KR20120079389A - Transgenic chicken containing 3d8 scfv gene and producing method thereof - Google Patents

Abstract

PURPOSE: A transgenic chicken and a method for producing the same are provided to ensure anti-viral ability and to be used as a model animal for preventing viral diseases. CONSTITUTION: A vector for transformation contains a promoter which is operable in birds and a 3D8 scFv gene which is operatively linked to the promoter. The promoter is a chicken beta-actin gene promoter. The 3D8 scFv gene further contains a reporter gene. The reporter gene is HA-tag. A method for producing a transgenic chicken comprises: a step of preparing the vector; a step of transducing the vector to chicken fertilized eggs; and a step of hatching the fertilized eggs.

Description

Transgenic chicken containing 3D8 scFv gene and method for producing the same

The present invention relates to a transgenic chicken and a method of manufacturing the same. More specifically, the beta-actin (CBA) gene promoter and 3D8 single-chain variable fragment (scFv) gene in chickens using recombinant lentivirus in chicken blastocyst stage-X. The present invention relates to a transgenic chicken, to which a gene having HA-tag (3D8 scFv-HA) bound thereto is introduced, and a method of manufacturing the same.

With the current risk of human transmission of the avian influenza virus, organizations in the US, such as USDA, CDC, and WHO, have established a pathology for infectious avian influenza virus and established a bird flu database in North America. In order to study the system of early bird flu alert system and to develop vaccines, the government is pushing ahead. In Japan, many research institutes continue to track the genetic and serological changes of migratory birds and bird flu viruses that cause bird flu in Japan every year. In Korea, the Centers for Disease Control and Veterinary Research and Quarantine are conducting research on avian influenza virus to prevent bird flu and diagnose early onset.

The method of minimizing the damage caused by avian influenza is the development of a vaccine, but in the case of avian influenza virus, there are many technical variants, so there are technical limitations in developing a perfect vaccine. Anti-viral agents such as Tamiflu and Relenza have been used as countermeasures, but the incidence of avian influenza virus that is resistant to anti-viral agents is steadily increasing, and there are problems such as decreased efficacy and side effects. Development of viral agents and development of avian influenza virus resistant transgenic chickens capable of verifying this are urgently needed. To date, there have been no studies on the development of avian influenza virus resistant transgenic chickens in Korea.

The 3D8 scFv gene is a mouse-derived antibody gene. Unlike general antibodies that recognize an antigen and remove the antigen by an antigen-antibody reaction, the 3D8 scFv antibody has the ability to specifically hydrolyze nucleic acids. It is expected to have antiviral activity against viruses invading from outside.

These characteristics are considered to be applicable to chickens that cause direct economic loss when infected with avian influenza. When producing transgenic chickens expressing the 3D8 scFv protein, model traits resistant to viruses including avian influenza The 3D8 scFv is expected to be used as a conversion chicken (control group related to viral diseases such as bird flu), a model animal for verifying the virus risk measure when a new strain of virus emerges, and a new animal for verifying a new virus. The development of transgenic chickens carrying the genes is required.

Under these technical backgrounds, the inventors have made intensive efforts to produce transgenic chickens expressing 3D8 scFv protein with nucleic acid hydrolysis ability.

After all, it is an object of the present invention to provide a vector for transformation expressing the 3D8 scFv protein.

Another object of the present invention is to provide a transgenic chicken expressing the 3D8 scFv protein.

Another object of the present invention is to provide a method for producing a transgenic chicken expressing the 3D8 scFv protein.

According to an aspect of the present invention, a promoter operable in an algae and a transformation vector in which a 3D8 scFv gene is coupled to be operable to the promoter may be provided.

According to one embodiment, the promoter operable in the bird may be a beta-actin gene promoter of chicken.

According to one embodiment, the 3D8 scFv gene may further comprise a reporter gene.

According to one embodiment, the reporter gene may be HA-tag.

According to another aspect of the present invention, a fertilized egg transformed with the vector may be provided.

According to another aspect of the present invention, a chicken transformed with the vector may be provided.

According to another aspect of the invention, the step of preparing a promoter operable in the algae and the transformation vector is coupled to the 3D8 scFv gene to be operable to the promoter; Transducing the vector into a fertilized egg of a chicken; And hatching the fertilized egg may be provided a method of producing a transgenic chicken comprising the step of identifying the individual transformed in the first generation.

