MX2011002722A - Recombinant vaccine of porcine rubulavirus. - Google Patents

Abstract

Porcine rubulavirus from La Piedad Michoacán is the causal agent of the blue eye syndrome, a disease that causes a large mortality in piglets and serious neurological and reproductive damages in adult pigs. The present invention refers to a vaccine against Porcine rubulavirus, as well as to an equipment for the specific diagnosis against the protein HN of Porcine rubulavirus.

Description

RECOMBINANT VACCINE OF THE RUBULAVIRUS PORCINO FIELD OF THE INVENTION The present invention relates to the technical field of medical biotechnology; more particularly it is relative to the technical field of veterinary medicine; and more particularly to a recombinant vaccine based on the porcine Rubulavirus HN protein.

BACKGROUND Porcine Rubulavirus from La Piedad Michoacán is the causative agent of Blue Eye Syndrome, a disease that causes great mortality in piglets and serious neurological and reproductive damage to adult pigs (Moreno-Lopez J et al, 1996, Arch. Virol., 91; 221 -23t).

The clinical signs vary according to the age of the animals, in piglets it is characterized by rigidity in the hind limbs, muscle tremor and involuntary movements, typical of a nervous system infection, dying 48 hours later. In adult pigs, reproductive and nervous alterations are induced, mainly in pregnant females, where there is a regression to estrus, fetal mummification and a considerable increase in the number of piglets dead at birth. In males, alterations such as orchitis, epididymitis, and testicular atrophy have been observed (Ramírez-Mendoza H, Hernández-Jauregui P, Reyes-Leyva J, Zenteno E, Moreno-Lopez J, Kennedy S. Comp Pathol 1997; 117; (3): 237-52). i Porcine Rubularivus is a 14 kb negative chain RNA virus. Its nucleocapsid is helical and is included in an extremely fragile lipoprotein membrane derived from the infected cell. The RNA codes for 6 different proteins, 4 of them associated with RNA that together form the nuclecápside, and two others that are found in the lipid layer, the fusion protein (F) and the Hemagglutinin Neuraminidase (NH). The latter is perhaps the most important in the biology of the virus, because it has two functions: a neuraminidase activity responsible for the elimination of sialic acid groups that allows an approach with cellular receptors, and another hemagglutinant that recognizes, and binds specifically saccharide structures on the surface of the target cell.

Previous work has shown that the pathogenic capacity of the virus is due to the ability of the NH protein to specifically recognize structures containing a2, 3 sialyl lactose in target organs, such as the respiratory tract, nervous system and epididymis (Reyes-Leyva J, Espinosa B, Hernández J, Zenteno R, Vallejo V, Hernández-Jáuregui P, Zenteno E. Comp Biochem Physiol B Biochem Mol Biol. 1997; 1 18 (2): 327-32).

Due to the veterinary problem with Porcine Rubulavirus - the development of vaccines and diagnostic equipment for Blue Eye disease is necessary. In this regard, the present invention provides a porcine Rubulavirus recombinant vaccine useful for the prevention of infection of the Blue Eye, as well as for its diagnosis.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a Western-blot test of fractions 1 and 2 of the purification of porcine Rubulavirus HN protein.

DETAILED DESCRIPTION OF THE INVENTION The present invention will be described based on examples, which should be understood as illustrative of the invention and in no way limiting thereof.

Example 1. Obtaining Porcine Rubulavirus RNA for its amplification and cloning. In pig kidney cell cultures (PK15), the reference strain of Porcine Rubulavirus (PoRV) was propagated to obtain viral RNA. The extraction of the RNA was through three different extraction methods in order to obtain a better quality product. The first was following the protocol Trizol reagent obtaining values in the purity of the RNA of 1.7 to 1 .8 according to the difference observed in the optical densities verified at 260 and 280 nm. The two following methods consisted in the use of the Trizol protocol, complemented by the purification of the RNA by passing through the columns RNeasy Spin Colum or RNeasy Mini Spin Colum, respectively. The efficiency in the purification of RNA was high, with values of 2.0 under the same optical densities. Simultaneously, an internal control corresponding to the "cyclophilin housekeeping" detected by the real-time PCR method was incorporated into the RNA quality verification system, which makes it possible to demonstrate the integrity of the RNA obtained.

