CN1342088A

CN1342088A - Polypeptide comprising amino acid of AN N-terminal choline binding protein truncate, vaccine derived therefrom and uses thereof

Info

Publication number: CN1342088A
Application number: CN99810854A
Authority: CN
Inventors: E·I·托曼恩; H·R·马舒尔; T·M·维泽曼; L·S·约翰逊; S·科尼格
Original assignee: St Jude Childrens Research Hospital; MedImmune LLC
Current assignee: St Jude Childrens Research Hospital; MedImmune LLC
Priority date: 1998-04-07
Filing date: 1999-04-07
Publication date: 2002-03-27
Anticipated expiration: 2019-04-07
Also published as: WO1999051188A2; CA2326388A1; HUP0102306A1; NO20005081D0; AU3479699A; JP4739521B2; JP2002516662A; BR9909910A; AU764811B2; HUP0102306A3; CA2326388C; AU3479799A; WO1999051188A9; NZ507983A; WO1999051188A3; MXPA00009899A; KR100638503B1; PL193132B1; KR20010052244A; TR200003498T2

Abstract

This invention provides an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate in which the amino acid sequence is set forth in any of SEQ ID NOS: 1, 3-7, or 9-11, including fragments, mutants, variants, analogs, or derivatives, thereof. Also, this invention provides a isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate, wherein the amino acid is set forth in SEQ ID NO 24, wherein the polypeptide retains its native tertiary structure and methods of preparation. This invention provides an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate, wherein the polypeptide has lectin activity and does not bind to choline. This invention provides an isolated immunogenic polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate. This invention provides an isolated nucleic acid encoding a polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate. Lastly, this invention provides pharmaceutical compositions, vaccines, and diagnostic and therapeutic methods of use.

Description

Comprise the amino acid whose polypeptide of the terminal intercepting of choline binding protein AN-thing, by this polypeptide vaccines derived and application thereof

Invention field

The present invention generally relates to the polypeptide of the terminal intercepting of choline binding protein AN-thing.The present invention also relates to bacterial infection, particularly pneumococcal infection provides protective effect or excites the vaccine that produces protection antibody and be used to diagnose antibody and antagonist with this peptide species of antagonism of passive immunotherapy.The nucleic acid of this polypeptide and/or this polypeptide of encoding also can be as the competitive inhibitor of streptococcus pneumoniae bacterial adhesion.At last, the present invention relates to the Therapeutic Method that utilizes this polypeptide to treat.

Background of invention

Streptococcus pneumoniae is a gram positive bacteria, and it is to cause that aggressive infects main cause as septicemia, meningitis, otitis media and lobar pneumonia people such as (, NEJM 332:1280-1284,1995) Tuomanen.Streptococcus pneumoniae combines with last lower respiratory tract cell consumingly.As most of antibacterials, by with proteinic adhesion (Cundell, the D.﹠amp that finishes streptococcus pneumoniae and human cell in the mode of similar agglutinin that present of the bonded bacterium surface of Eukaryotic carbohydrate; Tuomanen, E. (1994) Microb Pathog 17:361-374).Streptococcus pneumoniae combines with the non-inflammatory epithelium can regard asymptomatic transportation as.The someone proposes, it is relevant with local generation inflammatory factor to change affecting conditions into, the number and the type (Cundell, people such as D. (1995) Nature377:435-438) of the receptor that the described factor can obtain on the human cell by the activation of human cell change.Streptococcus pneumoniae just has an opportunity to get the mastery and utilized these not adjust one of receptor in this new environment, platelet activating factor (PAF) receptor (Cundell, people such as D. (1995) Nature 377:435-438).In a few minutes that paf receptor occurs, streptococcus pneumoniae experience cohesiveness and the enhanced fluctuation of aggressive.For example by the soluble recepter analog suppress antibacterial and combining of active cell can block disease in animal model progress (Idanpaan-Heikkila, people such as I. (1997) J.Infect.Dis., 176:704-712).Effectively comprise especially the soluble-carbohydrate that is with or without additional sialic lacto-N-neotetraose in this, it external prevention streptococcus pneumoniae attached to the human cell on and prevent to move in vivo to be born in the lung.Choline binding protein: the candidate structure adherance because of

Streptococcus pneumoniae produce a class can with the bonded surface protein of bacterium surface, this combination is by non-covalent bonded with cell wall teichoic acid or fat teichoic acid.The streptococcus pneumoniae surface coverage a class CBPs (choline binding protein), and this albuminoid combines with phosphatidylcholine is non-covalent.CbpA is the choline binding protein that a kind of 75kD surface exposes, and shows embedded structure.Have unique N-stub area, i.e. the zone of proline rich is thereafter by 10 C-end regions of being responsible for forming in conjunction with the duplicate block of choline.

CbpA is a kind of adhesin (part) of glycoconjugates, comprises on the glycoconjugates to be present in the lip-deep receptor of eukaryotic cell.Sudden change with CbpA defective moves at nasopharynx and shows the virulence that reduces in the living underage rat model.This combination relates to the choline determinant of modifying teichoic acid and mediates by the feature choline binding zone among each member of this proteinoid.People such as Lopez in their autolytic enzyme research, found this choline binding zone and to its characteristic carried out determining fully (people (1987) Eur.J.Biochem such as Ronda, 164:621-624).Other albumen that contains this zone comprises the autolysin and the protective antigen of streptococcus pneumoniae phage; be streptococcus pneumoniae surface protein A (PspA) (Ronda; C. wait people (1987) Eur.J.Biochem; 164:621-624 and McDaniel; L.S.; Deng people (1992) Microb Pathog, 13:261-269).CbpA can not move and be born in the nasopharynx zone, and this zone is to be produced by its distinctive N-stub area with other type member who has jointly (C end) of CbpA and itself and the bonded activity of people's cell.Owing to move living process and advancing of disease depends on that the streptococcus pneumoniae as first step is attached on people's cell, for the blocking-up disease, may be critical by cross reacting antibody or by function with the competitive inhibition blocking-up N-stub area of the peptide in this district of simulation.The full content of the choline binding protein of anti-Pnu-Imune 23 being discussed in PCT International Application PCT/US97/07198 and this PCT being applied for is incorporated herein for reference.The vaccine of present anti-streptococcus pneumoniae uses the purified carbon hydrate of the pod membrane of 23 normal serum types of this antibacterial; but this vaccine only has 50% protective effect (people such as Shapiro; NJEM 325:1453,1991) and do not have immunogenicity below 2 years old.And therapeutical peptide can provide the selectivity of treatment in by the case of multiresistance organism infection.So the present invention has satisfied this secular needs by the protectiveness vaccine is provided.

Summary of the invention

The invention provides a kind of isolating polypeptide, this polypeptide comprises the aminoacid sequence of the terminal intercepting of choline binding protein AN-thing.Described polypeptide comprises SEQ ID NOS 1, and the aminoacid sequence described in 3-7 or the 9-11 comprises its fragment, mutant, variant, analog or derivant.And, the invention provides a kind of isolating polypeptide, this polypeptide comprises the aminoacid sequence with the terminal intercepting of SEQ ID NO 24 amino acid whose choline binding protein AN-thing, and wherein this polypeptide shows its tertiary structure and the method for preparing this polypeptide also is provided.Isolating polypeptide is applicable to and makes the animal and human to bacterial infection, preferably pneumococcal infection is produced immunization.

The present invention also relates to have activity of lectin but do not have the terminal intercepting of the N-thing of the active choline binding protein A of choline binding.And the N-end that the invention provides a kind of immunogenicity choline binding protein A intercepts thing or its fragment.

The present invention also relates to isolating nucleic acid, as recombinant DNA molecules or cloned genes or its degeneracy variant, mutant, analog or its fragment, the activity of its encode isolating polypeptide or its competitive inhibition polypeptide.Preferably, isolating nucleic acid (comprising degeneracy thing, variant, mutant, analog or its fragment) has SEQ ID NOS:12,14-17,19-22 or 23 described sequences.In the further embodiment of the present invention, the so definite recombinant DNA molecules or the global DNA sequence of clone gene can be connected on the expression control sequenc effectively, this expression control sequenc be directed in the suitable hosts.Therefore, the invention still further relates to the unicellular host that transforms with cloned genes or recombinant DNA molecules, described clone gene or recombinant DNA molecules comprise the DNA sequence of code book invention polypeptide, particularly DNA sequence or its fragment determined of sequence by mentioned earlier.

The antibody of antagonism isolated polypeptide comprises natural generation and antibody recombinant methods.These antibody can comprise polyclone and the monoclonal antibody by known genetic technique preparation, and (chimeric) antibody of bispecific and comprise being suitable for diagnosing and use and can regulate the antibody that bacterial adhesion has included but not limited to other function of competitor effect.

A further object of the present invention provides a kind ofly treats mammal and controls the amount of antibacterial or its subunit or active so that handle or prevention is invaded, the method for the spontaneous or negative consequence that the idopathy attitude is produced.The invention provides the pharmaceutical composition that in Therapeutic Method, uses, wherein comprise or based on isolating polypeptide, its subunit or it is in conjunction with counter pair.

At last, the invention provides pharmaceutical composition, vaccine and use their diagnosis and Therapeutic Method.

The accompanying drawing summary

Fig. 1. the diagram of choline binding protein A (CbpA) and reorganization intercepting thing R1 (the N-end from CbpA begins about amino acid/11 6 to aminoacid 321 as shown in Figure 2) and R2 (the N-end from CbpA begins about amino acid/11 6 to aminoacid 444 as shown in Figure 2).Zone A begins about amino acid/11 53 to aminoacid 321 from the N-end of CbpA aminoacid sequence as shown in Figure 2; Area B is to begin about aminoacid 270 to aminoacid 326 from the N-end of CbpA aminoacid sequence as shown in Figure 2; And zone C is to begin about aminoacid 327 to aminoacid 433 from the N-end of CbpA aminoacid sequence as shown in Figure 2.

Fig. 2 A-B. compares the nucleic acid of CbpA N-stub area and the homology of the various serotypes of aminoacid sequence.

Fig. 3. expression and the purification of reorganization R1 and R2.

Fig. 4. the result of passive protection in mice.The immune serum protection mice of antagonism reorganization R2 avoids exciting of lethal streptococcus pneumoniae.

Fig. 5. anti--R2 antibody is to adhering to the LNnT-HSA bag by the titration of the R6x on the flat board.

Fig. 6. have blocking-up streptococcus pneumoniae and LNnT-HSA bag by the titration of the anti--CbpA antibody of the anti--Cbp-A of dull and stereotyped adhesion activity and absorption.

Fig. 7. in mice, initiatively protect the result.The immune serum protection mice of antagonism reorganization R1 avoids the (excimer: 560cfu serotype 6B) that excites of lethal streptococcus pneumoniae.

Detailed Description Of The Invention

The present invention relates to a kind of polypeptide of separation, this polypeptide comprises the amino acid sequence of choline binding protein AN-end intercepting thing. This polypeptide is applicable to make animal to produce immunization to pneumococcal infection. These polypeptide or its fragments of peptides, when the adjuvant with suitable is prepared, be used for antagonism pneumococcus and antagonism and have the vaccine of other bacterium of cross reactivity albumen.

The invention provides a kind of polypeptide of separation, this polypeptide comprises the amino acid sequence of choline binding protein AN-end intercepting thing. In one embodiment, this polypeptide has SEQ ID NO 1,3-5, and 7 or the described arbitrary amino acid sequence of 9-11, comprise its fragment, mutant, variant, analog or derivative. In another embodiment, this polypeptide has amino acid KXXE (SEQ ID NO 6).

The invention provides a kind of polypeptide of separation, this polypeptide comprises the amino acid sequence of the described choline binding protein AN-of Fig. 2 end intercepting thing. In one embodiment, this polypeptide has the amino acid sequence of the described conserved region of Fig. 2. For example, conserved region includes but not limited to amino acid sequence 158-210; 158-172; 300-321; 331-339; 355-365; 367-374; 379-389; 409-427 and 430-447. Fig. 2 describes the nucleic acid of CbpA N-stub area of the present invention and the homology of the various serotypes of amino acid sequence.

In addition, the invention provides a kind of polypeptide of separation, this polypeptide comprises the amino acid sequence with the described amino acid whose choline binding protein AN-end intercepting thing of SEQ ID NO 24, and wherein said polypeptide shows its tertiary structure. In one embodiment, described polypeptide is its analog, fragment, mutant or variant. Described variant as shown in Figure 2. The present invention also provides a kind of polypeptide of separation, this polypeptide comprise have the described serotype 4 of Fig. 2 approximately 16 to approximately 474 bit aminos acid or the amino acid sequence of the corresponding amino acid whose choline binding protein AN-end intercepting thing of serotype 4 as described in Figure 2, wherein said polypeptide shows its tertiary structure. In one embodiment, tertiary structure is corresponding with the tertiary structure in being present in native protein.

The example of method for preparing described polypeptide is as follows: with the choline binding protein A of hydroxylamine cleavage total length, the corresponding amino acid of serotype R6x or serotype 4 in the amino acid asparagine (N) at 475 places of wherein said hydroxylamine cleavage choline binding protein A serotype R6x and serotype 4 or different serotypes shown in Figure 2, the N-end that produces thus choline binding protein A intercepts thing. The choline binding protein A that generation is blocked or its fragment and keep other method of natural tertiary structure (that is, the tertiary structure of total length choline binding protein A) to have described and be known for a person skilled in the art. Because described polypeptide keeps its tertiary structure, the polypeptide of separation is applicable to as immunogene, the animal and human be infected to bacterium, and preferred pneumococcus produces immunization.

