JPS60145089A - Human antibody gene fragment and hybrid dna - Google Patents

Abstract

PURPOSE:To human antibody gene fragment, consisting of specific units containing genes on the basis of a site on the 5' terminal base side of a cleavage site by a restriction enzyme EcoRI and a base sequence, and capable of expressing human H chain proteins. CONSTITUTION:A human antibody gene fragment consisting of the following units (A) and (B) and a base sequence; (1) units (units A) containing V genes, D genes and J genes in the order mentioned within the range of about 0.33Kb from a site cleavable by a restriction enzyme AatII at about 1.6Kb from the 5' terminal base (hereinafter abbreviated to 5') to 5' and within the range of about 0.04Kb to the 3' terminal base (hereinafter abbreviated to 3') on the basis of the site of 5' which is a cleavage site by a restriction enzyme EcoRI of human chromosomic DNA containing antibody genes, (2) units (units B) containing Cr1 genes within the range of about 0.69Kb from a site cleavable by a restriction enzyme PstI at about 7.9Kb from 5' to 5' and about 1.02Kb to 3', (3) a base sequence having promoter function on the 5' side of the units A and (4) a base sequence having enhancer function between the units A and B.

Description

[Detailed description of the invention]

(a) Industrial application field The present invention relates to human antibody gene fragments and hybrid DNA.
It is related to A. More specifically, it relates to human antibody gene fragments capable of expressing human 1' chain protein. (b) Prior Art Conventionally, human immunoglobulin has been partially denatured from γ-globulin isolated from human blood and used as an immunological preparation for patients with LF. The problem is that human antibodies are dependent on human blood, which is difficult to obtain in large quantities in a stable manner, and that it is therefore difficult to constantly obtain homogeneous and safe immune antibodies. Nl' by genetically manipulating the protein
There is no denying that if it were possible to do this, it would be extremely advantageous in all aspects of pharmaceutical manufacturing. -C, Regarding the genes related to the constant region (Cr) in the human immunoglobulin chain, it has been elucidated that there are four types of class genes: Cry. Among them, Cry and Or
, + 0) lyso') The structure of the ras'r'n gene is (
), K rawinkel and l, It, I<a
bbittS et al.” -1he F M r30J 0
Llrna l” Volume 1, April <1982> I 4
Published on pages 03 to 407. Also, for the mekre Atide sequence of C r + i keiden-r, 1, noay W, F 1lison, 13e old + ct
t J, 13erson and i eory I-'
, H00(l et al. ``Nucleic Aci(
lsRcseardb” ]jO Volume 13th (198
2) Go to page 4071 and publish on page 4079. However, the above report does not suggest that the method for expressing human pile body protein is necessary.
) There is no C, which is described as equivalent to C with NΔdrl. (0) The purpose of the invention is to express the 1-1 chain protein or to provide the DJ function X1 human antibody transmission fragment. be. Another object of the present invention is to provide a hybrid DNA that follows the above-mentioned DNΔ...i fragment. (d) Structure of the Invention According to the research conducted by the present inventors, the purpose of the present invention is to use the restriction enzyme EC0R1 of human chromosome 1 NA containing an antibody gene.
The base at the 5i' end of the cleavage site (hereafter simply 5'
Based on the position of (1) 5' to the cleavable part of the restriction enzyme AatTIt of approximately 1,13 Kb (medium Kf] JM group, hereinafter the same). about 0.33 Kb towards the 3' terminal base (hereinafter simply referred to as 3')
In this order, add the V gene in the range of b, 1) gene and J) J) J to the restriction enzyme psB of about 7.9K b from 5' to the restriction enzyme psB in this order.
, about 0,69 towards 5' from the cuttable part.
. A unit (unit 13) containing a Cr1 gene in the range of 3' (approximately 1.02 KI toward D) (3) a nucleotide sequence that has a promoter function on the 5' side of the unit Δ, and (4) said 1 -1i 67△ and the 11-13 nucleotide sequence I+! Such a human antibody of the present invention is achieved by incorporating a pre-antibody fragment into the human antibody J'-19i 1'+ by providing NA. expresses the human 11 chain (Uself (j, V fit:).Next, if it is easier to understand Kijiaki's Hi1~Anti-141 topden-j'-rlJi piece, To confirm, use Attachment 14 drawings (explain). Follow! i I′
J, le')'J IIJi part (+/-:)')A
D 3' between (, 2 (r (1・jpe) each instep (G/
And 0, each Hanawa's 'jll' is 1 dye type f+' → tkoh\shi!
