JP2004537953A - New compound - Google Patents

Abstract

The present invention relates to BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066, and BASB071 polypeptides, and BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, and BASB065, BASB065, and BASB065, BASB065, and BASB066, BASB065, and BASB065, and BASB065, and BASB066 and BASB065, and BASB066 and BASB065. Techniques for producing such polypeptides using techniques are provided. The present invention also provides methods for diagnosis, prognosis, and treatment using them.

Description

【０３４９】
【化２】

【０３５０】
【化３】

【０３５１】
【化４】

【０３５２】
【化５】

【０３５３】
【表１】

【０３５４】
寄託した材料
ナイセリア・メニンギティディス血清型Ｂ株を含む寄託物を１９９７年６月２２日にアメリカ基準培養コレクション（この明細書では“ＡＴＣＣ”と表記）に寄託し、寄託番号１３０９０が与えられた。この寄託物はナイセリア・メニンギティディス（アルブレヒトとゴーン）と表記され、ナイセリア・メニンギティディス単離体から構成された、凍結乾燥された１．５〜２．９ｋｂの挿入体ライブラリーである。この寄託物は、Ｉｎｔ． Ｂｕｌｌ． Ｂａｃｔｅｒｉｏｌ． Ｎｏｍｅｎｃｌ． Ｔａｘｏｎ．、第８巻、１〜１５ページ、１９５８年に記載されている。
【０３５５】
ナイセリア・メニンギティディス菌株寄託物は、この明細書では、“寄託された菌株”または“寄託された菌株のＤＮＡ”と呼ぶ。
【０３５６】
寄託された菌株は、完全長ＢＡＳＢ０５１、ＢＡＳＢ０５７、ＢＡＳＢ０６０、ＢＡＳＢ０６１、ＢＡＳＢ０６３、ＢＡＳＢ０６５、ＢＡＳＢ０６６、及びＢＡＳＢ０７１遺伝子を含んでいる。寄託された菌株に含まれているポリヌクレオチドの配列と、それによってコードされるポリペプチドのアミノ酸配列は、この明細書における配列の記述と何らかの問題が起こったときに照合する対象となる。
【０３５７】
菌株の寄託は、特許手続き上の微生物の寄託の国際的承認に関するブダペスト条約に基づいてなされた。菌株は、特許発行の際に一般に対して公開されてそれが取り消されることはなく、しかもその際の制約または条件もない。寄託された菌株は当業者の便宜のために提供されるだけであり、３５ＵＳＣ§１１２のもとで必要とされるように、寄託が効力発生のために必要とされることを認めるものではない。[0001]
Technical field to which the invention belongs
The present invention relates to polynucleotides (as used herein, "BASB051 polynucleotide", "BASB057 polynucleotide", "BASB060 polynucleotide", "BASB061 polynucleotide", "BASB063 polynucleotide", "BASB065 polynucleotide", "BASB066" Polynucleotides "and" BASB071 polynucleotides ", polypeptides encoded by these polynucleotides (herein," BASB051 "," BASB057 "," BASB060 "," BASB061 "," BASB063 ", "BASB065", "BASB066", and "BASB071" or "BASB051 polypeptide", "BASB057 polypeptide", "BA B060 polypeptide "," BASB061 polypeptide "," BASB063 polypeptide "," BASB065 polypeptide "," BASB066 polypeptide ", and" BASB071 polypeptide "), and recombinant materials and methods for producing them. It is about. In another aspect, the present invention also relates to methods of using these polypeptides and polynucleotides. Uses include, for example, vaccines against bacterial infections. In yet another aspect, the present invention relates to a diagnostic method for detecting infection with a given pathogen.
Background of the Invention
Neisseria meningitidis (N. meningitidis) is a gram-negative bacterium often isolated from the human upper respiratory tract. This bacterium can cause invasive bacterial diseases such as bacteremia and meningitis. There are geographical, seasonal, and yearly differences in the occurrence of meningitis (Schwartz, B., Moore, PS, Bloom, CV, Clin. Microbiol. Rev., Vol. 2 ( Supplement), pages S18-S24, 1989). In mild climate countries, the disease is usually caused by serotype B strains and occurs at a rate of 1 to 10 per 100,000 population per year, but sometimes even higher (Katsumaru). Ski, EB (1997), Commun. Dis. Rep. Rev., Vol. 7, pages R55-59, 1995; Scholten, RJPM, Bielmer, HA, Poole. Mann, JT, et al., Clin. Infect. Dis., 16, 237-246, 1993; Cruz, C., Paves, G., Aguilar, E. et al., Epidemiol. Infect., 105. Vol. 119-126, 1990).
[0002]
In central Africa, where predominantly serotype A meningococci predominate, the rate of infection can reach 1000 per 100,000 population per year (Schwartz, B., Moore, PS et al. , Bloom, CV, Clin. Microbiol. Rev., 2nd Supplement, S18-S24, 1989). Overall, most of meningitis is caused by meningococci of serotypes A, B, C, W-135, Y, and a tetravalent A, C, W-135, Y polysaccharide vaccine is available (Arnan, J., Armandon, F., Minar, MC, Rafe, C., J. Biol. Stand., 10, 335-339, 1982).
[0003]
Polysaccharide vaccines are currently being improved to be chemically linked to transport proteins (Lieberman, JM, Chu, SS, Wong, VK, et al., JAMA, vol. 275). Pp. 1499-1503, 1996).
[0004]
No serotype B vaccine is available. This is because B capsular polysaccharide was found not to be immunogenic. This is likely due to structural similarities to host components (Weil, FA, Artenstein, MS, Blunt, ML, et al., J. Infect. Dis., 126, 514-522, 1972; Finne, JM, Reinonen, M., Mekelle, PM, Lancet, 2, 355-357, 1983).
[0005]
Efforts have been made over the years and vaccines based on the outer membrane of N. meningitidis have been developed (De Moraes, JC, Perkins, B., Camargo, MC, et al., Lancet, vol. 340). Bujung, G., Hoybee, EA, Grossby, JK et al., Lancet, 338, 1991). The vaccine was found to be effective in 57-85% of older children (above 4 years) and adolescents.
[0006]
There are many bacterial outer membrane components in this vaccine. For example, PorA, PorB, Rmp, Opc, Opa, FrpB. However, the contribution of these components to the observed protective effects needs to be further clarified. It has been shown that other outer membrane components of bacteria, such as TbpB and NspA, may be involved in immune defense by using animal or human antibodies (Martin, D., Cadüd). Lisoro, L., Met-Wilmott, C., Dumas, P., N., Amel, J., Brodow, BR, J. Exp. Med., 185, 1173-1183, 1997; Et al., Inf. Immun., 63, 884-890 (1995)). The mechanism of immune defense may be related to antibody-mediated bacterial activity and opsonophagocytosis.
[0007]
Bacteremia model animals have been used to combine all the mechanisms mediated by antibodies (Saukkonen, K., Reynonen, M., Abdilai, H., Pourman, JT, Vaccine, No. 7, 325-328, 1989). It is generally accepted that late complement component-mediated bactericidal mechanisms are critical for immunity to meningitis (Ross, SC, Rosenthal, PJ, Barberik, HM). , Densen, P., J. Infect. Dis., 155, 1266-1275, 1987).
[0008]
The frequency of infection with Neisseria meningitidis has increased dramatically over the past few decades. This is due to the emergence of bacteria that are resistant to many antibiotics and an increasing number of people with weak immune systems. It is no longer uncommon for Neisseria meningitidis to be isolated that is resistant to some or all of the standard antibiotics. This phenomenon has created a medical need that has never been encountered, and has created a demand for new antibacterial agents, vaccines, drug screening methods, and diagnostic methods to detect this organism.
Summary of the Invention
The present invention relates to BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066, and BASB071. Among them, BASB051, BASB057, BASB060, BASB061, BASB061, BASB0, BASB0, BASB065, BASB061 The present invention relates to BASB060, BASB061, BASB063, BASB065, BASB066, and BASB071 polynucleotides, and recombinant materials and methods for producing them. In another aspect, the present invention also relates to methods of using these polypeptides and polynucleotides. Methods of use include, inter alia, the prevention and treatment of microbial diseases. In yet another aspect, the present invention relates to a diagnostic method for detecting a disease or abnormality associated with a microbial infection. For example, it relates to a method for detecting the expression or activity of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066, and BASB071 polypeptide or polynucleotide.
[0009]
Various alterations and modifications in the spirit and scope of the invention disclosed herein will become readily apparent to those skilled in the art from reading the following and other portions of this specification.
Description of the invention
The present invention relates to BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066, and BASB071 polypeptides and polynucleotides, which are described in detail below. The invention especially relates to the nucleotide sequences given by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 and the amino acids given by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066, and BASB071 each having an array. It is understood that the sequences listed below in the sequence listing as "DNA" represent specific examples of one embodiment of the present invention. One of skill in the art would know that such sequences can generally be used in polynucleotides (including ribopolynucleotides).
Polypeptide
According to one aspect of the invention, in this specification "BASB051", "BASB057", "BASB060", "BASB061", "BASB063", "BASB065", "BASB066", and "BASB071" or "BASB051 polypeptide". Neisseria meningi, called "BASB057 polypeptide", "BASB060 polypeptide", "BASB061 polypeptide", "BASB063 polypeptide", "BASB065 polypeptide", "BASB066 polypeptide", and "BASB071 polypeptide". In addition to polypeptides of Tidis, there are provided biologically, diagnostically, prophylactically, clinically, or therapeutically effective variants of these polypeptides and compositions comprising them.
[0010]
According to the present invention, the following is further provided.
(A) comprises an amino acid sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, and most preferably at least 97-99% identical or completely identical to SEQ ID NO: 2, An isolated polypeptide.
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 1 over the entire length of SEQ ID NO: 1 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that corresponds to:
(C) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete match with the amino acid sequence of SEQ ID NO: 2 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that encodes the polypeptide.
[0011]
The BASB051 polypeptide given in SEQ ID NO: 2 is a BASB051 polypeptide derived from Neisseria meningitidis strain ATCC13090.
[0012]
The present invention also provides immunogenic fragments of the BASB051 polypeptide. That is, a continuous portion of a BASB051 polypeptide having the same or substantially the same immunological activity as the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 is provided. In essence, this fragment (if necessary when bound to a carrier) can enhance the immune response that recognizes the BASB051 polypeptide. The immunogenic fragment may include, for example, a BASB051 polypeptide lacking an N-terminal leader sequence, and / or a transmembrane region, and / or a C-terminal anchor region. In one preferred embodiment, the immunogenic fragment of BASB051 according to the invention has at least 85%, preferably at least 90%, more preferably at least 95%, identity to SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, Most preferably, at least 97-99% comprises substantially all of the extracellular region of the matched polypeptide.
[0013]
According to the present invention, the following is further provided.
(A) comprises an amino acid sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, and most preferably at least 97-99% identical or completely identical to SEQ ID NO: 4, An isolated polypeptide.
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that corresponds to:
(C) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete match with the amino acid sequence of SEQ ID NO: 4 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that encodes the polypeptide.
[0014]
The BASB057 polypeptide given in SEQ ID NO: 4 is a BASB057 polypeptide derived from Neisseria meningitidis strain ATCC13090.
[0015]
The present invention also provides immunogenic fragments of the BASB057 polypeptide. That is, a continuous portion of a BASB057 polypeptide having the same or substantially the same immunological activity as the polypeptide comprising the amino acid sequence of SEQ ID NO: 4 is provided. In short, this fragment (if necessary when bound to a carrier) can improve the immune response that recognizes the BASB057 polypeptide. The immunogenic fragment may include, for example, a BASB057 polypeptide lacking an N-terminal leader sequence, and / or a transmembrane region, and / or a C-terminal anchor region. In one preferred embodiment, the immunogenic fragment of BASB057 according to the invention has at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity with SEQ ID NO: 4 over the entire length of SEQ ID NO: 4, Most preferably, at least 97-99% comprises substantially all of the extracellular region of the matched polypeptide.
[0016]
According to the present invention, the following is further provided.
(A) comprises an amino acid sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, and most preferably at least 97-99% identical or completely identical to SEQ ID NO: 6, An isolated polypeptide.
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 5 over the entire length of SEQ ID NO: 5 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that corresponds to:
(C) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete match with the amino acid sequence of SEQ ID NO: 6 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that encodes the polypeptide.
[0017]
The BASB060 polypeptide given in SEQ ID NO: 6 is a BASB060 polypeptide derived from Neisseria meningitidis strain ATCC13090.
[0018]
The present invention also provides immunogenic fragments of the BASB060 polypeptide. That is, a continuous portion of a BASB060 polypeptide having the same or substantially the same immunological activity as the polypeptide comprising the amino acid sequence of SEQ ID NO: 6 is provided. In essence, this fragment (if necessary when bound to a carrier) can improve the immune response that recognizes the BASB060 polypeptide. The immunogenic fragment may include, for example, a BASB060 polypeptide lacking an N-terminal leader sequence, and / or a transmembrane region, and / or a C-terminal anchor region. In a preferred embodiment, the immunogenic fragment of BASB051 according to the present invention has at least 85%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 6 over the entire length of SEQ ID NO: 6, Most preferably, at least 97-99% comprises substantially all of the extracellular region of the matched polypeptide.
[0019]
According to the present invention, the following is further provided.
(A) comprises an amino acid sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, and most preferably at least 97-99% identical or completely identical to SEQ ID NO: 8, An isolated polypeptide.
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 7 over the entire length of SEQ ID NO: 7 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that corresponds to:
(C) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete match with the amino acid sequence of SEQ ID NO: 8 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that encodes the polypeptide.
[0020]
The BASB061 polypeptide given in SEQ ID NO: 8 is a BASB061 polypeptide derived from Neisseria meningitidis strain ATCC13090.
[0021]
The present invention also provides immunogenic fragments of the BASB061 polypeptide. That is, a continuous portion of the BASB061 polypeptide having the same or substantially the same immunological activity as the polypeptide comprising the amino acid sequence of SEQ ID NO: 8 is provided. In essence, this fragment (if necessary when bound to a carrier) can improve the immune response that recognizes the BASB061 polypeptide. The immunogenic fragment may comprise, for example, a BASB061 polypeptide lacking an N-terminal leader sequence and / or a transmembrane region and / or a C-terminal anchor region. In one preferred embodiment, the immunogenic fragment of BASB051 according to the invention has at least 85%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8, Most preferably, at least 97-99% comprises substantially all of the extracellular region of the matched polypeptide.
[0022]
According to the present invention, the following is further provided.
(A) comprises an amino acid sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, and most preferably at least 97-99% identical or completely identical to SEQ ID NO: 10, An isolated polypeptide.
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 9 over the entire length of SEQ ID NO: 9 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that corresponds to:
(C) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete match with the amino acid sequence of SEQ ID NO: 10 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that encodes the polypeptide.
[0023]
The BASB063 polypeptide given in SEQ ID NO: 10 is a BASB063 polypeptide derived from Neisseria meningitidis strain ATCC13090.
[0024]
The present invention also provides immunogenic fragments of the BASB063 polypeptide. That is, a continuous portion of a BASB063 polypeptide having the same or substantially the same immunological activity as the polypeptide comprising the amino acid sequence of SEQ ID NO: 10 is provided. In essence, this fragment (if necessary when bound to a carrier) can enhance the immune response that recognizes the BASB063 polypeptide. The immunogenic fragment may include, for example, a BASB063 polypeptide lacking an N-terminal leader sequence, and / or a transmembrane region, and / or a C-terminal anchor region. In one preferred embodiment, the immunogenic fragment of BASB063 according to the invention has at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity with SEQ ID NO: 10 over the entire length of SEQ ID NO: 10, Most preferably, at least 97-99% comprises substantially all of the extracellular region of the matched polypeptide.
[0025]
According to the present invention, the following is further provided.
(A) comprises an amino acid sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, and most preferably at least 97-99% identical or completely identical to SEQ ID NO: 12, An isolated polypeptide.
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 11 over the entire length of SEQ ID NO: 11 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that corresponds to:
(C) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete match with the amino acid sequence of SEQ ID NO: 12 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that encodes the polypeptide.
[0026]
The BASB065 polypeptide given in SEQ ID NO: 12 is a BASB065 polypeptide derived from Neisseria meningitidis strain ATCC13090.
[0027]
The present invention also provides immunogenic fragments of the BASB065 polypeptide. That is, a continuous portion of a BASB065 polypeptide having the same or substantially the same immunological activity as the polypeptide comprising the amino acid sequence of SEQ ID NO: 12 is provided. In essence, this fragment (if necessary when bound to a carrier) can improve the immune response that recognizes the BASB065 polypeptide. The immunogenic fragment may comprise, for example, a BASB065 polypeptide lacking an N-terminal leader sequence and / or a transmembrane region and / or a C-terminal anchor region. In one preferred embodiment, the immunogenic fragment of BASB065 according to the invention has at least 85%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 12 over the entire length of SEQ ID NO: 12, Most preferably, at least 97-99% comprises substantially all of the extracellular region of the matched polypeptide.
[0028]
According to the present invention, the following is further provided.
(A) comprises an amino acid sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, and most preferably at least 97-99% identical or completely identical to SEQ ID NO: 14, An isolated polypeptide.
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 13 over the entire length of SEQ ID NO: 13 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that corresponds to:
(C) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete match with the amino acid sequence of SEQ ID NO: 14 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that encodes the polypeptide.
[0029]
The BASB066 polypeptide given in SEQ ID NO: 14 is a BASB066 polypeptide derived from Neisseria meningitidis strain ATCC13090.
[0030]
The present invention also provides immunogenic fragments of the BASB066 polypeptide. That is, a continuous portion of a BASB066 polypeptide having the same or substantially the same immunological activity as the polypeptide comprising the amino acid sequence of SEQ ID NO: 14 is provided. In essence, this fragment (if necessary when bound to a carrier) can enhance the immune response that recognizes the BASB066 polypeptide. The immunogenic fragment may include, for example, a BASB066 polypeptide lacking an N-terminal leader sequence, and / or a transmembrane region, and / or a C-terminal anchor region. In one preferred embodiment, the immunogenic fragment of BASB066 according to the invention has at least 85%, preferably at least 90%, more preferably at least 95%, identity to SEQ ID NO: 14 over the entire length of SEQ ID NO: 14, Most preferably, at least 97-99% comprises substantially all of the extracellular region of the matched polypeptide.
[0031]
According to the present invention, the following is further provided.
(A) comprises an amino acid sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, and most preferably at least 97-99% identical or completely identical to SEQ ID NO: 16, An isolated polypeptide.
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 15 over the entire length of SEQ ID NO: 15 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that corresponds to:
(C) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete match with the amino acid sequence of SEQ ID NO: 16 A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that encodes the polypeptide.
[0032]
The BASB071 polypeptide given in SEQ ID NO: 16 is a BASB071 polypeptide derived from Neisseria meningitidis strain ATCC13090.
[0033]
The present invention also provides immunogenic fragments of the BASB071 polypeptide. That is, a continuous portion of a BASB071 polypeptide having the same or substantially the same immunological activity as the polypeptide comprising the amino acid sequence of SEQ ID NO: 16 is provided. In essence, this fragment (if necessary when bound to a carrier) can improve the immune response that recognizes the BASB071 polypeptide. The immunogenic fragment may include, for example, a BASB071 polypeptide lacking an N-terminal leader sequence and / or a transmembrane region and / or a C-terminal anchor region. In one preferred embodiment, the immunogenic fragment of BASB071 according to the invention has at least 85%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 16 over the entire length of SEQ ID NO: 16, Most preferably, at least 97-99% comprises substantially all of the extracellular region of the matched polypeptide.
[0034]
A fragment is a polypeptide having an amino acid sequence that entirely matches a portion, but not all, of any amino acid sequence of any polypeptide of the present invention. The fragments may be "independent" as in the case of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066, and BASB071. That is, it may be contained in a larger polypeptide to form a part or a region thereof. Most preferably, it forms a single contiguous region within a larger single polypeptide.
[0035]
Preferred fragments include, for example, truncated polypeptides containing a portion of the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or variants thereof. Specific examples include contiguous residue sequences containing amino and / or carboxyl terminal amino acid sequences. Degraded forms of the polypeptides of the invention produced by or in the host cell are also preferred. Even more preferred are fragments having structural or functional properties, such as α-helix and α-helix forming regions, β-sheet and β-sheet forming regions, turns and turn-forming regions, coils and coil-forming regions, hydrophilic regions, Examples include a hydrophobic region, an α amphipathic region, a β amphipathic region, a flexible region, a surface forming region, a substrate binding region, and a fragment containing an antibody highly indicating region.
[0036]
Preferred fragments further include an amino acid sequence having at least 15, 20, 30, 40, 50, or 100 contiguous amino acids from the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16. Or at least 15, 20, 30, 40, 50 or 100 truncated or removed from the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 And an isolated polypeptide comprising an amino acid sequence having a series of amino acids.
[0037]
The polypeptide fragments of the present invention can be used to produce the corresponding full-length polypeptide by peptide synthesis. Thus, this fragment can be used as an intermediate to produce a full-length polypeptide of the invention.
[0038]
Particularly preferred are substitutions, deletions or additions of several amino acids, e.g. 5-10, 1-5, 1-3, 1-2, or 1 amino acid, in any combination. Mutant.
[0039]
A polypeptide or immunogenic fragment of the invention may be in the form of the "mature" protein or may be part of a larger protein such as a precursor or fusion protein. It is often desirable to include additional amino acid sequences, including secretory or leader sequences, prosequences, additional histidine residues and other sequences useful for purification, and additional sequences to maintain stability during recombinant production. is there. Furthermore, it is conceivable to add an exogenous polypeptide, lipid tail or polynucleotide sequence to improve the immunity of the finally obtained molecule.
[0040]
According to one aspect, the invention relates to a polypeptide of the invention or a fragment thereof, and various parts of the constant regions of the heavy or light chains of immunoglobulins (IgG, IgM, IgA, IgE) of different subclasses. Genetically engineered soluble fusion proteins. Preferred as immunoglobulins are the constant parts of the heavy chains of human IgG, especially IgG1, in which fusion takes place in the hinge region. In a particular embodiment, the Fc portion can be removed simply by incorporating a cleavage sequence that can be cleaved with blood coagulation factor Xa.