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c or t <220> <221> variants <222> (531) <223> a, g, c or t <220> <221> variants <222> (534) <223> a, g, c or t <220> <221> variants <222> (537) <223> a, g, c or t <220> <221> variants <222> (540) <223> a, g, c or t <220> <221> variants <222> (546) <223> a, g, c or t <220> <221> variants <222> (549) <223> a, g, c or t <220> <221> variants <222> (555) <223> a, g, c or t <220> <221> variants <222> (558) <223> a, g, c or t <220> <221> variants <222> (564) <223> a, g, c or t <220> <221> variants <222> (570) <223> a, g, c or t <220> <221> variants <222> (576) <223> a, g, c or t <220> <221> variants <222> (579) <223> a, g, c or t <220> <221> variants <222> (588) <223> a, g, c or t <220> <221> variants <222> (594) <223> a, g, c or t <220> <221> variants <222> (597) <223> a, g, c or t <220> <221> variants <222> (609) <223> a, g, c or t <220> <221> variants <222> (615) <223> a, g, c or t <220> <221> variants <222> (618) <223> a, g, c or t <220> <221> variants <222> (624) <223> a, g, c or t <220> <221> variants <222> (633) <223> a, g, c or t <220> <221> variants <222> (637) <223> a, g, c or t <220> <221> variants <222> (642) <223> a, g, c or t <220> <221> variants <222> (651) <223> a, g, c or t <220> <221> variants <222> (657) <223> a, g, c or t <220> <221> variants <222> (663) <223> a, g, c or t <220) <221> variants <222> (672) <223> a, g, c or t <220> <221> variants <222> (675) <223> a, g, c or t <220> <221> variants <222> (678) <223> a, g, c or t <220> <221> variants <222> (681) <223> a, g, c or t <220> <221> variants <222> (684) <223> a, g, c or t <220> <221> variants <222> (687) <223> a, g, c or t <220> <221> variants <222> (690) <223> a, g, c or t <220> <221> variants <222> (699) <223> a, g, c or t <220> <221> variants <222> (702) <223> a, g, c or t <220> <221> variants <222> (708) <223> a, g, c or t <220> <221> variants <222> (711) <223> a, g, c or t <220> <221> variants <222> (717) <223> a, g, c or t <220> <221> variants <222> (723) <223> a, g, c or t <220> <221> variants <222> (726) <223> a, g, c or t <220> <221> variants <222> (732) <223> a, g, c or t <220> <221> variants <222> (735) <223> a, g, c or t <220> <221> variants <222> (738) <223> a, g, c or t <220> <221> variants <222> (741) <223> a, g, c or t <220> <221> variants <222> (744) <223> a, g, c or t <220> <221> variants <222> (747) <223> a, g, c or t <220> <221> variants <222> (750) <223> a, g, c or t <220> <221> variants <222> (756) <223> a, g, c or t <220> <221> variants <222> (759) <223> a, g, c or t <220> <221> variants <222> (765) <223> a, g, c or t <220> <221> variants <222> (771) <223> a, g, c or t <220> <221> variants <222> (780) <223> a, g, c or t <220> <221> variants <222> (783) <223> a, g, c or t <220> <221> variants <222> (789) <223> a, g, c or t <220> <221> variants <222> (792) <223> a, g, c or t <220> <221> variants <222> (804) <223> a, g, c or t <220> <221> variants <222> (807) <223> a, g, c or t <220> <221> variants <222> (810) <223> a, g, c or t <220> <221> variants <222> (813) <223> a, g, c or t <220> <221> variants <222> (816) <223> a, g, c or t <220> <221> variants <222> (819) <223> a, g, c or t <220> <221> variants <222> (822) <223> a, g, c or t <220> <221> variants <222> (825) <223> a, g, c or t <220> <221> variants <222> (828) <223> a, g, c or t <220> <221> variants <222> (831) <223> a, g, c or t <220> <221> variants <222> (834) <223> a, g, c or t <220> <221> variants <222> (837) <223> a, g, c or t <220> <221> variants <222> (840) <223> a, g, c or t <220> <221> variants <222> (846) <223> a, g, c or t <220> <221> variants <222> (849) <223> a, g, c or t <220> <221> variants <222> (855) <223> a, g, c or t <220> <221> variants <222> (861) <223> a, g, c or t <220> <221> variants <222> (867) <223> a, g, c or t <220> <221> variants <222> (870) <223> a, g, c or t <220> <221> variants <222> (873) <223> a, g, c or t <220> <221> variants <222> (879) <223> a, g, c or t <220> <221> variants <222> (888) <223> a, g, c or t <220> <221> variants <222> (891) <223> a, g, c or t <220> <221> variants <222> (894) <223> a, g, c or t <220> <221> variants <222> (897) <223> a, g, c or t <220> <221> variants <222> (903) <223> a, g, c or t <220> <221> variants <222> (906) <223> a, g, c or t <220> <221> variants <222> (912) <223> a, g, c or t <220> <221> variants <222> (915) <223> a, g, c or t <220> <221> variants <222> (924) <223> a, g, c or t <220> <221> variants <222> (930) <223> a, g, c or t <220> <221> variants <222> (936) <223> a, g, c or t <220> <221> variants <222> (939) <223> a, g, c or t <220> <221> variants <222> (942) <223> a, g, c or t <220> <221> variants <222> (948) <223> a, g, c or t <220> <221> variants <222> (951) <223> a, g, c or t <220> <221> variants <222> (954) <223> a, g, c or t <220> <221> variants <222> (957) <223> a, g, c or t <220> <221> variants <222> (960) <223> a, g, c or t <220> <221> variants <222> (966) <223> a, g, c or t <220> <221> variants <222> (972) <223> a, g, c or t <220> <221> variants <222> (975) <223> a, g, c or t <220> <221> variants <222> (978) <223> a, g, c or t <220> <221> variants <222> (987) <223> a, g, c or t <220> <221> variants <222> (996) <223> a, g, c or t <220> <221> variants <222> (999) <223> a, g, c or t <220> <221> variants <222> (1002) <223> a, g, c or t <220> <221> variants <222> (1017) <223> a, g, c or t <220> <221> variants <222> (1020) <223> a, g, c or t <220> <221> variants <222> (1023) <223> a, g, c or t <220> <221> variants <222> (1026) <223> a, g, c or t <220> <221> variants <222> (1029) <223> a, g, c or t <220> <221> variants <222> (1032) <223> a, g, c or t <220> <221> variants <222> (1035) <223> a, g, c or t <220> <221> variants <222> (1038) <223> a, g, c or t <220> <221> variants <222> (1050) <223> a, g, c or t <220> <221> variants <222> (1053) <223> a, g, c or t <220> <221> variants <222> (1056) <223> a, g, c or t <220> <221> variants <222> (1059) <223> a, g, c or t <220> <221> variants <222> (1062) <223> a, g, c or t <220> <221> variants <222> (1065) <223> a, g, c or t <220> <221> variants <222> (1068) <223> a, g, c or t <220> <221> variants <222> (1071) <223> a, g, c or t <220> <221> variants <222> (1074) <223> a, g, c or t <220> <221> variants <222> (1080) <223> a, g, c or t <220> <221> variants <222> (1086) <223> a, g, c or t <220> <221> variants <222> (1092) <223> a, g, c or t <220> <221> variants <222> (1101) <223> a, g, c or t <220> <221> variants <222> (1107) <223> a, g, c or t <220> <221> variants <222> (1110) <223> a, g, c or t <220> <221> variants <222> (1116) <223> a, g, c or t <220> <221> variants <222> (1119) <223> a, g, c or t <220> <221> variants <222> (1125) <223> a, g, c or t <220> <221> variants <222> (1131) <223> a, g, c or t <220> <221> variants <222> (1134) <223> a, g, c or t <220> <221> variants <222> (1140) <223> a, g, c or t <220> <221> variants <222> (1149) <223> a, g, c or t <220> <221> variants <222> (1152) <223> a, g, c or t <220> <221> variants <222> (1155) <223> a, g, c or t <220> <221> variants <222> (1164) <223> a, g, c or t <220> <221> variants <222> (1167) <223> a, g, c or t <220> <221> variants <222> (1170) <223> a, g, c or t <220> <221> variants <222> (1176) <223> a, g, c or t <220> <221> variants <222> (1182) <223> a, g, c or t <220> <221> variants <222> (1188) <223> a, g, c or t <220> <221> variants <222> (1194) <223> a, g, c or t <220> <221> variants <222> (1206) <223> a, g, c or t <220> <221> variants <222> (1209) <223> a, g, c or t <220> <221> variants <222> (1212) <223> a, g, c or t <220> <221> variants <222> (1215) <223> a, g, c or t <220> <221> variants <222> (1218) <223> a, g, c or t <220> <221> variants <222> (1224) <223> a, g, c or t <220> <221> variants <222> (1236) <223> a, g, c or t <220> <221> variants <222> (1239) <223> a, g, c or t <220> <221> variants <222> (1242) <223> a, g, c or t <220> <221> variants <222> (1251) <223> a, g, c or t <220> <221> variants <222> (1257) <223> a, g, c or t <220> <221> variants <222> (1263) <223> a, g, c or t <220> <221> variants <222> (1266) <223> a, g, c or t <220> <221> variants <222> (1275) <223> a, g, c or t <220> <221> variants <222> (1278) <223> a, g, c or t <220> <221> variants <222> (1281) <223> a, g, c or t <220> <221> variants <222> (1284) <223> a, g, c or t <220> <221> variants <222> (1290) <223> a, g, c or t <220> <221> variants <222> (1296) <223> a, g, c or t <220> <221> variants <222> (1299) <223> a, g, c or t <220> <221> variants <222> (1308) <223> a, g, c or t <220> <221> variants <222> (1323) <223> a, g, c or t <220> <221> variants <222> (1329) <223> a, g, c or t <220> <221> variants <222> (1332) <223> a, g, c or t <220> <221> variants <222> (1335) <223> a, g, c or t <220> <221> variants <222> (1338) <223> a, g, c or t <220> <221> variants <222> (1341) <223> a, g, c or t <220> <221> variants <222> (1344) <223> a, g, c or t <220> <221> variants <222> (1353) <223> a, g, c or t <220> <221> variants <222> (1362) <223> a, g, c or t <220> <221> variants <222> (1365) <223> a, g, c or t <220> <221> variants <222> (1368) <223> a, g, c or t <220> <221> variants <222> (1371) <223> a, g, c or t <220> <221> variants <222> (1374) <223> a, g, c or t <220> <221> variants <222> (1380) <223> a, g, c or t <220> <221> variants <222> (1383) <223> a, g, c or t <220> <221> variants <222> (1386) <223> a, g, c or t <220> <221> variants <222> (1389) <223> a, g, c or t <220> <221> variants <222> (1392) <223> a, g, c or t <220> <221> variants <222> (1401) <223> a, g, c or t <220> <221> variants <222> (1404) <223> a, g, c or t <220> <221> variants <222> (1410) <223> a, g, c or t <220> <221> variants <222> (1425) <223> a, g, c or t <220> <221> variants <222> (1428) <223> a, g, c or t <220> <221> variants <222> (1434) <223> a, g, c or t <220> <221> variants <222> (1437) <223> a, g, c or t <220> <221> variants <222> (1449) <223> a, g, c or t <220> <221> variants <222> (1452) <223> a, g, c or t <220> <221> variants <222> (1455) <223> a, g, c or t <220> <221> variants <222> (1470) <223> a, g, c or t <220> <221> variants <222> (1476) <223> a, g, c or t <220> <221> variants <222> (1479) <223> a, g, c or t <220> <221> variants <222> (1482) <223> a, g, c or t <220> <221> variants <222> (1488) <223> a, g, c or t <220> <221> variants <222> (1494) <223> a, g, c or t <220> <221> variants <222> (1497) <223> a, g, c or t <220> <221> variants <222> (1506) <223> a, g, c or t <220> <221> variants <222> (1509) <223> a, g, c or t <220> <221> variants <222> (1518) <223> a, g, c or t <220> <221> variants <222> (1521) <223> a, g, c or t <220> <221> variants <222> (1524) <223> a, g, c or t <220> <221> variants <222> (1536) <223> a, g, c or t <220> <221> variants <222> (1542) <223> a, g, c or t <220> <221> variants <222> (1566) <223> a, g, c or t <220> <221> variants <222> (1572) <223> a, g, c or t <220> <221> variants <222> (1575) <223> a, g, c or t <220> <221> variants <222> (1578) <223> a, g, c or t <220> <221> variants <222> (1581) <223> a, g, c or t <220> <221> variants <222> (1584) <223> a, g, c or t <220> <221> variants <222> (1587) <223> a, g, c or t <400> 10 atggcngtng tntaytayyt nytnytngcn ggnytnathg cntgywsnca ygcnytngcn 60 gcnggnacnc cngcnytngg ngaygaymgn ggnmgnccnt ggccngcnws nytngcngcn 120 ytngcnytng ayggnaaryt nmgnacngay wsnaaygcna cngcngcngc nwsnacngay 180 ttyggnaaya thacnwsngc nytnccngcn gcngtnytnt ayccnwsnws nacnggngay 240 ytngtngcny tnytnwsngc ngcnaaywsn acnccnggnt ggccntayac nathgcntty 300 mgnggnmgng gncaywsnyt natgggncar gcnttygcnc cnggnggngt ngtngtnaay 360 atggcnwsny tnggngaygc ngcngcnccn ccnmgnatha aygtnwsngc ngayggnmgn 420 taygtngayg cnggnggnga rcargtntgg athgaygtny tnmgngcnws nytngcnmgn 480 ggngtngcnc cnmgnwsntg gaaygaytay ytntayytna cngtnggngg nacnytnwsn 540 aaygcnggna thwsnggnca rgcnttymgn cayggnccnc arathwsnaa ygtnytngar 600 atggaygtna thacnggnca yggngaratg gtnacntgyw snaarcaryt naaygcngay 660 ytnttygayg cngtnytngg nggnytnggn carttyggng tnathacnmg ngcnmgnath 720 gcngtngarc cngcnccngc nmgngcnmgn tgggtnmgnt tygtntayac ngayttygcn 780 gcnttywsng cngaycarga rmgnytnacn gcnccnmgnc cnggnggngg nggngcnwsn 840 ttyggnccna tgwsntaygt ngarggnwsn gtnttygtna aycarwsnyt ngcnacngay 900 ytngcnaaya cnggnttytt yacngaygcn gaygtngcnm gnathgtngc nytngcnggn 960 garmgnaayg cnacnacngt ntaywsnath gargcnacny tnaaytayga yaaygcnacn 1020 gcngcngcng cngcngtnga ycargarytn gcnwsngtny tnggnacnyt nwsntaygtn 1080 garggnttyg cnttycarmg ngaygtngcn taygcngcnt tyytngaymg ngtncayggn 1140 gargargtng cnytnaayaa rytnggnytn tggmgngtnc cncayccntg gytnaayatg 1200 ttygtnccnm gnwsnmgnat hgcngaytty gaymgnggng tnttyaargg nathytncar 1260 ggnacngaya thgtnggncc nytnathgtn tayccnytna ayaarwsnat gtgggaygay 1320 ggnatgwsng cngcnacncc nwsngargay gtnttytayg cngtnwsnyt nytnttywsn 1380 wsngtngcnc cnaaygayyt ngcnmgnytn cargarcara aymgnmgnat hytnmgntty 1440 tgygayytng cnggnathca rtayaaracn tayytngcnm gncayacnga ymgnwsngay 1500 tgggtnmgnc ayttyggngc ngcnaartgg aaymgnttyg tngaratgaa raayaartay 1560 gayccnaarm gnytnytnws nccnggncar gayathttya ay 1602 <210> 11 <211> 32 <212> DNA <213> Corn <400> 11 tgggaattcc atggggagat ggtgacgtgc tc 32 <210> 12 <211> 33 <212> DNA <213> Corn <400> 12 gccgtcccac atggatttgt tgagggggta gac 33 <210> 13 <211> 37 <212> DNA <213> Corn <400> 13 ctcaacaaat ccatgtggga cgacggcatg tcggcgg 37 <210> 14 <211> 39 <212> DNA <213> Corn <400> 14 gcggtctaga tctaactaaa acatgcatgg gctatcatc 39 <210> 15 <211> 33 <212> DNA <213> Corn <400> 15 atgggaattc catggggaga tggtgacgtg ctc 33 <210> 16 <211> 39 <212> DNA <213> Corn <400> 16 gcggtctaga tctaactaaa acatgcatgg gctatcatc 39 <210> 17 <211> 17 <212> DNA <213> Corn <400> 17 ccggttttgg taccggt 17 <210> 18 <211> 17 <212> DNA <213> Corn <400> 18 catgaccggt accaaaa 17 <210> 19 <211> 22 <212> DNA <213> Corn <400> 19 gacaccattc caagcatacc cc 22 <210> 20 <211> 22 <212> DNA <213> Corn <400> 20 gttctcacta gaaaaatgcc cc 22 ...

"Polypeptide R2" means a choline binding protein A (CbpA) serotype type 4 (see Figure 1) of the N-terminal amino acid sequence was taken polypeptide having the following sequence: ENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESYAKSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAVSKFEKDSSSSSSSDSSTKPEASDTAKPNKPTEPGEKVAEAKKKVEEAEKKAKDQKEEDRRNYPTITYKTLELEIAESDVEVKKAELELVKVKANEPRDEQKIKQAEAEVESKQAEATRLKKIKTDREEAEEEAKRRADAKEQGKPKGRAKRGVPGELATPDKKENDAKSSDSSVGEETLPSPSLKPEKKVAEAEKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAESDVEVKKAELELVKEEAKEPRNEEKVKQAKAEVESKKAEATRLEKIKTDRKKAEEEAKRKAAEEDKVKEKPA (SEQ ID NO 1)

Encoding choline binding protein A (CbpA) serotype type 4 N-terminus of the polypeptide was taken DNA sequence R2: GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGAAAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGAGATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAAAATAGTGGGTGAGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGCTCTAGTTAACGAGTTGAACAACATTAAGAACGAGTATTTGAATAAAATAGTTGAATCAACCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAAAAGTAGATGAAGCTGTGTCTAAGTTTGAAAAGGACTCATCTTCTTCGTCAAGTTCAGACTCTTCCACTAAACCGGAAGCTTCAGATACAGCGAAGCCAAACAAGCCGACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAACGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAGAGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTGAAGAAGCAGAAGAAGAAGCTAAACGAAGAGCAGATGCTAAAGAGCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTGAAGAAACTCTTCCAAGCCCATCCCTGAAACCAGAAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCGAAACGAGGAAAAAGTTAAGCAAGCAAAAGCGGAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAAAATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCAGCAGAAGAAGATAAAGTTAAAGAAAAACCAGCTG (SEQ ID NO 12). CbpA amino acid sequence of serotype 4:. (SEQ IDNO 2) encoding CbpA serotype 4 DNA sequence of the amino acid sequence: GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGAAAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGAGATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAAAATAGTGGGTGAGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGCTCTAGTTAACGAGTTGAACAACATTAAGAACGAGTATTTGAATAAAATAGTTGAATCAACCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAAAAGTAGATGAAGCTGTGTCTAAGTTTGAAAAGGACTCATCTTCTICGTCAAGTTCAGACTCTTCCACTAAACCGGAAGCTTCAGATACAGCGAAGCCAAACAAGCCGACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAACGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAGAGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTGAAGAAGCAGAAGAAGAAGCTAAACGAAGAGCAGATGCTAAAGAGCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAACACCTGATAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTGAAGAAACTCTTCCAAGCCCATCCCTGAAACCAGAAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGgCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCGAAACGAGGAAAAAGTTAAGCAAGCAAAAGCGGAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAAAATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCAGCAGAAGAAGATAAAGTTAAAGAAAAACCAGCTGAACAACCACAACCAGCGCCGGCTCCAAAAGCAGAAAAACCAGCTCCAGCTCCAAAACCAGAGAATCCAGCTGAACAACCAAAAGCAGAAAAACCAGCTGATCAACAAGCTGAAGAAGACTATGCTCGTAGATCAGAAGAAGAATATAATCGCTTGACTCAACAGCAACCGCCAAAAACTGAAAAACCAGCACAACCATCTACTCCAAAAACAGGCTGGAAACAAGAAAACGGTATGTGGTACTTCTACAATACTGATGGTTCAATGGCGACAGGATGGCTCCAAAACAAtGGCTCAtGGTAcTACcTCAACAGCAATGGCGCTATGGCGACAGGATGGCTCCAAAACAATGGTTCATGGTACTATCTAAACGCTAATGGTTCAATGGCAACAGGATGGCTCCAAAACAATGGTTCATGGTACTACCTAAACGCTAATGGTTCAATGGCGACAGGATGGCTCCAATACAATGGCTCATGGTACTACCTAAACGCTAATGGTTCAATGGCGACAGGATGGCTCCAATACAATGGCTCATGGTACTACCTAAACGCTAATGGTGATATGGCGACAGGTTGGGTGAAAGATGGAGATACCTGGTACTATCTTGAAGCATCAGGTGCTATGAAAGCAAGCCAATGGTTCAAAGTATCAGATAAATGGTACTATGTCAATGGCTCAGGTGCCCTTGCAGTCAACACAACTGTAGATGGCTATGGAGTCAATGCCAATGGTGAATGGGTAAACTAA (SEQ ID NO 13)....

