MX2011013050A - Method for modifying the permeability of drugs in the corneal epithelium by using retinoids as modulators of tight junctions. - Google Patents

Abstract

The invention provides methods for modifying the force of the tight junctions located between the corneal epithelium cells of mammals, preferably of human or species of veterinary interest, by using compounds selected from the retinoid family, the analogues and/or derivatives thereof, for allowing or facilitating the transepithelial penetration of the drug of ophthalmologic interest into the intraocular tissues.

Description

METHOD FOR OBTAINING PROTEINS RECOMBIN BEFORE IMMUNOGENIC CAPACITY IN Pichia pastoris AND ITS USE IN U A i I PREPARATION OF VACCINES AGAINST INFLUENCE VIRUSES FIELD OF THE INVENTION The present application is in the field of genetic engineering and biotechnology and in particular in the production of vaccines against the influenza virus AH1N1.

OBJECT OF THE INVENTION The present patent application has as objects of invention: • A method to produce recombinant proteins (antigens) with immunogenic capacity in Pichia pastoris, resulting in a system of artificial biosynthesis of the viral particles of the virus capsid in the form of recombinant proteins; I • A first and second nucleotide sequences coding for the protein section encoding a first and second amino acid sequences respectively; • A first and second amino acid sequences with antigenic activity, designed by genetic engineering and its recombinant expression in Pichia pastoris; and the • Use of the first and second amino acid sequences, referred to as SEQ: ID No. 3 and SEQ ID No. 4, respectively; as vaccines or active ingredients for the development of vaccines against the Influenza AH1N1 virus or viral infections associated with this virus.

• Use of those of the amino acid sequences identified as: SEQ ID NO: 3 and SEQ ID NO: 4, as antigen in diagnostic tests.

BACKGROUND Each year the Influenza A virus causes infections in humans with varying degrees of severity depending on the immunity acquired by the host against the specific strain of the virus. i Three to five million people experience severe illness and from 0.25 to 0.5 million people die from this type of influenza annually, (WHO EBl 11/10). In the United States, more than 50,000 patients die annually due to infectious outbreaks of influenza and its consequences (Thompson WW, et al 2003).

The influenza virus manages to circumvent the immunity of the host by the accumulation of point mutations in its surface glycoproteins, Hemagglutinin (HA) and in some cases in Neuraminidase (NA) or by rearrangements of the segments of different viruses that co-infect the host. same cell leading to a new strain during assembly with HA and NA proteins not previously seen in the same virus, which in the worst case can lead to a pandemic, (Bragstad at al 2008).

During the last 100 years have been considered 3 pandemics of this type, prior to the reappearance of strain AH1N1 during 2009 in Mexico, these 3 pandemics mentioned are: the Spanish in 1918 produced by the strain AH1N1, the Asian eti 1957 produced by the strain AH2N2 and in 1968 the Hong Kong influenza strain AH3N2, it is assumed that each new pandemic and / or infectious outbreak emerges from its reservoirs I natural, birds, as in the case of bird flu that unleashed in recent years in Asia. : In April 2009 an epidemiological emergency was declared in Mexico (Fraser C, et (2009) Science 324 (5934): 1557-1561), after a few weeks in June 2009 a level IV alert was declared by the World Health Organization (WHO), the costs were estimated at 12000 lives at the end 2009 in North America (Mexico, USA, Canada). The Influenza virus is a pathogen that belongs to the family of Orthomyxoviridae, there are 3 types of Influenza A, B, C and are distinguished by antigenic differences in the proteins of the virus, mainly in the nucleoproteins and the matrix proteins, these 3 types of virus differ in their pathogenicity and their genomic organization, in humans the most common viruses are those of Influenza A and Influenza B, which produce disease. i In the particular case of Influenza A viruses are subdivided, in Garios subtypes according to the surface antigens found in their viral capsid, HA and NA, in influenza A viruses there are 16 subtypes of HA (H1-H16) and 9 subtypes of NA (N1-N9) that have been reported to date. Its genome consists of segmented RNA chain (Eight fragments).