Example 2. Cloning of the HN genes. Using RT-PCR, the genomic sequences encoding the HN protein were amplified. They were cloned in E. coli plasmids and after analyzing them by automatic sequencing reaction, the primers that amplify the fragments were designed. These primers contain at their 3 'end restriction sites that aid in the cloning process.

The primers selected for the first step, amplification by RT-PCR of HN were designed according to the sequences reported erf the database of the GEN-Bank. The amplification of eight RNA samples from the reference strain PoRV-LPMV-1984 was performed by an RT-PCR test, thus obtaining the DNA complementary strand (cDNA). The cDNA was amplified by the use of specific primers previously designed to obtain the segment that codes for the HN protein, using the enzyme pfUltra Hi-Fi polymerase, thus obtaining a product with better genetic quality. The analysis of the PCR products was carried out using 0.8% agarose gel bleed with ethidium bromide, where only the amplification of a cDNA sample from the eight samples worked was observed, with a product of 2000 base pairs approximately indicative that the designed primers functioned to obtain the expected DNA segment.

Example 3. Purification of recombinant proteins. For the expression of the purified recombinant protein, the expression kit for recombinant proteins was selected in E. coli, which have six histidines (6xH1s) in their amino acid sequence. The kit is called Qiaexpressionist and is from Qiagen laboratories. It uses expression plasmids like pQE30L, which is the one chosen for this application, since it contains in its sequence Cough promoters necessary for expression, the start codon, the first 9 codons that encode the amino acid sequence HHHHHHRGB and a region of insertion based on restriction sites. The cloning of a fragment in this plasmid generates a recombinant protein that expresses at one of its 6xHis ends, which are recognized by agarose beads with NiNTA and are adhered.

Example 4. Cloning in the vector. After evaluating the advantages and disadvantages of the baculovirus expression system, it was decided to adapt the protocol for the expression of HN in the cell wall of Lactococcus lactis. The system is based on the expression in L. lactis, which uses expression plasmids containing the maintenance genes in E. coli and L. lactis, which correspond to selection genes for antibiotic resistance, promoter region of expression in L lactis and genetic sequences that direct the expression of fragments cloned in the plasmid (site of insertion of the antigen to be expressed of interest) in the cell wall, by means of signaling of export to the cell wall and anchoring to the cell wall, mediated by the enzymatic system of sortase .

During this stage of the project, studies were carried out on the sequence of the gene in question, and the protocol of the methodology to be followed was designed. The fragment obtained would be cloned into expression plasmids in which restriction enzyme recognition sites would be incorporated. Finally, an expression system based on a bacterial cell line of E. coli was selected using the pGDual plasmid for the insertion of the sequence of the gene coding for the HN protein of Rubulavirus.

From this sample the purification of the PCR product was carried out with the Wizard SV Gel kit, to initiate a PCR with the restriction enzymes (Eam 1 1041 and tango buffer) which allowed the preparation of the DNA fragment for its binding to the Plasmid pDual GC Expression Vector, which was selected due to its characteristic for the expression of high levels of protein in mammalian cells or in bacterial cells. For the effects of plasmid ligation to DNA, the enzyme T4 DNA ligase was used, the product obtained was incorporated by electroporation (1800 volts) to the bacterial cells X-1 to insert the DNA into the vector. Transformed bacteria were seeded in petri dishes with LB medium supplemented with Kanamycin (30 mg / l), to allow bacterial growth (37 ° for 24 hrs). It was verified if the developed colonies had incorporated the plasmid, by means of a PCR reaction with the primers specific for the HN protein, testing 17 colonies, observing that all of these presented the insert.