R1 is a DNA sequence encoding the polypeptide is: GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGAAAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGAGATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAAAATAGTGGGTGAGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGCTCTAGTTAACGAGTTGAACAACATTAAGAACGAGTATTTGAATAAAATAGTTGAATCAACCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAAAAGTAGATGAAGCTGTGTCTAAGTTTGAAAAGGACTCATCTTCTTCGTCAAGTTCAGACTCTTCCACTAAACCGGAAGCTTCAGATACAGCGAAGCCAAACAAGCCGACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAACGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAGAGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTGAAGAAGCAGAAGAAGAAGCTAAACGAAGAGCAGATGCTAAAGAGCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTGAAGAAACTCTTC (SEQ ID NO 14).

% 22% 20peptide% 20C% 2FR2% 20% 22% 20means% 20to% 20comprise% 20the% 20polypeptide% 20of% 20duplicate% 20block% 20C% 20in% 20R2% 3B% 20% E5% 85% B6% E4% B8% AD % E9% 87% 8D% E5% A4% 8D% E5% 8C% BAC% E5% 85% B7% E6% 9C% 89% E8% 83% 86% E7% A2% B1% E7% BB% 93% E5 % 90% 88% E8% 9B% 8B% E7% 99% BDA% 20% 20% 20 (% 20CbpA% 20)% 20% E8% A1% 80% E6% B8% 85% E5% 9E% 8B4% E5 % 9E% 8B% E7% 9A% 84N-% E6% 9C% AB% E7% AB% AF% E6% 88% AA% E5% 8F% 96% E7% 89% A9% E7% 9A% 84327% E4% BD% 8D% E5% 88% B0433% E4% BD% 8D% E6% B0% A8% E5% 9F% BA% E9% 85% B8% E5% BA% 8F% E5% 88% 97% 2C% E5% 85% B6% E5% 85% B7% E6% 9C% 89% E4% B8% 8B% E5% 88% 97% E5% BA% 8F% E5% 88% 97% EF% BC% 9AKPEKKVAEAEKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAESDVEVKKAELELVKEEAKEPRNEEKVKQAKAEVESKKAEATRLEKIKTDRKKAEEEAKRKA% 20% 20% 20 (% 20SEQ% 20ID% 20NO% 204% 20)% 20.% E5% A4% 9A% E8% 82% BDC% 2FR2% E7% 9A% 84DNA% E5% BA% 8F% E5% 88% 97AAACCAGAAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCGAAACGAGGAAAAAGTTAAGCAAGCAAAAGCGGAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAAAATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCA% 20 % 20% 20 (% 20SEQ% 20ID% 20NO% 2015% 20)

" polypeptide A/R2 " means the polypeptide that comprises duplicate block A in R2, wherein duplicate block A has 153 of the terminal intercepting of N-thing of choline binding protein A (CbpA) serotype 4 types to 269 amino acids sequences, it has following sequence: TEPGEKVAEAKKKVEEAEKKAKDQKEEDRRNYPTITYKTLELEIAESDVEVKKAEL ELVKVKANEPRDEQKIKQAEAEVESKQAEATRLKKIKTDREEAEEEAKRRADA (SEQ ID NO 5). and as shown in Figure 1, the A district of polypeptide R2 is identical with A district among the R1.

The DNA sequence dna of coded polypeptide A/R2 is: ACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTGAGAA AAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCATTACTTACA AAACGCTTGAACTTGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGCTT GAACTAGTAAAAGTGAAAGCTAACGAAC CTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAGAGTAA ACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTGAAGAA GCAGAAGAAGAAGCTAAACGAAGAGCAGATGCT (SEQ ID NO 16).

Can change or modify the homogeneity of one or more amino acid residues or position to comprise variant, for example lack thing, comprise the residue that lacks than the whole specific residues of protein, substituent, wherein one or more specific residues are replaced and addition product by other residue, and wherein one or more amino acid residues are added into the end or the mid portion (see figure 2) of described polypeptide.These molecules comprise: mix for by selected nonmammalian host expresses " preferably " codon; The cleavage site of restriction endonuclease is provided; And provide other initial, the terminal or intermediary DNA sequence that promotes to be easy to expression vector establishment.Specifically, following is the example of aminoacid replacement in the serotype 4, and it includes but not limited to: 154 E is replaced by K; 155 P is replaced by L; 156 G is replaced by E; 157 E is replaced by K; 181 K is replaced by E; 182 D is replaced by A; 187 R is replaced by Y, H or L; 194 I is replaced by N; 200 E is replaced by D; 202 E is replaced by D; 209 E is replaced by K; 212 K is replaced by E; 218 V is replaced by L; 220 V is replaced by K or E; 221 K is replaced by E; 223 N is replaced by D or K; 225 P is replaced by S, T or R; 227 D is replaced by N; 228 E is replaced by K; 229 Q is replaced by E, G or D; 230 K is replaced by T; 232 K is replaced by N; 235 E is replaced by K; 236 A is replaced by E; 237 E is replaced by K; 240 S is replaced by N; 241 K is replaced by E; 242 Q is replaced by K; 249 K is replaced by E; 250 K is replaced by N; 257 E is replaced by Q or K; 263 A is replaced by L; 264 K is replaced by E; 265 R is replaced by N; 266 R is replaced by I; 267 A is replaced by K or V; 258 D is replaced by T; 269 A is replaced by D; 291 A is replaced by T, V, P, G or X; 294 G is replaced by G, A or E; 295 V is replaced by D or A; 295 P is replaced by L or F; 299 L is replaced by P or Q; 328 P is replaced by S; 329 E is replaced by G; 340 E is replaced by A; 343 K is replaced by E or D; 347 E is replaced by K; 349 D is replaced by A; 354 R is replaced by H; 366 E is replaced by D; 375 E is replaced by K; 378 K is replaced by E; 390 E is replaced by G; 391 P is replaced by S; 393 N is replaced by D; 397 V is replaced by I; Replaced by Q with 408 K.

"Polypeptide R2 serotype-R6x" means containing choline binding protein A (CbpA) serotype R6x taken was the N-terminal 16-444 of the amino acid sequence of a polypeptide having the following sequence: ENEGSTQAATSSNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVALNIKLSAIKTKYLRELNVLEEKSKDELPSEIKAKLDAAFEKFKKDTLKPGEKVAEAKKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVKEAELELVKEEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKRKADAKLKEANVATSDQGKPKGRAKRGVPGELATPDKKENDAKSSDSSVGEETLPSSSLKSGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTLDLEIAESDVKVKEAELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAEEEAKRKAAEEDKVKEKPA (SEQ ID NO 7 ) serotype R6x R2 encoding the polypeptide DNA sequence: GAAAACGAAGGAAGTACCCAAGCAGCCACTTCTTCTAATATGGCAAAGACAGAACATAGGAAAGCTGCTAAACAAGTCGTCGATGAATATATAGAAAAAATGTTGAGGGAGATTCAACTAGATAGAAGAAAACATACCCAAAATGTCGCCTTAAACATAAAGTTGAGCGCAATTAAAACGAAGTATTTGCGTGAATTAAATGTTTTAGAAGAGAAGTCGAAAGATGAGTTGCCGTCAGAAATAAAAGCAAAGTTAGACGCAGCTTTTGAGAAGTTTAAAAAAGATACATTGAAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAATACTTACAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTAAAGAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAAtCTCGAAACGAGGGCACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAACAtCAAGACAGAtCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCAGATGCTAAGTTGAAGGAAGCTAATGTAGCGACTTCAGAtCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTGAAGAAACTCTTCCAAGCTCATCCCTGAAATCAGGAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGACCTTGAAATTGCTGAGTCCGATGTGAAAGTTAAAGAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCGAGACGAGGAAAAAATTAAGCAAGCAAAAGCGAAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAACATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCAGCAGAAGAAGATAAAGTTAAAGAAAAACCAGCTG (SEQ ID NO 17) CbpA amino acid sequence of serotype R6x: ENEGSTQAATSSNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVALNIKLSAIKTKYLRELNVLEEKSKDELPSEIKAKLDAAFEKFKKDTLKPGEKVAEAKKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVKEAELELVKEEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKRKADAKLKEANVATSDQGKPKGRAKRGVPGELATPDKKENDAKSSDSSVGEETLPSSSLKSGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTLDLEIAESDVKVKEAELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAEEEAKRKAAEEDKVKEKPAEQPQPAPATQPEKPAPKPEKPAEQPKAEKTDDQQAEEDYARRSEEEYNRLTQQQPPKTEKPAQPSTPKTGWKQENGMWYFYNTDGSMATGWLQNNGSWYYLNANGAMATGWLQNNGSWYYLNANGSMATGWLQNNGSWYYLNANGAMATGWLQYNGSWYYLNSNGAMATGWLQYNGSWYYLNANGDMATGWLQNNGSWYYLNANGDMATGWLQYNGSWYYLNANGDMATGWVKDGDTWYYLEASGAMKASQWFKVSDKWYYVNGSGALAVNTTVDGYGVNANGEWVN (SEQ ID NO 8). encoding the amino acid sequence of CbpA of serotype R6x DNA sequence: GAAAACGAAGGAAGTACCCAAGCAGCCACTTCTTCTAATATGGCAAAGACAGAACATAGGAAAGCTGCTAAACAAGTCGTCGATGAATATATAGAAAAAATGTTGAGGGAGATTCAACTAGATAGAAGAAAACATACCCAAAATGTCGCCTTAAACATAAAGTTGAGCGCAATTAAAACGAAGTATTTGCGTGAATTAAATGTTTTAGAAGAGAAGTCGAAAGATGAGTTGCCGTCAGAAATAAAAGCAAAGTTAGACGCAGCTTTTGAGAAGTTTAAAAAAGATACATTGAAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAATACTTACAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTAAAGAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAAtCTCGAAACGAGGGCACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAACAtCAAGACAGAtCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCAGATGCTAAGTTGAAGGAAGCTAATGTAGCGACTtCAGAtCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTGAAGAAACTCTTCCAAGCTCATCCCTGAAATCAGGAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGACCTTGAAATTGCTGAGTCCGATGTGAAAGTTAAAGAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCGAGACGAGGAAAAAATTAAGCAAGCAAAAGCGAAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAACATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCAGCAGAAGAAGATAAAGTTAAAGAAAAACCAGAAGAACAACCACAACCAGCGCCGGCTACTCAACCAGAAAAACCAGCTCCAAAACCAGAGAAGCCAGCTGAACAACCAAAAGCAGAAAAAACAGATGATCAACAAGCTGAAGAAGACTATGCTCGTAGATCAGAAGAAGAATATAATCGCTTGACTCAACAGCAACCGCCAAAAACTGAAAAACCAGCACAACCATCTACTCCAAAAACAGGCTGGAAACAAGAAAACGGTATGTGGTACTTCTACAATACTGATGGTTCAATGGCAACAGGATGGCTCCAAAACAACGGTTCATGGTACTATCTAAACGCTAATGGTGCTATGGCGACAGGATGGCTCCAAAACAATGGTTCATGGTACTATCTAAACGCTAATGGTTCAATGGCAACAGGATGGCTCCAAAACAATGGTTCATGGTACTACCTAAACGCTAATGGTGCTATGGCGACAGGATGGCTCCAATACAATGGTTCATGGTACTACCTAAACAGCAATGGCGCTATGGCGACAGGATGGCTCCAATACAATGGCTCATGGTACTACCTCAACGCTAATGGTGATATGGCGACAGGATGGCTCCAAAACAACGGTTCATGGTACTACCTCAACGCTAATGGTGATATGGCGACAGGATGGCTCCAATACAACGGTTCATGGTATTACCTCAACGCTAATGGTGATATGGCGACAGGTTGGGTGAAAGATGGAGATACCTGGTACTATCTTGAAGCATCAGGTGCTATGAAAGCAAGCCAATGGTTCAAAGTATCAGATAAATGGTACTATGTCAATGGCTCAGGTGCCCTTGCAGTCAACACAACTGTAGATGGCTATGGAGTCAATGCCAATGGTGAATGGGTAAACTAA (SEQ ID NO 18)....

"Peptide C/R2 serotype R6x" refers to comprise the polypeptide (referring to Fig. 2) of duplicate block C in R2; wherein the repeat region C has a choline binding protein A (CbpA) serotype R6x intercept the N-terminus compounds 327-433 of the amino acid sequence which has the following sequence: KSGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTLDLEIAESDVKVKEAELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAEEEAKRKA (SEQ ID NO 10) serotype R6x C/R2 polypeptide DNA sequence: AAATCAGGAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGACCTTGAAATTGCTGAGTCCGATGTGAAAGTTAAAGAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCGAGACGAGGAAAAAATTAAGCAAGCAAAAGCGAAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAACATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCA (SEQ ID NO 20).

% 22% 20polypeptide% 20A% 2FR2% 20serotype% 20R6x% 20% 22% 20refers% 20to% 20comprise% 20the% 20polypeptide% 20 (referring% 20to% 20Fig.% 202)% 20of% 20duplicate% 20block% 20A% 20in% 20R2 % 3B% 20% E5% 85% B6% E4% B8% AD% E6% 89% 80% E8% BF% B0% E9% 87% 8D% E5% A4% 8D% E5% 8C% BAA% E5% 85 % B7% E6% 9C% 89% E8% 83% 86% E7% A2% B1% E7% BB% 93% E5% 90% 88% E8% 9B% 8B% E7% 99% BDA% 20% 20% 20 (% 20CbpA% 20)% 20% E8% A1% 80% E6% B8% 85% E5% 9E% 8BR6x% E7% 9A% 84N-% E6% 9C% AB% E7% AB% AF% E6% 88% AA% E5% 8F% 96% E7% 89% A9% E7% 9A% 84155% E4% BD% 8D% E5% 88% B0265% E4% BD% 8D% E7% 9A% 84% E6% B0% A8% E5% 9F% BA% E9% 85% B8% E5% BA% 8F% E5% 88% 97% 2C% E5% 85% B6% E5% 85% B7% E6% 9C% 89% E4% B8% 8B% E5% 88% 97% E5% BA% 8F% E5% 88% 97% EF% BC% 9APGEKVAEAKKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVKEAELELVKEEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKRKADA% 20% 20% 20 (% 20SEQ% 20ID% 20NO% 2011% 20)% 20% E7% BC% 96% E7% A0% 81% E5% A4% 9A% E8% 82% BDA% 2FR2% E8% A1% 80% E6% B8% 85% E5% 9E% 8BR6x% E7% 9A% 84DNA% E5% BA% 8F% E5% 88% 97% EF% BC% 9ACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAATACTTACAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTAAAGAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAAtCTCGAAACGAGGGCACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAACAtCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCAGATGCT% 20% 20% 20 (% 20SEQ% 20ID% 20NO% 2021% 20)% 20.

The present invention relates to a kind of isolating polypeptide, wherein this isolating polypeptide comprises its fragment, mutant, variant or analog by being formed as SEQ ID NOS22 or 23 described aminoacid sequences, or derivant.SPSLKPEKKVAEAEKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAESDVEVKK AELELVKEEAKEPRNEEKVKQAKAEVESKKAEATRLEKIKTDRKKAEEEAKRKAAE EDKVKEKPA (SEQ ID NO 22; Serotype 4; The 323-434 position) or PSSSLKSGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTLDLEIAESDVKVK EAELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAEEEAKRKAA EEDKVKEKRA (SEQ ID NO 23; Serotype R6x; The 322-434 position).

" polypeptide B/R2 " is meant that 270 of the terminal intercepting of the N-that comprises the described choline binding protein A of Fig. 2 (CbpA) serotype 4 types thing are to 326 amino acids polypeptide of sequence." polypeptide B/R2 serotype-R6x " is meant that 264 of the terminal intercepting of the N-that comprises the described choline binding protein A of Fig. 2 (CbpA) serotype R6x thing are to 326 amino acids polypeptide of sequence.The present invention's research has the polypeptide of the described A of Fig. 1, B, C, A+B, B+C, A+C region amino acid sequence.

In addition, the invention provides a kind of isolating polypeptide, this polypeptide comprises the aminoacid sequence of the terminal intercepting of the N-thing of choline binding protein A, and wherein said polypeptide has aminoacid KXXE (SEQ IDNO 6).

The present invention relates to comprise the terminal polypeptide that intercepts the aminoacid sequence of thing of choline binding protein AN-, wherein said aminoacid sequence is set forth among Fig. 2.In one embodiment, described polypeptide has an aminoacid sequence, and this sequence is the described conserved region of Fig. 2.For example, conserved region includes, but are not limited to aminoacid sequence 158-172; 300-321; 331-339; 355-365; 367-374; 379-389; 409-427; And 430-447.Fig. 2 has set forth the nucleic acid in N-terminal district of the various serotype CbpA that the present invention studied and the homology of aminoacid sequence.

The invention provides a kind of isolating polypeptide, this polypeptide comprises from the aminoacid sequence of the terminal intercepting of the N-of choline binding protein A, wherein said polypeptide have activity of lectin and not with choline binding.In one embodiment, described polypeptide have SEQ ID NO 1,3-5,7 or 9-11 described in arbitrary aminoacid sequence, comprise its fragment, mutant, variant, analog, or derivant.

