envelope glycoprotein, wherein the epitope peptide consists of the sequence shown in SEQ ID NO. 1-25.
It is another object of the present invention to provide a gene encoding the dominant epitope peptide.
The invention also aims to provide application of the dominant epitope peptide in preparation of Ebola virus epitope vaccines.
The fourth purpose of the invention is to provide the application of the gene in preparing the Ebola virus epitope vaccine.
The fifth purpose of the invention is to provide an Ebola virus epitope vaccine prepared by using the dominant epitope peptide, and the active ingredients of the vaccine are derived from the dominant epitope peptide.
The sixth purpose of the invention is to provide an Ebola virus epitope vaccine prepared by using the gene, and the active ingredients of the Ebola virus epitope vaccine come from the gene.
Compared with the prior art, the invention has the following beneficial effects:
the potential epitope derived from EBOV GP (gene number: AY354458.1) is predicted and verified by an ELISpot test. And the protective properties of the strain in different isolates around the world are subjected to various in-depth analyses so as to verify the selected dominant isolate. We further predicted the binding affinity of these epitopes to the appropriate sites of the respective MHC class I molecules and their immunogenicity, which will ultimately help to understand the immunobiology of EBOV GP and to refine epitope vaccine design against this virus in the future.
The dominant epitope peptides of the present invention can be viewed as viral subunits, i.e., highly conserved regions required for virosomal function, that are effective in stimulating cellular immune responses in the body. The polypeptide vaccine can be designed accordingly, the strategy aims to inoculate the minimum structure consisting of definite antigens to excite effective specific cellular immune response, has incomparable advantages of the traditional vaccine, is safe and convenient to use, and has wide application prospect in the antiviral process.
Drawings
FIG. 1 is a graph of five prediction tools predicting the number of high affinity dominant epitopes to EBOV GP in mouse and human MHC class I subtypes.
FIG. 2 is a hierarchical cluster analysis of all predicted 9 peptides of EBOV GP, with the direction of the arrow indicating the affinity from strong to weak.
FIG. 3 shows the predicted binding affinities of different MHC class I subtypes for 9 peptides, wherein the ordinate indicates 668 peptides of 9 and the number indicates the position of the first amino acid of the 9 peptide in the GP sequence; the abscissa is the mouse and human MHC class I subtype, and the direction of the arrow indicates the affinity from strong to weak.
FIG. 4 shows the MHC class I restricted dominant epitope conservation analysis of EBOV GP.
FIG. 5 is a model of the docking of interspecies conserved dominant epitopes with corresponding MHC class I molecules.
FIG. 6 shows the screening results of 9 peptide dominant epitopes verified to be immunogenic by ELISpot assay.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
Example 1
Obtaining dominant epitope peptide of Ebola virus envelope glycoprotein
Step 1: amino acid sequence search:
the glycoprotein of zaire ebola virus (GP, accession number: AY354458.1) was obtained from the NCBI GenBank database as input to various bioinformatics tools for epitope prediction, conservation analysis, molecular docking and experiments.
Step 2: epitope prediction:
for epitope prediction of H2-Db, H2-Dd, H2-Kb, H2-Kd, H2-Kk and H2-Ld, web-based Tools are recommended, such as the IEDB method (http:// Tools. idb. org/mhci /), SMMPMBEC from immune epitope databases (http:// Tools. org/mhci /), Net MHCpan 4.1(http:// www.cbs.dtu.dk/services/Net MHCpan /), SYFPEITHI (http:// www.syfpeit hi. de/bin/CSverr. dll/EpitopRection. htm) and Rakpep (http:// Tools. ucm. es/Tools/htops. html. htm) for prediction. The predicted epitope was selected to be the first 2% of the total predicted epitope. Finally, epitopes predicted by at least three prediction tools are selected for subsequent in-depth analysis.