From the previous results, three colonies are selected to carry out the purification of the clone and incorporate the obtained plasmid into specialized cells in the expression of proteins (KRX cells). The above is performed by electroporation, using only two colonies (1 and 3), sowing this type of cells again in two petri dishes with LB medium and kanamycin, for 24 hr. at 37 ° C. Of the colonies that grew, two were selected for each plate, and the expression of the protein was verified, by cultivating 150 μ? of bacterial culture (1 twentieth) in 150 ml of liquid medium LB. It remained in agitation until the bacteria reached the growth phase, and in the exponential phase (minimum 3 hrs), the bacterial growth factor IPTG, L-Rhamose, was added. The material was centrifuged (4000 rpm, for 10 min) to work exclusively with the bacteria (pellet), and by means of a lysis and purification process with the His Buffer kit, the protein was purified for evaluation by Western-Blot, using a monoclonal anti-HN, specific for porcine Rubulavirus, observing a positive, well-defined reaction on the paper membrane, with the presence of a band of an approximate molecular weight of 63 k corresponding to the HN protein derived from the nucleotide sequence expressed in SEQ ID NO. 1.

The procedure described above was also performed with the NP protein, except that the expression was carried out in BHK-21 cells, with which three chemical factors for cell transformation were evaluated (Lipofectamine 2000, FueGENE 6 and ExGEN), with three different concentrations of 0.5, 1 and 2 g of DNA, observing that the best expression of the NP protein was with Lipofectamine at 24 and 48 hrs. of incubation and at a concentration of 1 and 2 \ ig of DNA, as observed by Western-blot, where only positive reaction with the monoclonal anti NP is presented, and a reaction very weak at the concentration of Q.5 pg of DNA. We also observed a ligand expression at the concentration of 2 μg at 24 hrs using FueGene 6.

Example 5. Obtaining the recombinant HN protein. A first batch of the recombinant HN protein was prepared, which was analyzed by means of a digital micro-chip system Agilent Technologies, where a concentration of 80 μ? / ??? was determined, which was used in the immunization of pregnant mice and sows . The analysis showed a purity of 90% of the protein obtained, detected at a molecular weight of approximately 63 kDa.

Also, a second batch of recombinant HN protein was prepared, obtaining a concentration of 45,010 μg / ml, this batch was used for the standardization of the ELISA tests.

This second batch of protein was evaluated by Western-blot tests and revealed with the Chemistry-Luminescence analysis system (Amersham), demonstrating the specificity of the protein in positive and negative control sera for porcine Rubulavirus, indicative of its capacity and specificity of antigenic recognition.

Example 6. "In vitro" evaluation of the immunogenic recognition of the recombinant products obtained from the Porcine Rubulavirus. The "in vitro" evaluation of the antigenic recognition was carried out with the fragments and the complete protein of HN, by means of a Western Blot test using a specific monoclonal anti HN, thus determining that the recombinant products generated are feasible for use in the tests of antigenicity "in vivo". Simultaneously, in a similar way we worked with the NP proteins, with which the ELISA-differential tests were developed.

The antigenicity of the HN protein was determined by Western blot detecting a 63 k band. The antigenicity of the NP protein was determined under the same procedure. A batch of NP protein was obtained for its use in the ELISA tests programmed for the third stage of the project and sera from positive and negative control pigs were analyzed.

Example 7. Location of free farms of Porcine Rubulavirus. A farm was evaluated in Metepec, State of Mexico, which was serologically negative to porcine Rubulavirus. The animals that were used in the third and fourth stages for the immunogenicity tests were purchased in said farm. ^ Example 8. Evaluation of the immunogenicity of the experimental recombinant vaccine in laboratory animals. In order to obtain an adjuvant suitable for use in the immunogenicity tests of the recombinant product, four groups of 5 BALB / c mice were inoculated intramuscularly and subcutaneously with a dose of 200 μ? of a suspension of 4 different adjuvants based on saponins in a concentration of 15 mg / 10 ml. They were observed for 10 days after inoculation in their physiological constants such as temperature, nervous signs, behavior and mortality. The animals inoculated with two types of saponins showed a 100% mortality at 24 and 96 hours after inoculation. A third group showed a tolerance of 20% with a mortality of 80% at 96 hours after inoculation. The fourth group showed a tolerance of 80% and mortality of 20% on day 5 after inoculation. For this reason, it was decided to use the latter in the evaluation of the immunogenicity of the recombinant vaccine in mice.