[0041]
Further, the present invention relates to a method for preparing the fusion protein by genetic engineering, and to screening, diagnosis, and treatment of drugs using the fusion protein. Yet another aspect of the invention also relates to a polynucleotide encoding the fusion protein. Illustrative examples of fusion protein technology can be found in International Patent Application Nos. WO94 / 29458 and WO94 / 22914.
[0042]
The expression level of this protein in an expression system can be improved by chemically binding or expressing it as a recombinant fusion protein, as compared with a non-fusion protein. The fusion partner may serve to provide a T helper epitope (immune fusion partner), preferably a T helper epitope recognized by humans, or may express the protein more efficiently than the original recombinant protein (expression). Enhancer). Preferably, the fusion partner will be both an immunological fusion partner and an expression enhancer partner.
[0043]
Fusion partners include protein D from Haemophilus influenzae and the non-structural protein NS1 (hemagglutinin) from influenza virus. Another fusion partner is a protein known as LytA. It is preferable to use the C-terminal part of this protein. LytA, when N-acetyl-L-alanine amidase and the amidase LytA (encoded by the LytA gene (Gene, Vol. 43, pp. 265-272, 1986)), have specific binding to the peptidoglycan backbone. It is derived from Streptococcus pneumoniae, which synthesizes autolysin that degrades chemically. The C-terminal region of the LytA protein is involved in the affinity for choline and analogs of choline such as DEAE. Utilizing this property, a plasmid expressing C-LytA of Escherichia coli has been developed which is useful for expression of the fusion protein. Purification of hybrid proteins having a C-LytA fragment at the amino terminus is described in Biotechnology, Vol. 10, pp. 79-798, 1992. It is possible to use a repeating portion at the C-terminus of the LytA protein starting from residue number 178, for example, residue numbers 188 to 305.
[0044]
The present invention also includes variants of the above polypeptides. That is, polypeptides that differ only in conserved amino acid substitutions compared to a reference standard are included. In addition, a conserved amino acid substitution means that one residue is replaced with another residue having similar characteristics. Typical examples of such substitutions are substitutions of alanine, valine, leucine and isoleucine; substitution of serine for threonine; substitution of acidic residues aspartic acid and glutamic acid; substitution of asparagine for glutamine; Arginine substitution; substitution of the aromatic residue phenylalanine for tyrosine.
[0045]
The polypeptide of the present invention can be prepared by any appropriate method. Such polypeptides include isolated natural polypeptides, recombinant polypeptides, synthesized polypeptides, and polypeptides made by combining these methods. Methods for preparing such polypeptides are well-known in the art.
[0046]
Most preferably, the polypeptides of the present invention are derived from Neisseria meningitidis, but are also preferably those obtained from other organisms of the same genus in the same genus. The polypeptides of the present invention may be obtained, for example, from organisms of the same taxonomic family or order.
Polynucleotide
One object of the present invention is to provide a polynucleotide encoding a BASB051 polypeptide, particularly a polynucleotide encoding a polypeptide designated herein as BASB051.
[0047]
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding a BASB051 polypeptide comprising the full length gene and having the sequence given in SEQ ID NO: 1, or a region encoding a variant thereof. I have.
[0048]
The BASB051 polynucleotide provided in SEQ ID NO: 1 is a BASB051 polynucleotide derived from Neisseria meningitidis strain ATCC13090.
[0049]
According to yet another aspect of the present invention there is provided an isolated nucleic acid molecule encoding and / or expressing a BASB051 polypeptide and polynucleotide, particularly a Neisseria meningitidis BASB051 polypeptide and polynucleotide. . Such nucleic acid molecules include, for example, untreated RNA, ribozyme RNA, mRNA, cDNA, genomic DNA, B-DNA, Z-DNA. In yet another embodiment of the present invention, there are biologically, diagnostically, prophylactically, clinically, or therapeutically effective polynucleotides and polypeptides, variants thereof, and compositions comprising them.
[0050]
Another aspect of the invention encodes a BASB051 polypeptide having the deduced amino acid sequence of SEQ ID NO: 2, closely related to the isolated polynucleotide comprising at least one full-length gene Polynucleotides and variants thereof.
[0051]
In a particularly preferred embodiment of the present invention, there is provided a BASB051 polypeptide derived from Neisseria meningitidis, comprising or consisting of the amino acid sequence of SEQ ID NO: 2, or a variant thereof.
[0052]
Using the information in this specification, such as the polynucleotide sequence given in SEQ ID NO: 1, the book encoding BASB051 polypeptide can be obtained using standard cloning and screening methods and then obtaining full-length clones. The polynucleotide of the invention can be obtained. As a standard cloning method and screening method, there is a method of cloning and sequencing chromosomal DNA from bacteria using Neisseria meningitidis cells as a starting material. To obtain a polynucleotide sequence of the present invention, such as the polynucleotide sequence given in SEQ ID NO: 1, a library of clones of chromosomal DNA of Neisseria meningitidis in E. coli or other suitable host Is generally probed with a radiolabeled oligonucleotide derived from the subsequence, preferably a 17-mer or longer. The clones having the same DNA as the probe DNA can then be identified using stringent hybridization. By sequencing the individual clones thus identified through hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence, the polynucleotide sequence is extended in both directions, The sequence of the full-length gene can be determined. Conveniently, such sequencing can be performed, for example, using denatured double-stranded DNA prepared from a plasmid clone. Suitable methods are described in Maniatis, T., Flitch, E .; F. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) (especially "Hybridization Screening 1.90 and Sequencing of denatured double-stranded DNA template 13.70). Genomic DNA can be directly sequenced to obtain the sequence of the full-length gene. As evidence of the present invention, each polynucleotide provided in SEQ ID NO: 1 was found in a DNA library derived from Neisseria meningitidis.
[0053]
In addition, each DNA sequence provided in SEQ ID NO: 1 contains an open reading frame encoding a protein having approximately the same number of amino acid residues as provided in SEQ ID NO: 2. The estimated molecular weight of this protein can be calculated using the molecular weight values of amino acid residues well known to those skilled in the art.
[0054]
The polynucleotide of SEQ ID NO: 1 encodes the polypeptide of SEQ ID NO: 2 between the start codon of nucleotide number 1 of SEQ ID NO: 1 and the stop codon starting at nucleotide number 802.
[0055]
According to still another aspect of the present invention, there is provided an isolated polynucleotide comprising: or an isolated polynucleotide consisting of:
(A) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 1 over the entire length of SEQ ID NO: 1 A polynucleotide sequence that matches, or
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or 100 over SEQ ID NO: 2 over the entire length of SEQ ID NO: 2 Polynucleotide sequence encoding a polypeptide with a% exact match.
[0056]
The polynucleotide encoding the polypeptide of the present invention includes a labeled probe or a detectable probe comprising the sequence of SEQ ID NO: 1 or a fragment thereof, including homologs and orthologs from species other than Neisseria meningitidis, or Under stringent hybridization conditions (eg, at a temperature of 45 to 65 ° C. and a SDS concentration of 0.1 to 1%) using a labeled probe or a detectable probe containing the sequence of Screening of such libraries and isolating genomic clones containing the full-length gene and / or its polynucleotide sequence.
[0057]
According to the present invention, there is provided a polynucleotide sequence corresponding to the entire length of the coding sequence (open reading frame) in SEQ ID NO: 1. The invention further provides a coding sequence for a mature polypeptide or a fragment thereof, as well as a coding sequence for a mature polypeptide or a fragment having another coding sequence in the reading frame. Another coding sequence is, for example, a sequence encoding a leader or secretory sequence, a preprotein sequence, a proprotein sequence, a preproprotein sequence. A polynucleotide of the invention may also include at least one non-coding sequence. By way of example, at least one non-coding 5 'or 3' sequence, such as a sequence that is transcribed but not translated, a termination signal (such as a ρ-dependent termination signal or a ρ-independent termination signal), a ribosome binding. Sites, Kozak sequences, mRNA stabilizing sequences, introns, polyadenylation signals, and the like, but are not limited thereto. The polynucleotide sequence may also include additional coding sequences that code for additional amino acids. For example, it can encode a marker sequence that facilitates purification of the fused polypeptide. In some embodiments of the invention, the marker sequence is a hexahistidine peptide (Genz et al., Proc. Natl. Acad. Sci. USA, 86, 821-824) provided in a pQE vector (Qiagen). 1989), or the HA peptide tag (Wilson et al., Cell, 37, 767, 1984). Either can help to purify the polypeptide sequence to be fused. The polynucleotide of the present invention also includes a polynucleotide having a structural gene and a sequence originally associated with the structural gene and controlling the expression of the gene. However, the polynucleotide contained in the polynucleotide of the present invention is not limited to this.
[0058]
The nucleotide sequence encoding the BASB051 polypeptide of SEQ ID NO: 2 may be the same as the polypeptide coding sequence contained in nucleotide numbers 1-801 of SEQ ID NO: 1, respectively. Alternatively, it may be a sequence encoding the polypeptide of SEQ ID NO: 2, as a result of the redundancy (degeneracy) of the genetic code.
[0059]
The expression "polynucleotide encoding a polypeptide" is used herein to refer to a polypeptide of the invention, in particular a bacterial polypeptide, more particularly a Neisseria meningi having the amino acid sequence given by SEQ ID NO: 2. A polynucleotide comprising a sequence encoding a Tidis BASB051 polypeptide. The expression may be a single contiguous or discontinuous region encoding a polypeptide (eg, an integrated phage, an integrated insert, an integrated vector sequence, a polynucleotide interrupted by an integrated transposon sequence, Or polynucleotides interrupted for RNA editing, genomic DNA rearrangement), as well as additional polynucleotides that may also contain coding and / or non-coding sequences. .
[0060]
The present invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the deduced amino acid sequence of SEQ ID NO: 2. Fragments of a polynucleotide of the invention can be used, for example, to synthesize a full-length polynucleotide of the invention.
[0061]
Furthermore, according to a particularly preferred embodiment, of the amino acid sequence of the BASB051 polypeptide given by SEQ ID NO: 2, some amino acid residues, for example 5 to 10, 1 to 5, 1 to 3, 2 Provided is a polynucleotide encoding a BASB051 mutant in which one or zero amino acid residues have any combination of substitutions, changes, deletions, and / or additions. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of the BASB051 polypeptide.
[0062]
Further, in accordance with a preferred embodiment of the present invention, a polynucleotide that is at least 85% identical to a polynucleotide encoding a BASB051 polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, and at least 85% complementary to the polynucleotide. And polynucleotides are provided. In this regard, polynucleotides that are at least 90% identical over the entire length are particularly preferred, and among such particularly preferred polynucleotides, those that are at least 95% identical are particularly preferred. Further, among polynucleotides having at least 95% identity, polynucleotides having at least 97% identity are more preferred, among which at least 98% identity, at least 99% identity are more preferred, and at least 99% identity. Are even more preferred.
[0063]
According to a preferred embodiment, there is provided a polynucleotide encoding a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA of SEQ ID NO: 1.
[0064]
According to some preferred embodiments of the present invention, there are provided polynucleotides that hybridize to a BASB051 polynucleotide sequence, eg, a polynucleotide of SEQ ID NO: 1, particularly under stringent conditions.
[0065]
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences described herein. In this regard, the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As used herein, the terms "stringent conditions", "stringent hybridization conditions" refer to hybridization that occurs only when at least 95%, and preferably at least 97%, matches between sequences. Specific examples of stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, Culturing overnight at 42 ° C. in a solution containing 20 μg / ml denatured and cleaved salmon sperm DNA, then washing the hybridization support at about 65 ° C. in 0.1 × SSC. Things. Hybridization and washing conditions are well known and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In particular in Chapter 11. Solution hybridization can also be performed on the polynucleotide sequences of the present invention.
[0066]
According to the invention, furthermore, a stringent hybridization of a suitable library containing the complete gene for the polynucleotide sequence given by SEQ ID NO: 1 with a probe having this polynucleotide sequence given by SEQ ID NO: 1 or a fragment thereof Provided is a polynucleotide consisting of a polynucleotide sequence obtained by screening under conditions and isolating the polynucleotide sequence, or a polynucleotide comprising such a polynucleotide sequence. Fragments useful for obtaining such polynucleotides include, for example, probes and primers described in detail elsewhere in this specification.
[0067]
The polynucleotide assay of the present invention is described elsewhere in this specification. For example, the polynucleotide of the present invention is used as a probe for hybridization of RNA, cDNA, and genomic DNA, and the full-length encoding BASB051 is used. It is possible to isolate a cDNA or genomic clone, or to isolate another gene having a high degree of matching with the BASB051 gene, particularly a cDNA or genomic clone having a high degree of sequence matching. The probe will generally contain at least 15 nucleotide residues or base pairs. The probe preferably contains at least 30 nucleotide residues or base pairs, and may contain at least 50 nucleotide residues or base pairs. Particularly preferred probes will contain more than 20 and less than 30 nucleotide residues or base pairs.
[0068]
The coding region of the BASB051 gene can be isolated by synthesizing an oligonucleotide probe using the DNA sequence given in SEQ ID NO: 1 and conducting screening using this probe. Therefore, a library of cDNA, genomic DNA, or mRNA is screened using a labeled oligonucleotide having a sequence complementary to the gene of the present invention, and it is confirmed to which member of the library the probe hybridizes.
[0069]
One object of the present invention is to provide a polynucleotide encoding a BASB057 polypeptide, in particular a polynucleotide encoding a polypeptide designated herein as BASB057.
[0070]
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding a BASB057 polypeptide comprising the full length gene and having the sequence given in SEQ ID NO: 3, or a region encoding a variant thereof. I have.
[0071]
The BASB057 polynucleotide provided by SEQ ID NO: 3 is a BASB057 polynucleotide derived from Neisseria meningitidis strain ATCC13090.
[0072]
According to yet another aspect of the present invention there is provided an isolated nucleic acid molecule encoding and / or expressing a BASB057 polypeptide and polynucleotide, in particular a Neisseria meningitidis BASB057 polypeptide and polynucleotide. . Such nucleic acid molecules include, for example, untreated RNA, ribozyme RNA, mRNA, cDNA, genomic DNA, B-DNA, Z-DNA. In yet another embodiment of the present invention, there are biologically, diagnostically, prophylactically, clinically, or therapeutically effective polynucleotides and polypeptides, variants thereof, and compositions comprising them.
[0073]
Another aspect of the invention encodes a BASB057 polypeptide having the deduced amino acid sequence of SEQ ID NO: 4, and is closely related to an isolated polynucleotide comprising at least one full-length gene Polynucleotides and variants thereof.
[0074]
In a particularly preferred embodiment of the present invention, there is provided a BASB057 polypeptide derived from Neisseria meningitidis, comprising or consisting of the amino acid sequence of SEQ ID NO: 4, or a variant thereof.
[0075]
Using the information in this specification, such as the polynucleotide sequence provided in SEQ ID NO: 3, standard cloning and screening methods can be used, followed by obtaining a full-length clone to obtain a book encoding the BASB057 polypeptide. The polynucleotide of the invention can be obtained. As a standard cloning method and screening method, there is a method of cloning and sequencing chromosomal DNA from bacteria using Neisseria meningitidis cells as a starting material. To obtain a polynucleotide sequence of the present invention, such as the polynucleotide sequence given by SEQ ID NO: 3, a library of chromosomal DNA clones of Neisseria meningitidis in E. coli or other suitable host Is generally probed with a radiolabeled oligonucleotide derived from the subsequence, preferably a 17-mer or longer. The clones having the same DNA as the probe DNA can then be identified using stringent hybridization. By sequencing the individual clones thus identified through hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence, the polynucleotide sequence is extended in both directions, The sequence of the full-length gene can be determined. Conveniently, such sequencing can be performed, for example, using denatured double-stranded DNA prepared from a plasmid clone. Suitable methods are described in Maniatis, T., Flitch, E .; F. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) (especially "Hybridization Screening 1.90 and Sequencing of denatured double-stranded DNA template 13.70). Genomic DNA can be directly sequenced to obtain the sequence of the full-length gene. As evidence of the present invention, each polynucleotide provided in SEQ ID NO: 1 was found in a DNA library derived from Neisseria meningitidis.
[0076]
In addition, each DNA sequence provided in SEQ ID NO: 3 contains an open reading frame encoding a protein having approximately the same number of amino acid residues as provided in SEQ ID NO: 4. The estimated molecular weight of this protein can be calculated using the molecular weight values of amino acid residues well known to those skilled in the art.
[0077]
The polynucleotide of SEQ ID NO: 3 encodes the polypeptide of SEQ ID NO: 4 between the start codon of nucleotide number 1 of SEQ ID NO: 3 and the stop codon starting at nucleotide number 1402.
[0078]
According to still another aspect of the present invention, there is provided an isolated polynucleotide comprising: or an isolated polynucleotide consisting of:
(A) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 A polynucleotide sequence that matches, or
(B) at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 97-99% identity or 100 over the entire length of SEQ ID NO: 4 Polynucleotide sequence encoding a polypeptide with a% exact match.
[0079]
The polynucleotide encoding the polypeptide of the present invention may be a labeled probe or a detectable probe comprising the sequence of SEQ ID NO: 3 or a fragment thereof, including homologs and orthologs from species other than Neisseria meningitidis, or Under stringent hybridization conditions (eg, at a temperature of 45 to 65 ° C. and a SDS concentration of 0.1 to 1%) using a labeled probe or a detectable probe containing the sequence of Screening of such libraries and isolating genomic clones containing the full-length gene and / or its polynucleotide sequence.
[0080]
According to the present invention, there is provided a polynucleotide sequence corresponding to the entire length of the coding sequence (open reading frame) in SEQ ID NO: 3. The invention further provides a coding sequence for a mature polypeptide or a fragment thereof, as well as a coding sequence for a mature polypeptide or a fragment having another coding sequence in the reading frame. Another coding sequence is, for example, a sequence encoding a leader or secretory sequence, a preprotein sequence, a proprotein sequence, a preproprotein sequence. A polynucleotide of the invention may also include at least one non-coding sequence. By way of example, at least one non-coding 5 'or 3' sequence, such as a sequence that is transcribed but not translated, a termination signal (such as a ρ-dependent termination signal or a ρ-independent termination signal), a ribosome binding. Sites, Kozak sequences, mRNA stabilizing sequences, introns, polyadenylation signals, and the like, but are not limited thereto. The polynucleotide sequence may also include additional coding sequences that code for additional amino acids. For example, it can encode a marker sequence that facilitates purification of the fused polypeptide. In some embodiments of the invention, the marker sequence is a hexahistidine peptide (Genz et al., Proc. Natl. Acad. Sci. USA, 86, 821-824) provided in a pQE vector (Qiagen). 1989), or the HA peptide tag (Wilson et al., Cell, 37, 767, 1984). Either can help to purify the polypeptide sequence to be fused. The polynucleotide of the present invention also includes a polynucleotide having a structural gene and a sequence originally associated with the structural gene and controlling the expression of the gene. However, the polynucleotide contained in the polynucleotide of the present invention is not limited to this.
[0081]
The nucleotide sequence encoding the BASB057 polypeptide of SEQ ID NO: 4 may be the same as the polypeptide coding sequence contained in nucleotides 1-1401 of SEQ ID NO: 3, respectively. Alternatively, it may be a sequence encoding the polypeptide of SEQ ID NO: 4 as a result of the redundancy (degeneracy) of the genetic code.
[0082]
The expression "polynucleotide encoding a polypeptide" is used herein to refer to a polypeptide of the invention, in particular a bacterial polypeptide, more particularly a Neisseria meningi having the amino acid sequence given by SEQ ID NO: 4. A polynucleotide comprising a sequence encoding a Tidis BASB057 polypeptide. The expression may be a single contiguous or discontinuous region encoding a polypeptide (eg, an integrated phage, an integrated insert, an integrated vector sequence, a polynucleotide interrupted by an integrated transposon sequence, Or polynucleotides interrupted for RNA editing, genomic DNA rearrangement), as well as additional polynucleotides that may also contain coding and / or non-coding sequences. .
[0083]
The invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the deduced amino acid sequence of SEQ ID NO: 4. Fragments of a polynucleotide of the invention can be used, for example, to synthesize a full-length polynucleotide of the invention.
[0084]
Furthermore, according to a particularly preferred embodiment, of the amino acid sequence of the BASB057 polypeptide given by SEQ ID NO: 4, some amino acid residues, for example 5-10, 1-5, 1-3, 2 Provided is a polynucleotide encoding a BASB057 variant in which 1, 0 amino acid residues have any combination of substitutions, changes, deletions, and / or additions. Particularly preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of BASB057 polypeptide.
[0085]
Further, in accordance with a preferred embodiment of the present invention, a polynucleotide that matches at least 85% over its entire length with a polynucleotide encoding a BASB057 polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, and is complementary to the polynucleotide. And polynucleotides are provided. In this regard, polynucleotides that are at least 90% identical over the entire length are particularly preferred, and among such particularly preferred polynucleotides, those that are at least 95% identical are particularly preferred. Further, among polynucleotides having at least 95% identity, polynucleotides having at least 97% identity are more preferred, among which at least 98% identity, at least 99% identity are more preferred, and at least 99% identity. Are even more preferred.
[0086]
According to a preferred embodiment, there is provided a polynucleotide encoding a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA of SEQ ID NO: 3.
[0087]
According to some preferred embodiments of the present invention, there are provided polynucleotides that hybridize to a BASB057 polynucleotide sequence, eg, a polynucleotide of SEQ ID NO: 3, particularly under stringent conditions.
[0088]
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences described herein. In this regard, the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As used herein, the terms "stringent conditions", "stringent hybridization conditions" refer to hybridization that occurs only when at least 95%, and preferably at least 97%, matches between sequences. Specific examples of stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, Culturing overnight at 42 ° C. in a solution containing 20 μg / ml denatured and cleaved salmon sperm DNA, then washing the hybridization support at about 65 ° C. in 0.1 × SSC. Things. Hybridization and washing conditions are well known and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In particular in Chapter 11. Solution hybridization can also be performed on the polynucleotide sequences of the present invention.
