阅读说明：本技术 一种表达prrsv游离受体重组腺相关病毒及其制备方法和用途 (Recombinant adeno-associated virus for expressing PRRSV free receptor and preparation method and application thereof ) 是由 夏文龙 孙怀昌 刘晓明 吴植 于 2021-09-01 设计创作，主要内容包括：本发明涉及生物技术及动物疫病防控领域,具体公开了一种表达PRRSV游离受体重组腺相关病毒的制备方法,首先构建获得了GV461-SRCR59-Fc/Sn4D-Fc重组病毒载体,然后通过三质粒转染法包装获得重组腺相关病毒rAAV-CD163-Sn。本发明制备得到的融合表达CD163和Sn游离受体的重组腺相关病毒rAAV-CD163-Sn,为阻断PRRSV感染提供了新的生物制剂,也为PRRSV新型疫苗的研发打下了基础。(The invention relates to the field of biotechnology and animal epidemic disease prevention and control, and particularly discloses a preparation method of recombinant adeno-associated virus expressing PRRSV free receptor, which comprises the steps of firstly constructing and obtaining a GV461-SRCR59-Fc/Sn4D-Fc recombinant virus vector, and then packaging by a three-plasmid transfection method to obtain the recombinant adeno-associated virus rAAV-CD 163-Sn. The recombinant adeno-associated virus rAAV-CD163-Sn fusion-expressed CD163 and Sn free receptor prepared by the invention provides a new biological agent for blocking PRRSV infection and lays a foundation for the research and development of PRRSV novel vaccines.)

1. A preparation method of recombinant adeno-associated virus expressing PRRSV free receptor is characterized by comprising the following steps:

construction of S1, GV461-SRCR59-Fc/Sn4D-Fc recombinant viral vector:

firstly, designing a primer pair to amplify an SRCR59-Fc sequence, inserting an amplified product between BamHI and SalI enzyme cutting sites of a GV461 vector, and extracting a recombinant plasmid GV461-SRCR 59-Fc;

then designing a primer pair to amplify the Sn4D-Fc sequence, recovering an amplification product, inserting the amplification product between HindIII and BglII enzyme cutting sites of GV461-SRCR59-Fc, and extracting a recombinant virus vector GV461-SRCR59-Fc/Sn 4D-Fc;

s2, recombinant adeno-associated virus packaging: packaging the recombinant adeno-associated virus by adopting three plasmids, namely GV461-SRCR59-Fc/Sn4D-Fc, pAAV-Helper and pAAV-RC, according to a three-plasmid transfection method to obtain the recombinant adeno-associated virus rAAV-CD 163-Sn;

s3, collecting, concentrating and purifying the recombinant adeno-associated virus obtained in S2.

2. The method for preparing recombinant adeno-associated virus expressing PRRSV free receptor according to claim 1, wherein in S1, the gene sequence of the upstream primer in the primer pair amplifying the SRCR59-Fc sequence is shown as SEQ ID No.1, and the gene sequence of the downstream primer is shown as SEQ ID No. 2.

3. The method for preparing recombinant adeno-associated virus expressing PRRSV free receptor according to claim 2, wherein in S1, the gene sequence of the upstream primer in the primer pair for amplifying the Sn4D-Fc sequence is shown as SEQ ID No.3, and the gene sequence of the downstream primer is shown as SEQ ID No. 4.

4. The method for preparing recombinant adeno-associated virus expressing PRRSV free receptor according to claim 3, wherein the Sn4D-Fc sequence is fused with the sequence of splicing peptide P2A, the Sn4D gene sequence of the first 4 immunoglobulin-like domains of Sn and the gene sequence of porcine IgG Fc fragment.

5. The method of claim 4, wherein the SRCR59-Fc sequence fuses a porcine IgG heavy chain signal peptide sequence, a porcine CD163 molecule 5 th to 9 th cysteine rich domain SRCR59 gene sequence and a porcine IgG Fc fragment gene sequence.

