JPS5980615A - Dna and its use - Google Patents

Abstract

PURPOSE:To provide a DNA containing a gene coding the surface antigen precursor of an adw-type hepatitis virus B (HBV), useful for transforming animal cell, and producing HBV surface antigen (HBsAg) protein using said cell. CONSTITUTION:The Dane's particle DNA of an adw-type HBV having a single chain at a part thereof is converted to the complete double-chain structure, and bonded by using EcoRI with pBR322 incised by the same EcoRI enzyme. Escherichia coli is transformed by using the bonded product, cultured, and extracted to obtain the plasmid pBR322-EcoRI/HBV933 having HBV DNA. A straight chain HBV DNA is obtained from the plasmid, cyclized, incised with TaqI, and bonded with a fragment obtained by incising pBR322 with ClaI. The bonded product is integrated in the cell of Escherichia coli, the cell is cultured in a conventional medium, and the plasmid is extracted therefrom. The cell of Escherichia coli containing said bonded product is differentiated and cultured, and the bonded product is separated from the cell, purified, and incised with e.g. TaqI. The objective DNA containing preHBsAg gene can be obtained by removing pBR322 from the above product.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to DNA and its uses. More specifically, the present invention relates to a DNA containing a gene encoding a surface antigen precursor of adw hepatitis B virus and uses thereof.

h) Hepatitis B virus is a type of DNA virus.
It is caused by infection with HBV (hereinafter sometimes abbreviated as HBV). HB V is known as a den particle, and the surface of this virus particle contains 11 B.
V surface antigen (hereinafter abbreviated as HBsAg), double-stranded circular DNA with a partially single-stranded portion inside the particle, HB
v core antigen (HBcAg), H]3V e antigen (HB
eAg), and the activity of endogenous DNA synthase has been detected as an enzyme that repairs the single-stranded portion of double-stranded DNA. HBV DNA has approximately 3200 t=prb pairs with a 0+ amount of 2.1×106 Daltons. HBe
The antigenicity of Ag is centered on the common antigen a, adr, adw,
Four types of HBV, ayr and ayw, are known, and the DNA of the λyw type HBV has already been cloned.
Its DNA sequence has been determined.

Furthermore, attempts have been made to link this HBsAg gene downstream of various promoters and express it in E. coli, but recently, using yeast, ayw type) (BsAg
It has been reported that the gene was successfully expressed [P,
Valenz+xela et al,, Natu
re, 298.

347 (1982)).

On the other hand, for gene expression using animal cells, TK
(Thymidine Kinase) gene and the gene of interest are introduced into TK-deficient cells, cells are selected by a method that selects only cells with TK relics, and examples of gene expression in these cells (globin gene and TK ji 'Mixed inoculation with l gene) has been reported (Proc, Natl.

Acad, Sci, USA, 70.5684 (1979
) ) has been. Using this method, ayw type f
(Cotransformation of mouse TK-deficient cells using a plasmid made by ligating two molecules of 13V DNA and cloning into pBR322 and the TK gene.
orma-tion) Saze, ayw type HBsAg
Reports expressing (Proc, Natl, Acad
, Sci, USA, 77, 4549 (1980)
] or 5V-40 gene by combining the HBsAg gene,
Report on production of HT3sAg particles by inoculating monkey kidney cells (Proc, Natl, Acad, 8ci,
USA, Z8.

2606 (1981)).

The present inventors expressed the gene encoding the adw hepatitis B virus surface antigen precursor (preHBsAg) in mouse TK.
We succeeded in producing HB FI A g protein by expressing it in deficient 41L cells, and completed the present invention through further research.

That is, the present invention provides animal cells transformed with a DNA containing a gene encoding adw-type prellBsAg, 11DNA, and adw-type f (
The present invention provides a method for producing 13sAz protein.

The preHBsAg gene in the present invention includes the first
In the figure, DNA sequence 2866-3200-1-835
Those shown are preferred. In this Figure 1, D
NA sequence 155-835 is D of HBsAg gene
NA sequence, DNA sequence 2001-2458 is HB
This is the DNA sequence of the cAg gene, which is the DNA sequence 2 described above.
866-3200-1-835 is preHBsAg7I
] This is the DNA sequence of the gene.

