JP3803752B2 - DNA encoding porcine complement inhibitor - Google Patents

Description

【００２９】

【図面の簡単な説明】
【図１】プラスミドｃＤＮＡライブラリーの概要を示す図である。
【図２】本発明のｐＭＣＰｃＤＮＡを含むＪＹ２５細胞について、補体セレクション法による生存率を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to DNA encoding porcine complement inhibitors. More specifically, the present invention relates to a DNA encoding an inhibitor protein that suppresses porcine complement activity, a porcine complement inhibitor protein expressed therefrom, and a screening method for a complement inhibitor gene.
[0002]
[Prior art]
In recent years, organ transplantation has been widely performed in various countries, and rejection of organ transplantation from human to human has been solved by the development of particularly excellent immunosuppressive agents (such as cyclosporine and FK506). However, the shortage of organ donors (donors) is a serious problem. Because of these problems, transplantation of animal organs to humans, that is, xenotransplantation, has been studied. For example, in Europe and the United States, about 3500 heart transplants are performed annually, but the number of cases is less than about 20 to 30% of patients requiring heart transplants. The use of human relatives (for example, primates such as baboons and chimpanzees) as donor animals is problematic because of the ethical point due to the small number of donor animals and high intelligence. On the other hand, there are few problems when livestock is used as a donor animal. In particular, pigs are bred in large quantities and easy to supply, the size of pig organs is close to that of human organs, and there are advantages such as the establishment of strain preservation, etc. Research has been conducted on transplanting pig organs into humans.
Among the rejection reactions that occur when transplanting porcine organs to humans, acute rejection due to cellular immunity involving MHC is related to distantly related species in pigs and humans. Because it is not, it is predicted that it will not occur easily. Moreover, even if such rejection occurs, it is considered that it can be avoided by using the above-described excellent immunosuppressive agent.
However, antibodies against pigs already exist in human blood (called natural antibodies). As a result, when porcine tissues are transplanted into humans, natural antibodies in human blood recognize porcine tissues (antigens) to form antigen-antibody complexes, which are then complemented by humans. The body is activated and the activated human complement necroses the transplanted porcine tissue (rejection). Such a phenomenon occurs immediately (within 1 hour) after transplantation, and is therefore called hyperacute rejection.
[0003]
Drugs that suppress hyperacute rejection due to complement activation have not been developed. Human tissue is not damaged by human complement. This is because a factor that suppresses the activation reaction of complement is expressed in human tissues, and this factor is called a complement inhibitor (or complement regulatory factor). Three complement inhibitors are important: DAF (Decay accelating factor, CD55), MCP (Membrane cofactor protein, CD46), and CD59. DAF and MCP are thought to prevent complement activation by enhancing decay of C3b and C3 / C5 convertase, and CD59 inhibiting C9 step.
Complement inhibitors are species specific, and porcine complement inhibitors can block porcine complement activity, but not human complement activity. Therefore, when porcine tissue is transplanted into humans and the human complement system is activated, porcine complement inhibitors cannot suppress it. As a result, porcine tissue transplanted into humans will be necrotic.
[0004]
In order to solve the problems in transplanting a porcine organ to a human as described above, a human complement inhibitor may be expressed in advance in a porcine organ by a genetic engineering method. When transplanting a porcine heart, a human complement inhibitor may be expressed in advance in porcine vascular endothelial cells.
From such a point of view, research on transgenic pigs incorporating a human complement inhibitor gene (transgenic pigs) has been actively conducted.
[0005]
[Problems to be solved by the invention]
As described above, xenotransplantation has been studied using transgenic pigs incorporating human complement inhibitors. At present, the promoter used for producing this transgenic pig is derived from a human complement inhibitor or a virus. However, it is considered that the complement inhibitor promoter derived from pigs is more effective for the expression of complement inhibitors in pigs. Therefore, in order to obtain the above promoter, it is first necessary to obtain a porcine complement inhibitor cDNA. However, so far, the cDNA of porcine complement inhibitor is not known.
