KR101395347B1 - Hn protein of newcastle disease virus and the diagnostic composition for newcastle disease containing the same - Google Patents

Abstract

The present invention relates to the diagnosis of Newcastle disease (ND), a malignant epidemic of chicken.
According to the present invention, a recombinant baculovirus expression vector containing a Newcastle disease virus (NDV) haemagglutinin-neuraminidase (HN) gene is transfected into an insect cell and NDV HN protein expressed in the insect cell And NDV antigen test for hemagglutination inhibition (HI) test containing the same, it is possible to eliminate the risk of contamination or disease transmission that may occur during handling of living NDV, An NDV antigen diagnostic solution can be mass produced in an economical manner.

Description

TECHNICAL FIELD [0001] The present invention relates to a HN protein of Newcastle disease virus and an antigen diagnostic test for HI method including the HN protein,

The present invention relates to the diagnosis of Newcastle disease (ND), a malignant infection of chicken, and more particularly to a recombinant baculovirus expression vector containing a gene for a newcastle disease virus (NDV) HN (haemagglutinin-neuraminidase) The present invention relates to NDV HN protein expressed in insect cells and to a NDV antigen diagnostic solution for hemagglutination inhibition (HI) test comprising the same.

ND is a typical malignant epidemic of a highly contagious and lethal poultry. Since its first outbreak in the UK in the early 1920s, ND has caused economic damage to the poultry industry worldwide, including in Asia, Africa and Europe. Infected chickens show respiratory symptoms, digestive symptoms, genital symptoms and neurological symptoms. In particular, if ND occurs in a poultry farm where immunization has not been established by vaccination, more than 90% of chickens may die within two weeks.

NDV is a pathogen causing ND and is phylogenetically classified as Avian paramyxovirus type 1 (APMV-1) in Paramyxoviridae Avulavirus. NDV is classified as velogenic, mesogenic, and lentogenic NDV on the basis of virulence in chickens. The World Animal Health Organization (OIE) estimates that the pathogenicity index (ICPI) in a 1-day-old chick is 0.7 or more, or that the amino acid sequence that forms the segment of the NDV F protein has a polybasic sequence The infection of NDV is defined as ND.

Infectious NDV with little or no chicken mortality is not considered a Newcastle disease. In addition, these toxin-type NDVs can prevent ND damage by inducing an immune response with little or no damage from chicken, and some of the virulent viruses are used for ND vaccine production.

NDV classifies only one serotype so far, so any NDV strain can prevent Newcastle disease, and any NDV strain can be used to produce a diagnostic fluid. NDV has a wide variety of genotypes or genetic subgenotypes. The NDV genotypes are largely divided into class 1 and class 2 genotypes, of which the genetic viruses belonging to class II are the ones causing damage to the chicken. Class 2 type is composed of at least nine genotypes (I-type to I-type) to date. Both genotypes I and II are mostly NDVs that are isolated from wild birds or species other than birds, and do not cause damage to chickens. In genotype III NDV belonging to genotype IX is mainly isolated from chickens and predominantly viruses that cause severe damage to chickens. Currently, viruses belonging to the genotype I and genotype II of the class II type are used for the ND diagnostic solution or the vaccine production.

ND can be diagnosed by detecting NDV in infected chickens or by detecting NDV specific antibodies. Currently, enzyme linked immunosorbent assay (ELISA) or hemagglutination inhibition reaction (HI) method is mainly used as a method of detecting NDV antibody. In particular, HI is a standard antibody test that is generally used for ND antibody test because NDV can test the antibody by using the property of agglutinating the chicken erythrocytes so that it can be easily tested without professional facilities or equipments. The NDV diagnostic antigen currently used in HI is produced by inoculating infected NDV into chicken eggs (fertilized eggs) and mass-proliferating the virus, and then preparing the virus in the form of formalin or beta-propiolactone, to be. In this way, antigen production requires a minimum of two weeks of antigen production, including the virus growth period (5 days to 1 week), viral inactivation and inactivation confirmation (1 week minimum).