, -6 (there is. J3 in Figure 1 (, J' and 03' are 4' Schicht staining (book 1)
<'1 to 1 by I) true 1) - cut out including r... end 9 of the lino cut part of 1: t's lj lit is small) between ' and 3' (,1 approx. 21K b < 21K
■qsa'Cihru4 of b-I-IKb), cut out the middle of this [fJi I'i's AaL II and C The place indicated by the arrow is the limit element △
U atn 1. IJ can be cut off ((L position,
Approximately 1,6K b (1,6K lJ± 0.1
It is in the 11) place @. 45 in this invention 111th place 84
, about 0.33 Kb (0,33K
b 4: 0.02 Kb) from point 3' to h about 0
．． (14Kb (0,04Kb -!-0,005Kb
) to the point of approximately 0.37Kb (0.37Kit-70.025KIl). Among these 15th place △ are V gene, 1) iNl Rokuko and J
'+M genes are included, and they are arranged in C order from 5' to descendants. A3 Rir V gene, D gene, JW gene in Mokufuto Akira. Cr+3i11butsu, promoter and -'nhanli are the jshinbutsuji or nucleotide sequences explained below, respectively. The gene that encodes it. D Ei transmission: Located between the J gene and Vil Buddha,
A gene that encodes the most diverse part of the immunoglobulin molecule. 1 example (, [J, S cl+
1llin(1,B, (':levinger.,], N4. 1)avi Oand l, 1
ood. N aHIlie, 283.3! i (1980) Reference IJ Shobutsu j': IF variable region definition of immunoglobulin l-1 norin molecule 1; IF (gene that loads the connection part with the i region. , 13. 1vlax and one, leder, Natur
e. 2841, 370<1979> Reference ICr+iM gene: CO○1-1 end☆J of immunoglobulin 11 chain molecule:
From about 3;
One of the ruru genes. rl is l (indicates the 11 chains of 10 lysoclass 1.)] IJt-ter: I) Transfer from NA to RNA A
Signal for starting U (nucleotide sequence that causes Ikuno. John Hanley: l) N△ Silk change brings about the transcription tl+ rate from IchizoII T-Iter upstream of V gene r. Togeru tlIl+Kiwo Ijzuru 1n base array. For example, Satoshi Chokaido AJ1. , Honjo I (1, modern chemistry,
-υRho, 62 (1983)] In addition, the location indicated by an arrow pst■ in FIG. , 5' to about 7.9K +1 (7,
9Kb±0.5Kb). The present invention

[No unit] 3 is about 0.69 Kb (0
, 69 Kb ± 0,02 Kb) towards the 3' point approximately 1.02 Kb (1,02 Kb ± 0,03 K
b ) to the point of approximately 1.71 K+1 (1,71Kb
±0.05 Kb). This unit B
is the so-called constant region
Contains a part called Crt, starting from 5' and CH
+ area, 1) area. It contains the CH2 region and the Ct-13 region in this order. Furthermore, the 3' side end of this unit B includes a stop godon. Furthermore, in Figure 1, P indicates a nucleotide sequence that exists on the 5' side of unit A and has a promoter function.
shows the nucleotide sequence that controls the enhancer function present in unit A and the intermediate [3]. iIll:
A base sequence (one) that makes the difference! 1st place △, J-Hanley function to the right +3. j Jjl sequence ('') and unit B (order C114).
Add the 100% protein expressing the 11-chain protein, and add this to the l
@le is integrated into another vector L'U Hyzolid D
It is possible to get NA. Such a vector (for example, a vector for E. coli such as Ch on4A, cl+aron 2-8.
2,l113R32,'+,I) Colon Tagawa 1 lasmid hector like Ml-39:+)jJt3N(1,111
1W I-like dried grass α1 river j rasmid vector: Yl
1113. pJl+3 219. Y I'<
117. Y f again p16゜Y Ill! +, Y
Ill: (Plasmid vector for yeast such as 2:
1lsV2-(1111, pKsVlo like O) animal cell lice 1; l\ctor: 111! 136
Examples of such vectors include F-mid vectors for animal cells such as -806, which are shown as examples in the previous page.