Human major leukocyte antigen (HLA) -class I subtype allele epitopes such as HLA-A1 (HLA-A2601, -A3001, -A3002), HLA-A2 (HLA-A0201, -A0203, -A0206, -A6802), HLA-A3 (HLA-A0301, -A1101, -A3001, -A3101, -A3301, -A6801), HLA-A24 (HLA-A2301, -A2402, -A3201), HLA-B7 (HLA-B0702, -B0103501, -B5101, -B5301), HLA-B8 (HLA-B0801), HLA-B15-A44 (HLA-B080), HLA-B44 (HLA-A0101), HLA-B0203, HLA-B44) and HLA-B0206, -B4402, -B4403) and HLA-B58 (HLA-B5701, -B5801), cumulatively covering MHC class I subtypes of more than 97% of the population, epitope prediction was performed using the same tools as described above. Finally, all the first 2% epitopes of the respective subtypes predicted by these tools were selected and subjected to subsequent studies.
For mouse MHC class I subtypes, 3 occurrences and above in 5 prediction software are considered dominant epitopes. For human HLA class I subtypes, all 9 peptides present in the first 2% of predicted outcomes were considered dominant epitopes. As described above, bioinformatic analyses were performed using a variety of computational tools to predict potential MHC class I epitopes with different binding affinities across EBOV envelope glycoproteins. Mouse H-2 subtype (H2-Db, H2-Dd, H2-Kb, H2-Kd, H2-Kk and H2-Ld) and major HLA class I subtype alleles were analyzed. The 9-peptide affinity prediction for the EBOV envelope glycoprotein was derived from 668 peptides. Figure 1 lists the number of dominant epitopes produced by each prediction tool. After recalculation to exclude repeat 9 peptides, we obtained 42 dominant epitopes in the H-2 subtype and 301 dominant epitopes in the HLA class I subtype. Among the H-2 subtypes, the number of dominant epitopes of H2-Db and H2-Kk is the largest, and the dominant epitopes are 12 peptides 9. Among HLA class I subtypes, HLA-B7 has the most dominant epitope number, which is 90 peptides 9, and is far more than other 8 HLA class I subtypes.
Step 3: polypeptide synthesis:
the corresponding polypeptide was synthesized according to the predicted polypeptide sequence, and all 9 peptides used in the experiment were completed by Shanghai Qiangyao Biotech Co., Ltd., purity was 95%, 1 mg/peptide. After the synthesized polypeptide powder is centrifuged, 20 mu L of dimethyl sulfoxide (DMSO) is added to dissolve the polypeptide powder, 480 mu L of sterile ddH2O is added to the solution, and the mixture is frozen and stored in a refrigerator at the temperature of 20 ℃ below zero for later use.
Step 4: computer analysis:
(1)9 peptide clustering analysis: 668 peptides were subjected to clustering analysis to study the similarity of different MHC class I molecules when presenting antigen. After normalization processing is carried out on NetMHCpan data, bidirectional hierarchical clustering is carried out by using a pheasap software package (version 1.0.12), and expression values are represented by a heat map. FIG. 2 shows the differences between different MHC class I subtypes. The 33 MHC class I molecules were divided into 3 groups, including the H-2 subtype group, the HLA class I subtype group and the cross-reactive group. In the H-2 group, H2-Dd, H2-Db and H2-Kb presented antigen more similarly. In the cross-reactive group, there was a distinct regional difference. H2-Kd scored similarly to HLA-a24(HLA-a × 2301, -a × 2402); H2-Ld was more similar to HLA-B7 (HLA-B0702, -B3501, -B5101, -B5301) scores; H2-Kk antigen presentation gave similar results to HLA-B44 (HLA-B4001, -B4402, -B4403). Whereas within the group of HLA class I subtypes for the same 9 peptide, scores do not differ greatly between different HLA class I subtypes, indicating that there is also a small difference in their ability to present the same 9 peptide.
(2) Binding affinity analysis: to explore the binding strength between the 9 peptides and MHC class I subtypes, a hierarchical heat map of 33 MHC class I subtypes and 668 epitopes was plotted with GraphPad Prism 8.0.1 (fig. 3) to show the relationship between them. Data comes from the ranking of NetMHCPan. This metric is not affected by the inherent deviation of certain molecules from higher or lower average predicted affinities and is therefore a better indicator.
