JP4006511B2 - DNA amplification method, gene encoding repeated amino acid sequence - Google Patents

Description

【０１３１】
この表１より、O-型糖鎖の導入によって、糖鎖の構造に応じたレクチン反応性が認められ、このことから、FGF-1 分子に O-型糖鎖の機能を付与できたものと考えられた。
【０１３２】
〔試験例９〕
生物学的安定性の解析
試験例１で調製したムチンボックスを導入したＦＧＦ（ＣＭ１０）をＩＣＲマウス尾静脈血管内に投与し、一定時間ごとに尾を切断して、浸潤する血液を一定量採取した。これに血液凝固阻止因子を添加した後、ＳＤＳ変性電気泳動に供し、抗ＦＧＦ-１抗体を用いた免疫染色を行った。得られた結果を、デジタル化処理することで、血流中に存在するＦＧＦの量を定量解析した。
【０１３３】
結果を図９に示す。図９のAは、各時間毎に採取した試料を、免疫染色で解析した結果を示す。(a) は無処理のCM10、(b) はシアリダーゼ処理したCM10、(c) はシアリダーゼ処理後O-グリコシダーゼ処理したCM10の結果を示す。また、図９のBは、免疫染色の結果を定量的に解析した結果を、CM1 の結果を含めて示す。
【０１３４】
その結果、糖鎖を持たないCM10 分子を基準にすると、シアリル化されたO-型糖鎖を持つCM10 は血中滞在時間が延長し、シアリダーゼ処理によってシアル酸を除去したCM10は、逆に血中滞在時間が短縮することが判明した、これにより、糖鎖の構造を修飾することで、薬剤の体内安定性を制御できる可能性を示した。
【０１３５】
〔試験例１０〕
物理的安定性（非特異的吸着性）の解析
一般に、ＦＧＦ類縁タンパク質は疎水結合により、非特異的吸着が激しいことが知られている。試験例１で調製したムチンボックスを導入したＦＧＦ（CM10）は、糖鎖の導入により親水性の向上が図られているため、非特異的吸着の低減が予想された。
【０１３６】
そこで、ムチンボックスを導入したＦＧＦあるいは大腸菌で生産させたＦＧＦ−１（rec FGF）について、一定濃度(400 ng/μl) にそろえてガラス試験管に分注し、これを４℃、あるいは３７℃に保持し、経時的に一定量(100 μl)を採取した。採取した試料は−８０℃に急速冷凍し保存した。これらの試料について、抗ＦＧＦ−１抗体を用いたELISAによってＦＧＦ含量を定量解析した。
【０１３７】
結果を図１０に示す。図１０のAは４℃で保温した場合、図１０のBは37℃で保温した場合の結果である。いずれの温度においても糖鎖を導入したCM10 分子のガラス壁面への非特異的吸着は、単純蛋白質FGF-1 よりも低減していることが判明した。
【０１３８】
〔試験例１１〕
シアリルルイスＸ抗原の発現
実施例７で調製されたクローンにおいては、ムチンボックスを導入したＦＧＦがシアリルルイスＸ抗原を発現しているものと予想された。そこで、シアリルルイスＸ抗原の機能を解析する目的で、これをリガンドとするタンパク質であるＥ−セレクチンへの親和性を検討した。
【０１３９】
遺伝子組み換えによって生産した分泌型Ｅ−セレクチンをプレートに固定化し、実施例７の馴化培地を添加した。洗浄後、抗ＦＧＦ−１抗体を用いたELISA様の分析により、Ｅ−セレクチンに結合したムチンボックスを導入したＦＧＦを定量分析した。その結果、当該細胞が生産するＦＧＦは、Ｅ−セレクチンに対して親和性を有していることが確認された。
【０１４０】
【発明の効果】
以上、詳細に説明したように、本発明に係るＤＮＡ増幅方法は、通常のＰＣＲによって増幅が困難な塩基配列を確実に増幅することができる。特に、本方法によれば、通常のＰＣＲによって増幅が困難な塩基配列として、複数のアミノ酸からなるペプチドの繰り返し配列をコードする塩基配列を確実に増幅することができる。また、本方法では、特別な装置、器具及び試薬等を必要とせず、汎用されている装置、器具及び試薬のみを用いて所望の塩基配列を増幅することができる。本発明によれば、タンパク質の機能カセットを重複して作成することが可能となり、これによってタンパク質機能の大幅な改質に寄与することが可能となる。
【０１４１】
【配列表】

【０１４２】
【配列表フリーテキスト】
配列番号１は合成されたDNA断片である。
配列番号２はO-糖鎖付加配列のモチーフである。
配列番号３はムチンボックスをコードするAカセットである。
配列番号４はムチンボックスをコードするCカセットである。
配列番号５はムチンボックスをコードするGカセットである。
配列番号６はムチンボックスをコードするTカセットである。
配列番号７〜２０はプライマーである。
配列番号２１は合成されたDNA断片である。
配列番号２２、２３はプライマーである。
配列番号２４は合成されたDNA断片である。
配列番号２５、２６はプライマーである。
配列番号２７、３２、３３は合成されたDNA断片である。
配列番号３４はキメラタンパク質である。
配列番号３５〜４２はプライマーである。
配列番号４３は合成されたDNA断片である。
配列番号４４〜４６はプライマーである。
配列番号４７〜４９は合成されたDNA断片である。
配列番号５０はO-糖鎖付加配列のモチーフであり、第１番目及び第４番目の「Xaa」は任意のアミノ酸を示している。
【図面の簡単な説明】
【図１】分泌型 FGF の N-末端に導入するためのクラスター状ムチン型糖鎖付加配列をコードする cDNA の合成戦略の模式図である。
【図２】分泌型 FGF の N-末端に導入するためのクラスター状ムチン型糖鎖付加をコードする cDNA の合成過程におけるアガロースゲル電気泳動の写真である。
【図３】分泌型 FGF の C-末端に導入するためのクラスター状ムチン型糖鎖付加をコードする cDNA の合成戦略の模式図である。
【図４】分泌型 FGF の N-末端、C-末端にクラスター状ムチン型糖鎖付加を導入した分泌型FGF-1（CM10、NM10）の模式図である。
【図５】 CM10、NM10のSDS-PAGE 電気泳動の写真、および、各種酵素処理したCM10、NM10のSDS-PAGE 電気泳動の写真である。
【図６】 CM10、NM10のヒト臍帯静脈由来血管内皮細胞に対するDNA合成促進活性を示す特性図である。
【図７】 CM10及びこれを各種グリコシダーゼ処理した試料のヘパリンセファロースカラムからの溶出パターンを示すSDS-PAGE 電気泳動の写真である。
【図８】 CM10の各種蛋白質分解酵素に対する抵抗性試験の結果を示すSDS-PAGE 電気泳動の写真である。
【図９】CM10のマウス血中での安定性試験の結果を示す特性図である。
【図１０】 CM10の非特異的吸着性に関する評価試験の結果を示す特性図である。[0001]
BACKGROUND OF THE INVENTION
The present invention, for example, a DNA amplification method for amplifying a nucleotide sequence that is difficult to amplify by ordinary PCR, such as a gene encoding a repeated sequence of a peptide consisting of a plurality of amino acids, by the method, The present invention relates to an amplified gene and a protein obtained by expressing the gene.
[0002]
[Prior art]
The PCR (polymerase chain reaction) method is an important technique indispensable in the field of molecular biology. According to the PCR method, it is possible to easily amplify a target gene even from a very small amount of a gene sample, and it is also possible to create an artificial gene sequence. However, as is clear from the principle, it is difficult to amplify a sequence containing many homologous sequences by the PCR method, and there has been almost no report on the synthesis of an artificial gene encoding a peptide sequence containing repeats. As this cause, it is conceivable that the primer used causes misannealing with respect to the template DNA.
[0003]
The only reported example is a method in which primers are immobilized on beads and PCR is performed on a solid phase (Kenneth E. Dombrowski, and Stephen E. Wright, Nucleic Acid Research, 1992, Vol. 20, No. 24 6743). -6744). However, this technique is hardly used because it requires complicated operations and requires an operation different from the protocol of the normal PCR method. Even when this method is used, there have been no reports of high frequency and high density repetitions to date.
[0004]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a method for amplifying a DNA fragment that has been difficult to amplify by equipment and techniques widely used in ordinary PCR methods, and in particular, a DNA encoding a peptide repeat sequence. It aims at providing the method of amplifying a fragment.
[0005]
[Means for Solving the Problems]
In the DNA amplification method according to the present invention that achieves the above-described object, a pair of primers are designed so as to sandwich the first region in the base sequence to be amplified and to anneal to each other at the 3 ′ end, and these pairs of primers are used. By performing a PCR reaction, the step of synthesizing the first region (hereinafter referred to as the first step), the second region partially overlapping the first region of the base sequence, and the 3 ′ end A pair of primers are designed to anneal with each other, and a PCR reaction is performed using the pair of primers to synthesize the second region (hereinafter referred to as the second step), and these steps are synthesized. A step of synthesizing a base sequence including the first region and the second region by performing a PCR reaction using the annealed first region and the second region as a template ( Lower, it is characterized in that it has a third step) and.
[0006]
In the first step, in the PCR reaction, the amplification reaction is performed by annealing the 3 ′ terminal sides of each primer. That is, in the first step, one primer uses the other primer as a template, and the other primer performs an amplification reaction using one primer as a template. Thus, in the first step, the first region sandwiched between the pair of primers can be amplified. Similarly, in the second step, one primer can use the other primer as a template and the other primer can amplify the second region by performing an amplification reaction using one primer as a template.
[0007]
In the third step, a PCR reaction is performed using the first region amplified in the first step and the second region amplified in the second step as a template. Here, since the first region and the second region have overlapping regions, annealing is performed in this region in the PCR reaction. In the third step, a desired base sequence including the first region and the second region can be amplified by performing a PCR reaction using an arbitrarily set primer.
[0008]
Further, the DNA amplification method according to the present invention may be a gene in which the base sequence to be amplified encodes a repeated sequence of a peptide composed of a plurality of amino acids.
Furthermore, in the DNA amplification method according to the present invention, the repeating sequence of the peptide may be a sugar chain acceptor.
