KR20060023384A - Methods of manufacturing recombinant antibody from hepatitis a virus and applications thereof - Google Patents

Abstract

The present invention relates to a method for producing an antibody against hepatitis A virus and its application, wherein the VP1 gene, which is a capsid protein related to the inhibition of viral infection, is obtained from hepatitis A virus isolated from a hepatitis A virus infected patient and then recombined with E. coli. By producing a large amount of protein, and using it as an antigen to produce an immune antibody containing anti-hepatitis A virus antibody from mammals including cows, to provide a preventive and functional material for hepatitis A virus, such as acute hepatitis It is about how to produce.

Hepatitis A virus, antibody, production method, application, infection, isolation, capsid protein, VP1 gene

Description

Method of manufacturing antibody against hepatitis A virus and its application {Methods of Manufacturing Recombinant Antibody from Hepatitis A Virus and Applications Thereof}             

1 is a photograph of recombinant capsid antigen VP1 gene amplification.

2 is a photograph of recombinant capsid antigen VP1 protein expressed in E. coli.

Figure 3 is a photograph showing the virus infection inhibitory effect in animal cell experiments by cow, egg yolk, pig blood antibodies through the vaccine.

The present invention relates to a method for producing an efficient antigen for producing an antibody having an inhibitory effect against hepatitis A virus infection and to the production of super-antibodies using the same. More specifically, the VP1 gene, which is a capsid protein involved in the inhibition of viral infection, is obtained from hepatitis A virus isolated from a hepatitis A virus infected patient and then recombined into E. coli to produce a large amount of protein. The present invention relates to a method for producing an anti-hepatitis A virus-containing immune antibody from a mammal, including a prophylactic functional material against hepatitis A virus, which causes acute hepatitis.

Infectious hepatitis caused by viruses in the human body is mainly known five types, A, B, C, D, E. Among these five types of hepatitis, hepatitis B, C and D are transmitted mainly by the blood of virus infected patients, while hepatitis A and E are orally infected through contaminated water or food.

Hepatitis A is the most frequent hepatitis among the five types of viral hepatitis described above. In Hong Kong, Asia, the number is higher, and 67% to 83% of hepatitis is diagnosed as hepatitis A.

Hepatitis A virus is a small virus of about 27 nm in the picona virus group. It has a single helical RNA as a gene. May invade the human body. Polio, cold, conjunctivitis, meningitis, or foot-and-mouth virus in animals belong to this group of viruses. Unlike rotavirus, which has numerous serotypes, hepatitis A virus is known to have only one serotype worldwide.

Hepatitis A becomes more severe as the patient ages. For example, jaundice, one of the symptoms of hepatitis, shows only about 10% of children under 6 years old, but 40% to 50% of people over 6 years old and 14 years old, 70 people over 14 years old. Jaundice can be seen in hepatitis patients between 80% and 80%. Therefore, the mortality rate is higher in adults. Hepatitis A mortality is reported as 1.8% in patients over 50 years of age. The overall mortality rate is 0.4%. Once invaded into the body by oral virus spreads throughout the body through the incubation period of about 30 days. In a systemic infection, the patient then releases the virus to the feces so that the feces act as a major source of viral transmission. The spread of hepatitis A is easily transmitted by contact with patients, by family members, by kindergartens, nursing homes, or cooks, including contaminated food (mainly restaurant kitchens, cafeteria, or group meals), or inadequate preparation by individuals in incubation periods. It is easily spread through freshly cooked seafood, raw fish, shellfish, contaminated drinks, and feces contaminated pools or rivers. Globally, the spread of hepatitis A is closely related to socioeconomic level, hygiene and hygiene. In developing countries, almost all children under 9 years of age have antibodies to the virus. Most children do not have clinical symptoms, so children play an important role in the spread of hepatitis A virus. .

In the production of antibodies, colostrum contains not only an antiviral component but also a significant amount of immunoglobulins. Immunoglobulins in mammals of all species are transmitted from the mother to the offspring, giving newborns passive immunity against many types of pathogens. Vaccines can also be administered in pigs to produce antibodies through pig blood. In recent years, the development of antibodies having immunity to specific antigens has been actively conducted by administering vaccines to laying hens.