From this point of view, the present inventors performed expression cloning of a porcine complement inhibitor cDNA. As a result, the cells were given resistance to porcine complement and obtained cDNA having high homology to human MCP. In this process, a new cDNA library screening method was found.
The present invention has been made based on such findings, and an object of the present invention is to provide a porcine complement inhibitor cDNA, a porcine complement inhibitor protein expressed therefrom, and a screening method for the complement inhibitor gene.
[0006]
[Means for Solving the Problems]
The present invention made to solve the above problems
(1) DNA consisting of the base sequence shown in SEQ ID NO: 1 or a part of this base sequence;
(2) DNA consisting of the 59th to 1147th sequences of the base sequence shown in SEQ ID NO: 1 or DNA having this sequence;
(3) A porcine complement inhibitor consisting of the amino acid sequence represented by SEQ ID NO: 2;
(4) DNA encoding the amino acid sequence represented by SEQ ID NO: 2 or DNA having this base sequence;
(5) A method for screening a clone having a complement inhibitor gene comprising introducing a cDNA library into a host, adding an antibody and a complement component to the host, and isolating a surviving host;
About.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
The base sequence represented by SEQ ID NO: 1 according to the present invention is a cDNA encoding a porcine complement inhibitor (hereinafter referred to as pMCP cDNA), and such cDNA can be prepared, for example, by the following method.
[0008]
First, mRNA is prepared. mRNA can be prepared according to conventional methods, for example, using normal methods such as guanidinium thiocyanate method, hot phenol method, lithium salt from cells, tissues, etc. expressing porcine complement inhibitors. Poly (A) ⁺ RNA (mRNA) can be prepared by extracting total RNA using a method or the like and passing the obtained RNA through an oligo (dT) cellulose column. In this step, it is preferable to use porcine vascular endothelial cells, which are considered to be high expression sites of complement inhibitors, as the extracted cells. Porcine vascular endothelial primary culture strain or porcine vascular endothelial established cell line PAE cells (J. Biol Chem. 262 , 4098, 1987) is preferably used.
[0009]
Next, the synthesis of cDNA from poly (A) ⁺ RNA is performed by a conventional method (for example, Gubler et al., Gene, 25 , 263, 1983) or a commercially available cDNA synthesis kit (for example, Pharmacia, Amersham, For example, manufactured by Boehringer Mannheim).
The obtained cDNA is separated into long base pairs as necessary, and then inserted into a phage vector λgt11 according to a conventional method, or a cDNA library is prepared by inserting it into an appropriate plasmid vector. .
[0010]
The pMCP cDNA is cloned from the cDNA library thus prepared. Although cloning of pMCP cDNA can be performed by a conventional plaque hybridization method, colony hybridization method, a method using a labeled specific antibody, etc., the present inventors have found a new screening method based on anti-complement activity. This method is preferably used.
That is, after inserting the cDNA library into an appropriate cell, an antibody (antiserum) against the cell and a complement component (serum of the target species, in this case porcine serum) are added to stimulate complement activity and By selecting cells having somatic action, cells containing the target cDNA can be cloned. More specifically, as shown in the Examples below, a plasmid vector library was introduced into human lymphoblast JY25 cells (see J. Immunology 141 , 4283, 1988) according to a conventional method, and an antibiotic-containing medium Preselect cells by culturing in Since the plasmid vector contains an anti-antibiotic gene, cells into which the vector has been introduced can survive and proliferate even in an antibiotic-containing medium. Preselection with an antibiotic-containing medium is performed to enhance cell selectivity, but may be omitted in some cases.
Then, anti-JY25 antibody (antiserum) and complement component (serum of the target species, in this case porcine serum) are added to the preselected cells to stimulate complement activity. A cell into which a plasmid containing the pMCP gene has been introduced expresses a complement inhibitor in the cell and inhibits activation of complement, so that the cell can survive. By repeating the selection with the antibiotic-containing medium and the selection with complement, a clone of cells containing the target cDNA (pMCP cDNA) can be obtained.