[0009]
Furthermore, in the DNA amplification method according to the present invention, the sugar chain acceptor may be a mucin-type sugar chain acceptor. By using a sugar chain acceptor as a repeat peptide sequence as a biologically meaningful mucin-type sugar chain acceptor, a basic technology for introducing an additional sequence of a clustered mucin-type sugar chain can be established.
Furthermore, in the DNA amplification method according to the present invention, it is preferable that the pair of primers includes a base sequence designed to encode the amino acid sequence of the repeated peptide with different degenerate codons.
[0010]
On the other hand, the gene according to the present invention encodes a protein that has been amplified by any of the DNA amplification methods described above and has a function as an acceptor of a sugar chain.
Further, the gene according to the present invention is obtained by introducing a gene encoding the above protein into the 3 ′ end and / or 5 ′ end of a gene encoding another protein, or at any place in the protein coding region. It preferably encodes a protein containing a region having a function as a chain acceptor. Here, Xaa-Thr-Pro-Xaa-Pro represented by Ala-Thr-Pro-Ala-Pro (SEQ ID NO: 2) is a protein having a function as an acceptor of the sugar chain (SEQ ID NO: 50, Xaa is arbitrary) It is preferable to include a repetitive sequence of a peptide generalized by
[0011]
Furthermore, the gene according to the present invention may be a growth factor in which the other protein has heparin-binding properties.
Furthermore, the recombinant expression vector according to the present invention has any of the above genes.
Furthermore, the transformant according to the present invention is obtained by transforming using a recombinant expression vector.
[0012]
Furthermore, the method for producing a glycoprotein according to the present invention comprises culturing or cultivating the transformant, and collecting the glycoprotein from the obtained culture or cultivated product.
Furthermore, the glycoprotein of the present invention is obtained by expressing the above gene.
Furthermore, the pharmaceutical composition according to the present invention comprises the glycoprotein as an active ingredient.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
In the following description, a gene encoding a “repeat sequence of a peptide serving as an acceptor of a mucin-type sugar chain”, which is an example of a gene encoding a peptide repeat sequence, is amplified, and the amplified gene encodes another protein. An example of introduction into a gene will be described. However, the DNA amplification method according to the present invention is not limited to the method of amplifying the base sequence, and is applied when amplifying a DNA base sequence that is difficult to amplify by a general PCR method. Examples of a DNA base sequence that is difficult to amplify by a general PCR method include those that have a repetitive sequence in the base sequence and that the primer used is easy to misanneal.
[0014]
1. Amplification of the gene encoding the peptide repeat sequence
1) Base sequence design
As an example of a gene encoding a peptide repeat sequence, a gene encoding an amino acid sequence (for example, 10 repeats) obtained by repeating a “peptide sequence serving as an acceptor of a mucin-type sugar chain” is designed.
[0015]
First, for example, the base sequence described in SEQ ID NO: 1 is designed. That is, as shown in FIG. 1, this base sequence is added to a region that repeatedly encodes the amino acid sequence of a peptide that serves as an acceptor of a mucin-type sugar chain (hereinafter referred to as a mucin region) and to the 5 ′ end of the mucin region. And a region encoding the N-terminal side of fibroblast growth factor (hereinafter referred to as FGF (fibroblast growth factor)) on the 3 ′ end side of the mucin region. In addition, this base sequence has sequences cleaved by a restriction enzyme such as Eco RI on the 5 ′ end side and 3 ′ end side.
[0016]
In addition, the amino acid sequence of the peptide serving as the acceptor of the mucin-type sugar chain is Xaa-Thr-Pro-Xaa-Pro (SEQ ID NO: 50, Xaa is arbitrary as in Ala-Thr-Pro-Ala-Pro (SEQ ID NO: 2)). And an O-linked sugar chain-modified sequence that serves as an action site for N-acetylgalactosamyltransferase-I. The base sequence represented by SEQ ID NO: 1 is designed to encode the amino acid sequence of a peptide that repeats Ala-Thr-Pro-Ala-Pro (SEQ ID NO: 2) 10 times.
[0017]
Here, Ala, Thr and Pro are all defined by the first base and the second base of the codon. Therefore, the third base can be arbitrarily determined in the codons of Ala, Thr and Pro. For example, the base sequence encoding Ala-Thr-Pro-Ala-Pro includes gcaacaccagcacca (SEQ ID NO: 3) as A cassette, gccacccccgccccc (SEQ ID NO: 4) as C cassette, gcgacgccggcgccg (SEQ ID NO: 5) as G cassette and T cassette As gctactcctgctcct (SEQ ID NO: 6). Then, using these A cassette, C cassette, G cassette and T cassette, the mucin region in the base sequence represented by SEQ ID NO: 1 is designed as shown in FIG. In the mucin region in FIG. 1, [A], [C], [G], and [T] indicate an A cassette, a C cassette, a G cassette, and a T cassette, respectively.
[0018]
2) Primer design
In order to amplify the base sequence represented by SEQ ID NO: 1 by the PCR method, a primer represented by an arrow in FIG. 1 is designed. For example, a pair of primers that sandwich the first half of the base sequence (referred to as the first region) and overlap by about 10 mer on the 3 ′ end side, and a second half of the base sequence (referred to as the second region) are sandwiched, A pair of primers overlapping about 10 mer on the 3 ′ end side is designed. Note that the first region and the second region are defined so as to overlap each other by about 20 mer.
[0019]
Specifically, primer # 225 (SEQ ID NO: 7) and primer # 235 (SEQ ID NO: 8) are designed as a pair of primers that sandwich the first region, and primer # is used as a pair of primers that sandwich the second region. 226 (SEQ ID NO: 9) and primer # 236 (SEQ ID NO: 10) are designed.
[0020]
Primer # 235 is the same sequence as the first to 61st bases in SEQ ID NO: 1, and primer # 225 is a sequence complementary to the sequence from the 52nd base to 106th base in SEQ ID NO: 1. . Therefore, primer # 225 and primer # 235 have base sequences complementary to each other on the 3 ′ end side. Primer # 226 has the same sequence as bases 85 to 139 in SEQ ID NO: 1, and primer # 236 is a sequence complementary to the sequence from base 130 to base 207 in SEQ ID NO: 1. . Therefore, primer # 226 and primer # 236 have base sequences complementary to each other on the 3 ′ end side.
[0021]
3) PCR
In this method, two-stage PCR is performed to amplify the gene represented by SEQ ID NO: 1. In the first-stage PCR, only the first region is amplified using primer # 225 and primer # 235, and only the second region is amplified using primer # 226 and primer # 236. In other words, in the first stage PCR, the first step of amplifying only the first region and the second step of amplifying only the second region are performed independently of each other. In the second stage PCR, the entire gene represented by SEQ ID NO: 1 is amplified using the first and second regions amplified in the first stage PCR as templates.
[0022]
In the first step in the first stage, a PCR reaction is performed according to a normal protocol except that no template DNA is present. That is, in the first cycle of the PCR reaction in the first step, primer # 225 and primer # 235 are annealed to each other on the 3 ′ end side, and thereafter, extension reaction from primer # 235 is performed using primer # 225 as a template. The extension reaction from primer # 225 is performed using primer # 235 as a template.
[0023]
In the second and subsequent cycles of the PCR reaction in the first step, an extension reaction is performed in the same manner as in the first cycle, or an extension reaction is performed using an already synthesized fragment as a template. Also at this time, since primer # 225 and primer # 235 are used, misannealing is prevented and only the first region can be amplified.
[0024]
In the second step, as in the first step, a PCR reaction is performed according to a normal protocol except that no template DNA is present. That is, in the first cycle of the PCR reaction in the second step, primer # 226 and primer # 236 are annealed to each other on the 3 ′ end side, and then an extension reaction from primer # 236 is performed using primer # 226 as a template. An extension reaction from primer # 226 is performed using primer # 236 as a template.
[0025]
In the second and subsequent cycles of the PCR reaction in the second step, an extension reaction is performed in the same manner as in the first cycle, or an extension reaction is performed using an already synthesized fragment as a template. Also at this time, since primer # 226 and primer # 236 are used, misannealing is prevented and only the second region can be amplified.
[0026]
In the second-stage PCR, the first region and the second region are used as template DNAs, and primer # 237 (SEQ ID NO: 11) and primer # 238 (SEQ ID NO: 12) indicated by arrows in FIG. Perform each reaction according to the protocol. Here, since the first region and the second region are defined so as to overlap by about 20 mer, annealing is performed at the overlapping portion. The entire base sequence represented by SEQ ID NO: 1 can be amplified by using primer # 237 and primer # 238. Hereinafter, the amplified base sequence is referred to as “amplified fragment”.
[0027]
In these first and second stage PCRs, it is preferable to use a DNA polymerase having a high proofreading activity. Thus, even if an unexpected misanneal occurs, it can be corrected by the 3 ′ exonuclease activity of the DNA polymerase.
[0028]
On the other hand, in the first-stage PCR and the second-stage PCR, it is preferable to perform optimization by appropriately setting the temperature conditions, time conditions, and number of reaction cycles in each of the DNA denaturation reaction, annealing reaction, and extension reaction.
Thus, according to this method, a gene encoding a peptide repeat sequence can be amplified by performing two-step PCR using the primers designed in 2) above. In particular, a gene encoding a peptide repeatedly containing a biologically significant mucin-type glycosylation sequence as its amino acid sequence can be amplified by the method described above. Thereby, a basic technology for introducing an additional sequence of a clustered mucin-type sugar chain can be established.
[0029]
2. Above 1. Of amplified fragments prepared in step 1 into other genes
Examples of a method for introducing an amplified fragment into another gene include the method described in T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p239 (1982). By introducing the amplified fragment into another gene according to this method, a linked gene obtained by linking the amplified fragment to another gene can be prepared.
[0030]
As the other gene, a gene encoding a protein secreted through a normal secretion pathway is preferable. Examples of this protein include factors belonging to the FGF family, closely related factors, or other proteins having heparin-binding properties but no structural similarity to the former. Specific examples of other proteins described herein include, but are not limited to, heparin-binding epidermal growth factor-like factor (HB-EGF) and platelet-derived growth factor (PDGF). . As specific examples of factors belonging to the FGF family or closely related factors, FGF-1 to 23 are known.