Therefore, in the present invention, in the production of antibodies against hepatitis A virus, the hepatitis A virus capsid protein VP1 is mass-produced by genetic engineering technology to simplify the production of antigens, and thus, a specific high immune antibody It aims to provide a method of production.

The above task is to obtain a large amount of protein by obtaining the VP1 gene, which is a capsid protein related to the inhibition of viral infection, from the hepatitis A virus isolated from hepatitis A virus infected patients, and recombining it into E. coli, and using it as an antigen to produce cow's milk. The object is achieved by producing a method for producing an anti-hepatitis A virus-containing immune antibody from a mammal, including a prophylactic functional material for hepatitis A virus, such as acute hepatitis.

That is, the present invention devises a method for producing VP1 protein in Escherichia coli through genetic manipulation of VP1, a capsid protein known as an antigen for making neutralizing antibodies in hepatitis A virus. In addition, by optimizing the conditions for improving the expression rate through experiments and developing a vaccine through the development of the technology to produce anti-A virus virus with high immunity, genetic engineering of genes with neutralizing ability of hepatitis A virus The method is to develop a method for producing an antibody with hepatitis A virus that has an anti-infective effect by producing antigen and highly immunologically.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated.

1. Isolation and Culture of Hepatitis A Virus

Excretion from hepatitis A patients was collected, diluted, and infected with animal cells, and cultured until lesions appeared. Cells were collected, frozen, thawed three times, and centrifuged to separate a small amount of supernatant, stored at -70 ° C, and used for gene amplification, analysis, and culture. (See Example 1).

2. Amplification of Hepatitis A Virus Antigen Gene

Total RNA from cells infected with hepatitis A virus was extracted, oligonucleotide primers prepared for synthesizing cDNA from RNA and reverse transcriptase were synthesized using cDNA, followed by PCR using another primer. Antigen RNA genes were amplified and converted to dsDNA (see Example 2).

3. Escherichia coli Metastasis and Sequence Analysis of Hepatitis A Virus Antigen Gene

The antigen gene converted to cDNA should be inserted into the cloning vector plasmid, which was then cleaved using appropriate restriction enzymes and inserted into the plasmid. A large amount of plasmid was prepared from E. coli transformed with this plasmid and then used as the parent of all experiments. The nucleotide sequence of the cloned antigen gene was analyzed but determined in both directions so that there was no error in sequencing (see Example 3).

4. Expression of Recombinant Capsid Protein VP1 in Escherichia Coli

After sequencing, the antigen gene was cloned. Gene expression was attempted after the transformed E. coli was selected. Protein electrophoresis was performed to confirm whether the expressed protein is an antigenic protein (see Example 4).

5. Vaccine Preparation and Immunization Using Recombinant Capsid Protein VP1

The expressed recombinant antigen protein was partially purified after centrifugation. Vaccines were prepared using the appropriate amount of antigen. Cows were planned to be immunized four times at two-week intervals, until two months before they were able to obtain enough antibody titers, and two weeks later. Specific antibody values delivered to each animal were measured using molecular immunological methods. (See Example 5).

6. Inhibitory effect of hepatitis A virus

In vitro experiments were carried out through animal cell experiments to determine the viral infection inhibition ability of antibodies obtained by immunization. First, the hepatitis A virus was infected with animal cells, and then the antibody was inhibited as a result of treatment with the antibody against recombinant capsid antigen VP1 (see Example 7).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are not intended to limit the scope of the present invention, it is possible for the ordinary change by those skilled in the art within the scope of the technical idea of the present invention.

Example 1

Isolation of Hepatitis A Virus

To prepare hepatitis A virus for use as an antigen, Vero E6 cells with monolayers formed in cylindrical barrels were passaged using a specimen obtained from a patient until lesions were observed. When lesions appeared, thaw freeze was repeated to recover the virus and stored at -70 ° C.