This paper employed " polypeptide with activity of lectin " is meant a kind of with non-covalent bond and the bonded polypeptide of carbohydrate, peptide or protein." adhesin " defined herein is meant that antibacterial combines with human cell's non-covalent bond or enough stablizes to stand the secretion of washing." in conjunction with LNnT " defined herein is meant that the substrate that wraps up in conjunction with lacto-N-neotetraose is more than albumin-binding-contrast.

The invention provides a kind of isolating immunogenic polypeptide, this polypeptide comprises from the aminoacid sequence of the N-terminal intercepting of choline binding protein A.The present invention predicts that described immunogenic polypeptide has the arbitrary aminoacid sequence described in SEO ID NOS 1,3-7 or the 9-11, comprises its fragment, mutant, variant, analog, or derivant.The invention provides a kind of isolating polypeptide, this polypeptide comprises the described aminoacid sequence that intercepts from the N-terminal of choline binding protein A of Fig. 2.In one embodiment, described polypeptide has the aminoacid sequence of the described conserved region of Fig. 2.

The present invention relates to comprise the analog of the polypeptide of above-mentioned aminoacid sequence.Described analogue polypeptide may have the N-terminal methionine or the terminal polyhistidyl of N-of the N that do not connect or be connected to the polypeptide that comprises described aminoacid sequence or COOH end.

In another embodiment, the present invention has studied the fragments of peptides of the polypeptide that is produced by the proteolytic digestion product of described polypeptide.In another embodiment, be connected with one or more chemical parts on the described polypeptide derivative.In another embodiment, described chemical part is a water-soluble polymer.In another embodiment, described chemical part is a Polyethylene Glycol.In another embodiment, described chemical part be single, two, three or tetrapegylated.In another embodiment, described chemical part is the terminal monopegylated of N-.

It is useful especially that Polyethylene Glycol (PEG) is connected on the chemical compound, because PEG has very low toxicity (people such as Carpenter, 1971) to mammal.For example, U.S.'s approval utilizes the PEG adduct of adenosine deaminase to treat human serious merging acquired immunodeficiency syndrome.Combining second benefit that obtains with PEG is to reduce the immunogenicity and the antigenicity of heterobifunctional compound effectively.For example, human protein's PEG adduct can be used for treating the disease of other mammalian species and does not bring out serious immunoreactive danger.The compounds of this invention can be sent so that the host immune response that reduces or prevent to resist described chemical compound or resist the cell that can produce described chemical compound with the microencapsulation device.The compounds of this invention also can be to be wrapped in microencapsulation form such as the liposome delivery in the film.

Many PEG activity forms that are applicable to the protein direct reaction have been described.Be used for comprising with the PEG reagent of protein amino reaction the active ester or the carbonic acid ester derivative of carboxylic acid, particularly wherein leaving group is the chemical compound of N-hydroxy-succinamide, right-nitrophenol, imidazoles or 1-hydroxyl-2-Nitrobenzol-4-sulphonic acid ester.The PEG derivant that comprises maleimide amino or halo acetyl group is the reagent that is applicable to the modification of protein free sulfhydryl groups.Similarly, the PEG reagent that comprises amino hydrazine or acid hydrazide can be used for and aldehyde reaction, and described aldehyde is to be produced by the periodate oxidation of carbohydrate group in the protein.

In one embodiment, the amino acid residue of polypeptide described herein preferably exists with " L " isomeric forms.In another embodiment, the alternative any L-amino acid residue of residue so that " D " isomeric forms exists needs only the functional characteristic that described polypeptide can keep needed activity of lectin.NH ₂Be meant free amine group in the aminoterminal existence of polypeptide.COOH is meant the free carboxy in the c-terminus existence of polypeptide.The employed abbreviation of this paper is consistent with the polypeptide name of standard, J.Biol.Chem., 243:3552-59 (1969).

Should be noted that all amino acid residue sequences of this paper are all represented by formula, the left side of wherein said formula and right orientation all be at aminoterminal on the conventional direction of c-terminus.In addition, should be noted that at amino acid residue sequence and begin or the peptide bond that is connected with other one or more amino acid residue sequences represented in the dash that ends up.

Utilize the synthetic polypeptide of known solid phase, liquid phase or polypeptide condensation technology or its combined preparation can comprise natural and alpha-non-natural amino acid.Be used for the Boc (N that the synthetic aminoacid of peptide can be standard ^αThe N of-amido protecting ^α-tert-butoxycarbonyl) amino-acid resin; deprotection, neutralization, coupling and washing methods (1963 by standard in the initial solid phase method of Merrifield; J.Am.Chem.Soc.85:2149-2154) synthetic; or by Carpino and Han (1972, J.Org.Chem.37:3403-3406) the alkali labile N that at first describes ^α9-fluorenyl methoxy carbonyl (Fmoc) aminoacid of-amido protecting.Therefore, polypeptide of the present invention can comprise D-aminoacid, D-aminoacid and the amino acid whose combination of L-and various " designer " aminoacid that is used to pass on special nature (for example Beta-methyl aminoacid, C _α-methylamino acid and N _α-methylamino acid etc.).Synthesizing amino acid comprises the ornithine that is used for synthetic lysine, is used for the fluorophenylalanine and the nor-leucine that is used for synthetic leucine or isoleucine of synthetic styrene-acrylic propylhomoserin.In addition, by specifying specific amino acids, can produce alpha-helix, β-corner, βZhe Die, γ-corner and cyclic peptide at specific coupling step.

On the one hand, peptide of the present invention can comprise special aminoacid at the C-end, and it is in conjunction with CO ₂H or CONH ₂Side chain is to simulate free glycine or glycine-amide group.The other method of considering this special residue be have form by joint or with the D or the L amino acid analogue of the side chain of beadlet bonding.In one embodiment, false free C-terminal residue can be D or L optical configuration; In another embodiment, can use the racemic mixture of D and L isomer.

In other embodiments, in the N-terminal residue that pyroglutamic acid can be used as peptide is included in.Although pyroglutamic acid is unable to undergo the check by Edman degraded institute calling sequence,, will keeps enough non-pyroglutamic acid peptides on the beadlet and be used for order-checking by replacement being only limited to the peptide of 50% on the given beadlet with the terminal pyroglutamic acid of N-.Those of ordinary skill can recognize easily that this technology can be used for any order-checking of mixing the peptide of the residue that tolerates the Edman degraded at the N-end.Hereinafter describe other method that shows required active each peptide in detail.When having specific N-end group in 50% peptide, the specific activity that contains the peptide of protected N-end group such as pyroglutamic acid will easily obtain proving by the activity of relatively complete (100%) protected peptide with not protected (0%) peptide.

In addition, estimate that the peptide of the present invention's preparation has the architectural characteristic of better definition, and use peptide mimics and peptide analog key such as ester bond to prepare peptide with new features.In another embodiment, can produce reduction peptide bond (that is R, ₁-CH ₂-NH-R ₂, R wherein ₁And R ₂Be amino acid residue or sequence) peptide.Can be used as the dipeptides subunit and introduce the reduction peptide bond.Such molecule will tolerate peptide bond hydrolysis, as, proteinase activity.This class peptide will provide has unique function and active part, as, owing to being had toleration, metabolism decomposition or proteinase activity prolonged the intravital half-life.And affined peptide shows that enhanced functional activity is known (Hruby, 1982, Life Sciences 31:189-199 in some system; People such as Hruby, 1990, Biochem is J.268:249-262); The invention provides the be tied method of peptide of a kind of production, this peptide mixes random sequence in all other positions.

The peptide that can synthesize affined, cyclic or rigidization, condition be insert aminoacid or amino acid analogue at least two positions of peptide sequence can the crosslinked chemical functional group who forms cross-linking agent with this peptide of constraint after processing, cyclisation or rigidization to provide.When inducing aminoacid, revolution will help cyclisation when mixing.Can make the crosslinked amino acid whose example of peptide is that the cysteine that forms disulphide, the aspartic acid that forms lactone or Lactose enzyme and chelating agen such as γ-carboxyl-glutamic acid (Gla) are (Bachem) with the chelating transition metal and form cross-linking agent.By improvement Zee-Cheng and Olson (1980, Biophys, Biochem.Res.Commun.94:1128-1132) described synthetic method can prepare the γ-carboxyl-glutamic acid of protection.Can by as the oxidation cysteine residues with form disulphide or add metal ion with form chelate handle contain in the peptide sequence at least two can be crosslinked amino acid whose peptide, make peptide crosslinked and form the peptide of affined, cyclic or rigidization.The invention provides the strategy of systems produce cross-linking agent.For example, if 4 cysteine residues are mixed in the peptide sequence, can use different protecting groups (Hiskey, 1981, in The Peptides:Analysis, Synthesis, Biology, Vol.3, Gross and Meienhofer, eds., Academic Press:New York, 137-167 page or leaf; People such as Ponsanti, 1990, Tetrahedron 46:8255-8266).The first pair of cysteine can be by deprotection and oxidation, then can be with second group of deprotection and oxidation.In the method, can form the cross-linking agent of the curing of particular group.In addition, can mix a pair of cysteine with a pair of amino acid analogue of checking so that make cross-linking agent have different chemical characteristics.

Following nonclassical amino acid can be attached in the peptide so that introduce the motif of specific conformation: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (people such as Kazmierski, 1991, J.Am.Chem.Soc.113:2275-2283); (2S, 3S)-methylphenylalanine, (2S, 3R)-methylphenylalanine, (2R, 3S)-methylphenylalanine and (2R, 3R)-methylphenylalanine (Kazmierski and Hruby, 1991, Tetrahedron Lett); The amino naphthane of 2--2-carboxylic acid (Landis, 1989, Ph.D.Thesis, University of Arizona); Hydroxyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (people such as Miyake, 1989, J.Takeda Res.Labs.43:53-76); B-carboline (D and L) (Kazmierski, 1988, Ph.D.Thesis, University of Arizona); HIC (histidine isoquinolinecarboxylic acid) (people such as Zechel, 1991, Int.J.Pep.Protein Res.43); And HIC (histidine ring urea) (Dharanipragada).

Following amino acid analogue and peptide mimics can be introduced in the peptide to induce or to help forming special secondary structure: LL-Acp (LL-3-amino-2-propenone-6-carboxylic acid), a kind of dipeptide analog (people such as Kemp who induces β-corner, 1985, J.Org.Chem.50:5834-5838); Induce the analog (people such as Kemp, 1988, Tetrahedron Lett.29:5081-5082) of beta sheet; Induce the analog (people such as Kemp, 1988, Tetrahedron Lett.29:5057-5060) of β-corner; Induce alpha-helix analog (people such as Kemp, 1988, TetrahedronLett.29:4935-4938); Induce γ-corner analog (people such as Kemp, 1989, J.Org.Chem.54:109-115) and the analog that provides by following list of references: Nagai and Sato, 1985, Tetrahedron Lett.26:647-650; People such as DiMaio, 1989, J.Chem.Soc.Perkin Trans.1687 page or leaf; And Gly-Ala corner analog (people such as Kahn, 1989, Tetrahedron Lett.30:2317); Amido link is with joining thing (people such as Jones, 1988, Tetrahedron Lett.29:3853-3856); Tetrazolium (people such as Zabrocki, 1988, J.Am.Chem.Soc.110:5875-5880); DTC (people such as Samanen, 1990, Int.J.ProteinPep.Res.35:501-509); With people such as Olson, 1990, people such as J.Am.Chem.Sci.112:323-333 and Garvey, 1990, the analog of instructing among the J.Org.Chem.56:436.Conformation restriction analogies β-corner and β-projection have been described in the United States Patent (USP) 5440013 (being published in August 8 nineteen ninety-five) of Kahn.

The present invention also provides the modification or the deriving method of polypeptide of the present invention or peptide.The modification of peptide is known to those of ordinary skill, and comprises phosphorylation, carboxy methylation and acidylate.Modification can be undertaken by chemistry or enzyme mode.The peptide derivant that can prepare on the other hand, glycosylation or fatty acidylate.In the art, the preparation method of the peptide of glycosylation or fatty acidylate is known.Also can prepare the fatty acyl group peptide derivant.Such as but not limited to, free amine group (the N-end or lysyl-on) can be by acidylate, as myristoylation.In another embodiment, can be-(CH with comprising structural formula ₂) nCH ₃The aminoacid of aliphatic lateral chain introduce in the peptide.This and other the peptide-fatty acid conjugates that is suitable for the present invention's use is disclosed in British patent GB8809162.4, International Patent Application PCT/AU89/00166 and the list of references above 5.

In the nucleic acid of this peptide species of coding, can suddenly change and make specific codon change into the codon of coding different aminoacids.This sudden change is undertaken by changing minimum as far as possible nucleotide usually.Such replacement sudden change can be carried out and make in the gained protein aminoacid in nonconservative mode (promptly, by belong to other the amino acid whose codon of amino acids with specific size or characteristic change into belong to the peculiar codon of another kind of other aminoacid) or change in conservative mode (that is, changing into the peculiar codon of the aminoacid that belongs to identical category) by belonging to other amino acid whose codon of amino acids with specific size or characteristic.This conservative change makes the less variation of generation on the proteinic 26S Proteasome Structure and Function of gained usually.Non-conservative change more likely changes the proteinic structure of gained, activity or function.Will be understood that the sequence that the present invention includes the conservative change that contains not obvious change gained protein active or binding characteristic.Amino acid whose replacement can be selected from such amino acid whose other member in the sequence.For example, nonpolar (hydrophobicity) aminoacid comprises alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The aminoacid that contains aromatic ring structure is phenylalanine, tryptophan and tyrosine.Polar neutral amino acid comprises glycine, serine, threonine, cysteine, tyrosine, agedoite and glutamine.Positively charged (alkalescence) aminoacid comprises arginine, lysine and histidine.Electronegative (acidity) aminoacid comprises aspartic acid and glutamic acid.Do not wish performance molecular weight or isoelectric point, IP that this variable effect is determined by polyacrylamide gel electrophoresis.

Particularly preferred being substituted by:

-Lys replaces Arg and Arg replaces Lys, and such replacement can keep positive charge;

-Glu replaces Asp and Asp replaces Glu, and such replacement can keep negative charge;

-Ser replaces Thr, and such replacement can keep free-OH; With

-Gln replaces Asn, and such replacement can keep free NH ₂

Synthetic DNA sequence allows to make things convenient for the gene of construction expression analog or " mutain ".People such as Noren, Science, 244:182-188 (in April, 1989) have described the conventional method in the alpha-non-natural amino acid fixed point introducing protein.This method can be used for producing the analog based on alpha-non-natural amino acid.

In the present invention, can use molecular biology, microbiology and the recombinant DNA technology of this area routine.These technology have been proved absolutely in the literature.For example, referring to people such as Sambrook, " Molecular Cloning:A Laboratory Manual " (1989); " CurrentProtocols in Molecular Biology " I-III rolls up [Ausubel, R.M., ed. (1994)]; " Cell Biology:A Laboratory Handbook " I-III rolls up [J.E.Celis, ed. (1994)]; " Current Protocols in Immunology " I-III rolls up [Coligan, J.E., ed. (1994)]; " Oligonucleotide Synthesis " (M.J.Gait ed.1984); " Nucleic AcidHybridization " [B.D.Hames ﹠amp; S.J.Higgins eds. (1985)]; " TranscriptionAnd Translation " [B.D.Hames ﹠amp; S.J.Higgins eds. (1984)]; " Animal CellCulture " [R.I.Freshney, ed. (1986)]; " Immobilized Cells AndEnzymes " [IRL Press, (1986)]; B.Perbal, " A Practical Guide To Molecular Cloning " (1984).

In other embodiments, in the N-terminal residue that pyroglutamic acid can be used as peptide is included in.Although pyroglutamic acid is unable to undergo the check by Edman degraded institute calling sequence,, will keep the peptide of enough non-pyroglutamic acids on the beadlet by replacement being only limited to the peptide of 50% on the given beadlet with the terminal pyroglutamic acid of N-.Those of ordinary skill can recognize easily that this technology can be used for any order-checking of mixing the peptide of the residue that tolerates the Edman degraded at the N-end.Hereinafter describe other method that shows required active each peptide in detail.When having specific N-end group in 50% peptide, the concrete activity that contains the peptide of protected N-end group such as pyroglutamic acid can will easily obtain proving by the activity of relatively complete (100%) protected peptide with the peptide of not protected (0%).

The chemical part that is used for derivatization: the chemical part that is suitable for derivatization can be selected from water-soluble polymer.Selected polymer should be water miscible in case the composition of its connection can not be deposited in aqueous environments as, in the physiological environment.Preferably, use for the treatment of finished product preparation, polymer will be pharmaceutically useful.Those skilled in the art can select required polymer according to the consideration such as whether polymer/composition conjugate will use in treatment, if be used for the treatment of, then to consider required dosage, circulation time, to proteolysed toleration and other factors.For one or more compositions of the present invention, it is confirmable using assay method provided herein.

For example, the optional copolymer of water-soluble polymer, carboxymethyl cellulose, glucosan, polyvinyl alcohol, polyvinylpyrrolidone, poly--1 from Polyethylene Glycol, ethylene glycol/propylene glycol, 3-dioxolane, poly--1,3,6-trioxane, ethylene/copolymer-maleic anhydride, polyamino acid (homopolymer or randomcopolymer) and glucosan or poly-(n-vinyl pyrrolidone) Polyethylene Glycol, propylene glycol homopolymer, poly(propylene oxide)/ethylene oxide copolymer, polyoxyethylene polyhydric alcohol and polyvinyl alcohol.The Polyethylene Glycol propionic aldehyde is because its stability in water may have advantage aborning.