Animal cells used for expression include adw type p
Any cell that can express the reHBsAg gene and produce IIBsAg protein may be used, such as mouse TK-deficient cells; in this case,
DNA sequence 2429-3200-1-835-1989
L7 is preferred.

D containing the adw type preHBsAg gene of the present invention
NA can be obtained, for example, by cloning adw type HBV DNA. For example, as shown in Fig. 2, after converting adw-type HB V particle DNA, which has a single-stranded portion in part, into complete diNHill DNA by DNA polymerase reaction, restriction fI element E
Cut with c o RI. On the other hand, pBR322DNA as a plasmid was also cut with the same enzyme EcoRI, and both were ligated at the EcoRI site, resulting in Escherichia coli 71776.
By transforming the strain, culturing and extracting the ad
Plasmid pBR322- containing w-type HBV DNA
FicoRI/HBV933 can be separated.

This plasmid was cut four times with EcoRI, and adw
Type HB V DNA for example in agarose gel 7
(, 4T1 electrophoresis or polyacrylamide spikes) Separate by v electrophoresis. The linear f(BV
DNA, for example 'I' 4 D N A! Make a ring using J gauze. This circular HBV DNA (I
) was cut with Taq I, while C1a of pBR322
I cut and T4 DNA! Binding in the presence of J gauze (
I) Let. Note that circular DNA (I) has only one Taq I cleavage site, and similarly, C1a of pBR322
There is also one I cleavage site. Taq I and C1a
The cohesive end nucleotide sequences resulting after I cleavage are identical and can be ligated by T4 DNA ligase. Depending on the case, a linker can also be combined.

The above-mentioned conjugate (I) is incorporated into E. coli, for example, E. coli χ1776 strain, by a method known per se, and cultured in a medium known per se. A drug such as ampicillin may be added to the medium to facilitate selection of transformed strains.

After culturing the selected colonies, methods known per se, such as the Birnboim-Doly method (Nucleic
Acids Re8egch, 7 r 1513 (
1979):) to extract the buwosmid, measure the molecular weight using, for example, agarose sedge, ``W-expressing or acrylamide sedge'' V electrophoresis, and extract the conjugate (I).
It is possible to distinguish between E. coli containing T) and E. coli containing pBR322.

After culturing E. coli containing the conjugate (11), Birnb
The target preHBsAg gene is purified by purifying the bound product (■[) using the oim-Doly method or cesium chloride-equilibrium density gradient centrifugation, and cutting it with, for example, Taq I to remove pBR322 DNA. The containing DNA (I[) is obtained. This DNA (III)
It is also possible to generate even smaller DllA fragments (1) by treating with restriction enzymes.

The obtained DNA (][) can be inoculated into animals by itself, but in order to make it easier to confirm the transformed strain, co-transformation can be performed using a marker such as TK. leCProc.

Natl, Acad-8ci, USA, 50+56
8 (1963)] is better.

If a transformed strain having DNA (I[) is cultured for several days to several days, M nail l(BsAg protein) will be produced and accumulated in the broth.

The HBsAg protein produced and accumulated in the culture medium can be separated and collected by concentrating and drying the culture solution as it is and separating it together with the cells, but after separating the cells, an organic solvent or strong acid may be added to the supernatant, or pH 8178 to the isoelectric point, or by a conventional method such as salting out.
, Abbott Laboratories). Here, even if the HBsAg gene is contained, pre
HB, those in which the aAg gene is not complete, such as Eao
RI DNA fragment and BamftI DNA fragment are II
It was found that B a A g protein was not produced.

The f(BsAg protein obtained according to the present invention) can be used as a vaccine for hepatitis T3 virus in the same manner as HBsAg obtained by conventional methods.

Example (1) Preparation of adw type HB V DNA ad
W type HB V CPhoenix Laborator
ies Divi-Hichi Chimpanzee (early 9 weight 12
100s+1 plasma whose blood was positive for HBsAg and the presence of Den particles was confirmed by electron microscopy.
! Collect this and put it into Spinco 5W27 rotor (Bec
kman.

Centrifuge (24,00 Orp) using
m, i 6 hours, 4°C) to precipitate adw HBV, and then add 20 mM Tris-HCI (pH 7,6).
30 g/Klt'! /ffi.