[0089]
According to the invention, furthermore, a suitable library containing the complete gene for the polynucleotide sequence given in SEQ ID NO: 3 is subjected to stringent hybridization using a probe having this polynucleotide sequence given in SEQ ID NO: 3 or a fragment thereof. Provided is a polynucleotide consisting of a polynucleotide sequence obtained by screening under conditions and isolating the polynucleotide sequence, or a polynucleotide comprising such a polynucleotide sequence. Fragments useful for obtaining such polynucleotides include, for example, probes and primers described in detail elsewhere in this specification.
[0090]
The polynucleotide assay of the present invention is described elsewhere in this specification. For example, the polynucleotide of the present invention is used as a probe for hybridization of RNA, cDNA, and genomic DNA, and the full-length encoding BASB057 is used. It is possible to isolate a cDNA or genomic clone, or to isolate another gene having a high degree of matching with the BASB057 gene, in particular, a cDNA or genomic clone having a high degree of sequence matching. The probe will generally contain at least 15 nucleotide residues or base pairs. The probe preferably contains at least 30 nucleotide residues or base pairs, and may contain at least 50 nucleotide residues or base pairs. Particularly preferred probes will contain more than 20 and less than 30 nucleotide residues or base pairs.
[0091]
The coding region of the BASB057 gene can be isolated by synthesizing an oligonucleotide probe using the DNA sequence shown in SEQ ID NO: 3 and performing screening using this probe. Therefore, a library of cDNA, genomic DNA, or mRNA is screened using a labeled oligonucleotide having a sequence complementary to the gene of the present invention, and it is confirmed to which member of the library the probe hybridizes.
[0092]
One object of the present invention is to provide a polynucleotide encoding a BASB060 polypeptide, particularly a polynucleotide encoding a polypeptide designated herein as BASB060.
[0093]
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding a BASB060 polypeptide comprising the full length gene and having the sequence given in SEQ ID NO: 5, or a region encoding a variant thereof. I have.
[0094]
The BASB060 polynucleotide provided in SEQ ID NO: 5 is a BASB060 polynucleotide derived from Neisseria meningitidis strain ATCC13090.
[0095]
According to yet another aspect of the present invention there is provided an isolated nucleic acid molecule encoding and / or expressing a BASB060 polypeptide and polynucleotide, particularly a Neisseria meningitidis BASB060 polypeptide and polynucleotide. . Such nucleic acid molecules include, for example, untreated RNA, ribozyme RNA, mRNA, cDNA, genomic DNA, B-DNA, Z-DNA. In yet another embodiment of the present invention, there are biologically, diagnostically, prophylactically, clinically, or therapeutically effective polynucleotides and polypeptides, variants thereof, and compositions comprising them.
[0096]
Another aspect of the invention encodes a BASB060 polypeptide having the deduced amino acid sequence of SEQ ID NO: 6, closely related to an isolated polynucleotide comprising at least one full-length gene. Polynucleotides and variants thereof.
[0097]
In a particularly preferred embodiment of the present invention, there is provided a BASB060 polypeptide derived from Neisseria meningitidis and comprising or consisting of the amino acid sequence of SEQ ID NO: 6, or a variant thereof.
[0098]
Using the information in this specification, such as the polynucleotide sequence provided in SEQ ID NO: 5, the book encoding BASB060 polypeptide can be obtained using standard cloning and screening methods and then obtaining full-length clones. The polynucleotide of the invention can be obtained. As a standard cloning method and screening method, there is a method of cloning and sequencing chromosomal DNA from bacteria using Neisseria meningitidis cells as a starting material. To obtain a polynucleotide sequence of the present invention, such as the polynucleotide sequence given in SEQ ID NO: 5, a library of chromosomal DNA clones of Neisseria meningitidis in E. coli or other suitable host Is generally probed with a radiolabeled oligonucleotide derived from the subsequence, preferably a 17-mer or longer. The clones having the same DNA as the probe DNA can then be identified using stringent hybridization. By sequencing the individual clones thus identified through hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence, the polynucleotide sequence is extended in both directions, The sequence of the full-length gene can be determined. Conveniently, such sequencing can be performed, for example, using denatured double-stranded DNA prepared from a plasmid clone. Suitable methods are described in Maniatis, T., Flitch, E .; F. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) (especially "Hybridization Screening 1.90 and Sequencing of denatured double-stranded DNA template 13.70). Genomic DNA can be directly sequenced to obtain the sequence of the full-length gene. As evidence of the present invention, each polynucleotide provided in SEQ ID NO: 5 was found in a DNA library derived from Neisseria meningitidis.
[0099]
In addition, each DNA sequence provided in SEQ ID NO: 5 contains an open reading frame encoding a protein having approximately the same number of amino acid residues as provided in SEQ ID NO: 6. The estimated molecular weight of this protein can be calculated using the molecular weight values of amino acid residues well known to those skilled in the art.
[0100]
The polynucleotide of SEQ ID NO: 5 encodes the polypeptide of SEQ ID NO: 6 between the start codon of nucleotide number 1 of SEQ ID NO: 5 and the stop codon starting at nucleotide number 418.
[0101]
According to still another aspect of the present invention, there is provided an isolated polynucleotide comprising: or an isolated polynucleotide consisting of:
(A) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 5 over the entire length of SEQ ID NO: 5 A polynucleotide sequence that matches, or
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or 100 over SEQ ID NO: 6 over the entire length of SEQ ID NO: 6 % A polynucleotide sequence encoding an exactly matched polypeptide.
[0102]
The polynucleotide encoding the polypeptide of the present invention may be a labeled probe or a detectable probe consisting of the sequence of SEQ ID NO: 5 or a fragment thereof, including homologs and orthologs from species other than Neisseria meningitidis, or Under stringent hybridization conditions (eg, at a temperature of 45 to 65 ° C. and a SDS concentration of 0.1 to 1%) using a labeled probe or a detectable probe containing the sequence of Screening of such libraries and isolating genomic clones containing the full-length gene and / or its polynucleotide sequence.
[0103]
According to the present invention, there is provided a polynucleotide sequence corresponding to the entire coding sequence (open reading frame) in SEQ ID NO: 5. The invention further provides a coding sequence for a mature polypeptide or a fragment thereof, as well as a coding sequence for a mature polypeptide or a fragment having another coding sequence in the reading frame. Another coding sequence is, for example, a sequence encoding a leader or secretory sequence, a preprotein sequence, a proprotein sequence, a preproprotein sequence. A polynucleotide of the invention may also include at least one non-coding sequence. By way of example, at least one non-coding 5 'or 3' sequence, such as a sequence that is transcribed but not translated, a termination signal (such as a ρ-dependent termination signal or a ρ-independent termination signal), a ribosome binding. Sites, Kozak sequences, mRNA stabilizing sequences, introns, polyadenylation signals, and the like, but are not limited thereto. The polynucleotide sequence may also include additional coding sequences that code for additional amino acids. For example, it can encode a marker sequence that facilitates purification of the fused polypeptide. In some embodiments of the invention, the marker sequence is a hexahistidine peptide (Genz et al., Proc. Natl. Acad. Sci. USA, 86, 821-824) provided in a pQE vector (Qiagen). 1989), or the HA peptide tag (Wilson et al., Cell, 37, 767, 1984). Either can help to purify the polypeptide sequence to be fused. The polynucleotide of the present invention also includes a polynucleotide having a structural gene and a sequence originally associated with the structural gene and controlling the expression of the gene. However, the polynucleotide contained in the polynucleotide of the present invention is not limited to this.
[0104]
The nucleotide sequence encoding the BASB060 polypeptide of SEQ ID NO: 6 may be the same as the polypeptide coding sequence contained in nucleotide numbers 1-417 of SEQ ID NO: 1, respectively. Alternatively, it may be a sequence encoding the polypeptide of SEQ ID NO: 6, as a result of the redundancy (degeneracy) of the genetic code.
[0105]
The expression "polynucleotide encoding a polypeptide" is used herein to refer to a polypeptide of the invention, in particular a bacterial polypeptide, and more particularly a Neisseria meningi having the amino acid sequence given by SEQ ID NO: 6. It refers to a polynucleotide comprising a sequence encoding a Tidis BASB060 polypeptide. The expression may be a single contiguous or discontinuous region encoding a polypeptide (eg, an integrated phage, an integrated insert, an integrated vector sequence, a polynucleotide interrupted by an integrated transposon sequence, Or polynucleotides interrupted for RNA editing, genomic DNA rearrangement), as well as additional polynucleotides that may also contain coding and / or non-coding sequences. .
[0106]
The present invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the deduced amino acid sequence of SEQ ID NO: 6. Fragments of a polynucleotide of the invention can be used, for example, to synthesize a full-length polynucleotide of the invention.
[0107]
Furthermore, according to a particularly preferred embodiment, of the amino acid sequence of the BASB060 polypeptide given by SEQ ID NO: 6, some amino acid residues, for example 5-10, 1-5, 1-3, 2 Provided is a polynucleotide encoding a BASB060 variant in which one or zero amino acid residues have any combination of substitutions, changes, deletions, and / or additions. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of the BASB060 polypeptide.
[0108]
Further, in accordance with a preferred embodiment of the present invention, a polynucleotide that matches at least 85% over its entire length with a polynucleotide encoding a BASB060 polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, and is complementary to the polynucleotide. And polynucleotides are provided. In this regard, polynucleotides that are at least 90% identical over the entire length are particularly preferred, and among such particularly preferred polynucleotides, those that are at least 95% identical are particularly preferred. Further, among polynucleotides having at least 95% identity, polynucleotides having at least 97% identity are more preferred, among which at least 98% identity, at least 99% identity are more preferred, and at least 99% identity. Are even more preferred.
[0109]
According to a preferred embodiment, there is provided a polynucleotide encoding a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA of SEQ ID NO: 5.
[0110]
According to some preferred embodiments of the present invention, there are provided polynucleotides that hybridize to a BASB060 polynucleotide sequence, eg, the polynucleotide of SEQ ID NO: 5, especially under stringent conditions.
[0111]
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences described herein. In this regard, the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As used herein, the terms "stringent conditions", "stringent hybridization conditions" refer to hybridization that occurs only when at least 95%, and preferably at least 97%, matches between sequences. Specific examples of stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, Culturing overnight at 42 ° C. in a solution containing 20 μg / ml denatured and cleaved salmon sperm DNA, then washing the hybridization support at about 65 ° C. in 0.1 × SSC. Things. Hybridization and washing conditions are well known and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In particular in Chapter 11. Solution hybridization can also be performed on the polynucleotide sequences of the present invention.
[0112]
According to the invention, furthermore, a suitable library containing the complete gene for the polynucleotide sequence given in SEQ ID NO: 5 is subjected to stringent hybridization using a probe having this polynucleotide sequence given in SEQ ID NO: 5 or a fragment thereof. Provided is a polynucleotide consisting of a polynucleotide sequence obtained by screening under conditions and isolating the polynucleotide sequence, or a polynucleotide comprising such a polynucleotide sequence. Fragments useful for obtaining such polynucleotides include, for example, probes and primers described in detail elsewhere in this specification.
[0113]
The polynucleotide assay of the present invention is described elsewhere in this specification. For example, the polynucleotide of the present invention is used as a probe for hybridization of RNA, cDNA, and genomic DNA, and the full-length encoding BASB060 is used. It is possible to isolate a cDNA or genomic clone, or to isolate another gene having a high degree of matching with the BASB060 gene, in particular, a cDNA or genomic clone having a high degree of sequence matching. The probe will generally contain at least 15 nucleotide residues or base pairs. The probe preferably contains at least 30 nucleotide residues or base pairs, and may contain at least 50 nucleotide residues or base pairs. Particularly preferred probes will contain more than 20 and less than 30 nucleotide residues or base pairs.
[0114]
The coding region of the BASB060 gene can be isolated by synthesizing an oligonucleotide probe using the DNA sequence given in SEQ ID NO: 5 and conducting screening using this probe. Therefore, a library of cDNA, genomic DNA, or mRNA is screened using a labeled oligonucleotide having a sequence complementary to the gene of the present invention, and it is confirmed to which member of the library the probe hybridizes.
[0115]
One object of the present invention is to provide a polynucleotide encoding a BASB061 polypeptide, particularly a polynucleotide encoding a polypeptide designated herein as BASB061.
[0116]
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding the BASB061 polypeptide comprising the full length gene and having the sequence given in SEQ ID NO: 7, or a region encoding a variant thereof. I have.
[0117]
The BASB061 polynucleotide provided by SEQ ID NO: 7 is a BASB061 polynucleotide derived from Neisseria meningitidis strain ATCC13090.
[0118]
According to yet another aspect of the present invention there is provided an isolated nucleic acid molecule encoding and / or expressing a BASB061 polypeptide and polynucleotide, particularly a Neisseria meningitidis BASB061 polypeptide and polynucleotide. . Such nucleic acid molecules include, for example, untreated RNA, ribozyme RNA, mRNA, cDNA, genomic DNA, B-DNA, Z-DNA. In yet another embodiment of the present invention, there are biologically, diagnostically, prophylactically, clinically, or therapeutically effective polynucleotides and polypeptides, variants thereof, and compositions comprising them.
[0119]
Another aspect of the invention encodes a BASB061 polypeptide having the deduced amino acid sequence of SEQ ID NO: 8, and is closely related to an isolated polynucleotide comprising at least one full-length gene Polynucleotides and variants thereof.
[0120]
In a particularly preferred embodiment of the present invention, there is provided a BASB061 polypeptide derived from Neisseria meningitidis, comprising or consisting of the amino acid sequence of SEQ ID NO: 8, or a variant thereof.
[0121]
Using the information in this specification, such as the polynucleotide sequence provided in SEQ ID NO: 7, the book encoding BASB061 polypeptide can be obtained using standard cloning and screening methods and then obtaining full-length clones. The polynucleotide of the invention can be obtained. As a standard cloning method and screening method, there is a method of cloning and sequencing chromosomal DNA from bacteria using Neisseria meningitidis cells as a starting material. To obtain a polynucleotide sequence of the present invention, such as, for example, the polynucleotide sequence provided in SEQ ID NO: 7, a library of clones of chromosomal DNA of Neisseria meningitidis in E. coli or other suitable host Is generally probed with a radiolabeled oligonucleotide derived from the subsequence, preferably a 17-mer or longer. The clones having the same DNA as the probe DNA can then be identified using stringent hybridization. By sequencing the individual clones thus identified through hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence, the polynucleotide sequence is extended in both directions, The sequence of the full-length gene can be determined. Conveniently, such sequencing can be performed, for example, using denatured double-stranded DNA prepared from a plasmid clone. Suitable methods are described in Maniatis, T., Flitch, E .; F. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) (especially "Hybridization Screening 1.90 and Sequencing of denatured double-stranded DNA template 13.70). Genomic DNA can be directly sequenced to obtain the sequence of the full-length gene. As evidence of the present invention, each polynucleotide provided in SEQ ID NO: 7 was found in a DNA library derived from Neisseria meningitidis.
[0122]
In addition, each DNA sequence provided in SEQ ID NO: 7 contains an open reading frame encoding a protein having approximately the same number of amino acid residues as provided in SEQ ID NO: 8. The estimated molecular weight of this protein can be calculated using the molecular weight values of amino acid residues well known to those skilled in the art.
[0123]
The polynucleotide of SEQ ID NO: 7 encodes the polypeptide of SEQ ID NO: 2 between the start codon of nucleotide number 1 of SEQ ID NO: 7 and the stop codon starting at nucleotide number 514.
[0124]
According to still another aspect of the present invention, there is provided an isolated polynucleotide comprising: or an isolated polynucleotide consisting of:
(A) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 7 over the entire length of SEQ ID NO: 7 A polynucleotide sequence that matches, or
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or 100 over the entire length of SEQ ID NO: 8 % A polynucleotide sequence encoding an exactly matched polypeptide.
[0125]
The polynucleotide encoding the polypeptide of the present invention includes a labeled probe or a detectable probe comprising the sequence of SEQ ID NO: 7 or a fragment thereof, including homologs and orthologs from species other than Neisseria meningitidis, or Under stringent hybridization conditions (eg, at a temperature of 45 to 65 ° C. and a SDS concentration of 0.1 to 1%) using a labeled probe or a detectable probe containing the sequence of Screening of such libraries and isolating genomic clones containing the full-length gene and / or its polynucleotide sequence.
[0126]
According to the present invention, there is provided a polynucleotide sequence corresponding to the entire coding sequence (open reading frame) in SEQ ID NO: 7. The invention further provides a coding sequence for a mature polypeptide or a fragment thereof, as well as a coding sequence for a mature polypeptide or a fragment having another coding sequence in the reading frame. Another coding sequence is, for example, a sequence encoding a leader or secretory sequence, a preprotein sequence, a proprotein sequence, a preproprotein sequence. A polynucleotide of the invention may also include at least one non-coding sequence. By way of example, at least one non-coding 5 'or 3' sequence, such as a sequence that is transcribed but not translated, a termination signal (such as a ρ-dependent termination signal or a ρ-independent termination signal), a ribosome binding. Sites, Kozak sequences, mRNA stabilizing sequences, introns, polyadenylation signals, and the like, but are not limited thereto. The polynucleotide sequence may also include additional coding sequences that code for additional amino acids. For example, it can encode a marker sequence that facilitates purification of the fused polypeptide. In some embodiments of the invention, the marker sequence is a hexahistidine peptide (Genz et al., Proc. Natl. Acad. Sci. USA, 86, 821-824) provided in a pQE vector (Qiagen). 1989), or the HA peptide tag (Wilson et al., Cell, 37, 767, 1984). Either can help to purify the polypeptide sequence to be fused. The polynucleotide of the present invention also includes a polynucleotide having a structural gene and a sequence originally associated with the structural gene and controlling the expression of the gene. However, the polynucleotide contained in the polynucleotide of the present invention is not limited to this.
[0127]
The nucleotide sequence encoding the BASB061 polypeptide of SEQ ID NO: 8 may be the same as the polypeptide coding sequence contained in nucleotides 1-513 of SEQ ID NO: 7, respectively. Alternatively, it may be a sequence encoding the polypeptide of SEQ ID NO: 8, as a result of the redundancy (degeneracy) of the genetic code.
[0128]
The expression "polynucleotide encoding a polypeptide" is used herein to refer to a polypeptide of the invention, in particular a bacterial polypeptide, more particularly a Neisseria meningi having the amino acid sequence given by SEQ ID NO: 8. A polynucleotide comprising a sequence encoding a Tidis BASB061 polypeptide. The expression may be a single contiguous or discontinuous region encoding a polypeptide (eg, an integrated phage, an integrated insert, an integrated vector sequence, a polynucleotide interrupted by an integrated transposon sequence, Or polynucleotides interrupted for RNA editing, genomic DNA rearrangement), as well as additional polynucleotides that may also contain coding and / or non-coding sequences. .
[0129]
The invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the deduced amino acid sequence of SEQ ID NO: 8. Fragments of a polynucleotide of the invention can be used, for example, to synthesize a full-length polynucleotide of the invention.
[0130]
Furthermore, according to a particularly preferred embodiment, in the amino acid sequence of the BASB061 polypeptide given by SEQ ID NO: 8, some amino acid residues, for example 5-10, 1-5, 1-3, 2 Provided is a polynucleotide encoding a BASB061 variant in which one or zero amino acid residues have any combination of substitutions, changes, deletions, and / or additions. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of the BASB061 polypeptide.
[0131]
Further, in accordance with a preferred embodiment of the present invention, a polynucleotide that matches at least 85% over its entire length with a polynucleotide encoding a BASB061 polypeptide having the amino acid sequence set forth in SEQ ID NO: 8, and is complementary to the polynucleotide. And polynucleotides are provided. In this regard, polynucleotides that are at least 90% identical over the entire length are particularly preferred, and among such particularly preferred polynucleotides, those that are at least 95% identical are particularly preferred. Further, among polynucleotides having at least 95% identity, polynucleotides having at least 97% identity are more preferred, among which at least 98% identity, at least 99% identity are more preferred, and at least 99% identity. Are even more preferred.
[0132]
According to a preferred embodiment, there is provided a polynucleotide encoding a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA of SEQ ID NO: 7.
[0133]
According to some preferred embodiments of the present invention, there are provided polynucleotides that hybridize to a BASB061 polynucleotide sequence, eg, the polynucleotide of SEQ ID NO: 7, especially under stringent conditions.
[0134]
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences described herein. In this regard, the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As used herein, the terms "stringent conditions", "stringent hybridization conditions" refer to hybridization that occurs only when at least 95%, and preferably at least 97%, matches between sequences. Specific examples of stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, Culturing overnight at 42 ° C. in a solution containing 20 μg / ml denatured and cleaved salmon sperm DNA, then washing the hybridization support at about 65 ° C. in 0.1 × SSC. Things. Hybridization and washing conditions are well known and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In particular in Chapter 11. Solution hybridization can also be performed on the polynucleotide sequences of the present invention.
[0135]
According to the present invention, further, a suitable library containing the complete gene for the polynucleotide sequence given by SEQ ID NO: 7 is subjected to stringent hybridization using a probe having this polynucleotide sequence given by SEQ ID NO: 7 or a fragment thereof. Provided is a polynucleotide consisting of a polynucleotide sequence obtained by screening under conditions and isolating the polynucleotide sequence, or a polynucleotide comprising such a polynucleotide sequence. Fragments useful for obtaining such polynucleotides include, for example, probes and primers described in detail elsewhere in this specification.