[0031]
By introducing the amplified fragment into another gene, a peptide repeat sequence can be introduced into another protein. At this time, by appropriately setting the position at which the amplified fragment is introduced, a repetitive peptide sequence can be introduced at a predetermined position in another protein.
[0032]
When the amplified fragment shown in SEQ ID NO: 1 is introduced, a region encoding the N-terminal side of FGF is added to the 3 ′ terminal side, so that the amplified fragment is converted into a gene encoding FGF (hereinafter referred to as the following procedure). , Referred to as the FGF gene).
[0033]
That is, a PCR reaction is performed using the amplified fragment (SEQ ID NO: 1) and the FGF gene (SEQ ID NO: 31) as a template, for example, using # 237 (SEQ ID NO: 11) and # 630 (SEQ ID NO: 16) as primers. In this PCR reaction, a region encoding the N-terminus of FGF is present on the 3 ′ end side of the amplified fragment, and this region and the region encoding the N-terminus of FGF are annealed. Then, the amplified fragment and the FGF gene can be linked by synthesizing full-length DNA by an extension reaction from these primers.
[0034]
In addition, since the amplified fragment represented by SEQ ID NO: 1 has a region encoding a part of the secretory signal sequence on the 5 ′ end side, the amplified fragment is converted into a gene encoding the secretory signal sequence according to the following procedure. (Hereinafter referred to as a secretory signal gene).
[0035]
That is, a PCR reaction is performed using the amplified fragment (SEQ ID NO: 1) and the secretory signal gene (SEQ ID NO: 30) as a template, for example, using # 105 (SEQ ID NO: 17) and # 238 (SEQ ID NO: 12) as primers. In this PCR reaction, a region encoding the C-terminus of the secretory signal sequence is present on the 5 ′ end side of the amplified fragment, and this region and the region encoding the C-terminus of the secretory signal sequence are annealed. Then, the amplified fragment and the secretory signal gene can be linked by synthesizing full-length DNA by an extension reaction from these primers.
[0036]
Furthermore, since the linking gene between the amplified fragment and the FGF gene and the linking gene between the amplified fragment and the secretory signal gene obtained here have the amplified fragment as a common region, using these two linked genes as a template, for example, By performing a PCR reaction using # 105 (SEQ ID NO: 17) and # 630 (SEQ ID NO: 16) as primers, a gene in which a secretory signal gene, an amplified fragment, and an FGF gene are linked can be prepared.
[0037]
By adding a part of the 3 ′ end of the FGF gene to the 5 ′ end of the amplified fragment, the amplified fragment can be linked to the 3 ′ end of the FGF gene according to the above-described method (see FIG. 3). .
The obtained linked gene can be replicated and maintained in the host by introducing it into a vector such as a commonly used plasmid. Any vector can be used as long as it can be replicated and maintained in the host. Examples thereof include pBR322 and pUC18 derived from E. coli, and pET-3c constructed based on these.
[0038]
3. Expression of amplified genes
1) Construction of expression vector
2. An expression vector can be constructed by ligating the ligated gene obtained in (1) downstream of a promoter in a vector suitable for expression. When constructing an expression vector, a mucin region and a region containing a base sequence encoding another protein (hereinafter referred to as “region containing a base sequence encoding a mucin-type glycosylated protein”) are cut out. By linking this to the downstream of the promoter in a vector suitable for expression, an expression vector can be obtained.
[0039]
A region containing a base sequence encoding a mucin-type glycosylated protein has ATG as a translation initiation codon at the 5 ′ end and TAA, TGA or TAG as a translation termination codon at the 3 ′ end. May be. Furthermore, in order to express the protein encoded by the region, a promoter is connected upstream thereof. The promoter may be any promoter as long as it is appropriate for the host used for gene expression. When the host to be transformed is Bacillus subtilis, SP01 promoter, SP02 promoter, penP promoter and the like, and when the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like can be mentioned. When the host is an animal cell, examples include SV40-derived promoters and retrovirus promoters.
[0040]
Any plasmid can be used as a plasmid into which a recombinant DNA having a base sequence encoding a mucin-type glycosylated protein constructed in this way can be replicated and maintained in a host cell. Examples thereof include vectors constructed based on pBR322, pUC18, etc. derived from E. coli. Examples of the method for incorporation into a plasmid include the method described in T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p. 239 (1982).
[0041]
2) Preparation of transformant
A transformant can be obtained by introducing the expression vector constructed in 1) above into an appropriate host cell. The host cell may be any host cell having a glycosylation pathway, such as Bacillus subtilis (eg, Bacillus subtilis DB105), yeast (eg, Pichia pastoris, Saccharomyces cerevisiae), animal cell (eg, COS cell, CHO cell, BHK cell, NIH3T3 cell, BALB / c3T3 cell, HUVE cell, LEII cell), insect cell (for example, Sf-9 cell, Tn cell) and the like can be exemplified.
[0042]
The transformation may be performed by a method generally performed for each host cell or any method that can be applied. For example, if the host cell is yeast, the yeast can be made into a competent cell by the lithium method or other methods, and the constructed expression vector can be introduced by the temperature shock method or electroporation method. If the host cell is an animal cell, the constructed expression vector can be introduced into cells in the growth phase or the like by the calcium phosphate method, lipofection method or electroporation method.
[0043]
4). Creation of repetitive amino acid sequence-added proteins
1) Production of repetitive amino acid sequence-added proteins
For example, a mucin-type glycosylated protein is produced by culturing the transformant obtained as described above in a medium. When the transformant is cultured, a medium generally used for each host is used as the medium used for the culture. Or, if it is not general, it may be an applicable medium. As an example, if the host is yeast, YPD medium or the like is used. If the host is an animal cell, use Dulbecco's MEM supplemented with animal serum. Culturing is performed under conditions generally used for each host. Moreover, it is sufficient if the condition is applicable even if it is not general. As an example, if the host is yeast, it is carried out at about 25 to 37 ° C. for about 12 hours to 2 weeks, and if necessary, aeration or stirring can be added. If the host is an animal cell, at about 32-37 ° C, 5% CO ₂ It can be performed at 100% humidity for about 24 hours to 2 weeks. If necessary, the gas phase conditions can be changed and stirring can be added.
[0044]
In order to obtain a mucin-type glycosylated protein from the culture of the transformant as described above, the protein released into the culture solution can be directly recovered from the supernatant after centrifugation. In addition, when extracting from cultured cells or cells, the target protein is removed from the cells by culturing the cells or cells by homogenizer, French press, ultrasonic wave, lysozyme and / or freeze-thawing. And the protein can be obtained from the soluble fraction. In addition, when the target protein is contained in the insoluble fraction, a method in which the cells or cells are destroyed, the insoluble fraction is collected by centrifugation, and is made soluble by a buffer solution containing guanidine hydrochloride or the like and collected. In addition, there is a method in which cells or cells are directly destroyed with a buffer solution containing a protein denaturant such as guanidine hydrochloride, and the target protein is eluted outside the cells.
[0045]
In order to purify the mucin-type glycosylated protein from the supernatant, known separation / purification methods can be combined appropriately. These known separation and purification methods include salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, Isoelectric focusing etc. can be used. Furthermore, when the mucin-type glycosylated protein has heparin-binding properties, an affinity chromatography method using heparin sepharose as a carrier can be applied.
[0046]
As long as the activity of the mucin-type glycosylated protein thus obtained is not impaired, dialysis and lyophilization can be performed to obtain a dry powder. Furthermore, adding serum albumin or the like as a carrier for storage is effective for preventing adsorption of mucin-type glycosylated proteins to the container. The coexistence of a trace amount of a reducing agent in the purification process or storage process is suitable for preventing the oxidation of mucin-type glycosylated proteins. Examples of the reducing agent include β-mercaptoethanol, dithiothreitol, glutatin and the like.
[0047]
2) Addition of sugar chain
Mucin-type glycosylated proteins can also be linked to sugar chains by chemical methods. As a specific method, a method based on any one of the following a) and b) or a combination thereof may be considered.
[0048]
a). First, these sugar chains are completed by a biological method, a chemical synthesis method, or an appropriate combination thereof. At that time, an appropriate protein-binding residue can be introduced at the sugar chain terminal. For example, an aldehyde group is formed by reducing and partially oxidizing the reducing end of the completed sugar chain, and this is bonded to an amino group in the protein to form an amide bond, thereby completing the binding between the sugar chain and the protein.
[0049]
b). First, an aldehyde group is formed by reducing and partially oxidizing the reducing end of a monosaccharide or an appropriate protein-binding residue bound to the monosaccharide, and by forming an amide bond with an amino group in the protein, The combination of monosaccharide and protein is completed. A sugar chain is completed by binding a further monosaccharide or sugar chain to a functional group such as a hydroxyl group of this monosaccharide. For this binding, a biological method, a chemical synthesis method, a method in which these are appropriately combined, or the like can be considered.
[0050]
3) Use of the produced mucin-type glycosylated protein
The novel mucin-type glycosylated protein obtained in 2) above is given a new function by addition of a sugar chain while retaining the physiological activity inherent in the protein itself. Specifically, modification of in vivo kinetics due to high molecular weight resulting from glycosylation, selective distribution of organs in vivo by glycan recognition proteins in vivo, and tissue specific by presentation of bioactive glycan epitopes It can be applied to targeting.
[0051]
Furthermore, the obtained mucin-type glycosylated protein is excellent in stability such as heat resistance, acid resistance, alkali resistance, and proteolytic enzyme resistance, and non-specific adsorption can be reduced by increasing hydrophilicity. Expected to bring. Therefore, by using mucin-type glycosylated proteins for pharmaceuticals, before administration to living bodies, physical stability such as acid resistance and alkali resistance is improved, and loss prevention by reducing adsorption to containers is prevented. In addition, after administration to a living body, it can improve biological stability and can be applied to organ / tissue-specific targeting and pharmacokinetic modification.
[0052]
Furthermore, among mucin-type glycosylated proteins, those having heparin-binding properties (hereinafter referred to as “mucin-type glycosylated heparin-binding proteins”) are enhanced by covalently binding sugar chains. Therefore, it can be used as a medicine. Here, high function means that the activity of the target protein is improved. An example of high functionality is that by covalently binding a sugar chain to a protein, the activity remaining after treatment with heat, acid and alkali is higher than that of a protein not having a sugar chain covalently bonded. It is done.