As can be seen from the figure, the epitope binding strength exhibited a regional distribution overall, in which the 1-28, 163-190, 217-244, 352-379, 568-595 and 649-668 epitopes had strong binding affinity to the MHC class I molecule, while the 109-136, 298-325, 379-460 and 595-649 epitopes had weak binding affinity to the MHC class I molecule.
(3) Immunogenicity analysis: immunogenicity analysis was performed on all predicted 9 peptide epitopes in an R studio environment using the PAAQD2.0 software package. Immunogenicity is only related to the polypeptide sequence, not to the spatial structure. If the assay result probability >0.5, the peptide is considered immunogenic, otherwise it is not. High affinity polypeptides may not be able to sufficiently induce an immune response. In addition to being immunoreactive, an antigen should also be immunogenic. All EBOV GPs were analyzed using the PAAQD software package to predict immunogenicity of the 9 peptides, judging whether they are immunogenic according to their prediction probability. If the likelihood >0.5, the peptide is considered immunogenic. Thus, 309 of 668 EBOV GP predicted 9 peptides to be immunogenic. Specifically, 148 peptides out of 301 HLA class I subtype dominant epitopes were immunogenic and 25 peptides out of 42H-2 subtype dominant epitopes were immunogenic.
(4) Conservation assay
To determine the conservation of the predicted epitopes between protein sequences of different EBOV strains, we performed ebola virus interspecies and intraspecies conservation analyses on all the selected dominant predicted epitopes using the BLASTP tool. The criterion for intraspecies conservation is Zaire Ebola virus (taxid:186538) in addition to Zaire Ebola virus (1995) (taxid: 128951). The criterion for interspecies conservation is an Ebola virus (taxid:186536) other than the Zaire Ebola virus (taxid: 186538). In the analysis results, if the E value < E-5, the peptide is considered to be conserved. Dominant epitopes can therefore be divided into four broad categories according to conservation status: interspecific-intraspecies-; interspecific-intraspecies +; interspecific + intraspecies-; and interspecies + intraspecies +.
Multiple sequence alignments determine the conservation status of the EBOV GP dominant epitope. Based on the conservation analysis of pan-MHC class I restricted epitopes, FIG. 4 lists the statistics of the conservation analysis of all dominant epitopes.
For the analysis statistics of pan MHC class I dominant epitopes, it can be seen from FIG. 4 that pan HLA class I restricted dominant epitopes are more conserved than H-2 restricted dominant epitopes. This is because the results in HLA I summarize the superfamily in many alleles, and the identification of H-2 restricted epitopes requires the approval of the 3/5 algorithm. Meanwhile, it is clear that the dominant epitope shows strong interspecies conservation but weak intraspecies conservation.
(5) peptide-MHC molecule docking
HPEPDOCK is a novel network server, and can simulate molecule docking by inputting a 9-peptide dominant epitope sequence and an MHC class I molecule RDB format file, so that a docking model is obtained. Relevant MHC class I molecule 3D structural data { HLA-A1[ HLA-A0206 (3OXR) ], HLA-B7[ HLA-B0702 (5EO1), HLA-B3501 (1A9E), HLA-B5101 (1E28), HLA-B5301 (1A1N) ], HLA-B8[ HLA-B QR 0801 (4P) ], HLA-B15[ HLA-B1501 (1XR9), HLA-A0101 (4NQV) ], HLA-B44[ HLA-B4402 (3)) ], H2-Ld (6L9M), H2-Kb (6JQ3), and H2-Db 5961 (5EO 1). Docking of each 9 peptide with the same MHC class I molecule resulted in 100 mock docking structures, but the top 10 mock docking structures were considered the most important predictors in silico.