Example 2

Amplification of the Hepatitis A Virus Capsid Antigen VP1 Gene

The monolayer-formed Vero E6 cells were infected with the virus and cultured until the lesion effect appeared. When the lesion effect appeared, the cells except the supernatant were collected, Trizol (1 ml) was added to dissolve the cells, and the solution was added to Chloroform (0.2 ml) and shaken vigorously for 3 minutes. The RNA precipitate was recovered by centrifugation, dried and dissolved in RNase free distilled water to separate RNA.

Viral RNA 7ul, 3ul 30% DMSO, 2ul antisense primer (VP1: 5'-ACGAGTCCCTTTCTCAACAGT-3 ') was added for cDNA synthesis to dominate the hepatitis A virus capsid protein and heated at 95 ° C for 10 minutes. To this was added 5 ul 1ST STRAND BUFFER 4 ul, 0.1 M DTT 2 ul, 10 mM dNTPs 1 ul, 1 ul Supercript II 1 ul to react at 42 ℃ for 1 hour to synthesize cDNA.

Synthesized cDNA 1 ul, 2.5 ul sense primer (VP1: 5'-ATGGATGTTACCACACAG-3 '), 2,5 ul antisnese primer, 10 mM dNTPs 1ul, 50 mM MgCl2 1.5 ul, 10X PCR buffer 5ul, Tag polymerase 0.3 ul Water 36.2 ul was added and mixed vigorously. The mixture was heat treated at 94 ° C. for 5 minutes, and then treated 30 times at 94 ° C. for 30 seconds, 52 ° C. for 30 seconds, and 72 ° C. for 1 minute, and finally at 72 ° C. for 7 minutes to amplify the gene. The amplified product was injected into 1% agarose gel, electrophoresed, stained with ethidium bromide, and photographed under ultraviolet light (FIG. 1).

Example 3

Transfer of Amplified Antigen Genes

The amplified gene was cut with BamH1 restriction enzyme and cloned into the cloning vector True Blue vector. The vector was transferred to E. coli DH5α and plated in LB solid medium containing X-gal and ampicilin to form a crack. Characteristic colonies were selected to isolate plasmids, and digested with restriction enzyme BamH1 to confirm that the inserted gene was the desired antigen gene. After culturing the identified clones in the same liquid medium, the plasmids are isolated, cleaved with BamH1 enzyme, purified purely of the desired antigen gene, and cloned into the expression vector pCH433 BamH1 restriction enzyme cleavage site. This vector was transferred to E. coli DH5α, and colonies grown on LB solid medium containing ampicillin were selected to isolate plasmids. The separated plasmid was cut with a restriction enzyme to confirm that the inserted gene was a gene of a desired size, and to confirm whether the gene went in the normal direction. Plasmids of the identified strains were isolated and finally analyzed for nucleotide sequences of the inserted genes to confirm gene transfer.

The hepatitis A virus capsid antigen VP1 gene sequence is shown in SEQ ID NO: 1, and the hepatitis A virus capsid antigen VP1 amino acid sequence is shown in SEQ ID NO: 2.

[SEQ ID NO: 1]

