CELL-SURFACE POLYPEPTIDE GENE

Abstract: A DNA coding for cell-surface polypeptides of Porphyromonas gingivalis , and a recombinant DNA containing the above DNA integrated thereinto. A microorganism containing the recombinant DNA can produce cell-surface polypeptides of periodontal disease bacteria, useful for preventing and diagnosing periodontal diseases, in a large quantity.

Description: This invention relates to a cell surface polypeptide of Porphyromonas (Bacteroides) gingivalis (another name; periodontopathic organism: hereinafter, referred to as Porphyromonas gingivalis) having a molecular weight of about 72-kDa (kilodalton) (hereinafter abbreviated as periodontopathic organism cell surface polypeptide), a DNA encoding the polypeptide, a recombinant DNA wherein the DNA is integrated and a microorganism containing the recombinant DNA. The DNA obtained in the invention can be utilized as a reagent, namely as a probe for detecting Porphyromonas gingivalis. Further, the periodontopathic organism cell surface polypeptide obtained in the invention and produced by a microorganism can be utilized for a diagnosis of patients suffering with periodontal diseases and having antibodies against Porphyromonas gingivalis , as an antigen for preparing an antibody for diagnosing patients suffering with periodontal diseases and having Porphyromonas gingivalis or as a prophylactic vaccine for periodontal diseases. It is reported by Yoshimura et al. in Infect. Immun., 57 , 3646 (1989) that since the periodontopathic organism cell surface polypeptide exhibits strong immune response to the sera of patients of periodontal diseases, infecting with Porphyromonas gingivalis, antibodies against the polypeptide existing in the sera of the patients can be detected using it. Further, it is reported by Ogawa et al. in Nippon Saikin-gaku Zasshi, 44 , 329 (1989), by Yoshimura et al. in J. Bacteriol., 160, 949 (1984) and by Yamaji et al. in Nisshishushi, 33 , 349 (1991) that the immune response of the polypeptide is different from that of the known fimbrial protein from the known periodontopathic organism having a molecular weight of 41 kDa, and thus the periodontopathic organism cell surface polypeptide of the invention is different from the ciliary protein. As to the structure of the periodontopathic organism cell surface polypeptide, its amino acid composition is reported by Yoshimura et al. in Infect. Immun. 57 , 3646 (1989), and the amino-terminal amino acid sequence of a purified polypeptide thereof is reported by Yoshimura et al. in FEMS Microbiol. Lett., 92 , 47 (1992). Elucidated amino acids are 40 amino acids, and when we compare 40 amino acid with an amino acid number estimated from the molecular weight of the periodontopathic organism cell surface polypeptide, the rate is only under 10 %. Further, concerning with a DNA encoding the periodontopathic organism cell surface polypeptide, it is reported by Yoshimura et al. in FEMS Microbiol. Lett., 92 , 47 (1992) that its part was obtained. However the base sequence of the gene is not elucidated at all. The periodontopathic organism cell surface polypeptide is recovered from Porphyromonas gingivalis cells, but its supply amount is limited because of difficulty of the culture of Porphyromonas gingivalis. Thus development of an industrial process for supplying the periodontopathic organism cell surface polypeptide is desired. Infection and Immunity, Vol. 60, No. 7, p. 2926-2935 and FEMS Microbiology Letters, 92, 47-56 describe a purified 75 kDa cell surface component isolated from Porphyromonas gingivalis which, however, differs from the polypeptide of the present invention. Disclosure of Invention According to the invention, there are provided a cell surface polypeptide of Porphyromonas gingivalis having an amino acid sequence shown in SEQ ID NO : 1 (Numbers 1 - 558), a DNA encoding the cell surface polypeptide of Porphyromonas gingivalis having the