JPH1192406A - Nucleic acid preparation containing gold colloid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject preparation capable of inducing a high immunization in a smaller amount than those of conventional vaccines due to the strong drug delivery system effect of a gold colloid and useful for the immunization of target animals by including the gold colloid and a genetic expression force set. SOLUTION: This nucleic acid preparation preferably useful for the vaccination of poultry (especially fowl) is obtained by mixing a gold colloid with an expression force set in a ratio suitable for a target animal. The gold colloid is obtained by mixing gold chloride with trisodium citrate in a constant ratio and has particle diameters of <=100 nm, preferably 1-100 nm, especially preferably 45-100 nm. The expression force set is obtained by binding a gene encoding an autoimmune disease antibody, a cancer-specific antigen or cytokinin to the downstream of an improved chicken B-actin gene promoter in a ratio suitable for the target animal.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a nucleic acid preparation comprising colloidal gold and a gene expression cassette as main components. More specifically, the present invention relates to a nucleic acid preparation mainly containing a mixture of a colloidal gold and a gene expression cassette in which a gene encoding a protein is connected downstream of a promoter, and an immunization method comprising administering the mixture to a living body.

[0002]

2. Description of the Related Art Conventionally, live vaccines using an attenuated live pathogen or inactivated vaccines using an inactivated pathogen have been used for vaccination of poultry. Comparing inactivated and live vaccines, inactivated vaccines are generally safer but have a shorter duration of effect. Conversely, live vaccines are superior in efficacy and persistence, although side effects are feared. Although there are two types of vaccines having different characteristics as described above, when the pathogen is successfully attenuated, the efficacy is more important than the safety and a live vaccine is often used. However, in recent years, the safety of live vaccines has been questioned, as it has been reported that aseptic meningitis has developed due to live measles vaccination.

[0003] In addition to the safety issues, live vaccines
1) When the host has already acquired immunity against the vaccine strain or the same type of pathogen as the vaccine strain, the inoculated vaccine strain is easily eliminated from the host, and a sufficient vaccine effect cannot be obtained. 2) For the same reason, even if a transfer antibody is present, the sufficient effect of the live vaccine is not exhibited. 3) Interference occurs between live vaccines, so that it is difficult to produce a multivalent live vaccine by mixing them, and there are also disadvantages related to the effects of the vaccine.

On the other hand, in order to carry out efficient vaccination, attempts have been made to produce highly effective multivalent live vaccines utilizing the characteristics of live vaccines (Taylor J. et al., AV).
IAN DISEASE 40, 173-180, 1996, Sonderweijer PJA
et al., Vaccine 11, 349-358, 1993). This multivalent live vaccine utilizes a virus genome of a live vaccine as a vector, and is mainly composed of a virus vector in which a protective antigen gene of another pathogen is integrated into the virus genome. However, since the basic form of the virus vector vaccine is a live vaccine, it cannot be denied that the vaccine virus may be excreted from the inoculated individual and become a new source of infection as in the case of the conventional attenuated live vaccine.

[0005] Under such circumstances, a completely new immunity in which a gene encoding a protective antigen incorporated into the gene expression cassette is directly expressed in the living body by administering the gene expression cassette (plasmid) into the living body. A method was attempted and it was reported that this method provided sufficient protection against chicken influenza and rabies virus (Momtgomery, DNA Cell. Biol., 12, 7).
77-783 (1993): Influenza / mouse, Robinson, Va
ccine, 11, 957-961: influenza / mouse, chicken).

[0006] Vaccines using gene expression cassettes (hereinafter also referred to as DNA vaccines) are different from conventional live attenuated vaccines and virus vector vaccines. Since reduction does not occur theoretically, it can be expected that a multivalent DNA vaccine can be easily produced.

[0007] However, the effect of DNA vaccination reported so far is that, for example, in the case of chicken influenza, 100 μg / dose plasmid is administered simultaneously and twice from three routes (intravenous, subcutaneous and intraperitoneal). For the first time, they achieved only 50% defense (Robinson, Vaccine, 11,957-961 (199
3)). In addition, in mouse experiments, influenza was first protected by administering 200 μg of plasmid three times (Momtgomery, DNA Cell. Biol.
12, 777-783 (1993)). When the plasmid is administered alone as described above, its uptake is due to endocytosis, and most of the plasmid stays between cells, so that only a very small amount is actually taken up into cells.