ATGGATGTTACCACACAGGTTGGAGACGATTCAGGGGGTTTTTCAACAACAGTTTCTACAGAGCAGAATGTTCCTGATCCCCAAGTCGGCATAATAACCATGAGGGACCTAAAAGGGAAAGCCAATAGGGGGAAGATGGATGTTTCAGGAGTGCAAGCACCTGTGGGAGCTATTACAACAATTGAGGATCCAGTTTTAGCAAAGAAAGTGCCTGAGACATTTCCTGAATTGAAGCCTGGAGAGTCCAGACATACATCAGATCACATGTCTATTTATAAATTCATGGGAAGGTCTCATTTTCTGTGTACTTTTATCTTCAATTCAAATAATAAAGAGTACACATTTCCAATAACTTTGTCTTCAACTTCTAATCCTCCTCATGGTTTACCATCAACATTAAGGTGGTTTTTCAATTTGTTTCAGTTGTATAGAGGACCATTGGATTTGACAATTATTATCACAGGAGCCACTGATGTAGATGGTATGGCCTGGTTTACTCCAGTAGGCCTTGCTGTCGACACCCCTTGGGTGGAAAAGGAGTCAGCTTTGTCTATTGATTATAAAACTGCCCTTGGAGCTGTTAGATTTAATACAAGAAGAACAGGGAACATTCAGATTAGATTGCCATGGTATTCTTATTTGTATGCCGTGTCTGGAGCGTTGGATGGCTTGGGAGATAAGACAGATTCCACATTTGGATTGGTTTCTATTCAGATTGCAAATTACAATCATTCTGATGAATATTTGTCCTTTAGTTGTTACTTGTCTGTCACAGAACAATCAGAGTTTTATTTTCCTAGAGCTCCATTGAATTCAAATGCTATGTTGTCCACTGAGTCTATGATGAGTAGAATTGCAGCTGGAGACTTGGAGTCATCAGTGGATGATCCTAGATCAGAGGAGGACAGAAGATTTGAG AGTCATATAGAATGTAGGAAACCATACAAAGAATTGAGATTGGAGGTTGGGAAACAAAGACTCAAATATGCTCAGGAAGAG TTGTCA

[SEQ ID NO: 2]

M D V T T Q V G D D S G G F S T T V S T E Q N V P D P Q V G I I T M R D L K G K A N R G K M D V S G V Q A P V G A I T T I E D P V L A K K V P E T F P E L K P G E S R H T S D H M S I Y K F M G R S H F L C T F I F N S N N K E Y T F P I T L S S T S N P P H G L P S T L R W F F N L F Q L Y R G P L D L T I I I T G A T D V D G M A W F T P V G L A V D T P W V E K E S A L S I D Y K T A L G A V R F N T R R T G N I Q I R L P W Y S Y L Y A V S G A L D G L G D K T D S T F G L V S I Q I A N Y N H S D E Y L S F S C Y L S V T E Q S E F Y F P R A P L N S N A M L S T E S M M S R I A A G D L E S S V D D P R S E E D R R F E S H I E C R K P Y K E L R L E V G K Q R L K Y A Q E E L S

Example 4

Production of Capsid Antigen Protein in Escherichia Coli

Escherichia coli transfected with the vector containing the antigen gene was incubated overnight in LB medium (yeast extract 5g, tryptone peptone 10g, NaCl 10g / 1L) and inoculated in 1% in a large amount of copper medium. When the absorbance (600 nm) of the culture reached 0.5, isopropyl-b-D-thiogalatoside was added to a final concentration of 0.5 mM, and shaking culture was continued for 4 hours. The microbial cells were collected by centrifugation of the culture solution, diluted with distilled water, mixed with electrophoretic loading buffer, heated at 95 ° C. for 5 minutes, and then electrophoresed to confirm the expressed antigen protein.

Partial tablets were suspended in 25% Sucrose, 50 mM Tris, pH 8.0, and added 10 mg of lysozyme and maintained for 10-15 minutes in refrigerated state. Then 2XRIPA-TET buffer solution was added and sonicated after 5 minutes. The solution was centrifuged (15000 g, 20 minutes) to give the supernatant as the antigen. Some of the supernatants were mixed with SDS-PAGE sample buffer, boiled and loaded into gels, followed by electrophoresis and coomassive staining to confirm protein expression.

Figure 2 shows a capsid antigen VP1 protein picture expressed in E. coli.

Example 5

Vaccination and Immunization of Capsid Antigen Proteins

A vaccine using 10% Aluminum hydroxide gel was produced using the recombinant antigen prepared in Example 4, and immunized to the cow two months before the expected birth. After birth, each was collected and stored by date. After the titer test, ELISA was used to collect the high titers from day 1 to day 3. In the case of laying hens, the adjuvant is a mixture of Drakeol, which is a kind of mineral oil, and an emulsifier, and Span 85 and Tween 85 are mixed in an ratio of 54:46 as an emulsifier. Was used. Drakeol, emulsifier, and antigen were mixed and emulsified in a ratio of 9: 1: 8, and then intramuscularly injected four times at two week intervals until sufficient antibody titer was obtained.