amino acid sequence shown in SEQ ID NO : 1 (Numbers 1 - 558), a recombinant DNA which is a DNA having integrated thereinto the DNA encoding the cell surface polypeptide of Porphyromonas gingivalis having the amino acid sequence shown in SEQ ID NO : 1 (Numbers 1 - 558), and a microorganism having the recombinant DNA which is the DNA having integrated thereinto the DNA encoding the cell surface polypeptide of Porphyromonas gingivalis having the amino acid sequence shown in SEQ ID No. 1 (Numbers 1 - 558). Fig. 1 is a drawing illustrating the process of creating a recombinant plasmid containing a DNA encoding the periodontopathic organism cell surface polypeptide. Detailed Description of Invention The DNA and recombinant DNA of the invention are prepared according to the following general process. The whole DNA is prepared from Porphyromonas gingivalis according to the method of Meyer et al. (Cell, 30 , 45 (1982)) and the method of Maniatis et al. (Molecular Cloning Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, published in 1989 (hereinafter referred to as Mol. clon.)), and the DNA is inserted into a vector DNA such as a plasmid DNA of Escherichia coli to give a library of recombinant DNAs. From the recombinant DNA library, a recombinant DNA having a DNA encoding the periodontopathic organism cell surface polypeptide is selected. The culture broth of Porphyromonas gingivalis is centrifuged, the cells are collected, lysozyme, proteinase K and sodium dodecyl sulfate (SDS) are added thereto, and the cells are ruptured and dissolved. After phenol extraction, phenol-chloroform extraction and chloroform extraction, DNAs are recovered by ethanol precipitation. The extracted DNAs are treated with 5 to 100 units/ml, preferably 10 units/ml of a restriction enzyme KpnI in a suitable solution, e.g. a solution containing 10 mM Tris-HCl buffer (e.g. pH 7.5), MgCl 2 (e.g. 6 mM) and NaCl (e.g. 10 mM), and then NaCl is added so as to be 100 mM and the DNA is treated with 5 to 100 units/ml, preferably 10 units/ml of a restriction enzyme XhoI. The DNAs after the treatment are fractionated using low melting point agarose gel electrophoresis (Lars Wieslander: Anal. Biochem., 98 , 305 (1979)), and fragments of about 3.4 kilobases are recovered and integrated into a vector. As the vector Blue-script, pUC18, pUC19, etc. can for example be used. When Blue-script is used, the vector DNA is treated with 5 to 100 units/ml, preferably 10 units/ml of a restriction enzyme KpnI in a suitable solution, e.g. a solution containing 10 mM Tris-HCl buffer (e.g. pH 7.5), MgCl 2 (e.g. 6 mM) and NaCl (e.g. 10 mM), and then NaCl is added so as to be 100 mM and the DNA is treated with 5 to 100 units/ml, preferably 10 units/ml of a restriction enzyme XhoI to cleave the KpnI and XhoI sites of the vector DNA. These DNAs are fractionated using low melting point agarose gel electrophoresis (Lars Wieslander: Anal. Biochem., 98 , 305 (1979)), and a fragment of about 2.9 kilobases is recovered. This vector DNA fragment and the previously prepared DNA fragments are incubated for a definite time (e.g. 2 hours) at an appropriate temperature (e.g. 16°C) using a DNA ligation kit (produced by Takara Shuzo Co.). Thus recombinant DNAs are obtained. Using the obtained recombinant DNA, Escherichia coli , for example an XL1-Blue strain, an LE 392 strain, DH 5 strain or a JM 83 strain is transformed, for example according to the method of Scott et al. (Katsuya Shigesada: Saibo Kogaku, 2 , 616 (1983)). When Blue-script is used as the vector, since the ampicillin-resistant gene exists on the recombinant DNAs, the transformed Escherichia coli strains exhibit ampicillin resistance. Hereinafter, a method for selecting a strain having a novel recombinant DNA carrying the periodontopathic organism cell surface polypeptide gene from the ampicillin-resistant (Ap r ) transformants is described. Namely, the obtained recombinant DNAs are fixated on