I won't tell you what to do. Examples are listed below, including human 1 to staining 1 DNA isolation 1
゜Creation of a human gene library, screening of human antibody genes, creation of restriction endonuclease cleavage maps of human antibody genes, recloning of human antibody genes, and Expression within cells will be explained in detail. Example 1. (Isolation of human chromosomal DNA) Crush 08 human 1~cultured cells A Rl-177 strain 3xi with a crow bar,
In the presence of 2% SDS, Protearche K (manufactured by Sigma) r:
: After treatment, 10ml 1vl Tris 1-ICR
Add saturated phenol (pH 8, 0> -1mM LDTΔ aqueous solution) and separate the J-Nol phase (from the aqueous phase by stretching).
9(111I7.5>-100mMNa C9-5m
C was dialyzed against an aqueous solution of M l ball D'l-△. After treatment with ribonucleno7-zeΔ (manufactured by Sigma) and phenol extraction, 10mM Tris 1-IC# (
1) Dialyzed against I-l 8.0> -1mM EltΔ aqueous solution, about 1.2 mg of human chromosome IJ'NΔ
Obtained IN, [31in and one, W. 5tarford, Nuclic Ac1ds Re
s,,3. 2303 (1976)]. Example 2 (Human Gene Library) 150 μ9 of the human chromosomal DNA obtained in Example 1 was partially digested with the restriction endonuclease EcoRI (manufactured by Takara Shuzo) according to the method shown in Example 4 described below, and then digested with a sucrose density gradient. Centrifugation [sucrose 10-40% (wt/vol),
28,000 r, p, m,
3μ is 1q. Next, this l) NΔ cutoff 0.8μ7 and cl+aron4Δ vector [[2, R, Blact
ncr, +3. G. W! I I IallS, Δ, E, B 1ec
l+I, K, l), -1' l+ompson
,I-1,E,Faber,I-,A,Furlong
, l). LJ, Qrunward, l), 0. Kiefr
r, l, ), Q. 1vloorC, J, W, 3clu+mm, F, l
, 3beldonarHI Q, Sm! Lllll!
eS, 3science, [1G, 161<1977), 1. Which concatenation (j, charon 4A vector Rigbt-old m and 1.-e (L -a I'
A hybrid DNA was obtained in which human-derived 1) NA was inserted between m/j. ld! The ligation reaction was carried out using 66mM Tris-1-1cf (pl-17,
6) -6,611IM MO (+2-10111M didiothreitol-1mM ATP in aqueous solution at 11°C
, which lasted 12 hours. Obtained 1 = Hybrid [)N8, Auto) VitrO packaging [A, Be
aker and M, Golcl, Proc. Natl, Acad, Sci, u, s,, 72.
581 (1975)] was carried out, and a human gene library (1,8X 10"PFU/μ9.human DNA
containing 99% or more of A). Example 3. (Screening for human antibody genes) CI+aro containing human-derived DNA obtained in Example 2
A collection of n4A phages (gene library) was infected with E. coli strain E392 to form plaques. Clones containing human antibody genes are BQntOn and oaV.
is plaque hybridization method [W, D
, Benton a'and R.W., Davis,
3science, Yu9G, 180 (1977)] was used to prepare [32p]-II-identifying human antibody H chain J3m.
I chose 1 Butsuko. Charon containing human antibody genes
Adjustment of NΔ from 4A phage is l”llo
mas and [)aVis people LM, 'l'ho
mas and l'<, W, '1) avis, J
. M (11,13iol, 91. 315 (1974
) Food pA? ] NiJ: I did VJ. . Example 4. (i1i + 1 limit - 1 - Ndomekrea L'1
Example 3 (C11a1 containing human antibody genes isolated by 1υ)
'o11 4A I) N△ 171g up to 1lill]-Buffer for nuclease agitation 'LI day co I
<I, M lug,', S pt+I, X
baI, Xl+ol digestion C is! iomM 1-ri
s-)-1cj(pl-17,4)-100mM Na
CR-10m, M Mg SO4 aqueous solution, Δatl
I, Aval,, 13mm l-11. 1-1 indm, Ps, pvtH digestion with 10mM-rriS-1-1cI 17,5)
-60 + 11M Na (J-7mM
-HCf (l1l-17,4) -10mM M(]
SO0-1111M Jig A jig upper aqueous solution (1-I DaI, S ma1 digestion is 101
11M 1'ris-F+(J(+)l-I B,O
) -2011M KCl--7111M M(l C
fz -7mM 2-mercaptoethanol aqueous solution,
Each was used. ]20μρ, restriction endonuclease (Δa (■: Toyobo, Avai: Besenda, Hoop Laboratories, 5phI: New England Biolabs, others: Takara Shuzo) 2 Unit was added and digestion was carried out at 37°C for over 1 hour.When performing digestion with two types of restriction endonucleases, first treatment with restriction endonuclease C, which acts at a low salt concentration, was then carried out with the specified restriction endonuclease C. The salt concentration was increased to 0.2 μl after digestion with restriction endonuclease, which acts at higher salt concentrations.