Next, under the same conditions (SW270-tar, 24+00Orpm+
HBv was washed by centrifugation for 16 hours at 4 hours, serum component gold was removed, and HBV was concentrated. Add this precipitate to 5yxl of 20
Suspended in mM Tris-HCI (pH 7,6), D
It was stored at -20°C until used for the NA synthesis reaction.

The method of Kaplan et al. (J, Virol, 12.995 (1973)) was carried out using the above-described sediment sail 8at.
According to the reaction solution containing H-dTTP (160mM Tr
is-HCI (pH 7,5), 40mM MgC
l2.120mM NH4Cl, 0.3% 2-mercaptoethanol, 0.5% Nonidet P-40, 1,0m
M dATP, dCTP.

The single-stranded portion of the 11 B V DNA was made double-stranded by incubating it in dGTP for 16 hours at 37°C [Intervi:rology, to, 254 (
197B)). At that time, 3H-dTT to the acid-insoluble fraction
The DNA synthesis reaction was confirmed using P incorporation as an indicator. The obtained reaction surface was placed in an ultracentrifuge (Beckman L
Centrifuge (5-50 i'-ter 5W55)
+ 000 rpm + 6 hours, 4°C) and
Precipitate and collect HB V after NA synthesis reaction 7.1 ap
l mo'Ex solution for DNA extraction (0.02M Trj,
5-HCI (pH 7,6), 0.02M ET)TA
, 1% SDS, proteinase K (11'y/ml
)) K was suspended and incubated at 37t for 16 hours. Next, 01 M Tris-HCl (pH 7,6
), 0. LMNaCl and 0. Old) IM ED
11 in TA;] Deproteinized twice with 1 #Il of phenol (J, Virol, Neto 368 (197
7)], take the aqueous layer and add 0°2M N8.
C1, add 2.5 times the amount of 99% ethanol, -20
HB V DNA was precipitated at <0>C.

By the above operation, a complete double strand a with the single stranded part repaired
dw type HBV DNA was obtained.

(2) Cloning of adw type HBV DNA The double-stranded adwgJ HBV obtained by the above (1)
I) Restriction enzyme ICc o R that recognizes a specific site on the NA strand and generates a single-stranded cohesive end to cleave the DNA.
100 mM Tris-HCI (pH 7
,5), 7mM MgC12,5Q mM NaCI
37°C in the presence of L and 7mM 2-mercaptoethanol.
The adw type HBv I) NAEcoRI fragment was obtained. On the other hand, a plasmid pBR322 from a large bacteria was used as a cloning vector. pBR322DN
After A was digested with EcoRI to obtain linear DNA, the 5'-terminal phosphoric acid was removed by alkaline phosphatase treatment (5science, 196.1313 (197
7) l). Adw type HB V D
NA and plasmid DNA each have EcoR at both ends.
It has a sticky end of a medium chain produced by I digestion. These a
, dw type H'B VDNAEcoRI fragment and pBR32
21i: mixed with coRI fragment and 66 mM Tri
s-1(C1 (pH 7,6).

6, 6mM MgCl2. 14°C in the presence of 10mM dithiothlate-Ill and 0.4mM ATP
Then '1' dDNA ligase was applied to join the DNA.

7QmM Mn(J2.30mM CaCl2.40m
Mf'! Solution W1 consisting of sodium t' acid. (pH5,
6) and incubate at 90°C for 20 minutes (; rlMo
l, Biol, 96.

495 (1975)') L, Escherichlp L coli suspended in the same solution 100///
11776 strains (ATCCA31244 iU, s, P,
41.90495) (5×10 bacteria) was added with 5 Ill of the reaction solution obtained above, and incubated at 0° C. for 60 minutes.

Next, this E. coli was treated with 2071 g/#t of ampicillin.