a nitrocellulose filter, and associated with a synthetic DNA probe having a DNA sequence estimated from the amino acid sequence of the periodontopathic organism cell surface polypeptide, and ones strongly associating therewith are selected (the method of Grunstein-Hogness, Proc. Natl. Acad. Sci., USA., 72 , 3961 (1975)). The probe DNA is synthesized by a usual triester method (J. Am. Chem. Soc., 97 , 7327 (1975)). The selection with the synthetic DNA probe can further be ascertained according to the method of Southern et al. (J. Mol. Biol., 98 , 503 (1975)), and by this method a recombinant DNA having the DNA fragment encoding periodontopathic organism cell surface polypeptide can be identified. An example of the thus obtained recombinant DNAs is a plasmid pCSP 409. This plasmid can be used as a supply source of the DNA encoding the periodontopathic organism cell surface polypeptide. Further, a DNA encoding the periodontopathic organism cell surface polypeptide is cut out from the novel recombinant DNA of the invention, the DNA is integrated into an expression vector DNA, the resultant recombinant DNA is introduced into a microorganism, the resultant transformed microorganism is cultured to form and accumulate the periodontopathic organism cell surface polypeptide in the culture broth, and the polypeptide is recovered. Thereby the polypeptide can be prepared. As the recombinant DNA containing the DNA encoding the periodontopathic organism cell surface polypeptide, -the above plasmid pCSP 409 can-be mentioned as a suitable example. As the expressing vector DNA, any one can be used so long as it can express the inserted DNA in the microorganism. Preferably, there can be used a vector DNA which has a suitable promoter, e.g. the promoter of the tryptophan (trp) system, the lactose (lac) system, the PL system or the like, into the down-stream of which a DNA can be inserted, and further wherein the interval between the contained Shine-Dalgarno sequence (hereinafter abbreviated as SD sequence) and the translation initiation codon (ATG) is adjusted to a suitable distance, e.g. 6 to 18 base pairs. As a specifically preferred vector DNA, plasmid pGEL1 can be mentioned. An Escherichia coli containing pGEL1 is deposited with National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (Bikoken) on October 6, 1984 as Escherichia coli IGEL1 (FERM BP-629). Recombination between the DNA encoding the polypeptide and the vector DNA can be conducted by a method described in Mol. clon., namely using a general recombinant DNA method to digest both DNAs with a restriction enzyme and conduct ligation with T4DNA ligase. Purification of DNA fragments formed by the restriction enzyme digestion is conducted by low melting point agarose gel electrophoresis (L. Wieslander: Anal. Biochem., 98 , 305 (1979), hereinafter referred to as LGT method), etc. The ligation reaction of the DNA fragments is conducted at 5 to 25°C, preferably 16°C for 15 minutes to 16 hours, using a DNA ligation kit (produced by Takara Shuzo Co.), or at 1 to 37°C (preferably 3 to 20°C) for 15 minutes to 72 hours (preferably 2 to 20 hours) in a reaction solution containing 2 to 200 mM (preferably 10 to 40 mM) of Tris-HCl (pH 6.1 to 9.5, preferably pH 7.0 to 8.0), 2 to 20 mM (preferably 5 to 10 mM) of MgCl 2 , 0.1 to 10 mM (preferably 0.5 to 2.0 mM) of ATP and 1 to 50 mM -(preferably 5 to 10 mM) of dithiothreitol, using 0.3 to 10 units of T4DNA ligase. The resultant recombinant DNA is introduced into a microorganism, if necessary using the transformation method of Cohen et al. (S.N.Cohen et al.: Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)). As the microorganism, any one can be used so long as it can express the DNA encoding the periodontopathic organism cell surface polypeptide of the invention, and form and accumulate the polypeptide in the culture broth. Preferably used is Escherichia coli . Isolation of the DNA encoding the