MX2010005637A - Modified polyhedrin, and polyhedras and biotechnological products obtained from same. - Google Patents

Abstract

The invention relates to a fragment of the polyhedrin sequence that allows any peptide or protein containing said fused sequence to be incorporated into the crystal of the polyhedra and to form part thereof. Moreover, any type of sequence, such as vaccine sequences, can be introduced into the crystal independently of the disease against which protection is sought. Depending on the import sequence used, short or prolonged release crystals might be generated, which allows the generation of continuous controlled release vaccines. The point mutations of the polyhedrin can be used to change the shape, size and stability of the polyhedra crystals. Similarly, the crystal allows the functional properties of the proteins of interest incorporated therein to be maintained. The latter applies to the use of enzymes of biotechnological interest, which can be maintained at ambient temperature inside the crystal for prolonged periods without losing the functional properties thereof.

Description

"MODIFIED POLYHYDRINE, POLYHYDRATES AND PRODUCTS BIOTECHNOLOGICAL OBTAINED FROM THE SAME FIELD OF THE INVENTION The present invention is related to the techniques used in genetic engineering for obtaining biotechnological products such as proteins, vaccines, enzymes, molecular markers, etc. and more particularly, it is related to the protein known as polyhedrin which includes point mutations in its chain, as well as the polyhedra and biotechnological products obtained from said mutated polyhedrin. These mutations are introduced in specific places within the first 58 or 110 amino acids, counted from the amino terminus of the polyhedrin. Likewise, these 58 amino acids are identified, as the minimum sequence of import to the polyhedra crystal, and the sequence 1 0 (which contains the amino acids 1-58 in its sequence) can also be used. The use of 58 (SEQ ID NO: 1, of the annexed sequences document) or 110 (SEQ ID NO: 2, of the annexed document sequences) first amino acids of the polyhedrin (and mutations contained therein) fused to proteins of biotechnological interest , allow the incorporation of said proteins to the crystal of the polyhedra and the subsequent release of the proteins of interest in a regulated manner. The mutations introduced in the polyhedrin allow to control the morphology and size of the polyhedra crystals when it is expressed in cells of insect. Within the biotechnological products that can be obtained from the present invention, vaccines for human or veterinary use are found that are resistant to changes in ambient temperature, so they do not require refrigeration and are easy to store. From the present invention, it is also possible to obtain polyhedra crystals containing enzymes of biotechnological interest, which retain their enzymatic activity (even contained within the polyhedra crystal), as demonstrated herein. Thus, depending on the sequence used to produce the fusion proteins (amino acids 1-58 or 1-1 10 of the polyhedrin) and of the mutations contained in these sequences, it is possible to obtain crystals of rapid or prolonged release, such and as demonstrated in this document.

BACKGROUND OF THE INVENTION Baculoviruses are viruses enveloped in a lipid bilayer containing a double-stranded circular DNA genome of approximately 80 to 180 kilobases (kb) (Smith, GE, and Summers, MD (1978).) Analysis of baculovirus genomes with restriction endonucleases. Virology 89, 517-527). These viruses belong to the Baculoviridae family and more than 500 species of baculoviruses obtained from more than 600 species of insects have been described (Blissard, GW, and Rohrmann, GF (1990).) Baculovirus diversity and molecular biology Annual review of entomology 35 , 127-155).

Baculoviruses exclusively infect arthropods of the order Lepidoptera (butterflies and larvae), Hymenoptera (flies) and Diptera (flies and mosquitoes) as well as some crustaceans of the order Decapoda (shrimp) (Couch, JA (1974).) An enzootic nuclear polyhedrosis virus of pink shrimp: ultrastructure, prevalence, and enhancement, Journal of invertebrate pathology 24, 311-331); and, (Blissard, G.W., and Rohrmann, G.F. (1990)); (Herniou, E.A., Olszewski, J.A., Cory, J.S., and O'reilly, D.R. (2003), The genome sequence and evolution of baculoviruses, Annual review of entomology 48, 211-234).