[0008] In order to solve the above-mentioned problem of inefficient vaccination, DNA vaccines require expensive gold powder (0.5-3 μm).
Attempts have been made to induce an immune response with a nucleic acid preparation for immunization to which m) has been attached (EF Fynan, ProNAS, 90, 114).
78-11482 (1993)). However, this method requires the use of a special and expensive inoculation device called a gene gun that uses high-pressure gas or explosives at the time of administration, and is not practical as a method for immunizing a large number of animals in view of technology and cost. Absent. Furthermore, the method of attaching a plasmid to gold particles is also very complicated, and cannot be said to be a practical method for targeting a large number of animals.

[0009] In addition, DDS such as liposomes has been studied in order to efficiently incorporate a DNA vaccine into cells (M. Yokoyama., FEMS Immunology and Medical MDS).
icrobiology, 14, 221-230 (1996), Sakaguchi et al.,
Vaccine, 14, 747 (1996)), it can be said that this method is also impractical in terms of cost.

[0010]

As described above, although some immunization methods for directly administering a plasmid into the body have already been attempted, an animal immunization method which can withstand practical use in terms of efficacy and dosage form has been tried. There is no report so far, and the development of a practical and effective immunization method, which replaces the conventional method, is desired.

[0011] The present invention provides a nucleic acid preparation comprising a mixture of a gene expression cassette constructed to express in vivo a gene encoding a protective antigen against infection and other functional proteins, and fine particles of colloidal gold; An object of the present invention is to provide a method for immunizing a subject animal using a nucleic acid preparation.

[0012]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above object, and as a result, have found that the chicken Newcastle disease virus (NDV) F gene is located downstream of the chicken β-actin gene promoter. It was found that chicks or the like to which a mixture of the incorporated plasmid and colloidal gold particles were administered acquired higher NDV protection ability than the case where no colloidal gold was mixed.
In addition, they found that chicks and the like to which a mixture of a plasmid in which chicken infectious bursal disease virus VP2 gene was incorporated in the same manner as described above and colloidal gold particles obtained a higher neutralization titer. It was completed.

[0013] Accordingly, the present invention provides a gene expression cassette in which the NDVF gene is incorporated downstream of the chicken β-actin gene promoter, a vaccine against NDV infection containing colloidal gold as a main component, and a downstream of the chicken β-actin gene promoter. And a vaccine against IBDV infection, which contains colloidal gold as a main component, and a gene expression cassette incorporating the IBDV VP2 gene. Hereinafter, the present invention will be described in more detail.

[0014] The present invention is characterized by a mixture of a gene expression cassette and colloidal gold constructed so that a protective antigen against infection can be expressed in vivo. By administering this mixture to a subject animal, high immunity against a protective antigen can be imparted.

[0015] A gene expression cassette in which a protective antigen against infection is incorporated so as to function in vivo is available from Sambrook.
Et al. (Molecular Cloning, A Laboratory Ma
nual Second Edition. Cold Spring Harbor Laboratory
Press, NY, 1989). That is, the gene encoding the protective antigen against the target infectious disease is cloned,
This is prepared by constructing a DNA fragment in which this is linked downstream of a promoter that functions in an animal body, and inserting the DNA fragment into a plasmid.

[0016] As the promoter, any promoter can be used as long as it functions in an animal body, and preferably, a chicken β-actin gene promoter is used. More preferably, improved chicken β-actin gene promoters (JP-A-2-56891 and JP-A-3-68097), which have been improved in various ways to enhance expression efficiency, are used.