[0136]
The polynucleotide assay of the present invention is described elsewhere in this specification. For example, the polynucleotide of the present invention is used as a probe for hybridization of RNA, cDNA, and genomic DNA, and the full-length encoding BASB061 is used. It is possible to isolate a cDNA or genomic clone, or to isolate another gene having a high degree of matching with the BASB061 gene, particularly a cDNA or genomic clone having a high degree of sequence matching. The probe will generally contain at least 15 nucleotide residues or base pairs. The probe preferably contains at least 30 nucleotide residues or base pairs, and may contain at least 50 nucleotide residues or base pairs. Particularly preferred probes will contain more than 20 and less than 30 nucleotide residues or base pairs.
[0137]
The coding region of the BASB061 gene can be isolated by synthesizing an oligonucleotide probe using the DNA sequence given by SEQ ID NO: 7 and conducting screening using this probe. Therefore, a library of cDNA, genomic DNA, or mRNA is screened using a labeled oligonucleotide having a sequence complementary to the gene of the present invention, and it is confirmed to which member of the library the probe hybridizes.
[0138]
One object of the present invention is to provide a polynucleotide encoding a BASB063 polypeptide, particularly a polynucleotide encoding a polypeptide designated herein as BASB063.
[0139]
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding the BASB063 polypeptide comprising the full length gene and having the sequence given by SEQ ID NO: 9, or a region encoding a variant thereof. I have.
[0140]
The BASB063 polynucleotide provided by SEQ ID NO: 9 is a BASB063 polynucleotide derived from Neisseria meningitidis strain ATCC13090.
[0141]
According to yet another aspect of the present invention there is provided an isolated nucleic acid molecule encoding and / or expressing a BASB063 polypeptide and polynucleotide, particularly a Neisseria meningitidis BASB063 polypeptide and polynucleotide. . Such nucleic acid molecules include, for example, untreated RNA, ribozyme RNA, mRNA, cDNA, genomic DNA, B-DNA, Z-DNA. In yet another embodiment of the present invention, there are biologically, diagnostically, prophylactically, clinically, or therapeutically effective polynucleotides and polypeptides, variants thereof, and compositions comprising them.
[0142]
Another aspect of the invention encodes a BASB063 polypeptide having the deduced amino acid sequence of SEQ ID NO: 10, closely related to the isolated polynucleotide comprising at least one full-length gene. Polynucleotides and variants thereof.
[0143]
In a particularly preferred embodiment of the present invention there is provided a BASB063 polypeptide, derived from Neisseria meningitidis, comprising or consisting of the amino acid sequence of SEQ ID NO: 10, or a variant thereof.
[0144]
Using the information in this specification, such as the polynucleotide sequence given in SEQ ID NO: 9, the standard encoding BASB063 polypeptide can be obtained using standard cloning and screening methods and then obtaining full-length clones. The polynucleotide of the invention can be obtained. As a standard cloning method and screening method, there is a method of cloning and sequencing chromosomal DNA from bacteria using Neisseria meningitidis cells as a starting material. To obtain a polynucleotide sequence of the present invention, such as the polynucleotide sequence given in SEQ ID NO: 9, a library of chromosomal DNA clones of Neisseria meningitidis in E. coli or other suitable host Is generally probed with a radiolabeled oligonucleotide derived from the subsequence, preferably a 17-mer or longer. The clones having the same DNA as the probe DNA can then be identified using stringent hybridization. By sequencing the individual clones thus identified through hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence, the polynucleotide sequence is extended in both directions, The sequence of the full-length gene can be determined. Conveniently, such sequencing can be performed, for example, using denatured double-stranded DNA prepared from a plasmid clone. Suitable methods are described in Maniatis, T., Flitch, E .; F. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) (especially "Hybridization Screening 1.90 and Sequencing of denatured double-stranded DNA template 13.70). Genomic DNA can be directly sequenced to obtain the sequence of the full-length gene. As evidence of the present invention, each polynucleotide provided in SEQ ID NO: 9 was found in a DNA library derived from Neisseria meningitidis.
[0145]
In addition, each DNA sequence provided in SEQ ID NO: 9 contains an open reading frame encoding a protein having approximately the same number of amino acid residues as provided in SEQ ID NO: 10. The estimated molecular weight of this protein can be calculated using the molecular weight values of amino acid residues well known to those skilled in the art.
[0146]
The polynucleotide of SEQ ID NO: 9 encodes the polypeptide of SEQ ID NO: 2 between the start codon of nucleotide number 1 of SEQ ID NO: 9 and the stop codon starting at nucleotide number 814.
[0147]
According to still another aspect of the present invention, there is provided an isolated polynucleotide comprising: or an isolated polynucleotide consisting of:
(A) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 9 over the entire length of SEQ ID NO: 9 A polynucleotide sequence that matches, or
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or 100% over SEQ ID NO: 10 over the entire length of SEQ ID NO: 10 % A polynucleotide sequence encoding an exactly matched polypeptide.
[0148]
Polynucleotides encoding the polypeptides of the present invention include labeled or detectable probes consisting of the sequence of SEQ ID NO: 9 or fragments thereof, including homologs and orthologs from species other than Neisseria meningitidis, or Under stringent hybridization conditions (eg, at a temperature of 45 to 65 ° C. and a SDS concentration of 0.1 to 1%) using a labeled probe or a detectable probe containing the sequence of Screening of such libraries and isolating genomic clones containing the full-length gene and / or its polynucleotide sequence.
[0149]
According to the present invention, there is provided a polynucleotide sequence which corresponds to the coding sequence (open reading frame) in SEQ ID NO: 9 over its entire length. The invention further provides a coding sequence for a mature polypeptide or a fragment thereof, as well as a coding sequence for a mature polypeptide or a fragment having another coding sequence in the reading frame. Another coding sequence is, for example, a sequence encoding a leader or secretory sequence, a preprotein sequence, a proprotein sequence, a preproprotein sequence. A polynucleotide of the invention may also include at least one non-coding sequence. By way of example, at least one non-coding 5 'or 3' sequence, such as a sequence that is transcribed but not translated, a termination signal (such as a ρ-dependent termination signal or a ρ-independent termination signal), a ribosome binding. Sites, Kozak sequences, mRNA stabilizing sequences, introns, polyadenylation signals, and the like, but are not limited thereto. The polynucleotide sequence may also include additional coding sequences that code for additional amino acids. For example, it can encode a marker sequence that facilitates purification of the fused polypeptide. In some embodiments of the invention, the marker sequence is a hexahistidine peptide (Genz et al., Proc. Natl. Acad. Sci. USA, 86, 821-824) provided in a pQE vector (Qiagen). 1989), or the HA peptide tag (Wilson et al., Cell, 37, 767, 1984). Either can help to purify the polypeptide sequence to be fused. The polynucleotide of the present invention also includes a polynucleotide having a structural gene and a sequence originally associated with the structural gene and controlling the expression of the gene. However, the polynucleotide contained in the polynucleotide of the present invention is not limited to this.
[0150]
The nucleotide sequence encoding the BASB063 polypeptide of SEQ ID NO: 10 may be the same as the polypeptide coding sequence contained in nucleotides 1-813 of SEQ ID NO: 9, respectively. Alternatively, it may be a sequence encoding the polypeptide of SEQ ID NO: 10, as a result of the redundancy (degeneracy) of the genetic code.
[0151]
The expression "polynucleotide encoding a polypeptide" is used herein to refer to a polypeptide of the invention, in particular a bacterial polypeptide, more particularly a Neisseria meningi having the amino acid sequence given in SEQ ID NO: 10. A polynucleotide comprising a sequence encoding a Tidis BASB063 polypeptide. The expression may be a single contiguous or discontinuous region encoding a polypeptide (eg, an integrated phage, an integrated insert, an integrated vector sequence, a polynucleotide interrupted by an integrated transposon sequence, Or polynucleotides interrupted for RNA editing, genomic DNA rearrangement), as well as additional polynucleotides that may also contain coding and / or non-coding sequences. .
[0152]
The present invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the deduced amino acid sequence of SEQ ID NO: 10. Fragments of a polynucleotide of the invention can be used, for example, to synthesize a full-length polynucleotide of the invention.
[0153]
Furthermore, according to a particularly preferred embodiment, of the amino acid sequence of the BASB063 polypeptide given by SEQ ID NO: 10, some amino acid residues, for example 5-10, 1-5, 1-3, 2 Provided is a polynucleotide encoding a BASB063 variant in which 1, 0 amino acid residues have any combination of substitutions, changes, deletions, and / or additions. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of the BASB063 polypeptide.
[0154]
Further, in accordance with a preferred embodiment of the present invention, a polynucleotide that matches at least 85% over its entire length with a polynucleotide encoding a BASB063 polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, and a polynucleotide complementary to the polynucleotide. And polynucleotides are provided. In this regard, polynucleotides that are at least 90% identical over the entire length are particularly preferred, and among such particularly preferred polynucleotides, those that are at least 95% identical are particularly preferred. Further, among polynucleotides having at least 95% identity, polynucleotides having at least 97% identity are more preferred, among which at least 98% identity, at least 99% identity are more preferred, and at least 99% identity. Are even more preferred.
[0155]
According to a preferred embodiment, there is provided a polynucleotide encoding a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA of SEQ ID NO: 9.
[0156]
According to some preferred embodiments of the present invention, there are provided polynucleotides that hybridize to a BASB063 polynucleotide sequence, eg, the polynucleotide of SEQ ID NO: 9, particularly under stringent conditions.
[0157]
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences described herein. In this regard, the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As used herein, the terms "stringent conditions", "stringent hybridization conditions" refer to hybridization that occurs only when at least 95%, and preferably at least 97%, matches between sequences. Specific examples of stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, Culturing overnight at 42 ° C. in a solution containing 20 μg / ml denatured and cleaved salmon sperm DNA, then washing the hybridization support at about 65 ° C. in 0.1 × SSC. Things. Hybridization and washing conditions are well known and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In particular in Chapter 11. Solution hybridization can also be performed on the polynucleotide sequences of the present invention.
[0158]
According to the invention, furthermore, a suitable library containing the complete gene for the polynucleotide sequence given by SEQ ID NO: 9 is subjected to stringent hybridization using a probe having this polynucleotide sequence given by SEQ ID NO: 9 or a fragment thereof. Provided is a polynucleotide consisting of a polynucleotide sequence obtained by screening under conditions and isolating the polynucleotide sequence, or a polynucleotide comprising such a polynucleotide sequence. Fragments useful for obtaining such polynucleotides include, for example, probes and primers described in detail elsewhere in this specification.
[0159]
The polynucleotide assay of the present invention is described elsewhere in this specification. For example, the polynucleotide of the present invention is used as a probe for hybridization of RNA, cDNA, and genomic DNA, and the full-length encoding BASB063 is used. It is possible to isolate a cDNA or genomic clone, or to isolate another gene having a high degree of matching with the BASB063 gene, particularly a cDNA or genomic clone having a high degree of sequence matching. The probe will generally contain at least 15 nucleotide residues or base pairs. The probe preferably contains at least 30 nucleotide residues or base pairs, and may contain at least 50 nucleotide residues or base pairs. Particularly preferred probes will contain more than 20 and less than 30 nucleotide residues or base pairs.
[0160]
The coding region of the BASB063 gene can be isolated by synthesizing an oligonucleotide probe using the DNA sequence given by SEQ ID NO: 9 and performing screening using this probe. Therefore, a library of cDNA, genomic DNA, or mRNA is screened using a labeled oligonucleotide having a sequence complementary to the gene of the present invention, and it is confirmed to which member of the library the probe hybridizes.
[0161]
One object of the present invention is to provide a polynucleotide encoding a BASB065 polypeptide, in particular a polynucleotide encoding a polypeptide designated herein as BASB065.
[0162]
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding the BASB065 polypeptide comprising the full length gene and having the sequence given in SEQ ID NO: 11, or a region encoding a variant thereof. I have.
[0163]
The BASB065 polynucleotide provided by SEQ ID NO: 11 is a BASB065 polynucleotide derived from Neisseria meningitidis strain ATCC13090.
[0164]
According to yet another aspect of the present invention there is provided an isolated nucleic acid molecule encoding and / or expressing a BASB065 polypeptide and polynucleotide, particularly a Neisseria meningitidis BASB065 polypeptide and polynucleotide. . Such nucleic acid molecules include, for example, untreated RNA, ribozyme RNA, mRNA, cDNA, genomic DNA, B-DNA, Z-DNA. In yet another embodiment of the present invention, there are biologically, diagnostically, prophylactically, clinically, or therapeutically effective polynucleotides and polypeptides, variants thereof, and compositions comprising them.
[0165]
Another aspect of the invention encodes a BASB065 polypeptide having the deduced amino acid sequence of SEQ ID NO: 12, closely related to the isolated polynucleotide comprising at least one full-length gene. Polynucleotides and variants thereof.
[0166]
In a particularly preferred embodiment of the present invention, there is provided a BASB065 polypeptide derived from Neisseria meningitidis, comprising or consisting of the amino acid sequence of SEQ ID NO: 12, or a variant thereof.
[0167]
Using the information in this specification, such as the polynucleotide sequence given in SEQ ID NO: 11, the standard encoding cloning of the BASB065 polypeptide can be accomplished using standard cloning and screening methods and then obtaining full-length clones. The polynucleotide of the invention can be obtained. As a standard cloning method and screening method, there is a method of cloning and sequencing chromosomal DNA from bacteria using Neisseria meningitidis cells as a starting material. To obtain a polynucleotide sequence of the present invention, such as the polynucleotide sequence given in SEQ ID NO: 11, a library of clones of chromosomal DNA of Neisseria meningitidis in E. coli or other suitable host Is generally probed with a radiolabeled oligonucleotide derived from a subsequence, preferably a 17-mer or longer. The clones having the same DNA as the probe DNA can then be identified using stringent hybridization. By sequencing the individual clones thus identified through hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence, the polynucleotide sequence is extended in both directions, The sequence of the full-length gene can be determined. Conveniently, such sequencing can be performed, for example, using denatured double-stranded DNA prepared from a plasmid clone. Suitable methods are described in Maniatis, T., Flitch, E .; F. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) (especially "Hybridization Screening 1.90 and Sequencing of denatured double-stranded DNA template 13.70). Genomic DNA can be directly sequenced to obtain the sequence of the full-length gene. As evidence for the present invention, each of the polynucleotides provided in SEQ ID NO: 11 was found in a DNA library derived from Neisseria meningitidis.
[0168]
In addition, each DNA sequence provided in SEQ ID NO: 11 contains an open reading frame encoding a protein having approximately the same number of amino acid residues as provided in SEQ ID NO: 12. The estimated molecular weight of this protein can be calculated using the molecular weight values of amino acid residues well known to those skilled in the art.
[0169]
The polynucleotide of SEQ ID NO: 11 encodes the polypeptide of SEQ ID NO: 12 between the start codon of nucleotide number 1 of SEQ ID NO: 11 and the stop codon starting at nucleotide number 715.
[0170]
According to still another aspect of the present invention, there is provided an isolated polynucleotide comprising: or an isolated polynucleotide consisting of:
(A) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 11 over the entire length of SEQ ID NO: 11 A polynucleotide sequence that matches, or
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or 100 over SEQ ID NO: 12 over the entire length of SEQ ID NO: 12 % A polynucleotide sequence encoding an exactly matched polypeptide.
[0171]
Polynucleotides encoding the polypeptides of the present invention include labeled or detectable probes consisting of the sequence of SEQ ID NO: 11 or fragments thereof, including homologs and orthologs from species other than Neisseria meningitidis, or Under stringent hybridization conditions (eg, at a temperature of 45 to 65 ° C. and a SDS concentration of 0.1 to 1%) using a labeled probe or a detectable probe containing the sequence of Screening of such libraries and isolating genomic clones containing the full-length gene and / or its polynucleotide sequence.
[0172]
According to the present invention, there is provided a polynucleotide sequence which corresponds to the coding sequence (open reading frame) in SEQ ID NO: 11 over its entire length. The invention further provides a coding sequence for a mature polypeptide or a fragment thereof, as well as a coding sequence for a mature polypeptide or a fragment having another coding sequence in the reading frame. Another coding sequence is, for example, a sequence encoding a leader or secretory sequence, a preprotein sequence, a proprotein sequence, a preproprotein sequence. A polynucleotide of the invention may also include at least one non-coding sequence. By way of example, at least one non-coding 5 'or 3' sequence, such as a sequence that is transcribed but not translated, a termination signal (such as a ρ-dependent termination signal or a ρ-independent termination signal), a ribosome binding. Sites, Kozak sequences, mRNA stabilizing sequences, introns, polyadenylation signals, and the like, but are not limited thereto. The polynucleotide sequence may also include additional coding sequences that code for additional amino acids. For example, it can encode a marker sequence that facilitates purification of the fused polypeptide. In some embodiments of the invention, the marker sequence is a hexahistidine peptide (Genz et al., Proc. Natl. Acad. Sci. USA, 86, 821-824) provided in a pQE vector (Qiagen). 1989), or the HA peptide tag (Wilson et al., Cell, 37, 767, 1984). Either can help to purify the polypeptide sequence to be fused. The polynucleotide of the present invention also includes a polynucleotide having a structural gene and a sequence originally associated with the structural gene and controlling the expression of the gene. However, the polynucleotide contained in the polynucleotide of the present invention is not limited to this.
[0173]
The nucleotide sequence encoding the BASB065 polypeptide of SEQ ID NO: 12 may be the same as the polypeptide coding sequence contained in nucleotide numbers 1-714 of SEQ ID NO: 11, respectively. Alternatively, it may be a sequence encoding the polypeptide of SEQ ID NO: 12, as a result of the redundancy (degeneracy) of the genetic code.
[0174]
The expression "polynucleotide encoding a polypeptide" is used herein to refer to a polypeptide of the invention, in particular a bacterial polypeptide, and more particularly a Neisseria meningi having the amino acid sequence given by SEQ ID NO: 12. A polynucleotide comprising a sequence encoding a Tidis BASB065 polypeptide. The expression may be a single contiguous or discontinuous region encoding a polypeptide (eg, an integrated phage, an integrated insert, an integrated vector sequence, a polynucleotide interrupted by an integrated transposon sequence, Or polynucleotides interrupted for RNA editing, genomic DNA rearrangement), as well as additional polynucleotides that may also contain coding and / or non-coding sequences. .
[0175]
The invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the deduced amino acid sequence of SEQ ID NO: 12. Fragments of a polynucleotide of the invention can be used, for example, to synthesize a full-length polynucleotide of the invention.
[0176]
Furthermore, according to a particularly preferred embodiment, of the amino acid sequence of the BASB065 polypeptide given by SEQ ID NO: 12, some amino acid residues, for example 5-10, 1-5, 1-3, 2 Provided is a polynucleotide encoding a BASB065 variant in which 1, 0 amino acid residues have any combination of substitutions, changes, deletions, and / or additions. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of the BASB065 polypeptide.
[0177]
Further, in accordance with a preferred embodiment of the present invention, a polynucleotide that matches at least 85% over its entire length with a polynucleotide encoding a BASB065 polypeptide having the amino acid sequence set forth in SEQ ID NO: 12, and is complementary to the polynucleotide And polynucleotides are provided. In this regard, polynucleotides that are at least 90% identical over the entire length are particularly preferred, and among such particularly preferred polynucleotides, those that are at least 95% identical are particularly preferred. Further, among polynucleotides having at least 95% identity, polynucleotides having at least 97% identity are more preferred, among which at least 98% identity, at least 99% identity are more preferred, and at least 99% identity. Are even more preferred.
[0178]
According to a preferred embodiment, there is provided a polynucleotide encoding a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA of SEQ ID NO: 11.
[0179]
According to some preferred embodiments of the present invention, there are provided polynucleotides that hybridize to a BASB065 polynucleotide sequence, eg, a polynucleotide of SEQ ID NO: 11, particularly under stringent conditions.
[0180]
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences described herein. In this regard, the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As used herein, the terms "stringent conditions", "stringent hybridization conditions" refer to hybridization that occurs only when at least 95%, and preferably at least 97%, matches between sequences. Specific examples of stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, Culturing overnight at 42 ° C. in a solution containing 20 μg / ml denatured and cleaved salmon sperm DNA, then washing the hybridization support at about 65 ° C. in 0.1 × SSC. Things. Hybridization and washing conditions are well known and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In particular in Chapter 11. Solution hybridization can also be performed on the polynucleotide sequences of the present invention.
[0181]
According to the invention, furthermore, a stringent hybridization of a suitable library containing the complete gene for the polynucleotide sequence given in SEQ ID NO: 11 with a probe having this polynucleotide sequence given in SEQ ID NO: 11, or a fragment thereof, Provided is a polynucleotide consisting of a polynucleotide sequence obtained by screening under conditions and isolating the polynucleotide sequence, or a polynucleotide comprising such a polynucleotide sequence. Fragments useful for obtaining such polynucleotides include, for example, probes and primers described in detail elsewhere in this specification.
[0182]
The polynucleotide assay of the present invention is described elsewhere in this specification. For example, the polynucleotide of the present invention is used as a probe for hybridization of RNA, cDNA, and genomic DNA, and the full-length encoding BASB065 is used. It is possible to isolate a cDNA or a genomic clone, or to isolate another gene having a high degree of matching with the BASB065 gene, particularly a cDNA or genomic clone having a high degree of sequence matching. The probe will generally contain at least 15 nucleotide residues or base pairs. The probe preferably contains at least 30 nucleotide residues or base pairs, and may contain at least 50 nucleotide residues or base pairs. Particularly preferred probes will contain more than 20 and less than 30 nucleotide residues or base pairs.
[0183]
The coding region of the BASB065 gene can be isolated by synthesizing an oligonucleotide probe using the DNA sequence provided in SEQ ID NO: 11 and performing screening using the probe. Therefore, a library of cDNA, genomic DNA, or mRNA is screened using a labeled oligonucleotide having a sequence complementary to the gene of the present invention, and it is confirmed to which member of the library the probe hybridizes.
[0184]
One object of the present invention is to provide a polynucleotide encoding a BASB066 polypeptide, particularly a polynucleotide encoding a polypeptide designated herein as BASB066.
[0185]
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding a BASB066 polypeptide comprising the full length gene and having the sequence given in SEQ ID NO: 13, or a region encoding a variant thereof. I have.