On the other hand, there are baculovirus species capable of infecting insects of the order Coleoptera, Neuroptera, Thysanura and Trichoptera; however, there is still no molecular characterization of such baculoviruses (Herniou, EA, Olszewski, JA, Cory, JS, and O'reilly, DR (2003); (Van Oers, MM, And Vlak, JM (2007). genomics, Current drug targets 8, 1051-1068).

In general, baculoviruses are harmless to humans and mammals in general. The Baculoviridae family is classified into two genera according to the sixth report of the International Committee on Taxonomy of Viruses (ICTV, International Committee on Taxonomy of Viruses), Nucleopolyhedrovirus (NPVs) and Granuloviruses (GVs) (Jehle, JA, Blissard, GW, Bonning, BC, Cory, JS, Herniou, EA, Rohrmann, GF, Theilmann, DA, Thiem , SM, And Vlak, JM (2006) On the classification and nomenclature of baculoviruses: a proposal for revision, Archives of virology 151, 1257-1266). The classification is based on the characteristics Structures of bodies formed by a protein matrix called occlusion bodies (OBs, occlusion bodies) or polyhedra (Summers, M.D., And Smith, G.E. (1975), Comparative studies of baculovirus granulins and polyhedrins, Intervirology 6, 168-180).

The OBs or polyhedra are mainly formed by a protein of 29 kilo Daltons (kDa) encoded in the viral genome called polyhedrin, which is synthesized in large quantities during the late phase of infection and accumulates in the nucleus of infected cells where it forms macromolecular crystals called polyhedra (Rohrmann, GF (1986), Polyhedrin structure, The Journal of general virology 67 (Pt 8), 1499-1513).

NPVs produce large polyhedra (1.5 μ? T? Long) in the nucleus of the infected cell, which contain a large amount of virions (baculovirus) inside them (Slack, J., And Arif, BM (2007 The baculoviruses occlusion-derived virus: virion structure and function Advances in virus research 69, 99-165). The polyhedra selectively incorporates baculoviruses, such that it does not contain genetic material or proteins of the insect host cell. The mechanisms of so selective polyhedra importation are unknown to date (Slack, J., And Arif, BM (2007) .The baculoviruses occlusion-derived virus: virion structure and function.Advances in virus research 69, 99-165) . In the present proposal, the amino acid sequence 1 -58 of the polyhedrin is identified as the minimum sequence necessary to incorporate proteins or peptides of interest to the polyhedra crystal efficiently and selectively.

Baculoviruses have been used exhaustively for the production of recombinant proteins of human and animal origin. Said proteins can be used for diagnostic or therapeutic purposes. In addition, proteins of viral origin have been produced for viruses that affect humans and livestock, using the insect virus production system. This has been achieved by introducing copies of the genes to be expressed within the baculovirus genome, and using insect cells to produce the recombinant proteins in a soluble form (not incorporated in the polyhedra crystal).

Because baculoviruses do not affect humans or mammals in general, this system has been approved by the FDA (Food and Drug Administration) as a bioinsecticide. For this purpose wild baculoviruses (without genome modifications) or recombinant baculoviruses have been used, which express several toxins for various pests and insects that affect crops of human interest.

Emphasizing polyhedrin, this is a protein encoded within the genome of the insect virus, which consists of a sequence of 243 amino acids. This protein is undoubtedly the most expressed by the insect, once infected host cells (about 20% of the protein produced by the virus is polyhedrin), which makes it a very useful target to produce large amounts of proteins recombinants.

Polyhedrin forms paracrystals with polyhedral shape in insect cells (so-called polyhedra). Hundreds of people are trapped inside of free baculoviruses. The large number of polyhedra that are formed inside the insect cell end up lysing it, and the polyhedra are released into the environment. Viruses occluded within the polyhedra resist inclement weather for years. Viruses occluded within the polyhedra survive for up to 10 years, being perfectly infective. This indicates that viruses are protected within the polyhedra crystal, safeguarding their integrity and viability for years, even at room temperature.

In the patent documents US 4,870,023, EP 0,349,594 A4, and JP 2003- 319778, the use of the polyhedra of a virus is described. As mentioned above, there are about 500 different viruses within the large family of baculoviruses. The three previous documents are directed to the use of proteins or peptides fused to the wild sequence of the polyhedrin, that is, for these fusions the complete sequence of 243 amino acids is used, which does not allow to have an adequate control over the morphology of the produced polyhedra and only allows the production of monovalent polyhedra particles, that is, it only allows the fusion of a sequence of a single type of peptide or a protein to the polyhedrin. In short, the wild-type polyhedrin sequence has no modifications in its DNA sequence.