A 5% bromophenol blue/50% glycerol aqueous solution was added, and 0.8% to 2.5% agarose gel electrophoresis was performed. The agarose used was Sigma's type ■ for electrophoresis. As electrophoresis buffer 1-, 40 n+
M, Tris-CH3COOH(DH8,0)-1
An mM EDTA aqueous solution was used. In a vertical gel with a thickness of 2 oboro, at a voltage of 6 to 9 v/c IR,
Electrophoresis was performed for 1.5 to 3 hours. During this electrophoresis, l) As a molecular weight marker of NΔ cut J″1,
- Di's I) NA was digested by 1-1 ind m'C by Ringer Nunnheim! M) was used. After electrophoresis, transfer the DNA in the agarose gel to 2 μfj
/ m (1-1 budium sonide aqueous solution is dyed,
This gel was irradiated with long wavelength ultraviolet light to observe the digestion pattern. By analyzing the digestion patterns of various restriction endonucleases and combinations of two restriction endonucleases U, we determined the relative positions of the cut points of each restriction endonuclease, as described below. Determine the relationship lζ. Example 5. [Recloning of human antibody genes (human antibody gene recloning)
Hybrid DNA of 8,2K b-l-1ind containing Cr+ gene and Escherichia coli plasmid pF3R 322;
NA 3/19 was indl according to the method of Example 4.
[digested with l'ea, agar l'-suguru electrophoresis running gel'ea
0.8%). 8.2 containing Cr+ gene
Cut out a band that covers a portion of the DNA of K I), and apply the agarose gel fragment to 3x FJ (vol, 7wt).
of 8M NaCfO4 aqueous solution. CMII and Thomas' glass filter method [C, W, C11
en and C, Δ. Thomas Jr., A nal, 3 ic
+chem, 10 Takumi 339 (1980)], the DNA fragment of 8.21 (b) was recovered by agarose gel. On the other hand, the E. coli plasmid I'1BR3221
μ! 7 was digested with Hindll according to Example 4, alkaline phosphatase (E, colic
75) Add 0.5 unit of (manufactured by Takara Shuzo) and heat to 45℃.
The reaction was carried out for 1 hour. After the reaction was completed, fluorine and enol extraction was repeated three times in order to deactivate and remove alkaline phosphatase in the reaction solution. 1indt of 1IBR322 obtained in this way
The n/alkaline small sphatase treatment solution is mixed with an aqueous solution of the 8,2 Kb Hind m fragment recovered from the gel, and after ethanol precipitation, it is dissolved in 50 μl of freezing reaction buffer (see Example 2). 2 units of T4-DNA
Add ligase and react at 11°C for 12 hours to obtain hybrid DNA All1. Transformation of E. coli strain C600 was carried out using the usual CaCjz method [
, M., V., Norgard, K., Keen a.
