JPS62100291A - Gene fragment and plasmid - Google Patents

Abstract

PURPOSE:To obtain plasmid of chimera antibody development type, by transducing a gene fragment containing range V and range J in kappa (kappa) chain gene of mouse immunoglobulin and a gene fragment containing range C in kappa(kappa) chain gene of human immunoglobulin to a plasmid. CONSTITUTION:Gene fragment A which contains range V and range J in kappa(kappa) chain gene of mouse immunoglobulin and has about 4.2Kbp length and gene fragment B which contains range C in kappa (kappa) chain gene of human immunoglobulin and about 2.6Kbp length are introduced to a plasmid in an order wherein B-1 side of the gene fragment B is arranged at the A-2 side of the gene fragment A, to give a plasmid of development type.

Description

Detailed Description of the Invention a. Industrial Application Field The present invention relates to a gene fragment containing the V region and J region present in the (kappa) chain gene of mouse immunoglobulin;
This invention relates to a gene fragment containing the C region present in the (kappa) chain gene of human immunoglobulin and a novel expression purple plasmid into which these gene fragments are introduced.

b. Prior Art With the recent rapid development of genetic manipulation technology, the elucidation of immunoglobulin genes has progressed, and numerous reports have been made.

Conventionally, it has been difficult to obtain antigen-specific human monoclonal antibodies. On the other hand, for mice, it was monoclonal with antigen specificity.

C8 Purpose of the Invention Therefore, the present inventor aimed to elucidate the kappa chain gene in immunoglobulins, and in particular to elucidate the gene fragments containing the ■region and J region in the chain gene of mouse immunoglobulin, as well as the kappa chain gene in mouse immunoglobulin. As a result of research aimed at developing type 5 plasmids, we have achieved the present invention. did.

d1 Structure of the Invention According to the research of the present inventor, the purpose of the present invention is (1)
This is achieved by providing the gene fragments (2) and the plasmids (3) and (4).

(1) A gene fragment (A) having a length of about 4.2K bl) containing the ■region and J region in the (kappa) chain gene of the mouse immunoglobulin represented by the restriction enzyme cleavage map shown in Figure 1.

(2) C in the (kappa) chain gene of human immunoglobulin represented by the restriction enzyme cleavage map shown in Figure 2.
Gene fragment (B) with a length of approximately 2.6 K bp including the region.

(3) (i) The gene fragment (A> and (ti))
The gene (B) is A-2 in the gene fragment (A)
A new plasmid was introduced in the order in which the B-1 side of the gene fragment (B) is arranged on the side.

(41(+) said gene fragment (A>, (i) said gene fragment (B) j5 and (b) human immunoglobulin Hf
A gene fragment (C) containing the enhancer base sequence in A31 Buddha is introduced, and the gene fragment (A) and the gene fragment (B) are inserted into the Δ-2 side of the gene fragment (A>). Fragment (B) is a novel plasmid introduced in the order in which the [13-2 side is sequenced.

The gene fragment (A>) and the gene fragment (B) of the present invention
These are placed on the A-2 side of the gene fragment (△),
By combining the gene fragments (B) in the order in which the B-1 side is arranged, a chain can be formed in a mouse-human chimera, and the chain in this chimera consists of the V (variable region) of mouse immunoglobulin. ) and human immunoglobulin C (constant region) can be used to form a mouse-human chimeric antibody gene.

It is generally known that V region genes have low antigenicity, and chimeric antibodies formed from mouse-derived V regions and human-derived C regions have been shown to be useful in the treatment of various diseases, such as the treatment of various cancers. )■I can get it.

The gene fragment <A) in the present invention can be obtained by the method described in the Examples below, and is about 4.2
It has a length of K bp. And this gene fragment (Δ)
As shown in Figure 1, the restriction enzyme BomHI (
A-1) and l-1indlI[(△-2) are fragments that have cleavable sites at both ends, including 2 sites that can be cleaved with the restriction enzyme PstI and 1 site that can be cleaved with the restriction enzyme PstI. Owned.

The gene fragment (B) can also be obtained by the method described in the Examples below, and is approximately 2.6K bl).
What's good about it? The lever gene fragment (B) is
As shown in Figure 2, both ends contain the restriction enzyme FCORI.
Each of these sites has two sites that can be cleaved by the restriction enzyme 5aCI.

Thus, in the present invention, the gene cleavage (A) and gene fragment (B) are defined as (A-) in the gene (A).
2) side, that is, the l-1indl site, in an order such that the (B-1) side of the gene fragment (B) is arranged and then introduced into the plasmid to obtain an expression type plasmid.