In the document Identification of an amino acid essential to the normal assembly of Autographa californica nuclear polyhedrosis virus polyhedra. J Virol. 1986 May; 58 (2): 684-688. E B Carstens, A Krebs, and C E Gallerneault, discloses the sequence of the mutant polyhedrin called M5, with the wild polyhedrin sequence of Autographa californica. The M5 point mutation is caused by the substitution of leucine instead of the original proline at site 58, and it was found that it is responsible for the cubic appearance of the produced polyhedra, it was found that these recombinants that have this point mutation have a low production This mutation corresponds to nucleotide 172 of the polyhedrin gene, the change is 5 -GGATCC-3 'to 5 -GGATCT-3' in M5. However, in said document the complete sequence of the polyhedra is used, unlike the present proposal. Likewise, nothing is described about the stability of the proteins incorporated in the cubic crystal versus the wild crystal (original). Nor is there mention of the ability to control the release times of the protein of interest introduced into the polyhedra. The above can only be achieved using the fragments mentioned in this document.

The fusion of proteins of interest to the complete polyhedrin gene in order to produce vaccines are mentioned in the documents Recombinant breast carcinoma-associated mucins expressed in a baculovirus system containing a tumor specific epitope. Immunotechnology. 1998 Oct; 4 (2): 97-105. Hu P, Wright SE. In this document the complete polyhedrin gene is used and without point mutations.

The documents that are mentioned below use the promoter of the polyhedrin gene for the production of antigens or proteins of interest. These antigens are not incorporated into the polyhedra crystal and only the promoter sequence of the polyhedrin contained in the insect genome is used for the production of antigens in insect cells. European patent application EP0260090 03/16/1988. Expression of hepatitis B viral antigens from recombinant baculovirus vectors. U.S. Patent 4,745,051 issued 05/17/1988 The Texas A &M University System. Method for producing a recombinant baculovirus expression vector. European patent application EP0385394 02/27/1990. Res, Ass Biotech Agricult Chem Polyhedrin gene and genetic engineering thereof.

The documents mentioned below use the complete polyhedrin gene to produce fusion proteins, this does not allow to regulate the amount of protein integrated in the crystal, nor modulate the release of the protein of interest (rapid or prolonged release), which is only achieved using sequences 1-58 and 1-100 with or without incorporation of the mutations described herein. Patent in Japan JP06339382, 08/04/1994. HOKKO CHEM IND CO LTD. Polyhedrin gene and its use. Patent US 7,432,347 B2. Patent US 7,619,060 B2.

The use of the complete polyhedrin sequence as it has been proposed and described in the aforementioned documents does not allow the release of the proteins or peptides fused to said sequence, because the polyhedra is more stable and durable. Nor does it allow to control the amount of protein of interest incorporated within the crystal of the polyhedra. Likewise, the protein or peptide fused to the Complete polyhedrin sequence is difficult to process to recover the biotechnological product of interest.

The use of the first 1-58 or 1-110 amino acids of the polyhedrin sequence fused to the protein of interest (as described in the present document) allows a more homogeneous incorporation of the protein of interest to the crystal of the polyhedra, and facilitates the controlled release of the protein of interest once it is introduced into the organism, as demonstrated in the present document (Figures 4A and 4B). The point mutations described in the present proposal allow regulating the shape of the crystal (cubic or polyhedral) and thereby modulate the release times of the protein of interest (Figures 3 and 4).

In relation to this fact and in a general way, it can be mentioned that in the biotechnology industry more stable products with longer shelf life are required than those currently known, for example, it can be mentioned that a general problem of vaccines , is that many of them require refrigeration and can not be taken to remote places such as rural and sparsely populated areas, new vaccines are needed that can have a long shelf life and still be effective when applied. Similar problems occur with other biotechnological products such as enzymes, etc.