5011 (1/stl of thymidine and 20"μ9/
*LB agar medium containing diaminopimelic acid (17!
9 Bactodoryptone 10g, Bacto Yeast Extract 5
9, containing NaC1109 and 0.8 g of glucose) and cultured at 37°C for 2 days. In the resulting ampicillin-resistant colonies, pB containing foreign DNA
E. coli harboring the R322 pfusmid was selected by the "toothpick" method (5science, 195, 393 (1977)). That is, a colony taken from a toothpick was added to 1001 IJ of solubilization solution (1% SDS
, 10% glycerol, 0.02 MEDTA, 0.04
% xylene cyano-IV), heat treated at 7°t for 10 minutes, and then spiked with 1% agarose gel.
Colonies with plasmids larger than pBR322 were selected and collected. This completed the cloning of adw type HBV DNA.

E. coli containing this recombinant DNA was treated with 20 μf/me ampicillin, 50 μf Al thymidine and 20 μg/me
grown in LB medium containing ml of diaminopimelic acid,
170 μ9 during the logarithmic growth phase. Chloramphenicol was added to the mixture so that the concentration of chloramphenicol was increased, and the culture was continued for several hours to amplify the plasmid DNA. Next, lysozyme, EDT
A, 8Dβ was used to disrupt bacteria and plasmid DNA
was separated and purified by cesium chloride density gradient centrifugation (J, Bact, 121).

354 (1975)]. b1) Rareda plasmid DNA
When digested with 1i:coRI, all 3.2 kbp DN
A and a 4.3 kbp DNA. 4.3kbp
D1? A is vector T) NA of pBR322
Therefore, 3.2 kbp of I)NA was cloned as the adti type fl BVI I)NA. When this ar1w type HBV 3.2kbp DNA is digested with the restriction enzyme Bam1l, approximately 1.8kbp D
NA, 1.4 kbp DNA and 30 bp DNA
It turned out to be disconnected. H obtained as above
Recombination of BV3.2kbp DNA and pBR322DNA I) NA(pBR322-FcoRI/HBV
The large intestine rooster χ17 '76 strain with χl 776
/pT3R322-EcoRI/) It is named TT3V933.

Plasmid pBR322-EcoRI/HB obtained above
150μq of V933 was added to the reaction solution of 300μF [100mM
Tris = HC1, pH 7.5, 7mM MgCl
2.50mM NaCl, 7mM 2-mercaptoethanol 150 units H:coR (Takara Shuzo)] 33
After incubating for 7116 hours, using a 1% agarose gel with buffer solution (100mM Tris-11CI, pH 8, 3, 1
80mAJ in 00mM boric acid, 2mM EDTA:)
Electrophoresis was performed for 3 hours. After electrophoresis, the gel piece containing the 3.2 kbp DNA fragment was sealed in a dialysis tube, submerged in the electrophoresis buffer, and the DNA fragment was electrophoresed from the gel (J , Mo1. BioL, S, O, 119
(1977)), and the fluid inside the dialysis tube was extracted with phenol and further extracted with ether. NaCl was added to a concentration of 0.2 M, followed by twice the amount of cold ethanol to precipitate the DNA at -20°C. .

6011g of 3.2kbp DNA fragment to 2501g
Reactions of 11 i, e C66mM Tris-HCI,
pH7.6, 6.6mM MgCl2, lQ
mM dithiothreitol, 1mM A'l'P,
After circularization by reacting overnight at 14°C in T4 DNA ligase, 1% agarose gel electrophoresis was performed, and the DNA was eluted from the gel piece containing the 3.2 kbp circular DNA. , phenol extraction and ether extraction, and add cold ethanol to extract the DNA at -20°C.
It is precipitated.

30 μq of the obtained 3.2 kbp ff3-like DNA was
μl of reaction solution (10mM Tris-HCI, pl7
．． 5.10mM 2-mercaptoethanol + 10m
4MgG12.100mM NaCl, 30k”
ツI・TaqI (Bethesda Re5e>ch
Laboratories) Cut in 1E for 5 hours at 65°C, perform electrophoresis on 1% agarose gel, extract with phenol and ether, add cold ethanol and incubate at 120°C.
Linear DNA (Δ) was precipitated at °C.

On the other hand, the 201tQ DBR322 is 20μ! reaction solution (
50mM NA, C1, 6mM Tris-HCI,
pH 7,9, 6mM MgCl2. lOO119/
me bovine serumalbumin
+ 20 unit C1a I (Bethesda
Resea,ch Laboratories ))
] for 3 hours at 37°C to obtain linear DNA.
Add 65 t of alkaline phosphatase.
; The reaction was continued for 1 hour to remove the 5' terminal phosphoric acid group.