For the immunogenicity tests, five groups of freshly weaned BALB / c mice were formed, with 10 animals each. Group 1 was immunized with 1 pg of HN and group 2 with 5 pg of HN, both inoculated subcutaneously. Groups 3 and 4 were administered with the same doses of HN but added with 200 μ? of saponins as adjuvant and applied intramuscularly and group 5 consisted of the negative control group. A second stimulus was applied to the fourth week. Serum samples were collected at 7, 14, 21, 28 and 35 days after inoculation and evaluated by the ELISA technique. The response was similar in the four groups evaluated, regardless of dose, route or adjuvant, antibodies were only detected in sera diluted at a concentration of 1: 2 by the ELISA technique.

The animals inoculated with two types of saponins showed a 100% mortality at 24 and 96 hours after inoculation. The saponin administered in the third group showed a tolerance of 20% with a mortality of 80% at 96 hours after inoculation. While the saponin administered to the fourth group showed a tolerance of 80% and mortality of 20% on day 5 after inoculation. The mice inoculated with the HN protein and the number four saponin had a positive immune response detecting in all of them presence of antibodies by the ELISA technique, regardless of dose, route or type of adjuvant.

Example 9. Evaluation of the immunogenicity of the recombinant vaccine in specific pathogen free pigs. For the evaluation of the recombinant vaccine in pigs, of the HN protein, we worked with the ISCOM immuno-stimulant or saponin adjuvant. The recombinant vaccine was evaluated, by means of the experimental immunization in 5 pregnant sows, applying two doses 4 and 2 weeks before delivery intramuscularly, with a concentration of 350 μ9 / 3 ??? of HN protein in each application. After the immunization, the temperature of the animals and the area of application were evaluated in order to determine some macroscopic alteration in the skin or inflammation, as well as variations in body temperature derived from the reaction to vaccination. Additionally, a pregnant, non-immunized sow was handled as control of the experiment.

After vaccination, no apparent changes were observed in the area where the vaccine was applied. Febrile reactions did not occur in any of the sows immunized until one day before parturition, in particular, after the second application of the vaccine. The vaccine, which corresponds to the period during which, may present some adverse reaction to vaccination. The observations made are indicative that the evaluated vaccine does not produce any febrile reaction in the pregnant sows, since the temperature range remained between 39-39.5 ° C.

After delivery, the IgG Immunoglobulin concentration, specific for the porcine Rubulavirus HN protein in colostrum, was determined. using the Capture Diagnostic Kit of anti-IgG, adapted to the recombinant protein HN. The average concentration detected of IgG-anti HN, present in colostrum was 3.6 pg / ml of IgG, at the time of delivery, which decreased considerably at 24 hrs after delivery (1.67 pg / ml on average). The negative control sow did not present specific IgG levels and remained with values of 0.

The concentration of IgG, was estimated by means of an ELISA test, applying a linear regression, R2 = 0.8143 with the following equation: ^ 0. 0043x - 0,1676 Where x = observed optical density This same test was applied for all determinations, considering for the analysis a minimum regression value of 0.80 in relation to the IgG standard curve, established from a maximum concentration of 1. μg / ml, and 7.8 μg / ml as a minimum concentration from serial double dilutions.

The humoral response in sows was evaluated 28 days before parturition from the first immunization, where it was observed that IgG levels, specific for HN, were detected from day 21 post-vaccination (4.60, 13.561 , 1, 835, 4,530, 2,720 μg ml, respectively). At 28 days post-vaccination the IgG levels were 12.835, 16,930, 2.942, 10.450, and 12.356 μ9 / ???, respectively. Finally, at 30 days post-vaccination the IgG levels were 15,167, 13,536, 2,194, 14,378, and 12,458 μg / ml, respectively.