In addition, conventional diagnostic antigens have a problem of contamination because they treat viable viruses that are infectious as well as a problem in the production of diagnostic antigens when the supply of the cells is not smooth due to the occurrence of diseases or insufficient supply. Therefore, there is a need for a new manufacturing process capable of manufacturing and supplying an antigen diagnostic solution more quickly and safely, which has improved the conventional antigen production method, in case of emergency in which a large amount of diagnostic solution is urgently required due to disease epidemic.

Therefore, the present inventors found that HN protein itself has a characteristic of coagulating chicken erythrocytes when NDV HN protein produced at high concentration in insect cells is used, and HI test for NDV antibody test is possible by using it. The NDV HN protein was synthesized by using Baculovirus, and HI test was conducted using the NDV HN protein. As a result of comparison with the conventional HI test using NDV as an antigen, it was confirmed that the NDV antibody can be significantly tested, .

Prior arts relating to diagnosis and vaccination of Newcastle disease include Korean Patent Registration No. 10-0437888 (detection of Newcastle disease virus using primers for detecting Newcastle disease virus and detection of the virus using the primers, pathogenicity and genotyping method), Korean Patent Registration No. 10- 0801180 (attenuated recombinant Newcastle disease virus and Newcastle disease vaccine containing it).

It is an object of the present invention to provide a method for producing a recombinant baculovirus expression vector containing the NDV HN gene and a recombinant baculovirus expression vector produced by the method.

Another object of the present invention is to transfect insect cells with a recombinant baculovirus expression vector containing the NDV HN gene and to detect the NDV HN protein expressed in the insect cells and the NDV antigen diagnostic solution .

In order to achieve the above object, the present invention provides a method for producing a virus, comprising the steps of (1) extracting viral genomic RNA from Newcastle diease virus (NDV) lasota strain, and (2) (3) the cDNA synthesized by the above step (2), the forward primer of SEQ ID NO: 1, and the reverse primer of SEQ ID NO: ) And a reverse primer of SEQ ID NO: 2 using PCR to synthesize DNA and (4) DNA synthesized by the above step (3) is treated with Xba1 and HindIII to extract a DNA fragment ; And (5) inserting the 4 virus poly a DNA fragment extracted by the step bekyul polyhedrin promoter pFastBac ^TM 1 multi-cloning site (multicloning) of an expression vector with a (P _PH); consists of, the The forward primer of SEQ ID NO: 1 contains the NDV HN gene which is characterized by the insertion of a HindIII restriction endonuclease site at the 5 'end of the ATG above the Xba1 restriction enzyme site and the reverse primer of SEQ ID NO: 2 at the 5' end of the stop codon The present invention provides a method for producing a recombinant baculovirus expression vector.

Also provided is a recombinant baculovirus expression vector comprising the NDV HN gene produced by the method for producing the recombinant baculovirus expression vector.

(1) transforming a recombinant baculovirus expression vector containing the NDV HN gene into DH10B _AC E. Coli cells containing Bacmid, followed by a recombinant process with Bacimid; and (2) (3) transfecting the recombinant Bacimid extracted in step (2) into insect cells (spodoptera frugiperda 9 cells, Sf9 cells); and (4) Expressing the NDV HN protein in an insect cell transfected by the step; And (5) extracting the NDV HN protein expressed by the step (4) from the insect cells through a cell disruption solution and an ultrasonic treatment. The NDV HN protein using the recombinant baculovirus expressing the NDV HN protein Of the present invention.

Also provided is a NDV HN protein produced by the method for producing the NDV HN protein.

The present invention provides a NDV antigen diagnostic solution for testing for the inhibition of hemagglutination (HI method) comprising NDV HN protein prepared by the above-mentioned method for producing NDV HN protein.