6. The method for preparing the recombinant adeno-associated virus expressing PRRSV free receptor according to claim 5, wherein in S2, the specific packaging process of the recombinant adeno-associated virus rAAV-CD163-Sn is as follows:

respectively absorbing 10 mu g of each of the three plasmids GV461-SRCR59-Fc/Sn4D-Fc, pAAV-Helper and pAAV-RC, mixing, adding 1mL of 0.3M CaCl₂Uniformly mixing, adding the mixture into 1mL of HBS solution, dropwise adding the mixed solution into a culture dish containing AAV-293 cells with 70% -80% confluence, uniformly mixing, placing the mixture in a cell culture box for 6 hours, then removing the culture solution in the culture dish, replacing the culture solution with 10mL of fresh culture solution, placing the culture solution in the cell culture box for continuous culture for 66-72 hours, collecting the cells and the culture solution, alternately freezing and thawing in a refrigerator at-80 ℃ and a water bath at 37 ℃, repeating the freezing and thawing for four times, centrifuging for 10 minutes at 10000 Xg, removing cell fragments, and collecting centrifugal supernatant, wherein the supernatant is recombinant adeno-associated virus rAAV-CD 163-Sn.

7. The method of claim 6, wherein the collection, concentration and purification of the recombinant adeno-associated virus (S3) comprises:

adding 40% PEG8000 solution to rAAV-CD163-Sn virus solution to the final concentration of 8%, placing on ice for 2 hours, mixing uniformly every 15 minutes, centrifuging for 30 minutes at 2500 Xg, resuspending the precipitate with PBS, then centrifuging for 30 minutes at 3000 Xg of the resuspension solution, transferring the supernatant to a 15mL centrifuge tube, adding Benzonase nuclease to the final concentration of 50U/mL, digesting to remove residual plasmid DNA, incubating for 30 minutes at 37 ℃, filtering the virus solution with a 0.45 μm filter, and purifying the filtered virus solution by CsCl density gradient centrifugation.

8. A recombinant adeno-associated virus rAAV-CD163-Sn produced by the production method according to claim 7.

9. A biological agent comprising the recombinant adeno-associated viral rAAV-CD163-Sn of claim 8.

10. Use of the recombinant adeno-associated virus rAAV-CD163-Sn according to claim 8 or the biological agent according to claim 9 for preparing a medicament against PRRSV infection.

Technical Field

The invention belongs to the field of biotechnology and animal epidemic disease prevention and control, and particularly relates to recombinant adeno-associated virus for expressing PRRSV free receptor, a preparation method and application thereof.

Background

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) belongs to the family of arteriviruses, the genus of arteriviruses, is an enveloped, single-stranded, positive-stranded RNA virus with a full length of about 15kb and is capable of causing Porcine Reproductive and Respiratory Syndrome (PRRS) characterized primarily by sow reproductive disorders, increased neonatal piglet mortality, and respiratory symptoms in pigs of various ages. Since the PRRS is discovered for the first time in 1987 in the United states, the PRRS continuously erupts and epidemics all over the world, viruses are isolated for the first time in 1996 in China, and the high pathogenic strain (HP-PRRSV) is developed in 2006 and erupts in most areas of the country, in recent years, PRRSV NADC30-like strains with high homology with the strain NADC30 in Henan, Sichuan, Zhejiang and other areas of China are discovered in succession, and the PRRSV is difficult to purify and eradicate, is one of the most important infectious diseases of pig farms at present, and brings huge economic loss to the pig farming. The disease is mainly prevented and controlled by virtue of inactivated and attenuated vaccine immunization at present, and an inactivated vaccine has better safety, but the immunity stimulated by the inactivated vaccine is mainly humoral immunity, so that the protective effect is slow, the duration is short, and the immune effect is not ideal; the attenuated vaccine has a good protective effect on homologous strains, but has a poor protective effect on heterologous strains, and has the risks of virulence reversion and virus recombination. Therefore, the traditional vaccine is difficult to effectively control the spread of PRRS, and the prevention and control situation is not optimistic.