Deproteinize the reaction solution with phenol, and remove the DNA with cold ethanol.
(B) was precipitated.

E. coli strain 71776 was transformed using the following methods.

Next, this E. coli was treated with 20 μQ/me of ampicillin, 50
Plate on LB agar medium (described above) containing 71Q/ml thymidine and 20 μs/ml diaminopimelic acid,
The cells were cultured at 37°C for 2 days. Resistance to ampicillin occurred
p:13R3 with foreign DNA in the colony
22 pfusmid (pBR322-Taq I/HBV9
99) was selected by the "toothpick method" (described above). That is, transfer U-sized colonies on a toothpick to 100 μl of solubilization solution (1% SDS, 10
% Glycerol, 0.02M EDTA, 0.04
% xylene cyano-)v), 70t:,1
After heat treatment for 0 minutes, a 1% agarose gel was run on a 1% agarose gel, and colonies containing a 7.4 kbp plasmid were selected and collected.

Plasmid pBR322-Ta, qI/HBv999 into which the adw type HBV DNA obtained above was integrated
One platinum loopful of the recombinant with diaminopimelic acid 100 // Ql*l was dispensed into a 200 scrap flask.
.

Inoculate 30 ml of LB medium containing 20 μ9/wl of thymidine and 12°5 μQ/ml of thymidine and incubate at 37°C.
The cells were incubated overnight with shaking at 200 rpm. This culture? &2
5 m/ into LB 4yeJ500 g/ containing diaminopimelic acid 100 μQ/ml and thymidine 207197me dispensed into 11 flasks and 3'1.
After shaking culture for 4 hours + 20 Or p m,
Chloramphenicol was added at a concentration of 170 μQ/mtVc, and culture was continued for an additional 6 to 8 hours to amplify plasmid DNA. 8. 3g of cultured rit cultured under these same conditions. OOOrpm, 4
℃, centrifuged for 5 minutes, and the resulting bacterial cells were diluted with physiological saline for 2
Wash twice and add 30txt buffer (50mM TrisH)
CI (pH8,0), 25'16 (W/V)S
'j--Crose]. This is Bgl's 5"
'f/#/lysozyme solution was added, kept in water for 5 min, and then diluted with 12 ml of 0.2 M Na2 EDTA (pH 8
-0), 15-l of 5 M NaCl and Bgl in 1()% SDS were added, left to stand for 1 hour or more, and centrifuged at 18.00 rpm for 30 minutes at 4°C. Ethidium bromide (EtBr
) to lOO~200μq/1/', cesium chloride (C
sC1) was added to a density of 1.57-1.58 g/i+m3, and CsC1 was heated for 20 t:, 48 hours at 40.00 Orpm with a Beckman 5 Q Ti rotor.
-EtBr density gradient centrifugation. The plasmid DNA bands were collected and further subjected to Cs under the same conditions.
It was purified by C1-EtBr density gradient centrifugation. After centrifugation, plasmid DNA1i! 11 min was collected and an equal volume of isoamyl alcohol was added to remove EtBr, followed by buffer (10 mM Tris-HCl, pH 8).
, 0, 1mM EDTA) to obtain pBR322-Taq I/HBV999 DNA.

(3) 3QQ71g of pBR32 obtained in (2)
2-Taq I/HBV999 in reaction solution C100mM
Na1l, l 9mM 2-mercaptoethanol,
10mM MgCl2.10mM Tris-HC
After treatment at 65° C. for 3 hours in 300 units Taq I, pH 7.5, the mixture was subjected to 1% agarose gel electrophoresis. After eluting the DNA from the gel containing the 3.2 kbp DNA fragment, phenol extraction and ethanol precipitation were performed to obtain a pre-HBsAg-Taq I DNA fragment.

(4) 100μ prelTB obtained in (3)
Add the sAg-TaqI DNA fragment to the reaction solution C60mM N
aC1.

2mM 2-mercaptoethanol-IV, 7mM
MgCl2, 10mM Tris-HCI, pH
7,5,100 units) Bgl II (Takara Shuzo)] at 37°C for 3 hours, then p
A reHBsAg-Bgll DNA fragment was obtained.