The foreign genes incorporated into the gene expression cassette include (1) genes encoding protective antigens against various infectious diseases such as viral diseases, bacterial diseases, and parasitic diseases, for example, NP or HA of influenza virus. (Ulmer JB et al., Science, 259, 1745-1749 (199
3)), glycoprotein of rabies virus (Xiang ZQ et al., Vir
olgy, 199, 132-144 (1994)), the envelope protein of human immunodeficiency virus and Rev (Wang B. et al., DNA cell
Biol., 12, 799-805 (1993), Okuda K. et al., Aids Re.
s. Human Retrovir. 11, 132-140 (1995)), herpes simplex virus glycoprotein B or D (Manickan E. et al.,
J. Immnol., 155, 259-265 (1995), Ghisi H., Antivira
l Res., 28, 147-157 (1995), McClements WL., Proc. N
atl. Acad. Sci. USA, 93, 11414-11420 (1996), Bourne
N., J. Inf. Dis., 173, 800-807 (1996)), a glycoprotein of bovine herpes virus (Cox G., J. Virol., 67, 799-8).
05 (1993)), the surface antigen of hepatitis B virus (Davis HL e
t al., Human Mol. Genet., 2, 1847-51 (1993)) and core proteins (Kuhober A et al., J. Immunol., 156, 3687-369).
5 (1996)), the nucleocapsid of hepatitis C virus (Maj
or ME et al., J. Virol., 69, 5798-5805 (1995), Laggi
ng LM et al., J. Virol., 69, 5859-5863 (1995)), L1 of papillomavirus (Donnelly JJ et al., J. Infe
ct. Dis., 173, 314-320 (1996)), the human T-cell leukemia virus (HTLV-1) virus envelope (Agad).
janyan MG. et al., In Vaccine 94, 47-538 (1994)),
Cytomegalovirus virus segment pp65
(Pande H. et al., Scand. J. Infect. Dis., S99, 117-
120 (1995), the sporozoite protein of the malaria parasite (Sedega
h M et al., Proc. Natl. Acad. Sci. USA, 91, 9866-9.
870 (1994)), a paramyosin protein from Schistosoma japonicum (Yang
W. et al., Biochem. Biophys. Res Commun., 212, 102.
9-1039 (1995)), and (2) a gene encoding a cancer-specific antigen, for example, an idiotype antibody of B cell lymphoma (Hawkins RE. Et al., Blood, 8
3, 3279-3288 (1994)), carcinoembryonic antigen (Conry RM. Et a
l., Cancer Res., 54, 1164-1168 (1994)), human immunoglobulin V region (Watanabe A. et al., J. Immunol., 15
1, 2871-2876 (1993)), MHC class I molecules (Geissler
EK. Et al., J. Immunol., 152, 413-421 (1994)), and (3) a T-cell receptor beta chain having a therapeutic effect on autoimmune diseases such as rheumatoid arthritis. Encoding gene (Williams WV., Et al., Immu
nol. Res., 13, 145-153 (1994)), (4) (1)-
Proteins that are administered together with the DNA vaccine of (3) and enhance its efficacy, such as genes encoding cytokines such as interleukin 2, interleukin 4, and granulocyte macrophage colony stimulating factor (Geis
sler M. et al., J. Immunol., 158, 1231-1237, (199
7)) Preferably, a gene encoding a protective antigen against chicken infection corresponding to (1) is used. More preferably, an F gene which is a protective antigen against NDV or VP2 which is a protective antigen against IBDV is used. Genes are used.

[0018] A multivalent DNA vaccine is prepared by mixing various plasmids into which a DNA fragment comprising a promoter and a protective antigen gene has been inserted, or constructing a plasmid into which a DNA in which various DNA fragments are ligated is inserted. Can be produced.

As a plasmid for inserting such a large DNA, for example, a phagemid vector (pUC119) capable of cloning a DNA of up to about 20 kb and stably expressing it can be mentioned. More specifically, a chicken β-actin gene promoter (about 1.4 kb) was inserted into pUC119, and a gene (3.2 kb) encoding a capsid protein of chicken infectious bursal disease virus was further downstream thereof.
(1.7 kb), a gene encoding a spike protein of chicken infectious bronchitis virus (4.2 kb) and a polyA addition signal can be stably integrated.

