Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-20
2003-04-22
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C536S023500, C536S023600, C514S04400A, C514S021800, C422S067000
Reexamination Certificate
active
06551779
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to polypeptides, DNAs coding for said polypeptides, recombinant vectors containing said DNAs, host cells transformed with said recombinant vectors, a method for preparing said polypeptides by cultivating said host cells, and use thereof.
BACKGROUND OF THE INVENTION
Helicobacter pylori
(which has previously been known as
Campylobacter pylori
) is a Gram-negative bacillus discovered from a chronic gastritis biopsy specimen as a spiral bacterium in 1983 [J. R. Warren and B. Marshall,
Lancet, i,
1273 (1983)].
After the discovery thereof, the significance of this bacterium to chronic gastritis and duodenal ulcer has attracted attention, and investigation thereof has become one of important themes biologically and medically. In particular, elucidation of the mechanisms of cell injuries leading to diseases and developments of new inspection methods have been gained attention.
Methods for detecting
Helicobacter pylori
(hereinafter briefly referred to as HP) include cultivating methods, urease tests, expiratory tests, histological inspections and serological methods. In particular, the serological methods, in which anti-HP antibodies in the sera are detected, are excellent in that they are noninvasive and a large amount of specimens can be treated.
Various anti-HP antibodies are contained in the sera, so that measurement sensitivity and specificity thereof vary depending on the preparation methods and kind of antigens used for antibody measurement. As a result, the clinical significance of assaying the anti-HP antibodies are also various.
For example, detection methods using lysates of all HP cells as antigens for detection of the anti-HP antibodies [B. J. Rathbone,
Lancet, ii,
1217 (1985)] sometimes cause false positive judgement due to cross reaction antibodies with other Campylobacter, or conversely, false negative judgement caused by interaction between antigens or denaturation in antigen preparation stages.
B. J. Rathbone reports that antigens partially purified with acidic glycine extract from cells are improved in both specificity and sensitivity than lysates of all HP cells [B. J. Rathbone, “Serological Response to
Helicobacter pylori”, Eur. J. Gastroenterol. Hepatol.,
4 (Supple. 1), S21-S23 (1992)]. Furthermore, he reports that methods using urease, etc. highly purified by high performance liquid chromatography (HPLC), etc. as antigens for detection have a tendency to enhance specificity, but to reduce sensitivity because of genetic diversity between HP strains.
Further, of antibodies corresponding to various antigen components of HP, antibodies were screened which were important in relation to utility on serum diagnosis and pathology of gastropathy and duodenal diseases. von Wulffen et al. report that IgG and IgA antibodies are useful; and that particularly, antibodies to 110-KD and 22-KD antigens characteristically appear in the pathology of these diseases [H. von Wulffen, J. Heesemann, G. W. Bulzow et al.,
J. Clin. Microbiol.,
24, 716-720 (1986)]. C. S. Goodwin et al. conduct ELISA using acidic glycine extract as an antigen, and report that main antigen molecules are 84 KD, 33 KD, 28 KD and 25 KD, in addition to 64 KD, 62 KD and 57 KD.[C. S. Goodwin, E. Blincow, G. Peterson et al.,
J. Infect. Dis.,
155, 488-494 (1987)]. Hirschl et al. report that a 128-KD antigen is useful. A. R. Stacey, D. G. Newell et al. fractionate respective antigen components by HPLC, and report that antibodies to HP-derived urease are useful on serum diagnosis [A. R. Stacey, P. R. Hawtin and D. G. Newell,
Eur. J. Clin. Microbiol. Infect. Dis.,
9, 732-737 (1990)]. D. J. Evans et al. establish ELISA using a 600-KD or more high molecular weight cell-associated protein as an antigen [D. J. Evans Jr., D. G. Evans, D. X. Grahm et al.,
Gastroenterology,
96, 1004-1008 (1986)].
On the other hand, T. Sugiyama et al. try to detect anti-HP antibodies in the blood by use of HP-derived antigens purified by monoclonal antibodies. As a result, they report that an antigen named “CP2 antigen” (molecular weight: about 60 KD) is excellent in detection specificity of HP, and that the antibody titer thereof has a high correlation with the pathology of gastritis [T. Sugiyama et al.,
Gastroenterology,
101, 77-83 (1991) and Toshiroh Sugiyama et al.,
Nippon Shokakibyo Gakkaisi,
85, 1128 (1988)].
In order to actually obtain the HP antigens by the conventional methods, it has hitherto been necessary to isolate the antigens directly from HP cells. However, in order to obtain the HP cells, it is necessary to use expensive liquid media containing 10% horse sera under microaerobic conditions and to conduct culture for a long period of time of about 5 days. Further, the content of the target antigens in all the cells is so small that purification by separation is difficult, resulting in difficulty of obtaining purified antigens in amounts as large as industrially available.
Also for the CP2 antigen which is considered to be particularly useful as a specific antigen, its purification by separation has been difficult and complicated.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide amino acid sequences showing the primary structure of HP-derived polypeptides, DNAs coding for said polypeptides, vectors containing said DNAs, and transformants into which said vectors are introduced, considering the necessity of obtaining genes of the HP-derived polypeptides and enabling mass production by genetic engineering techniques, in order to increase the industrial availability of the HP-derived polypeptides utilized as antigens (CP2 antigen) which mainly make possible specific and quantitative inspection of HP.
Another object of the present invention is to provide a method for preparing said polypeptides.
A further object of the present invention is to provide use of said polypeptides.
Other and further objects of this invention will be apparent from the following description.
As a result of intensive investigation for attaining the above-mentioned objects, the present inventors have succeeded in cloning CP2 antigen genes of HP, and determining the primary structure of the polypeptides of the present invention, the CP2 antigens, from their nucleotide sequences, thus competing the present invention.
That is, the present invention provides:
(1) A polypeptide having an amino acid sequence represented by SEQ ID NO 4 SEQ ID: 5 or SEQ ID NO:6, or a part thereof;
(2) A DNA coding for an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or a part thereof;
(3) A DNA having a nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or a part thereof;
(4) A recombinant vector comprising the DNA described in (2) or (3);
(5) A host cell transformed with the recombinant vector described in (4);
(6) A method for producing the polypeptide described in (1) comprising cultivating the host cell described in (5) in a medium and collecting the polypeptide from the resulting culture product;
(7) A reagent for assay of an anti-
Helicobacter pylori
antibody comprising the polypeptide described in (1);
(8) A reagent for detection of a
Helicobacter pylori
gene comprising a DNA described in (2) or (3), or a DNA complementary to said DNA, and has a property of hybridizing to a
Helicobacter pylori
gene;
(9) A reagent for detection of a
Helicobacter pylori
gene by polymerase chain reaction (PCR) method comprising at least two oligonucleotide primers which are selected from the group consisting of the DNAs described in (2) or (3), and complemental DNAs thereof; and
(10) The reagent of (9) wherein the reagent comprising two oligonucleotide primers, and said oligonucleotide primers have the following characteristics:
(i) one primer hybridyzing a strand of
Helicobacter pyloli
gene and another primer hybridyzing a strand complementary to the strand, and
(ii) being capable of giving an extention product by synthesis, and said extent
Hirayasu Kazunari
Kawabata Tomohisa
Sugiyama Tosiro
Tanaka Takumi
Edwards & Angell LLP
Neuner George W.
Portner Ginny Allen
Smith Lynette R. F.
Sugiyama Tosiro
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