nd J, J. lvlonaham, Qene, 3.279 (19
78)] was carried out. In other words, 5Id
, L-broth (1% 1-ribin, 0.5% yeast extract, 0.5% NaCR. 111-17.2) was inoculated with an 18-hour culture of E. coli strain C600, and Avoid growing at an optical density of 0.3C. Transfer the bacterial cells to cold magnesium buffer [0
, IM Na CR-5111M M(l CR2-5
mM Tris-1-I CR ('l juice 17.6.0
C92-250mM
Resuspend in K (J-5111M MCI Cr2-5m1y114-LICR (pl+ 7.6.0°C)) and let it flow for 25 minutes at 0'C'c. /1(+'ci' in calcium buffer)
fl condense, hybrid DNA aqueous solution and 2;1(
yOl, : vol,)il'+! After keeping this mixture at 0°C for 60 minutes, 1 mQ of LBG-
Broth (1% human broth, 0.5% yeast 12J L =
l varnish, 1% NaCR, 0.08% G/L, TJ −
vinegar. pH 7.2) and cultured at 37°C for 1 hour. The culture is inoculated into selective medium (one broth plate containing 30 μg ampicillin/#Il!) at 1()0 μρ/plate. The plates are incubated overnight at 37°C to grow the transformants. DNA was prepared from the obtained colonies using a known method, and the desired hybrid DNA was confirmed by Aga L1 gel electrophoresis. [Restriction enzyme map of rib clones] Na clones prepared in the experiments of Examples 4 and 5, namely, I) TJ I B, I) TJ I BR
, FITJ2. l]TJ4. Restriction enzyme maps of pTJ5 and pTJ7 are shown in the attached FIGS. 2 and 3. Figure 2 shows the fragment of human chromosomal DNA containing the antibody gene cut by the restriction enzyme EC0RI, with the 5' position designated as EcoRI-(1,) and the 3' position designated as EcoRI-( Figure 2 also shows the positions where the DNA fragment can be cleaved with the restriction enzymes Xba and XhoI.
Cut each small fragment into four small fragments, and divide each small fragment into a plasmid vector for E. coli.
Small Igo. Seibutan Rizoku (1-N name cut...i part ro (
Hi1~chromosome part) 1-(1111'L-h fEcoll(1) +v
l-1int, l]Il-(1) approximately 3,4KbII
D-l', b lli+1(1m-(11←l l
ind III - (2) Approximately 3.6 Kl]
/U i! There is something C that was given to me. ] (+111JIJ3" 1: coRl-(+1 <-> If 1ncl 1
1-(]l DNA fragment J'〈approximately 3,4Kl+) for E. coli 1~hector pIN<322 (more than p+3+<322) [-COR]←111
Insert between old + m and l, :ra. The detailed 01 restriction enzyme map of this rib clone is shown in II-+1 in Figure 3. )It was shown to. 1112i [pl-J 7] front ffi[! l--(1) [prJ3] 13am
HI -m ←BamLI I - (21 DNA small I
IJi? pF-3R322 Ll' (approximately 0.8 Kb)
I inserted it into 13amHI Rei 1~. The restriction enzyme map for this strain is shown in Figure 3, 1-(2).
lko. II-(pTJ2) Hind In-(11+-+ t-1ind lt
I-(2) 1] Approximately 3. GKb) to p
l3 +'< 322 no l-11ncl m'') i1
-Inserted into. The restriction enzyme map of this rhizogen [-1-in] is shown in Figure 3H. 111-Δ(pTJIB) Hind III-(21-+N ind 1lI-
(3) DNA small size 11i (approximately 8.2Kb) p1
Inserted into 11 ind III 1~ of 322 on the 3rd floor. The restriction enzyme map of this libk1''-in is shown in m-A of FIG. In this figure 3 ■-△ UPstJ
-(between 31 and ll ind III -(between 31,
Have you confirmed that there are 3 to 4 Pst J Zai 1? 'l J l 131) above ■1-Δ and IFil h method 'cp + 3 + < 322, but the insertion method is b, which is the opposite of ■-Δ. The restriction enzyme map of is shown in l1l-13 in Figure 3!ζ1゜In △-(]] [Ill' J 5 ] above 11]
-Δ(pl' J l B > P s L J-[2
1 → [-) St l', - (31 small DNA fragments p
In l31, 322's 1) b inserted into stl 1-. A diagram of the 111th limit of this process is shown in Figure 3 (Ill-A-+1). 1[1-△ (2) l l) -1J±■11cho18kiII-△(1)l J l 13>smell (,1li
14 between nd[1(3) and p s l-+31
i rul l1l- [31 closest 1) sll reit to P st J-+21 was deleted. The restriction enzyme map of this rib clone is shown in Figure 3.
) /j. Example 6 (Expression of intracellular C of DNA fragmentation J!l) Δf<
Approximately 1 x 0110 H77 cells were incubated with hot cells and treated with Norucresol.