Example 6

Anti-Hepatitis A Virus Capsid Antibody Antibody Titer Comparison

Colostrum collected from cows born after immunization as in Example 5 was collected by date. In addition, pigs with sufficient titers, piglets and egg yolks collected from laying hens were collected. Antibody titer against hepatitis A virus was measured by ELISA as follows. The obtained antibody was diluted 2 ⁿ times in succession with phosphate buffer (PBS, pH 7.4) to obtain a sample solution. In order to confirm the titer of the antibody, hepatitis A virus capsid protein VP1 was suspended in a coating buffer, and 100 μl of the hepatitis A virus was deposited on a 96well plate and attached overnight at 4 ° C. The attached antigen was washed with PBST (50mM PBS, 0.05% tween 20), and the antibody solution was diluted twice in succession from 1:10 and then dispensed in 100 μl and reacted at 37 ° C. for 2 hours. After completion of the reaction, washed with PBST, goat-anti-bovine IgG HRP, goat-anti-pig IgG HRP, goat-anti-chicken IgY diluted 1: 1,000, respectively, 100 μl was dispensed for 1 hour at 37 ℃ I was. After completion of the reaction, the reaction was washed with PBST, and then 100 μl of substrate solution (OPD peroxidase substrate, Sigma) was reacted at 37 ° C., and 1M H ₂ SO ₄ was added to stop the reaction. After the reaction was stopped, the reaction value was checked using an ELISA reader to measure the titer of the antibody (OD _{490 nm} > 1.0). As a result, it showed the highest titer in the first colostrum and decreased with time. After the third day, the potency decreased rapidly. In addition, the colostrum was also stable in a series of processes of cooling, thawing, homogenization, sterilization and freeze drying of colostrum. Similarly, high titers were observed in pig blood and egg yolk.

Example 7

Inhibitory effect of virus infection in vitro

In order to confirm the inhibitory effect of hepatitis A virus infection using the antibody identified in Example 10, the following experiment was performed using animal cells. First, Vero E6 cells were grown to monolayer and then infected with hepatitis A virus. At this time, after adding the antibody (1: 100) to be tested, the medium was removed the next day, washed with PBS, and then fixed for 30 minutes after adding 10% formalin. After washing with PBS again, 0.5% triton X-100 / PBS was added and allowed to stand for 5 minutes, and then anti-Hepatitis A virus antibody (1: 250) / PBS / 1% BSA solution was added and allowed to stand at room temperature for 30 minutes. . After washing twice with PBA (1% BSA / PBS), HRP-conjugated goat anti-mouse IgG (1: 500) / PBA was added and allowed to stand at room temperature for 30 minutes. Finally, a mixture of 4 ml of 3-amino-9ethylcarbazole dissolved in 1 ml N, N-dimethylformamide, 1.5 ml, 3.5 ml, and 5 µl of 0.05 M sodium acetate buffer and hydrogen peroxide was treated. Stained infected cells were identified. As a result, treatment with antibodies to recombinant fusion antigens has been shown to inhibit infection from hepatitis A virus.

Figure 3 shows the hepatitis A virus infection inhibitory effect in in vitro animal cell experiments through the antibody, Figure 3a is non-infection, Figure 3b is a virus infection, Figure 3c is a virus + antibody.

Example 8

Fermented milk production using anti-A virus antibody

Using skim milk powder, the raw milk obtained by adjusting the nonfat milk solid content to 17% by weight was sterilized at 135 ° C for 3 seconds. The sterilized crude oil was cooled to 37 ° C., and then inoculated with Lactobacillus ashidophilus HY 2177 and Lactobacillus casei HY 2743 at a concentration of 10 ⁶ cfu / ml and incubated until pH 4.5. After completion of the culture, the culture was cooled. Meanwhile, 0.5 wt% of antibody, 1.0 wt% of juice concentrate, 2.0 wt% of dietary fiber, 0.5 wt% of glucose, 1.0 wt% of oligosaccharide, and 0.03 wt% of vitamin were mixed and dissolved in purified water to prepare a syrup. The syrup thus prepared was sterilized at 65 ° C. for 30 minutes, cooled, mixed with the culture solution at a predetermined ratio, homogenized by stirring, and packaged in a container to prepare fermented milk. As a result of the sensory test, the fermented milk prepared as described above did not feel foreign body caused by the capsule and showed good results in flavor, physical properties and overall taste.