According to this method, the cDNA of porcine complement inhibitors (pMCP and other porcine complement inhibitors) can be easily collected. In addition to pigs, complement inhibitors of various animals can be collected by applying this method to cDNA libraries of various animals.
[0011]
Isolation of pMCP cDNA (or a cDNA fragment containing a part thereof) from the thus selected clone can be performed in accordance with a conventional cDNA isolation method. Further, if necessary, the cDNA is subcloned, and pMCP cDNA ( Or a part thereof) may be isolated. When the obtained DNA is cDNA containing a part of pMCP cDNA, clones having full-length pMCP cDNA can be selected by screening a cDNA library using the DNA as a probe.
The base sequence of the obtained pMCP cDNA can be determined using, for example, a conventional method such as the dideoxy method or a commercially available DNA sequencer (for example, manufactured by Applied Biosystems).
[0012]
According to the above method, pMCP cDNA can be cloned and its base sequence can be determined. The pMCP cDNA obtained in the examples described later has a base length of 1365 bp (SEQ ID NO: 1) and is a region encoding a porcine complement inhibitor. Was 1089 bp. Among them, the translation region of about 600 bp showed high homology (about 70%) with human MCP cDNA. The total number of amino acids of the porcine complement inhibitor deduced from the nucleotide sequence of SEQ ID NO: 1 was 363 residues (SEQ ID NO: 2).
[0013]
The pMCP cDNA thus obtained is useful for producing a porcine complement inhibitor because a sufficient amount of porcine complement inhibitor can be produced by a conventional genetic engineering method using the pMCP cDNA. For example, a porcine complement inhibitor can be obtained by incorporating pMCP cDNA into an appropriate vector, preparing an expression vector, and culturing a recombinant obtained by introducing the vector into an appropriate host. A DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 2 or a DNA fragment containing the same may be used as the DNA fragment incorporated into the vector.
As the vector, those having a promoter, SD sequence, terminator, enhancer, various markers and the like necessary for expression can be used as necessary, and examples of the host include bacteria such as Escherichia coli and Bacillus subtilis. In addition, microorganisms such as yeast, animal and plant cells, and the like can be used. In addition, the type of sugar chain of the target peptide and the glycosylation degree differ depending on the type of host cell; the N and / or C terminal amino acid sequence of the precursor peptide expressed in the host cell It is well known to those skilled in the art that peptides having various terminal amino acid sequences can be obtained by being processed by peptidase or the like.
[0014]
Further, the porcine complement inhibitor of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 2 has an action of blocking the activation of porcine complement, and also blocks the activation of human complement. It is useful as a drug that inhibits complement activation.
In addition, regarding the porcine complement inhibitor of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 2, a part of the amino acid sequence is deleted or substituted with another amino acid as long as it is substantially the same as the inhibitor. Other amino acid sequences may be partially inserted, one or more amino acids may be bonded to the N-terminal and / or C-terminal, or sugar chains may be similarly deleted or substituted.
[0015]
【The invention's effect】
As described above, by using the pMCP cDNA of the present invention, a porcine complement inhibitor can be produced by genetic engineering, and a sufficient amount of porcine complement inhibitor can be provided. Since the amino acid sequence of the porcine complement inhibitor can be determined from the pMCP cDNA base sequence, it is possible to synthesize DNA encoding the porcine complement inhibitor from this amino acid sequence. Production is possible. According to the genetic engineering method, not only the amino acid sequence of a naturally occurring protein but also the base sequence is modified so that one or more amino acid substitutions, insertions, deletions, and other proteins A fusion protein can be produced. Furthermore, the pMCP cDNA of the present invention is useful for analyzing the promoter region of porcine complement inhibitors.
Further, according to the screening method of the present invention, cDNAs of complement inhibitors from various animals can be easily collected.
[0016]
【Example】
EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these examples.