I have about 5 tags. This RNA4 m'J
As a result of applying Qligodl-7J-y and C purification, 14071 g of r+oly(Δ) RNA was obtained. This -1-otal RN△10119,
Using 500 ng of ll0IV(Δ) RNA, 2.01(b
p I3am-1-1ind■Fragmen i-2 x
io' cpm (, Northern l+y
When bridizatin was applied, a band of 1'' strand 111RNA was detected at the 18S position, and a band for (-1 antimembrane-type mRNA) was detected at the 233 position.Also, using the same block, C1ΔRH77totalRNΔ
EcoRI completely disassembled fragment 1 to 30U[11e
rllbVb r i d i 7a 1], two bands were obtained at approximately 21 Kbp and 9 Kbp, and 2 flail alleles were detected. The obtained human 1-1-1 chain genetic result (HI Gl) EC0R
I fragment, about 21 Kbp into P, Berg a) Open vector psV-2gDt 13 coR1-+
) Incorporated into Ih, two types of 1 lasmid I with different directions
]5V-2, HIGI-a doll5V-2, 1-11
G1-b was created. Restriction enzyme maps of these two types of 1 lasmids are shown in FIG. 11-knotted silk changing Hector-DSV-2111G1
was transformed into Escherichia coli M
Later, J last board 1 ~ was increased by 11T/Muno 1+] and 1ζ. Next, by resobu l\ processing Czo 1''
1 - turned into shirast. Lymphoid cells 2 x 106 cells and 1!7 g (11-cillast were added to 50% P
4000 (1,5 mff) Cells were fused for ~7 min, then centrifuged after dilution of MLM Ig.
I) Remove I-G. E+' to +1 I EI
RI) I'vl 1 complete medium C111 was cultured and then transferred to a selective medium. Choice 1E', 2:10γ/t for ground
r+1! :Linboon, 1!1γ/mQ hypoA
-rin, 6T/mQ mycol+l+cnoli
Contains c acid. After 211 consecutive days of C metium exchange, 2 weeks later, my
col+l+c++ol ic acid resistant ml
1. -1 Knee is called lζ. 11 Nore I, -J+1-1 F r O(l U (
! II CV is shown in Table 1 (3゜margin below) Table 1 = 10 knees 24well plate 5 x 10 per well
The number C of the wells that will fly when the wells are sown is shown. When the above colonies were examined for detection of human 1-1-1 chain in C1 superinfection by ELISA method, it was found that ll5V21
-l IG Ia for 171[, psV
2NI G l l) +3 -C is p3L no I'C''
1 colony, 2 colonies in N5-1
The 11fI protein was secreted. Next, C
When ytoplasmicstailliTlg was performed, it was found that pSV2, which was not distributed in the J.
-HIG 1b-P3U 1-11 chain protein was detected in the cytoplasm in the 10[inch] knee. The surface of 104 cells of 210 Knee (C-3), in which human 1-1 chain protein was detected only in the cytoplasm, was treated with 17:1' anti-human I.
Fluorescent staining with ~1uGl21-10,
According to the rules, 10' fl! It was found that human 11 chains were present on the cell surface in 30% of the cells of the human body. To C33 S Meb A nin C1 internal 1abe
l L/, anti-L: using l-I U G, l'ro
tain Δ SO11118rO3[! 1113
(-] 7 Furmacia) 0hi l-1-1 chain l) I'e
Cip mouth ale L//C'b's Nitsui"CS I)
S P A G [three C inspection ri J L, I Kodokoro! The secreted type 1-1 chain is present in i4K, and each of the formed I-portions is secreted in 68K, and the secretory type is secreted in 11 cells, and the membrane 41 chain is also exposed. 4.1λl if+i in the tube<t 1J2 Akizoe f, 1 chain 1 diagram t, L II'L shokiden 5' included 1~chromosome 1
] N△ restriction fermentation f 1. (H old 1 shows J, 17 cut sites and (in between (,, white, middle and measure sequence). 21'A and 33 are l of Figure )N△ to 111
91.
Figure 1 shows the 11 limit anesthesiology map, .