[0186]
The BASB066 polynucleotide provided by SEQ ID NO: 13 is a BASB066 polynucleotide derived from Neisseria meningitidis strain ATCC13090.
[0187]
According to yet another aspect of the present invention there is provided an isolated nucleic acid molecule encoding and / or expressing a BASB066 polypeptide and polynucleotide, particularly a Neisseria meningitidis BASB066 polypeptide and polynucleotide. . Such nucleic acid molecules include, for example, untreated RNA, ribozyme RNA, mRNA, cDNA, genomic DNA, B-DNA, Z-DNA. In yet another embodiment of the present invention, there are biologically, diagnostically, prophylactically, clinically, or therapeutically effective polynucleotides and polypeptides, variants thereof, and compositions comprising them.
[0188]
Another aspect of the invention encodes a BASB066 polypeptide having the deduced amino acid sequence of SEQ ID NO: 14, and is closely related to an isolated polynucleotide comprising at least one full length gene Polynucleotides and variants thereof.
[0189]
In a particularly preferred embodiment of the present invention, there is provided a BASB066 polypeptide derived from Neisseria meningitidis, comprising or consisting of the amino acid sequence of SEQ ID NO: 14, or a variant thereof.
[0190]
Using the information in this specification, such as the polynucleotide sequence provided in SEQ ID NO: 13, the standard encoding cloning of the BASB066 polypeptide can be accomplished using standard cloning and screening methods and then obtaining full-length clones. The polynucleotide of the invention can be obtained. As a standard cloning method and screening method, there is a method of cloning and sequencing chromosomal DNA from bacteria using Neisseria meningitidis cells as a starting material. To obtain a polynucleotide sequence of the present invention, such as the polynucleotide sequence given in SEQ ID NO: 13, a library of chromosomal DNA clones of Neisseria meningitidis in E. coli or other suitable host Is generally probed with a radiolabeled oligonucleotide derived from the subsequence, preferably a 17-mer or longer. The clones having the same DNA as the probe DNA can then be identified using stringent hybridization. By sequencing the individual clones thus identified through hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence, the polynucleotide sequence is extended in both directions, The sequence of the full-length gene can be determined. Conveniently, such sequencing can be performed, for example, using denatured double-stranded DNA prepared from a plasmid clone. Suitable methods are described in Maniatis, T., Flitch, E .; F. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) (especially "Hybridization Screening 1.90 and Sequencing of denatured double-stranded DNA template 13.70). Genomic DNA can be directly sequenced to obtain the sequence of the full-length gene. As evidence for the present invention, each of the polynucleotides provided in SEQ ID NO: 13 was found in a DNA library derived from Neisseria meningitidis.
[0191]
In addition, each DNA sequence provided in SEQ ID NO: 13 contains an open reading frame encoding a protein having approximately the same number of amino acid residues as provided in SEQ ID NO: 14. The estimated molecular weight of this protein can be calculated using the molecular weight values of amino acid residues well known to those skilled in the art.
[0192]
The polynucleotide of SEQ ID NO: 13 encodes the polypeptide of SEQ ID NO: 2 between the start codon of nucleotide number 1 of SEQ ID NO: 13 and the stop codon starting at nucleotide number 1174.
[0193]
According to still another aspect of the present invention, there is provided an isolated polynucleotide comprising: or an isolated polynucleotide consisting of:
(A) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 13 over the entire length of SEQ ID NO: 13 A polynucleotide sequence that matches, or
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or 100 over SEQ ID NO: 14 over the entire length of SEQ ID NO: 14 % A polynucleotide sequence encoding an exactly matched polypeptide.
[0194]
The polynucleotide encoding the polypeptide of the present invention may be a labeled probe or a detectable probe comprising the sequence of SEQ ID NO: 13 or a fragment thereof, including homologs and orthologs from species other than Neisseria meningitidis, or Under stringent hybridization conditions (eg, at a temperature of 45 to 65 ° C. and a SDS concentration of 0.1 to 1%) using a labeled probe or a detectable probe containing the sequence of Screening of such libraries and isolating genomic clones containing the full-length gene and / or its polynucleotide sequence.
[0195]
According to the present invention, there is provided a polynucleotide sequence which corresponds to the coding sequence (open reading frame) in SEQ ID NO: 13 over its entire length. The invention further provides a coding sequence for a mature polypeptide or a fragment thereof, as well as a coding sequence for a mature polypeptide or a fragment having another coding sequence in the reading frame. Another coding sequence is, for example, a sequence encoding a leader or secretory sequence, a preprotein sequence, a proprotein sequence, a preproprotein sequence. A polynucleotide of the invention may also include at least one non-coding sequence. By way of example, at least one non-coding 5 'or 3' sequence, such as a sequence that is transcribed but not translated, a termination signal (such as a ρ-dependent termination signal or a ρ-independent termination signal), a ribosome binding. Sites, Kozak sequences, mRNA stabilizing sequences, introns, polyadenylation signals, and the like, but are not limited thereto. The polynucleotide sequence may also include additional coding sequences that code for additional amino acids. For example, it can encode a marker sequence that facilitates purification of the fused polypeptide. In some embodiments of the invention, the marker sequence is a hexahistidine peptide (Genz et al., Proc. Natl. Acad. Sci. USA, 86, 821-824) provided in a pQE vector (Qiagen). 1989), or the HA peptide tag (Wilson et al., Cell, 37, 767, 1984). Either can help to purify the polypeptide sequence to be fused. The polynucleotide of the present invention also includes a polynucleotide having a structural gene and a sequence originally associated with the structural gene and controlling the expression of the gene. However, the polynucleotide contained in the polynucleotide of the present invention is not limited to this.
[0196]
The nucleotide sequence encoding the BASB066 polypeptide of SEQ ID NO: 14 may be the same as the polypeptide coding sequence contained in nucleotide numbers 1-1173 of SEQ ID NO: 13, respectively. Alternatively, it may be a sequence encoding the polypeptide of SEQ ID NO: 14, as a result of the redundancy (degeneracy) of the genetic code.
[0197]
The expression "polynucleotide encoding a polypeptide" is used herein to refer to a polypeptide of the invention, in particular a bacterial polypeptide, and more particularly a Neisseria meningi having the amino acid sequence given by SEQ ID NO: 14. It refers to a polynucleotide comprising a sequence encoding a Tidis BASB066 polypeptide. The expression may be a single contiguous or discontinuous region encoding a polypeptide (eg, an integrated phage, an integrated insert, an integrated vector sequence, a polynucleotide interrupted by an integrated transposon sequence, Or polynucleotides interrupted for RNA editing, genomic DNA rearrangement), as well as additional polynucleotides that may also contain coding and / or non-coding sequences. .
[0198]
The invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the deduced amino acid sequence of SEQ ID NO: 14. Fragments of a polynucleotide of the invention can be used, for example, to synthesize a full-length polynucleotide of the invention.
[0199]
Furthermore, according to a particularly preferred embodiment, several amino acid residues in the amino acid sequence of the BASB066 polypeptide given by SEQ ID NO: 14, for example 5-10, 1-5, 1-3, 2 Provided is a polynucleotide encoding a BASB066 variant in which 1, 0 amino acid residues have any combination of substitutions, changes, deletions, and / or additions. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of the BASB066 polypeptide.
[0200]
Further, in accordance with a preferred embodiment of the present invention, a polynucleotide that matches at least 85% over its entire length with a polynucleotide encoding a BASB066 polypeptide having the amino acid sequence set forth in SEQ ID NO: 14, and is complementary to the polynucleotide. And polynucleotides are provided. In this regard, polynucleotides that are at least 90% identical over the entire length are particularly preferred, and among such particularly preferred polynucleotides, those that are at least 95% identical are particularly preferred. Further, among polynucleotides having at least 95% identity, polynucleotides having at least 97% identity are more preferred, among which at least 98% identity, at least 99% identity are more preferred, and at least 99% identity. Are even more preferred.
[0201]
According to a preferred embodiment, there is provided a polynucleotide encoding a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA of SEQ ID NO: 13.
[0202]
According to some preferred embodiments of the present invention, there are provided polynucleotides that hybridize to a BASB066 polynucleotide sequence, eg, the polynucleotide of SEQ ID NO: 13, particularly under stringent conditions.
[0203]
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences described herein. In this regard, the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As used herein, the terms "stringent conditions", "stringent hybridization conditions" refer to hybridization that occurs only when at least 95%, and preferably at least 97%, matches between sequences. Specific examples of stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, Culturing overnight at 42 ° C. in a solution containing 20 μg / ml denatured and cleaved salmon sperm DNA, then washing the hybridization support at about 65 ° C. in 0.1 × SSC. Things. Hybridization and washing conditions are well known and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In particular in Chapter 11. Solution hybridization can also be performed on the polynucleotide sequences of the present invention.
[0204]
According to the invention, furthermore, a suitable library containing the complete gene for the polynucleotide sequence given by SEQ ID NO: 13 is subjected to stringent hybridization using a probe having this polynucleotide sequence given by SEQ ID NO: 13 or a fragment thereof. Provided is a polynucleotide consisting of a polynucleotide sequence obtained by screening under conditions and isolating the polynucleotide sequence, or a polynucleotide comprising such a polynucleotide sequence. Fragments useful for obtaining such polynucleotides include, for example, probes and primers described in detail elsewhere in this specification.
[0205]
The polynucleotide assay of the present invention is described elsewhere in this specification. For example, the polynucleotide of the present invention is used as a probe for hybridization of RNA, cDNA, and genomic DNA, and the full-length encoding BASB066 is used. It is possible to isolate a cDNA or genomic clone, or to isolate another gene having a high degree of matching with the BASB066 gene, particularly a cDNA or genomic clone having a high degree of sequence matching. The probe will generally contain at least 15 nucleotide residues or base pairs. The probe preferably contains at least 30 nucleotide residues or base pairs, and may contain at least 50 nucleotide residues or base pairs. Particularly preferred probes will contain more than 20 and less than 30 nucleotide residues or base pairs.
[0206]
The coding region of the BASB066 gene can be isolated by synthesizing an oligonucleotide probe using the DNA sequence given by SEQ ID NO: 13 and performing screening using this probe. Therefore, a library of cDNA, genomic DNA, or mRNA is screened using a labeled oligonucleotide having a sequence complementary to the gene of the present invention, and it is confirmed to which member of the library the probe hybridizes.
[0207]
One object of the present invention is to provide a polynucleotide encoding a BASB071 polypeptide, particularly a polynucleotide encoding a polypeptide designated herein as BASB071.
[0208]
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding a BASB071 polypeptide comprising the full length gene and having the sequence given in SEQ ID NO: 15, or a region encoding a variant thereof. I have.
[0209]
The BASB071 polynucleotide provided by SEQ ID NO: 15 is a BASB071 polynucleotide derived from Neisseria meningitidis strain ATCC13090.
[0210]
According to yet another aspect of the present invention there is provided an isolated nucleic acid molecule which encodes and / or expresses a BASB071 polypeptide and polynucleotide, in particular a BASB071 polypeptide and polynucleotide of Neisseria meningitidis. . Such nucleic acid molecules include, for example, untreated RNA, ribozyme RNA, mRNA, cDNA, genomic DNA, B-DNA, Z-DNA. In yet another embodiment of the present invention, there are biologically, diagnostically, prophylactically, clinically, or therapeutically effective polynucleotides and polypeptides, variants thereof, and compositions comprising them.
[0211]
Another aspect of the invention encodes a BASB071 polypeptide having the deduced amino acid sequence of SEQ ID NO: 16, closely related to the isolated polynucleotide comprising at least one full-length gene Polynucleotides and variants thereof.
[0212]
In a particularly preferred embodiment of the present invention, there is provided a BASB071 polypeptide derived from Neisseria meningitidis, comprising or consisting of the amino acid sequence of SEQ ID NO: 16, or a variant thereof.
[0213]
Using the information in this specification, such as the polynucleotide sequence provided in SEQ ID NO: 15, standard cloning and screening methods are used, followed by obtaining full-length clones to obtain a book encoding BASB071 polypeptide. The polynucleotide of the invention can be obtained. As a standard cloning method and screening method, there is a method of cloning and sequencing chromosomal DNA from bacteria using Neisseria meningitidis cells as a starting material. To obtain a polynucleotide sequence of the present invention, such as the polynucleotide sequence given in SEQ ID NO: 15, a library of clones of chromosomal DNA of Neisseria meningitidis in E. coli or other suitable host. Is generally probed with a radiolabeled oligonucleotide derived from the subsequence, preferably a 17-mer or longer. The clones having the same DNA as the probe DNA can then be identified using stringent hybridization. By sequencing the individual clones thus identified through hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence, the polynucleotide sequence is extended in both directions, The sequence of the full-length gene can be determined. Conveniently, such sequencing can be performed, for example, using denatured double-stranded DNA prepared from a plasmid clone. Suitable methods are described in Maniatis, T., Flitch, E .; F. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) (especially "Hybridization Screening 1.90 and Sequencing of denatured double-stranded DNA template 13.70). Genomic DNA can be directly sequenced to obtain the sequence of the full-length gene. As evidence of the present invention, each of the polynucleotides provided in SEQ ID NO: 15 was found in a DNA library derived from Neisseria meningitidis.
[0214]
In addition, each DNA sequence provided in SEQ ID NO: 15 contains an open reading frame encoding a protein having approximately the same number of amino acid residues as provided in SEQ ID NO: 16. The estimated molecular weight of this protein can be calculated using the molecular weight values of amino acid residues well known to those skilled in the art.
[0215]
The polynucleotide of SEQ ID NO: 15 encodes the polypeptide of SEQ ID NO: 2 between the start codon of nucleotide number 1 of SEQ ID NO: 15 and the stop codon starting at nucleotide number 805.
[0216]
According to still another aspect of the present invention, there is provided an isolated polynucleotide comprising: or an isolated polynucleotide consisting of:
(A) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or complete SEQ ID NO: 15 over the entire length of SEQ ID NO: 15 A polynucleotide sequence that matches, or
(B) at least 85% match, preferably at least 90% match, more preferably at least 95% match, more preferably at least 97-99% match or 100 over SEQ ID NO: 16 over the entire length of SEQ ID NO: 16 % A polynucleotide sequence encoding an exactly matched polypeptide.
[0217]
The polynucleotide encoding the polypeptide of the present invention includes a labeled probe or a detectable probe consisting of the sequence of SEQ ID NO: 15 or a fragment thereof, including homologs and orthologs from species other than Neisseria meningitidis, or Under stringent hybridization conditions (eg, at a temperature of 45 to 65 ° C. and a SDS concentration of 0.1 to 1%) using a labeled probe or a detectable probe containing the sequence of Screening of such libraries and isolating genomic clones containing the full-length gene and / or its polynucleotide sequence.
[0218]
According to the present invention, there is provided a polynucleotide sequence corresponding to the entire coding sequence (open reading frame) in SEQ ID NO: 15. The invention further provides a coding sequence for a mature polypeptide or a fragment thereof, as well as a coding sequence for a mature polypeptide or a fragment having another coding sequence in the reading frame. Another coding sequence is, for example, a sequence encoding a leader or secretory sequence, a preprotein sequence, a proprotein sequence, a preproprotein sequence. A polynucleotide of the invention may also include at least one non-coding sequence. By way of example, at least one non-coding 5 'or 3' sequence, such as a sequence that is transcribed but not translated, a termination signal (such as a ρ-dependent termination signal or a ρ-independent termination signal), a ribosome binding. Sites, Kozak sequences, mRNA stabilizing sequences, introns, polyadenylation signals, and the like, but are not limited thereto. The polynucleotide sequence may also include additional coding sequences that code for additional amino acids. For example, it can encode a marker sequence that facilitates purification of the fused polypeptide. In some embodiments of the invention, the marker sequence is a hexahistidine peptide (Genz et al., Proc. Natl. Acad. Sci. USA, 86, 821-824) provided in a pQE vector (Qiagen). 1989), or the HA peptide tag (Wilson et al., Cell, 37, 767, 1984). Either can help to purify the polypeptide sequence to be fused. The polynucleotide of the present invention also includes a polynucleotide having a structural gene and a sequence originally associated with the structural gene and controlling the expression of the gene. However, the polynucleotide contained in the polynucleotide of the present invention is not limited to this.
[0219]
The nucleotide sequence encoding the BASB071 polypeptide of SEQ ID NO: 16 may be the same as the polypeptide coding sequence contained in nucleotides 1-804 of SEQ ID NO: 15, respectively. Alternatively, it may be a sequence encoding the polypeptide of SEQ ID NO: 16, as a result of the redundancy (degeneracy) of the genetic code.
[0220]
The expression "polynucleotide encoding a polypeptide" is used herein to refer to a polypeptide of the invention, in particular a bacterial polypeptide, more particularly a Neisseria meningi having the amino acid sequence given by SEQ ID NO: 16. A polynucleotide comprising a sequence encoding a Tidis BASB071 polypeptide. The expression may be a single contiguous or discontinuous region encoding a polypeptide (eg, an integrated phage, an integrated insert, an integrated vector sequence, a polynucleotide interrupted by an integrated transposon sequence, Or polynucleotides interrupted for RNA editing, genomic DNA rearrangement), as well as additional polynucleotides that may also contain coding and / or non-coding sequences. .
[0221]
The invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the deduced amino acid sequence of SEQ ID NO: 16. Fragments of a polynucleotide of the invention can be used, for example, to synthesize a full-length polynucleotide of the invention.
[0222]
Furthermore, according to a particularly preferred embodiment, of the amino acid sequence of the BASB071 polypeptide given by SEQ ID NO: 16, some amino acid residues, for example 5-10, 1-5, 1-3, 2 Provided is a polynucleotide encoding a BASB071 variant in which one or zero amino acid residues have any combination of substitutions, changes, deletions, and / or additions. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of the BASB071 polypeptide.
[0223]
Further, in accordance with a preferred embodiment of the present invention, a polynucleotide that matches at least 85% over its entire length with a polynucleotide encoding a BASB071 polypeptide having the amino acid sequence set forth in SEQ ID NO: 16, and is complementary to the polynucleotide. And polynucleotides are provided. In this regard, polynucleotides that are at least 90% identical over the entire length are particularly preferred, and among such particularly preferred polynucleotides, those that are at least 95% identical are particularly preferred. Further, among polynucleotides having at least 95% identity, polynucleotides having at least 97% identity are more preferred, among which at least 98% identity, at least 99% identity are more preferred, and at least 99% identity. Are even more preferred.
[0224]
According to a preferred embodiment, there is provided a polynucleotide encoding a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA of SEQ ID NO: 15.
[0225]
According to some preferred embodiments of the present invention, there are provided polynucleotides that hybridize to a BASB071 polynucleotide sequence, eg, the polynucleotide of SEQ ID NO: 15, particularly under stringent conditions.
[0226]
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences described herein. In this regard, the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As used herein, the terms "stringent conditions", "stringent hybridization conditions" refer to hybridization that occurs only when at least 95%, and preferably at least 97%, matches between sequences. Specific examples of stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, Culturing overnight at 42 ° C. in a solution containing 20 μg / ml denatured and cleaved salmon sperm DNA, then washing the hybridization support at about 65 ° C. in 0.1 × SSC. Things. Hybridization and washing conditions are well known and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In particular in Chapter 11. Solution hybridization can also be performed on the polynucleotide sequences of the present invention.
[0227]
According to the present invention, furthermore, a stringent hybridization of a suitable library containing the complete gene for the polynucleotide sequence given by SEQ ID NO: 15 with a probe having this polynucleotide sequence given by SEQ ID NO: 15 or a fragment thereof A polynucleotide comprising a polynucleotide sequence obtained by screening under conditions and isolating the polynucleotide sequence, or a polynucleotide comprising such a polynucleotide sequence is provided. Fragments useful for obtaining such polynucleotides include, for example, probes and primers described in detail elsewhere in this specification.
[0228]
The polynucleotide assay of the present invention is described elsewhere in this specification. For example, the polynucleotide of the present invention is used as a probe for hybridization of RNA, cDNA and genomic DNA, and the full-length encoding BASB071 is used. It is possible to isolate a cDNA or a genomic clone, or to isolate another gene having a high degree of matching with the BASB071 gene, particularly a cDNA or genomic clone having a high degree of sequence matching. The probe will generally contain at least 15 nucleotide residues or base pairs. The probe preferably contains at least 30 nucleotide residues or base pairs, and may contain at least 50 nucleotide residues or base pairs. Particularly preferred probes will contain more than 20 and less than 30 nucleotide residues or base pairs.
[0229]
The coding region of the BASB071 gene can be isolated by synthesizing an oligonucleotide probe using the DNA sequence shown in SEQ ID NO: 15 and performing screening using this probe. Therefore, a library of cDNA, genomic DNA, or mRNA is screened using a labeled oligonucleotide having a sequence complementary to the gene of the present invention, and it is confirmed to which member of the library the probe hybridizes.
[0230]
There are several well-known methods utilized by those skilled in the art to obtain full-length DNA or to extend short DNA. For example, a method based on the cDNA end rapid amplification (RACE) method (see, for example, Flowman et al., PNAS USA, Vol. 85, pp. 8998-9002, 1988). Recently, this technology has been improved to, for example, the Marathon (registered trademark) method (Clontech Laboratories), and the search for cDNAs longer than before has been greatly simplified. In the Marathon® method, cDNA is prepared from mRNA extracted from a selected tissue and an “adapter” sequence is ligated to each end. Next, nucleic acid amplification (PCR) is performed using a combination of gene-specific and adapter-specific oligonucleotide primers to amplify the "missing" 5 'end of the DNA. Next, "nested" primers, ie, primers designed to anneal in the amplified product (typically adapter-specific primers that further anneal the 3 'end in the adapter sequence, or selected primers) The PCR reaction is repeated using a gene-specific primer that further anneals the 5 'end of the gene sequence. The product of this reaction is then analyzed by DNA sequencing and independent full-length PCR is performed by directly binding the product to the DNA present, or using new sequence information to design the 5 'primer. By performing the above, a full-length DNA can be constructed and a complete sequence can be obtained.