In this case, the gene fragments (A) and (B) may be linked as they are, or may be linked via another gene unless there is a particular problem.

In addition, when the gene fragments (A) and (B) are introduced into a plasmid as described above, the gene fragment (C) containing the enhancer base sequence in the H chain of the human immunoglobulin gene
By introducing a combination of these, it is possible to obtain a plasmid with higher expression efficiency. This gene fragment (C) having an enhancer function can be introduced into various positions in the plasmid, for example, it may be on the (A-1) side of the gene fragment (A), or it may be inserted into the gene fragment (A). It may be located between (A> and (B), or roughly anywhere on the (B-2) side of gene fragment (B). It can also perform its functions when introduced with direction. In particular, when gene fragment (C) is introduced into the (A-1) side of gene fragment (A) with the immunoglobulin gene and its reading direction aligned, an excellent plasmid with high expression efficiency can be obtained.
11.

The enhancer fragment (C) used in the present invention was discovered and previously proposed by some of the inventors of the present invention. JP-A-60-1209
It is disclosed in Japanese Patent No. 91. Enhancement j! disclosed in this bulletin! 11 gene fragment (C
) can be used as

Vectors for introducing the gene fragment of the present invention include:
Various things can be mentioned. Such vectors include, for example, charon 4A, charon28λgt
77F-divector for E. coli such as WEs/λB: p
Plasmid vectors for E. coli such as BR322, Dr3R325, pMB9; o UB 110. +
11 Plasmid vector for Bacillus subtilis such as HWI: YE
p13゜p JDB 219. YRp7. YRp1
6, Yip5゜Y [plasmid vector for yeast such as p32; PS2neo, DSV2gpt
, o KSV-10: plasmid vectors for animal cells such as D Ti B 6-806; however, these plasmid vectors are shown merely as examples; It is a plasmid and can be used. Preferred among these are plasmid vectors for E. coli and plasmid vectors for animal cells.

As described above, according to the present invention, a gene fragment (A) containing the V J5 and J region in the mouse immunoglobulin κ chain gene and a gene fragment (B) containing the C region in the human immunoglobulin chain gene can be provided. Expression type plasmids into which these gene fragments have been introduced are also provided.

The present invention will be described in detail below with reference to Examples.

Example 1 Human cultured cells A R+-177 strain 3 x 108 cells were crushed with a glass rod, treated with protease K (manufactured by Sigma) in the presence of 2% SDS (4), and then treated with Ion MT.
ris-toICj (pH 8,0) -1mM
Phenol saturated with aqueous EDTA was added. Decompose the water tank and phenol layer by centrifugation (phenol extraction)
, the aqueous phase was diluted with 20 m M TrlS H(J (D H7
, 5> -100mM Na (J-dialyzed against 5mM EDTA aqueous solution. After filling with ribonuclease A (manufactured by Sigma) 911 and performing phenol extraction, the aqueous phase was diluted with 10mM Tris -1-ICf (1) H
8,0)-1mM EDTA aqueous solution to remove approximately 1,211+jj) human chromosomal DNA [
N, B11n and D, W, 5tafr
ord.

Nucleic Ac1ds Res, , 3
．． 2303 (1976)].

Example 2 (Creation of human gene library) The human chromosomal DNA obtained in Example 1 was cut with C restriction enzyme FcoR1 (Takara Shuzo) according to the method shown in Example 7, which will be described later, and then subjected to agarose gel electrolysis. Perform electrophoresis and collect 2Kb to 3Kb
A DNA fragment corresponding to the above was recovered using the electronic method. Next, λc)tW of this DNA fragment
This was ligated with the E SλB vector (Amerjam) to obtain a hybrid DNA in which human-derived DNA was inserted between the right arm and left arm of the λatW E SλB vector.

T4-DNA ligase (Bethesda Research) was used for ligation.
The ligation reaction was carried out using 66m M T
ris-HCj (pH7,6>-6,6m M
MoCl3-1010M dithiothreitol-111
The test was carried out in a 1M ATP aqueous solution at 11°C for 12 hours. For the obtained hybrid DNA, in Vi
trO packaging was performed to create a human gene library (using Amajam Git).