Polymer can have molecular weight arbitrarily, and can be branched or not branched.With regard to Polyethylene Glycol, for easy operating and preparation, preferred molecular weight at about 2kDa to (term " about " be illustrated in the preparation of Polyethylene Glycol the described molecular weight of some molecular proportions heavier other are then lighter) between about 100kDa.Can use other big or small Polyethylene Glycol, this depends on that required treatment overall picture is (as, required lasting release period, to bioactive influence, if any, the difficulty of operation, antigenic have or not or size and Polyethylene Glycol to other known effect of human cytokines or analog).

So the quantity of the polymer molecule that connects is variable, and those skilled in the art can determine its influence to function.Can carry out single derivatization to identical or different chemical part (for example, polymer is as the Polyethylene Glycol of Different Weight), perhaps derivatization can be carried out two, three, four or some combination.The ratio of polymer molecule and one or more component molecules is variable, and their concentration in reactant mixture also is variable.In general, best than (from the efficient of reaction, do not have excessive unreacted one or more compositions and polymer) will determine by various factors, as required the derivatization degree (as, single, two, third-class), the molecular weight of selected polymer, polymer be branched or not branched and reaction condition.

Peg molecule (or other chemical part) should be connected on one or more compositions, and described composition is considered to proteinic functional or antigenicity is regional influential.For a person skilled in the art, many operable methods of attachment are arranged, as referring to being incorporated herein EP0401384 for reference (PEG is coupled on the G-CSF), also can be referring to people such as Malik, 1992, Exp.Hematol.20:1028-1035 (reported with tresyl chloride and made GM-CSFpegylation).For example, Polyethylene Glycol can come covalent bond by reactive group such as free amine group or carboxyl through amino acid residue.Reactive group be those can with the bonded group of activated polyglycol molecule.Amino acid residue with free amine group comprises the amino acid residue of lysine residue and N-end; Amino acid residue with free carboxy comprises the amino acid residue of asparagicacid residue, glutaminic acid residue and C-end.Sulfydryl also can be used as the reactive group that connects peg molecule.For therapeutic purposes, it is preferred being connected on the amino, as is connected on N-end or the lysine group.

The invention provides a kind of isolating nucleic acid, its coding has the polypeptide of the aminoacid sequence that contains the terminal intercepting of choline binding protein AN-thing.The invention provides a kind of isolating nucleic acid, its coding contains the polypeptide of the aminoacid sequence of the terminal intercepting of choline binding protein AN-thing as described in Figure 2.In one embodiment, nucleic acid is SEQ ID NOS 12, and 14-17 and 19-21 are described, comprise its fragment, mutant, variant, analog or derivant.Nucleic acid is DNA, cDNA, genomic DNA, RNA.In addition, isolating nucleic acid can be connected on the promoter of rna transcription effectively.Expectation uses this nucleic acid to come the competitive inhibition activity of lectin.

" carrier " is a kind of replicon, as plasmid, phage or cosmid.Can be connected so that duplicate the sections that is connected with another DNA sections.

" DNA " refers to the polymerized form of the deoxyribonucleotide (adenine, guanine, thymus pyrimidine or cytosine) that exists with its sub-thread form or bifilar helix.This term only refers to the firsts and seconds structure of molecule, and it is not restricted to any specific tertiary structure.Therefore, this term comprises distrand DNA, particularly finds in linear DNA molecule (as restricted fragment), virus, plasmid and chromosome.In the discussion of specific distrand DNA molecular structure, this paper can be according to only describing sequence along the normal convention that provides sequence on 5 ' to the 3 ' direction of the non-transcribed thigh of DNA (that is, have with the mRNA homologous sequence thigh).

When transcribing and translating of DNA sequence controlled and regulated to expression control sequenc, DNA sequence " is connected " effectively to expression control sequenc.Term " effectively connect " be included in the DNA sequence front of being expressed have suitable initial signal (as, ATG) and keep correct frame to allow under the control of expression control sequenc, expressing DNA sequence and to produce required product by this dna sequence encoding.The gene that inserts if desired in the recombinant DNA molecules does not contain suitable initial signal, then such initial signal can be inserted in this gene front.

In addition, the present invention also provides the carrier that comprises above-mentioned nucleic acid molecules.Promoter can be or be equal to antibacterial, yeast, insecticide or mammiferous promoter.In addition, carrier can be plasmid, cosmid, yeast artificial chromosome (YAC), phage or eucaryon viral DNA.

Can use other the numerous carrier framework known in the art that can be used for expressing protein.These carriers include but not limited to: adenovirus (AV), adeno associated virus (AAV), simian virus 40 (SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Moloney muroid leucovirus, DNA delivery system, i.e. liposome and expression plasmid delivery system.In addition, a class carrier comprises by the deutero-DNA element of virus, and virus is as bovine papilloma virus, polyoma virus, baculovirus, retrovirus or Semliki Forest virus.These carriers can be purchased or be assembled by means commonly known in the art by described sequence.

The present invention also provides the host carrier system of producing polypeptide, and it comprises the carrier of suitable host cell.Proper host cell includes but not limited to prokaryotic cell or eukaryotic cell, as bacterial cell (comprising gram-positive cell), yeast cells, fungal cell, insect cell and zooblast.Many mammiferous cells all can be used as the host, include but not limited to l cell NIH 3T3, Chinese hamster ovary celI, HeLa cell, Ltk cell, Cos cell etc.

In expressing DNA sequence of the present invention, can adopt the combination of various host/expression vectors.For example, useful expression vector can be made up of the sections of chromosome, non-chromosome and synthetic DNA sequence.Suitable carriers comprises SV40 and known bacterial plasmid, as escherichia coli plasmid col E1, pCR1, pBR322, pMB9 and derivant thereof, the derivant of plasmid such as RP4; Phage DNA S is as the various derivants of phage, as NM989 and other phage DNA, as M13 and thread sub-thread phage DNA; Yeast plasmid is as 2 μ plasmid or derivatives thereofs; Operable carrier in the eukaryotic cell is as operable carrier in insecticide or mammalian cell; By the deutero-carrier of the combination of plasmid and phage DNA, as being modified with the plasmid that utilizes phage DNA or other expression control sequenc etc.

Can in these carriers, use the sequence of various expression control sequencs-control and the expression of its DNA sequence that effectively is connected-express DNA sequence of the present invention.These operable expression control sequencs comprise control zone, glycerol 3-phosphate acid kinase or other glycolytic ferment of the main operon of early stage or late promoter as SV40, CMV, cowpox, polyoma or adenovirus, lac system, trp system, TAC system, TRC system, LTR system, phage and promoter region, fd coat protein promoter, acid phosphatase (as, the sequence and the various combination thereof of the promoter of promoter Pho5), yeast α-mating factor and other known control prokaryotic cell or eukaryotic cell or its viral gene expression.

Various unicellular host cells also can be used to express DNA sequence of the present invention.These hosts can comprise known eucaryon and prokaryotic hosts, as escherichia coli, Rhodopseudomonas, bacillus, streptomyces, fungus such as zymic bacterial strain and zooblast such as CHO, R1.1, B-W and L-M cell, African green monkey kidney cell (as, COS1, COS7, BSC1, BSC40 and BMT10), insect cell (as, SF9) and tissue culture in people's cell and plant cell.

Should be appreciated that not every carrier, expression control sequenc and host can both bring into play good equally function for expressing DNA sequence of the present invention.Even identical all hosts of expression system can not bring into play good equally function.Yet without departing from the present invention, those skilled in the art do not need too much experiment just can select suitable carriers, expression control sequenc and host to finish required expression.For example, when selecting carrier, must consider the host, because carrier must be brought into play function in the host.Also should consider copy number, control copy number purpose ability and coded any other the proteic expression of carrier of carrier, as the antibiotic marker thing.

When selecting expression control sequenc, will consider various factors usually.For example, these factors comprise system relative intensity, its control ability and with the specific dna sequence of being expressed or the compatibility of gene, particularly consider potential secondary structure.To select suitable unicellular host by considering following factors: as, the host's of the product that the DNA sequence that they are expressed with the requirement of the ability of the compatibility of selected carrier, its secretion characteristic, its correct folded protein, its fermentation and to quilt the is coded toxicity and the difficulty or ease of expression product purification.

The present invention further provides a kind of method for preparing polypeptide, it comprises makes the polypeptide that above-mentioned host carrier system is grown and recovery is produced under the condition that suitable permission polypeptide produces.

The present invention further provides a kind of can isolating polypeptide of specific recognition or antibody bonded with it.Described antibody can be monoclonal antibody or polyclonal antibody.In addition, described antibody can be with detectable label labelling, but described label is a label radioactive colorimetric, fluorescence or luminous.Described traget antibody can be monoclonal antibody or polyclonal antibody.In one embodiment, described traget antibody is the traget antibody of purification.The method of traget antibody is well known in the art.

The example of term " antibody " comprises antibody naturally occurring and that non-natural exists.Specifically, term " antibody " comprises polyclone and monoclonal antibody and fragment thereof.In addition, term " antibody " comprises chimeric antibody and complete synthesis antibody and fragment thereof.Described antibody includes, but are not limited to polyclone, monoclonal, chimeric, strand, Fab fragment and Fab expression library.

Can use polyclonal antibody that the whole bag of tricks known in the art prepares polypeptide or derivatives thereof or analog (for example, referring to antibody--laboratory manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press:Cold Spring Harbor, NewYork, 1988).In order to prepare antibody, can make it by immunity by the CbpA or derivatives thereof (for example fragment or fusion rotein) of giving various host animal injection interceptings, described host animal includes, but are not limited to rabbit, mice, rat, sheep, goat etc.In one embodiment, can be with conjugation of polypeptides to immunogenic carrier, for example on bovine serum albumin (BSA) or the key hole  hemocyanin (KLH).Depend on host's kind, various adjuvants can be used for the enhance immunity reaction.

In order to prepare monoclonal antibody or its fragment, its analog or derivatives thereof, can use produce antibody molecule by any continuous cell line in culture technology (for example, referring to antibody--laboratory manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press:Cold Spring Harbor, New York, 1988).These technology include, but are not limited to initial hybridoma technology (1975 by Kohler and Milstein exploitation, Nature 256:495-497) and trioma technology, people B-quadroma technology (people such as Kozbor, 1983, today immunology 4:72) and the EBV-hybridoma technology (people such as Cole of preparation human monoclonal antibodies, 1985, monoclonal antibody and treatment of cancer, Alan R.Liss, Inc., pp.77-96).In another embodiment of the present invention, can in germfree animal, utilize state-of-the-art technology (PCT/US90/02545) manufacture order clonal antibody.According to the present invention, people's antibody can use and can pass through end user's hybridoma (people such as Cote, 1983, Proc.Natl.Acad.Sci.U.S.A.80:2026-2023) or by transform human B cell (people such as Cole in external use EBV virus, 1985, monoclonal antibody and treatment of cancer, AlanR.Liss, Inc., pp.77-96) obtain.In fact, according to the present invention, can use the passing through of being developed will be to the get up technology (people such as Morrison of preparation " chimeric antibody " of polypeptide gene with specific mouse antibodies molecule and gene splicing with suitable bioactive human antibody molecules, 1984, J.Bacteriol.159-870; People such as Neuberger, 1984, Nature 312:604-608; People such as Takeda, 1985, Nature 314:452-454); Described antibody comprises within the scope of the present invention.Described people or humanization chimeric antibody are preferred for treating human diseases (as described below), because people or humanized antibody react than heteroantibody induction of immunity, particularly the anaphylactoid probability of itself is much smaller.Another embodiment of the present invention is utilize to make up (the people such as Huse of the technology described in the Fab expression library, 1989, Science 246:1275-1281) come to identify fast and easily to have required specific monoclonal Fab fragment, or derivatives thereof or analog to polypeptide.

The antibody fragment that comprises the antibody molecule idiotype can produce by known technology.For example, these fragments include but not limited to: the F that can produce by the pepsin digestion of antibody molecule (ab ') ₂Fragment; Can be by reduction F (ab ') ₂Fab ' the fragment that segmental disulphide bridges produces is handled the Fab fragment that antibody molecule produces with passing through with papain and Reducing agent.

In production of antibodies, can screen required antibody by technology known in the art, as, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immunoradiometric assay, the plain reaction of gel diffusion deposition, immunodiffusion mensurations, original position immunoassay (for example using gold colloidal, enzyme or labelled with radioisotope), Western blotting, precipitation, CA (as, gel CA, hemagglutination mensuration), complement is in conjunction with mensuration, immunofluorescence assay, a-protein mensuration and immunoelectrophoresis mensuration etc.In one embodiment, detect antibodies by detecting former the labelling on the antibody.In another embodiment, detect former antibody by detecting secondary antibody or reagent with combining of former antibody.In another embodiment, labelling secondary antibody.Known in this area have panimmunity to detect bonded method and within the scope of the present invention in measuring.

In order to detect serviceable indicia substance markers antibody, label such as enzyme, fluorogen, chromophore, radiosiotope, dyestuff, gold colloidal, emulsion particle and chemiluminescence agent external.In addition, in order to detect in vivo, available as radiosiotope (preferred technetium or iodine); Magnetic resonance shift reagen (as gadolinium and manganese); Or radiopaque reagent comes traget antibody.

The most frequently used label is radioelement, enzyme, when being exposed to ultraviolet light following time the chemical drugs etc. of fluorescence is arranged in these researchs.Many fluorescent materials all are known and can be used as label.They comprise as fluorescein, rhodamine, auramine, texas Red, AMCA indigo plant and fluorescein.Concrete test material is the anti-rabbit antibody for preparing in goat and put together through isothiocyanate and fluorescein.The also available radioelement of polypeptide or use enzyme labelling.Can come the detection of radioactive labels thing by present available method of counting.Preferred isotope can be selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I and ¹⁸⁶Re.

Equally also can use the enzyme labelling thing and can detect by colorimetric, spectrophotometric, fluorescence spectrophotometry, electric current or the air-flow technology of any present employing.By with reactions such as bridging molecules such as carbodiimides, isothiocyanate, glutaraldehyde, enzyme and selected granule are puted together.Operable many enzymes all are known and can use in these methods.Preferably peroxidase, beta-Glucuronidase, β-D-glucosidase, beta-D-galactosidase, urase, glucoseoxidase add peroxidase and alkali phosphatase.Mention the marker material and the method that disclose candidate in United States Patent (USP) 3654090,3850752 and 4016043 by way of example.

In another embodiment of the present invention, can prepare be suitable for that the medical expert uses be purchased test kit to determine predetermined target cell and combine existing of active ability or to lack with under a cloud in conjunction with active or predetermined.According to experimental technique discussed above, a class test kit comprises the polypeptide of labelling or its at least in conjunction with counter pair, for example its specific antibody and directions for use, and certainly, this depends on institute's choosing method, as " emulative ", " sandwich ", " DASP " etc.This test kit also can comprise peripheral reagent such as buffer agent, stabilizing agent etc.

Therefore, can prepare proves the antibacterial that helps being scheduled in conjunction with the existence of active cell or the test kit of ability, and it comprises:

(a) the immuno-chemical reaction composition of at least a labelling of scheduled volume, this composition obtains by polypeptide of the present invention or its specificity directly or indirectly are connected on the detectable in conjunction with counter pair;

(b) other reagent; With

(c) explanation of the described test kit of use.

The invention provides antagonist or blocker, it includes but not limited to: fragments of peptides, analogies, nucleic acid molecules, ribozyme, polypeptide, micromolecule, carbohydrate molecule, monosaccharide, oligosaccharide or antibody.The present invention also comprises competitive blocking-up or suppresses pneumococcal medicament.The invention provides a kind of medicament, it comprises inorganic compound, nucleic acid molecules, oligonucleotide, organic compound, peptide, peptide simulated compound or suppresses the protein of this polypeptide.

The invention provides a kind of vaccine, it comprises and has SEQ ID NOS:1,3-7,9-11, the polypeptide of aminoacid sequence described in 22 and 23 and pharmaceutically acceptable adjuvant or carrier.Described polypeptide can comprise the aminoacid sequence of the terminal intercepting of the described choline binding protein AN-of Fig. 2 thing.The invention provides a kind of vaccine, it comprises polypeptide and pharmaceutically acceptable adjuvant or carrier, and described polypeptide has the aminoacid sequence that comprises the described reserved area of Fig. 2.For example, the reserved area includes but not limited to aminoacid sequence 158-172; 300-321; 331-339; 355-365; 367-374; 379-389; 409-427 and 430-447.The invention provides a kind of vaccine, it comprises isolating nucleic acid and the pharmaceutically acceptable adjuvant or the carrier of this polypeptide of encoding.

Can induce resisting gram-positive bacteria by carrying out immunity (vaccination) with the polypeptide or derivatives thereof of immune commercial weight or fragment and adjuvant, particularly pneumococcal Active immunity, wherein said polypeptide or its antigenic derivant or fragment are the antigen components of described vaccine.

Can with the mixture of adjuvant of preparation vaccine in preparation polypeptide or derivatives thereof of the present invention or fragment.Preferably, derivant or its fragment as the polypeptide of the present invention of described vaccine antigen component is adhesin.More preferably, be total or total with the closely-related bacteria culture antigen of bacterial strain with all or a lot of Gram-positive strains as the polypeptide of described vaccine antigen component or peptide derivant or its fragment.Most preferably, the antigen component of described vaccine is an adhesin, and it is a kind of common antigen.