[0017]
Example 1
1) Extraction of total RNA from porcine vascular endothelial cells and purification of poly (A) ⁺ RNA Porcine vascular endothelial cell primary culture strain or PAE cells (1 × 10 ⁸ cells) were added to 10 15 cm dishes. 5.5M GTC (guanidine thiocyanate) solution was added to the 3 ml / dish. At this time, the solution is viscous due to the large amount of DNA, but the DNA is shredded by repeating suction and discharge of the solution 20 to 30 times with a syringe (30 ml) with an 18-gauge injection needle to lower the viscosity. I let you.
Next, the residue was removed by high-speed centrifugation (800 rpm), and the supernatant was layered on a CsTFA solution (17 ml) placed in a polyallomer tube (40 ml) and ultracentrifuged overnight. The upper GTC layer and the intermediate DNA layer were gently removed and the rest was discarded. The tube was turned upside down to remove excess moisture, and 2 cm from the bottom of the tube was cut out and placed on ice. The RNA at the bottom of the tube was scraped with the tip of a blue chip and dissolved in 600 μl of 4M GTC solution. Centrifuge lightly to remove insoluble material. 15 μl of 1M acetic acid and 450 ml of ethanol were added per 600 μl, cooled at −20 ° C. for 3 hours or more, and then centrifuged in a microtube (4 ° C.) for 10 minutes.
[0018]
The supernatant was removed and RNA was quickly dissolved in 1 ml of freshly prepared TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Centrifuge lightly and collect the supernatant. Total RNA was prepared by adding 13 μl of 2M NaCl per 330 μl of supernatant and cooling at −20 ° C. for several hours.
Separation and purification of poly (A) ⁺ RNA (mRNA) from total RNA was performed using a Clontech mRNA purification kit (in principle, affinity chromatography using an oligo (dT) -cellulose column). That is, the column filled with dT resin is washed several times with the washing buffer, and then the sample is placed thereon, washed again with the washing buffer, and then RNA (mRNA) attached to the dT resin with the elution buffer is removed. After elution and ethanol precipitation, it was used to prepare cDNA.
[0019]
2) Preparation of cDNA library Distilled water was added to the mRNA (4 μg) obtained above to make 17 μl and heated at 68 ° C. for 5 minutes. After cooling on ice for 10 minutes, the following was added:
x5 First Strand Buffer 6 μl
0.1M DTT 3 μl
RNasin 1 μl
Linker primer (1.6 μg / μl) 3.3 μl
dNTP solution (10 mM) 2 μl
After the addition, the mixture was rapidly stirred, then left at 43 ° C. for 2 minutes, Superscript II was added, and the mixture was incubated at 43 ° C. for 1 hour, and then placed on ice.
Next, the following was added to the above reaction solution on ice.
ddH ₂ O 134.3 μl
x5 Second Strand Buffer 48 μl
dNTP solution (10 mM) 4 μl
0.1M DTT 9 μl
Leave on ice for 5 minutes and add the following:
DNA polymerase I (5U / μl) 12 μl
RNaseH (1.5U / μl) 2.7 μl
After the addition, the mixture was rapidly stirred, incubated at 16 ° C. for 150 minutes, and 4 μl of T4 DNA polymerase (3 U / μl) was added. Incubated at 16 ° C for 15 minutes and then at 37 ° C for 10 minutes. Extraction was performed with 250 μl of phenol / chloroform, and extraction was further performed by adding 100 μl TE to phenol / chloroform. The extracts were combined, and the following was added thereto.
3M sodium acetate 40 μl
Carrier (Glycogen) 10 μl
100 ml ethanol 1 ml
After addition, incubate overnight at -20 ° C (or 1 hour at -80 ° C), then centrifuge (15,000 rpm for 20 minutes), wash with 70% ethanol, centrifuge (15,000 rpm for 5 minutes), 15 μl Dissolved in TE.
[0020]
The following was added to the lysate obtained above (4 μl) on ice.
x10 ligation buffer 2 μl
SalI adapter (1μg / μl) 1.2 μl
ddH ₂ O 9.8 μl
10 units of T4 DNA ligase (approx. 3 μl)
After the addition, it was incubated at 8 ° C. overnight, and inactivated by heating at 70 ° C. for 30 minutes.