According to another aspect of the invention, the cells of chickens transformed with the vector of any one of claims 1 to 4 after infection with the virus, characterized in that for detecting the detection amount of the nuclear protein (NP) of the virus A virus detection method can be provided.

Since the study has anti-viral activity in chickens, it can be used as a model animal in the research field for the prevention of viral diseases such as avian influenza.

1 is a schematic diagram of a lentiviral vector in which a HA-tag (3D8 scFv-HA) is coupled to a promoter and 3D8 scFv gene of a beta-actin (CBA) gene of a chicken.
2 shows a schematic diagram of a lentiviral vector insert having a promoter of the CBA gene and a 3D8 scFv-HA gene.
FIG. 3 shows the result of Western blot analysis using HA-tag after infection of 293T cells with a recombinant virus having a CBA gene promoter and a 3D8 scFv-HA gene.
4 is a result of semen PCR analysis of candidate transgenic chickens (G0).
5 is a blood PCR analysis of candidate transgenic chickens (G1).
Figure 6 shows the results of the second generation transgenic chicken Southern analysis.
7 is a result of 3D8 scFv-HA mRNA expression analysis in the blood of transgenic chicken (G1) (RT-PCR).
8 shows antiviral activity of fetal fibroblasts of the third generation transgenic chicken (G2).

In the present invention, a method of producing a second generation transgenic chicken (G1) into which a promoter of a beta-actin gene and a 3D8 scFv-HA gene were introduced. The anti-viral activity of the transgenic chickens developed in this study provides the possibility of being used as a model animal for research on viral diseases such as avian influenza.

Hereinafter, the present invention will be described in more detail.

The present invention is a second generation having a CBA promoter and a 3D8 scFv-HA gene by microinjecting a recombinant lentivirus into a chicken blastocyst stage embryo using a recombinant lentivirus having a beta-actin gene promoter and a 3D8 scFv-HA gene. Provided is a method for producing a transgenic chicken (G1).

The present invention provides expression vector construction and recombinant lentiviral production, verification of expression vector expression at the cellular level, viral microinjection and fertilized egg artificial culture, production of first generation candidate transgenic eggs, selection of first generation transgenic chicken (G0) Production of second generation transgenic chickens (G1) with genes, and finally 3D8 scFv-HA expression validation in selected second generation transgenic chickens and antiviral activity validation in fetal fibroblasts of transgenic chickens.

According to an aspect of the present invention, a promoter operable in an algae and a transformation vector in which a 3D8 scFv gene is coupled to be operable to the promoter may be provided.

According to one embodiment, the promoter may be a beta-actin gene of chicken.

According to one embodiment, the 3D8 scFv gene may further comprise a reporter gene, the kind of which is generally a reporter gene, or other substance used to confirm the presence of the transformed gene in the field of molecular biology It doesn't matter.

According to one embodiment, the reporter gene may be HA-tag.

According to another aspect of the present invention, a fertilized egg transduced with the vector may be provided.

According to another aspect of the present invention, a chicken transformed with the vector may be provided.

According to yet another aspect of the present invention, there is provided a promoter operable in a chicken and a transformation vector having a 3D8 scFv gene coupled to be operable to the promoter; Transducing the vector into a fertilized egg of a chicken; And hatching the fertilized egg may be provided a method of producing a transgenic chicken comprising the step of identifying the individual transformed in the first generation.

According to another aspect of the invention, a virus detection method characterized in that after infecting the cells of chickens transformed with the vector with a virus, the detection amount of the nuclear protein (NP) of the virus can be provided. have.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example One. Lentivirus  Vector construction and recombinant virus production

The present invention aims to construct a lentiviral vector in which a 3D8 scFv-HA gene is coupled to a promoter region of approximately 1.7 kb in the beta-actin gene of chicken. First, the beta-actin gene promoter region and 3D8 scFv-HA gene of chicken were amplified by PCR to obtain DNA fragments of approximately 1.7 and 0.8 kb, respectively. The amplified genes were constructed with lentiviral vectors using molecular biological methods. 1 is a schematic diagram of a lentiviral vector in which a HA-tag (3D8 scFv-HA) is coupled to a promoter and 3D8 scFv gene of a beta-actin (CBA) gene of a chicken. The constructed viral vector confirmed that the promoter region and 3D8 scFv-HA gene were cloned correctly by sequencing (see SEQ ID NO: 1). Figure 2 is a schematic diagram showing the position of EcoRI and XhoI used in the beta actin gene and 3D8 scFv gene, HA-tag structure and linkage of the chicken inserted into the vector, the sequence is shown in SEQ ID NO: 1 . In addition, the cloned 3D8 scFv-HA gene confirmed that the codons that accurately indicate the start (ATG) and end (TAG) of protein synthesis existed.