Can pass through methods known in the art, the application or the dna vector transport protein of for example transfection, electroporation, micro-injection, transduction, cell fusion, deae dextran, calcium phosphate precipitation, fat transfection (lysosome fusion), particle gun, the carrier that will comprise the present invention and with nucleic acid be the vaccine of principal agent is introduced the host of needs (for example referring to people such as Wu, 1992, J.Biol.Chem.267:963-967; Wu and Wu, 1988, J.Biol.Chem.263:14621-14624; People such as Hartmut, the Canadian patent application 2,012,311 of asking in March 15 nineteen ninety).

Described vaccine can be by any parenteral route administration, and described parenteral route includes, but are not limited to intramuscular, intraperitoneal, intravenous etc.Preferably because ideal vaccination result be illustrate with antigen and therefore with the immunoreation of Pathogenic organisms, so directly or by targeting or select viral vector to be administered to lymphoid tissue indirectly, for example lymph node or spleen are ideal.Because immunocyte is constantly duplicating, so they are to be the ideal target cell of the nucleic acid vaccine of principal agent with the retroviral vector, because retrovirus needs replicating cell.

Can suspect to be subjected to gram positive bacteria preference chain coccus, and more preferably being treated animal of pneumococcal infection and make this animal obtain passive immunity by antiserum, polyclonal antibody or the neutralizing monoclonal antibody that will resist polypeptide of the present invention.Although passive immunity can not bring secular protective effect, it may be to treat the valuable instrument of not vaccinated curee's bacterial infection as yet.For the treatment of Gram-positive antibiotics resistance bacterial strain, passive immunity is a particular importance, because there is not other treatment to utilize.Preferably, the antibody that is used for passive immunotherapy that is given is autoantibody.For example,, be reduced to minimum in order to make immunoreactive probability at antibody if described curee is the people, preferably, described antibody be obtain by human body or " humanized ".Initiative of the present invention or passivity vaccine or give adhesin and can be used for protection and treated animal and make it avoid gram positive bacteria, preference chain coccus, and more preferably pneumococcal infection.

The invention provides a kind of pharmaceutical composition, it comprises a certain amount of polypeptide described herein and pharmaceutically suitable carrier or diluent.

For example, this pharmaceutical composition that is used to stop streptococcus pneumoniae to be attached to mucomembranous surface can comprise the proteinic antibody in antiagglutinin district and/or the superfluous agglutinin district of solubility.Adhere to the initial step that to block infection by arbitrary mechanism blocking-up, reduce the effect of giving birth to that moves thus.This turns over again and reduces interpersonal propagation and prevent symptomatic advancing of disease.

The invention provides a kind of immunoreactive method that touches or infected pneumococcal curee of inducing, it comprises and gives described curee a certain amount of pharmaceutical composition, induction of immunity reaction thus.

The invention provides a kind of curee of prevention by the method for pneumococcal infection, it comprises and gives the pharmaceutical composition that a certain amount of prevention of described curee streptococcus pneumoniae adheres to effective dose, prevents pneumococcal infection thus.

The invention provides a kind of curee of prevention by the method for pneumococcal infection, it comprises and gives the pharmaceutical composition that a certain amount of prevention of described curee comprises antibody and pharmaceutically suitable carrier or diluent, prevents pneumococcal infection thus.

The invention provides a kind of method that has infected or touched pneumococcal curee for the treatment of, it comprises that giving described curee treats effective dose vaccine of the present invention, treats described curee thus.

The invention provides a kind of inhibition has touched or has infected pneumococcal curee's host cell and moved living method, it comprises and gives that described curee is a certain amount of to be comprised by SEQ IDNOS 1,3-5,7 or 9-11 described in the pharmaceutical composition of the polypeptide formed of aminoacid sequence, induction of immunity reaction thus.Described blocking-up moves the therapeutic peptide of the effect of giving birth to and is sent by respiratory mucosa.The pharmaceutical composition that comprises the polypeptide of forming by aminoacid sequence described in Fig. 2.

This paper employed " pharmaceutical composition " be meant by for example stop streptococcus pneumoniae to move to give birth to provide therapeutic effect or benefit, the polypeptide products of the present invention of treatment effective dose and suitable diluent, antiseptic, solubilizing agent, emulsifying agent, adjuvant and/or carrier.The amount that provides described disease therapeuticing effect and dosage regimen is provided this paper employed " treatment effective dose ".Described compositions is liquid or lyophilizing or with the exsiccant preparation of other method and comprise by various buffer agent inclusions (Tris-HCl for example, acetate, phosphate), the diluent that pH and ion concentration are formed, albumin or the gelatin of additive as stoping the surface to absorb, detergent (polysorbas20 for example, Tween 80, Pluronic F68, bile salt), solubilizing agent (glycerol for example, Polyethylene Glycol), antioxidant (ascorbic acid for example, sodium pyrosulfite), antiseptic (for example, thimerosal, benzyl alcohol, parabens), filler or tension regulator (lactose for example, mannitol), polymer such as Polyethylene Glycol are covalently bound to protein, mix polymer such as polylactic acid with complexing of metal ion or with described material, polyglycolic acid, in the particular formulations of hydrogel etc. or be added on it, perhaps be added to liposome, microemulsion, micelle, the single or multiple lift vesicle, on erythrocyte ghost or the spheroplast.Described compositions will influence the interior release rate of physical state, dissolubility, stability, body and the interior clearance rate of body of therapeutic agent.The selection of compositions will depend on proteinic physics or the chemical property with therapeutic activity.For example, may need to contain the preparation of detergent by the deutero-product of film conjunction type active matter.Controlled release or slow releasing composition are included in the preparation in the lipotropy storage storehouse (for example fatty acid, wax, oil).The present invention also comprises by the microparticle compositions of polymer (for example poloxamer or poloxamines) bag quilt and is coupled on tissue specificity receptor, part or the antigenic antibody or be coupled to active matter on the part of tissue specificity receptor.Other embodiment of the present composition comprises that protectiveness coating, protease inhibitor or the penetration enhancers of particulate form are used for various approach and comprise non-intestinal, lung, nose and oral administration.

In addition, this paper employed " pharmaceutically suitable carrier " well known to a person skilled in the art and include, but are not limited to 0.01-0.1M and the phosphate buffer of preferred 0.05M or 0.8% saline.Therefore, described pharmaceutically suitable carrier can be water or non-aqueous solution, suspension and emulsion.Examples of non-aqueous is propylene glycol, Polyethylene Glycol, vegetable oil such as olive oil and injectable organic ester such as ethyl oleate.Aqueous carrier comprises water, alcohol/aqueous solution, suspension or emulsion, comprises saline and buffering medium.The parenteral carrier comprises sodium chloride solution, woods Ge Shi glucose, glucose and sodium chloride, lactic acid Ringer's mixture or fixing oil.Intravenous vehicles comprises that liquid and supplementary, electrolyte replenisher such as those are electrolyte replenisher of principal agent etc. with woods Ge Shi glucose.Also can there be antiseptic and other additive such as antimicrobial, antioxidant, placebo, noble gas etc.

Term " adjuvant " is meant that enhancing is to antigenic immunoreactive chemical compound or mixture.Adjuvant can serve as the tissue bank of slow released antigen and also can serve as the lymphsystem activator of non-specific enhance immunity reaction (people such as Hood, Immunology, Second Ed., 1984, Beniamin/Cummings:Menlo Park, California, p.348).Usually, under the situation that does not have adjuvant, carry out primary excitation with antigen separately and can not cause body fluid or cell immune response.Adjuvant includes, but are not limited to complete Freund's adjuvant, incomplete Freund's adjuvant, Saponin, inorganic gel such as aluminium hydroxide, surfactant such as LYSOLECITHIN SUNLECITHIN A, Pluronic polyhydric alcohol, polyanion, peptide, oil or hydrocarbon emulsion, key hole  hemocyanin, dinitrophenol,DNP and the people's adjuvant that comes in handy such as BCG (bacillus calmette-guerin vaccine) and spillikin bacillus (Corynebacteriumparvum).Preferably, described adjuvant is pharmaceutically useful.

Controlled release or slow releasing composition are included in the preparation in the lipotropy storage storehouse (for example fatty acid, wax, oil).The present invention also comprises by the microparticle compositions of polymer (for example poloxamer or poloxamines) bag quilt and is coupled on tissue specificity receptor, part or the antigenic antibody or be coupled to chemical compound on the part of tissue specificity receptor.Other embodiment of the present composition comprises that protectiveness coating, protease inhibitor or the penetration enhancers of particulate form are used for various approach and comprise non-intestinal, lung, nose and oral administration.

When administration, chemical compound is removed from mucomembranous surface or circulation rapidly usually and therefore can be caused fugitive relatively pharmacological activity.So, require often to give heavy dose of relatively bioactive compound and keep curative effect.The chemical compound that known copolymer by covalently bound water-soluble polymer such as Polyethylene Glycol, Polyethylene Glycol and polypropylene glycol, sodium carboxymethyl cellulose, glucosan, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are modified obviously shows the half-life longer than corresponding unmodified chemical compound (people such as Abuchowski, 1981 after intravenous injection; People such as Newmark, 1982; With people such as Katre, 1987).This modification also can increase the dissolubility of described chemical compound in aqueous solution, eliminates aggregation, the physics that strengthens chemical compound and chemical stability and reduce the immunogenicity and the reactivity of chemical compound greatly.As a result, compare with the unmodified chemical compound, can by give still less number of times or the more described polymer-chemical compound adduct of low dosage obtain biologic activity in the needed body.

Dosage: enough amounts include, but are not limited to be approximately 1 μ g/kg-1000mg/kg.Described amount can be 10mg/kg.The pharmaceutically acceptable form of compositions comprises pharmaceutically suitable carrier.

As mentioned above, the invention provides the therapeutic combination that comprises pharmaceutical composition, wherein said pharmaceutical composition comprises carrier, vaccine, polypeptide, nucleic acid and antibody, anti-antibody and active in being attached to the medicament on the host cell with streptococcus pneumoniae competition cause of disease.

The preparation that comprises the therapeutic combination of active component is well known in the art.Typically, described compositions can be prepared into polypeptide aerosol or injectable liquid solution or the suspension that is delivered to nasopharynx part, yet, also can be prepared into and be applicable to and before injection, be dissolved in or be suspended in solid form in the liquid.Said preparation also can be emulsified.Usually, with active treatment composition and mixed with excipients pharmaceutically acceptable and can be compatible with active component.For example Shi Yi excipient is water, saline, glucose, glycerol, ethanol etc. and combination thereof.In addition, if desired, described compositions can comprise the auxiliary substance of a small amount of enhanced activity composition effectiveness such as wetting agent or emulsifying agent, pH buffer agent.

Active component can be mixed with the therapeutic combination that exists with neutral pharmaceutical acceptable salt.Officinal salt comprises acid-addition salts (forming with the free amine group of polypeptide or antibody molecule), and it is and mineral acid example hydrochloric acid or phosphoric acid, or organic acid forms as acetic acid, oxalic acid, tartaric acid, mandelic acid etc.The salt that is formed by free carboxy also can be derived from the hydroxide of inorganic base such as sodium, potassium, ammonium, calcium or ferrum or organic base such as 2-aminopropane., trimethylamine, 2-ethyl amido alcohol, histidine, procaine etc.

The compositions that when being " A ", comprises " A " (wherein " A " is single protein, dna molecular, carrier etc.) when the described protein of about at least 75% (weight) in the compositions, DNA, carrier (depending on the category of kind under A and the B) is substantially free of " B " (wherein " B " comprises one or more contaminating protein matter, dna molecular, carrier etc.).Preferably, in compositions, " A " comprises the A+B of about at least 90% (weight), most preferably, and about at least 99% (weight).

Term as used herein " treatment effective dose " be meant enough be reduced by at least about 15%, preferably about at least 50%, more preferably about at least 90% and most preferably prevent the obvious clinically insufficient amount of host's activity, function and reaction aspect.In addition, the treatment effective dose enough causes host's obviously improvement of disease clinically.In the context of the present invention, the host response aspect can not proved by lasting or the infection that spreads germs completely.The minimizing of the host's obviously improvement of disease removing from move living host cell of minimizing, antibacterial of comprising antibacterial load clinically, the fever relevant or inflammation or the minimizing of relevant any symptom with bacterial infection with infection.

According to the present invention, can pass through non-intestinal, see through mucosa, for example through port, nose, lung or rectum, the perhaps composition of transdermal introducing therapeutic combination of the present invention.Preferably, route of administration is non-intestinal, for example by intravenous injection and also include, but are not limited in intra-arterial, intramuscular, intradermal, subcutaneous, intraperitoneal, the ventricle and the intracranial administration.Preferably through port or lung are sent and are activated mucosal immunity; Because moving usually, streptococcus pneumoniae gives birth in nasopharynx and lung mucosa, so mucosal immunity may be effective especially prophylactic treatment.When in therapeutic combination of the present invention, using, term " unit dose " is meant the suitable physically discrete unit that is used for the people as single dose, constituent parts is included as the active substance and the needed diluent of the scheduled volume that produces required therapeutic effect and calculate, i.e. carrier.

In another embodiment, described reactive compound can particularly send in the liposome at vesicle (referring to Langer, science 249:1527-1533 (1990); People such as Treat, liposome in infectious disease and treatment for cancer, Lopez-Berestein and Fidler (editor), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid, pp.317-327; Referring to above).

In another embodiment, described treatment chemical compound can be sent in controlled release system.For example, can utilize venoclysis, implantable osmotic pumps, percutaneous plaster, liposome or other administering mode to give described polypeptide.In one embodiment, can use pump (referring to Langer, as mentioned above; Sefton, CRC Crit.Ref.Biomed.Eng.14:201 (1987); People such as Buchwald, surgery 88:507 (1980); People such as Saudek, N.Engl.J.Med.321:574 (1989)).In another embodiment, can use macromolecular material (referring to the medical application of controlled release, Langer and Wise (editor), CRC publishing house, Boca Raton, Florida (1974); The bioavailability of controlled release drug, medicine design and performance, Smolen and Ball (editor), Wiley, New York (1984); Ranger and Peppas, J.Macromol.Sci.Rev.Macromol.Chem.23:61 (1983); Also referring to people such as Levy, science 228:190 (1985); People such as During, Ann.Neurol.25:351 (1989); People such as Howard, J.Neurosurg.71:105 (1989)).In another embodiment, controlled release system can be placed on the treatment target, promptly brain near, therefore only need the described system of fraction dosage (for example referring to Goodson, the medical application of controlled release, as mentioned above, vol.2, pp.115-138 (1984)).Preferably, controlled-release device is introduced treatment target be not suitable for immune activation position or tumor near.Other controlled release system (science 249:1527-1533 (1990)) has been discussed in the comment of Langer.

Preferably, be the people to give above-mentioned active component as the treatment target of the effective therapeutic scheme of bacterial infection, but also can be any animal.Therefore, those of ordinary skill in the art can both understand rapidly, the method of the invention and pharmaceutical composition are particularly suitable for giving any animal, mammal and include, but are not limited to domestic animal such as cat or dog particularly, farm-animals as but be not restricted to cattle, horse, goat, sheep and pig, wild animal (no matter be open-air or at the zoo in), zoologize as mice, rat, rabbit, goat, sheep, pig, Canis familiaris L., cat etc., promptly use for the veterinary.

In Treatment and composition for of the present invention, provide the active component of treatment effective dose.The treatment effective dose can determine that according to patient's characteristics (age, body weight, sex, situation, complication, other disease etc.) this is to well known to a person skilled in the art method by the general medical science worker.In addition, along with carrying out other conventional study, the data more specifically that the various patient's various diseases appropriate dosage levels of relevant treatment will occur, and according to the treatment situation, by the age of therapist and general health situation, those of ordinary skill can be determined the dosage that suits.Usually, for intravenous injection or infusion, dosage can be lower than intraperitoneal, intramuscular or other route of administration.The drug dosage schedule table can be dependent on circulating half-life and employed preparation and changes.The described compositions of treatment effective dose is given in the mode compatible with dosage particles.The accurate amount of the required active component that gives depends on doctor's judgement and is that each individuality is peculiar.Yet appropriate dosage can be approximately per kilogram of body weight 0.1-20 every day, preferably approximately is 0.5-10 and more preferably a few microgram active components of 1-and depend on route of administration.The suitable first administration and the scheme of booster injection also are changeable, but it is characterized in that after first administration, one hour or many hours at interval, and by injection or other route of administration give repeatedly to measure subsequently.Perhaps, the method that can consider continuous intravenous infusion is enough to keep the micromolar concentration of 10 nanomoles-10 in the blood.

Give with other chemical compound: with regard to the treatment of bacterial infection, can give active component of the present invention with one or more pharmaceutical compositions that is used for the treatment of bacterial infection, described pharmaceutical composition includes, but are not limited to (1) antibiotic; (2) the soluble-carbohydrate inhibitor of bacterial adhesion element; (3) other micromolecular inhibitor of bacterial adhesion element; (4) inhibitor of bacterial metabolism, transhipment or conversion; (5) the molten born of the same parents' of antibacterial stimulus object or (6) are at antibacterial antibody on other bacterial antigens or vaccine.Other effective active component comprises antiinflammatory such as steroidal or nonsteroidal anti-inflammatory drug.Administration (giving of active component for example of the present invention and antibiotic cocktail) simultaneously, perhaps administration successively.