PRRSV has strict cell tropism, invading the porcine monocyte-macrophage system mainly through a receptor-mediated endocytosis mechanism, of which alveolar macrophages (PAMs) are the first choice, and the PRRSV receptors present on PAM are mainly four: CD163 molecules, Sialoadhesin (Sn), Heparan Sulfate (HS) and Non-muscle myosin heavy chain ii-a (NMMHC-ii a), whereas CD163 and Sn are key receptors mediating PRRSV infection of PAM cells and the binding regions for CD163, Sn receptors and PRRSV are well defined, CD163 is the 5 th to 9 th scavenger receptor cysteine rich domain (SRCR59) and Sn is the first 4 immunoglobulin-like domains (Sn 4D). The virus free receptor is characterized in that an artificially constructed soluble virus receptor and a cell receptor are used for competitively binding the virus, so that the virus free receptor plays a role similar to a neutralizing antibody, and the essence is to block the binding action of the virus and a specific receptor on a target cell. Because the virus receptor is encoded by animal host genome, is highly conservative and is not easy to mutate, the virus infection blocking effect of the free receptor is more stable and reliable than the neutralizing antibody induced by the traditional vaccine, and the free receptor has been successfully used for anti-infection research of the viruses such as Coxsackie virus, mouse hepatitis virus, human coronavirus and the like. Two recombinant adenoviruses (rAdV) expressing CD163 and Sn free receptors have been constructed in the laboratory at the previous stage respectively, and the two free receptors can be successfully expressed and synergistically inhibited from PRRSV infection, however, the expression time of the two free receptors mediated by the rAdV in pigs is short, the rAdV has high immunogenicity, the generation of a carrier specific antibody is not beneficial to the expression of a target gene, and the duration of the free receptors in the pigs cannot be prolonged through multiple vaccinations, so that a delivery carrier needs to be reselected.

At present, PRRSV vaccines are mainly inactivated vaccines and attenuated vaccines, and have the defects of poor cross protection effect, potential virus recombination, strong virulence reversion and the like, and virus receptors are encoded by host cells, are highly conserved and are not easy to mutate.

Disclosure of Invention

The invention aims to provide a recombinant adeno-associated virus expressing PRRSV free receptors and a preparation method and application thereof, and the prepared recombinant adeno-associated virus rAAV-CD163-Sn fusing and expressing CD163 and Sn free receptors provides a new biological agent for blocking PRRSV infection and lays a foundation for the research and development of PRRSV novel vaccines.

The invention provides a preparation method of recombinant adeno-associated virus expressing PRRSV free receptor, which comprises the following steps:

construction of S1, GV461-SRCR59-Fc/Sn4D-Fc recombinant viral vector:

firstly, designing a primer pair to amplify an SRCR59-Fc sequence, inserting an amplified product between BamHI and SalI enzyme cutting sites of a GV461 vector, and extracting a recombinant plasmid GV461-SRCR 59-Fc;

then designing a primer pair to amplify the Sn4D-Fc sequence, recovering an amplification product, inserting the amplification product between HindIII and BglII enzyme cutting sites of GV461-SRCR59-Fc, and extracting a recombinant virus vector GV461-SRCR59-Fc/Sn 4D-Fc;

s2, recombinant adeno-associated virus packaging: packaging the recombinant adeno-associated virus by adopting three plasmids, namely GV461-SRCR59-Fc/Sn4D-Fc, pAAV-Helper and pAAV-RC, according to a three-plasmid transfection method to obtain the recombinant adeno-associated virus rAAV-CD 163-Sn;

s3, collecting, concentrating and purifying the recombinant adeno-associated virus obtained in S2.

Further, in S1, the gene sequence of the upstream primer in the primer pair for amplifying the SRCR59-Fc sequence is shown as SEQ ID NO.1, and the gene sequence of the downstream primer is shown as SEQ ID NO. 2.

Further, in S1, the gene sequence of the upstream primer in the primer pair for amplifying the Sn4D-Fc sequence is shown as SEQ ID NO.3, and the gene sequence of the downstream primer is shown as SEQ ID NO. 4.

Further, the Sn4D-Fc sequence fuses a splicing peptide P2A sequence, Sn4 immunoglobulin-like domain Sn4D gene sequence before Sn and a pig IgG Fc fragment gene sequence.