Example 3 (Screening for human immunoglobulin chain genes) λgt containing the human-derived DNA obtained in Example 2 above
A collection of 'WEsλB phages (gene library) was infected with E. coli strain LE392 to form plaques. The clone containing the human immunoglobulin chain gene is B
enton and 1) avis plaque hybridization method [W, [,), [3 enton and
R.W. Davis, 5science, 1
96. 180 (1977)] using [32
p]-labeled mouse immunoglobulin C. λGTwc containing 1'1 gene in human immunoglobulin
Preparation of DNA from λB phage was performed according to the method of Thomas and Avis [M, Thoias and
R,W, [)avis, J.

Mol: , Biol, , 91. 315 (1
974)] I did it.

Example 4 (Mouse chromosomal DNA monomer) Mouse hybridoma NL-11
odiun+ Iauryl 5ulf'ate
) After treatment with protease K (manufactured by Sigma) in the presence of
DNA was extracted by adding water-saturated phenol, the aqueous phase was separated by centrifugation, and tOmM Tris-H (Jp H
7,4,0,1m M Na CO, 0.1a+
Dialyzed against MEDTA (TNE) buffer. After treatment with ribonuclease A (Sigma) and repeated antiphenol extraction, 1.2 rng of mouse chromosomal DNA was obtained by dialysis with TNE buffer (81 rng described in Example 1).
(see reports of in and 3 tafford).

Example 5 (Creation of mouse gene library) Example 4
Using the restriction enzyme HindI[I(
After complete decomposition using Takara Shuzo), initial density gradient centrifugation [Original 1
0-40% (wt/vol).

28000r, p, m, X15 hours, 20℃
] to obtain DNA fragments corresponding to 4 Kb to 9 Kb.

Next, this DNA fragment 0.45 μ9 was ligated with HindII [7-me of Charon28 vector DNA (Bethesda Research Laboratories) using T4-DNA ligase, and in vitro using a kit from Amerjam.
Mouse NL-
One gene library was obtained at 4 x 106 PFU/μ.

Example 6 (Screening for chain genes in mouse immunoglobulin) E. coli strain IE392 was infected with the Charon28 phage containing mouse NL-1-derived DNA obtained in Example 5, and subjected to plaque hybridization (W,
D, Benton snd R,W.

Davis, 5science196 180(
(1977) was used to select a 72p-labeled mouse antibody using the chain J gene.

Cha-ron containing suppressor gene in mouse immunoglobulin
Preparation of DNA from phage 28 was performed by Thomas and Qa.
vis method (described above).

Example 7 (Creation of restriction enzyme cleavage map) 1 μg of the phage DNA containing the human and mouse chain genes obtained in Examples 3 and 6 was added to restriction enzyme cleavage buffer F.
- [Eco R1, MluI, Xbal.

For cutting, 50 m M Tris-HCffl (1
) H724) -100mM Na (J-10m
MM9804 aqueous solution, BamHI, Hin
d m, PstT, 10mM Tris for cleavage
-HCF (D tot 7,5)
-60mM Na (J -7mM
Mg(J) 2 aqueous solution was cleaved with ACCI and 5acI using 101M Tris-H(J (pH 7,4
)-10111MMCI SO4-111M dithiothreitol aqueous solution, and
0mM Tris-1icf (DH8,0)
20m M KCf-711M IVHI C1z
-111M2-mercaptoethanol aqueous solution was used in each case. Restriction enzymes (Acc and Sac: manufactured by New England Biolabs, others manufactured by Takara Shuzo) were dissolved at 20 μm. 2 units were added and digestion was carried out at 37°C for over 1 hour. When cutting with two types of restriction enzymes, first treat with a restriction enzyme that acts at low salt concentrations, then increase the salt concentration to a specified concentration, and then treat with a restriction enzyme that acts at higher salt concentrations. Processed.

After cleavage with restriction enzymes, 4 μρ of 0.25% bromophenol blue/50% glycerol aqueous solution was added.
8% to 2.5% agarose gel electrophoresis was performed. The agarose used was Sigma's type ■ for electrophoresis.

As electrophoresis buffer, 40mM Tr
is -CH3C0OH(pH8,0) -1
An mM EDTA aqueous solution was used. At a voltage of 6-9 V/+a++, 1.5-
Electrophoresis was performed for 3 hours. During this electrophoresis, DNA
As a molecular weight marker for the fragment, λ phage DNA was
-1indn [digested with Boehringer Mannheim]. After electrophoresis, the DNA in the agarose gel was stained with a 2 μ9/d ethidium bromide aqueous solution, and the gel was irradiated with long wavelength ultraviolet rays to observe the cutting pattern.

By analyzing the cleavage patterns of each υ1 restriction enzyme alone and a combination of two restriction enzymes, the relative positional relationship of each restriction enzyme cleavage point was determined.