According to the present invention, the NDV antigen diagnostic fluid containing the NDV HN protein prepared according to the present invention can be used as a substitute for the conventional NDV antigen diagnostic fluid when the specificity and sensitivity of the test for testing Newcastle Disease antibody are evaluated Unlike the conventional method of producing viruses proliferated in bovine sponges, the new antigen diagnostic solution according to the present invention is produced by expressing only the Newcastle disease virus HN protein produced using recombinant baculovirus through insect cells Since the NDV antigen diagnostic fluid is prepared, there is an effect of eliminating the risk of environmental pollution or disease transmission in the handling of live NDV.

In addition, since the NDV antigen diagnostic solution is produced through a complicated process such as virus propagation, virus detection, virus inactivation, and inactivation confirmation test using an expensive sterile chicken egg, Or more. On the other hand, in the case of producing the antigen diagnostic solution according to the present invention, not only an expensive sterile chicken seed is used but also an antigen diagnostic solution can be manufactured within one week since the expression protein is produced by cell culture and the manufacturing cost can be saved, There is an economical advantage in manufacturing a diagnostic liquid.

On the other hand, since it is possible to mass-produce diagnostic liquid easily in a general laboratory, it is possible to produce and supply NDV antigen diagnostic fluid quickly and safely even if it is difficult to supply sterilized chicken eggs due to infectious disease or vaccine production.

FIG. 1 is a schematic diagram of a recombinant baculovirus expression vector containing NDV HN gene. FIG.
FIG. 2 is a schematic diagram of a process for preparing an expression vector containing NDV HN gene. FIG.
FIG. 3 is a result of western blot analysis of NDV HN protein expressed in insect cells using NDV chicken immunized sera.
4 is a schematic diagram of a process for preparing a NDV antigen diagnostic fluid containing NDV HN protein.
FIG. 5 shows the result of comparing the NDV HN protein antigen extracted with the insect cells and the culture supernatant by the HI method.

Hereinafter, the present invention will be described in detail.

The present invention relates to a recombinant baculovirus expression vector containing NDH HN gene and a method for producing NDV HN protein by culturing insect cells transfected therewith. Further, the present invention relates to a NDV antigen diagnostic solution for HI assay comprising the NDV HN protein.

According to the present invention, a method for producing a NDV antigen diagnostic solution is a method for producing a diagnostic solution containing a recombinant NDV HN protein produced using recombinant baculovirus. Since the produced NDV HN protein has a characteristic of agglutinating chicken erythrocytes, NDV HN protein concentration in the diagnostic antigen can be quantified by measuring the concentration of NDV antigen solution prepared by using chicken erythrocytes in buffer solution and measuring the hemagglutination . In addition, when the NDV antigen test solution is reacted with avian serum containing NDV-specific antibody, an antigen-antibody reaction is caused to inhibit (inhibit) chicken erythrocyte aggregation. If there is no NDV specific antibody in the avian serum, the antigen-antibody reaction does not occur and the hemagglutination inhibition reaction does not occur. This principle can be used to determine the presence of NDV-specific antibodies in the sera from birds. In addition, NDV-specific antibodies can be quantified by measuring the presence or absence of aggregation of the blood after reacting the sera diluted with the buffer solution with the antigen diagnostic solution. Therefore, the NDV antibody titer can be quantified by the HI method in the avian serum using the NDV antigen diagnostic solution of the present invention.

The principle and action of producing the NDV antigen diagnostic solution according to the present invention are as follows. In other words, when insect cells infected with the recombinant beculovirus containing the entire NDV HN gene are cultured, the recombinant NDV HN protein contains the trans-membrane domain and is distributed more intensively in the insect cells than the culture medium. In addition, it is desirable to rapidly extract HN protein because the HN protein produced by the proteolytic enzyme is easily denatured if it takes a long time to extract the produced protein. It is also preferable to add a protease inhibitor to prevent the denaturation of the HN protein during the storage of the HN protein.