Recombinant lentivirus was produced in 293FT virus packaging cells using a lentivirus vector having a beta-actin gene promoter region of the chicken and a 3D8 scFv-HA gene. The titer of the recombinant lentivirus produced was indirectly quantified by Western blot analysis of the amount of gag protein, a recombinant virus surface protein.

Example  2. Verification of expression vector expression at the cellular level

3 shows that 293T cells derived from human kidneys were infected with a recombinant virus having a CBA promoter and a 3D8 scFv-HA gene, and 3D8 scFv-HA protein expression was confirmed by HA-tag Western analysis. Column 1 of FIG. 3 represents a positive control group, column 2 represents a negative control group, and column 3 represents 3D8 scfv-HA. The observations show that the lentiviral vectors you built work correctly at the cellular level.

Example  3. Virus microinjection and fertilized egg artificial culture

The fertilized egg (stage-X) used in the present invention used fertilized eggs that were bred by the brown practical system possessed by the National Livestock Science Institute. The artificial culture of fertilized eggs was carried out according to the method established in the National Livestock Science Research Center. First, eggs that were about 3g heavier than fertilized eggs were selected as secondary surrogate eggs, and the pointed portions of the eggs were cut to 3.5cm in diameter, and then all egg whites and egg yolks inside the eggs were discarded and used. Embryos selected prior to virus microinjection were excised and transferred to surrogate egg shells, and microscopically injected recombinant lentiviral microorganisms containing approximately 1 μl of beta-actin (CBA) promoter region and 3D8 scFv-HA gene using capillary tubes under a microscope. It was. After the filling of the surrogate egg shell completely by using the prepared culture solution, it was sealed with a wrap and incubated for 2 days in an incubator of 90 ° every 11 minutes in an incubator having a temperature of 37.6 ° C. and a humidity of 65% -70%. After the 3 days of secondary culture, the third surrogate egg shell was selected as a fresh egg about 25g heavier than the weight of the fertilized egg and cut the buttocks into 4cm in diameter and used as surrogate egg shell. The fertilized egg and its contents were transferred to the prepared surrogate egg shell, sealed with a wrap, and incubated for 15 days while incubating at 30 ° every 30 minutes in an incubator having a temperature of 37.6 ° C. and a humidity of 65% -70%. After incubation was incubated without the process of egg transfer. After injecting recombinant lentiviral with a CBA promoter region and 3D8 scFv-HA gene into 380 fertilized eggs and artificial culture using surrogate egg eggs, 200 candidate transgenic eggs successfully hatched and survived for more than a week after hatching. The number of transition chicks was 187. In addition, the control group that had only artificial culture using surrogate egg shell without microinjection of virus hatched 20 chicks in all 27 fertilized eggs. This shows that the fertilized egg culture system of chicken using surrogate egg was operated normally. Overall fertilized egg culture and the production of candidate transformed eggs using surrogate egg method are shown in Table 1.

group
Stage-X embryos (n)
Number of Embryos Survived After Culture
Hatched population
(%)

Week 1 Survival Chick (%) 4 days
(%) 11 days
(%) 19 days
(%) 3D8 scFv 380 328
(86.3) 299
(78.6) 288
(75.7) 200
(52.6) 187
(48.2) Control group 27 - - - 20
(74.1) -