Further, the SRCR59-Fc sequence fuses a porcine IgG heavy chain signal peptide sequence, a porcine CD163 molecule 5 th to 9 th cysteine-rich domain SRCR59 gene sequence and a porcine IgG Fc fragment gene sequence.

Further, in S2, the recombinant adeno-associated virus rAAV-CD163-Sn specifically packages as follows:

respectively absorbing 10 mu g of each of the three plasmids GV461-SRCR59-Fc/Sn4D-Fc, pAAV-Helper and pAAV-RC, mixing, adding 1mL of 0.3M CaCl₂Uniformly mixing, adding the mixture into 1mL of HBS solution, dropwise adding the mixture into a culture dish containing AAV-293 cells with 70% -80% confluence degree, uniformly mixing, placing the mixture in a cell culture box for 6 hours, then removing culture solution in the culture dish, replacing the culture solution with 10mL of fresh culture solution, placing the culture solution in the cell culture box for continuous culture for about 66-72 hours, converting most cells from an adherent state into round and falling off, collecting the cells and the culture solution, and placing the cells and the culture solution in a refrigerator at minus 80 ℃ and a water bath at 37 ℃ for continuous cultureAnd (3) alternately freezing and thawing for four times, centrifuging for 10 minutes at 10000 Xg, discarding cell debris, and collecting centrifugal supernatant, wherein the supernatant is the recombinant adeno-associated virus rAAV-CD 163-Sn.

Further, in S3, the collecting, concentrating and purifying process of the recombinant adeno-associated virus specifically comprises:

adding 40% PEG8000 solution to rAAV-CD163-Sn virus solution to the final concentration of 8%, placing on ice for 2 hours, reversing and mixing evenly once every 15 minutes, centrifuging for 30 minutes at 2500 Xg, resuspending the precipitate with PBS, subsequently centrifuging for 30 minutes at 3000 Xg of the resuspension solution, transferring the supernatant to a 15mL centrifuge tube, adding Benzonase nuclease to make the final concentration of 50U/mL, digesting to remove residual plasmid DNA, incubating for 30 minutes at 37 ℃, filtering the virus solution with a 0.45 μm filter, and purifying the filtered virus solution by CsCl density gradient centrifugation.

The invention also provides the recombinant adeno-associated virus rAAV-CD163-Sn prepared by the preparation method.

The invention also provides a biological preparation of the recombinant adeno-associated virus rAAV-CD163-Sn

The invention also provides application of the recombinant adeno-associated virus rAAV-CD163-Sn or the biological agent in preparation of a medicine for resisting PRRSV infection.

Compared with the prior art, the invention has the following beneficial effects:

1. after the recombinant adeno-associated virus rAAV-CD163-Sn prepared by the invention is transduced into PK-15 cells, the secretory expression of two free receptors, namely SRCR59-Fc and Sn4D-Fc can be detected, and the recombinant adeno-associated virus rAAV-CD163-Sn can be used for prevention and control research of porcine reproductive and respiratory syndrome viruses.

2. The invention takes recombinant adeno-associated virus (rAAV) as a delivery vector, fuses and expresses a Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) CD163 molecule and a sialic acid adhesin (Sn) free receptor, and provides a novel biological agent for blocking PRRSV infection.

3. The recombinant adeno-associated virus rAAV-CD163-Sn with fusion expression of CD163 and Sn free receptors, which is prepared by the invention, can successfully express two free receptors with biological activity on the in vitro cell level, can be developed as a novel biological preparation for resisting PRRSV infection, provides a novel biological preparation for blocking PRRSV infection, and lays a foundation for the research and development of novel PRRSV vaccines.

Drawings

FIG. 1 is an amplification diagram of the gene sequences of two free receptors of the invention, SRCR59-Fc and Sn 4D-Fc;

FIG. 2 is a diagram showing the double restriction enzyme digestion of the recombinant viral vector of the present invention;

FIG. 3 is a transmission electron microscope image of recombinant viral particles prepared in the present invention;

FIG. 4 shows the Western-blot detection results of two free receptors (SRCR59-Fc and Sn4D-Fc) in the present invention.