BRIEF DESCRIPTION OF THE INVENTION In order to solve the problems of the current technique with respect to the use of the complete polyhedrin sequence in its wild form, progressive deletions of the wild-type polyhedrin gene were made in order to identify the minimum sequence of incorporation into the crystal of the polyhedrin. polyhedra Two partial sequences were identified which comprise amino acids 1-58 and 1-110 of the polyhedrin sequence. The first aspect of the invention, therefore, describes the use of sequence 1-58 to produce crystals of rapid absorption, which implies a release of the protein or peptide of interest to the blood system in the first 10 days of its intramuscular injection. (Figure 4B). The use of sequence 1-110 produces polyhedra crystals of prolonged release, allowing the release of the protein or peptide of interest to the blood system at 14 days after its intramuscular injection (Figure 4A). Mutations introduced into these polyhedrin fragments allow an extra level of control in the release of the protein or peptide of interest into the bloodstream.

Specifically, the mutation at position 25 consists of introducing an Aspartic Acid (GAT) instead of a Glycine (GGT) from the wild-type polyhedrin sequence. This mutation produces crystals of cubic form (Figure 3A) and 5 times of larger size compared with the crystal produced without the mutation referred to above. These new crystals allow a release of the protein or peptide of interest to the blood system within the first 24 hours of its intramuscular injection, when the mutation is introduced in sequence 1-58 (Table 1). If sequence 1-110 is used, the mutation described above allows a release to the bloodstream within the first 5 days of its intramuscular injection (Figure 4B).

TABLE 1. Mean times of fluorescence duration (in days) in vivo of polyhedra containing the green fluorescent protein as a reporter, which was incorporated into the crystal of the polyhedra using the sequences described below. The average times were obtained from in vivo studies as shown in Figure 4 of the present application.

While the mutation at position 59, consists of the introduction of a Leucine (CTT) instead of the Proline (CCT) of the wild sequence. Said point mutation allows the crystals formed to be cubic in shape, and the protein or peptide of interest contained in the crystal to be released into the blood system within the first 7 days when the sequence 1-110 is used (Table 1). Table 1 describes the type of sequence of polyhedrin used (with and without the mutation described) and the average times of release into the bloodstream.

More specifically, the polyhedrin fragment (1-58 or 1-110 with or without point mutations described) is fused, by genetic engineering techniques such as the polymerase chain reaction (PCR) and the ligation of DNA fragments to plasmids , with an amino acid sequence of the protein, peptide or biotechnological product of interest in its form of genetic material so that from this fusion polyhedra containing the product of interest are generated when expressed in insect cells, where the amino acid sequence it does not lose its function.

A second aspect of the invention is to provide a polyhedra with controlled morphology whose crystals are formed by the fusion protein to allow the prolonged release of the protein, peptide or biotechnological product of interest. In this sense, in the same polyhedra it is possible to incorporate fusion proteins whose portion corresponding to the amino acid sequence is of a different nature. The crystals of the polyhedra are different from one another in their portion of the amino acid sequence. For example, by the present invention it is possible that the same polyhedra intended to produce vaccines may contain fusion proteins for more than one disease, that is, a multivalent vaccine (against several diseases) is achieved.

The use of any of the polyhedrins developed by the authors of the present invention has the advantage of reducing the number of viruses encapsulated in the expressed polyhedra, which is very convenient because polyhedra of very high purity and free of baculoviruses are obtained, which are clearly useful for diverse biotechnological applications.

Particularly, in the development of the present invention it was found that the use of a fragment of the polyhedrin with the first 58 amino acids thereof fused to some amino acid sequence of a biotechnological product of interest allows a 10 day late release of said product, while the use of a fragment with the first 110 amino acids allows a late release of 14 days (Table 1).

In the case of using the modified polyhedrin sequence with amino acids 1-110, about 100,000 copies of the fusion protein are obtained per unit of polyhedra crystal, ie it is a crystal with a higher content of peptide or protein of interest. Whereas, with the use of the polyhedrin sequence in its amino acids 1-58, about 10-50 times fewer copies (ie, about 200 to 1000 proteins of interest per crystal) of the fused proteins or peptides are obtained said polyhedrin sequence.