[0053]
For example, a clustered mucin-type sugar chain added to FGF has an effect of regulating the physiological function of FGF. The physiological function of FGF specifically refers to promoting or suppressing the proliferation of fibroblasts, vascular endothelial cells, myoblasts, chondrocytes, osteoblasts, and glial cells. Therefore, mucin-type glycosylated FGF is effective for promoting cell growth and regeneration of tissues such as liver, wound healing, nerve function regulation, and fibroblast growth regulation, and various diseases such as fiber It is useful for the prevention and treatment of blastoma, hemangioma, osteoblastoma, neuronal death, Alzheimer's disease, Parkinson's disease, neuroblastoma, amnesia, dementia, myocardial infarction, and as a hair growth agent, hair restorer, etc. Is also available.
[0054]
The mucin-type glycosylated heparin-binding protein obtained as described above uses pharmaceutically acceptable solvents, excipients, carriers, adjuvants and the like, and in accordance with conventional methods for preparation of preparations, solutions and lotions And pharmaceutical compositions such as aerosols, injections, powders, granules, tablets, suppositories, intestinal solvents and capsules. In the pharmaceutical composition, the content of the mucin-type glycosylated heparin-binding protein that is an active ingredient may be about 0.0000000001 to 1.0% by weight. The pharmaceutical composition can be safely administered parenterally or orally to mammals such as humans, mice, rats, rabbits, dogs and cats. The dosage of the pharmaceutical composition may be appropriately changed depending on the dosage form, administration route, symptoms, etc., but when administered to mammals including humans, for example, mucin glycosylated heparin-binding protein is added in an amount of 0.0001-100 mg. It is exemplified that it is applied to the affected area several times a day.
[0055]
In the above-described example, the heparin-binding protein is described as an example, but other natural proteins can be enhanced by covalently binding mucin sugar chains. In the above description, cDNAs encoding clustered mucin-type glycosylated sequences with various repeat frequencies can be prepared by changing the primers used for the PCR reaction to appropriate sequences. In addition, in the above description, in the cloning described in “1. Amplification of a gene encoding a repetitive amino acid sequence”, not only a target-size clone but also a minor by-product is selected and cloned, whereby various repetitive operations are performed. A cDNA encoding a frequency clustered mucin-type glycosylation sequence can be prepared. Examples of a gene encoding a protein containing such a cDNA sequence are shown in SEQ ID NO: 48 and SEQ ID NO: 49.
[0056]
【Example】
The present invention will be described more specifically with reference to the following examples. However, these examples are for illustrative purposes and do not limit the technical scope of the present invention.
[0057]
[Example 1]
Synthesis of a cDNA encoding the amino acid sequence of a peptide serving as an acceptor of a mucin-type sugar chain introduced into the N-terminus of secretory FGF (hereinafter referred to as “mucin box”) (see FIG. 1)
1) Synthesis of the first half of cDNA encoding mucin box
PCR (Polymerase Chain Reaction: Polymerase chain reaction) was used in a reaction system in which # 225 (SEQ ID NO: 7) and # 235 (SEQ ID NO: 8) were used as primers in the absence of template, and each of them was present at a concentration of 8 pmol / μl. ) As the reaction conditions, a cycle of 94 ° C. (30 seconds), 68 ° C. (30 seconds), and 72 ° C. (30 seconds) was repeated 30 times. The reaction products were separated by agarose gel electrophoresis (FIG. 2, lane 1), and a specifically amplified 106 base pair band was extracted and purified by ethanol precipitation. The obtained DNA fragment contains the base sequence shown in SEQ ID NO: 1.
[0058]
2) Synthesis of the second half of cDNA encoding mucin box
In the absence of template, # 226 (SEQ ID NO: 9) and # 236 (SEQ ID NO: 10) were used as primers, and PCR was performed in a reaction system in which these were present at a concentration of 8 pmol / μl, respectively. As reaction conditions, a cycle of 94 ° C. (30 seconds), 68 ° C. (30 seconds), and 72 ° C. (30 seconds) was repeated 30 times. The reaction products were separated by agarose gel electrophoresis (FIG. 2, lane 2), and a specifically amplified 123 base pair band was extracted and purified by ethanol precipitation. The obtained DNA fragment contains the base sequence shown in SEQ ID NO: 1.
[0059]
3) Synthesis of full-length cDNA encoding mucin box
The DNA fragments (106 base pair fragments and 123 base pair fragments) obtained in 1) and 2) above were mixed and used as templates, and primers # 237 (SEQ ID NO: 11) and # 238 (SEQ ID NO: 12) PCR was performed in a reaction system in which each of them was present at a concentration of 8 pmol / μl. As the reaction conditions, a cycle of 94 ° C. (30 seconds), 60 ° C. (30 seconds), and 72 ° C. (30 seconds) was repeated 30 times. The reaction products were separated by agarose gel electrophoresis (FIG. 2, lane 3), and a specifically amplified 207 base pair band was extracted and purified by ethanol precipitation. The obtained DNA fragment has the base sequence shown in SEQ ID NO: 1.
[0060]
This 207 base pair fragment encodes the N-terminal side of the FGF as a sequence for introduction into the N-terminal of the FGF-1 portion of the secretory FGF at the 3 ′ end of the coding strand encoding the mucin box. The base sequence to be inserted is inserted. In addition, this 207 base pair fragment is used as a sequence for introduction into the N-terminus of the FGF-1 portion of the secretory FGF at the 5 ′ end of the coding strand encoding the mucin box. A base sequence encoding the C-terminal side is inserted. The 207 base pair fragment has Eco RI sites inserted at both ends as restriction enzyme sites for incorporation into the vector.
[0061]
4) Cloning of cDNA encoding mucin box
The DNA fragment obtained in 3) above was completely digested with Eco RI, and the PCR product was end-treated. This reaction product was purified using a DNA fragment purification kit (Roche Diagnostics High Pure PCR Product Purification Kit). The obtained purified DNA fragment was mixed with a vector pretreated with Eco RI and CIAP, pBluescript II (KS +), and subjected to a DNA ligation reaction. Using this reaction product, E. coli strain DH5α was transformed. The obtained transformant was seeded on an agarose medium in the presence of ampicillin, X-Gal, and IPTG, and primary colonies were selected by selecting the grown white colonies.
[0062]
By using the selected colonies and performing direct PCR using vector primers # 115 (SEQ ID NO: 19) and # 116 (SEQ ID NO: 20), selecting a clone that gives the expected 295 base pair band, Secondary screening was performed.
The colony obtained by the secondary screening was liquid-cultured in ampicillin-containing LB medium, and the plasmid was roughly purified from the grown cells by the alkaline method. This plasmid was digested with Eco RI, and the excised DNA fragment was analyzed by agarose gel electrophoresis. A clone giving a band of 195 base pairs, which is the target size, was selected for the tertiary screening. For several selected clones, the plasmid was purified again, and the inserted sequence of the plasmid was decoded by cycle sequencing.
[0063]
A clone with a sequence that matches the expected sequence was selected and named mucx10@N-ter./pBS/cl.9/000524. Research Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology, May 29, 2000 Is deposited at FERM P-17881. The DNA sequence of this clone is shown in SEQ ID NO: 21. Moreover, the result of digesting the plasmid of this clone with Eco RI and analyzing the excised DNA fragment by agarose gel electrophoresis is shown in “FIG. 2, lane 4”.
[0064]
[Example 2]
Synthesis of cDNA encoding mucin box to be introduced into C-terminal of secretory FGF (see Fig. 3)
1) Synthesis of the first half of cDNA encoding mucin box
In the absence of the template, PCR was performed in a reaction system using # 224 (SEQ ID NO: 22) and # 225 (SEQ ID NO: 23) as primers and present at a concentration of 8 pmol / μl, respectively. As the reaction conditions, a cycle of 94 ° C. (30 seconds), 55 ° C. (30 seconds), and 72 ° C. (30 seconds) was repeated 25 times. The reaction product was separated by agarose gel electrophoresis, and a specifically amplified 110 base pair band was extracted and purified by ethanol precipitation. The obtained DNA fragment contains the base sequence shown in SEQ ID NO: 24.
[0065]
2) Synthesis of the second half of cDNA encoding mucin box
In the absence of template, # 226 (SEQ ID NO: 9) and # 227 (SEQ ID NO: 23) were used as primers, and PCR was performed in a reaction system in which these were each present at a concentration of 8 pmol / μl. As the reaction conditions, a cycle of 94 ° C. (30 seconds), 55 ° C. (30 seconds), and 72 ° C. (30 seconds) was repeated 25 times. The reaction product was separated by agarose gel electrophoresis, and a 107 bp band specifically amplified was extracted and purified by ethanol precipitation. The obtained DNA fragment contains the base sequence shown in SEQ ID NO: 24.
[0066]
3) Synthesis of full-length cDNA encoding mucin box
The DNA fragments obtained in 1) and 2) (110 base pair DNA fragments and 107 base pair DNA fragments, respectively) were mixed and used as templates, and primers # 230 (SEQ ID NO: 25) and # 231 (sequences) No. 26) was used, and PCR was performed in a reaction system in which these were present at a concentration of 8 pmol / μl. As the reaction conditions, a cycle of 94 ° C. (30 seconds), 50 ° C. (30 seconds), and 72 ° C. (30 seconds) was repeated 25 times. The reaction product was separated by agarose gel electrophoresis, and a specifically amplified 195 base pair band was extracted and purified by ethanol precipitation. The obtained DNA fragment has the base sequence shown in SEQ ID NO: 24.
[0067]
In this 195 base pair fragment, the base sequence encoding the C-terminal side of the FGF is inserted into the 5 ′ end of the coding strand encoding the mucin box as a sequence for introduction into the C-terminal of the secretory FGF. Has been. In this 195 base pair fragment, a base sequence for instructing the end of protein translation is inserted at the 3 ′ end of the coding strand encoding the mucin box. Further, this 195 base pair fragment has Eco RI sites inserted at both ends as restriction enzyme sites for incorporation into the vector.