Each RNA fragment is formed in conjunction with the nucleoproteins and the polymerase group consisting of PA, PB1 and PB, the so-called ribonucleoprotein complex (RNP), in the complex viral particles 8 of this type are surrounded by a capsid of the protein matrix MI.

The virus in these 8 fragments of RNA of negative polarity in its genome, contains the necessary instructions for the expression and assembly of the necessary resources for I form new viruses, each segment encoding one or two unique viral proteins. t In addition the two surface glycoproteins, Hemagglutinin (HA); and Neuraminidase (NA) are the major determinants of the virulence of the disease, the HA protein forms the major part of the glycoprotein surface of the influenza virus and is responsible for the union between the viral capsids and the sialic acid present in the cell membranes and their receptors in the host. This protein is a 225 kD trimer formed by 3 identical monomers of 75 kD each, each monomer consisting of HA1 polypeptides of 50 kD and HA2 and 25 kD.

It is important, then, that in order to develop an effective and stable vaccine similar to the original viral particles, these are folded and glycosylated in a similar way that they happen when assembling in human cells, for this, the cells in which they are required are required. If the vaccine is produced, have an analogous capacity to form this type of link.

Vectors (plasmids) can be used to express, and manipulate, any DNA sequence of interest, in this way they code for proteins, enzymes and antigens purposes, of various types, such as therapeutic or vaccine development.

,, Then a virus and / or particles encoded by DNA sequences of the same, particularly those viruses with recombinant capacity by the segmentation of their genome as Influenza viruses can be integrated into a vector i and thus be produced in a microorganism on a large scale, without neglecting the appropriate selection to the profile of the virus according to the host that parasites and in this way present the appropriate transformations after the translation and expression of the recombinant pfoteine so that it is functionally and structurally analogous to the original. . i One of the disadvantages of the production of vaccines by this strategy is that it is necessary to isolate the virus, know its genome either from RNA or AUN and from this develop the vector, however in the case of the Influenza virus. At H1N1, the specific sequences are known, from those reported during the infectious outbreak in Mexico in April 2009.

The integrity of the selection marker, the detection of the envelope gene of the I virus, as well as its sequencing and comparison with the original sequence are the fundamental elements to be developed to determine the influence of a high number of generations after the gene recombination in said expression systems.

Mutations at the level of the genetic sequence produced by the cells can be incorporated and maintained, giving as a final result a protein whose characteristics can be adverse in their subsequent use (International Conference on i '; Harmonization: Final Guideline on Quality of Biotechnological Products: Analysis of the Expression Construct in Cells Used for Production of rDNA-Derived Protein Products. ICH Secretariat !, Geneva, Switzerland, February 1996). i ? Pichia pastoris is a methylotrophic yeast, whose ability to grow in the presence of methanol, is genotypically given by the AOXl and AOX2 genes, grows well with substrates such as monosaccharides, alcohols and amino acids (Wolf 1996), which makes it an ideal organism to evaluate the production of proteins of biomedical interest at an industrial level; however, the production of these proteins due to their high added value, fully justify the use of minimal means such as those found in the literature (Lin Cereguino et al 2000).

In the eighties, a strain of Pichia pastoris was developed that grows at high cell densities (Phillips Petroleum Co., Wegner G. H. 1983). Since 1988 many pharmaceutical and biotechnology companies have licensed the technology and its technological platform of Pichia pastoris (Cregg et al., 1993, Lin Cereguino et al 2000, Gellis $ in G. 2000). I · The wild strain, original of Pichia pastoris, X-33, has been genetically modified to obtain derivations, with particular characteristics, such is the case of the GS1 15 strain that does not express the histinidol dehydrogenase protein (His-) and by Genetic Engineering can phenotypically select clones whose recombinant genes of interest can be expressed efficiently and marked by selective growth in a medium without Histidine.