The HI method, the reagent, and the reaction conditions used in the NDV antigen diagnostic liquid manufacturing process can be conventionally known in the art. Here, the NDV HN protein is known in the art and can be used regardless of the virus strain.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Construction of Expression Vector Containing NDV HN Gene

The viral genomic RNA was extracted from NDV lasota strain using the RNeasy RNA extraction kit (Qiagen) according to the manufacturer's manual. The cDNA of the NDV HN protein gene was synthesized by adding the extracted RNA and reverse primer of SEQ ID NO: 2 to a tube of RT primix kit (Bioneer) for cDNA synthesis, and the first strand cDNA was synthesized according to the manufacturer's manual. Then, the first strand cDNA, the forward primer of SEQ ID NO: 1, and the reverse primer of SEQ ID NO: 2 were inserted into a tube of PCR premix kit (Bioneer) for Polymerase Chain Reaction (PCR) PCR was carried out by repeating 30 cycles in a PCR amplifier (PE 9600; Perkin Elmer) using 95 ° C for 30 seconds, 53 ° C for 1 minute and 72 ° C for 1 minute and 30 seconds as one cycle, Finally, DNA synthesis was performed at 72 ° C for 7 minutes. For the above-mentioned PCR, 1,754 nucleic acids were amplified so as to include 1,734 nucleic acids constituting the HN gene among all the genes of the NDV lasota strain registered with the NCBI (Genbank) registration number AY845400. Primers were prepared.

SEQ ID NO: 1: 5'-GC TCTAGA GCATGGACCGCGCCGTTAGCC-3 '

SEQ ID NO: 2: 5'-CC AAGCTTG GCTAGCCAGACCTGGCTTCTC- 3 '

Specifically, the primer of SEQ ID NO: 1 has an Xba 1 restriction enzyme action site upstream of the ATG at the 5 'end (in the sequence, , And the primer of SEQ ID NO: 2 was inserted at the 5 'end with a Hind III restriction site (underlined in the sequence) upstream of the end codon. The amplified HN gene cDNA fragment was inserted into a pCR-PCR vector (Invitrogen, USA) without restriction enzyme treatment and cloned to prepare "pCR / NDVHN", and the resultant was subjected to an automatic base sequence analyzer (ABI 377, USA) The nucleotide sequence of the inserted gene was compared with the nucleotide sequence disclosed in NCBI (Genbank) Registration No. AY845400 to confirm whether or not the HN gene cDNA was correctly inserted. The pCR / NDVHN vector of Example 1 was treated with Xba 1 and Hind III to extract a 1,748 bp DNA fragment. Then, the extracted NDV HN DNA fragment was inserted into a multicloning site of a pFastBac ^™ 1 expression vector having a beculovirus polyhedrin promoter (P _PH ) to obtain a recombinant baculovirus expression vector (hereinafter referred to as "pFB / NDVHN"Quot;). FIG. 1 is a schematic diagram illustrating a series of procedures for preparing an expression vector containing NDV HN gene.

Example 2. Production of recombinant baculovirus expressing NDV HN protein

Bekyul recombinant viruses that express the NDV HN protein was produced through the process of FIG. 2 in accordance with the method of using a Baculovirus espression system kit (Life Science Inc.) provided by the manufacturer. That is, the recombinant baculovirus expression vector pFB / NDVHN of Example 1 was transformed into DH10BAC E. coli cells containing Bacmid and subjected to bacmid and recombination. The recombinant Bacimid was then extracted and transfected into insect cells ( Spodoptera frugiperda 9 cells, hereinafter referred to as "Sf9 cells") to produce recombinant baculovirus. Then, the culture supernatant of the transfected cells was harvested and plaque assay was conducted to isolate recombinant baculovirus (hereinafter referred to as " rBac / NDVHN ") expressing NDV HN protein.