[0068]
4) Cloning of cDNA encoding mucin box
The DNA fragment obtained in 3) above was completely digested with Eco RI, and the PCR product was end-treated. This reaction product was purified using a DNA fragment purification kit (Roche Diagnostics High Pure PCR Product Purification Kit). The obtained purified DNA fragment was mixed with a vector pretreated with Eco RI and CIAP, pBluescript II (KS +), and subjected to a DNA ligation reaction. Using this reaction product, E. coli strain DH5α was transformed. The obtained transformant was seeded on an agarose medium in the presence of ampicillin, X-Gal, and IPTG, and primary colonies were selected by selecting the grown white colonies.
[0069]
By using the selected colonies and performing direct PCR using vector primers # 115 (SEQ ID NO: 19) and # 116 (SEQ ID NO: 20), selecting a clone that gives the expected 283 base pair band, Secondary screening was performed.
[0070]
The colony obtained by the secondary screening was liquid-cultured in ampicillin-containing LB medium, and the plasmid was roughly purified from the grown cells by the alkaline method. This plasmid was digested with Eco RI, and the excised DNA fragment was analyzed by agarose gel electrophoresis. A clone giving a band of 183 base pairs, which is the target size, was selected and used as the tertiary screening. For several selected clones, the plasmid was purified again, and the inserted sequence of the plasmid was decoded by cycle sequencing.
[0071]
A clone with a sequence that matches the expected sequence was selected and named mucx10@C-ter./pBS/cl.I2-3/000524. On May 29, 2000, Biotechnology Industry Deposited at the Technical Research Institute at FERM P-17882. The DNA sequence of this clone is shown in SEQ ID NO: 27.
[0072]
[Example 3] Synthesis of cDNA encoding secreted FGF
Here, as a secretory FGF, a chimeric protein (SEQ ID NO: 34) consisting of an N-terminal 40 amino acid residue (secretory signal sequence) of mouse-derived FGF-6 and human-derived FGF-1 (without a secretory signal sequence). ) Was synthesized.
[0073]
1) Preparation of mouse FGF-6 cDNA fragment
Using FGF-6 cDNA (SEQ ID NO: 28) prepared from mouse brain as a template, # 1048 (5'-GCG TCG ACC CAC CAT GTC CCG GGG AGC AGG ACG TGT TCA GGG CAC GCT GCA GGC TCT CGT CTT C-3 PCR reaction was performed using ') (SEQ ID NO: 13) and # 968 (5'-GCG ATA TCC AGT AGC GTG CCG TTG GCG CG-3') (SEQ ID NO: 14) as primers. The specifically amplified 138 base pair band was separated by electrophoresis, extracted, and then double cut with EcoR V and Sal I. The resulting 130 base pair band was separated and extracted and used for the ligation reaction shown below. This 130 base pair fragment contains a region encoding an amino acid sequence indicating a secretion signal in mouse FGF-6.
[0074]
2) Preparation of human FGF-1 gene fragment
Using FGF-1 cDNA (SEQ ID NO: 29) prepared from human liver cells as a template, # 967 (5'-GCG TCG ACA GCG CTA ATT ACA AGA AGC CCA AAC TC-3 ') (SEQ ID NO: 15) and # 630 PCR reaction was performed using (5′-CCG AAT TCG AAT TCT TTA ATC AGA AGA GAC TGG-3 ′) (SEQ ID NO: 16) as a primer. The specifically amplified 434 base pair band was separated by electrophoresis, extracted, and then double-cut with EcoR I and Sal I. The obtained band of 418 base pairs was separated and extracted, and this was inserted into a pBluescript II (KS +) cloning vector (2934 base pairs) double-cut with EcoR I and Sal I, and FGF-1α / pBluescript II ( KS +) was obtained (3352 base pairs). The obtained FGF-1α / pBluescript II (KS +) was sequentially digested with Aor51H I and Sal I, and the resulting band of 3344 base pairs was separated and extracted and used for the ligation shown below. This 3344 base pair fragment contains a region encoding human FGF-1.
[0075]
3) Creation of chimeric genes
The 130 base pair fragment obtained in 1) above and the 3344 base pair fragment obtained in 2) above were subjected to a DNA ligation reaction to obtain an N-FGF-6 / 1α-IV / pBluescript II (KS +) vector. (3474 base pairs, SEQ ID NO: 32). The 130 base pair fragment has a codon encoding an asparagine residue to which an N-type sugar chain is added.
[0076]
4) N-FGF-6 / 1α <NQ> Chimeric gene creation
Here, site-directed mutagenesis is performed on the N-FGF-6 / 1α-IV / pBluescript II (KS +) vector obtained in 3) above, and the asparagine residue added by the N-type sugar chain is mutated to glutamic acid. It was. That is, first, using N-FGF-6 / 1α-IV / pBluescript II (KS +) vector as a template, # 105 (5'-GCG TCG ACC CAC CAT GTC-3 ') (SEQ ID NO: 17) and # 124 (5 PCR reaction was performed using '-GCG ATA TCC AGT AGC GTG CCT TGG GCG CG-3') (SEQ ID NO: 18) as a primer. A specifically amplified 138 base pair band was separated by electrophoresis, extracted, and then double cut with EcoR V and Sal I. The resulting 130 base pair band was subjected to DNA ligation reaction together with the 3344 base pair fragment obtained in 2) above, and N-FGF-6 / 1α The <NQ> / pBluescript II (KS +) vector was obtained (3474 base pairs, SEQ ID NO: 33). N-FGF-6 / 1α In the <NQ> / pBluescript II (KS +) vector, a codon encoding an asparagine residue added by an N-type sugar chain in a 130 base pair fragment is mutated to a codon encoding glutamine.
[0077]
Select a clone of the N-FGF-6 / 1α-IV / pBluescript II (KS +) vector Sal I / EcoR I digestion product obtained here and cloned into the expression vector pMEXneo, and select FGF-6 / 1α-IV. Named /pMEXneo/cl.15-1/DH5α/970903, deposited on September 10, 2000 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology at FERM P-16408. The DNA sequence of this clone is shown in SEQ ID NO: 32.
[0078]
N-FGF-6 / 1α Select a clone of <NQ> / pBluescript II (KS +) vector Sal I / EcoR I digestion product cloned into the expression vector pMEXneo and select FGF-6 / 1α-IV '(NQ) /pMEXneo/cl.153- It was named 1 / DH5α / 970903 and was deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology on September 11, 2000 at FERM P-16418. The DNA sequence of this clone is shown in SEQ ID NO: 33.
[0079]
Example 4
Synthesis of cDNA encoding a protein having a mucin box introduced at the N-terminus of secretory FGF (shown in FIG. 4B)
Here, using the cDNA encoding the mucin box obtained in Example 1, a cDNA for creating a protein having a mucin box introduced at the N-terminus of a secretory FGF (shown in FIG. 4A) is prepared. did.
[0080]
1) Synthesis of cDNA encoding the signal sequence of secretory FGF
Here, a cDNA encoding a signal sequence of secretory FGF was synthesized. N-FGF-6 / 1α PCR was performed using the <NQ> / pBluescript II (KS +) vector as a template and # 115 (SEQ ID NO: 19) and # 241 (SEQ ID NO: 35) as primers. The reaction products were separated by agarose gel electrophoresis, and a specifically amplified 160 base pair band was extracted.
[0081]
2) Synthesis of cDNA encoding mucin box region
Here, a cDNA encoding a mucin box region was synthesized. PCR was performed using the mucx10@N-ter./pBS/cl.9/000524 plasmid prepared in Example 1 as a template and # 239 (SEQ ID NO: 36) and # 240 (SEQ ID NO: 37) as primers. . The reaction products were separated by agarose gel electrophoresis, and a specifically amplified 196 base pair band was extracted.
[0082]
3) Synthesis of cDNA encoding FGF-1 region of secretory FGF
Here, cDNA encoding a region having heparin-binding property in secretory FGF was synthesized. N-FGF-6 / 1α PCR was performed using the <NQ> / pBluescript II (KS +) vector as a template and # 242 (SEQ ID NO: 38) and # 116 (SEQ ID NO: 20) as primers. The reaction products were separated by agarose gel electrophoresis, and a specifically amplified 447 base pair band was extracted.
[0083]
4) Synthesis of full-length cDNA encoding a protein with mucin box introduced at the N-terminus of secretory FGF
The DNA fragments obtained in the above 1), 2) and 3) (160 base pairs, 196 base pairs and 447 base pairs, respectively) were mixed to form a template, and # 213 (SEQ ID NO: 39) and # 116 (SEQ ID NO: 20) ) As a primer. As the reaction conditions in this PCR, a cycle of 94 ° C. (30 seconds), 55 ° C. (30 seconds), and 72 ° C. (60 seconds) was repeated 25 times.
[0084]
The PCR reaction products were separated by agarose gel electrophoresis, and a specifically amplified 746 base pair band was extracted. This 746 base pair fragment contains a signal sequence coding region, a mucin box coding region inserted downstream thereof, and a heparin-binding region inserted downstream thereof (coding human FGF-1). To be included). That is, the full length of cDNA encoding a protein having a mucin box introduced at the N-terminus of secretory FGF was synthesized here.
[0085]
5) Cloning of cDNA encoding a protein with mucin box introduced at the N-terminus of secretory FGF
The DNA fragment obtained in 4) above was completely digested with Eco RI, and the end treatment of the PCR product was performed (701 base pairs). Subsequently, this reaction product was purified using a DNA fragment purification kit (Roche Diagnostics High Pure PCR Product Purification Kit). The obtained purified DNA fragment was mixed with a pCAD vector pretreated with Eco RI and CIAP and subjected to a DNA ligation reaction. Using this recombinant vector, E. coli strain DH5α was transformed. The obtained transformant was seeded on an agarose medium in the presence of ampicillin, and a primary screening was performed by selecting the grown colonies.
[0086]
By using the selected colonies and performing direct PCR using primers # 174 (SEQ ID NO: 40) and # 212 (SEQ ID NO: 41), a clone that gives the expected 807 base pair band is selected. Next screening. Furthermore, by performing direct PCR using primers # 174 (SEQ ID NO: 40) and # 102 (SEQ ID NO: 42), by selecting a clone that gives the expected band of 704 base pairs, The correct one was selected.