The following were then added to this solution (20 μl):
x10 NotI buffer (Toyobo H) 4 μl
x10 Triton 4 μl
x100 BSA 0.4 μl
ddH ₂ O 5.6 μl
NotI (500U / μl) 4 μl
After the addition, it was incubated at 37 ° C. for 2 hours, and heat-inactivated at 70 ° C. for 30 minutes.
[0021]
The following were added to the above solution.
1M NaCl 7 μl
ddH ₂ O 22 μl
Glycogen (10μg / μl) 1 μl
After the addition, it is attached to a small centrifugal gel filtration device (3,000 rpm for 3 minutes), desalted with a Millipore filter (UFCP3 TK50), centrifuged (10,000 rpm for 5 minutes), the supernatant is removed, and 100 μl TE is added. And centrifuged (10,000 rpm for 15 minutes). After removing the supernatant, 100 μl TE was further added and centrifuged (10,000 rpm for 15 minutes). The supernatant was removed, and 100 μl (1/10) TE was added and centrifuged (10,000 rpm for 20 minutes). After removing the supernatant, it was dispersed in 30 μl (1/10) TE, and the filter was turned upside down and centrifuged (5,000 rpm twice for 5 seconds) to obtain cDNA.
[0022]
The following samples were mixed on ice.
cDNA 30 μl
Expression vector [pMSF + (HygroB)] 21 μl
x10 ligation buffer 12 μl
ddH ₂ O 56 μl
T4 DNA ligase (4.6U / μl) 1.5 μl
After mixing, the mixture was left at 8 ° C. overnight, deactivated by heating at 70 ° C. for 30 minutes, and desalted with a Millipore filter (UFCP3 TK50). Thereafter, the mixture was centrifuged (10,000 rpm for 15 minutes), the supernatant was removed, 100 μl TE was added, and the mixture was centrifuged (10,000 rpm for 15 minutes). After removing the supernatant, 100 μl TE was further added and centrifuged (10,000 rpm for 15 minutes). The supernatant was removed, and 100 μl (1/10) TE was added and centrifuged (10,000 rpm for 20 minutes). After removing the supernatant, it was dispersed in 30 μl (1/10) TE, the filter was turned upside down and centrifuged (5,000 rpm for 5 seconds) to obtain 30 μl of cDNA library (1.2 × 10 ⁶ clones, average size: 1.5 kbp). The outline of the structure of the obtained cDNA library is shown in FIG.
[0023]
3) human lymphoblastoid cells JY25 cells in cloning <br/> logarithmic growth phase pMCPcDNA the (1x10 ^8/10 cuvettes) was dispersed in HeBS (800 [mu] l), which the above cDNA library (20 [mu] l / 10 cuvettes) In addition, electroporation (250 V 960 μF) was performed. The cells were washed with 200 ml of D.C. It was dispersed in MEM [containing 20% FCS, IT (insulin / transferrin), PS (penicillin / streptomycin)] and cultured for 24 hours. Then D. containing 400 μg / ml hygromycin B. It was re-dispersed in MEM (containing 10% FCF, IT, PS), cultured for 10 to 14 days (1 × 10 ⁷ cells), and then washed with PBS.
Washed cells were subjected to complement selection. That is, the cells are labeled with D. MEM (containing 10 ml FCS + 10 ml pig serum + 80 ml D.MEM + 1 ml anti-JY25 antibody + 667 μg anti-DAF antibody + 100 μg anti-MCP antibody) was added and incubated at 37 ° C. for 2 hours. After the reaction, The cells were washed once with MEM, and the number of viable cells was counted by trypan blue staining (viable cell number: 0.1 to 1%). An anti-DAF antibody and an anti-MCP antibody were added to enhance selectivity. After washing live cells with PBS, 100 ml Re-dispersed in MEM (10% FCS) and cultured for 24 hours.
Thereafter, the culture in the above-mentioned hygromycin B-containing medium and complement selection were repeated twice (the number of viable cells finally became 20%).