By way of example, and in the case that vaccines are desired, the technique of the present invention allows regulating the number of copies and the amount of immunological protection induced in a vaccine containing the crystals. The present invention can be used to obtain any type of vaccine against viruses, bacteria and parasites. The use of more of a protein of interest by glass allows to produce polyvalent vaccines that protect against different viruses and bacteria in the same vaccine or crystal.

In a further aspect of the invention there is provided a method for producing polyhedra, the method comprising fusing an amino acid sequence to a polyhedrin fragment as defined above, expressing said fusion proteins in a baculovirus, preferably Autographa califorica and infecting cells of insect to obtain polyhedra and recover by means of a single centrifugation step.

The present invention is also directed to biotechnological products comprising a polyhedrin fragment as defined above, said products, being vaccines, enzymes, proteins, etc. As shown in Figure 5, the enzymes contained within the polyhedra crystal, using fusions of the amino acids 1-58 or 1-110 to the enzyme of interest, allow to produce enzymes incorporated within the polyhedra crystal, which maintain its enzymatic activity for several years.

One of the advantages of the present invention is that it is scalable, which allows the production of large quantities of the desired biotechnological product, with a high degree of purity and at a low cost of production and purification (Figure 2).

BRIEF DESCRIPTION OF THE FIGURES The novel aspects that are considered characteristic of the present invention will be established with particularity in the appended claims. However, the same invention in conjunction with other objects and advantages thereof, will be better understood in the following detailed description of certain preferred embodiments of the invention, when read in conjunction with the accompanying drawings, in which: Figure 1 shows the transfer plasmids for the production of recombinant baculoviruses including the polyhedrin of the present invention.

Figure 2 shows the recombination procedure with linearized viral genome and the recovery of the expressed polyhedra.

Figure 3A shows three photographs obtained by confocal microscopy (left), electron scanning microscopy (center) and light transmission microscopy (right) of insect cells expressing a cube-shaped polyhedra particle.

Figure 3B shows three photographs by confocal microscopy (left), electron scanning microscopy (center) and light transmission microscopy (right) of insect cells expressing polyhedra without mutation in the polyhedrin sequence.

Figure 4 shows an image obtained by fluorescence equipment for in vivo imaging of a rat before it was injected with the green fluorescent protein (GFP) fused to a fragment of the modified polyhedrin (shown in the panel without nanovaccine).

Figures 4A and 4B show a pair of images obtained by live imaging of a rat with intramuscular injection of approximately 10,000 polyhedra obtained from the modified polyhedrin sequence (amino acids 1-110 in Figure 4A and 1-58 in Figure 4B ) fused to the green fluorescent protein (GFP) used as a reporter.

Figure 5 illustrates the enzymatic activity of 2 enzymes fused to the polyhedra using the import sequence 1-110. In this figure the enzymatic activity is measured for the enzyme luciferase (Figure 5A) and beta-galactosidase (Figure 5B). These results demonstrate that it is possible to selectively introduce 2 enzymes of completely different structures and activities to the polyhedra crystal, using the import sequence 1-110. Moreover, these enzymes maintain their post-substrate selectivity and their enzymatic activity even within the crystal. This is the first time that the incorporation of enzymes to the crystal of the polyhedra and how its enzymatic specificity and activity is maintained.

DETAILED DESCRIPTION OF THE INVENTION Mutations in the wild-type polyhedrin sequence are introduced by polymerase chain reaction (PCR) with oligonucleotides containing the change or changes to be introduced, which they are techniques well known to those skilled in the art. SEQ ID NO: 1 and SEQ ID NO: 2 respectively show the nucleotide sequence corresponding to the fragments comprising amino acids 1 to 58, and 1 to 10 of the wild-type polyhedrin.

More particularly, in position 59 the mutation consists of introducing a Leucine (CTT) in place of the Proline (CCT) of the wild-type sequence. While the mutation at position 25 consists of introducing an Aspartic Acid (GAT) instead of a Glycine (GGT) from the wild-type polyhedrin sequence. Said point mutations in places 25 and / or 59 allow controlling the size and shape of the expressed polyhedra crystal, as well as its stability, particularly the mutation in place 59 results in the production of cubic-shaped polyhedra crystals.