[0087]
The selected colonies were liquid-cultured in ampicillin-containing LB medium, and the plasmid was roughly purified from the grown cells by the alkaline method. This plasmid was digested with Eco RI, and the excised DNA fragment was analyzed by agarose gel electrophoresis. A clone giving a band of 701 base pairs, which is the target size, was selected and used as the tertiary screening. For several clones selected in the tertiary screening, the plasmid was purified again and the inserted sequence of the plasmid was decoded by cycle sequencing.
[0088]
A clone having a sequence that matches the expected sequence was selected and named NM10: mucx10@Nter.secFGF/pCAD/cl.N-6/000524. Deposited at the Industrial Technology Research Institute with FERM P-17879. The DNA sequence of this clone is shown in SEQ ID NO: 43.
[0089]
Example 5
Synthesis of cDNA encoding a protein having a mucin box introduced at the C-terminus of secretory FGF (shown in FIG. 4C)
Here, using the cDNA encoding the mucin box obtained in Example 2, a cDNA for creating a protein having a mucin box introduced into the C-terminus of secretory FGF (shown in FIG. 4A) is prepared. did.
[0090]
1) Synthesis of cDNA encoding almost the entire length of secretory FGF
N-FGF-6 / 1α PCR was performed using <NQ> / pBluescript II (KS +) vector as a template and # 213 (SEQ ID NO: 39) and # 234 (SEQ ID NO: 44) as primers. The reaction products were separated by agarose gel electrophoresis, and a specifically amplified 551 base pair band was extracted.
[0091]
2) Synthesis of cDNA encoding mucin box
Here, a cDNA encoding a mucin box region was synthesized. PCR was carried out using the mucx10@C-ter./pBS/cl.I2-3/000524 plasmid prepared in Example 2 as a template and # 232 (SEQ ID NO: 45) and # 233 (SEQ ID NO: 46) as primers. went. The reaction products were separated by agarose gel electrophoresis, and a specifically amplified 195 base pair band was extracted.
[0092]
3) Synthesis of the full-length cDNA of a protein with a mucin box introduced at the C-terminal of secretory FGF
PCR was performed using the DNA fragments obtained in 1) and 2) above as a template by mixing # 213 (SEQ ID NO: 39) and # 233 (SEQ ID NO: 46) as primers. As the reaction conditions in this PCR, a cycle of 94 ° C. (30 seconds), 55 ° C. (30 seconds), and 72 ° C. (30 seconds) was repeated 25 times.
[0093]
The PCR reaction products were separated by agarose gel electrophoresis, and a specifically amplified 721 base pair band was extracted. This 721 base pair fragment contains a signal sequence encoding region, a heparin binding region inserted downstream thereof (region encoding human FGF-1), and a mucin box inserted downstream thereof. It is included. That is, the full length of cDNA encoding a protein having a mucin box introduced into the C-terminal of secretory FGF was synthesized here.
[0094]
4) Cloning of cDNA of protein with mucin box introduced into C-terminal of secretory FGF
The DNA fragment obtained in the above 3) was completely digested with Eco RI, and the end treatment of the PCR product was performed. Subsequently, this reaction product was purified using a DNA fragment purification kit (Roche Diagnostics High Pure PCR Product Purification Kit). The obtained purified DNA fragment was mixed with a pCAD vector pretreated with Eco RI and CIAP and subjected to a DNA ligation reaction. Using this recombinant vector, E. coli strain DH5α was transformed. The obtained transformant was seeded on an agarose medium in the presence of ampicillin, and a primary screening was performed by selecting the grown colonies.
[0095]
By using the selected colonies and performing direct PCR using primers # 174 (SEQ ID NO: 40) and # 212 (SEQ ID NO: 41), a clone that gives the expected 807 base pair band is selected. Next screening. In addition, by performing direct PCR using primers # 174 (SEQ ID NO: 40) and # 102 (SEQ ID NO: 42), by selecting a clone that gives the expected 554 base pair band, The correct one was selected.
[0096]
The selected colonies were liquid-cultured in ampicillin-containing LB medium, and the plasmid was roughly purified from the grown cells by the alkaline method. This plasmid was digested with Eco RI, and the excised DNA fragment was analyzed by agarose gel electrophoresis. A clone giving a band of 701 base pairs, which is the target size, was selected and used as the tertiary screening. For several clones selected in the tertiary screening, the plasmid was purified again and the inserted sequence of the plasmid was decoded by cycle sequencing.
[0097]
A clone having a sequence that matches the expected sequence was selected and named CM10: mucx10@Cter.secFGF/pCAD/cl.D-5/000524. On May 29, 2000, Biotechnology, AIST Deposited at the Industrial Technology Research Institute at FERM P-17880. The DNA sequence of this clone is shown in SEQ ID NO: 47.
[0098]
Example 6
Expression of protein with mucin box introduced at N-terminal or C-terminal of secreted FGF
1) Gene transfer into animal cells
NM10: mucx10@Nter.secFGF/pCAD/cl.N-6/000524 plasmid obtained in Example 4 or CM10: mucx10@Cter.secFGF/pCAD/cl.D-5/000524 obtained in Example 5 The plasmid was transfected into CHO-K1 cells (Chinese hamster ovary cell K1 substrain) by the lipofection method, and the transfected cells were selected by culturing in the absence of thymidine and hypoxanthine.
[0099]
2) Gene amplification
Methotrexate (MTX) was added to the obtained culture solution of the gene-transferred cells to amplify the transgene. The MTX concentration was sequentially increased, and finally clones that survived in the presence of 1000 nM MTX were selected. Among these selected clones, a clone having a mucin box introduced at the N-terminus was named NM10 / CHO, and a clone having a mucin box introduced at the C-terminus was named CM10 / CHO.
[0100]
3) Mass production of target protein by selected clones
NM10 / CHO and CM10 / CHO were each increased until the culture dish was almost full, and the amount of protein produced was increased by replacing the medium with a serum-free medium. The medium was changed every day, and the conditioned medium obtained was centrifuged at low speed, and the supernatant was stored at 4 ° C.
[0101]
Example 7
Expression of sialyl Lewis X antigen
The clones prepared in Example 6 were further introduced with core 2N-acetylglucosamine transferase and fucose transferase 7 to express sialyl Lewis X antigen.
[0102]
1) Gene transfer of core 2N-acetylglucosamine transferase
The human core 2N-acetylglucosaminyltransferase gene was cloned by PCR according to a conventional method and incorporated into the pZeoSV2 (+) vector. The gene was introduced into the NM10 / CHO cells or CM10 / CHO cells prepared in Example 6 by the above-described method, and the transgene-expressing cells were selected based on zeocin resistance. These clones were named NM10 / C2GN-T / CHO and CM10 / C2GN-T / CHO, respectively.
[0103]
2) Gene transfer of fucose transferase 7
The human fucose transferase 7 gene was cloned by PCR according to a conventional method and incorporated into the pMEXneo vector. The gene was introduced into NM10 / C2GN-T / CHO or CM10 / C2GN-T / CHO by the method described above, and a transgene-expressing cell was selected based on geneticin resistance. These clones were named NM10 / C2GN-T / FT7 / CHO and CM10 / C2GN-T / FT7 / CHO, respectively.
[0104]
<Test example>
Various tests as described below were performed using the cell lines obtained in Example 6 or Example 7 described above, and the characteristics of the protein into which the mucin box was introduced were evaluated.
[Test Example 1]
Analysis of secreted proteins
1) Crude purification of secreted protein using affinity for heparin
Heparin affinity column chromatography was performed using the conditioned medium obtained in Example 6, and the FGF-containing fraction was roughly purified. In the heparin affinity column chromatography, first, heparin sepharose beads were added to the conditioned medium obtained in Example 6 and stirred at 4 ° C. overnight. Next, beads settled by standing were packed in a column and washed thoroughly with a buffer solution having a physiological salt concentration. Thereafter, the protein bound to the beads was eluted with a buffer containing 2.5 M salt. The buffer from which the protein was eluted was monitored by absorbance at 280 nm, and the FGF content of each fraction was measured by ELISA using an anti-FGF-1 antibody.
[0105]
As a result, it was confirmed that 5-8 μg / ml of FGF-1 antigenic molecules (referred to as NM10 and CM10, respectively) were secreted in both NM10 / CHO and CM10 / CHO. No molecule having FGF-1 antigenicity was detected in the conditioned medium of CHO cells into which the gene was not introduced (detection sensitivity: 100 ng / ml).
[0106]
2) SDS denaturing electrophoresis
The FGF-containing fraction obtained as described above was subjected to SDS-denaturing electrophoresis. The protein was denatured by boiling the sample with a buffer for electrophoresis (containing SDS and 2-mercaptoethanol), and electrophoresis was performed in the presence of SDS using a 12.5% acrylamide gel. After the separated protein is electrically transferred to a nitrocellulose membrane, anti-FGF-1 monoclonal antibody and horseradish peroxidase-labeled anti-mouse IgG antibody, or biotin-labeled anti-FGF-1 monoclonal antibody and horseradish peroxidase-labeled streptavidin And were detected by chemiluminescence.
[0107]
The results are shown in FIG. NM10 and CM10 were both found to have a molecular weight close to 35 kDa (lanes 1 and 2, respectively), whereas secretory FGF-1 had a molecular weight of 18.5 kDa (lane 5). These were larger than the molecular weight of 21 kDa of the molecules (NM1, CM1) in which the mucin-type glycosylation sequence was introduced only once into secretory FGF-1 (lanes 3 and 4, respectively).
Since the difference between the molecular weight of secreted FGF-1 and the molecular weight of NM10 and CM10 cannot be explained only by the increase in the molecular weight of the protein backbone, it was expected that NM10 and CM10 had glycosylation.