Example 9

Preparation of dietary supplement using anti-A virus antibody

Health supplements such as lactic acid bacteria foods and formal preparations were prepared using the prepared colostrum antibodies. 20% by weight of antibody and 25% by weight of Lactobacillus ashidophilus dried powder, 10% by weight of oligosaccharide, 10% by weight of anhydrous glucose, 5% by weight of fructose, 2% by weight of vitamin C, 8% by weight of fruit powder, 5% of aloe % And 15% by weight of dietary fiber were mixed and packed in a fixed amount in sticks or bottles.

Example 10

Preparation of special nutrition products using anti-hepatitis A virus

Special nutritional products such as baby food and formula were prepared using the prepared capsule antibody. First, about 30 kinds of raw materials such as protein, refined vegetable fat, vitamins, sugars and iron salts are mixed in skim milk in a liquid state and standardized to meet the components of special nutritional products. After heating to about 80 ℃ using a vacuum concentrator, it is concentrated so that the solid content is 45-50%. It was hot-air dried in a spray dryer, mixed with 5% by weight of the antibody and other raw materials in powder form such as vitamins and lactose, filled with nitrogen and packed in cans or sticks.

As described above, a vaccine using the hepatitis A virus capsid VP1 antigen according to the present invention is prepared to produce a high titer of anti-hepatitis A virus antibody, and the hepatitis A virus, which is the cause of the group infection, can be suppressed using the vaccine. It is possible to prepare food and application compositions that can be used, it is expected that the use and application in the related fields.