[0231]
The polynucleotides and polypeptides of the present invention can be used, for example, as research reagents and materials for discovering therapeutic methods and diagnostic methods for diseases, especially human diseases. This is described further herein in the context of polynucleotide assays.
[0232]
The polynucleotide of the present invention is an oligonucleotide derived from the sequences of SEQ ID NOS: 1 to 16. These polynucleotides are utilized in the methods described herein, preferably in PCR, to determine whether all or a portion of the herein identified polynucleotides are transcribed in bacteria in the infected tissue. Can be used for It is believed that such sequences can also be used to diagnose the stage and type of infection of the pathogen.
[0233]
According to the present invention, a polypeptide in which an amino acid is added to the amino terminus or carboxyl terminus of a mature protein, or a polypeptide comprising amino acids in the mature polypeptide (the mature form has, for example, two or more polypeptide chains) Are also provided. Such sequences have a variety of functions, among others, playing a role in processing proteins from precursors to mature forms, enabling protein transport, and increasing the half-life of proteins. And may be shorter or easier to handle the protein for assay or production. As is common in living organisms, the added amino acids may be removed from the mature protein by cellular enzymes.
[0234]
For each and every polynucleotide of the invention, a complementary polynucleotide is provided. Preferably, these complementary polynucleotides are completely complementary to each polynucleotide of interest.
[0235]
Precursor proteins may be inactive due to the fusion of one or more prosequences to the mature protein. Upon removal of the prosequence, such inactive precursors are generally activated. Removal of some or all of the prosequence can activate it. Generally, such precursors are called proproteins.
[0236]
In describing some polynucleotides of the present invention, the notation "N" can be used in addition to the standard notation for nucleotides, A, G, C, T / U. "N" means that any of the four nucleotides of DNA or RNA may appear at a designated position in the DNA or RNA sequence. Except where it is preferred that N is not a nucleic acid that, when read in combination with the position of an adjacent nucleotide in the correct reading frame, has the effect of generating a premature stop codon in that reading frame.
[0237]
In short, the polynucleotides of the present invention can encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), and one or more prosequences that are not leader sequences of the preprotein. Mature protein precursor, or preproprotein. A preproprotein is the precursor of a proprotein, which includes a leader sequence and one or more prosequences. The prosequence is generally removed in a processing step to produce an active, mature form of the polypeptide.
[0238]
According to one aspect of the present invention, there is provided a method of using the polynucleotide of the present invention for treating or preventing a disease, particularly for gene immunization.
[0239]
When using the polynucleotide of the present invention for gene immunization, it may be preferable to use an appropriate delivery method. For example, a method of directly injecting plasmid DNA into muscle (Wolf et al., Hum. Mol. Genet., Vol. 1, p. 363, 1992; Manthorpe et al., Hum. Gene. Ther., Vol. 4, p. 419, 1983), delivery of DNA complexed with a specific protein carrier (Wu et al., J. Biol. Chem., 264, 16985, 1989), DNA-calcium phosphate precipitation method (Benvenisti and Richhef). , PNAS USA, Vol. 83, pp. 9551, 1986), a method of encapsulating DNA in various forms of liposomes (Kaneda et al., Science, 243, 375, 1989), particle bombardment (Tan et al., Nature). 356, 152, 1992; Eisen Brown Other methods include DNA Cell Biol., Vol. 12, p. 791, 1993), and in vivo infection using a cloned retroviral vector (Sieger et al., PNAS USA, 81, 5849, 1984). .
Vectors, host cells, expression systems
The present invention relates to vectors comprising one or more polynucleotides of the present invention, host cells genetically modified with the vectors of the present invention, and the production of polypeptides of the present invention by recombinant techniques. In order to produce such a protein using RNA derived from the DNA structure of the present invention, a cell-free translation system can also be used.
[0240]
The recombinant polypeptide of the present invention can be prepared based on a genetically modified host cell containing an expression system by a method well known to those skilled in the art. Therefore, as still another aspect, the present invention provides an expression system comprising one or more polynucleotides of the present invention, a host cell genetically modified using the expression system, and a polypeptide of the present invention obtained by recombinant techniques. Production.
[0241]
In order to produce the polypeptide of the present invention by genetic recombination, the expression system or a part thereof, or the polynucleotide of the present invention is incorporated into host cells by genetic engineering. Polynucleotides are introduced into host cells using methods described in standard laboratory manuals. Experimental guides include, for example, Davis et al., "Basic Methods in Molecular Biology" (1986), Sambrook et al., "Molecular Cloning: A Laboratory Manual," Second Edition (Cold Spring Harbor Laboratory). Press, Cold Spring Harbor, New York, 1989). Methods include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic. There is introduction, infection.
[0242]
Representative examples of suitable host cells include the bacterial cells streptococci, staphylococci, enterococci, E. coli, E. coli, streptomyces, cyanobacteria, and Bacillus subtilis. (Bacillus subtilis), Moraxella catarrhalis, Haemophilus influenzae, and Neisseria meningitidis (cells of fungi); basidlomycete), Candida albicans, Aspergillus cells; insect cells Drosophila S2, Spodoptera Sf9 cells; animal cells CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1, Bowes (Bowes) Melanoma cells; gymnosperms and angiosperms that are plant cells.
[0243]
A variety of expression systems can be used to produce the polypeptides of the present invention. Such vectors include, in particular, chromosome-derived vectors, episomal-derived vectors, and virus-derived vectors. Specifically, a vector derived from a bacterial plasmid, a vector derived from a bacteriophage, a vector derived from a transposon, a vector derived from a yeast episome, a vector derived from an insertion element, a vector derived from a yeast chromosome element, a baculovirus, a papovavirus such as SV40, In addition to vectors derived from viruses such as vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, picornavirus, retrovirus, and alphavirus, vectors derived from combinations thereof, such as plasmids and bacteriophage genetic elements Vectors derived from certain cosmid and phagemid combinations are included. The expression system construct may include a region that regulates expression and controls development. Generally, expression can be achieved using any system or vector suitable for maintaining, propagating, expressing and / or expressing the polypeptide in the host. The appropriate DNA sequence can be inserted into the expression system using any of a variety of well-known general methods. Such methods are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, supra.
[0244]
In eukaryotic recombinant expression systems, appropriate secretion signals are incorporated into the expressed polypeptide to secrete the translated protein into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment there is a possibility. This signal can be endogenous to the polypeptide or a heterologous signal.
[0245]
The polypeptides of the present invention can be recovered from recombinant cell culture and purified by well-known methods. Known methods include, for example, precipitation with ammonium sulfate or ethanol, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography. Can be Most preferably, purification utilizes ionic metal affinity chromatography (IMAC). If the polypeptide is denatured during intracellular synthesis, isolation and purification, the protein can be refolded using known methods to recreate the active conformation.
[0246]
The expression system may be a live recombinant microorganism such as a virus or a bacterium. The gene of interest can be inserted into the genome of a living recombinant virus or bacterium. When a living vector is inoculated with the living vector and infected, the antigen is expressed in the living body and an immune response is induced. Examples of viruses and bacteria used for this purpose include poxviruses (eg, vaccinia, fowlpox, canarypox), alphaviruses (Sindbis virus, Semliki forest virus, Venezuelan equine encephalitis virus), adenovirus, adeno-associated virus , Picornavirus (poliovirus, rhinovirus), herpes virus (varicella-zoster virus, etc.), Listeria, Salmonella, Neisseria, and BCG. These viruses and bacteria can be toxic and can be attenuated in various ways to obtain a live vaccine. Such live vaccines are also part of the present invention.
Diagnostics, prognosis, serotyping, mutation detection
The present invention also relates to the use of the polynucleotide and polypeptide BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 of the present invention as a diagnostic reagent. Diagnosis of disease, staging of disease, infection by detecting BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotides and / or polypeptides in eukaryotes, especially mammals, especially humans. A method for diagnosing the response of a living body to a drug will be provided. Eukaryotes, in particular mammals, especially humans, among which humans infected or suspected of being infected with an organism containing the gene or protein BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071. Can be detected at the nucleic acid or amino acid level by various well-known methods, as well as the methods described herein.
[0247]
Polypeptides and polynucleotides for prognosis, diagnosis, or other analysis can be obtained from the body material of the putatively infected and / or infected individual. Polynucleotides from any other source, particularly DNA or RNA, can be used for direct detection, or can be enzymatically amplified by PCR or any other method before performing the analysis. RNA, especially mRNA, cDNA and genomic DNA can be used in a similar manner. By performing the amplification, the characterization of the species or strain of the organism that has infected the individual or the organism that resides within the individual can be achieved by analyzing the serotype of a polynucleotide selected from the organism. Detecting deletions and insertions by size changes when the amplified product is compared to a serotype of a reference sequence selected from the relevant organism, preferably a different species of the same genus, or a different strain of the same species. it can. Point mutations can be identified by hybridizing the amplified DNA to a labeled BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotide sequence. Perfectly matched sequences, or a significant proportion of sequences, are only incompletely detected by digestion with DNAse or RNAse for DNA and RNA, respectively, or by detecting differences in melting temperature or regeneration kinetics. Discrepancies or a significant percentage of the discrepancies can be distinguished. Polynucleotide sequence differences can also be detected as changes in the mobility of polynucleotide fragments in gels during electrophoresis when compared to a reference sequence. In that case, a modifier may or may not be used. Polynucleotide differences can also be detected by direct sequencing of DNA or RNA. See, for example, Myers et al., Science, 230, 1242, 1985. Sequence changes at specific positions can also be revealed by nuclease protection assays, such as RNase, V1, S1 protection assays, and chemical cleavage methods. See, for example, Cotton et al., Proc. Natl. Acad. Sci. USA 85: 4397-4401, 1985.
[0248]
In another embodiment, oligonucleotide probes comprising a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 nucleotide sequence or a fragment thereof are arranged in an array, for example, for gene mutation, serotype, classification, identification. Can be effectively screened. Because of the well-known and general nature of array technology, it can be applied to a variety of molecular genetics issues such as gene expression, gene linkage, gene variability, and the like (see, for example, Qi et al., Science, 274, 610, 1996).
[0249]
Therefore, as another aspect, the present invention relates to a diagnostic kit including:
(A) the polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 or a fragment thereof;
(B) a nucleotide sequence complementary to the sequence of (a);
(C) a polypeptide of the invention, preferably a polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, or a fragment thereof;
(D) an antibody against the polypeptide of the present invention, preferably an antibody against the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16.
[0250]
It will be appreciated that all such kits (a), (b), (c), (d) contain substantial components. Such kits are used, inter alia, for diagnosing a disease or susceptibility to a disease.
[0251]
The present invention also relates to the use of the polynucleotide of the present invention as a diagnostic reagent. When the polynucleotide of the present invention, preferably the polynucleotide of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 is mutated to cause under-expression, over-expression, or altered expression, a disease or pathogenicity is caused. The detection of such mutations is a diagnostic tool that can be used to diagnose disease, predict disease prognosis, determine disease stage, confirm susceptibility to disease, and to clarify such matters. can do. Organisms that have mutated such polynucleotides, especially infected organisms in such a condition, can be detected at the polynucleotide level by a variety of methods, such as those described elsewhere in this specification. .
[0252]
Cells from organisms having mutations or polymorphisms (allelic mutations) in the polynucleotides and / or polypeptides of the invention are detected at the polynucleotide or polypeptide level by various methods, eg, to determine serotype. You can also. For example, RT-PCR can be used to detect mutations in RNA. It is particularly preferred to combine RT-PCR with an automated detection system, for example GeneScan. PCR can also be performed on RNA, cDNA, and genomic DNA. As an example, mutations can be identified and analyzed using PCR primers complementary to a polynucleotide encoding BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide.
[0253]
According to the invention there is further provided a primer wherein 1, 2, 3 or 4 nucleotides have been removed from the 5 'and / or 3' end. The primer may be used, in particular, for DNA and / or RNA of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 isolated from a sample derived from an individual, for example, a sample derived from a substance constituting the body. Can be used to amplify. When a polynucleotide isolated from an infected individual is amplified using this primer, the sequence of the polynucleotide can then be determined in a variety of ways. In this way, mutations in the polynucleotide sequence are detected, and based on the mutations, diagnosis and / or prognosis of the infection state or its stage or progress, serotyping and / or classification of the infectious agent Can be performed.
[0254]
The present invention further provides a method for diagnosing a disease, preferably for diagnosing a bacterial infection, more preferably for diagnosing an infection caused by Neisseria meningitidis. This method comprises expressing a polynucleotide having the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 from a sample derived from an individual, for example, a sample derived from a substance constituting the body. Includes measuring the level rise. Increase or decrease in expression of a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotide may be measured using any method known to those skilled in the art as a method for quantifying a polynucleotide. Can be. Such methods include, for example, amplification, PCR, RT-PCR, RNase protection, Northern blot, spectroscopy, and other hybridization methods.
[0255]
Further, the diagnostic method of the present invention may be used to detect overexpression of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide by comparing with a control normal tissue sample, for example, to determine the presence of infection. Can be detected. Assays that can be used to measure levels of a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide in a sample derived from a host, eg, a body-derived material. Is well known to those skilled in the art. Such assays include radioimmunoassays, competitive binding assays, Western blot analysis, antibody sandwich assays, antibody detection, and ELISA.
[0256]
The polynucleotides of the present invention can be used as components of a polynucleotide array, preferably as components of a high density array or grid. This high-density array is particularly useful for diagnosis and prognosis. For example, using a collection of spots, each containing one or more polynucleotides of the present invention in addition to a different gene, using probes obtained from or derived from substances making up the body, Probing can be performed using hybridization or nucleic acid amplification to determine whether a particular polynucleotide sequence or related sequences are present in an individual's body. The presence of such may indicate the presence of a pathogen, in particular Neisseria meningitidis, and may provide a diagnosis and / or prognosis of the disease or disease progression. May help to clarify. Grids containing multiple variants of the polynucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 are preferred. Also preferred are grids comprising multiple variants of the polynucleotide sequence encoding the polypeptide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16.
antibody
Antibodies immunospecific for such polypeptides or polynucleotides can be produced using the polypeptides and polynucleotides of the present invention, or variants thereof, or cells expressing the same, as immunogens.
[0257]
According to some preferred embodiments of the present invention, there are provided antibodies against BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptides or polynucleotides.
[0258]
Antibodies against the polypeptide or polynucleotide of the present invention express the polypeptide and / or polynucleotide of the present invention, or one or both fragments having an epitope, or one or both analogs, or one or both of them. The cells can be obtained by administering the cells to an animal, preferably a non-human animal, according to general protocols. For preparing monoclonal antibodies, any conventional technique which provides antibodies produced by continuous cell line cultures can be used. There are various methods, for example, Kohler, G, Milstein, C.E. Cosbour et al., Immunology Today, Vol. 4, p. 72, 1983; Kohl et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Lis, 1985. Nature, 256, 495-497, 1975. ) On pages 77-96.
[0259]
A single-chain antibody against the polypeptide or polynucleotide of the present invention can be produced using a single-chain antibody production method (US Pat. No. 4,946,778). In addition, transgenic mice, or other organisms or animals, such as mammals, can be used to express immunospecific humanized antibodies against the polypeptide or polynucleotide of the present invention.
[0260]
Alternatively, the phage display method is used to screen the PCR-amplified v-gene of human-derived lymphocytes screened to treat anti-BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071. Antibody genes having binding activity to a polypeptide of the invention can be selected from a repertoire or from a library that has never been tested (Mcaccafati et al., Nature, 348, 552-554, 1990). Years; Marks et al., Biotechnology, Vol. 10, pp. 779-783, 1992). The affinity of these antibodies can also be improved, for example, by chain shuffling (Clackson et al., Nature, 352, 628, 1991).
[0261]
Clones expressing the polypeptide or polynucleotide of the present invention can be isolated or identified using the antibodies described above, and the polypeptide or polynucleotide can be purified, for example, by affinity chromatography.
[0262]
Thus, in particular using BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide or BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB066 against BASF061 or BASB066 against BASB051 or BASB066 against BASB051. can do.
[0263]
As polypeptide variants, antigen, epitope, or immunity equivalent variants constitute a particular aspect of the present invention.
[0264]
It is preferred that the antibodies or variants thereof be modified to reduce immunity in the individual. For example, if the individual is a human, it is most preferred that the antibody be "humanized" and that the complementarity-determining regions of the antibody derived from the hybridoma be grafted on a human monoclonal antibody. This is described, for example, in Jones et al., Nature, Vol. 321, 522-525, 1986, or Tempest et al., Biotechnology, Vol. 9, 266-273, 1991.
Antagonists and agonists-assays and molecules
The polypeptides and polynucleotides of the present invention can also be used to evaluate the avidity of small molecule substrates and ligands in, for example, cells, cell-free preparations, compound libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands, or may be artificial products having similar structures or functions. See, for example, Corrigan et al., The Latest Protocols in Immunology, Vol. 1 (2), Chapter 5, 1991.
[0265]
By the screening method, the binding of the candidate compound to the polypeptide or polynucleotide, the cell or membrane having the polypeptide or polynucleotide, or the fusion protein of the polypeptide is determined using a label directly or indirectly associated with the candidate compound. It can be easily measured. In the screening method, competition with a labeled competitor compound may occur. Furthermore, by using an appropriate detection system for cells containing the polypeptide or polynucleotide in these screening methods, it is possible to test whether the candidate compound generates a signal by activating or suppressing the polypeptide or polynucleotide. it can. Inhibitors of activity are generally analyzed in the presence of a known agonist to observe the effect of that agonist on activation due to the presence of the candidate compound. Using a constitutively active polypeptide and / or a constitutively expressed polypeptide and polynucleotide to test whether a candidate compound inhibits activation of the polypeptide or polynucleotide in the presence of an agonist or inhibitor By doing so, it is possible to screen for an inverse agonist or an inverse inhibitor. Furthermore, the screening method involves mixing the candidate compound with a solution containing the polypeptide or polynucleotide of the present invention to form a mixture, and in the mixture BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide and / or Alternatively, it may include only measuring the activity of the polynucleotide and comparing the BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide and / or polynucleotide activity of the mixture to a reference. It is possible. Fusion proteins, for example, fusion proteins made from an Fc protein and BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptides, as previously described, may be antagonists of the polypeptides of the invention, or phylogenetically. And / or can be used in high-throughput screening assays to identify antagonists of a functionally related polypeptide (D. Bennett et al., J. Mol. Recognition, 8, 52-58, 1995). Y .; K. Johansson et al., J. Biol. Chem., 270 (16), 9449-9471, 1995).
[0266]
Polynucleotides, polypeptides, and antibodies that bind and / or interact with the polypeptides of the present invention can also be used to design screening methods to detect the effect of additional compounds on mRNA and / or polypeptide production in cells. Can be used. For example, an ELISA can be constructed by standard methods in the art to measure secreted or cell-associated levels of polypeptide using monoclonal and polyclonal antibodies. This can be used to find agents (referred to as antagonists and agonists, respectively) that may suppress or enhance the production of polypeptides from appropriately treated cells or tissues.
[0267]
According to the present invention, compounds that promote (agonist) or inhibit (antagonist) the action of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide or polynucleotide, in particular, bacterial growth inhibitory and / or bactericidal properties Also provided are compound screening methods for identifying compounds. This screening method can include high throughput techniques. For example, to screen for agonists or antagonists, a mixture for a synthetic reaction comprising a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide and a product obtained by labeling a substrate or ligand of the polypeptide, a method for screening cells, The preparation of a compartment (eg, a membrane, a cell envelope, a cell wall), or any of these, can be used to prepare candidate compounds that may be agonists or antagonists of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071. Culture in the absence or presence of The ability of a candidate compound to agonize or antagonize a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide is due to reduced binding of a labeled ligand or reduced production of a product from such a substrate. Appears. Molecules that bind without inducing the effects of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptides are most likely to be good antagonists. Molecules that bind well, and in some cases increase the rate of production of a product from a substrate, increase signaling, and increase the activity of a chemical channel are agonists. The rate and level of contextual product production, signal transduction, and chemical channel activity from the substrate can be readily detected by using a reporter system. Reporter systems that may be useful in this regard include those in which a substrate labeled for colorimetry has been converted to a product, the activity of a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotide or polypeptide. Reporter genes that are involved in the alteration of, such as, but not limited to, binding assays known in the art.
[0268]
Another example of an assay for agonists of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 is an example of an assay for BASB051, BASB057, BASB060, BASB066, BASB066, BASB066, BASB066, BASB066, BASB066, BASB066, BASB061 BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or a molecule that binds BASB071, BASB051, BASB057, BASB060, BASB061, BASB0, BSB0, or BSB063 Natural group Or a competitive assay that is combined with that mimic ligand, or substrate or ligand. When BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 are labeled with a radioactive substance or a compound for colorimetry, BASB051, BASB057, BASB060, BASB060, BASB060, BASB060 Because the number of BASB063, BASB065, BASB066 or BASB071 can be accurately determined, the effects of potential antagonists can be evaluated.
[0269]
Potential antagonists include, inter alia, small organic molecules, peptides, polypeptides, antibodies that bind to the polynucleotides and / or polypeptides of the invention, thereby inhibiting or extinguishing their activity or expression. . Potential antagonists bind to the same site on the binding molecule but do not elicit the activity elicited by BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071, thereby causing BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 Eliminates the binding of polypeptides and / or polynucleotides and excludes BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptides and / or polynucleotides or / and expressed polynucleotides. Prevent small organic molecules, peptides, closely related Polypeptides such that the protein, antibodies are also possible.
[0270]
Potential antagonists also include small molecules that bind to and occupy the binding site of the polypeptide, thereby blocking the binding of molecules that bind cells and inhibiting normal biological activity. Examples of small molecules include, but are not limited to, small organic molecules, peptides, peptide-like molecules. Other potential antagonists include antisense molecules (for a description of this molecule, see Okano, J. Neurochem. 56: 560, 1991; "As an antisense inhibitor of gene expression" Oligodeoxynucleotides, CRC Press, Boca Raton, Florida, 1988). Preferred potential antagonists include BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or a compound related to BASB071, BASB051, BASB057, BASB060, BASB061, BASB063, BASB063, BASB066 or BASB065, BASB066. .