The colloidal gold used in the present invention comprises chloroauric acid and trisodium citrate in a certain ratio, for example, 0.1%.
1% per 100 ml of 1 mg / ml chloroauric acid aqueous solution
By mixing 1.1 ml of sodium citrate,
Very easily and inexpensively prepared (Geoghegan et al,
J. Histochem. Cytochem., 25, 1187-1200 (1977), Goodm
ann et al, J. maicroscopy, 123, 201-213 (1981), De
Waele et al, J. Histochem. Cytochem., 31, 376-381
(1983), Roth et al., J. Histochem. Cytochem., 30,
691-696 (1982)). According to this method, a gold colloid composed of fine particles of about 1/5 or less of the above-mentioned gold powder can be obtained. In the present invention, the particle size is 1 nm to 100 nm.
Are used. Preferably, 45 nm
Gold colloids in the range of １００100 nm are used.

[0021] The nucleic acid preparation of the present invention is prepared by mixing the gold colloid with the gene expression cassette described above. The mixing ratio of the gene expression cassette and the gold colloid in the nucleic acid preparation is determined by sequentially selecting a quantitative ratio suitable for each subject animal. The nucleic acid preparation thus obtained is one that confers high immunity to a subject animal, and alone or together with a suitable stabilizer that can be administered as a drug,
It is used as a DNA vaccine by administering it to an animal.

[0022] The animal to be inoculated is not particularly limited as long as it is an animal that has been normally vaccinated. Birds, mammals and humans including poultry such as pigs, cattle and chickens can be administered to humans. Application of is also possible.
Particularly, in the examples of the present invention, the effects are confirmed using chickens. However, the effects are not particularly limited to chickens, and it is considered that the effects of the present invention can be obtained in various animals.

[0023]

According to the present invention, there is provided a nucleic acid preparation in which a gene expression cassette and gold colloid are mixed.

[0024] The DNA vaccine containing the nucleic acid preparation of the present invention as a main component is a conventional DNA vaccine because the colloidal gold of the present invention has an effect as a powerful drug delivery system.
Higher immunity can be induced with a small amount compared to a vaccine. Further, since the nucleic acid preparation is microparticles, it can be administered by injection as in the case of ordinary vaccination. Hereinafter, the present invention will be described more specifically with reference to examples.

[0025]

EXAMPLES Example 1: Construction of F protein expression plasmid Plasmid pCAGn-mcs-poly (A) having a chicken β-actin gene promoter (Japanese Patent Application No. Hei 8-
271369) was digested with a restriction enzyme HindIII, treated with phenol / chloroform and precipitated with ethanol to obtain an opened plasmid. The open end of this plasmid is TaK
Using aRa DNA Blunting Kit (Takara Shuzo Co., Ltd.), blunting was performed according to the attached protocol, followed by dephosphorylation treatment with bacterial alkaline phosphatase (Takara Shuzo Co., Ltd.). For the NDV-F gene, XLIII10 constructed and reported by Sato et al.
From H, those recovered by partial digestion with restriction enzymes BsmI and BbeI, agarose electrophoresis, phenol / chloroform treatment and ethanol precipitation were used. The NDV
-F gene was replaced with TaKaRa DNA Blunting
Kit was used to insert the downstream of the chicken β-actin gene promoter to construct an expression plasmid pCAG (F) to which the NDV-F gene was bound. The outline of this expression plasmid is shown in FIG.

Example 2 Construction of VP2 Protein Expression Plasmid Eagle MEM (Nissui Co., Ltd.) containing a live vaccine virus (manufactured by Institute for Chemistry and Serological Therapy) containing 3% of fetal calf serum
Were inoculated with chicken fetal embryo fibroblasts and cultured. The culture supernatant was subjected to ultracentrifugation at 22 Krpm for 2 hours using 40% sucrose as a cushion (Hitachi, Ltd., RPS40T) to obtain virus particles as sedimentation. The virus genome R was obtained from the concentrated virus particles by phenol chloroform treatment and ethanol precipitation.
NA was recovered. Next, cDNA encoding VP2 was cloned by RT-PCR. For RT-PCR, TaKaRa RNA LA PCR Ki
Using t (AMV) (Takara Shuzo Co., Ltd., RR012A) and a synthetic DNA (Takara Shuzo Co., Ltd.) having the nucleotide sequence shown in SEQ ID NO: 1 and 2: 2 in the Sequence Listing, the procedure was performed according to the attached protocol. The sequence of the synthetic DNA is based on the previously reported base sequence (Hudson PJ, Nuc. Acid. Res., 14, 5001-501).
2 (1986)). The amplified cDNA was converted to XhoI
After digestion with pCAGn-mcs-po, a termination codon was added and the cDNA was previously blunted and dephosphorylated.
ly (A) was inserted to construct a plasmid expressing the IBDV-VP2 gene, pCAG (VP2).