[0108]
3) Digestion with various carbohydrate degrading enzymes
As described above, using a protein roughly purified by heparin affinity column chromatography, acetate buffer (pH 5.0), sialidase derived from Arthrobacter ureafaciens were added, and the mixture was incubated at 37 ° C. overnight. The enzyme reaction was stopped by heating at 0 ° C. for 3 minutes. This was analyzed by SDS denaturing electrophoresis as in 2). Alternatively, phosphate buffer (pH 7.8) and peptide-N-glycosidase F (PNGase) derived from Flavobacterium were added and incubated overnight at 37 ° C, and then the enzyme reaction was stopped by heating at 100 ° C for 3 minutes. . This was similarly analyzed by SDS denaturing electrophoresis.
[0109]
In addition, using the sialidase-treated sample, add phosphate buffer (pH 6.4) and O-glycosidase derived from Streptococcus pneumonia, incubate at 37 ° C overnight, and then heat at 100 ° C for 3 minutes to carry out the enzyme reaction. stopped. This was analyzed by SDS denaturing electrophoresis as described above.
The results are shown in FIGS.
[0110]
FIG. 5B shows the result of sialidase treatment. Compared to NM10 and CM10 before the enzyme treatment (lanes 1 and 3 respectively), the molecular weight on SDS-PAGE of NM10 and CM10 after the enzyme treatment (lanes 2 and 4 respectively) was changed. From this, it was concluded that NM10 and CM10 are sialic acid-containing molecules.
[0111]
FIG. 5C shows the result of the PNGase treatment. No changes were observed in the molecular weights on SDS-PAGE of NM10 and CM10 (lanes 1 and 3 respectively) before enzyme treatment and NM10 and CM10 (lanes 2 and 4 respectively) after enzyme treatment. However, it was found that a minor band of CM10 (36 kDa, lane 3) disappeared by PNGase treatment (lane 4). As a result, NM10 is a molecule that does not contain an N-linked sugar chain, and CM10 also has a major fraction that does not contain an N-linked sugar chain, but some minor fractions are N-linked. It was concluded that the molecule contains sugar chains.
[0112]
5D and FIG. 5E show the results of treatment with O-glycosidase for each of NM10 (D in FIG. 5) and CM10 (E in FIG. 5). Compared to the untreated sample (lane 1), the mobility is decreased by sialidase treatment (lane 2), but no change is observed in the mobility only by O-glycosidase treatment (lane 3). On the other hand, when the sialidase-treated sample was treated with O-glycosidase, the molecular weight was greatly reduced (lane 4). This is based on the high substrate specificity that O-glycosidase cleaves only the unmodified Gal β 1-3 GalNAc structure. From this result, NM10 and CM10 are sialylated Gal β 1-3 It was revealed that it was modified with a sugar chain having a GalNAc structure.
Furthermore, the molecular weight obtained was consistent with the molecular weight obtained when ldlD cells, which are O-glycan biosynthesis mutants, were used as hosts (lane 5), indicating that NM10 and CM10 were O-type. It was confirmed that the molecule was modified with a sugar chain.
[0113]
[Test Example 2]
Composition analysis of amino acids and amino sugars
The sample roughly purified by heparin affinity column chromatography in the same manner as in Test Example 1 was diluted to reduce the salt concentration, and then subjected to heparin affinity column chromatography using HPLC again (Test Examples described later). 4). The salt concentration was increased linearly from 0.5 to 2.5 M, monitored by absorbance at 214 nm, and the FGF content of each fraction was measured by ELISA using an anti-FGF-1 antibody. The obtained FGF-containing fraction was separated by SDS denaturing electrophoresis and analyzed by silver staining. The results are shown in FIG. 7B (lane 1). As shown in FIG. 7B, FGF into which the mucin box was introduced was detected as a nearly single band. As a result, it was found that the FGF into which the mucin box was introduced was purified with high purity.
[0114]
A part of the sample purified to a high purity as described above was heated in 6N hydrochloric acid at 110 ° C. for 22 hours to hydrolyze the peptide bond. The protein content (concentration) was accurately quantified by analyzing this with an amino acid analyzer. In addition, the glycoside bond was hydrolyzed by heating the other part in 4N hydrochloric acid at 100 ° C. for 6 hours. The amino sugar content (concentration) was accurately quantified by quantitative analysis with HPLC.
[0115]
These amino acid quantitative analysis results and amino sugar quantitative analysis results were combined to calculate the number of sugar chains in one molecule of glycoprotein.
As a result, it was found that 10.0 moles of GalNAc residues exist in 1 mole of CM10 protein. In addition to the results of Test Example 1 above, CM10 has a sialylated Gal β 1-3 GalNAc structure. It was concluded that it was modified with 10 sugar chains.
[0116]
[Test Example 3]
DNA synthesis promoting activity
HUVEC (human umbilical cord-derived vascular endothelial cells) stops the cell cycle when a growth factor such as FGF is deficient even in the presence of 15% serum. To the HUVEC in such a state, FGF introduced with the mucin box prepared in Test Example 1 or secreted FGF (secFGF) produced in CHO cells was added, and 18 hours later, radiolabeled thymidine was taken up for 6 hours. Let During this time, the amount of newly synthesized DNA was calculated by measuring the radioactivity incorporated into the DNA, and evaluated as DNA synthesis promoting activity.
[0117]
The results are shown in FIG. 6A shows NM10, FIG. 6B shows CM10, and FIG. 6C shows DNA synthesis promoting activity of secreted FGF-1 against HUVEC. White squares are untreated samples, black squares are sialidase and O- The result of the sample which removed O-type sugar chain by glycosidase treatment is shown.
[0118]
As a result, the FGF (A in FIG. 6 and B in FIG. 6) into which the mucin box was introduced exhibited a sufficient decrease in specific activity compared to secFGF (C in FIG. 6), but had sufficient DNA synthesis promoting activity. It became clear that it was holding. In addition, by removing the O-type sugar chain, the activity is restored to the same level as secFGF (black square), so it is considered that the cause of the decrease in specific activity is the O-type sugar chain. It was.
[0119]
[Test Example 4]
Heparin affinity affinity chromatography
The affinity of heparin was examined for FGF (CM10) into which the mucin box obtained in Test Example 1 was introduced or FGF-1 produced by Escherichia coli. Heparin sepharose beads were added to the conditioned medium of FGF secreting cells into which the mucin box was introduced, and the mixture was stirred at 4 ° C. for 2 hours or more. The beads that settled by low-speed centrifugation were collected, washed thoroughly with PBS, and then the protein bound to the heparin-immobilized beads was eluted with PBS containing 2.5 M NaCl. Further, a phosphate buffer solution was added to the eluate to lower the salt concentration, and then the solution was again subjected to high performance liquid chromatography packed with heparin affinity affinity beads to elute the bound protein by a NaCl concentration gradient. .
[0120]
The results are shown in FIG. FIG. 7A shows the elution pattern of CM10 monitored by absorbance at 214 nm. Peak 1 is the elution peak of CM10, and the elution salt concentration corresponds to 1.3 M. On the other hand, FGF-1 derived from E. coli is eluted at the position of peak 2, that is, the salt concentration is 1.7 M. From this result, it was found that FGF (CM10) into which a mucin box was introduced had a lower affinity for immobilized heparin than the simple protein FGF-1.
[0121]
FIG. 7B shows the result of silver staining of the fraction at the position corresponding to peak 1 (lane 1) and peak 2 (lane 2) in the elution pattern of A in FIG. As a result, peak 1 is eluted as almost pure CM10, and the protein eluted as peak 2 has its O-type glycan-binding region decomposed due to natural degradation due to its molecular weight, and almost FGF-1 It was thought to correspond to a protein that consisted only of parts.
[0122]
[Test Example 5]
Thermal stability of FGF with mucin box
For FGF introduced with a mucin box and FGF-1 produced by Escherichia coli, the conditioned medium of the secretory cells was sufficiently dialyzed against PBS, and a part thereof was incubated in PBS kept at 56 ° C. or 70 ° C. for 30 minutes. After holding for 12 hours at room temperature, the sample was dialyzed again against PBS at 4 ° C. to prepare a sample. The heat stability of each sample was used as an index of stability by comparison with a sample subjected to a DNA synthesis promoting activity test of HUVEC after various treatments and dialyzed with PBS at 4 ° C. for 12 hours.
[0123]
When kept at room temperature for 12 hours, the activity of E. coli-derived FGF-1 was also protected by heparin, but the activity was retained in the FGF into which mucin box was introduced regardless of the presence or absence of heparin. In addition, when heat-treated, the FGF-1 introduced with a mucin box retains about 50% of the activity at 56 ° C. despite the fact that FGF-1 derived from Escherichia coli is almost inactivated. About 30% of the activity remained in the sample, which was considered to have improved heat resistance stability.
[0124]
[Test Example 6]
Acid and alkali resistance of FGF with mucin box
For FGF introduced with a mucin box and FGF-1 produced in E. coli, the conditioned medium of the secretory cells is sufficiently dialyzed against PBS, and a part thereof is citrate buffer pH 4.0 or pH 10.0. The sample was dialyzed in sodium carbonate buffer for 12 hours, dialyzed again against PBS at 4 ° C., and used as a sample. The stability of each sample was used for the HUVEC DNA synthesis promoting activity test after various treatments, and was used as an index of stability by comparison with a sample dialyzed with PBS at 4 ° C. for 12 hours.
[0125]
By these acid or alkali treatments, FGF-1 derived from E. coli almost loses its activity. On the other hand, FGF into which mucin box was introduced showed almost no decrease in activity even with acid treatment at pH 4.0 regardless of the presence of heparin, and an improvement in acid resistance stability was observed. In addition, FGF into which a mucin box was introduced retained about 50% of activity even after alkaline treatment at pH 10.0, and an improvement in alkali resistance stability was observed.
[0126]
[Test Example 7]
Anti-proteolytic enzyme stability of FGF with mucin box
The conditioned medium of FGF-secreting cells into which mucin boxes have been introduced is sufficiently dialyzed against PBS, and proteolytic enzyme thrombin (0.01%) or factor Xa (0.01%) is added to a part thereof, and the mixture is incubated at 37 ° C. for 1 hour. did. This was subjected to the above-mentioned SDS-denaturing electrophoresis, and the intensity of the remaining band was compared with the sample before the treatment, which was used as an index of stability. The results are shown in FIG.