<110> KOREA YAKULT COMPANY LIMITED <120> Methods of Manufacturing Recombinant Antibody from Hepatitis A          Virus and Applications Thereof <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1005 <212> DNA <213> VPone genes of Hepatitis A Virus antigen capsid protein <400> 1 atggatgtta ccacacaggt tggagacgat tcagggggtt tttcaacaac agtttctaca 60 gagcagaatg ttcctgatcc ccaagtcggc ataataacca tgagggacct aaaagggaaa 120 gccaataggg ggaagatgga tgtttcagga gtgcaagcac ctgtgggagc tattacaaca 180 attgaggatc cagttttagc aaagaaagtg cctgagacat ttcctgaatt gaagcctgga 240 gagtccagac atacatcaga tcacatgtct atttataaat tcatgggaag gtctcatttt 300 ctgtgtactt ttatcttcaa ttcaaataat aaagagtaca catttccaat aactttgtct 360 tcaacttcta atcctcctca tggtttacca tcaacattaa ggtggttttt caatttgttt 420 cagttgtata gaggaccatt ggatttgaca attattatca caggagccac tgatgtagat 480 ggtatggcct ggtttactcc agtaggcctt gctgtcgaca ccccttgggt ggaaaaggag 540 tcagctttgt ctattgatta taaaactgcc cttggagctg ttagatttaa tacaagaaga 600 acagggaaca ttcagattag attgccatgg tattcttatt tgtatgccgt gtctggagcg 660 ttggatggct tgggagataa gacagattcc acatttggat tggtttctat tcagattgca 720 aattacaatc attctgatga atatttgtcc tttagttgtt acttgtctgt cacagaacaa 780 tcagagtttt attttcctag agctccattg aattcaaatg ctatgttgtc cactgagtct 840 atgatgagta gaattgcagc tggagacttg gagtcatcag tggatgatcc tagatcagag 900 gaggacagaa gatttgagag tcatatagaa tgtaggaaac catacaaaga attgagattg 960 gaggttggga aacaaagact caaatatgct caggaagagt tgtca 1005 <210> 2 <211> 335 <212> PRT <213> VPone amino acids of Hepatitis A Virus antigen capsid protein <400> 2 Met Asp Val Thr Thr Gln Val Gly Asp Asp Ser Gly Gly Phe Ser Thr   1 5 10 15 Thr Val Ser Thr Glu Gln Asn Val Pro Asp Pro Gln Val Gly Ile Ile              20 25 30 Thr Met Arg Asp Leu Lys Gly Lys Ala Asn Arg Gly Lys Met Asp Val          35 40 45 Ser Gly Val Gln Ala Pro Val Gly Ala Ile Thr Thr Ile Glu Asp Pro      50 55 60 Val Leu Ala Lys Lys Val Pro Glu Thr Phe Pro Glu Leu Lys Pro Gly  65 70 75 80 Glu Ser Arg His Thr Ser Asp His Met Ser Ile Tyr Lys Phe Met Gly                  85 90 95 Arg Ser His Phe Leu Cys Thr Phe Ile Phe Asn Ser Asn Asn Lys Glu             100 105 110 Tyr Thr Phe Pro Ile Thr Leu Ser Ser Thr Ser Asn Pro Pro His Gly         115 120 125 Leu Pro Ser Thr Leu Arg Trp Phe Phe Asn Leu Phe Gln Leu Tyr Arg     130 135 140 Gly Pro Leu Asp Leu Thr Ile Ile Ile Thr Gly Ala Thr Asp Val Asp 145 150 155 160 Gly Met Ala Trp Phe Thr Pro Val Gly Leu Ala Val Asp Thr Pro Trp                 165 170 175 Val Glu Lys Glu Ser Ala Leu Ser Ile Asp Tyr Lys Thr Ala Leu Gly             180 185 190 Ala Val Arg Phe Asn Thr Arg Arg Thr Gly Asn Ile Gln Ile Arg Leu         195 200 205 Pro Trp Tyr Ser Tyr Leu Tyr Ala Val Ser Gly Ala Leu Asp Gly Leu     210 215 220 Gly Asp Lys Thr Asp Ser Thr Phe Gly Leu Val Ser Ile Gln Ile Ala 225 230 235 240 Asn Tyr Asn His Ser Asp Glu Tyr Leu Ser Phe Ser Cys Tyr Leu Ser                 245 250 255 Val Thr Glu Gln Ser Glu Phe Tyr Phe Pro Arg Ala Pro Leu Asn Ser             260 265 270 Asn Ala Met Leu Ser Thr Glu Ser Met Met Ser Arg Ile Ala Ala Gly         275 280 285 Asp Leu Glu Ser Ser Val Asp Asp Pro Arg Ser Glu Glu Asp Arg Arg     290 295 300 Phe Glu Ser His Ile Glu Cys Arg Lys Pro Tyr Lys Glu Leu Arg Leu 305 310 315 320 Glu Val Gly Lys Gln Arg Leu Lys Tyr Ala Gln Glu Glu Leu Ser                 325 330 335  

Claims (10)

In the production of hepatitis A virus antigens, some of the virus genes are cloned by genetic engineering techniques to produce vaccines. The method of claim 1, Method for producing hepatitis A virus inhibited colostrum by high immunity to cows before birth using the vaccine The method of claim 1, Method for producing hepatitis A virus inhibiting antibody into egg yolk by highly immunizing the laying hens using the vaccine The method of claim 1, Method of producing hepatitis A virus inhibiting antibody as pig blood by high immunity to swine using the vaccine Fermented milk containing an immune antibody prepared by the method of any one of claims 2 to 4 and inducing an active immune response A dietary supplement that contains an immune antibody prepared by the method of any one of claims 2 to 4 and induces an active immune response. A special nutrition product containing an immune antibody prepared by the method of any one of claims 2 to 4 to induce an active immune response Feed additive comprising an immune antibody prepared by the method of any one of claims 2 to 4 as an active ingredient Pharmaceutical additives comprising the immune antibody prepared by the method of any one of claims 2 to 4 as an active ingredient A food additive comprising an immune antibody prepared by the method of any one of claims 2 to 4 as an active ingredient.

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