[0271]
In yet another aspect, the invention includes polypeptides of the invention, or fragments thereof, and various portions of the constant regions of the heavy or light chains of various subclasses of immunoglobulins (IgG, IgM, IgA, IgE). The present invention relates to a recombinant soluble fusion protein. Preferred as immunoglobulins are the constant parts of the heavy chains of human IgG, especially IgG1, in which fusion takes place in the hinge region. According to a particular embodiment, the Fc part can be easily removed by incorporating a cleavage sequence that can be cut with blood coagulation factor Xa. The present invention further relates to a method for preparing the fusion protein by genetic engineering, and to screening, diagnosis, and treatment of drugs using the fusion protein. Yet another aspect of the invention also relates to a polynucleotide encoding such a fusion protein. Illustrative examples of techniques for fusion proteins can be found in International Patent Applications WO 94/29458, WO 94/22914.
[0272]
Each of the polynucleotide sequences described in this specification can be used for the discovery and development of antimicrobial compounds. Once expressed, the encoded protein can be used as a target for screening antimicrobial agents. In addition, the antisense sequence may be constructed using a polynucleotide sequence encoding the amino-terminal region of the encoded protein, a Shine-Dalgarno sequence, or a sequence that facilitates translation of individual mRNAs. Can be controlled.
[0273]
According to the present invention, the polypeptides, polynucleotides, agonists, antagonists of the present invention can be used to interact with one or more pathogens and a eukaryotic host, preferably a mammalian host, responsible for the pathogenesis after infection. There is also provided a use to prevent the initial physical interaction between them. In particular, the molecules of the present invention may be used to transform bacteria, especially Gram-positive and / or Gram-negative bacteria, into eukaryotic, preferably mammalian extracellular matrix proteins on indwelling devices, or wound extracellular matrix proteins. Used to prevent attachment; eukaryotic, preferably mammalian, extracellular matrix proteins and the bacteria BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB066 or BASB071 proteins that cause tissue damage. Used to prevent bacteria from attaching in between, and / or to prevent the onset of the disease from progressing normally due to infection due to implantation of an indwelling device or other means different from surgical methods. be able to.
[0274]
According to yet another aspect of the present invention there are provided BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 agonists and antagonists, preferably bacterial growth inhibitory and / or bactericidal agonists and antagonists.
[0275]
The agonists and antagonists of the present invention can be used, for example, for the prevention, suppression, and / or treatment of disease.
[0276]
In yet another aspect, the invention relates to mimotopes of the polypeptide of the invention. Mimotopes are peptide sequences that are very similar (in sequence or structure) to the native peptide and are recognized by antibodies that recognize the native peptide. Mimotopes can also enhance antibodies that recognize native peptides when combined with a suitable carrier.
[0277]
Peptide mimotopes can be designed for a particular purpose by adding, deleting, or substituting selected amino acids. Thus, the peptide can be modified so that it can be easily coupled to a protein carrier. For example, it may be desirable to include cysteine at the end for some chemical conjugation methods. In addition, the peptide bound to the protein carrier should have a hydrophobic end opposite to the bound end of the peptide so that this free end not bound to the peptide remains associated with the surface of the protein carrier. It would be desirable to do so. Doing so places the peptide in a conformation very similar to that of the peptide when in the context of the whole original molecule. For example, the peptide can be altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail. Also, D-stereoisomers can be added or substituted for one or more amino acids to make preferred derivatives, for example, improving the stability of the peptide.
[0278]
In addition, peptide mimotopes can be identified using an antibody capable of binding to the polypeptide of the present invention using a method such as a phage display method (EP 0 552 267 B1). This method yields a number of peptide sequences that are similar in structure to the native peptide, and thus can bind anti-natural peptide antibodies, but are not necessarily similar in sequence to the native polypeptide.
vaccine
Another aspect of the invention relates to a method of eliciting an immune response in an individual, especially a mammal, preferably a human. This method provides sufficient BASB051, BASB057, BASB060, BASB061 to generate an antibody and / or T-cell immune response to protect the individual from infection, particularly bacterial infection, especially Neisseria meningitidis. , BASB063, BASB065, BASB066 or BASB071 polynucleotides and / or polypeptides, or fragments or variants thereof, to the individual.
[0279]
The present invention also provides a method of slowing the rate of bacterial replication due to such an immune response. Yet another aspect of the invention relates to another method of eliciting an immune response in an individual. The method comprises providing a nucleic acid vector, sequence, or ribozyme to the individual to express a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotide and / or polypeptide, or a fragment or variant thereof. Indicating, and expressing BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotides and / or polypeptides, or fragments or variants thereof, in vivo, so that the disease is already in the individual, preferably a human. To protect the individual from the disease, whether or not it is occurring in the body, antibodies and / or T cells (eg, It includes operations to induce an immune response, such as generation of an immune response including emissions-producing T cells or cytotoxic T cells). One example of gene delivery is to quickly put the gene into the desired cell, such as a particle coating. Such nucleic acid vectors can include DNA, RNA, ribozymes, modified nucleic acids, DNA / RNA hybrids, DNA-protein complexes, or RNA-protein complexes.
[0280]
Yet another aspect of the invention is that it is capable of eliciting an immune response in an individual, preferably a human, when introduced into the individual, and wherein the individual has a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, An immunological composition that elicits an immune response against a BASB066 or BASB071 polynucleotide and / or a polypeptide encoded thereby. The immunogenic composition comprises a recombinant BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotide and / or a polypeptide encoded thereby, and / or the BASB051, BASB057, BASB060, BASB061, BASB061. , BASB065, BASB066 or BASB071 polynucleotides, polypeptides encoded thereby, or DNA and / or RNA encoding and expressing antibodies to other polypeptides of the invention. This immune response can be used for treatment or prevention. The immune response can also take the form of antibody immunity and / or cellular immunity, such as the form of cellular immunity arising from CTLs or CD4 + T cells.
[0281]
The BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide or fragment thereof can be fused to a coprotein or compound fragment. The coprotein or compound fragment, which may or may not itself produce antibodies, stabilizes the first protein to provide a fusion protein having antigenic and / or immunological properties, preferably protective properties. Alternatively, a modified protein can be produced. Thus, the recombinant fusion protein may be an antigenic coprotein such as, for example, Haemophilus influenzae, glutathione-S-transferase (GST), β-galactosidase, or any other comparison that solubilizes the protein and facilitates its production and purification. It is preferred that it further contain lipoproteins from the larger coproteins. In addition, the coprotein can act as an adjuvant in the sense of generally stimulating the immune system of an organism that accepts the protein. The coprotein can be attached to either the amino or carboxyl terminus of the first protein.
[0282]
In the vaccine composition of the present invention, the BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide and / or polynucleotide, a fragment thereof, a mimotope thereof, a mutant thereof are obtained by the above-mentioned live recombinant vector, for example, It may be present in a vector such as a live bacterial vector.
[0283]
For BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptides, non-living vectors, such as bacterial outer membrane vesicles, or "bubbles", are also suitable. OM vesicles are derived from the outer membrane of the bilayer membrane of Gram-negative bacteria, and are found in many Gram-negative bacteria including Chlamydia trachomatis and C. psittaci. (Tou, L. et al., FEMS Microbiol. Lett., 163, 223-228, 1998). Bacterial pathogens that have been reported to generate blebs include Bordetella pertussis, Borrelia burgdorferi, Brucella melitensis, Brucella visvis , Escherichia coli, Haemophilus influenza, Legionella pneumophila, Neisseria gonorrhea, Neisseria gonorrhose, Naseria goniasnos Ginosas, Gonias nosiné s. There is such Erushinia en terrorism coli Atlantica (Yersinia enterocolitica), but is not limited only to this.
[0284]
Bblebs have the advantage of being able to provide outer membrane proteins in their native conformation and are therefore particularly effective in vaccines. Bubbles can also be improved for vaccines by genetically modifying bacteria to alter the expression of one or more molecules located in the outer membrane. Thus, for example, the expression of a desired immune protein in the outer membrane, such as the expression of a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide is introduced or up-regulated. can do. Alternatively or additionally, unrelated outer membrane molecules (eg, unprotected antigens or immunodominant but variable proteins) or harmful outer membrane molecules (eg, toxic molecules such as LPS, Or molecules that may elicit an autoimmune response) can be down-regulated. These methods will be described in detail below.
[0285]
The non-coding flanking region of the BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene contains a regulatory region important for expression of that gene. This regulation occurs at both the transcriptional and translational levels. The sequence of these regions located upstream or downstream of the gene's open reading frame can be obtained by DNA sequencing. This sequence information can be used to determine potential regulatory motifs, such as various promoter elements, terminator sequences, inducible sequence elements, repressors, elements responsible for phase changes, Shine-Dalgarno sequences, Other types of regulatory motifs and sequences can be determined, as well as regulatory-related regions having the following structures:
[0286]
With this sequence information, the natural expression of the BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene can be changed. Up-regulation of gene expression can be achieved by altering the promoter, Shine-Dalgarno sequence, potential repressors, operator elements, or other elements of interest. Similarly, down-regulation of expression can be achieved by similar alterations. Also, by altering the phase mutation sequence, the expression of the gene can be placed under the control of the phase mutation or made independent of this regulation. In the alternative, the gene can be placed under the control of one or more inducing elements, so that expression can be controlled. Specific examples of such controls include, but are not limited to, induction by temperature shifts and the addition of inducible substrates, such as the addition of selected carbohydrates or derivatives thereof, microelements, vitamins, cofactors, metal ions, and the like. Not necessarily.
[0287]
Such changes can be introduced in several different ways. Alteration of sequences related to gene expression can be achieved by random mutating in vivo and selecting the desired phenotype. Another method consists in isolating the region of interest and then mutating the region randomly or by making substitutions, insertions or deletions by site-specific mutations. . The altered region can then be reintroduced into the bacterial gene by homologous recombination to evaluate its effect on gene expression. Another method utilizes knowledge of the sequence of the region of interest to replace or remove all or part of the natural regulatory sequence. In this case, the target regulatory region is separated or changed to include regulatory elements from another gene, a combination of regulatory elements from various genes, synthesized regulatory elements, or any other regulatory elements. Or a specific portion of the wild-type regulatory sequence is removed. These altered sequences can then be reintroduced into bacterial genes through homologous recombination. Preferred promoters that may be available for up-regulation of gene expression include promoters from Neisseria meningitidis or Neisseria gonorrhoeae porA, porB, lbpB, tbpB, p110, 1st, hpuAB; ompCD, copB UspA2; TbpB from Moraxella catarrhalis; p1, p2, p4, p5, p6, lpD, tbpB, D15, Hia, Hmwl, Hmw2 from Haemophilus influenzae. But not limited to this.
[0288]
According to one embodiment, expression of the gene causes the promoter of the gene to become stronger (through isolating the upper sequence of the gene, changing the sequence in vitro, and reintroducing the genome by homologous recombination). It can be changed by replacing with a promoter. Up-regulated expression can be achieved both in bacteria and in outer membrane vesicles selected (made by bacteria) from bacteria.
[0289]
According to another embodiment, the methods described above can be used to produce recombinant bacterial strains with improved properties for vaccine applications. Such strains have reduced toxicity, have increased expression of certain antigens, have knocked out (or have reduced expression of) genes that interfere with the immune response, or have regulated expression of immunodominant proteins Strain, and a strain in which the selection of outer membrane vesicles is regulated, but is not limited thereto.
[0290]
Therefore, according to the present invention, there is also provided an altered upstream region of the BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene. The altered upstream region contains a heterologous regulatory element that alters the expression level of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 protein located in the outer membrane. An upstream region according to this aspect of the invention includes a sequence upstream of the BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene. The upstream region begins just upstream of the BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene and usually extends from the ATG start codon to no more than about 1000 bp upstream of this gene. For genes located in polycistronic sequences (operons), the upstream region may begin immediately before the gene of interest or before the first gene in the operon. The altered upstream region according to this aspect of the invention preferably contains a heterologous promoter at a position 500-700 bp upstream of the ATG.
[0291]
Thus, the present invention provides a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide within the altered bacterial foam. Further according to the present invention there is provided a host cell capable of producing a membrane-based non-live vesicle vector. Further according to the present invention there is provided a nucleic acid vector comprising a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene having an altered upstream region comprising a heterologous regulatory element.
[0292]
Further according to the present invention there is provided a method for preparing host cells and bacterial blebs according to the present invention.
[0293]
The present invention further provides compositions, particularly vaccine compositions, as well as methods comprising a polypeptide and / or polynucleotide of the invention and an immunostimulatory DNA sequence. For immunostimulatory DNA sequences, see Sato, Y .; Science, Vol. 273, p. 352, 1996.
[0294]
According to the present invention, further, in a polynucleotide structure used in an immunity establishment experiment using a gene in a model animal infected with Neisseria meningitidis, the above-mentioned polynucleotide, or a specific fragment thereof, may be used as a bacterial cell. Also provided are methods of using fragments known to encode the non-variable region of the surface protein of the invention. Such experiments will be particularly useful in identifying protein epitopes capable of raising an immune response for prevention or treatment. By this method, monoclonal antibodies of special value derived from the required organs of animals that are resistant to or capable of eliminating the infection can be used to convert bacterial infections in mammals, especially humans, especially Neisseria It is believed that it will soon be ready for the development of a prophylactic or therapeutic method for Meningitidis infection.
[0295]
The invention also includes a vaccine formulation comprising an immunogenic recombinant polypeptide and / or polynucleotide of the invention and a suitable base, for example, a pharmaceutically acceptable carrier. Since polypeptides and polynucleotides can be degraded in the stomach, it is preferable to administer each of them parenterally. For example, it is administered subcutaneously, intramuscularly, intravenously, or intradermally. Formulations suitable for parenteral administration include aqueous, non-aqueous, sterile, which may contain antioxidants, buffer solutions, bacterial growth inhibiting compounds, solutes that render the formulation isotonic with the body fluid of the individual, preferably blood. Aqueous and non-aqueous sterile dispersions which may contain dispersing agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules or vials. The formulation may be stored in a lyophilized state, requiring only the addition of a sterile base immediately before use.
[0296]
The vaccine formulation of the present invention may further include an adjuvant system to improve the immunity of the formulation. Preferably, the adjuvant system selectively increases a TH1-type response.
[0297]
Immune responses can be broadly divided into two typical categories. It is a humoral-mediated immune response and a cell-mediated immune response (traditionally characterized by antibodies and cellular effector mechanisms for protection, respectively). These categories of responses are referred to as TH1-type responses (cell-mediated responses) and TH2-type responses (fluid-mediated responses).
[0298]
A typical TH1-type immune response would be characterized by the generation of antigen-specific, haplotype-limited cytotoxic T lymphocytes and a natural killer cell response. In mice, a TH1-type response is characterized by producing antibodies of the IgG2a subtype, whereas in humans, this response corresponds to an IgG1-type antibody. TH2-type immune responses are characterized by the production of various isotypes of immunoglobulins. For example, in a mouse, IgG1, IgA, and IgM are produced.
[0299]
It is thought that cytokines are driving behind these two types of immune responses as they evolve. A high level of TH1 type cytokine induces a cell-mediated immune response to a given antigen, while a high level of TH2 type cytokine induces a body fluid-mediated immune response to that antigen. Tends to be induced.
[0300]
The distinction between TH1-type and TH2-type immune responses is not exact. In effect, the individual will support an immune response that is described as being predominantly TH1 or predominantly TH2. However, it is often convenient to consider the cytokine family described by Mossman and Koffman in murine CD4 + ve T cell clones (Mossman, TR and Koffman, RL, "TH1 and TH2 cells. : Different patterns of lymphokine secretion leading to different functional properties ", Annual Review of Immunology, 7, 145-173, 1989). In general, a TH1-type response is accompanied by the production of cytokines IFN-γ and IL-2 by T lymphocytes. Other cytokines that are often directly involved in eliciting a TH1-type immune response, such as IL-12, are not produced by T cells. Conversely, TH2-type responses are accompanied by secretion of IL-4, IL-5, IL-6 and IL-13.
[0301]
Certain vaccine adjuvants are known to be particularly suitable for stimulating either TH1-type or TH2-type cytokine responses. In general, the best indicator of the TH1: TH2 balance of the immune response after vaccination or infection is to measure the production of TH1-type or TH2-type cytokines by T lymphocytes in vitro after restimulation with antigen; And / or measuring the ratio of IgG1: IgG2a in an antigen-specific antibody reaction.
[0302]
Thus, TH1-type adjuvants, when restimulated in vitro with antibodies, selectively stimulate the isolated populations of T cells to produce high levels of TH1-type cytokines, with CD8 + cytotoxic T lymphocytes, It is an adjuvant that promotes the development of both antigen-specific immunoglobulin responses associated with TH1-type isotypes.
[0303]
Adjuvants capable of selectively stimulating a TH1-type cellular response are described in International Patent Applications WO 94/00153, WO 95/17209.
[0304]
3de-O-acrylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is known from GB 2220211 (Libi). Chemically, it is a mixture of 3 de-O-acrylated monophosphoryl lipid A and 4, 5, or 6 acrylated chains, manufactured by Ribi Immunochem, Montana. A preferred form of 3de-O-acrylated monophosphoryl lipid A is disclosed in EP 0 689 454 B1 (SmithKline Beecham Biologicals).
[0305]
Preferably, the particles of the 3D-MPL are small enough and sterile filtered through a 0.22 micron membrane (EP 0 689 454).
[0306]
3D-MPL will contain from 10 μg to 100 μg, preferably 25 to 50 μg per dose. The antigen will then typically comprise 2 to 50 μg per dose.
[0307]
Another preferred adjuvant is QS21. This is an Hplc purified non-toxic fraction from Kiraya. In some cases, this may be mixed with 3de-O-acrylated monophosphoryl lipid A (3D-MPL) and further mixed with a base.
[0308]
A method for manufacturing QS21 is disclosed in U.S. Pat. No. 5,057,540.
[0309]
Non-reactive adjuvant formulations containing QS21 have been described in the past literature (WO 96/33739). Such formulations containing QS21 and cholesterol are known to work well as adjuvants that stimulate TH1 when formulated with an antigen.
[0310]
Yet another adjuvant that selectively stimulates a TH1-type cellular response is an immunomodulatory oligonucleotide. For example, an unmethylated CpG sequence is disclosed in WO 96/02555.
[0311]
Combinations of various adjuvants as described above that stimulate TH1 are also contemplated as adjuvants that selectively stimulate TH1 type cellular responses. For example, QS21 can be prescribed together with 3D-MPL. The ratio of QS21: 3D-MPL will typically be from 1:10 to 10: 1, but is preferably from 1: 5 to 5: 1 and often substantially 1: 1. . The preferred range of the QS21: 3D-MPL ratio for obtaining optimal synergy is 2.5: 1 to 1: 1.
[0312]
Preferably, the vaccine composition of the present invention also contains a base. The base is an oil-in-water emulsion or an aluminum salt such as aluminum phosphate or aluminum hydroxide.
[0313]
Preferred oil-in-water emulsions are metabolizable oils such as squalene, α-tocopherol, Tween 80 ™. In a particularly preferred aspect, the antigen in the vaccine composition of the invention is associated with QS21 and 3D-MPL in such an emulsion. Further, the oil-in-water emulsion may comprise Span 85 ™ and / or lecithin and / or tricaprylin.
[0314]
When administered to humans, the vaccine will typically contain QS21 and 3D-MPL per dose in a dose of 1 μg to 200 μg, for example 10 to 100 μg, preferably 10 μg to 50 μg per dose. Oil-in-water emulsions will typically contain 2-10% squalene, 2-10% alpha tocopherol, 0.3-3% Tween 80. Preferably, the ratio of squalene: α-tocopherol is equal to or less than 1. This is because this makes the emulsion more stable. The span 85 may be present at a 1% level. In some cases, it is desirable that the vaccines of the present invention further include a stabilizer.
[0315]
The non-toxic oil-in-water emulsion preferably contains a non-toxic oil, such as squalane or squalene, and an emulsifier, such as Tween 80, in a water-soluble base. The water-soluble base can be, for example, a phosphate buffer solution.
[0316]
Particularly potent adjuvant formulations comprising QS21, 3D-MPL, tocopherol in an oil-in-water emulsion are described in WO 95/17210.
[0317]
The present invention provides a multivalent vaccine composition comprising, in addition to the vaccine preparation of the present invention, other antigens, particularly antigens effective for treating cancer, treating autoimmune diseases and related diseases. Such a multivalent vaccine composition may include an adjuvant that induces TH-1 as described above.
[0318]
Although the invention has been described with respect to particular BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptides and polynucleotides, the present invention provides for naturally occurring polypeptides and fragments of polynucleotides, It is understood that similar recombinant polypeptides and polynucleotides with additions, deletions, or substitutions that do not substantially affect the immunological properties of the peptide or polynucleotide are also included.
[0319]
The antigen can also be supplied in the form of a whole bacterium (dead or live) or in the form of a subcellular fraction. This includes, of course, supplying Neisseria meningitidis itself.
Compositions, kits, administration
According to yet another aspect of the invention, a BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotide and / or BASB051, BASB057, BASB060 for administration to a single cell or multicellular organism. , BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptides are provided.
[0320]
The invention also relates to compositions comprising the polynucleotides and / or polypeptides described herein, or agonists or antagonists thereof. The polypeptides and polynucleotides of the present invention may be non-sterile or sterile for use on cells, tissues, or organisms with one or more bases, such as pharmacological agents suitable for administration to an individual. It can be used in combination with various bases. Such compositions comprise, for example, an amount added to the vehicle, ie, a therapeutically effective amount of a polypeptide and / or polynucleotide of the invention, and a pharmacologically acceptable base or excipient. Contains. Bases include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation must suit the mode of administration. The invention further relates to a diagnostic or pharmacological pack or kit comprising one or more containers filled with one or more components of the composition according to the invention.