BRIEF DESCRIPTION OF FIGURES Figure 1. Schematic sample of the plasmid pJ201-HA30762 showing the restriction sites and the components of the cloning vector containing the gene sequence SEQ ID No.1 optimized for expression in Pichia pastoris.

Figure 2. Restriction map pJ201-HA30780, this map shows the restriction sites and the components of the cloning vector that contains the gene sequence SEQ ID No. 2 optimized for expression in Pichia pastoris.

Figure 3. Restriction map pPIC9, this map shows the restriction sites for the SEQ ID No. 1 gene sequence.

Figure 4. Restriction map pPIC9, this map shows the restriction sites for the SEQ ID No. 2 gene sequence.

Figure 5. Protein modeling performed of the final structure of the amino acid sequence SEQ ID No. 3; Representation of Structure slats Folded B Representation of the hydrophobic surface.

Figure 6. Protein modeling performed of the final structure of the amino acid sequence SEQ ID No. 3; Representation of the hydrophobic surface.

Figure 7. Protein modeling performed of the final structure of the amino acid sequence SEQ ID No. 2; Representation of Folding structure slats.

Figure 8. Protein modeling performed of the final structure of the amino acid sequence SEQ ID No. 2; Representation of the hydrophobic surface.

Figure 9. Schematic of the procedure for the design and development of the recombinant vaccine. 1 DETAILED DESCRIPTION OF THE INVENTION The method for producing recombinant proteins (antigens) with immunogenic capacity, resulting in a system of artificial biosynthesis of viral particles of the virus capsid in the form of recombinant proteins, the reason for this patent application is represented in Figure 1, and comprises 3 phases that below; They describe.

I. Obtain (1) by molecular engineering a DNA sequence that gives Pichia pastoris the ability to produce viral antigens from Influenza A H1N1 MX 2009.

This phase is carried out in the following stages: a) Identify and select the amino acid sequence consisting of the NI AHI NI Influenza virus sequence from the infectious outbreak strain of 2009 in Mexico, reported in NCBI, genbank with the access number ACQ99617.1. b) Segment the sequence identified in a) according to its antigenic activity to obtain only a fragment of the amino acid sequence, capable of awakening the immune response in less time. It is important to note that the fragment of the amino acid sequence optionally is a monomer of the complete trimer identified as HA30762 or the globular region of the monomer, which we hereinafter identify as HA30780. c) Check the tertiary structure of the fragment obtained in b), preferably by bioinformatic modeling (see figure 6, 7, 8 and 9), optionally using Swiss model (c), when tertiary structure is not maintained, it is returned to the previous stage; but if it is maintained, proceed as in stage d). d) Add to the fragment of the proven amino acid sequence, a histidine tag, consisting of 6 histidines. Particularly, to the FRA30762 fragment the labeling is added at the amino terminal end, and the HA30780 fragment is added at the carboxyl end. Obtaining thus a fragment HA30762 axis 572 amino acids described as SEQ ID No. 3; and a HA30780 fragment described as SEQ ID No. 4 e) Optionally, a cut-off site for enterokinase is added to the HA30780 fragment between the histidine labeling and the amino acid sequence fragment. The cutting site consists of 5 amino acids that are Asp-AsplAsp-Asp-Lys. Obtaining thus a fragment HA30780 of 235 amino acids and its sequence is described as SEQ ID No. 4., f) Sequences SEQ ID No. 3 and SEQ ID No. 4 are translated to their respective DNA nucleotide sequences in a reading frame by preferential codons for Pichia pastoris, obtaining an optimized sequence for each translated sequence, g) To clone the sequence in the SHUTTLE vector pPIC9, first and second restriction sites are added to each of the translated sequences, from step f). Characteristically, for the sequence HA30762, the first restriction site Avrll is added, at the 5 'end; and the second Notl restriction site, is added? at the 3 'end, identifying the sequence obtained as SEQ ID No. 1 (see figure io).; j On the other hand, for sequence HA30780, the first Xhol test site is added, at the 5 'end; and the second Notl restriction site is added at the 3 'end, identifying the sequence obtained as SEQ ID No. 2 (see figure 11). h) Chemically and independently synthesize the sequences SEQ ID No. 1 and SEQ ID No. 2, in such a way that a nucleotide sequence synthesized within a cloning vector is obtained for each sequence, called pJ201: 54361 (see figure 2) and pJ201: 53226 (see figure 3) respectively. , i) Verify, by sequencing, the nucleotide sequence synthesized within each cloning vector. j) Biologically amplify each cloning vector of h), using as Escherichia coli replication system, optionally the strains TOP1QF 'and DH5a, preferably TOP 10F'. 'k) Recover the cloning vectors amplified in j), preferably by miniprep by alkaline lysis. 1) Obtain the sequences synthesized in the cloning vectors, by enzymatic digestion using restriction endonucleases mentioned g). : m) Cloning enzymatically the digested sequences 1) by means of biochemical ligation processes between a phosphate group and a 3 'end of the deoxyribonuclides dNTPs of the SHUTTLE vector pPIC9 and the synthesized sequences obtained in 1),! in such a way that a recombinant nucleotide sequence cloned within a SHUTTLE vector pPIC9, named pPIC9: HA307Ó2 (see figure 4) and pPIC9: HA30780 (see figure 5) respectively, is obtained for each ligation. n) Transforming into Escherichia coli cells optionally TOP 10F ', DHSct; using competent calcium cells and a heat shock method at 40 ° C for 20 seconds, to obtain cells of recombinant Escherichia coli that produces the construction cloned in m). , o) Amplify biologically and individually pPIC9: HA30762 and pPIC9: HA30780 obtained in m) by propagating the recombinant Escherichia coli cells obtained in n). | p) Retrieve independently, pPIC9: HA30762 and pPIC9: HA30780 amplified i in o) preferably by miniprep by alkaline lysis. q) Characterize pPIC9: HA30762 and pPIC9: HA30780 by restriction endonuclease sites, EcoRI, Xhol, Not and Avrll, from the DNA extracted by a plasmid Miniprep protocol amplified by Escherichia coli.