It is convenient to mention that during the development of the present invention, an exhaustive mutagenesis of the DNA sequences corresponding to the amino acids 1-58 and 1-110 of the polyhedrin was carried out, identifying that the aforementioned mutations at sites 25 and / or 59 for each sequence control the shape, size and stability of the polyhedra crystal in vivo.

For example, with the objective of producing a biotechnological product of prolonged release and low number of copies, what is done initially is to merge, using the genetic engineering techniques mentioned above, the amino acid sequence of said biotechnological product. of interest with the fragment of the polyhedrin comprising its amino acids 1 to 110.

For its part, in order to produce fast-release products, the desired sequence (the desired protein or peptide in its form of genetic material or DNA) is fused with the polyhedrin fragment comprising its components by means of the aforementioned genetic engineering techniques. amino acids 1 to 58 with the mutation in the amino acid 25 (Table 1).

These fusions between the modified polyhedrin sequence and the amino acid sequence of interest are cloned into a transfer plasmid, which contains, at its 5"end, the strong promoter of a baculovirus polyhedra, preferably, cloned from the viral genome. of Autographa califórnica (Figure 1).

More particularly, Figure 1 illustrates the plasmid used to transfer the gene of the desired product to baculovirus DNA, in order to produce recombinant baculoviruses expressing the desired product to the polyhedrin as a polyhedra with controlled morphology.

Figure 1 illustrates the use of the first 1 10 amino acids of the polyhedrin (PHi. O) fused to a gene of the product of interest. The other sequences illustrated with black arrows are required for the correct recombination with the viral genome. These sequences include the baculovirus 1629 gene, which is an important gene for producing recombinant baculoviruses that are capable of producing polyhedra.

As shown in Figure 2, the transfer plasmid (Figure 1), which was developed by the authors of the present invention, is mixed with the baculovirus viral genome (AcMNPV genome) to obtain recombinant baculoviruses that express the product of interest. These recombinant baculoviruses are stored and can be used several times to produce more biotechnological products. Said products are purified from sf9 insect cells.

This construct containing the fused sequences of interest is recombined with the linear viral genome (Figure 2), in order to produce recombinant viral genomes capable of producing modified polyhedra that express the product of interest.

For example, in the case of wanting to introduce different vaccines in the same polyhedra crystal, several transfer vectors can be used during the recombination, thus producing a polyvalent polyhedra against several diseases, or against the same disease using different vaccines thereof. pathogen.

The method of recombination is carried out by transfection of the genetic material of the transfer plasmid, with the genetic material of the linearized viral genome preferably of Autographa califorica.

The recombination takes place in Sf9 and / or H5 insect cells, which are grown in a laboratory flask with controlled temperature and humidity, using the methods and protocols detailed in the following examples, which are illustrative but not limiting of the invention.

Example 1.

Method for the production of recombinant baculoviruses.

The recombinant baculoviruses are generated by homologous recombination of the previously described plasmids (Figure 2) with the AcMNPV linear genomic DNA Bsu36l Bac-N-Blue (Invitrogen, Carlsbad, CA).

In all cases, the expression cassettes are incorporated in the polyhedrin locus within the AcMNPV Bsu36l Bac-N-Blue genome and in the sense orientation to the polyhedra promoter (Figure 1).

Both the transfer plasmids and the linear genomic DNA of AcMNPV are co-transfected by cationic lipids (CELLFECTINE®, Invitrogen Carlsbad, CA) into Sf21 or H5 cells.

One week later, the culture medium is harvested and diluted for plaque assays using low melting point agarose (Sigma, St Louis, MD, USA) and 5-bromo-4-chloro-3-indolyl- -D- galactoside (X-gal, Sigma, St. Louis, MD, USA) to detect the expression of the lacZ gene. After two rounds of purification, the baculoviruses are amplified for 7 days in Sf9 or H5 cells cultured in suspension.

Subsequently, the medium is centrifuged at 3000 rpm for 15 min and the supernatant is collected to perform plaque assays and to know the viral titer. These viruses are highly pure and are ready to produce recombinant polyhedra with integrated vaccines.

Example 2 Method for the production of polyhedra crystals containing vaccines of interest.