Figure 4 psVZ-HIGI-C'psVZ-1' IGl-b
Procedural amendment September 1983 (To the Commissioner of the Japan Patent Office 1, Indication of the case Patent application No. 1983-208383 2, Title of the invention Human antibody gene fragment and hybrid DNA 3, Relationship with the person making the amendment Patent application rr) 1-11 Minamihonmachi, Higashi-ku, Osaka (300) Teijin Ltd. Representative Sashibe Okamoto 5 Subject of amendment 6 Contents of amendment (1) The scope of the % allowance rust request will be corrected as shown in the attached sheet. (2) Specification No. 3 1-(Cr)J on page 4 line r(, Cr
)” is corrected. (3) "rcrt" on page 3, line 4 of the same page is corrected to rcrtJ. (4) “Cr7. Cr, and Cr, J” on page 3, line 5.
is corrected as r Crv , Crs and cr< J. (5) [Cr and Cr4J [c
rt and Cr, correct as J. (6) "rcr," on page 3, line 11, is corrected to rcrtJ. (7) On page 4, lines 6-7, [restriction enzyme EcoRI J is corrected as [restriction endonuclease EcoRI J]. (8) "Restriction enzyme AatllJ on page 4, line 11 of the same page is corrected as [restriction endonuclease AatIIj." (9) "Restriction enzyme PstIJ on page 4, line 16 of the same page is corrected as [restriction endonuclease PstIj." (lO) Correct rCr, J on page 4, line 18 to rCr, J. (1) Correct [restriction enzyme Eco
RI J and “restriction endonuclease Ecoi” respectively.
ζ■" and correct it. (12) [Restriction enzyme Aat[J on page 6, line 1] is changed to "restriction endonuclease AatlZJ." (13) "CrI gene" on page 6, line 13 is corrected to "Cr. gene." (]4) "Cr1 gene" on page 7, line 10 of the same page r Cr
. "Genes," he corrected. (15) "Press the restriction enzyme PstlJ with [restriction endonuclease Pat 1. J" on page 8, line 3. CH1lJj area when viewed from 5', h
``area, CH, area, and CH, area'' are corrected to ``includes a portion called r cr, and as viewed from 5', are C-eye area, h area, CH2 area, and CH3 area''. (17) “charon 4 A” on page 9, lines 9-10.
. Charon 28, j as “Charon 4 A
, eharon 28. Correct it with J. (18) On page 9, lines 17-18, ``1 for animal cells'' is revised to ``for plant cells-1''. (+9) Correct "stretching" on page 1, line 13, to read "C centrifugation". (20) Same No. J] Page 7 line ``Sucrose density gradient centrifugation [Sucrose l
0~40%-1 by sucrose density gradient centrifugation [sucrose 10~40%
%” and correct it. (21) l-char in the +1jjlO line and 17th line
on J respectively [Correct Charon J and PI. (22) r(32p)J on page 12, line 19 of the same page is r(”p
)l. (23) Same @ page 13] Correct the line ``r IS adjustment'' to ``1 adjustment''. (24) [Ba/IIJ on page 13, line 15] [Bgl
Correct it to IIJ. (25) 1 Bethenda Research of 2 to 3 banks of the 14th member
"Manufactured by Bethesda Research Laboratories, Inc." is corrected to "Manufactured by Bethesda Research Laboratories, Inc." (26) On page 15, line 5, "with ethidium bromide bath solution" is corrected to "with ethidium bromide aqueous solution." (27) rcr of 1.5Q lines 14 and 20, -1
Correct rcr+'J and K' respectively. (28) "For freezing reaction" in line 16, line 17 of the same statement [corrected to 1 for ligation reaction. (29) "Adjustment" on page 18, line 5 is corrected to "1 adjustment." (30) Correct ``[restriction enzyme map j of Zaphan] to 1 [restriction enzyme map of subclone]'' in line 8 of the 18th member. (31) Correct the 18th line 10 of the same sentence to read ``-Refloan-1 as 1 subclone''. (32) On page 18, line 12, "1-restriction enzyme map" is corrected to "1-restriction enzyme map". (33) "Restriction I; coRI" on page 18, lines 13-14.
Correct j to 1-restriction endonuclease EcoRI J. (34) Correct 1-restriction enzyme Xba J on page 18, line 17 to "restriction endonuclease) CbalJ."
d III J as 'restriction edonuclease γ-Hind
Correct it as III J. (36) p TJs J [pTJ
3 Correct it as j. (37) rpTJ2 f-pTJ2J on page 19, line 5
Correct it with J. (38) Correct “each subclone” in line 8 of member 19 to “each subclone” (39) Correct r (pTJ, ] J in line 10 of page 19 to r(pTJ3), j (40) Same No. 2 (1 page 1 line 1, 7 line, 12 line and 17
Correct the lines ``Restriction enzyme map 1'' to ``Restriction endosphrease breakpoint map.'' (41) [
-Restriction Enzyme Map'' is revised to 1 Restriction Endonuclease Breakpoint Map-1'. (42) 1” from page 21, line 20 to page 22, line 1” To
tal RNA, J is corrected as l total RNA J. (43) ``Betting the l-01i2od T column'' on page 22, lines 1-2'' is changed to ``Through the Oligo dT column.''