(5) pBR322-Taq I obtained in (2)
/HBV999100/i in reaction solution C60mM NaC
1,6mM 2))”)J7'F
/' , l Om M MgCl2 + 100/I9
/Ill bovl, ne serum album
n, 6 mM Tris-fICl, pH 8.0
, 50 units of AvaI (Takara Shuzo)] at 37°C for 8 hours, and then using 1% agarose gel/14 gas phoresis.
After phenol extraction and ethanol precipitation, 2.2kb
An AyaI DNA fragment of p was obtained.

(6) pBR322Taq I/obtained in (2)
HBV99910 Night 9 was added to the reaction solution (1mM dithimethreitol.

7mM MgG12.20mM Tris-HCI
, pH 7,4.

After reacting for 8 hours at 37°C in 50 units Hpa, ■ (Takara Shuzo), 1% agarose suge/I/11. Perform phenol extraction and ethanol precipitation using a rapid shaker.
2. : A 2 kbp HpaII DNA fragment was obtained.

Example Unoa/l/Fun petri dish (i! diameter 6 cm) MFM medium (minixum essence) containing KIO% bovine serum
mouse TK-deficient cells were cultured overnight at 37°C. After culturing, these cells (7 x 105
HS V-106 (Bet, hesd
aReseaFh Labora, purchased from tories) o, 2ttq and lQ7zg pre HBsAg
- Taq I DNA fragment and Gra, lla
The method of M et al. (Virology, '52.456 (19
73)] and inoculated. After culturing at 37°C for 4 hours, HAT medium [15 prefectures hypoxanthine.

MEM medium containing 1 μq/#ll aminopterin + 5 pQ/me thymidine] was used instead, and the culture was continued at 37°C.

The culture solution Q was exchanged once every 3 to 4 days, and the cells were further cultured at 37°C. After about 2 to 3 weeks, the generated individual colonies were transferred to a new microplate and cultured in 0.2*/HAT medium at 37°C for 7 to 140 minutes.
The presence of sAg protein was determined using the Austria Kit (described above). ■■■■■■■■preHB8Ag-Bgl
ll DNA fragment, linear Ec containing the HBsAg gene.

RIT) NA fragment, BamHI containing HBsAg gene
Similar experiments were conducted with DNA fragments (described in Japanese Patent Application No. 77020/1983).

As a result, as shown in Table 1, ■■■■■■■I■■
■■Entertainment■■preHBsAg-Taq IDN A
When transformed with the fragment and the preHBsAg-Bgl HD DNA fragment, HBsAg protein was produced and Ec.

It was found that no production of HBaAg protein was observed for the RI DNA fragment and the BamHI DNA fragment.

From the above facts, adw type H in mouse TK-deficient cells.
It was found that the preHBsAg gene is required for the expression of BsAg protein.

Table 1 DNAli loaf piece Australia ■ Nyoru cpmT
aq I 5450, 3182.5B14,
904,240Bgl]I 4332.26
23.220? , 1763.300]ucoRI
4G+ 40,145,474+ 124Ha
, mHI: 8.42, 50.60.55 (
44-A Book 4 (Evening) Table 2 shows the preHBsAg-Taq I mentioned above.
These are the results of measuring over time the amount of HBsAg protein produced (values measured using the Ausliava kit) and cell proliferation in a culture system of mouse L cells transformed with DNA fragments.

Table 2 1 0,23Xlo625 20.56xlO649 31,0Xl06110 4 1,7 X], O” 8135
2,1 Xl061662 (32,7xlO64394 73,2Xl067999 8 3,8 By mation, H.A.T. Culture mouse L cells that can now grow in culture medium* (37t; 1ftJ for 11 days) L
, 20ml of the culture supernatant was collected and centrifuged C24.
000 rpm (spincoter 5W27.10-tar), 18
After concentrating for 4I hours, cesium chloride equilibrium 1Ii
Gradient centrifugation was performed once [ ], OmM Tria-f (
C1, pH 7.6.

150mM Na1l, 1mM EDTA, C
sC1 111.25g/*t, 40.00Orpm
, 48 hours, 15°C (50T10-tar)]. Both obtained samples were tested using HT3s using Ausliava kit.
When the presence or absence of Ag protein was investigated, ], 2 Q/c
It was found that HBsAg protein was present at the density of m3.