Example 3 Preparation of Gold Colloid The gold colloid used in the present invention was prepared according to the method of Frens et al. (Frens, Nature Phys Sci, 241, 20-22 (197
3)). That is, 100 ml of distilled water is boiled, and chloroauric acid (Sigma G402) is rotated at high speed with a stirrer.
2) 10 mg was dissolved. To the solution, 1% trisodium citrate was quickly added dropwise while stirring with a stirrer, and after stirring for about 10 minutes, the reaction solution was left at room temperature to cool. The particle size of the gold colloid is citric acid 3
In this example, 1.1 ml and 0.5 ml of 1% trisodium citrate were added to prepare gold colloids having particle diameters of about 45 nm and about 100 nm, depending on the amount of sodium.

Example 4: Competition method EL for measuring anti-F antibody titer
The method of preparing the plate used in the ISA competition ELISA is described below. The Newcastle disease virus Ishii strain was inactivated with formalin and recovered by coarse centrifugation. The calcium and magnesium-free phosphate buffer (PBS)
(-)). This was dispensed into a 96-well plate (Dynatech Imlon 2) at 100 μl per well. After standing at 4 ° C. overnight, 3% PBS (-) containing 0.05% of polyoxyethylene sorbitan monolaurate (Tween 20, Nacalai Tesque 356-24) was added.
Washed twice. Next, 200 μl of PBS (−) containing 2% of bovine serum albumin (BSA, Sigma A-7030) was dispensed per well and allowed to stand at room temperature for 2 hours.
The plate prepared by the above procedure was used for the competitive ELISA.
It was used for.

[0029] The chicken serum collected was PB containing 5% skim milk (Snow Brand Milk Products) and 0.05% Tween 20.
It was diluted 10-fold with S (-) (hereinafter also referred to as antibody diluent), and 100 μl was added per well. After 1 hour at room temperature, PBS (-) containing Tween 20 in 0.05%
And washed. Then, the method of Yoshitake et al. (J. BIochem., 9
2, 1413-1424 (1982)) and peroxidase-labeled anti-F protein antibody # 83 (Sato et al, J. Gen. Virol., 7
1, 1199-1203 (1990)) was diluted to an arbitrary multiple with an antibody diluent, and 100 μl was added per well. After 1 hour at room temperature, PBS (-) containing Tween 20 in 0.05%
And washed. Then, 100 μl of a substrate consisting of citric acid, disodium hydrogen phosphate, OPD and sodium hydrogen peroxide was added to each well at 100 μl, protected from light, allowed to stand at room temperature for 1 hour, and 3 M sulfuric acid was added in 50 μl portions and mixed well. The reaction was stopped. 490/6 soon after
The absorbance at 50 nm was measured. The resulting value was divided by the absorbance value of the negative serum and multiplied by 100 to obtain the antibody titer.
The following were defined as F antibody positive.

Example 5 IBDV Neutralization Test In order to know the immune status due to pCAG (VP2) administration, a virus neutralization test was performed, and a neutralizing antibody titer in chicken serum was measured. The collected chicken serum was serially diluted from 10 times to 1280 times with an Eagle MEM medium on a 96-well plate.
There, IBDVK strain was added at ₁₀₀ TCID ₅₀ per well.
Add, vortex and mix at 37 ° C for 90
Left for a minute. Here, one chicken fibroblast (CEF)
50,000 cells were added per well, and the cells were further cultured at 37 ° C.
Three days later, the value of the maximum dilution concentration at which no cytopathic effect was observed was defined as the neutralization titer of the serum.