and revise it. (Inquiry) Same page 22, line 3 r Total )'<NA
Correct J to 1-total RNA J. (45) “P□-gu” on page 22, line 4 is corrected as “blow-boo 1.” (46) l Bam J on page 22, line 5 is corrected as “Bam
Correct Hl, J. (47) On page 22, line 10, 1-broke-1 is corrected to 1 probe. (48) Correct page 22, line 11 of the same statement to read ``Complete decomposition of 1-1 to 1-complete decomposition.'' (49) 22jj lines 17-20 of rpsv-2
Figure 4 shows the restriction enzyme maps of all the former GI-a, pSV-2, and HIGI-b plasmids and these two plasmids. J Wol-pSV-2-HI
GI-a and psv2-HIGI-b were created. The restriction endosphrease breakpoint maps of these two glazes of neulasmid are shown in Figure 4. , -1. (50) Revised IF to ``1 rearrangement'' in the 23rd Sada line 1 as ``1 rearrangement''. (51) “Transformation J” on page 23, line 2 of the same
(52) On page 23, lines 2-3 of the same statement, the word "shaped wing transformant" is corrected to "transformant." (53) “1-Medium exchange” in No. 23, line 13 of the same
I corrected it to ``Medium father replacement''. (54) $e1 on page 24 is corrected as follows. 1 Table 1 Number of colonies” (55) 1- from line IO to line F from the bottom of page 24
For pSV 2 HIGI a] colony. For psV2111Glb, p3UI-CI colony, for psV2-HIGI-a (
:lo=-. pSV 2- HH,"r' 3 in I-b
U] and one colony,” I am corrected. (56) liJ p424, bottom row 3, “pSV2
-111Glb-P3UI J r pSV2-HIG
Corrected as I-b-p3U11. (57) r Protein A in lines 6-7 of 2500
Sopbarose 4 B, I'Prote J
in A-5epbarose4BJ and d] Correct. (58) “Restriction enzyme EcoRIJ” on page 25, 14-j.
1-restriction endonuclease EcoRI J and nJ. (5) Restriction endonuclease breakpoint map-1
I am corrected. Scope of Claims] The 5'-end base (hereinafter abbreviated as 1.5') of the cleavage site by the restriction endonuclease EcoRI of human chromosome 1) NA containing the antibody gene (based on the direct position) (, lower h[2+1) Approximately 0.33 Kb from 5' to the part cleavable by restriction endonuclease Aatll of 1,6 I<b, 3' thin end base (hereinafter abbreviated as 3') ) towards the V gene in the range of approximately Q, 04 Kb, 1)
A unit containing the gene and the J gene in this order (unit A)
IJ 9 Kb, about 1 j) towards 3' Cr in the range of 2 Kb. Enthronement (unit B) containing the gene (3) A nucleotide sequence having a promoter function on the 5' side of the unit A, and (4) A salt N sequence having an enhancer function between the unit A and the unit B. Human antibody gene fragment consisting of unit and base sequence. 2. Norinodo DNA incorporating the gene fragment of the first claim above.

Claims (1)

[Claims] 1. Anti-hyper-staining containing 13 N to θ
From the following fil E)', approximately 1. GK b restriction enzyme △al
Cut by 1tli i'iJ from the functional part to 15 of 5' (1,33 Kb). It(r/ (to the middle) containing iK1 gene, D gene and J31 gene in this order
(2) b' to about 7.9Kl] restriction enzyme IT) 3t
J Nirunikiri 11i uJ function part to 5'l/)7'j about (+]9 Kb. Crti in the range of 3'(7)7'j to about 1.02 to l)
Medium level including lff Den I (unit 13) (:3) 107-tar + fi 1 on the 5' side of the medium level A
1 base sequence, and (4) a base sequence consisting of a base sequence that has the function [between 30] and a base sequence between the unit and the unit [between 30] and the base sequence. piece. 2. A hybrid DNA incorporating the gene fragment of claim 1 above.