Furthermore, this HB s A g? with an electron microscope. As a result of observing the white particles, it was found that the particles had a diameter of 20 to 2.2 nm.

[Brief explanation of drawings]

Figure 1 shows the entire DNA sequence of adw type HB'V DNA using EcoRI.
1 They are shown in order starting from the center of 0. Figure 2 shows pB
A schematic diagram of the construction of R322-EcoR/HBV933 is shown in Figure 3. pBR322-Taq I/HBV999
A schematic diagram of the construction of is shown. Figure 4 shows each 4B<DNA
The relationship between the fragment and the preHBsAg gene and the HBsAg gene is shown. In the drawing [4, dNTPj is dATP, cl'I'TP,
It represents four types, dcTP and dGTP, and in Fig. 4, the mark (*) indicates that the left and right sides are connected. [111'+i's 7+1:+(Port'l'j i l r
g history) 1 (12030lIo 5
010 20 30 40
50 [111:'+i's I;"1gI'C white,', has no gun history) 10 20 30 40
I'+'-H IJ iii between 50171
No Maki history CCT(:8GCI'(:TGTATCG
8G88GC(:TTAG8GTCTCCTGAGGC8'rTGc'rcAccT 2050C
G8G'rCG8GACATAGC'rC'I"rcG
GΔ8TCTCAGΔGGACTCG'rAACGAG
'rGGAC8CCA'r8CTGCACTCAGGC
88GCC8TTCTCTGCTGGGGGGAATT
G8'rG8C 2100GTGGi'
8TG8CGrG8GrCCGrTCGGTAAG8G
8CGACCCCCTTAACTACTG゛rcTA
(;CTACCTGGGTGGG'rAATA 8TT
TGCAAGATCCAGCATCCAGAG8'rC
2150AGATCGATGG8CC
C8CCC8rTΔTTA8M:GTTCTAGGTC
GTAGGTCTCTAGT8GT8G'rCAATT
A'rGTTAATACTAM:ATGGGTTTAA
AGATCAGGCAACTA 220
08'I'CATCAGTTA 8'rAC8ATi'
Ai'GATTGrACCCAAAT'r'rCTAG
TCCGTTGΔDing'r'rGTGGT'rTCAT8゛1゛8TC'rTGCCTTACTTT'rGGAA
GAG8GACTG'rACTchoG8 2
25o8ΔCACC88AC'rA'r8'rAG8A
CGGAΔ'rG8AAACCTTCTCTCTG8C
ATG88CTAT8'r'l'TGG'rCTC'l
"I"I'CGGΔGTGTGGATi'CGC:AC
TCC'rCCAGCCT8゛r8G8C
23001'A'l'8A8CCAG8GA8AG
CCTCACACCTAAGCGTGAGGG8GGTC
GGArATCTGC8CC888TGC:CCCTA
'rCT'rATCM(:ACTTCCGG88ACT
ACT(T'rGTTAGA 2350
GTGCI'TTACGGGG8T8G8A'rAGT
TGTGAAGGGCC'rTTGATGACAACAA
TCTCGACGGGG80C(;AG(;C8GGTC
CCCTAG8AG8AGAACTCCCTCGCCT
CGC 8G 2400GC'rGGCC
TGGC'rCCcrCCAGGGGATCTTCTTT
CTTG8GGGAGCGGAGC Bow rCACGCA
G8TCTC8Ai'CGCCGCGrCGC8G8A
GA'rCTCAATCTCGGGA八TC'rC
24501'GCGTC'l'AC;AG
rT8GCGGCGCAGCGTC'rTCTAGAG
TTAGAGCCC'r'rAGΔGAATGT'r8GTATTCCT'rG(;8CTCATAACj?r
CGGAAAACTT'rACGGCGCT'rTA
2500ri'Ac8ArC8rA8G
G8ACCTG8GTATTCCAGCCT'rTGA
ΔATGCCCCG8AAr intercountry i'I':! j'(
], no change) TTCCTCTAC8GTACCT
ATC;'r'rTAATCCTG8ATGGC888CTCC'rTCC'rTTC 255
088GGAGA'rG'rCΔrGG8TAG8A8TTAGGACTTACCG'r'rTGAGGA8C
G88ACCTAAGATTCATTT8CAAGAG
G8CATTATT8AT8GGTG'rC88CA8T'rTG'rG 2600GATTC
TAAGTA八ATG'rTCTCCTG'rAATA
8TTATCCACAGTTGTTAΔACACGGC
CCTCTCACTGTAAATGAAAAGΔG8A
G8TTG8AATT88TT8'rGCCTGC
2650CCGGAGAGTGΔCAT
TTACTTTTCTC'rTCTAACTTTTAAT
TAATACGGACGAACCTTATTATCCA