FIG. 3 shows the result of Western blotting NDV HN protein expressed in insect cells using NDV chicken immunized sera.

Example 3. Preparation of NDV antigen diagnostic solution for HI method test

The recombinant baculovirus of Example 2 was used to produce and extract NDV HN protein, and then the recombinant baculovirus was prepared as a Newcastle Disease diagnostic antigen for hemagglutination inhibition assay. The process for producing a NDV antigen diagnostic solution containing NDV HN protein as an active ingredient is shown in FIG.

(1) Newcastle disease virus HN protein expression step

The diagnostic antigen containing the recombinant NDV HN protein was prepared by the following method. Namely, recombinant baculovirus rBac / NDVHN at a concentration of 0.1 MOI was added to Sf9 cells monolayer cultured in a 175-cm2 cell culture flask and infected at 27 ° C for 90 minutes. The infected cells were then harvested from the tissue culture flask and then added to a spinner culture vessel cultured in 200 ml of Sf9 insect cells at a concentration of 2 x 10 ⁶ / ml and cultured at 27 ° C for 4 days.

(2) step of harvesting insect cells infected with baculovirus

After the incubation period of 4 days at 27 ° C after the infection with the recombinant beculovirus, the cytopathic effect was observed, the infected insect cells were harvested from the tissue culture flask and added to a 50 ml conical centrifuge tube. The collected cell cultures were centrifuged at 500 xg for 20 minutes using a centrifuge. After centrifugation, centrifugal supernatant was collected and removed, and only the infected insect cells were collected and harvested.

(3) Extraction of Newcastle Disease Virus HN protein from infected insect cells

To the harvested infected insect cells, a 1/20 volume of cell lysate solution was added to the insect cell culture solution, followed by reaction at room temperature for 5 minutes. In this case, protease inhibitor cocktail (Roche Molecular Biochemicals, Germany) is added to prevent destruction of NDV HN protein by proteolytic enzymes in the cell disruption process. The preparation of the cell disruption solution used for HN protein extraction of Newcastle disease virus is as follows.

0.01M phosphate buffered Saline, pH 7.4 ................. 100ml

Tween 20 .............................................. 50μl

Protease inhibitor cocktail ............................ 5 tablets

Then, ultrasonication was simply performed for 3 minutes at about 10 KHz with an ultrasonic processor (Soniprep150, Sanyo, Japan) in order to disrupt the completely undamaged infected insect cells. In order to prevent protein denaturation due to the thermal reaction during the ultrasonic treatment, the samples were subjected to ultrasonic treatment in ice water for 3 minutes at intervals of 30 seconds.

(4) Newcastle Disease Manufacturing Antigen Bleeding and Storage Step Extracted

The HN protein solution of Newcastle disease virus extracted from the above was centrifuged at 500 x g for 20 minutes using a centrifuge. After centrifugation, precipitated cell component precipitate was removed and only centrifugal supernatant was harvested and used as a diagnostic reagent for hemagglutination inhibition assay antigen. In this way, about 10 ml of antigen can be prepared with a 50 ml insect cell culture. At that time, the prepared antigen was dispensed in 0.5 ml portions and stored in a -70 ° C freezer until used in the experiment.

(5) Quantitative determination of the antigen amount of the Newcastle disease diagnostic antigen

Antigen quantification of new Newcastle Disease antigen was performed using hemagglutination (HA) method using chicken erythrocytes. The viral solution was diluted 2-fold with sterilized PBS in a 96-well V-shaped microplate and added in an amount of 25 μl per well. The same amount of 0.5% (v / v) chicken erythrocyte solution was added and left at room temperature for 40 minutes . Thereafter, the reciprocal of the maximum dilution factor for coagulating the chicken erythrocytes was determined in the hemagglutination (HA) antigens.