. Integrate (2) the DNA of the vectors pPIC9: HA30762 and pPIC9: HA30780 vector obtained in phase I to the genome of Pichia pastoris, to obtain upa Pichia pastoris recombinante with production capacity of viral antigen against AH1N1 México 2009, through the following stages : a) Recombine genetically by the preparation of the recombinant vectors pPIC9: HA30762 and pPIC9: HA30780 obtained in phase I, to achieve a homologous genetic recombination, digesting each vector independently i with the restriction endonuclease Sacl. b) Propagate Pichia pastoris to genetically transform, growing a line of Pichia pastoris GS 1 15, in a volume of 500 mL of YPD medium at 30 ± 2 ° C, giving it constant agitation at 300 ± 20 RPM until reaching an optical density at 590nm UV spectrum, of 1.5 absorbance units, c) Recover Pichia pastoris GS 1 15 cells in a pellet by centrifugation at 1500g or 5minutes / 4 ° C d) Prepare the cells of Pichia pastoris GS 1 15 recovered in c), this is done mendiante :; - a first wash with 500 mL of sterile deionized water at 4 ° C, centrifuging at 1500g for 5 minutes / 4 ° C, eliminating the supernatant by decantation; - a second wash with 250 mL of sterile deionized water at 4 ° C, centrifuging at 1500g for 5 minutes / 4 ° C, eliminating the supernatant by decantation; , I - a third wash with 20mL sorbitol 1M sterile at 4 ° C, after centrifuging at 1500g I for 5 minutes / 4 ° C, removing the supernatant by decanting to resuspend in lmL of sterile 1M sorbitol, obtaining a suspension of Pichia pastoris GS 1 15 cells ready for electroporation. e) Electroporate the Pichia pastoris GSl 15 cells obtained in d) independently with each vector, (pPIC9: HA30762 and pPIC9: HA30780) containing the cloned recombinant insert, using the following electroporation conditions 1500 V, 25uF, 200O of 5 -10mSecond, in an electric field of 7500V / cm in the cell for electroporation, of 0.2cm, in such a way that recombinant clones Pichia pastoris GSl 15, called Pp9HA30762 and Pp9HA30780 respectively. f) Stabilize the recombinant clones of Pichia pastoris GSl 15 (Pp9HA3Q762 and Pp9HA30780) by growing them in culture plates with dextrose-based regeneration medium (RDB) at 30 ± 2 ° C to stabilize the cells. G) Select the recombinant clones of Pichia pastoris GSl 15 that integrated into their genome the vector that can optionally be pPIC9: HA30762 or pPIC9: HA3078. The selection is carried out by exposing the recomposing clones Pp9HA30762 and Pp9HA30780 with a first selection marker, this consists in the plate culture of said clones in the absence of histidine (M), to propagate only the recombinant clones. Pichia pastoris GSl 15 that integrated the vector into its genome, later the propagated clones exposed to the first selection marker, are exposed to a second selection marker, which is methanol, and said exposure is carried out by plating with methanol medium minimum (MM), to propagate only the clones that integrated multiple copies of the vector producing a phenotype of high expression called Mut + 'h) Isolate the different phenotypes of recombination from the clones that integrated multiple copies of the vector producing a high expression phenotype denominated Mut + and a phenotype of low expression Muts. ! i) Characterize each phenotype by growing in a flask in medium with GÍice | ol with a gradient of methanol at 30 ± 2 ° C conferring constant agitation at 300 ± 2Ó RPM for 48 hours, showing periodically, preferably in intervals of one hour, storing independently at -20 ° C the supernatants obtained from the centrifugation of the samples at 1,000 RPM for 10 minutes, to then run them on a gel under denaturing conditions of polyacrylamide (SDS-PAGE) at 10%.