Detailed Description

The present invention is described in detail below with reference to the drawings and the specific embodiments, but it should be understood that the scope of the present invention is not limited by the specific embodiments. The test methods in the following examples, which are not specified in specific conditions, are generally conducted under conventional conditions, and the steps thereof will not be described in detail since they do not relate to the invention.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

The sources of the biological materials in the invention are as follows:

1. pShuttle-SRCR59-Fc and pShuttle-Sn4D-Fc vectors were constructed and stored in the laboratory (see the literature: Chen Y, Guo R, He S, et al. additive inhibition of pore regeneration and reactivity syndrome Virus infection with the soluble silica adhesives [ J ]. Virus Research,2014,179: 85-92.).

2. AAV Helper-Free systems (including GV461, pAAV-Helper, and pAAV-RC plasmids) are provided by Kjekay Gene technologies, Inc., Shanghai.

AAV-293 cells were provided by Kjekey Gene science and technology, Inc. of Shanghai.

Example 1

A method for preparing recombinant adeno-associated virus expressing PRRSV free receptor,

the target gene of the recombinant adeno-associated virus rAAV-CD163-Sn is fused by two free receptor coding sequences of CD163 and Sn. Wherein the CD163 free receptor coding sequence is SRCR59-Fc, which fuses a porcine IgG heavy chain signal peptide sequence, a porcine CD163 molecule cysteine-rich domain (SRCR59) gene sequence from 5 th to 9 th and a porcine IgG Fc fragment gene sequence; the Sn free receptor coding sequence is Sn4D-Fc, and the sequence fuses a splicing peptide P2A sequence, a gene sequence of the first 4 immunoglobulin-like domains (Sn4D) of Sn and a gene sequence of a pig IgG Fc fragment.

The preparation process comprises the following steps:

1. construction of recombinant viral vectors

(1) Amplification of SRCR59-Fc sequence: 1 pair of primers are designed according to a porcine CD163 cysteine-rich domain sequence (GenBank accession number: NM-213976) and a porcine IgG Fc fragment sequence (GenBank accession number: LOC396781), the nucleotide sequence of an upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 2:

SEQ ID NO.1：5’-AAGGATCCGTTGGAGGGGACATTCCCT-3’

SEQ ID NO.2：5’-GGGTCGACTCATTTACCCTGAGTCTTGGA-3’

the primers were synthesized by Shanghai Biotechnology engineering Co., Ltd, and PCR amplification was carried out using pShuttle-SRCR59-Fc vector plasmid as a template and PrimeSTAR Max DNA polymerase (purchased from Baozi medical science, Beijing) Co., Ltd.), the result of agarose gel electrophoresis showed that the SRCR59-Fc gene sequence was successfully amplified (FIG. 1), the amplified product was recovered and inserted between the BamHI and SalI sites of the GV461 vector, and recombinant plasmid GV461-SRCR59-Fc was extracted according to the protocol of plasmid extraction kit (purchased from QIAGEN Bio., Germany).

(2) Sn4D-Fc sequence amplification: 1 pair of primers was designed based on the porcine Sn first 4 immunoglobulin-like domain sequence (GenBank accession No.: NM-214346) and the porcine IgG Fc fragment sequence (GenBank accession No.: LOC 396781): the nucleotide sequence of the upstream primer is shown as SEQ ID NO.3, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4:

SEQ ID NO.3：5’-GGAAGCTTATGGACTTCCTGCTCCTGCT-3’

SEQ ID NO.4：5’-TTAGATCTTCATTTACCCTGAGTCTTGGA-3’

the primers were synthesized by Shanghai Biotechnology, Inc., PCR-amplified with pShuttle-Sn4D-Fc vector plasmid as template according to PrimeSTAR Max DNA polymerase (purchased from Baozi medical technology, Beijing) Co., Ltd.), agarose gel electrophoresis showed successful amplification of Sn4D-Fc gene sequence (FIG. 1), the amplified products were recovered and inserted between HindIII and BglII cleavage sites of GV461-SRCR59-Fc, and recombinant plasmid GV461-SRCR59-Fc/Sn4D-Fc was extracted according to the protocol of plasmid extraction kit (purchased from QIAGEN, Germany).