G8'rCAGGTAGTTAATCATTACTTC
C88ACC8080A1 275(1'
rTGGAATAAATAGGTCTAGTCCATCA
ATTAGTAATGA8GGTTTGGTCTGTA
TATT'rACA'rACTCTTTGGAAGGC
TGGTA'rTC'rA'rATA8GAGGGA88CCAC 2800ATAMTGTA
TGAGAMCCTTCCGACCATAAG8'rA
TAT'rC'rCCCTTTGσrGACG'rAG
CGCATCAT'rTTGCGGGTCACC8'r
ATTC'rTGGG8AC88G8GCT8C
2850'rccΔT(:GCGT8GTA
88ACGCCCAGTGGrATAAGAACCCT
TGTTCTCG8'rGATTCCTTCCCGA'
rC8TCAGTTGGACCCTGCATTCGGA
GCCA8CTC8ΔC8Δ 2950T
AAGG8AGGGCTAGT8GTCAAACCTG
G8CGTAAGCGTCGGTTG8GTTGTTA
TCCAG8TTGGG8CTTC8ACCCC8TC
8A (iGACC8CTGGCCAGC8GCC8AC
3000TAGG'rCTMCCCCT
GAAGTTGGGGG'rAGTTCCTGGTGAC
CGGTCGTCGG'r'rG1I+::+Lノ)
Ino'i. 'i (Go to 1F-)10
20 30 40 50
G[l;GTAGAG8GGTGG8(;Ai”rcT
c'rGTCAGTAGGAGT(;UG(itAL.
Ljl At. ul lpBR322-EcoRI/
HEIV933j43 diagram prjn5zz-1oql/HBV999 procedure
Amendment (method) % formula % 2, title of the invention J) NA and its use 3, relationship with the person making the amendment'1 ↑) Applicant fi Address 11i 1HI, Higashi-ku, Osaka-shi 2
Ding II 27 a Place name J4: (293) Take 1.1
1 Pharmaceutical Co., Ltd. Representative Kurabayashi Ikushibu 4, Agent 11 Location 1 temporary city, regular ward 1-Mikicho 2-chome I
-1] No. 7 85 Shin 5, date of correction order 813 Sum 5
February 220, 2008 (shipment date) B Figure 1 of the drawing subject to amendment 7 Contents of amendment as shown in attached sheet g List of attached documents (]) Attachment 1 or more copies

Claims (7)

[Claims] (1) DNA0 containing the gene encoding the surface antigen precursor of ady hepatitis B virus (2) DNA0 according to claim 1, which is for animal cell transformation. (3) DNA0 according to claim 1 or 2, which is obtained by cleaving adw hepatitis B virus double-stranded DNA with restriction enzymes Tat and I. (4) DNA0 according to claim 1 or 2, which is obtained by cleaving adw hepatitis B virus double-stranded DNA with restriction enzyme Bglll. (5) Animal cells transformed with DNA containing a gene encoding a surface antigen precursor of adw hepatitis B virus. (6) The method according to claim 5, wherein the DNA containing the gene encoding the surface antigen precursor of the ADW hepatitis B virus is inoculated into thymidine kinase-deficient animal cells using the thymidine kinase gene as a selection marker. animal cells. (7) The animal cell according to claim 5 or 6, which is a mouse L cell. (s) culturing animal cells transformed with DNA containing a gene encoding the surface antigen precursor of ADW hepatitis B virus to accumulate ADW hepatitis B virus surface antigen protein; a characterized by collecting
Method for producing dw hepatitis B virus surface antigen protein.