III. To produce (3) a first and second recombinant viral antigen of Imfiueriza AH1N1 México 2009 in Pichia pastoris GS115 obtained in phase II, by the following steps: a) Scale Mut and Mut phenotypes obtained in phase II, to bioreactor in a laboratory scale from 2L to 7L, through the independent propagation of Pichia pastoris GS1 15 Pp9HA30762 and Pp9HA30780 in a medium that has minimal salts, trace elements and glycerol as a carbon source, until reaching an optimum cellular density, at an optical density at a wavelength of at UV spectrum, 3.0 units. b) Expressing independently the viral antigens mentioned above as amino acid sequences (4) SEQ ID No. 3 and SEQ ID No. 4, the expression is carried out by gradients in the concentration of methanol at a differential feed rate of 3.6 mL / h / L up to 10.9mL / h / L in 64h Fed-Batch Reactor type, using as reference the growth at 30 ± 2 ° C, pH 5.0, and conferring constant agitation at 500 ± 20 RPM.

A following object of invention relates to a first and second nucleotide sequences that respectively encode a first and second amino acid sequences with antigenic activity designed by genetic Engineering and its recombinant expression in Pichia pastoris, both obtained by the method described above.

The first nucleotide sequence (SEQ ID No. 1) refers to a chain of nucleotides optimized for its recombinant expression in Pichia pastoris subjected to bioengineering from the Hemagglutinin protein sequence of Influenza virus AHlNl Cepa México 2009, to be added to the sequence of the viral protein, two digestion sites for the Avrll and Notl endonucleases, as well as 6 amino acids of Histidine at the amino terminal end in the final protein configuration.

Sequence characteristics I • Length: 1730 base pairs • Type: nucleic acid • Thread type: simple • Type of molecule: DNA • Description of the sequence: See SEQ ID No. 1 • Characterization: Restriction fragment length polymorphisms (RFLP), Sequencing.