Binding affinity was assessed as described above and 9 peptide-MHC class I docking was performed. After molecular docking, the binding conformation of MHC class I alleles and corresponding dominant epitopes, and respective binding energy and docking models are obtained. We selected 5 murine dominant epitopes with interspecies conservation for docking with the corresponding MHC class I molecules. Meanwhile, the 5 dominant epitopes are also the dominant epitopes of human HLA class I subtypes, and then the simulated 9 peptides are in butt joint with corresponding HLA class I molecules. Lower scores indicate better docking performance. Each 9-peptide docking resulted in 10 different mock docking structures.
As can be seen from the docking results table 1, the docking scores for 9 peptides GPCAGDFAF, GAFFLYDRL and TVIYRGTTF were lower for some human HLA class I subtypes than for the mouse H-2 allele, indicating that these 9 peptides may perform better in human immune responses. In contrast, the other two epitopes FHKEGAFFL and LPQAKKDFF showed opposite trends, expressing their preference for binding to the mouse H-2 allele. Figure 5 shows a docking model of human HLA class I molecules with lower docking scores with the corresponding 9 peptides, and a docking model of the 9 peptides with the corresponding mouse MHC class I molecules.
TABLE 1 results of the docking
Since peptide TELRTFSIL had no corresponding RDB file for its corresponding mouse MHC class I molecule, mock docking of this 9 peptide with mouse MHC class I molecules was not performed. The docking model was also included in the figure because the corresponding human HLA class I molecules were subjected to docking simulation.
Step 5: enzyme linked immunospot (ELISpot) assay validation:
artificially synthesized mouse high affinity 9 peptide was diluted with sterile PBS solution to a final concentration of 20 μ g/mL for ELISpot assay. IFN-gamma specific capture antibody was first diluted with sterile PBS to a final concentration of 5. mu.g/mL and added to a 96-well plate at a volume of 100. mu.L per well for overnight incubation at 4 ℃. The 96-well plate was then sealed with 10% fetal bovine serum in RPMI1640 medium for 2h at room temperature. After the collected mouse spleen cell suspension is subjected to red blood cell lysis, the spleen cells are resuspended in RPMI1640 medium containing 10% fetal bovine serum and 1% double antibody, and the ratio of each well is 1 × 106The number of splenocytes was added to a 96-well plate. Adding the diluted 9 peptide with the same volume, 37 ℃ and 5% CO2Incubate in incubator for 24 h. The negative control is RPMI1640 medium containing 10% fetal bovine serum and 1% double antibody, and the positive control is canavalin A (Con A) of 10. mu.g/mL. After incubation, plates were washed with double distilled water and PBST. After washing, 100. mu.L of biotinylated anti-IFN-. gamma.detection antibody was added to the wells at a final concentration of 2. mu.g/mL and incubated for 2h at room temperature. After washing with PBST, streptavidin-horseradish peroxidase (streptavidin-HRP) was added and incubated for 1h at room temperature. After PBST washing, AEC substrate was added for color development and the reaction was stopped by washing with water. And after the 96-well plate is air-dried, scanning and counting spots in the 96-well plate by using a CTL-immunoSpot plate reading machine. All results are shown as per 106Spot Forming Unit (SFU) mean for individual splenocytes. The bar graph was plotted using GraphPad Prism 8.0.1.
25 immunodominant epitope peptides (shown in SEQ ID NO. 1-25) of the H-2 restriction 9 peptide with immunogenicity are artificially synthesized and used for ELISpot test verification. The peptide was used to stimulate splenocytes from mice immunized with pVAX-GPEBOV plasmid, and IFN-. gamma.secretion was observed. ResultsShown as 10 in figure 66Spot Forming Unit (SFU) mean for individual splenocytes. It can be seen from the figure that the spleen cells of BALB/C and C3H mice can secrete IFN-gamma after being stimulated by 25 pieces of 9 peptides respectively. Interestingly, the amount of IFN-. gamma.secreted by splenocytes from BALB/C mice was greater than that secreted by splenocytes from C3H mice, whether stimulated by the H-2d or H-2k restricted epitopes.