[0127]
In the case of secreted FGF (secFGF), which is a simple protein produced in CHO cells, the intensity of the original band is reduced by the enzyme treatment as described above, and a new band that appears to be derived from the degradation product appears along with this. . However, in the case of FGF (in FIG. 8, “NM10” and “CM10”) into which a mucin box was introduced, almost no band corresponding to a degradation product was detected. From this, it was considered that FGF into which a mucin box was introduced had increased resistance to proteolytic enzymes (thrombin, factor Xa).
[0128]
[Test Example 8]
Analysis of affinity for sugar chain recognition protein using peanut agglutinin as an example
As described above, it was found that the FGF (NM10, CM10) into which the mucin box was introduced was modified with an O-type sugar chain. Therefore, lectin affinity column chromatography was performed for the purpose of examining whether or not the sugar chains introduced into these FGFs have a function. Wheat germ agglutinin (WGA) is known to recognize sialic acid at the sugar chain end, and its affinity is said to increase when sialic acid is present in agglomerated form. Peanut agglutinin (PNA) is a protein with known affinity for mucin-type sugar chains, and its affinity is said to increase when mucin-type sugar chains without sialic acid are present in agglomerated form. . Castor agglutinin (RCA120) is known to recognize a galactose residue at the end of the sugar chain, and its affinity is said to increase when galactose is present in agglomerated form. Therefore, these lectins were immobilized on FGF (CM10) and secreted FGF-1 (secFGF) into which mucin boxes were introduced, and CM1 used in Test Example 1, using a gel in which these agglutinins were immobilized on a sepharose carrier. The binding to the column was compared.
[0129]
Load various FGFs onto an immobilized lectin column, wash thoroughly, and contain the hapten sugars of each lectin (0.5 M N-acetylglucosamine (WGA), 0.3 M galactose (PNA), 0.5 M lactose (RCA120)) Bound protein was eluted using a buffer solution. The washing solution and the eluate were fractionated, and the FGF content present in each fraction was quantitatively analyzed by ELISA using an anti-FGF-1 antibody.
The results are shown in Table 1. In Table 1, “WGA” indicates wheat germ agglutinin, “PNA” indicates peanut agglutinin, and “RCA120” indicates castor agglutinin.
[0130]
[Table 1]

[0131]
According to Table 1, lectin reactivity corresponding to the structure of the sugar chain was observed by the introduction of the O-type sugar chain, and from this, the function of the O-type sugar chain could be imparted to the FGF-1 molecule. it was thought.
[0132]
[Test Example 9]
Analysis of biological stability
The FGF (CM10) into which the mucin box prepared in Test Example 1 was introduced was administered into the ICR mouse tail vein blood vessel, the tail was cut at regular intervals, and a certain amount of infiltrating blood was collected. After adding a blood coagulation inhibitor to this, it was subjected to SDS-denaturing electrophoresis, and immunostaining was performed using an anti-FGF-1 antibody. The obtained results were digitized to quantitatively analyze the amount of FGF present in the bloodstream.
[0133]
The results are shown in FIG. A of FIG. 9 shows the result of analyzing the sample collected every time by immunostaining. (a) shows untreated CM10, (b) shows sialidase-treated CM10, and (c) shows sialidase-treated and O-glycosidase-treated CM10. FIG. 9B shows the result of quantitative analysis of the result of immunostaining, including the result of CM1.
[0134]
As a result, when CM10 molecules without sugar chains are used as a reference, CM10 with sialylated O-glycans has a longer residence time in blood, while CM10 with sialic acid removed by sialidase treatment is It was found that the residence time in the medium was shortened, which indicated that the in vivo stability of the drug could be controlled by modifying the sugar chain structure.
[0135]
[Test Example 10]
Analysis of physical stability (nonspecific adsorption)
In general, it is known that non-specific adsorption of FGF-related proteins is intense due to hydrophobic bonds. The FGF (CM10) into which the mucin box prepared in Test Example 1 was introduced was improved in hydrophilicity by introduction of a sugar chain, and therefore nonspecific adsorption was expected to be reduced.
[0136]
Accordingly, FGF-1 (rec FGF) produced by mucin box-introduced FGF or E. coli was dispensed into a glass test tube at a constant concentration (400 ng / μl), and this was dispensed at 4 ° C. or 37 ° C. And a constant amount (100 μl) was collected over time. The collected sample was snap frozen at −80 ° C. and stored. About these samples, the FGF content was quantitatively analyzed by ELISA using an anti-FGF-1 antibody.
[0137]
The results are shown in FIG. 10A shows the results when the temperature is kept at 4 ° C., and FIG. 10B shows the results when the temperature is kept at 37 ° C. It was found that the nonspecific adsorption of the sugar chain-introduced CM10 molecule to the glass wall surface at any temperature was lower than that of the simple protein FGF-1.
[0138]
[Test Example 11]
Expression of sialyl Lewis X antigen
In the clone prepared in Example 7, it was predicted that the FGF into which the mucin box was introduced expressed the sialyl Lewis X antigen. Therefore, for the purpose of analyzing the function of sialyl Lewis X antigen, the affinity to E-selectin, which is a protein having this as a ligand, was examined.
[0139]
Secreted E-selectin produced by gene recombination was immobilized on a plate, and the conditioned medium of Example 7 was added. After washing, FGF in which a mucin box bound to E-selectin was introduced was quantitatively analyzed by ELISA-like analysis using an anti-FGF-1 antibody. As a result, it was confirmed that the FGF produced by the cells has affinity for E-selectin.
[0140]
【The invention's effect】
As described above in detail, the DNA amplification method according to the present invention can reliably amplify a base sequence that is difficult to amplify by ordinary PCR. In particular, according to this method, a base sequence encoding a peptide repeat sequence consisting of a plurality of amino acids can be reliably amplified as a base sequence that is difficult to amplify by ordinary PCR. Moreover, in this method, a special base, an instrument, a reagent, etc. are not required, but a desired base sequence can be amplified using only a widely used apparatus, an instrument, and a reagent. According to the present invention, protein functional cassettes can be created in duplicate, thereby contributing to significant modification of protein function.
[0141]
[Sequence Listing]

[0142]
[Sequence Listing Free Text]
SEQ ID NO: 1 is a synthesized DNA fragment.
SEQ ID NO: 2 is a motif of an O-glycosylation sequence.
SEQ ID NO: 3 is an A cassette encoding a mucin box.
SEQ ID NO: 4 is a C cassette encoding a mucin box.
SEQ ID NO: 5 is a G cassette encoding a mucin box.
SEQ ID NO: 6 is a T cassette encoding a mucin box.
Sequence number 7-20 is a primer.
SEQ ID NO: 21 is a synthesized DNA fragment.
SEQ ID NOs: 22 and 23 are primers.
SEQ ID NO: 24 is a synthesized DNA fragment.
SEQ ID NOs: 25 and 26 are primers.
SEQ ID NOs: 27, 32, and 33 are synthesized DNA fragments.
SEQ ID NO: 34 is a chimeric protein.
Sequence number 35-42 is a primer.
SEQ ID NO: 43 is a synthesized DNA fragment.
Sequence number 44-46 is a primer.
SEQ ID NOs: 47 to 49 are synthesized DNA fragments.
SEQ ID NO: 50 is a motif of an O-glycosylation sequence, and the first and fourth “Xaa” indicate arbitrary amino acids.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of a synthesis strategy of cDNA encoding a clustered mucin-type glycosylation sequence for introduction into the N-terminus of secretory FGF.
FIG. 2 is a photograph of agarose gel electrophoresis during the synthesis of cDNA encoding clustered mucin-type glycosylation for introduction into the N-terminus of secretory FGF.
FIG. 3 is a schematic diagram of a synthesis strategy of cDNA encoding clustered mucin-type glycosylation for introduction into the C-terminal of secretory FGF.
FIG. 4 is a schematic diagram of secreted FGF-1 (CM10, NM10) in which clustered mucin-type glycosylation is introduced at the N-terminal and C-terminal of secreted FGF.
FIG. 5 is a photograph of SDS-PAGE electrophoresis of CM10 and NM10, and a photograph of SDS-PAGE electrophoresis of CM10 and NM10 treated with various enzymes.
FIG. 6 is a characteristic diagram showing DNA synthesis promoting activity of CM10 and NM10 on human umbilical vein-derived vascular endothelial cells.
FIG. 7 is a photograph of SDS-PAGE electrophoresis showing the elution pattern from a heparin sepharose column of a sample obtained by treating CM10 and various glycosidases thereof.
FIG. 8 is a photograph of SDS-PAGE electrophoresis showing the results of a resistance test of CM10 against various proteolytic enzymes.
FIG. 9 is a characteristic diagram showing the results of a stability test of CM10 in mouse blood.
FIG. 10 is a characteristic diagram showing the results of an evaluation test on nonspecific adsorption of CM10.

Claims (4)

Designed to cover the first region in the base sequence of a gene encoding a repeated sequence of a peptide consisting of a plurality of amino acids and anneal to each other at the 3 ′ end to encode the repeated amino acid sequence with different degenerate codons By designing a pair of primers so as to include the base sequence and performing PCR using these pair of primers, these pair of primers anneal to each other at the 3 ′ end, and one of these pair of primers is the other template. As the extension reaction proceeds, and the step of synthesizing the first region;
A base designed to cover the second region in the base sequence of the gene partially overlapping with the first region, anneal to each other at the 3 ′ end, and encode repeated amino acid sequences with different degenerate codons By designing a pair of primers so as to include the sequence and performing PCR using these pair of primers, the pair of primers anneal to each other at the 3 ′ end, and one of the pair of primers is used with the other as a template. A step in which an extension reaction proceeds to synthesize the second region;
A step of synthesizing a base sequence including the first region and the second region by performing PCR using the annealed first region and second region synthesized in each of these steps as a template. DNA amplification method characterized by the above.   2. The DNA amplification method according to claim 1, wherein the repeating sequence of the peptide is a sugar chain acceptor.   3. The DNA amplification method according to claim 2, wherein the sugar chain acceptor is a mucin-type sugar chain acceptor. The DNA amplification according to any one of claims 1 to 3, wherein the peptide comprising a plurality of amino acids is a peptide comprising Xaa-Thr-Pro-Xaa-Pro (SEQ ID NO: 50, Xaa is any amino acid). Method.

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