The industrial application of this sequence SEQ ID No. 1 consists of providing the recombinant instructions for Pichia pastoris to encode and express the amino acid sequence SEQ ID No. 3. The first amino acid sequence SEQ ID No. 3 with antigenic activity is characterized by having : 1 a bioengineered polypeptide chain containing a monomeric fragment of the protein Hemagglutinin in its trimer form Influenza AHlNl Cepa México 2009, a labeling of 6 histidines at the amino terminus.

And the characteristics of the sequence are: • Length: 572 amino acid residues • Type: Pro tein • Type of Strand: simple • Type of molecule: protein ' • Description of the sequence: See SEQ ID No. 3 j • Characterization: 10% polyacrylamide gel electrophoresis (SDS-PAGE), Column Affinity Chromatography (IMAC).

The use of sequence SEQ ID No. 3 favors the development of vaccines or active ingredients for the development of vaccines against the Influenza AH1N1 virus or viral infections associated with this virus.

Another use of this sequence is as an antigen in immunochemical methods, preferably Western Blot and ELISA.

The second nucleotide sequence SEQ ID No.2 refers to a chain of nucleotides optimized for its recombinant expression in Pichia pastoris subjected to bio-engineering from the globular region sequence of the Hemagglutinin protein of Influenza virus AH1N1 Cepa México 2009, for add to the sequence of the viral protein, two digestion sites for the Xhol and Notl endonucleases, likewise 6 amino acids of Histidine at the carboxyl terminus and a restriction site for enterokinase of the KEX enxima immediately after the labeling of Histidines in the configuration. final protein.

Characteristics of the sequence • Length: 719 base pairs · Type: nucleic acid • Thread type: simple • Type of molecule: DNA • Sequence description: See SEC ID N ° 2 • Characterization: Restriction fragment length polymorphisms (RFLP), Sequencing.

The industrial application of this sequence SEQ ID No. 2 consists in providing the recombinant instructions for Pichia pastoris to encode and express the amino acid sequence SEQ ID No. 4.

The second amino acid sequence with antigenic activity is characterized because it has: -a polypeptide chain subjected to bioengineering that contains a monomeric fragment of the globular region of the Hemaglutinin protein in its trimer form of the Influenza AH1N1 virus Cepa México 2009, - a labeling of 6 histidines at the carboxyl end. ! And the characteristics of the sequence are: • Length: 235 amino acid residues • Type: Protein! • Type of Strand: simple · Type of molecule: protein í • Description of the sequence: See SEQ ID No. 4 • Characterization: 10% polyacrylamide gel electrophoresis (SDS-PAGE), Column Affinity Chromatography (IMAC).

I The use of sequence SEQ ID No. 4 favors the development of vaccines or active ingredients for the development of vaccines against the Influenza AH1N1 virus or viral infections associated with this virus.

Another use of this sequence is as an antigen in immunochemical diagnostic methods, preferably Western Blot and ELISA.

Claims (11)