For the production of the vaccines, insect cells are infected with the virus containing a wild copy of the polyhedrin at a multiplicity of infection of 1 and is combined with 5 multiplicities of infection of the virus carrying the fused polyhedra copy. 1-1 10 with the vaccine that is intended to be produced.

After one week of infection, the cells are centrifuged at 1500 rpm for 15 minutes and the recombinant polyhedra are collected. These are washed 3 times with saline and centrifuged 3 times more.

Example 3 Comparative example. Expression in insect cells of mutated polyhedrin fused to the green fluorescent protein.

Following the techniques mentioned in Example 1, a fusion protein of the modified polyhedrin was obtained in the amino acid 59 (as already described in the mutation in this position) and fused to the green fluorescent protein, this protein fusion resulted in the production of cubic polyhedra crystals with a diameter of 5 microns (see figure 3A). In this case, the import sequence 1-1 10 of the polyhedrin was used to form the crystal.

In contrast, if the wild-type sequence (without mutation in the amino acid 59) of the polyhedrin fused to said green fluorescent protein is used, the The resulting crystals are of the shape of a polyhedron and have a diameter of 1 miera (see figure 3B).

More particularly in Figure 3A, the upper panel shows 3 photographs obtained by confocal microscopy (left), electron scanning microscopy (center) and light transmission microscopy (right). The particle on the left shows fluorescence because it was incorporated as a reporter to the green fluorescent protein (GFP), which allows its location. In the photograph on the right, an insect cell is shown expressing a single cubic polyhedra particle. These cubic particles are obtained by the mutation explained above.

By way of comparison it can be seen that the three photographs of Figure 3B show the same sequence, but obtained without the mutation that induces a change of shape to cubic, the differences are very evident.

Example 4 In vivo analysis Using the methods described above, the green fluorescent protein (GFP) was fused to a modified polyhedrin fragment comprising its amino acids 1 to 110 as well as to a fragment comprising its amino acids 1 to 58 (SEQ 1 and SEQ 2 of the annexed document of sequences).

An image obtained by fluorescence equipment for in vivo imaging in a rat before (left panel) is shown in Figure 4A. denominated without nanovaccine) of receiving the injection of the fusion proteins mentioned in the previous paragraph. The central panel shows the fluorescence one day after injection of the 1-110 polyhedra and the right panel 10 days after the injection.

In Figures 4B the same rat is shown to which 50 microliters of a subcutaneous injection (about 10,000 polyhedra particles) were administered in the right and left groin area (the injection sites are indicated by white arrows) . The image shows the fluorescence over time (1 and 10 days of observation), illustrating the stability of the protein fused 1-110 inside the animal. This fused protein is of prolonged release, so the fluorescence decays very slowly, while the fused protein is incorporated into the bloodstream. Eventually all the fluorescence disappears from the animal, when all the fused protein is released. The release time can be controlled by the mutations that have been detailed in the present description.

Comparing Figures 4A and 4B it is evident that the sequence of polyhedra 1-110 maintains a fluorescence signal for more than 10 days in the injected animal, while the polyhedrin sequence comprising its amino acids 1-58 is maintained in the animal for less than 10 days, because the GFP protein used as a reporter of crystal stability is released more quickly.

In this way, using sequence 1-110, it is possible to produce biotechnological products of longer duration and prolonged release, and with the sequence of 1-58 products of shorter duration and faster release. Example 5 Stability of 2 enzymes incorporated in the polyhedra.

Figure 5A shows the enzymatic activity of the luciferase enzyme fused to the import sequence 1-110 without mutations. The enzymatic activity of the luciferase within the crystal is compared to the activity of the same free enzyme, which shows that substrate specificity and enzymatic activity remain intact within the polyhedra crystal. Figure 5B shows a second example using the enzyme beta-galactosidase fused to the import sequence 1-110 without mutations. The enzymatic activity of the beta-galactosidase enzyme within the crystal is compared to the activity of the same free enzyme, which shows that the substrate specificity and enzymatic activity remain intact within the polyhedra crystal, as occurs with the example shown in Figure 5A for another enzyme for biotechnological use.