Experimental Example 1. Antigen concentration of Newcastle Disease Diagnostic Fluid according to the novel invention

The Newcastle Disease Assay of the present invention had a Newcastle Disease virus HN protein with a hemagglutinating (HA) titer of 2 ¹³ per 25 μl of the production antigen. For the Newcastle Disease Antibody Test, an antigen solution of 4 (ie 2 ² ) HA titers per 25 μl of antigen assay is usually used in the HI assay. Because producing a diagnostic antigen of a 10ml solution to the 200ml culture medium, it is possible to manufacture the antigen solution to the total 5,120ml ^{(2 13/2} 2 HA titer x 10ml) required to use the HI method. Generally, considering that 2.5 ml of the antigen test solution is required to test five serum samples (two repeat test standards per sample) in one 96-well microplate, 10 ml of the antigen test solution Of the sample.

Figure 5 Conventionally, a conventional method for extracting an expressed protein and a novel method according to the present invention The NDV HN protein extracted was compared and quantitated by the HI method. As a result, NDV HN protein concentration of hemagglutination (HA) titer of 2 ³ per 25 μl of culture was obtained. Therefore, it is possible to prepare a solution of the antigen is needed to use the HI method by harvesting the cell culture medium 400ml ^{(2 3/2} 2 HA titer x 200ml). This is a low concentration corresponding to 7.8% (400 ml / 5120 ml) of the antigen diagnostic fluid prepared from the infected cells. Therefore, when an antigen diagnostic fluid is prepared by extracting from an infected cell by a novel method, a Newcastle antigen diagnostic fluid can be easily mass produced.

Experimental Example 2. Preservation of Newcastle Disease Antigen Diagnostic Solution by New Inventions

The inventors of the present invention investigated the stability of the Newcastle Disease antigen diagnostic solution at the storage temperature to maintain the effective amount of the antigen. Antigen measurement was carried out by the HA method of Example 3 above while keeping the prepared antigen at refrigeration (4 캜) temperature for 8 weeks. The Newcastle Disease diagnostic fluid prepared according to the present invention had 2 ¹³ (log 2) HA titers immediately after storage at refrigeration temperature and 2 ¹⁰ (log 2) HA titers after 12 weeks of storage. In addition, the conventional antigens prepared using the bovine serum had the HA titers of ²⁸ (log 2) immediately after storage at the refrigeration temperature and 2 ⁷ (log 2) of the HA titers after 12 weeks of storage. Therefore, when the antigenic liquid was prepared from the infected cells according to the present invention, it had sufficient antigen amount for use as a Newcastle antigen diagnostic liquid for at least 12 weeks at the refrigeration temperature.

[Table 1] The result of the preservation test of the Newcastle Disease Antigen Diagnostic Solution prepared by the present invention

EXPERIMENTAL EXAMPLE 3 Evaluation of the efficacy of the New Test Disease Diagnostic Solution according to the novel invention

Whether the test solution of the Newcastle disease antigen according to the present invention responded to the immunized serum of each NDV strain was examined. The immunized sera used for the evaluation of the efficacy of new antigens are the sera prepared by inoculating 20 strains of chicken, La Sota, B1, VG / GA and Ulster2C, which are used as vaccines, When the NDV antibody titer was measured as a new antigen, the HI antibody titer of 6.4 ± 2.76, 6.5 ± 1.64, 4.4 ± 2.76 and 4.6 ± 3.09, respectively, in the serum samples of La Sota, B1, VG / GA and Ulster2C log2 standard). When the antibody titers were measured using the conventional Newcastle Disease antiserum against the same immunized sera, the mean values of serum antibody titers were 6.2 ± 2.61, 6.1 ± 1.1.6, 4.5 ± 2.78 and 4.5 ± 2.78, respectively, in La Sota, B1, VG / GA and Ulster2C 4.9 ± 2.55 HI antibody titer (log2 standard). The correlation coefficient (r) between two antigens showed a very high correlation with the lowest of 0.930 (B1 immunized sera) to 0.969 (Ulster2C immunity sera) when the antibody titers of the same immunized sera were compared. On the other hand, when 100 Newcastle Disease negative serum samples were tested, both new and conventional antigens showed Newcastle Disease negative reaction. These results show that the test solution of Newcastle Disease Antigen according to the present invention has proved to be effective in conducting Newcastle Disease antibody test and can replace the conventional antigen.