Example  4. First generation candidate transgenic chickens G0 Genetic analysis

The first generation transgenic chicken (G0) produced by injecting a recombinant lentivirus into the embryonic stage embryos is produced in a 100% mosaic form. Therefore, a complete transgenic chicken can be said to be transgenic chicken when the genetic information necessary for transformation is transmitted through the gonad and the second generation transgenic chicken (G2) is produced. Due to this method characteristic, selection of the first generation transgenic chicken (G0) having a foreign gene carried in a recombinant virus in the gonad (semen) is an essential process. The semen analysis of males of candidate transgenic chickens produced by microinjecting a recombinant virus having a CBA promoter region and a 3D8 scFv-HA gene confirmed the presence of the target gene in 15 semen. 4 shows a part of the entire analysis process. In FIG. 4, Pc represents a positive control group, Nc represents a negative control group, and columns 4 and 11 represent transgenic chickens. Primers and PCR conditions used for PCR verification are as follows. Semen was artificially collected from candidate transgenic chickens that had mature maturation, and genomic DNA was isolated from them to obtain the following primers (CBA promoter F of SEQ ID NO: 2 (5-CCT CTG CTA ACC ATG TTC ATG CCT TC-3). And CBA-3D8 scFv R of SEQ ID NO: 3 (5-GCT AGT GAA TGT GTA TCC AGA AGC CTT-3)] and taq-polymerase (AccuPrime SuperMix II, Invitrogen) at 94 ° C-2 min, 94 ° C-45 PCR was performed under conditions of seconds, 62 ° C.-30 seconds, and 68 ° C.-20 seconds to select individuals having a target gene in semen.

Example  5. Selection of transgenic chickens with the injected gene

Two of the 15 transgenic chickens with foreign genes injected into semen were selected, which were considered to be highly transgenic, and their semen were artificially inseminated to normal females to generate second generation candidate transgenic chicks. Hatched. 5 μl of blood was collected from the wing veins of the candidate transgenic chicks to isolate genomic DNA for PCR genetic analysis. PCR genetic analysis was performed according to established methods, and the results are shown in FIG. 5. 5, Pc is a positive control group, Nc is a negative control group. Row 29 represents transgenic chickens. Finally, PCR analysis showed positive PCR results in 7 blood samples. Chickens initially selected by PCR method were re-extracted for extraction, genomic DNA was extracted, treated with EcoRI / XhoI restriction enzymes shown in FIG. 2, and the 3D8 scFv gene was Southern blot. The analysis was performed. The results are shown in FIG. In FIG. 6, Pc represents a positive control group, and rows 3 to 9 represent transgenic chickens. PCR and Southern analysis showed the same results.

The above results show that the transgenic DNA including the CBA gene promoter and the 3D8 scFv gene is not a mosaic but a complete transgenic chicken. Table 2 summarizes the production results of the first generation and second generation transgenic chickens. In short, semen was collected from two first-generation transgenic chickens and artificially fertilized each normal hen to produce 1456 fertilized eggs, from which 967 transgenic chickens were produced, and all individuals that successfully hatched. Blood collection, genomic DNA isolation, and PCR analysis were carried out in the field to finally produce 7 second generation transgenic chickens.

Table 2 shows the production results of the second generation transgenic chicken (G1).

ID Ranunculus Incubation water (%) Positive entity (G1) J21 (G0) 676 459 (67.9) 5 J186 (G0) 780 508 (65.1) 2

Example  6. Second Generation Transformation From chicken  3 D8 scFv - HA  Expression Verification and Anti-Functionality in Fetal Fibroblasts of Transgenic Chickens. Viral activity  Verification

7 is a result of confirming that the 3D8 scFv gene is expressed in the transgenic chickens by the mRNA extraction from the blood of the final selected transgenic chickens and the 3D8 scFv gene expression was examined by RT-PCR method. In FIG. 7, column 1 to column 3, where no RNA expression of the 3D8 scFv gene is shown, are the control group, and column 4 to column 10 represent the transgenic chicken.

Next, the semen of the selected second generation transgenic chicken (G1) was artificially inseminated in normal females to produce fertilized eggs, and fetal fibroblasts were isolated from culture embryos on day 10, followed by PCR genetic analysis. Only cells with the 3D8 scFv gene were selected. Selected fetal fibroblasts infected with low-pathogenic avian influenza with MOI 0.5 and sampled cells at each time to produce antibodies capable of binding to NP of avian influenza (anti-influenza nuclear protein (NP) antibody, Santa Cruz Cat # sc-101352). Flow cytometry was performed. The results are shown in FIG. Referring to FIG. 8, after 8 hours of reaction, No. 5 and No. 6 N-183-1. Compared to P196-1, the antiviral effect was observed at No. 4 and N165-3. In other words, it can be seen that the number of times 4 and N165-3 is lower than those of 5, 6, and N183-1 P196-1, which indicates that the virus is degraded and detected by the NP antibody. it means. This confirmed that the transgenic chicken according to the present invention has anti-viral activity.