Therefore, in specific embodiment, described therapeutic combination can further comprise active component and one or more following active components for the treatment of effective dose: antibiotic, steroid etc.Examples of formulations is as follows:

Preparation

Iv formulation I

Component mg/ml

Cefotaxime 250.0

Polypeptide 10.0

Glucose USP 45.0

Sodium sulfite USP 3.2

Disodiumedetate USP 0.1

Water for injection adds to 1.0ml iv formulation II in right amount

Component mg/ml

Ampicillin 250.0

Polypeptide 10.0

Sodium sulfite USP 3.2

Disodiumedetate USP 0.1

Water for injection adds to 1.0ml iv formulation III in right amount

Component mg/ml

Gentamycin (sulfate) 40.0

Polypeptide 10.0

Sodium sulfite USP 3.2

Disodiumedetate USP 0.1

Water for injection adds to 1.0ml iv formulation IV in right amount

Component mg/ml

Polypeptide 10.0

Glucose USP 45.0

Sodium sulfite USP 3.2

Disodiumedetate USP 0.1

Water for injection adds to 1.0ml iv formulation V in right amount

Component mg/ml

Polypeptide antagonist 5.0

Sodium sulfite USP 3.2

Disodiumedetate USP 0.1

Water for injection adds to 1.0ml in right amount

Therefore, in the particular instance that requires to reduce or suppress the infection that the antibodies by the antibacterial of bacteria mediated and host cell or its antibody or its part or this part causes, introduce the interaction that polypeptide is blocked antibacterial and host cell.

This paper also considers to send the polypeptide with activity of lectin and performance adhesin inhibitor of the present invention (or derivatives thereof) effect of the present invention by lung.Adhesin inhibitor (or derivatives thereof) is delivered in the mammiferous lung, and in lung, it can disturb antibacterial, and promptly streptococcus and preferred streptococcus pneumoniae combine with host cell.Found other report [people such as Adjei, drug research, 7:565-569 (1990) about the protein formulation sent by lung in this area; People such as Adjei, International Journal of Pharmaceutics, 63:135-144 (1990) (leuprorelin acetate); People such as Braquet, Journal of Cardiovascular Pharmacology, 13 (suppl.5); 143-146 (1989) (endothelin-1); People such as Hubbard, Annals of InternalMedicine, Vol.III, pp.206-212 (1989) (alpha1-antitrypsin); People such as Smith, J.Clin.Invest.84:1145-1146 (1989) (α 1-protease); People such as Oswein, " proteinic aerosolization effect ", Proceedings of Symposium on Respiratory Drug DeliveryII, Keystone, Colorado, March, (1990) (recombinant human somatropin); People such as Debs, J.Immunol.140:3482-3488 (1988) (interferon-and tumor necrosis factor); People such as Platz, United States Patent (USP) 5,284,656 (granulocyte colony-stimulating factors)].In the United States Patent (USP) 5,451,569 of nineteen ninety-five JIUYUE promulgations on the 19th, the method and composition that is used for by the lung delivering drugs has been described to people such as Wong.

All such devices all require to use the preparation that is suitable for distributing adhesin inhibitor (or derivatives thereof).Typically, each preparation is specific for employed type of device and employed common diluent, adjuvant and/or the carrier, can comprises and use suitable propellant material in treatment.Also can consider to use the carrier of liposome, microcapsule or microsphere, inclusion complex or other type.Depend on the type of chemical modification or the type of institute's operative installations, also can prepare in different preparations by the adhesin inhibitor that chemical method is modified.

Typically, be applicable to that the preparation that utilizes injecting type or ultrasonic type aerosol apparatus to use will comprise concentration with the about 0.1-25mg biological activity of every ml solution adhesin inhibitor and be dissolved in adhesin inhibitor (or derivatives thereof) in the water.Described preparation also can comprise buffer agent and monosaccharide (for example be used for stablizing the adhesin inhibitor and regulate osmotic pressure).Described nebulizer formulation also can comprise surfactant, be used to reduce or the adhesin inhibitor that causes by solution atomization when preventing to form aerosol at surface aggregation.

The preparation that utilizes the metered dose inhaler upon actuation device to use comprises powder in small, broken bits usually, and wherein said powder is included in the adhesin inhibitor (or derivatives thereof) of help low suspension in propellant of surfactant.Described propellant can be any conventional substances such as Chlorofluorocarbons (CFCs), HCFC, hydrogen fluorohydrocarbon or the hydrocarbon that is used for this purpose, comprises Arcton 11, dichlorodifluoromethane, dichloro-tetrafluoro ethanol and 1,1,1,2-tetrafluoroethane or its combination.Suitable surfactant comprises sorbitan trioleate and soybean lecithin.Oleic acid also can be used as surfactant.

The liquid aerosol formulation comprises adhesin inhibitor and dispersant in physiologically acceptable diluent.Dry powder aerosol preparation of the present invention is made up of the adhesin inhibitor and the dispersant of solid form in small, broken bits.For liquid or dry powder aerosol preparation, must be with described preparation aerosolization.That is, described preparation the liquid or solid microgranule be must be broken into and nasal passage or lung mucosa really be arrived so that guarantee the dosage of aerosolization.Term as used herein " aerosol microgranule " is used for description and is applicable to nose or lung administration, can get at the liquid or solid microgranule that reaches mucosa.Other considers that as the structure of delivery apparatus, other component and microgranule characteristics in the preparation be important.These features by the lung administration are well known in the art and the structure of the use of preparation, atomization method and delivery apparatus requires to carry out normal experiment by those of ordinary skills at the most.In specific embodiment, the dynamic diameter of middle number of described microgranule will be 5 microns or littler so that guarantee described drug microparticles arrival alveolar [Wearley, L.L., Crit.Rev.in Ther.Drug CarrierSystems 8:333 (1991)].

The inhaler and the Diskus of aerosol delivery systems such as pressurised metered dosage are disclosed in Newman, S.P., and aerosol and lung, Clarke, S.W. and Davia, D. (editor) is among the pp.197-22 and can unite use with the present invention.

In another embodiment, as hereinafter going through, aerosol formulation of the present invention can comprise other therapeutics or pharmacological activity component except the adhesin inhibitor, as includes, but are not limited to antibiotic, steroidal compounds, NSAID (non-steroidal anti-inflammatory drug) etc.

Liquid aerosol formulation: the invention provides and be used for the treatment of, for example the patient's of streptococcus, particularly pneumococcal infection aerosol formulation and dosage form by antibacterial.Common described dosage form comprises the adhesin inhibitor in pharmaceutically acceptable diluent.Pharmaceutically acceptable diluent includes, but are not limited to sterilized water, saline, buffered saline, glucose solution etc.In specific embodiments, the diluent that can in the present invention or pharmaceutical preparation of the present invention, use be the saline of phosphate-buffered or usually pH at the buffered saline solution or the water of 7.0-8.0 scope.

Liquid aerosol formulation of the present invention can not comprise or comprise pharmaceutically acceptable steroidal, diluent, solubilizing agent or emulsifying agent, surfactant and excipient.Described preparation can comprise carrier.Described carrier is to may be dissolved in the blood circulation and is physiologically acceptable macromole, wherein physiology can accept to be meant that those skilled in the art allow described carrier is injected to the patient as the part of therapeutic scheme, preferably, described carrier is metastable in blood circulation, has the suitable plasma clearance half-life.Described macromole includes, but are not limited to soybean lecithin, oleic acid and sorbitan trioleate, preferred sorbitan trioleate.

The preparation of the present embodiment also can comprise and is used to other medicament of keeping pH, stabilizing solution or being used to regulate osmotic pressure.The example of described medicament includes, but are not limited to salt such as sodium chloride or potassium chloride and carbohydrate such as glucose, galactose or mannose etc.

The present invention further considers the liquid aerosol formulation that comprises adhesin inhibitor and another kind of treatment active drug such as antibiotic, steroidal compounds, NSAID (non-steroidal anti-inflammatory drug) etc.

Aerosol dry powder formulations: also consider aerosol formulation of the present invention is prepared into the adhesin inhibitor that comprises powder form in small, broken bits and the dry powder formulations of dispersant.

The preparation that distributes by the powder inhalator device will comprise the dry powder in small, broken bits that contains adhesin inhibitor (or derivatives thereof) and also can comprise promote powder from device dispersion amount as accounting for the filler of weight of formulation 50-90%, as lactose, Sorbitol, sucrose or mannitol.The most advantageously, adhesin inhibitor (or derivatives thereof) should be prepared into mean diameter 10mm (or micron), most preferably the particulate form of 0.5-5mm is so that be delivered to the far-end lung most effectively.In another embodiment, described dry powder formulations can comprise the dry powder in small, broken bits that contains adhesin inhibitor, dispersant and filler.The filler of uniting use with preparation of the present invention can comprise lactose, Sorbitol, sucrose or the mannitol that promotes powder dispersion amount from device.

The present invention further considers the dry powder formulations that comprises adhesin inhibitor and another kind of treatment active drug such as antibiotic, steroidal compounds, NSAID (non-steroidal anti-inflammatory drug) etc.

The related oral administration solid dosage form of this paper is described in Remington ' sPharmaceutical Science usually, 18th Ed.1990 (Mark Publishing Co.Easton PA18042) the 18th chapter, and it is for reference to be introduced into this paper.The solid dosage form comprises tablet, capsule, pill, lozenge or dragee, cachet or piller.Liposome or proteinoid encapsulation also can be used to prepare the preparation (for example United States Patent (USP) 4,925, the proteinoid microsphere of report in 673) of the present composition.Liposomes enclose can be used and described liposome can be with various polymer-derived (for example United States Patent (USP) 5,013,556).Marshall, K. be at Modern Pharmaceutics the 10th chapter of being edited by G.S.Banker and C.T.Rhodes, described the possible solid dosage form of therapeutic agent in 1979, and it is for reference to be introduced into this paper.Usually, described preparation will comprise described component (or its form of modifying) by chemical method and be used for avoiding gastric environment to destroy and permission in the inert component of intestinal release of bioactive substances.

Especially, this paper also considers the oral dosage form of above-mentioned compositions derived therefrom.Described component can be modified by chemical method and be made that the described derivant of oral delivery is effective.Usually, described chemical modification is that at least one part is connected on the described component molecule, the hydrolysis of wherein said part possibility (a) Profilin; (b) take in the blood flow from stomach or intestinal.Also expectation increases the stability in the large of described component and increases the body circulation time.The example of described part comprises: the copolymer of Polyethylene Glycol, ethylene glycol and propylene glycol, carboxymethyl cellulose, glucosan, polyvinyl alcohol, polyvinylpyrrolidone and polyproline.Abuchowski and Davis, 1981, " soluble polymer-enzyme adduct " In:Enzymes as Drugs, Hocenberg and Roberts, editor, Wiley-Interscience, New York, NY, pp.367-383; People such as Newmark, 1982, J.Appl.Biochem.4:185-189.Spendable other polymer is poly--1,3-dioxolanes and poly--1,3,6-tioxocane.As mentioned above, medicinal preferably polyalkylene glycol moiety.

For described component (or derivatives thereof), discharging the position can be stomach, small intestinal (duodenum, jejunum or ileum) or large intestine.Those skilled in the art can obtain not dissolve under one's belt and discharge the preparation of described material at duodenum or other position of intestinal.Preferably, described release will be avoided the illeffects of gastric environment by protected protein matter (or derivatives thereof) or by bioactive substance being discharged into gastric environment in addition as in the intestinal.

In order to ensure obtaining sufficient gastric tolerability, the coating that can not be penetrated in the pH5.0 environment at least is essential.The example that is used as the more common inert component of casing is Cellulose acetotrimellitate (CAT), Hydroxypropyl Methylcellulose Phathalate (HPMCP), HPMCP 50, HPMCP55, poly-acetic acid O-phthalic vinyl acetate (PVAP), Eudragit L30D, Aquateric, Cellacefate (CAP), Eudragit L, Eudragit S and Lac.These coatings can use with the form of hybrid films.

Coating or coating mixture also can be used for being not ready for prevention by the destructive tablet of gastric environment.This can comprise sugar-coat or the coating that tablet is swallowed easily.Capsule can be by being used to send dried therapeutic agent, and promptly the duricrust of dry powder (as gelatin) is formed; For the therapeutic agent of liquid form, can use soft gelatin shell.The shell material of cachet can be thick starch or other edible paper.For the triturate of pill, lozenge, molded tablet or tablet, can use wet granulation technique.

Peptide therapeutics can be included in the preparation of being made up of tiny multiparticle with the piller form that granule or particle diameter are approximately 1mm.The preparation of the described material by the capsule administration also can be the filler of powder, compacting gently or even can be tablet.Can prepare therapeutic agent by pressing.

Also can comprise coloring agent and flavoring agent.For example, protein (or derivatives thereof) can be prepared into preparation (as by liposome or microsphere encapsulation), further be included in edible product then, as contain in the chilled beverage of coloring agent and flavoring agent.

Can dilute or increase the volume of therapeutic agent with inert material.These diluent can comprise the glucosan and the starch of carbohydrate, particularly mannitol, alpha-lactose, Lactis Anhydrous, cellulose, sucrose, modification.Some inorganic salt also can comprise calcium triphosphate, magnesium carbonate and sodium chloride as filler.Some diluent that can obtain by the commercial channel are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

In the therapeutic drug formulation of solid dosage form, can comprise disintegrating agent.Material as disintegrating agent includes, but are not limited to starch, comprises based on the commerce of starch disintegrating agent Explotab.Also can use sodium starch glycollate, amberlite, sodium carboxymethyl cellulose, over-expense chain starch, sodium alginate, gelatin, Fructus Citri tangerinae skin, acid carboxymethyl cellulose, natural sponge shape thing and bentonite.The disintegrating agent of another kind of form is insoluble cation exchange resin.Powdery natural gum can be used as disintegrating agent and binding agent and these can comprise that powdery natural gum is as agar, karaya or tragcanth.Alginic acid and sodium salt thereof also can be used as disintegrating agent.Binding agent can be used for therapeutic agent is bonded together and forms hard tablet and comprise material from natural product such as arabic gum, tragcanth, starch and gelatin.Other binding agent comprises methylcellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC).Polyvinylpyrrolidone (PVP) and hydroxypropyl emthylcellulose (HPMC) all can be used for therapeutic agent is granulated in alcoholic solution.

Can comprise lubricant in the preparation of therapeutic agent is used for preventing adhering in preparation preparation process.Can between therapeutic agent and mold wall, use one deck lubricant, and these lubricants include, but are not limited to stearic acid and comprise its magnesium salt and calcium salt, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oil and wax.Also can use aqueous solution lubricant such as sodium lauryl sulphate, Stepanol MG, various molecular weight polyethylene glycol, Carbowax 4000 and 6000.

Can add fluidizer, it can improve medicine mobile in preparation preparation process and help rearrangement in the tabletting process.Fluidizer can comprise starch, Pulvis Talci, burnt silicon dioxide and hydrated aluminosilicate.

In order to help therapeutic agent to be dissolved in the water environment, can add surfactant as wetting agent.Surfactant can comprise anionic detergent such as sodium lauryl sulphate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.Also can use cationic detergent and can comprise benzalkonium chloride or benzethonium chloride.Can be used as the potential nonionic detergent that surfactant is included in the preparation and comprise lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated

Oleum Ricini

10,50 and 60, glyceryl monostearate, Spheron MD 30/70 40,60,65 and 80, sucrose fatty acid ester, methylcellulose and carboxymethyl cellulose.These surfactants can be separately or form of mixtures in varing proportions be present in the preparation of protein or derivatives thereof.

The additive that for example strengthens polypeptide (or derivatives thereof) picked-up potentially is fatty acid such as oleic acid, linoleic acid plus linolenic acid.

Lung is sent: this paper also considers to send polypeptide of the present invention (or derivatives thereof) by lung.When sucking, described polypeptide (or derivatives thereof) is delivered in the mammiferous lung and covers the mucomembranous surface of alveolar.Other report of relevant this respect comprises people such as Adjei, 1990, and drug research, 7:565-569; People such as Adjei, 1990, International Journal of Pharmaceutics, 63:135-144 (leuprorelin acetate); People such as Braquet, 1989, Journal ofCardiovascular Pharmacology, 13 (suppl.5): 143-146 (endothelin-1); People such as Hubbard, 1989, Annals of Internal Medicine, Vol.III, pp.206-212 (a1-antitrypsin); People such as Smith, 1989, J.Clin.Invest.84:1145-1146 (a-1-protease); People such as Oswein, 1990, " proteinic aerosolization ", Proceedings ofSymposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human somatropin); People such as Debs, 1988, people such as J.Immunol.140:3482-3488 (interferon-g and tumor necrosis factor-alpha) and Platz, United States Patent (USP) 5,284,656 (granulocyte colony-stimulating factors).Nineteen ninety-five JIUYUE be presented in 19th in people's such as Wong the United States Patent (USP) 5,451,569 and described the method and composition that is used for by the lung delivering drugs.

Consider to use the various machinerys that are used for by lung delivering therapeutic agents product in the invention process, include, but are not limited to nebulizer, metered dose inhaler upon actuation and powder inhaler, all these is that those skilled in the art are familiar with.

Typically, be applicable to that the preparation that utilizes injecting type or ultrasonic type aerosol apparatus to use will comprise concentration with the about 0.1-25mg biological activity protein of every ml solution and be dissolved in polypeptide (or derivatives thereof) in the water.Described preparation also can comprise buffer agent and monosaccharide (for example be used for stable protein and regulate osmotic pressure).Described nebulizer formulation also can comprise surfactant, be used to reduce or the protein that causes by solution atomization when preventing to form aerosol at surface aggregation.