[0321]
The polypeptides, polynucleotides and other compounds of the present invention can be used alone or in combination with other compounds, eg, therapeutic compounds.
[0322]
The pharmacological compositions of the present invention can be administered in any effective and convenient way. Specific examples include, in particular, topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal administration.
[0323]
In therapy or as prophylaxis, the active agent can be administered to the individual as an injectable composition, for example, as a sterile, preferably isotonic, aqueous dispersion.
[0324]
In yet another aspect, the present invention dissolves a therapeutically effective amount of a polypeptide and / or polynucleotide, eg, a polypeptide and / or polynucleotide, agonist or antagonist peptide, or small molecule compound of the present invention. A pharmacological composition is provided which comprises the form in combination with a pharmacologically acceptable base or excipient. Bases include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to a pharmacological pack or kit comprising one or more containers filled with one or more components of the composition according to the invention. The polypeptides, polynucleotides and other compounds of the present invention can be used alone or in combination with other compounds, eg, therapeutic compounds.
[0325]
The composition will be in a form suitable for the route of administration, for example, systemic or oral. Preferred forms of systemic administration include injection, typically intravenous. Other injection routes are also possible, for example subcutaneous, intramuscular or intraperitoneal injection. Alternative means of systemic administration include transmucosal or transdermal administration using penetrants such as bile salts, fusidic acids, or other detergents. In addition, if the polypeptides or other compounds of the present invention can be made into enteric or encapsulated preparations, oral administration is also possible. Administration of these compounds can be topical and / or localized in the form of ointments, pastes, gels, solutions, powders and the like.
[0326]
For administration to mammals, especially humans, it is expected that the daily dose of the active agent will be from 0.01 mg / kg to 10 mg / kg, typically 1 mg / kg. In any case, the physician will decide the actual dose which will be most suitable for the individual. The dose will depend on the age, weight and response of the individual. The above doses are examples of the average case. Of course, there may be cases where higher or lower doses are preferred, which are also within the scope of the present invention.
[0327]
The required range of dosage depends on the peptide selected, the route of administration, the nature of the formulation, the nature of the patient's illness and the judgment of the physician. However, suitable doses are 0.1-100 μg / kg of patient weight.
[0328]
The vaccine composition is conveniently in injectable form. Conventional adjuvants can be used to increase the immune response. A suitable unit dose of vaccine is 0.5-5 μg of antibody per kg of body weight, and it is preferred that such dose be administered 1-3 times at 1-3 week intervals. At doses within the above ranges, adverse toxicological effects will not be observed with the compounds of the invention and will not prevent administration to individuals suitable for administering the compounds.
[0329]
However, given the variety of compounds available and the differing effects depending on the route of administration, the required range of dosage is expected to be wide. For example, in the case of oral administration, a higher dose may be required than by intravenous injection. Dose level differences can be adjusted by standard empirical work for optimization, as is well known in the art.
Sequence database, sequences stored on tangible media, algorithms
Polynucleotide and polypeptide sequences are valuable sources of information for determining two- and three-dimensional structures and for further identifying similar homology sequences. To store data in a computer-readable medium and use the stored data in a known macromolecular structure program, or to search a sequence database using a well-known search tool such as a GCG program package. Makes this very easy.
[0330]
The present invention also provides a method for analyzing a characteristic sequence or string, particularly a gene sequence or an encoded protein sequence. Preferred sequence analysis methods include, for example, sequence homology analysis methods such as coincidence analysis and similarity analysis, DNA, RNA, and protein structure analysis, sequence assembly branching analysis, sequence motif analysis, open reading frame determination, and nucleic acid base. Examples include calling, codon usage analysis, nucleobase trimming, and peak analysis of sequencing chromatograms.
[0331]
A computer-based method for identifying homology is provided. The method includes providing a first polynucleotide sequence comprising a polynucleotide sequence of the present invention in a computer readable medium; wherein the first polynucleotide sequence comprises at least one second polynucleotide or polypeptide sequence. Identifying the homology as compared to.
[0332]
Another computer-based method for identifying homology is also provided. The method comprises providing a first polypeptide sequence comprising a polynucleotide sequence of the present invention in a computer readable medium; the first polypeptide sequence comprising at least one second polynucleotide or polypeptide sequence. Identifying the homology as compared to.
[0333]
Any publications or references cited in this specification, including patents and patent applications, are specifically and individually incorporated by reference as if the individual publications or references were fully explained. As if incorporated by reference herein in its entirety. All other patent applications for which we claim priority over this patent application are hereby incorporated by reference in their entirety, as described above with respect to publications and references.
Definition
"Identity," as known in the art, is a relationship determined by comparing two or more polypeptide sequences, or, in some cases, two or more polynucleotide sequences. In the prior art, "match" also refers to the degree of sequence relatedness determined by string-to-string matches between multiple polypeptide sequences or, in some cases, multiple polynucleotide sequences. "Match" can be easily calculated using known methods. Such methods are described in, for example, the following documents, but are not limited thereto. Computational Molecular Biology, Resque, A. et al. M. Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Planning, Smith, D.C. W. Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M. And Griffin, H .; G. FIG. Ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G. et al. Academic Press, 1987; "Sequence Analysis Primers", Gribskov, M .; And Devreux, J. Ed., M. Stockton Press, New York, 1991; Carillo, H. and Lippman, D.M. SIAM J .; Applied Math. 48, 1073, 1988. The method of confirming the match is designed so that the match between the sequences to be tested is maximized. Further, the method of confirming the match is coded in a computer program that is available to everyone. Methods for using a computer program to confirm a match between two sequences include GAP programs in the GCG program package (Douvreux, J. et al., Nucleic Acids Research, Vol. 12, (1), p. 387, 1984). BLASTP, BLASTN (Arthur, SF et al., J. Mol. Biol., 215, 403-410, 1990), FASTA (Pearson and Lippman, Proc. Natl. Acad. Sci. USA, Vol. 85, pp. 2444-2448, 1988), but is not so limited. BLAST family programs are available to anyone from NCBI and other sources ("BLAST Manual", Altshull, S. et al., NCBI NLM NIH Bethesda, MD 20894; Altshull, S. et al., J. Mol. Biol., 215, 403-410, 1990). The well-known Smith Waterman algorithm can also be used to confirm a match.
[0334]
Parameters for comparing polypeptide sequences include the following.
Algorithm: Needleman and Wunsch, J. et al. Mol. Biol. 48, 443-453, 1970.
Comparison matrix: Henikov and Henikov, Proc. Natl. Acad. Sci. USA, Vol. 89, pages 10915-10919, BLOSSUM62, 1992,
Gap penalty: 8,
Gap length penalty: 2.
[0335]
A useful program containing these parameters is available to anyone as a "gap" program from the Genetics Computer Group (Madison, Wis.). The above parameters are the default parameters for comparing peptides (no penalty for end gaps).
[0336]
Parameters for comparing polynucleotides include:
Algorithm: Needleman and Wunsch, J. et al. Mol. Biol. 48, 443-453, 1970.
Comparison matrix: match = + 10, mismatch = 0
Gap penalty: 50,
Gap length penalty: 3.
Available as a "gap" program from the Genetics Computer Group (Madison, Wis.). These parameters are the default parameters for comparing nucleic acids.
[0337]
Preferred meanings of "match" for polynucleotides and polypeptides are given in (1) and (2) below, as appropriate.
[0338]
(1) An isolated polynucleotide wherein the polynucleotide of the embodiment further comprises a polynucleotide sequence at least 50, 60, 70, 80, 85, 90, 95, 97, or 100% identical to the reference sequence of SEQ ID NO: 1. Contains nucleotides. This polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1, or may have a predetermined number of nucleotide changes compared to the reference sequence. The change is either a deletion, a substitution (including a transition or transversion), or an insertion of at least one nucleotide. The change may also occur at the 5 'or 3' end of the reference nucleotide sequence, may occur at any position between these ends, may be individually scattered between nucleotides of the reference sequence, Alternatively, it may be interspersed in one or more contiguous groups in the reference sequence. The number of nucleotides that vary is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the integer defining the percent match divided by 100, and then subtracting the product from the total number of nucleotides in SEQ ID NO: 1. That is,
n_n≤x_n− (X_n・ Y)
Becomes Where n_nIs the number of nucleotides changed, x_nIs the total number of nucleotides in SEQ ID NO: 1, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97%, 1.00 for 100%. Symbol of product operation, x_nIf the product of y and y is non-integer, round to the nearest integer less than that value and then x_nSubtract from A change in the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 causes a nonsense mutation, missense mutation, or frameshift mutation in the coding sequence, whereby the polynucleotide after such change May change the polypeptide encoded by.
[0339]
For example, a polynucleotide sequence of the invention may correspond to the reference sequence of SEQ ID NO: 1. That is, the polynucleotide sequences may be 100% identical or the percent identity may be less than 100%, including up to a predetermined number of nucleic acid changes relative to the reference sequence. The change is either a deletion, a substitution (including a transition or transversion), or an insertion of at least one nucleotide. The change may also occur at the 5 'or 3' end of the reference polynucleotide sequence, or at any position between these ends, and may be individually interspersed between the nucleic acids of the reference sequence. , Or in one or more contiguous groups in the reference sequence. The number of changed nucleic acids for a given percent match is the total number of nucleic acids of SEQ ID NO: 1 multiplied by the integer defining the percent match divided by 100, and then the product is subtracted from the total number of nucleic acids of SEQ ID NO: 1. It depends on things. That is,
n_n≤x_n− (X_n・ Y)
Becomes Where n_nIs the number of changed nucleic acids, x_nIs the total number of nucleic acids of SEQ ID NO: 1, y is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, etc. And x_nIf the product of y and y is non-integer, round to the nearest integer less than that value and then x_nSubtract from
[0340]
(2) an isolated polypeptide wherein the polypeptide of the embodiment further comprises a polypeptide sequence at least 50, 60, 70, 80, 85, 90, 95, 97, or 100% identical to the reference sequence of SEQ ID NO: 2. Contains peptides. This polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2, or may have a predetermined number of amino acid changes compared to the reference sequence. The change is either a deletion, a substitution (including a conservative or a non-conservative substitution), or an insertion of at least one amino acid. This change may also occur at the amino or carboxyl terminus of the reference polypeptide sequence, may occur at any position between these termini, and may be individually scattered between the amino acids of the reference sequence, or It may be interspersed in one or more contiguous groups in the reference sequence. The number of changing amino acids is determined by multiplying the total number of amino acids of SEQ ID NO: 2 by an integer defining a percent identity divided by 100, and then subtracting the product from the total number of amino acids of SEQ ID NO: 2. That is,
n_a≤x_a− (X_a・ Y)
Becomes Where n_aIs the number of nucleotides changed, x_aIs the total number of amino acids of SEQ ID NO: 2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97%, 1.00 for 100%. Symbol of product operation, x_aIf the product of y and y is non-integer, round to the nearest integer less than that value and then x_aSubtract from
[0341]
For example, a polypeptide sequence of the invention may correspond to the reference sequence of SEQ ID NO: 2. That is, the polypeptide sequences may be 100% identical, or may contain up to a predetermined number of amino acid changes compared to the reference sequence, and the percent identity may be less than 100%. The change is either a deletion, a substitution (including a conservative or a non-conservative substitution), or an insertion of at least one amino acid. This change may also occur at the amino or carboxyl terminus of the reference polypeptide sequence, may occur at any position between these termini, and may be individually scattered between the amino acids of the reference sequence, or It may be interspersed in one or more contiguous groups in the reference sequence. The number of changing amino acids for a given percent match is the total number of amino acids in SEQ ID NO: 2 multiplied by the integer defining the percent match divided by 100, and then the product is subtracted from the total number of amino acids in SEQ ID NO: 2. It depends on things. That is,
n_a≤x_a− (X_a・ Y)
Becomes Where n_aIs the number of changed amino acids, x_aIs the total number of amino acids in SEQ ID NO: 2, y is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, etc. And x_aIf the product of y and y is non-integer, round to the nearest integer less than that value and then x_aSubtract from
[0342]
“Individual” as used herein in reference to an organism means a multicellular eukaryote. These include, but are not limited to, metazoans, mammals, sheep, bovines, apes, primates, and humans.
[0343]
"Isolated" means altered "by the hand of man" from the natural state. That is, if that happens in nature, it has changed from its original environment, has been isolated, or both. For example, if a polynucleotide or polypeptide present in the body of an organism is not "isolated," but the same polynucleotide or polypeptide is separated from substances that coexist in nature. , "Isolated" in the sense used in this specification. In addition, whether a polynucleotide or polypeptide introduced into a living organism by transformation, genetic engineering, or other recombinant techniques, if present, is alive or dead, even if it is still present in the organism. Regardless, it is "isolated."
[0344]
"Polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide. It may be unmodified RNA or DNA or modified RNA or DNA, including a single-stranded region and a double-stranded region.
[0345]
"Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains key properties. A typical variant of a polynucleotide differs in nucleotide sequence from a reference polynucleotide. Due to changes in the nucleotide sequence of the variant, the amino acid sequence of the polypeptide encoded by the reference polynucleotide may or may not be different. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, truncations in the polypeptide encoded by the reference sequence. This will be described below. A typical variant of a polypeptide differs in amino acid sequence from a reference polypeptide. Generally, differences are limited so that the sequences and variants of the reference polypeptide are closely similar overall, and that the sequence matches in many regions; The difference between the variant and the reference polypeptide may be any combination of one or more amino acid substitutions, additions, and deletions in the amino acid sequence. The substituted or inserted amino acid residue may be an amino acid encoded by the genetic code or an amino acid not encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring variant, such as an allelic variant, or a variant that is not known to occur naturally. Non-naturally occurring variants of a polynucleotide or polypeptide can be made by mutagenesis techniques or by direct synthesis.
[0346]
“Disease” means any disease caused by or associated with a bacterial infection, including, for example, upper respiratory tract infections, invasive bacterial diseases such as bacteremia and meningitis .
[0347]
Example
The following examples are performed in a standard manner that is well known and obvious to those skilled in the art. Unless another method is described in detail. The embodiments are merely illustrative, and the present invention is not limited thereto.
Example 1
Neisseria Meningitidis ATCC13090 In BASB051 gene
SEQ ID NO: 1 shows the BASB051 gene of Neisseria meningitidis strain ATCC13090. A translation of the BASB051 polynucleotide sequence is shown in SEQ ID NO: 2, which has significant similarity to Neisseria gonorrhoeae ComL lipoprotein. The BASB051 polypeptide contains a leader sequence characteristic of a signal sequence on a lipoprotein.
Example 2
Neisseria meningitidis strain ATCC13090 Inside BASB057 gene
The BASB057 gene of Neisseria meningitidis strain ATCC13090 is shown in SEQ ID NO: 3. A translation of the BASB057 polynucleotide sequence set forth in SEQ ID NO: 4 shows significant similarity to the Neisseria gonorrhoeae MtrE outer membrane lipoprotein. The BASB057 polypeptide contains a leader sequence characteristic of a lipoprotein signal sequence.
Example 3
Neisseria meningitidis strain ATCC13090 Inside BASB060 gene
The BASB060 gene of Neisseria meningitidis strain ATCC13090 is shown in SEQ ID NO: 5. A translation of the BASB060 polynucleotide sequence shown in SEQ ID NO: 6 did not show significant similarity to any of the known proteins. The BASB060 polypeptide contains a leader sequence characteristic of a lipoprotein signal sequence and has the characteristics of an outer membrane lipoprotein.
Example 4
Neisseria meningitidis strain ATCC13090 Inside BASB061 gene
The BASB061 gene of Neisseria meningitidis strain ATCC13090 is shown in SEQ ID NO: 7. A translation of the BASB061 polynucleotide sequence shown in SEQ ID NO: 8 shows significant similarity to the Neisseria meningitidis mlp gene product. The BASB061 polypeptide contains a leader sequence characteristic of a lipoprotein signal sequence and has the characteristics of an outer membrane lipoprotein.
Example 5
Neisseria meningitidis strain ATCC13090 Inside BASB063 gene
The BASB063 gene of Neisseria meningitidis strain ATCC13090 is shown in SEQ ID NO: 9. A translation of the BASB063 polynucleotide sequence shown in SEQ ID NO: 10 did not show significant similarity to any of the known proteins. However, the BASB063 polypeptide contains a leader sequence characteristic of a lipoprotein signal sequence and has the characteristics of an outer membrane lipoprotein.
Example 6
Neisseria meningitidis strain ATCC13090 Inside BASB065 gene
The BASB065 gene of Neisseria meningitidis strain ATCC13090 is shown in SEQ ID NO: 11. A translation of the BASB065 polynucleotide sequence shown in SEQ ID NO: 12 did not show significant similarity to any of the known proteins. However, the BASB065 polypeptide contains a leader sequence characteristic of the lipoprotein signal sequence and has the characteristics of an outer membrane lipoprotein.
Example 7
Neisseria meningitidis strain ATCC13090 Inside BASB066 gene
The BASB066 gene of Neisseria meningitidis strain ATCC13090 is shown in SEQ ID NO: 13. A translation of the BASB066 polynucleotide sequence set forth in SEQ ID NO: 14 shows significant similarity to the Neisseria meningitidis CtrA protein. The BASB066 polypeptide contains a leader sequence characteristic of a lipoprotein signal sequence and has the characteristics of a protein located on the outer membrane.
Example 8
Neisseria meningitidis strain ATCC13090 Inside BASB071 gene
The BASB071 gene of Neisseria meningitidis strain ATCC13090 is shown in SEQ ID NO: 15. A translation of the BASB071 polynucleotide sequence set forth in SEQ ID NO: 16 shows significant similarity to the Neisseria gonorrhoeae HisJ protein. The BASB071 polypeptide contains a leader sequence characteristic of a lipoprotein signal sequence.
[0348]
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[0349]
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[0350]
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[0351]
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[0352]
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[0353]
[Table 1]

[0354]
Deposited materials
A deposit containing the Neisseria meningitidis serogroup B strain was deposited with the American Type Culture Collection (designated herein as "ATCC") on June 22, 1997, and given deposit number 13090. This deposit is designated Neisseria meningitidis (Albrecht and Gone) and is a lyophilized 1.5-2.9 kb insert library composed of Neisseria meningitidis isolates. It is. This deposit is made by Int. Bull. Bacteriol. Nomencl. Taxon. 8, Vol. 1, pp. 1-15, 1958.
[0355]
The Neisseria meningitidis strain deposit is referred to herein as the "deposited strain" or "DNA of the deposited strain."
[0356]
The deposited strain contains the full-length BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066, and BASB071 genes. The sequence of the polynucleotide contained in the deposited strain and the amino acid sequence of the polypeptide encoded thereby are to be compared with the description of the sequence in this specification when any problem occurs.
[0357]
The deposit of the strain was made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure. The strain is open to the public upon issuance of the patent and is not revoked, and there are no restrictions or conditions in doing so. Deposited strains are provided only for the convenience of those skilled in the art and do not acknowledge that a deposit is required for efficacy as required under 35 USC §112. .

Claims (24)

An isolated polypeptide comprising an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. 2. The method according to claim 1, wherein the amino acid sequence is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. An isolated polypeptide. 2. The isolated polypeptide of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. An isolated polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16. An immunogenic fragment of the polypeptide according to any one of claims 1 to 4, wherein the immunogenic activity of the immunogenic fragment is SEQ ID NO: 2, 4, 6, 8, 10, An immunogenic fragment, which is substantially the same as that of 12, 14, or 16 polypeptides. A polypeptide having at least 85% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16 over the entire length of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16 respectively Or an isolated polynucleotide comprising a nucleotide sequence that is complementary to the isolated polynucleotide. A nucleotide sequence that is at least 85% identical over the entire length of the coding region to the nucleotide sequence encoding the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16; or is complementary to the isolated polynucleotide An isolated polynucleotide comprising a specific nucleotide sequence. A nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15 over the entire length of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15 respectively Or an isolated polynucleotide comprising a nucleotide sequence that is complementary to the isolated polynucleotide. 9. The isolated polynucleotide according to any one of claims 6 to 8, which is at least 95% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16. An isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15. SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 or a fragment thereof An isolated polynucleotide obtainable by screening an appropriate library under stringent hybridization conditions using a labeled probe having the formula: An expression vector or a living recombinant microorganism comprising the isolated polynucleotide according to any one of claims 6 to 12. 14. A host cell comprising the expression vector of claim 13, or at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. A subcellular fragment or membrane of this host cell that expresses an isolated polypeptide comprising the amino acid sequence of A method for producing a polypeptide comprising an amino acid sequence having at least 85% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. 15. The method of claim 14, comprising culturing the host cell of claim 14 under conditions sufficient to produce the peptide, and recovering the polypeptide from the medium. A method for expressing the polynucleotide according to any one of claims 6 to 12, wherein a host cell is transformed with an expression vector containing at least one of these polynucleotides, and Such a method, comprising culturing the host cell under conditions sufficient to express any one of the polynucleotides. A vaccine composition comprising an effective amount of the polypeptide according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier. A vaccine composition comprising an effective amount of the polynucleotide according to any one of claims 6 to 12 and a pharmaceutically acceptable carrier. 19. The vaccine composition according to claims 17 or 18, comprising at least one other Neisseria meningitidis antigen. An antibody immunospecific for the polypeptide or immunogenic fragment of any one of claims 1-5. A method for diagnosing Neisseria meningitidis infection, wherein the polypeptide according to any one of claims 1 to 5, or an antibody immunospecific for this polypeptide, comprises: Such a method, comprising identifying whether it is present in a biological sample from the suspected animal. Use of a composition comprising an immunologically effective amount of a polypeptide according to any one of claims 1 to 5 in the manufacture of a medicament for use in raising an immune response in an animal. Use of a composition comprising an immunologically effective amount of a polynucleotide according to any one of claims 6 to 12 in the manufacture of a medicament for use in raising an immune response in an animal. A therapeutic composition useful in treating humans infected with Neisseria meningitidis comprising at least one antibody against the polypeptide according to any one of claims 1 to 5 and a suitable pharmaceutical carrier. .