(3) Enzyme digestion identification of the recombinant virus vector: the recombinant viral vector GV461-SRCR59-Fc/Sn4D-Fc was subjected to double digestion with BamHI/SalI, and agarose gel electrophoresis revealed that the recombinant vector released the expected band of 2 entries, which were the plasmid band and the SRCR59-Fc band, respectively (FIG. 2). The GV461-SRCR59-Fc/Sn4D-Fc was double digested with HindIII/BglII, and agarose gel electrophoresis showed that the recombinant vector released the expected 2-entry band, which were the plasmid band and the Sn4D-Fc band, respectively (FIG. 2).

2. Recombinant adeno-associated virus packaging

The recombinant adeno-associated virus was packaged by three plasmid transfection methods, according to the AAV Helper-Free System protocol provided by the Shanghai Jikai Gene science Co. Respectively sucking 10 μ g of each of the three plasmids GV461-SRCR59-Fc/Sn4D-Fc, pAAV-Helper and pAAV-RC, placing the three plasmids into a 1.5mL centrifuge tube, and then adding 1mL of 0.3M CaCl₂Lightly blowing, beating and uniformly mixing; adding the mixed solution into a 15mL conical centrifuge tube containing 1mL of HBS solution, and gently blowing and beating the mixed solution uniformly;and dropwise adding the mixed solution into a culture dish containing AAV-293 cells with 70% -80% confluence degree, slightly shaking the culture dish to uniformly distribute the solution, placing the culture dish in a cell culture box for 6 hours, then removing the culture solution in the culture dish, replacing with 10mL of fresh culture solution, and placing in the cell culture box for continuous culture. And (3) about 66-72 hours, most cells are changed from the adherent state into round and shed, at the moment, the cells and the culture solution are collected, the freeze thawing is repeated for four times in a refrigerator at the temperature of-80 ℃ and a water bath at the temperature of 37 ℃, the centrifugation is carried out for 10 minutes at 10000 Xg, cell fragments are discarded, and centrifugal supernatant is collected, and the supernatant is the recombinant adeno-associated virus rAAV-CD 163-Sn.

3. Collecting, concentrating and purifying recombinant adeno-associated virus

Adding 40% PEG8000 solution into rAAV-CD163-Sn virus solution to final concentration of 8%, standing on ice for 2 hr while reversing and mixing once every 15 min, centrifuging at 2500 Xg for 30 min, discarding supernatant, and re-suspending the precipitate with PBS; subsequently, the resuspension solution was centrifuged at 3000 Xg for 30 minutes, the supernatant was transferred to a 15mL centrifuge tube, Benzonase nuclease (purchased from Merck, USA) was added to give a final concentration of 50U/mL, residual plasmid DNA was digested and removed, and after incubation at 37 ℃ for 30 minutes, the virus solution was filtered through a 0.45 μm filter, and the filtered virus solution was purified by CsCl density gradient centrifugation according to the instructions provided by Kjekay Gentechnology Co., Ltd, and glycerol was added to the purified virus solution to give a final concentration of 5%, and the mixture was stored in a-80 ℃ refrigerator.

4. Recombinant virus and free receptor identification

The prepared recombinant virus rAAV-CD163-Sn has the diameter of about 22nm and typical adeno-associated virus morphology (figure 3) through observation of a transmission electron microscope; rAAV-CD163-Sn is transduced into PK-15 cells (MOI is 100), cell culture is collected after 72h, freezing and thawing are carried out repeatedly for 3 times in a refrigerator at the temperature of-80 ℃ and a water bath at the temperature of 37 ℃, centrifugation is carried out for 15 minutes at 12000 Xg, supernatant is collected to carry out protein gel electrophoresis, Western-blot detection is carried out by using goat anti-porcine IgG, and the result shows that expected SRCR59-Fc and Sn4D-Fc protein bands can be observed (figure 4), which indicates that two kinds of expressed freeforms can be separated under the action of shearing peptide, have bioactivity and can be used for research related to anti-PRRSV infection.

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 also intended to include such modifications and variations.

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