Example 2
Application of dominant epitope peptide of Ebola virus envelope glycoprotein
The invention lays a foundation for the research of the EBOV novel genetic engineering vaccine through the prediction of dominant MHC class I epitope peptide on the EBOV GP, various computer analyses and ELISpot test verification.
The experimental research shows that: the dominant MHC class I epitope peptide on the EBOV GP can effectively stimulate the cellular immune response of the body.
In recent years, although vaccines related to EBOV have been developed at home and abroad, from the application point of view, the vaccines still have a plurality of defects, wherein the most important point is that the immune response of cells generated by an organism is not good enough, and certain potential safety hazards exist. If the EBOV GP dominant MHC class I epitope peptide screened by the method is applied to the stimulation of the cellular immune response reaction of an organism, the EBOV GP dominant MHC class I epitope peptide has the following advantages:
1. following viral infection, Antigen Presenting Cells (APCs) activate CD8+ T cells by MHC class I antigen presentation. In the case of MHC class I molecules, the antigen binding groove is blocked at both ends by conserved tyrosine residues, resulting in the size of the bound peptide being generally limited to 8-10 amino acid residues. Therefore, we selected a9 amino acid peptide as the predicted epitope. At the same time, the cellular immune response mediated by CTL plays an important role in combating viral infections. After the CTL recognizes virus-infected cells (target cells), the target cells are cracked and killed by releasing perforin and granzyme and passing through the FasL-Fas pathway, so that the effect of effectively eliminating virus infection in an organism is achieved. The level of a certain virus-specific CTL in the organism and the activity thereof are positively correlated with the virus-removing effect.
2. Previous studies have shown that neutralizing antibodies specifically bind to viruses preventing them from infecting host cells, and therefore most of the researchers have devoted much effort in the study of neutralizing antibodies that specifically bind to EBOV GP. However, for an infected host cell, CD8+ T cells are required to exert a cytotoxic effect to clear the virus of intracellular infection.
3. Peptide prediction methods were used to predict the binding affinity of each 9 peptides to specific MHC class I molecules. Currently, there are many different principles for algorithms for predicting peptide affinity, and therefore we use a variety of prediction methods to predict polypeptides with stronger binding affinity. At the same time, we performed an immunogenicity analysis on all predicted 9 peptides to eliminate high affinity non-immunogenic epitopes.
4. After the 9 peptide dominant epitope is subjected to the analysis of the conservation among and in ebola virus species, the dominant epitope can be simply classified. Compared with other epitopes, the dominant epitope conserved among species is more suitable for vaccine development and immune research. The interspecies-conserved dominant mouse epitopes were mock-docked with the corresponding mouse MHC class I and human HLA class I molecules. These epitopes were verified by ELISpot assay to be able to elicit immune responses in mouse splenocytes.
5. There are studies in which volunteers were immunized with EBOV GP vaccine and then collected Peripheral Blood Mononuclear Cells (PBMCs) for ELISpot testing and the volunteers were HLA typed. The results of the study show that the polypeptides TELRTFSIL and TVIYRGTTF can induce IFN-gamma release from human T cells. Both polypeptides are among the 25 dominant epitopes of the 9 peptides we predict. The mice were shown to share common EBOV GP epitopes with humans, suggesting that other mouse dominant epitopes predicted herein may be antigen presented by human HLA class I molecules.
In the research, the bioinformatics method is used for performing polypeptide affinity prediction, immunogenicity analysis and conservation analysis on the EBOV GP, and meanwhile, docking simulation of the polypeptide and MHC class I molecules and prediction results of experimental verification software are performed, so that another idea is provided for subsequent Ebola virus related immunology research, and the epitope-based EBOV epitope vaccine is favorably designed in the future. Meanwhile, the method for predicting the high-affinity peptide and carrying out subsequent correlation analysis can also be used for basic immunity research and infectious disease prevention and control of other pathogens. Furthermore, epitope prediction has also been documented as being useful in the treatment and intervention of cancer.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations.
Sequence listing
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