Before the challenge, all animals that ingested colostrum were evaluated to determine the concentration of IgG antibodies, specific against the porcine Rubulavirus HN protein. These evaluations showed that the IgG-antiHN concentration was on average 4.5 μg / ml before the challenge, as a result of having ingested colostrum from the vaccinated mothers. ^ The statistical analysis, carried out using the GRAPH-PAD PRISMA 4 program, showed that On day 7 of births, statistically significant differences were observed among the group of piglets from vaccinated mothers (independently of the adjuvant) against the control group (piglets from unvaccinated mothers).

The experimental challenge of the piglets (n = 36) was carried out 7 days after birth, with a viral dose of 106 TCID / ml. The animals were challenged with 1 mi intratran salt and the response was evaluated by? the determination of the concentration of IgG, where it is shown that there was a statistically significant difference in the specific IgG levels for the porcine Rubulavirus HN protein on day 7 and 14 after challenge among the group of animals from immunized mothers (group A and B) against the control group from the non-immunized mother (Control group), but not at day 30, where the passive humoral response of the piglets decreases and the primary response of the control group increases, this lack of difference manifested itself 30 days may be due to neutralization (antigen-antibody reaction) that occurs in piglets that have passive immunity with the viral antigen that was inoculated, in order to eliminate the viral infection.

In all the animals the temperature was measured daily after the challenge, during 14 days, to determine changes in this physiological constant, they were also observed daily to evaluate if there were changes in the behavior or presentation of clinical signs. The mean values of temperature that were detected, did not show significant differences in the two groups (controls and from immunized mothers), since they were maintained between the range of 39.1 and 39.28 ° C.

The control group from the unvaccinated sow was called "positive control", where 5 piglets were inoculated for 7 days. Simultaneously they joined the moment of challenge, 4 piglets 3 days old, this is because the age of the piglets represents an important factor for susceptibility to the virus. In the piglets inoculated at 7 days of age, no clinical signs were observed, nor was there any mortality in this group, only a slight elevation of the temperature of 40.32 ° C on the second post-challenge day and the presence of constant lacrimation. However, the humoral response to the virus (PoRV), evaluated by the concentration of IgG, was significantly significant, as was shown in previous paragraphs. In contrast to the piglets inoculated at 3 days of age, where a mortality of 25% was observed when the disease appeared in one of the four challenged animals, which showed the classic signology of the Blue Eye Syndrome as it was: progressive nervous signs, Incoordination, kicking, involuntary masticatory movements and death, although the animal showed not to lose the appetite, the diagnosis was confirmed by PCR tests in samples of medium brain and olfactory bulb.

All the animals were sacrificed. At necropsy, brain samples were collected, where the presence of viral RNA was detected, using the Real-time PCR and Nested-PCR test, to verify the infection and / or establishment of the virus in the brain. The results obtained showed that piglets from vaccinated mothers were able to neutralize the virus, however, it was possible to detect genetic material of the virus through real-time PCR tests. The above, evidence that the animals were infected with the virus during the experimental challenge. The results showed the presence of concentrations of 10 to 100 average copies, for the P gene of the porcine Rubulavirus.

Additionally, the humoral immune response of all the animals was evaluated by the haemagglutination inhibition test, observing antibody titers of 1: 8 from day 21 in the sows, which increased considerably at day 60 post-vaccination with an average title of 1: 128. Colostrum was also evaluated by this test obtaining titres = 1: 128, in the samples obtained immediately after delivery, compared to the unvaccinated control sow who remained negative.

In the piglets, the titers observed at day 7 after calving, were 1: 64 average, unlike the piglets born from the unvaccinated negative control sow, which were negative. The results obtained by this test (IH), where the complete virus was used, are indicative that the antibodies induced by the recombinant protein HN, developed in this project, are capable of 'stimulating specific anti-antibodies'. Porcine Rubulavirus, where these antibodies are also capable of recognizing virus strains with different antigenic sequence.