periodontopathic organism cell surface polypeptide from the Escherichia coli having the recombinant DNA for determining the base sequence of the DNA is conducted using the method of Birnboim et al. (H.C.Birnboim et al.: Nucleic Acids Res., 7 , 1513 (1979)), etc. The isolated recombinant DNA is digested with 1 to 10 kinds of restriction enzymes and then treated by agarose gel electrophoresis or polyacrylamide gel electrophoresis, and the cleavage sites are checked. The base sequence of the DNAs is determined according to Maxam-Gilbert method (Proc. Natl. Acad. Sci., 74 , 560 (1977)) or Sanger method using M13 phage (Sanger et al., Proc. Natl. Acad. Sci. USA, 74 , 5463 (1977); Amersham Co., M13 Cloning and Sequencing Handbook). Preparation of the periodontopathic organism cell surface polypeptide from the microorganism having the recombinant DNA wherein the DNA encoding the cell surface polypeptide of Porphyromonas gingivalis is integrated can be conducted by culturing the microorganism in a medium, and recovering and purifying the polypeptide accumulated in the culture broth or in the cells. As the medium, any one can be used so long as it is suitable for the groth of the microorganism and the production of the periodontopathic organism cell surface polypeptide, and synthetic media, natural media, etc. are used. There can be used as carbon sources glucose, fructose, lactose, glycerol, manni tol , sorbitol, etc., as nitrogen sources ammonium chloride, ammonium sulfate, Casamino acid, yeast extract, polypeptone, meat extract, Bacto-tryptone, etc., as other nutrients potassium dihydrogenphosphate, dipotassium hydrogenphosphate, sodium chloride, magnesium sulfate, vitamin B 1 , magnesium chloride, etc. Culture is conducted at a pH of 5.5 to 8.5 and a temperature of 18 to 40°C preferably by stirring culture. After the start of the culture, the periodontopathic organism cell surface polypeptide is accumulated in 5 to 90 hours. When a microorganism such as Escherichia coli accumulating the polypeptide intracellularly is used, the cells are collected from the culture broth by centrifugation, etc. and ruptured by ultrasonic treatment, etc. to give an extract. When a microorganism secreting the periodontopathic organism cell surface polypeptide extracellularly is used, the culture broth is used as an extract. Purification of the periodontopathic organism cell surface polypeptide from the extract can be conducted according to fractionation with a base or solvent, ion exchange chromatography, gel filtration, affinity chromatography, etc. Detection of the resultant periodontopathic organism cell surface polypeptide can be conducted according to immunoassay, particularly using Western blot technique (Proc. Natl. Acad. Sci. USA, 76 , 4350 (1979)), etc. Examples of the invention are set forth below. Example 1 (1) Preparation of the genomic DNA from Porphyromonas gingivalis : The genomic DNA was prepared as follows from Porphyromonas gingivalis according to the method of Meyer et al. (Cell, 30 , 45 (1982)). The cells of Porphyromonas gingivalis (5 g) (wet weight) was suspended in 10 ml of 50 mM Tris-HCl (pH 7.5) containing 50 mM EDTA and 50 mM NaCl, 2 mg/ml of lysozyme was added, incubation was conducted at 37°C for 10 minutes, 0.5 mg/ml of proteinase K and 1.5 % of SDS were added, and incubation was conducted at 50°C for 6 hours to solubilize the cells. An equal amount of a phenol solution was added to this homogenate and extraction was conducted three times, and then extraction was conducted twice with phenol-chloroform and further once with chloroform. After the extraction, DNA was recovered by ethanol precipitation. The DNA precipitate was dissolved in 2 ml of a Tris-HCl (pH 8.0) solution containing 1 mM EDTA, 2 µg/ml of RNase was added, and incubation was conducted at 65°C for 4 hours. Then, 200 µl of 3M sodium acetate solution and further equal amount of isopropyl alcohol were added and the