Therefore, by using the transgenic chicken of the present invention, the unknown avian influenza virus can be infected with the transgenic chicken, and the harmfulness of the bias can be measured, and various studies related to other viruses can be applied to the development of new drugs. There is an advantage that can be utilized for various purposes, such as.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

                         SEQUENCE LISTING <110> RURAL DEVELOPMENT ADMINISTRATION   <120> Transgenic chicken containing 3D8 scFv gene and producing method        the <130> NPF18717 <160> 3 <170> PatentIn version 3.2 <210> 1 <211> 2535 <212> DNA <213> Artificial <220> <223> Chick Beta Actin promoter + 3D8 scFv + HA <400> 1 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 60 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 120 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 180 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 240 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 300 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg 360 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 420 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg 480 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaaa ggtgcggcgg 540 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggggg cggcggcggc 600 ggccctataa aaagcgaagc ggggggcggg ggggagtcgc tgcgcgctgc cttcgccccg 660 ggccccgctc cgccgccgcc tcgggccgcc cgccccggtt ctgactgacc gcgttactcc 720 cacaggtgag cgggggggac ggcccttctc ctccgggttg taattagcgc ttggtttaat 780 gacggcttgt ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt 840 tgtgcggggg gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt 900 gcggctccgc gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc 960 tccgcagtgt gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct 1020 gcgaggggaa caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg 1080 gcgcgtcggt cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc 1140 cggcttcggg tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg 1200 ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg 1260 gggaggggcg cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc 1320 attgcctttt atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg 1380 cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt 1440 gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc 1500 cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac 1560 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc 1620 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt 1680 ctcatcattt tggcaaagaa ttctgcagtc gacggtaccg cgggcccggg atccgccacc 1740 atggaggtcc agctgcagca gtctggacct gagctggtaa agcctggggc ttcagtgaag 1800 atgtcctgca aggcttctgg atacacattc actagctatg ttatgcactg ggtgaagcag 1860 aagcctgggc agggccttga gtggattgga tatattaatc cttacaatga tggtactaag 1920 tacaatgaga agttcaaagg caaggccaca ctgacttcag acaaatcctc cagcacagcc 1980 tacatggagc tcagcagcct gacctctgag gactctgcgg tctattactg tgcaagaggg 2040 gcctataaaa ggggatatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 2100 tctagaggtg ggggcggttc gggtggcggg ggctcgggcg ggggtggctc agatcttgtg 2160 atgtcacagt ctccatcctc cctggctgtg tcagcaggag agaaggtcac tatgagctgc 2220 aaatccagtc agagtctgtt caacagtaga acccgaaaga actacttggc ttggtaccag 2280 cagaaaccag ggcagtctcc taaactgctg atctactggg catccactag ggaatctggg 2340 gtccctgatc gcttcacagg cagtggatct gggacagatt tcactctcac catcagcagt 2400 gtgcaggctg aagacctggc agtttattac tgcaagcaat cttattatca catgtatacg 2460 ttcggatcgg ggaccaagct ggaaataaaa ctcgagtcta gatacccata cgacgtccca 2520 gactacgcta gctag 2535 <210> 2 <211> 26 <212> DNA <213> artificial <220> <223> CBA promoter Forward <400> 2 cctctgctaa ccatgttcat gccttc 26 <210> 3 <211> 27 <212> DNA <213> artificial <220> <223> GCT AGT GAA TGT GTA TCC AGA AGC CTT <400> 3 gctagtgaat gtgtatccag aagcctt 27

Claims (8)

A promoter operable in an alga and a transformation vector in which a 3D8 scFv gene is coupled to be operable to the promoter.
The transformation vector of claim 1, wherein the promoter operable in the bird is a chicken beta-actin gene promoter.
The transformation vector of claim 1, wherein the 3D8 scFv gene further comprises a reporter gene.
The transformation vector according to claim 3, wherein the reporter gene is HA-tag.
A fertilized egg transformed with the vector of any one of claims 1 to 4.
Chickens transformed with the vector of any one of claims 1 to 4.
Preparing a vector operable in a bird and a transformation vector in which a 3D8 scFv gene is coupled to be operable to the promoter;
Transducing the vector into a fertilized egg of a chicken; And
Hatching the fertilized egg to identify the individual transformed in the first generation method of producing a transgenic chicken.
A method for detecting a virus, characterized by measuring a detection amount of a nuclear protein (NP) of the virus after infecting a cell of a chicken transformed with the vector of any one of claims 1 to 4.

Images