The restriction enzyme cleavage map of the mouse immunoglobulin chain gene fragment is shown in FIG. 1, and the restriction enzyme cleavage map of the human immunoglobulin chain gene fragment is shown in FIG.

Example 8 (Subcloning of chain C region gene into human immunoglobulin) λatE containing chain CgA region gene in human immunoglobulin
3 μ7 of WsλB phage DNA was digested with EC0RI according to the method of Example 7, and subjected to agarose gel electrophoresis. A 2.6 Kb DNA fragment containing the gene in C.
It was recovered from agarose gel using electro-elution method. On the other hand, plasmid DBR322 for E. coli
1μ9 was cleaved with EcoR4 according to Example 7, and alkaline phosphatase (E, coli
Add 0.5 units of c75) (manufactured by Takara Shuzo) to 4
The reaction was carried out at 5°C for 1 hour. After the reaction was completed, phenol extraction was repeated three times to deactivate and remove alkaline phosphatase in the reaction solution. Obtained in this way [1
The EcoRI/alkaline phosphatase treatment solution of BR322 is mixed with 2 and 6 recovered from the gel with the aqueous EcoRI fragment solution, and dissolved in 50 μΩ of ligation reaction buffer (see Example 2) to simulate nitinol precipitation. Add 2 units of T4-DNA ligase and react at 11°C for 12 hours to prepare hybrid DNA (9).

Transformation of E. coli DH1 strain was carried out using the usual CaC1z method [M
, V., Norgard, K. ) (een a
nd J.

J. Monahai, Gene, 3. 21
9 (1978)]. That is,
5Id, Escherichia coli D in Noshi medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl H7,2)
An 18 hour culture of strain H1 is inoculated and grown to an optical density at 600 nm of 0.3. The bacterial cells were soaked in cold magnesium buffer'-[0,IM Na Cj-5m
M MgCfz -5+iMTris -HCl (
IJ H7,6,0°C)] and washed twice in 2Id cold calcium buffer - [100mMCa Cf2
-250mM KCf-5mM M<l Cjz
-5mM Tris -H(J (+187.6.0℃)
] and left at 0°C for 25 minutes. The cells were then concentrated to this volume of 1710 in calcium buffer and mixed with an aqueous hybrid DNA solution 2:1 (VOI,
:vol, ) mix. This mixture was heated at 0 for 60 minutes.
After keeping at °C, 1 d of LBG medium (1% tryptone.

0.0, S% yeast extract, 1% NaCl, 0.08% glucose, pH 7.2) and cultured at 37°C for 1 hour. The culture solution was mixed with selective medium (ampicillin 30 μg/d
Inoculate 100 μp/plate into a culture medium plate (containing medium). The plate is first cultured at 31°C to grow the transformed strain. DNA was prepared from the obtained colonies using a known method, and subjected to agarose gel electrophoresis.
The desired hybrid DNA was confirmed.

FIG. 3 shows the obtained subclone pYN-)-ILC
The restriction enzyme cleavage map of is shown.

Example 9 (Subcloning of chain V-J region gene into mouse immunoglobulin) Ch containing chain V-J region gene into mouse immunoglobulin
Aron28 phage DNA was cleaved with Hindl according to the method of Example 7 and subjected to agarose gel electrophoresis. By the same method as in Example 8, -J was added to V.
A 6.5Kb DNA fragment containing the gene was added to DBR32.
2 HindI [[cloned into the p YN
-MLV1 and pYN-MLV2 were created.

FIG. 4 shows the obtained subclone pYN-MLVl
, l) Restriction enzyme cleavage map of YN-MLV2 is shown. BamHI containing 31 genes in V-J (A-1>-Hln
Hind m (A-2) of the d m (A-2) fragment is ligated closer to the EC0RI site of is [)YN-MLVI.

The one with the reverse orientation is ρYN-MLV2.

Example 10 (Preparation of mouse-human chimeric antibody gene) Chain V-J was added to the mouse immunoglobulin obtained in Example 9.
[)YN-MLVI containing the region gene was digested with 3arnHI and EcoRI according to the method of Example 7, and subjected to agarose gel electrophoresis.

The resulting V contains the gene in -J 4.21 (ECO of b
The RI-BMHI fragment was prepared in the same manner as in Example 8.
DSV2neo vector [P, J, 5outh-
ern and p., Bera, J., Mo.
1. All[)I.