CLAIMS 1. A method for obtaining recombinant proteins with immunogeogenic capacity in Pichia pastoris, characterized in that it comprises the following phases:
1. I. To obtain by means of molecular engineering a first and second sequence of recombinant DNA that gives Pichia pastoris the capacity to produce viral antigens of Influenza AH1N1 MX 2009; II. Integrate each recombinant DNA sequence of the vectors pPIC9: HA30762 and pPIC9: HA30780 obtained in phase I, to the genome of Pichia pastoris, to obtain a first and second strain of recombinant Pichia pastoris with capacity to produce a viral antigen for each vector , against AHlNl México 2009; III. To produce a first and second recombinant viral antigens of Influenza AHlNl México 2009 in the recombinant Pichia pastoris obtained in Fig. II. i
2. 2. A first amino acid sequence with antigenic activity obtained by genetic engineering and its recombinant expression in Pichia pastoris, characterized in that it is formed by a polypeptide, which contains the amino acid sequence NH2-D-O-C02H, where D is: the complete sequence of Hemagglutinin of the virus of the Influenza AHlNl of the Strain reported in Mexico in 2009, and O is: a labeling of 6 Histidines that later intervenes in the purification process.
3. 3. A first nucleotide sequence coding for the amino acid sequence of claim 2 characterized in that, in a DNA reading frame, this sequence named SEQ ID No. 1 is a nucleic acid sequence, particularly deoxyribonucleic acids containing 2 Restriction Sites described above Ayrll and Notl, a start codon, the complete nucleotide sequence reported infectious outbreak of April 2009 in Mexico of the Hemagglutinin, of 1 98bp and the labeling of 6 Histidines, all this sequence modified and optimized for its expression in Pichia pastoris by preferential codons.
4. 4. A first amino acid sequence with antigenic activity obtained by genetic engineering and its recombinant expression in Pichia pastoris, characterized in that it is formed by a polypeptide, which contains the amino acid sequence NH2-HM-R-C02H, where L is: a labeling of 6 Histidines that subsequently involved in the purification process, M is: the sequence for the restriction site KE of enterokinase DDDDK, R: the sequence of the globular region of the Hemagglutinin of the influenza virus AH1N1 of the strain reported in Mexico in the $ 200, from amino acid 63 to 286.
5. 5. A first nucleotide sequence encoding the amino acid sequence of claim 4 characterized in that the DNA sequence encoding the above recombinant protein sequence, in a DNA reading frame, this sequence named SEQ ID No. 2 is a nucleic acid sequence , particularly deoxyribonucleic containing 2 Restriction Sites described above Xhol and Notl, a start codon, the complete nucleotide sequence reported of the infectious outbreak of April 2009 in Mexico of the Hemagglutinin, of 1698bp and the labeling of 6 Histidines, and a site of KEX restriction for enterokinase whole sequence optimized for expression in Pichia pastoris, by preferential codons.
6. 6. A first recombinant clone generated from Genetic Engineering made to the methylotrophic yeast Pichia pastoris, which by result produces a biotechnological derivative that is not naturally required in the environment, if not by the intervention of the hand of man characterized because includes SEQ ID No. 1 in its genome as producers of a recimbinant antigen SEQ ID No. 3.
7. 7. A second recombinant clone generated from Genetic Engineering made to the methylotrophic yeast Pichia pastoris, which by result produces a biotechnological derivative that is not naturally required in the environment, if : it is not by the intervention of the hand of man characterized because it includes in its genome the sequence SEQ ID No. 2 as producers of a recombinant antigen SEQ ID No. 4. j
8. 8. The use of the amino acid sequence SEQ ID No. 3, as an active ingredient in vaccines or against the Influenza AH1N1 virus or viral infections associated with this virus.
9. 9. The use of the amino acid sequence SEQ ID No. 4, as an active ingredient in vaccines or against the Influenza AH1N1 virus or viral infections associated with this virus.
10. 10. The use of the amino acid sequence SEQ ID No. 3, as an antigen in immunochemical diagnostic methods, preferably Western Blot and ELISA. 1 1. The use of the amino acid sequence SEQ ID No. 4, as an antigen in immuno-chemical diagnostic methods, preferably Western Blot and ELISA. 18 PxJESUMEN I The present application is in the field of genetic engineering and biotechnology and in particular in the production of vaccines against influenza virus AH1N1, and refers to a method for obtaining recombinant proteins with immunogenic capacity in Pichia pastoris and its use in a preparation of vaccines against influenza viruses, dichó method includes the phases of: Obtain by means of molecular engineering a first and second sequence of recombinant DNA that confers to Pichia pastoris the capacity to produce viral antigens of Influenza AHlNl MX 2009; Integrate each DNA sequence Recombinant vectors pPIC9: HA30762 and pPIC9: HA30780 obtained in phase I, to the genome of Pichia pastoris, to obtain a first and second strain of recombinant Pichia pastoris with capacity to produce a viral antigen for each vector, against AHlN l Mexico 2009; To produce a first and second recombinant viral antigens from Influenza AHlNl México 2009 in the recombinant Pichia pastoris obtained in Fig. II.