[Table 2] HI Antibody Tests by New Newcastle Disease Diagnostic Fluid According to NDV Strain Immune Serum

EXPERIMENTAL EXAMPLE 4. Outdoor Newcastle Disease Test Using New Newcastle Disease Diagnostic Fluid

A Newcastle Disease Diagnostic Fluid according to the present invention was used to test serum samples collected from chickens raised in an outdoor farm and compared with the results of a Newcastle Disease antibody test as a conventional antigen for the same samples.

The Newcastle disease antibody test was performed by the HI method. The criteria for the positive test were 16 times (4 log2) or more as defined by the World Animal Health Organization (OIE). A Newcastle disease antibody test was performed on a total of 791 outdoor farm serum samples using the new Newcastle disease antigen test.

When the new antigen was tested for 232 new antibiotic-positive Newcastle disease antibodies, 223 were positive for antibodies and 9 for negative antibodies. The relative sensitivity of new antigens to conventional antigens was 96.1%. When 559 points, which were judged as Newcastle disease antibody negative by conventional antigen, were tested with a new antigen diagnostic solution, 534 points were judged as antibody-negative and 25 points were judged as antibody-positive. Relative specificity of new antigens relative to conventional antigens was 95.5 %. The kappa value of the two diagnostic antigens was 0.90 and the correlation coefficient was 0.927. These results indicate that HI testing with new antigens is useful for testing Newcastle Disease antibodies in field farms.

[Table 3] Comparison of HI antibody test between new antigen and conventional antigen for serum samples

Relative sensitivity = 96.1% (223/232), relative specificity = 95.5% (534/559), κ value = 0.90, correlation coefficient = 0.927

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (5)

(1) extracting virus genomic RNA from Newcastle diease virus (NDV) lasota strain;
(2) A reverse primer of SEQ ID NO: 2, in which a HindIII restriction endonuclease site was inserted at the 5 'end codon and RNA extracted by the above step (1), was used to amplify the NDV HN (haemagglutinin-neuraminidase) Synthesizing cDNA of the gene;
(3) cDNA synthesized by the above step (2), a forward primer of SEQ ID NO: 1 in which an Xba1 restriction enzyme action site is inserted on the ATG at the 5 'end and a HindIII restriction enzyme Amplifying the reverse primer of SEQ ID NO: 2 inserted with the action site by heat treatment at 95 DEG C, using PCR, and synthesizing DNA;
(4) extracting a DNA fragment of 1748 bp by treating the DNA synthesized by the step (3) with Xba1 and HindIII;
(5) inserting the 4 virus poly a DNA fragment extracted by the step bekyul polyhedrin promoter pFastBac 1 ^TM multi-cloning site (multicloning) of an expression vector with a (P _PH);
(6) transforming a recombinant baculovirus expression vector containing the NDV HN gene prepared in the step (5) into DH10B _AC E.coil cells containing Bacmid, followed by recombination with Bacmid;
(7) extracting the recombinant Bacmid by the step (6);
(8) transfecting insect cells with the recombinant Bacmid extracted in step (7);
(9) expressing the NDV HN protein in an insect cell transfected by the step (8); And
(10) extracting the NDV HN protein expressed by the step (9) from the insect cells through a cell disruption solution and an ultrasonic treatment, and culturing the NDV HN protein using the recombinant baculovirus expressing the NDV HN protein Gt;
delete delete A NDV HN protein produced by the method of claim 1.
A NDV antigen diagnostic liquid for the test for inhibiting hemagglutination inhibition (HI method) comprising the NDV HN protein prepared by the method of claim 1.

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