This example demonstrates that the enzymatic activity of biotechnological products is maintained even when the enzymes are contained within a crystal. These crystals were kept at room temperature for a period of 1 year before evaluating their enzymatic activity. The free enzymes of the crystal, if maintained without refrigeration, lose their enzymatic activity in a period of 4 hours. The above is of manifest when comparing the enzymatic activity of enzymes maintained for 4 hours at room temperature, which are shown in Figure 5. The above occurs with both enzymes studied, as shown in Figures 5A (enzyme luciferase) and 5B (enzyme beta-galctosidase). Note how the enzymatic activity of the free enzymes is indistinguishable from the enzymatic activity of the enzymes incorporated in the polyhedra. The standard deviations were not significant between both experimental conditions.

Although certain preferred embodiments of the present invention have been described and exemplified, it should be emphasized that numerous modifications to them are possible, such as the particular mutation performed in the polyhedrin, the sequence fused to said modified polyhedrin etc. Therefore, the present invention should not be considered as restricted except by what is required by the prior art and by the scope of the appended claims.

Claims (19)

1. A fragment of the polyhedrin protein characterized in that it comprises its first 58 or up to its first 110 amino acids, wherein the fragment has a tion at position 25 which consists of introducing an Aspartic Acid (GAT) instead of a Glycine (GGT) of the wild sequence of polyhedrin. While the tion at position 59, consists of the introduction of a Leucine (CTT) instead of the Proline (CCT) of the wild sequence.
2. The polyhedrin fragment according to claim 1, characterized in that it comprises amino acids 1 to 58.
3. The polyhedrin fragment according to claim 1, characterized in that it comprises amino acids 1 to 110.
4. A polyhedrin fusion protein, characterized in that it comprises a polyhedrin fragment as claimed in claim 1 fused to an amino acid sequence.
5. The polyhedrin fusion protein according to the claim 4, characterized in that said amino acid sequence is the sequence of a vaccine, a protein, an enzyme, a reporter, a marker or the sequence of any other biotechnological product.
6. The polyhedrin fusion protein according to the claim 5, characterized in that said amino acid sequence is from a vaccine.
7. 7. - A polyhedra characterized in that its crystals comprise a fusion protein as claimed in any of claims 4 to 6.
8. 8. - The polyhedra, according to claim 7, characterized in that in the same polyhedra, the crystals are different from one another in their portion of the amino acid sequence.
9. 9. - The polyhedra according to any of claims 7 to 8, characterized in that the polyhedra is expressed by a baculovirus.
10. 10. The polyhedra according to claim 9, characterized in that said baculovirus is Autographa califórnica.
11. 11. The polyhedra according to any of claims 7 to 10, characterized in that the polyhedra has a cubic shape.
12. 12. The polyhedra according to claim 11, characterized in that the polyhedra has a size of approximately 5 microns.
13. 13. The polyhedra according to any of claims 7 to 12, characterized in that when the polyhedra is formed from the modified polyhedrin fragment in its amino acids 1 to 1, it includes at least 100,000 copies of the fusion protein.
14. 14. -A polyhedra according to any of claims 7 to 13, characterized in that when the polyhedra is formed from the polyhedrin modified in its amino acids 1 to 58, 200 to 1000 copies of the fusion protein are obtained with respect to when the amino acid fragment 1 to 1 is used.
15. 15. - A polyhedra according to any of claims 7 to 13, is characterized by different release times of the biotechnological product once injected in vivo, as noted in Table 1.
16. 16. - A method for producing a polyhedra as claimed in any of claims 7 to 15, the method being characterized in that it comprises: a) fusing an amino acid sequence to a polyhedrin fragment as defined in claim 1; b) expressing said fusion proteins in a baculovirus to form polyhedra; Y, c) recover the polyhedra produced.
17. 17. - The use of a modified polyhedrin for the production of polyhedra whose crystals comprise a polyhedrin fragment as defined in claim 1 fused to an amino acid sequence of a biotechnological product that is desired to be obtained.
18. 18. - The use as claimed in claim 16, wherein said product is a vaccine, an enzyme or a protein.
19. 19. - Biotechnological products comprising a polyhedrin fragment as claimed in claim 1.