Example 6: F expression plasmid pCAG (F)
Immunization Experiment 1 20 pmol (about 100 μg) of pCAG (F) was added to 0.1
It was dissolved in ml of PBS (-) and mixed with 0.1 ml of colloidal gold (particle size: 45 nm). This is a group of 5 SPF
Two injections were made into the lower leg muscles of chickens at the age of 2 weeks and 4 weeks. Blood is collected every two weeks after inoculation until the age of 10 weeks.
The transition of the antibody titer was measured by a competitive ELISA. Table 1 shows the measurement results of the anti-F antibody titer by the competitive ELISA. In the group not using colloidal gold, the antibody was induced only in 2/5 birds, and the degree was about 70 in the competitive ELISA method. In the group using gold colloid, 2/5 weeks after the second immunization (at the age of 6 weeks), anti-F antibody at a level capable of sufficiently protecting Newcastle disease in 3/5 birds (37.7 to 65.8 by ELISA value of competition method) A value was observed. The effect persisted until at least 10 weeks of age.

[0032]

[Table 1]

Example 7: F expression plasmid pCAG (F)
Immunization experiment 2 20 pmol (about 100 μg) of pCAG (F) was added to 0.1
It was dissolved in 0.1 ml of PBS (-) and mixed with 0.1 ml of colloidal gold (particle size: 100 nm). This was intravenously administered to SPF chickens twice by injection at 2 weeks and 4 weeks of age. Blood was collected sequentially, and the anti-F antibody titer was measured by competitive ELISA. Table 2 shows the measurement results of the anti-F antibody titer by the competitive ELISA. In the group not using colloidal gold, antibodies were raised only in 3/5 birds, whereas in the group using gold colloids, an anti-F antibody titer that could sufficiently protect Newcastle disease was observed in 5/5 birds. Intravenous administration gave better results than intramuscular administration even without colloidal gold, but the addition of colloidal gold provided reliable and good immunity to all test chickens.

[0034]

[Table 2]

Example 8: Immunization experiment with pCAG (VP2) 20 pmol (about 100 μg) of pCAG (VP2) was dissolved in 0.1 ml of PBS (−), and mixed with 0.1 ml of colloidal gold (particle size: 100 nm). This was administered by two injections into the lower leg muscles of SPF chickens at the age of 2 weeks and 4 weeks. Table 3 shows the results of measuring the neutralizing antibody titer in chicken serum by the IBDV neutralization test. Antibodies were raised in 2/5 birds in the group not using gold colloid, whereas antibodies were raised in all birds in the group using gold colloid. Also in IBDV, favorable immunity was able to be given to all the test chickens by the addition of colloidal gold.

[0036]

[Table 3]

[0037]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 24 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence ATGGAACTCC TCCTTCTACA ACGC 24

[0038] SEQ ID NO: 2 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence CTCTTAACAC GCAGTCGAGG TTGT 24

[Brief description of the drawings]

FIG. 1 is a diagram showing the outline of an NDV F protein expression plasmid.

FIG. 2 is a diagram showing an outline of an IBDV VP2 protein expression plasmid.

Continuation of front page (72) Inventor Yukio Tokiyoshi 3-14-19 Wakaba, Kumamoto City, Kumamoto Prefecture

Claims (11)

[Claims] 1. A nucleic acid preparation containing a gold colloid and a gene expression cassette as main components. 2. The nucleic acid preparation according to claim 1, wherein the gold colloid has a particle size of 100 nm or less. 3. The nucleic acid preparation according to claim 1, wherein the gold colloid has a particle size of 1 to 100 nm. 4. The nucleic acid preparation according to claim 1, wherein the gold colloid has a particle size of 45 to 100 nm. The nucleic acid preparation according to any one of claims 1 to 4, wherein the gene expression cassette is an expression cassette for expressing a protective antigen against infection. The nucleic acid preparation according to any one of claims 1 to 4, wherein the gene expression cassette is an expression cassette for expressing an autoimmune disease antigen. The nucleic acid preparation according to any one of claims 1 to 4, wherein the gene expression cassette is an expression cassette for expressing a cancer-specific antigen. The nucleic acid preparation according to any one of claims 1 to 4, wherein the gene expression cassette is an expression cassette for expressing a cytokine. 9. A method for immunizing a subject animal, comprising using the nucleic acid preparation according to any one of claims 1 to 8. 10. The immunization method according to claim 9, wherein the target animal is poultry. 11. The immunization method according to claim 10, wherein the poultry is a chicken.