The humoral immune response was evaluated by the collection of sera at 0, 7, 14, 21 and 30 days post vaccination. Antibody levels detected by the haemagglutination inhibition test ranged between 1: 8 and 1: 16 hemagglutinating units (HU) from day 21 after immunization, increasing to a level of 1: 128 HU at day 30. The presence of specific IgG for the HN protein, through the ELISA assay developed in this project, detecting IgG concentrations from day 21 post-vaccination. The postvaccination physiological constants of the sows remained normal. Samples of colostrum were taken for analysis, where anti-IgG concentrations of 3.6 μg / ml of IgG were detected at the time of delivery, which decreased considerably at 24 hrs (1.5 μg / ml), which presented a titer de = 1: 128 UH in the hemagglutination inhibition test (HI). To the young (piglets) samples of serum were taken, to determine the humoral immunity acquired, due to the ingestion of colostrum. The titres obtained in piglets from vaccinated sows, at day 7 of birth were 1: 64 HU determined by the HI technique, with average concentrations of 4.5 μ / m \ IgG, specific for the porcine Rubulavirus HN protein. The statistical analysis showed statistically significant differences (P <0.05), when comparing the group of piglets that ingested colostrum from vaccinated mothers, with respect to the control group, composed of piglets from unvaccinated sows. Subsequently, the animals were challenged with a pathogenic strain of Porcine Rubulavirus (1 ml / intranasal route in each nostril). The response was evaluated by determining the concentration of IgG, which shows that on days 7 and 14 post-challenge, if there was a statistically significant difference (P <0.05) in the specific IgG levels for the HN protein Rubulavirus Porcine, when comparing the group of animals from immunized mothers (group A and B) against the control group from non-immunized mothers (Control group). Not so in the evaluations made on the 30th pos challenge, where the passive humoral response of the piglets decreases and the primary response of the control group increases. This difference that manifested at 30 days, may be due to neutralization (antigen-antibody reaction) that occurs in piglets that have passive immunity with the viral antigen that was inoculated, in order to eliminate the viral infection.

The results obtained showed that piglets from vaccinated mothers were able to neutralize the virus, however, it was possible to detect genetic material of the virus through real-time PCR tests. This shows that the animals were infected with the virus during the experimental challenge.

Example 10. Development and standardization of the ELISA test from the recombinant protein. Initially the ELISA test was standardized for use with serum samples obtained from experimentally immunized rats, using monoclonal antibodies specific for Porcine Rubulavirus. The minimum required concentration of HN protein for the ELISA was determined by a block titration using double dilutions from 1: 2 with an initial concentration of 6.5 pg of HN protein per ml. Likewise, the optimal concentrations of the conjugate and monoclonal antibody were determined under the block system. The results obtained determined that the optimal concentration for the use of monoclonal Jel was 1: 400 and of: 500 for the second antibody (Anti IgG of mouse) conjugated with peroxidase. TMB (tetramethylbenzidine) was used as the substrate according to the producer's recommendations. Incubation times were established for plate antigenation in 24 h at 4o and 60 minutes at 37 ° for the antibodies used respectively.

The cut-off point, sensitivity and specificity of the test were established by means of a 2x2 contingency table. In such a way that a cut-off point was established from 2 times the standard deviation of the negative control (0.085). 30 sera from immunized animals and 30 sera from negative mice were worked. Under these experimental conditions 100% specificity and 100% sensitivity were observed, these results showed to be satisfactory for the effect of the evaluation of the recombinant vaccine in laboratory animals.

Additionally, the differential ELISA test was developed and standardized from the recombinant NP protein. The necessary minimum concentration of NP protein was determined by means of an ¾ block titration, using double dilutions from 1: 2 with a concentration of 500 pg of NP protein per ml, establishing as optimum the concentration of 1: 500. Likewise, the optimal concentrations of the conjugate and monoclonal antibody were determined under the block system, being for the monoclonal 1: 500 and 1: 1000 for the second antibody (Anti IgG of mouse) conjugated with peroxidase. TMB (tetramethylbenzidine) was used as the substrate according to the producer's recommendations. Incubation times were established for plate antigenation in 24 h at 4o and 60 minutes at 37 ° for the antibodies used, respectively. With the development of this test, optical densities (OD) were obtained between 0.8 and 1.0 for positive reference sera and 0.065 to 0.1 OD for negative reference sera. The test was adapted to a reading system, based on the percentage of positivity with respect to a positive reference serum, in such a way that it allows the comparison between different kits, independently of the laboratory in which it is carried out. The system was adapted to a Software for reading the prototype plates of the developed kit.