[0024]
The viable cells (1 × 10 ⁶ ) obtained above were centrifuged (1,000 rpm for 5 minutes), and the cells were washed with PBS. Next, 400 μl of 0.6% SDS / 10 mM EDTA mixture was added, mixed well, and left at room temperature for 20 minutes. 100 μl of 5M NaCl was added and mixed well (cells turned white), left at 4 ° C. overnight, centrifuged (15,000 rpm for 15 minutes), and the supernatant was collected. To the resulting supernatant, phenol / chloroform (200 μl + 200 μl) was added and centrifuged (12,000 rpm for 2-3 minutes), and the supernatant was collected. To the supernatant, 10 μl (4 μg) of polyacrylamide was added, 1 ml of ethanol was further added, and the mixture was allowed to stand at −80 ° C. for 2 hours. After washing by adding 70% ethanol, the precipitate was dispersed in 20 μl TE.
[0025]
The cDNA (2 μl) obtained above was electroporated (2.5 kV 25 μF 400Ω) into 50 μl of E. coli (MC1061). 950 μl of SOC medium was immediately added to each cuvette (total 10 cuvettes) samples. Incubated for 1 hour at 37 ° C. with agitation (20 rpm). Incubated samples were handled as follows.
(1) A sample (15 μl sample / plate) was applied to the plate, and the plate was stored at 4 ° C.
(2) The remaining sample was added to 2 ml of TBK ⁺ and cultured overnight at 37 ° C. with stirring, followed by mini-purification (preparation of a small amount of plasmid DNA). Mini-purified cDNA (2 ml sample) was dispersed in 20 μl TE. In the obtained cDNA, 10 μl was cleaved with a restriction enzyme (Xhol + NotI), and the size of the cDNA was checked using an agarose gel. The remaining samples were electroporated into JY25 cells and selected by the complement selection method described above to determine the number of viable cells.
[0026]
After performing the above check, 40 colonies were picked from the high viability plate among the storage plates, and the colonies were cultured overnight in 2 ml of TKB ⁺ . The culture was mini-purified, cut with a restriction enzyme (Xhol + NotI), the insert size of each cDNA was checked, and clones were classified by size. Of these, cDNA and bulk cDNA (control), which seemed more appropriate than the length and frequency of cDNA, were electroporated again into JY25 cells and analyzed by the complement selection method described above. The result is shown in FIG. As shown in FIG. 2, the cell [JY25 (-pMCPcDNA)] into which the bulk cDNA was introduced has a very poor survival rate, but the cell [JY25 (+ pMCPcDNA)] containing the selected cDNA (pMCPcDNA) has a high survival rate. And was resistant to porcine complement.
[0027]
CDNA was isolated from cells showing high survival rate, cleaved with a restriction enzyme (Xhol + NotI), bound to pBSIIKS ⁺ , and the nucleotide sequence was determined according to a conventional method. As a result, the cDNA had a base sequence of 1365 bp (SEQ ID NO: 1), and the region encoding the porcine complement inhibitor was 1089 bp. Among them, the translation region of about 600 bp showed high homology (about 70%) with human MCP. The sequence of the porcine complement inhibitor deduced from the base sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2. The total amino acid number of the inhibitor was 363 residues.
[0028]
[Sequence Listing]

[0029]

[Brief description of the drawings]
FIG. 1 is a diagram showing an outline of a plasmid cDNA library.
FIG. 2 is a graph showing the survival rate by the complement selection method for JY25 cells containing the pMCP cDNA of the present invention.

Claims (4)

Nucleotide sequence or Ranaru DNA represented by SEQ ID NO: 1.         A DNA consisting of the 59th to 1147th sequences of the base sequence shown in SEQ ID NO: 1 or a DNA having this sequence.         A porcine complement inhibitor consisting of the amino acid sequence represented by SEQ ID NO: 2.         DNA encoding the amino acid sequence represented by SEQ ID NO: 2 or DNA having this base sequence.

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