deposited DNA was recovered by centrifugation. The resultant DNA (about 10 mg) was dissolved in 1 ml of a solution consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. (2) Insertion of the plasmid of Porphyromonas gingival is into a vector: Creation of a recombinant DNA was conducted according to Mol. clon. The outline of the steps was shown in Fig. 1. The above genomic DNA of Porphyromonas gingivalis (300 µg) was added to 300 µl of a solution consisting of 10 mM Tris-HCl (pH 7.5), 6 mM MgCl 2 and 10 mM NaCl, 500 units of KpnI (produced by Takara Shuzo Co., hereinafter all restriction enzymes are produced by Takara Shuzo Co. unless otherwise noted) was added, and reaction was conducted at 37°C for 6 hours. Then, NaCl was added so as to be 100 mM, 500 units of XhoI was added and reaction was conducted at 37°C for 3 hours. About 50 µg of fragments each of about 3.4 kb were obtained from this reaction solution according to LGT method. Further, 10 µg of a vector plasmid (Blue-script) DNA was added to 30 µl of a solution consisting of 10 mM Tris-HCl (pH 7.5), 6 mM MgCl 2 and 10 mM NaCl, 50 units of KpnI was added, and reaction was conducted at 37°C for 6 hours. Then, NaCl was added so as to be 100 mM, 50 units of XhoI was added and reaction was conducted at 37°C for 3 hours. About 5 µg of a fragment of about 2.9 kb was obtained from this reaction solution according to LGT method. This fragment was mixed with 30 µg of the genomic DNA of Porphyromonas gingivalis treated with the restriction enzymes and incubation was conducted at 16°C for 2 hours using a DNA ligation kit (produced by Takara Shuzo Co.) to give recombinant DNAs containing the genomic DNAs of Porphyromonas gingivalis. (3) Selection of recombinant DNAs containing the DNA encoding the periodontopathic organism cell surface polypeptide: An Escherichia coli strain XL1-Blue (STRATAGENE Co.) was transformed according to the method of Scott et al. (Katsuya Shigesada: Saibo Kogaku, 2 , 616 (1983)) using the recombinant DNA obtained in Example 2. The resultant about 10,000 colonies were fixated on nitrocellulose. A synthetic DNA represented by the following formula (SEQ ID NO : 2) corresponding to the sequence of 7 amino acids at the N-terminal site of the purified cell surface polypeptide (wherein the base symbols are based on the guideline for preparation of a specification containing base sequences or amino acid sequences) was labeled with digoxigenin (hereinafter referred to as DIG) at the 3'-terminus, using DIG Oligonucleotide 3'-End Labeling Kit, BOEHRINGER MANNHEIM GmbH according to the operation method in the attached manual. Thus, 3 strains wherein strong association with the DIG-labeled probe was observed at 40°C were selected (the method of Grunstein-Hogness, Proc. Natl. Acad. Sci. USA, 72 , 3961 (1975)). On the obtained 3 strains, association with the above probe was ascertained according to the method of Southern (J. Mol. Biol., 98 , 503 (1975)). These plasmids were designated pCSP409-A, B and C, and all of them were thought to contain a DNA encoding the periodontopathic organism cell surface polypeptide because they have the DNA sequence expected from the amino acid sequence of the purified periodontopathic organism cell surface polypeptide. The above pCSP409-A is hereinafter called pCSP409, and the whole nucleotide sequence of its translation region was determined according to Sanger method using M13 phage (Sanger et al., Proc. Natl. Acad. Sci. USA, 74 , 5463 (1977): Amersham Co. M13 Cloning and Sequence Handbook)) . Thus revealed base sequence and the amino acid sequence determined therefrom are shown in SEQ ID NO : 1. The Escherichia coli containing the recombinant plasmid wherein pCSP409 was integrated was deposited with Bikoken on March 23, 1994 as Escherichia coli CSP409 (accession number FERM BP-4614). (4) Production of the periodontopathic organism cell surface polypeptide from Escherichia coli CSP409 