Genet, 1. 327 (1982)]
Insert between oRI-BamHI and create plasmid pSV2n.
eo-MLV was produced. This is shown in FIG.

Next, chain C was added to human immunoglobumin obtained in Example 8.
The o YN-HLC containing the region gene was cut with EOORI according to the method of Example 7, and subjected to agarose gel electrophoresis. The resulting 2.6 Kb DNA fragment containing the gene in C was transformed into the above plasmid DSV2ne using the same method as in Example 8.

- Inserted into the EC0RI site of MLV. Among the obtained clones, EC0RI (B-
1)-EcoRI (B-2> fragment of EcoRI (B
-1) is linked to mouse ■ to -J on the gene side (
Select a plasmid whose reading direction matches that of the mouse V-J gene and the human C gene, and insert this plasmid into F
) It was named SV2neO-MHL. This is shown in FIG.

On the other hand, plasmid pTJ3 containing the human immunoglobulin γ1 chain gene described in JP-A-60-120991,
According to the method described in Example 7, it was cut with MluI and HDaI, and agarose gel electrophoresis revealed that MluI contains the human γ1 chain gene enhancer region of about 0.9 Kb.
-H1)al fragment was obtained. This DNA fragment was added to polymerase buffer (37n+M potassium phosphate, 6.
7111M MC1C1z, 111M 2-
Dissolved in mercaptoethanol, D H7,4), d
In the presence of NTP, DNA polymerase Flenow (kl
enow)・Fragment to New England. The ends were blunted by adding Biolabs). Furthermore, after ligating Ba1RHI linkers (Takara Shuzo) to both ends using T4-DNA ligase, Bam
Cut with HI and perform agarose gel electrophoresis to obtain approximately 0.9
Ba containing the human γ1 inhibitory gene enhancer region of Kb
A mf-II fragment was obtained. This DNA fragment was inserted into the Bam1-II site of the above plasmid pSV2neo-MHL, and plasmids p5V2neo-MHLEI and [)SV2 with different insertion orientations were inserted.
neo-MHLE2 was produced.

This is shown in FIG.

[Brief explanation of drawings]

Attached Figure 1 shows the restriction enzyme cleavage map of the gene fragment <A) of the present invention, Figure 2 shows the restriction enzyme cleavage map of the gene fragment (B) of the present invention, and Figure 3 shows the restriction enzyme cleavage map of the gene fragment <A) of the present invention. Figure 4 shows the restriction enzyme cleavage map of the subclone pYN-MLV1,1)YNI-ILc obtained in Example 9 of the present invention.
Figure 5 shows a restriction enzyme cleavage map of N-LV2, and Figure 5 shows a plasmid restriction enzyme cleavage map obtained in an example of the present invention. Patent Applicant Teijin Stock Co., Ltd. Representative Patent Attorney 1) Juna
Figure 3 shows 0.5 Kbp Do-1→ Figure 4 Figure 5-b (Region 5-〇5 rifbu () Procedural city correct writing method) February 1986 Nre 1:1

Claims (1)

[Scope of Claims] 1. A gene fragment having a length of about 4.2 Kbp and containing the V region and J region of the mouse immunoglobulin κ chain gene, which is represented by the restriction enzyme cleavage map shown in Figure 1. 2. Approximately 2.6 containing the C region in the human immunoglobulin κ chain gene represented by the restriction enzyme cleavage map shown in Figure 2.
A gene fragment with a length of Kbp. 3. (i) A gene fragment (A) having a length of approximately 4.2 Kbp containing the V region and J region in the mouse immunoglobulin κ chain gene represented by the restriction enzyme cleavage map shown in FIG. (ii) Approximately 20% containing the C region in the human immunoglobulin κ chain gene represented by the restriction enzyme cleavage map shown in Figure 2.
．． Gene fragment (B) having a length of 6 Kbp and a new plasmid 4, (i ) V region and J in the mouse immunoglobulin κ chain gene shown in the restriction enzyme cleavage map shown in Figure 1.
A gene fragment with a length of approximately 4.2 Kbp including the region (
A), (ii) Approximately 2 cells containing the C region in the human immunoglobulin κ chain gene shown in the restriction enzyme cleavage map shown in Figure 2.
．． A gene fragment (B) having a length of 6 Kbp and (iii) a gene fragment (C) containing an enhancer base sequence in a human immunoglobulin H chain gene are introduced, and the gene fragments (A) and (B) are B-1 in the gene fragment (B) on the A-2 side in the gene fragment (A)
Novel plasmids introduced in side-aligned order.