Example 11. Validation of the ELISA test with reference sera. The validation of the developed ELISA assays was performed by comparison with a commercial capture IgG kit, where a linear regression was established comparing the different optical densities obtained from a known concentration of IgG, starting at 1 pg / ml. For the evaluation of the trials, we always worked with a minimum regression coefficient of R2 = 0.80 and applying the corresponding equation in the analysis of all the serum samples evaluated.

The recombinant protein was diluted to a concentration of 1-2 pg / ml in carbonate buffer, are placed 100 μ? per well (in the wells corresponding to the samples and their respective targets). In addition, a 1: 100 dilution of the capture antibody (Goat anti-pig-affinity purified (1 mg / ml), Cat: A100-104) is made in carbonate buffer, 100 μ? in the wells corresponding to the standard curve and to its target (rows 1 and 12). Cover the plate and leave at 4o C overnight. The next day the plate is washed 3 times with washing buffer. The entire plate is blocked with 150 μ? / ???? ppn blocking buffer for 1 h at 37 ° C and with gentle shaking. Three washings are carried out with washing buffer, the excess buffer is removed. In no step of the technique should the plate be allowed to dry. Prepare a standard curve of IgG (Calibration protein: Porcine reference serum (16.5 mg / ml of IgG) Cat: RS10-107) starting at a concentration of 1000 ng / ml in dilution buffer, perform serial double dilutions, place 100 μ? in the wells corresponding to each point of the curve (row 12), while 100 μ ?, of dilution buffer (row 1) is placed in the target of the standard. In the wells corresponding to the samples, the problem samples are placed, starting from a dilution of 1: 50 (for serum) and making triple dilutions (4), whereas in the corresponding targets, 100 μ? of dilution buffer. Incubate the plate 1 h at 37 ° C with gentle shaking. Wash 5 times with wash buffer, remove excess buffer. Incubate with HRP secondary antibody (100 μ? /?) At a 1: 1250 dilution in dilution buffer, for 1 h at 37 ° C with gentle shaking. Wash 5 times with wash buffer, remove excess buffer. Prepare the TMB by diluting a mg tablet in 10ml of citrate / perborate buffer pH 5.0. Place 100 μ? / ????, incubate 2-5min. Perform frequent readings at 650 nm to monitor the development of the plate. When the most concentrated point of the curve reaches a D.O. of 0.700 nm the reaction is stopped by adding 100 μ? of the stop solution.

Using an ELISA reader read the plate at a wavelength of 450 nm for TMB. The cutoff for both the standard curve and for the samples is calculated by averaging the values of the corresponding targets, the standard deviation is obtained, this value is multiplied by three and the result is added to the average. This value is subtracted from the absorbances corresponding to the curve to obtain the net absorbances. The cutoff point obtained for the samples is subtracted in turn from the absorbances obtained to have the net absorbance values problem. With the values of known concentration and their respective net absorbances, a graph of absorbance vs concentration is constructed, a linear regression is made and the equation of the curve is obtained, where the absorbance values problem is substituted to obtain the concentrations for each dilution of each sample, later these values are multiplied by the dilution factor, the closest values are averaged out of the four dilutions of each sample and the final concentration is obtained.

Claims (3)

REVINDICATORY CHAPTER

1 . A porcine Rubulavirus recombinant vaccine, characterized in that it is encoded by the nucleotide sequence of SEQ ID NO. 1.

2. A porcine Rubulavirus vaccine composition, characterized in that it comprises: the recombinant vaccine according to claim 1, and an adjuvant.

3. A diagnostic equipment for determining the presence of specific antibodies with the HN protein of porcine Rubulavirus in pig serum, characterized in that it comprises: i) the recombinant protein encoded by the nucleotide sequence of SEQ ID NO. 1, ii) anti-igG secondary antibodies, and iii) means for determining the presence of the antigen-antibody interaction.