Escherichia coli CSP409 obtained in (3) was inoculated in 10 ml of MCG medium (containing 0.6 % disodium hydrogenphosphate, 0.3 % potassium dihydrogenphosphate, 0.5 % sodium chloride, 0.1 % ammonium chloride, 0.5 % glucose, 0.5 % Casamino acid, 1 mM magnesium sulfate and 4 µg/ml vitamin B 1 , pH 7.2), and cultured at 30°C for 7 hours. The resultant culture broth was inoculated in 50 ml of MCG medium and cultured at 30°C for 5 hours. The cells were recovered from the culture broth by centrifugation (CR-20 produced by Hitachi, Ltd., Rotor RPR16-516; 8,000 rpm, 10 minutes). The resultant cells was dissolved in the buffer of Remuli (pH 7.4; Nature, 227, 680 (1970)), and assay was conducted according to Western blot technique (Proc. Natl. Acad. Sci. USA, 76 , 4350 (1979)) using the serum of a patient suffering with a periodontal disease and the serum of a normal subject. As a result, a band exhibiting the periodontopathic organism cell surface polypeptide specific to the serum of a patient suffering with a periodontal disease was observed. Industrial Applicability By a microorganism containing a recombinant DNA wherein a DNA encoding the periodontopathic organism cell surface polypeptide of Porphyromonas gingivalis of the invention was integrated, the cell surface polypeptide of a periodontopathic organism useful for prophylaxis and diagnosis of periodontal diseases can be obtained in a large amount. Therefore, the invention can be utilized in diagnostic medicament, prophylactic vaccine, etc. of periodontal diseases. SEQUENCE LISTING SEQ ID NO : 1 SEQUENCE LENGTH : 1782 base pairs SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear MOLECULE TYPE : Genomic DNA ORIGINAL SOURCE ORGANISM : Porphyromonas (Bacteroides) gingivalis STRAIN : OMZ409 FEATURE Feature Key : -35 signal Location : 9..14 Identification Method : S Feature Key : -10 signal Location : 32..37 Identification Method : S Feature Key : CDS Location : 60..1752 Identification Method : S Feature Key : Sig peptide Location : 60..105 Identification Method : S Feature Key : mat peptide Location : 208..1752 Identification Method : E SEQUENCE DESCRIPTION SEQ ID NO : 2 SEQUENCE LENGTH : 20 SEQUENCE TYPE : nucleic acid STRANDEDNESS : single TOPOLOGY : linear MOLECULE TYPE : DNA FEATURE Feature Key : CDS Location : 1..20 Identification Method : E SEQUENCE DESCRIPTION

Claims: A DNA encoding a cell surface polypeptide of Porphyromonas gingivalis having an amino acid sequence shown in SEQ ID NO: 1 (numbers 1-558). A recombinant DNA which is a DNA having integrated thereinto a DNA encoding a cell surface polypeptide of Porphyromonas gingivalis having an amino acid sequence shown in SEQ ID NO: 1 (numbers 1-558). The recombinant DNA according to claim 2 wherein the recombinant DNA is plasmid pCSP409, which is obtainable from the deposited Escherichia coli CSP409 (FERM BP-4614) which contains plasmid pCSP409. A microorganism having a recombinant DNA which is a DNA having integrated thereinto a DNA encoding a cell surface polypeptide of Porphyromonas gingivalis having an amino acid sequence shown in SEQ ID NO: 1 (numbers 1-558). The microorganism according to claim 4 wherein the microorganism belongs to Escherichia coli. The Escherichia coli according to claim 5 wherein the Escherichia coli is CSP409 (accession number: FERM BP-4614). A cell surface polypeptide of Porphyromonas gingivalis having an amino acid sequence shown in SEQ ID NO: 1 (numbers 1-558).

Inventors: FUKUI MASANORI, HASEGAWA MAMORU, HOKUKOKU SHUSABURO, MORI HIDEHARU, OGAWA TOMOHIKO, YAMADA KEIKO, YASUDA KENJI, FUKUI, MASANORI, HASEGAWA, MAMORU, HOKUKOKU, SHUSABURO, MORI, HIDEHARU, OGAWA, TOMOHIKO, YAMADA, KEIKO, YASUDA, KENJI
Applicants: KYOWA HAKKO KOGYO KK, KYOWA MEDEX CO LTD, MEITO SANGYO KK, KYOWA HAKKO KOGYO CO., LTD., KYOWA MEDEX CO., LTD., MEITO SANGYO KABUSHIKI KAISHA
Application year: 1995
Main classification: C12N15
IPC codes: A61K38, C07K14, G01N33, C12N15, C12N1