The preparation that utilizes the metered dose inhaler upon actuation device to use comprises powder in small, broken bits usually, and wherein said powder is included in the polypeptide (or derivatives thereof) of help low suspension in propellant of surfactant.Described propellant can be any conventional material such as Chlorofluorocarbons (CFCs), HCFC, hydrogen fluorohydrocarbon or the hydrocarbon that is used for this purpose, comprises Arcton 11, dichlorodifluoromethane, dichloro-tetrafluoro ethanol and 1,1,1,2-tetrafluoroethane or its combination.Suitable surfactant comprises sorbitan trioleate and soybean lecithin.Oleic acid also can be used as surfactant.

The preparation that is distributed by the powder inhalator device will comprise the dry powder in small, broken bits that contains polypeptide (or derivatives thereof) and also can comprise promotion powder dispersion amount from device, for example account for the filler of the 50-90% of weight of formulation, as lactose, Sorbitol, sucrose or mannitol.The most advantageously, protein (or derivatives thereof) should be prepared into mean diameter is 10mm (or micron), and most preferably the particulate form of 0.5-5mm is so that be delivered to most effectively in the far-end lung.

Nose is sent: this paper also considers to send polypeptide (or derivatives thereof) by nose or nasopharynx.Send by nose and to make that after the therapeutic agent product is administered to nose polypeptide directly by upper respiratory tract mucosa, needn't make described product be deposited in the lung.Be used for comprising the preparation that forms with glucosan or cyclodextrin by the preparation that nose is sent.

The following example is used for illustrating more fully the preferred embodiments of the invention.Yet they do not limit the present invention in any way scope.

The peptide intercepting thing of experimental detail part embodiment 1: choline binding protein A (CbpA)

Generation comprises the terminal intercepting of CbpA N-segmental polypeptide (serotype 4).With PCR primer SJ533 and SJ537 amplification total length CbpA, design primer based on the deutero--terminal amino acid sequence of CbpA polypeptide.5 ' forward primer SJ533=5 ' GGC GGA TCC ATG GA (A, G) AA (C, T) GA (A, G) GG3 '.This degenerate primer is combined with BamHI and NcoI restriction site and ATG start codon by aminoacid sequence XENEG design.3 ' reverse primer SJ537=5 ' GCC GTC GAC TTA GTT TAC CCA TTC ACCATT GGC 3 '.This primer is combined with the SalI restriction site so that clone, and is combined with the natural termination codon from CbpA, and this primer is based on 4 types and R6x sequence.

Under 50 ℃ of annealing temperatures with hi-fi enzyme (Boehringer Mannheim) and with the genomic DNA generation PCR product of 30 causes of primer SJ533 and SJ537 amplification as template.Gained PCR product with QIA fast PCR purification kit (Qiagen, Inc.) purification, then with BamHI and the digestion of SalI restriction endonuclease and be cloned into pQE30 expression vector with BamHI, XbaI and the digestion of SmaI restriction endonuclease (Qiagen, Inc.) in.Polypeptide R2:

Utilization is positioned at the intercepting form that terminal naturally occurring PvuII site (nucleic acid 1228 of 4 type sequences), second duplicate block (that is, C district shown in Figure 1) produces the CbpA gene, only contains 5 ' part of this gene.In order to produce the intercepting clone, with PvuII and XbaI digestion full-length clone PMI580 (4 type) or PMI581 (R6x), the gained fragment is connected among the expression vector PQE30 and is transformed in the suitable hosts.Express and this protein of purification.In this embodiment, the termination codon that expression vector utilized is positioned at the downstream of insert, so bigger than the predicted size of insert owing to 5 ' end at cloning site has additional nucleic acid by expressed proteins.The aminoacid sequence of polypeptide R2 has been described in SEQ IDNO 1.Polypeptide R1:

Use similar strategy to express first duplicate block in the CbpA N-stub area, i.e. the A district of polypeptide R1.Here utilized the naturally occurring XmnI site (nucleic acid 856 of 4 type sequences) between two amino duplicate blocks.With XmnI and AatII digestion CbpA full-length clone PMI580.With AatII and SmaI digested vector pQE30.The fragment that connects two models again is transformed into and also screens the clone who inserts in the escherichia coli.Select a positive colony and by this mottle purification of recombinant proteins matter.

Use escherichia coli pQE30 carrier also to express and all polypeptide of purification with Qia expression system (Qiagen).Use anti-histidine antibody and protein-specific antibody, by the proteinic aminoterminal of host and Western analyzing and testing His labelling.The purification of R1 and R2:

For induce produce and purification from colibacillary recombinant protein, single colony is selected from antibacterial that the flat board that contains recombiant plasmid cultivates and contains in the LB buffer of 50 μ g/ml kanamycin and 100 μ g/ml ampicillin overnight incubation under 37 ℃ of temperature at 6.0ml.This 6ml culture is added to 1L to have among the antibiotic LB under the above-mentioned concentration.The jolting culture is up to A600=～0.400 under 37 ℃ of temperature.Adding 1M IPTG in this 1L culture is 1mM to ultimate density.Then 37 ℃ of following jolting cultures 3-4 hour.In J-6B type centrifuge, rotated this 1L culture 15 minutes with the 4000rpm rotating speed.Abandoning supernatant is also stored ball shape precipitation under-20 ℃ of temperature.

1L ball shape precipitation is suspended in again the 50mM NaH of 25ml ₂PO ₄, 10mM Tris, 6MGuCl, 300mM NaCl, among the pH8.0 (buffer A).This mixture at room temperature rotated 30 minutes and use miniature tip (Danbury CT) went up ultrasonic twice 30 seconds totally for Sonics and Materials, Inc. at the VibraCell ultrasonic processor with the output of 50%Cuty Cycle and 7.Mixture rotated 5 minutes with the 10K rotating speed in the JA20 rotary apparatus and takes out supernatant.Supernatant is loaded into the 10ml Talon that connects with GradiFrac system (Pharmacia Biotech, Upsala, Sweden), and (Clonetech, Palo Alto is CA) on the resin column.This post is washed with 100ml buffer A balance and with this buffer of other 200ml.Cumulative volume with 100ml uses 100%50mM NaH ₂PO ₄, 8M urea, 20mM MES, pH6.0 (buffer B) comes eluting as final target buffer according to the pH gradient.With about 30% buffer B elute protein.Collect eluting peak and merging.

For refolding, use Dialysis tubing at room temperature to dialyse about 3 hours with the PBS of 2L volume with 1400 molecular weight cutoff values.Then in the PBS at 2L under 4 ℃ of temperature with the sample dialysed overnight.Use the Centriprep-30 column spinner during protein concentrates, to finish other buffer-exchanged by also rotation is next again in the remaining thing that PBS is added to rotation.Use the BCA method of protein measurement to determine protein concentration and use the painted 4-20%SDS-PAGE gel of Coomassie to show purity (Fig. 3).Embodiment 2: the activity of lectin of polypeptide R1 and R2

LNnt is a kind of similar thing of carbohydrate that is present in the streptococcus pneumoniae receptor on the eukaryotic cell.Shown that the damaged streptococcus pneumoniae mutant of CbpA can not adhere on eukaryotic cell or the immobilization sugar, shown that CbpA is the adhesion part.CbpA is a kind of module albumen, and it can be divided into terminal functional areas of two district: N-and the terminal choline binding district (Fig. 1) of C-.Whether the biologic activity of analyzing polypeptide R1 and R2 concentrates in unique N end (mimic by R2) or its fragment (mimic by R1) with the activity of determining complete CbpA.Determined whether only N-end region (R2) comprises the agglutinin binding bioactive when lacking choline binding district (CBD).Use total length CbpA and polypeptide R2 (the intercepting thing in the CBD district in the disappearance proline rich domain beyond the PvuII site) to test.

In the mensuration with the known glycoconjugate that can be discerned by CbpA: LNnT-albumin, 3 ' sialyl lactose-albumin and negative control albumin bag are organized culture hole.Seal culture plate, washing and added total length CbpA polypeptide R2 or polypeptide R1 (0.8 μ g/ml) in 15 minutes with albumin then, need not wash then, added fluorescein-labeled R6 streptococcus pneumoniae in 30 minutes, washing and visual numeration adhere to antibacterial.

The combination of R6 and carbohydrate is that positive control and demarcation are 100% (table 1) under the situation of not adding any peptide.In three were independently tested, total length CbpA or polypeptide R2 suppressed the surface combination of streptococcus pneumoniae and LNnT bag quilt competitively.Compared with the control, total length CbpA suppression ratio is 71%, 64% and 63%; Polypeptide R2 suppression ratio is 65%, 53% and 74%.The activity that is equal to of CbpA and R2 shows that the choline binding district is optional for the LNnT activity of lectin of CbpA, and R2 is candidate's a LNnT agglutinin.

With respect to the combining of LNnT, compare with total length CbpA (74 and 66%), R2 does not suppress combine (79% and 101%) of streptococcus pneumoniae and 3 ' sialyl lactose.It is active to have lost sialic identification when this shows the CBD disappearance.By contrast, as if R1 has the sialic activity of identification, enjoys CbpA characteristic but obviously covered this specific character in R2.This shows that polypeptide is folded into the influence that functional areas are subjected to this polypeptide composition and length.Discovery sequence in other bacterial strain has slight variation (see figure 2).Supposing the sequence homology that has height between R1 and the R2, further may be that to need R1 and R2 or R1 and R2 simultaneously for activity of lectin be the slightly different agglutinin of two species specificity (± sialic acid).

The soluble form of table 1CbpA is to the bonded inhibitory action of R6 streptococcus pneumoniae and purification glycoconjugate

	????LNnT		3 ' sialyl lactose
	????LNnT		3 ' sialyl lactose		The Cbp form	The streptococcus pneumoniae number (SD) of every monolayer	The % contrast	The streptococcus pneumoniae number of every monolayer	% contrasts (every hole)
No peptide	?3282 ??2421(489) ??2210(350)	????100％	??2611 ??2115(125)	????100％	The Cbp form	The streptococcus pneumoniae number (SD) of every monolayer	The % contrast	The streptococcus pneumoniae number of every monolayer	% contrasts (every hole)
No peptide	?3282 ??2421(489) ??2210(350)	????100％	??2611 ??2115(125)	????100％	Total length CbpA	?2075 ??1740(167) ??1415(50)	??63，71，64	??1933 ??1405(240)	????74 ????66
Polypeptide R2	?2461 ??1288(672) ??1440(530)	??74，53，65	??2639 ??1670(420)	????101 ????79	Total length CbpA	?2075 ??1740(167) ??1415(50)	??63，71，64	??1933 ??1405(240)	????74 ????66
Polypeptide R2	?2461 ??1288(672) ??1440(530)	??74，53，65	??2639 ??1670(420)	????101 ????79	Polypeptide R1	?3002 ??2245(182) ??2500(310)	??91，92，112	??1052 ??1445(526)	????40 ????68

N=LNnt carries out 3 tests, and each 3 hole N=sialyl lactoses carry out 2 tests, and each 3 hole activity of lectin combine active dependency with cell

The surface molecular that contains carrier's cell of carbohydrate (glycoprotein and glycolipid) and antibacterial combines with these glycoconjugates by carbohydrate, although the skeleton of protein or fat is very different.Therefore, can adhere to human cell surface at the external polypeptide that carries antibacterial with activity of lectin.This direct association between lectin activity and the cell combination is known for streptococcus pneumoniae.For example, LNnt competitive inhibition streptococcus pneumoniae and the activated A549 human pneumonocyte's of TNF combines and stops streptococcus pneumoniae development in vivo.For the activity of lectin that confirms CbpA intercepting thing can reflect cell in conjunction with activity, CbpA and intercepting thing have been tested to streptococcus pneumoniae and the bonded inhibitory action of pneumonocyte (table 2).The adhesion of total length CbpA and polypeptide R2 competitive inhibition streptococcus pneumoniae and pneumonocyte, compared with the control, its suppression ratio is respectively 58% and 63%.Polypeptide R1 is invalid, and the LNnt that shows R2 is essential in conjunction with activity for streptococcus pneumoniae and combining of pneumonocyte and has also explained this relation.

Table 2

The R6 streptococcus pneumoniae combines with the activated human pneumonocyte's of TNF

	The A549 lung
	The A549 lung		The Cbp form	The streptococcus pneumoniae number (meansigma methods) of every monolayer	The % contrast
No peptide	????697，704，674 ????702，722 ????(700)	????100％	The Cbp form		The % contrast
No peptide	????697，704，674 ????702，722 ????(700)	????100％	Total length CbpA	????376，431 ????(403)	????58％
Polypeptide R2	????517，693 ????314，342，350 ????(443)	????63％	Total length CbpA	????376，431 ????(403)	????58％
Polypeptide R2	????517，693 ????314，342，350 ????(443)	????63％	Polypeptide R1	????696，642，552 ????(630)	????90％

Each test of N=is carried out 2 times, and each 2 or 3 hole LNnT activity of lectin depend on R2

The N-end region of CbpA comprises and each has about 110 amino acid whose two duplicate blocks (seeing Fig. 1, A district among the polypeptide R2 and C district).In order to study the Relative Contribution of two districts, comparing with R2 and total length CbpA of A district will only be contained to biological activity R1.When testing in adhere to measuring, polypeptide R1 fully suppresses and the adhesion of LNnT (wild type 91,92 and 112%).Yet, polypeptide R1 demonstrate to sialyl lactose be combined with some inhibitory action (

contrast

68 and 40%).This shows that it is candidate's LNnT agglutinin district that the activity of lectin of LNnT needs polypeptide R2 and R2.By contrast, as if R1 has the sialic activity of identification.The antibody blocking cell combination of CbpA N-end region:

The N-end region of supposing CbpA is in conjunction with cell, can stop or reverse the combining of glycoconjugate of antibacterial and cell and purification to the active interference of N-end region.A kind of such interference mechanism is an antibody.

The anti-CbpA R2 of table 3 antibody wraps by the inhibitory action of surface combination R6 streptococcus pneumoniae and LNnt

	The streptococcus pneumoniae number (sD) of every monolayer	% contrasts (meansigma methods)
	The streptococcus pneumoniae number (sD) of every monolayer	% contrasts (meansigma methods)	Antibody before the immunity	????198(64)；88(4)	????100％
The antibody of R2 intercepting thing	????56(11)；9(2)	????28％；10％	Antibody before the immunity	????198(64)；88(4)	????100％

The undiluted rabbit antibody of 5 μ l+5 μ l 2 * 10 ⁷The pre-culture of R6x under the room temperature 6 * 30 minutes, is added to the LNnT bag then and is adhered to mensuration in the hole.Shown twice independently experiment.

Tested the ability that the antiserum blocking-up streptococcus pneumoniae that results from CbpA reorganization N-end region (R2) adheres to LNnT.At room temperature cultivate anti-CbpA antiserum of rabbit polyclonal (5 μ l) and 5 μ l2 * 10 ⁷The antibacterial of labelling 30 minutes.Mixture was applied 30 minutes on immobilization LNnT, wash 3 times to remove unconjugated antibacterial with PBS then.Add standard deviation display result with microscope number and the bonded antibacterial of culture plate and with the meansigma methods in 6 holes.Result shown in the table 3 shows that the antiserum that produced at the R2 polypeptide blocked combining of streptococcus pneumoniae and LNnT.Fig. 5 suppresses streptococcus pneumoniae R6x and the bonded titration curve of model receptor LNnT with anti-CbpA R2 antibody before showing immunity.1: 100 and 1: 200 dilution anti-R2 surpass 70% to the adherent blocking-up rate of streptococcus pneumoniae.Further be diluted to 1: 400 and eliminated and show that this acts on specific activity.

Be used for preparation table 3 and the sero-fast CbpA of anti-CbpA shown in Figure 5 and be that CbpA at serotype 4 produces.The streptococcus pneumoniae R6x bacterial strain that uses in adhesion inhibition is measured is deutero-by serotype 2.The ability of antibody blocking heterologous serotype bacterial adhesion shows the cross protection activity between the serotype.This activity is in demand for effective vaccine immunogens.The activity of the terminal native conformation antibody of CbpA N-:

Press the described methods of people such as Rosenow, can be by its natural host (streptococcus pneumoniae) purification CbpA on the choline affinity column.In addition, the poly histidine mark can be connected to the end of gene so that extend the albumen of transcribing by several histidine residues.These residues are convenient to purification on the nickel affinity substrate.Opposite with short intercepting thing, the purification of full-length polypeptide helps keeping natural tertiary structure.Particularly also can keep its natural tertiary structure by these biochemical methods by the CbpA of escherichia coli or other host bacteria purification by streptococcus pneumoniae.As immunogen, the CbpA of natural folding produces antibody, and they are different from potentially by using, and the antibody that different folding intercepting thing immunity produces can take place.Similarly, can have as the CbpA of therapeutic agent and to be different from the tertiary structure that intercepts thing, described intercepting thing can improve it and block adherent ability.Considering based on these, may be favourable full length protein as allowing its natural tertiary structure that is folded into it to cut away C-terminal (CBD) production CbpA by biochemical method then.For example, handle and to separate N and C-terminal at the amino acid sites 475 cutting CbpA of serotype R6x and serotype 4 choline binding protein A with azanol.The N-terminal fragment is suitable for use as therapeutic agent or immunogen.

In addition, natural CbpA can be used as the antiserum of immunogen and active structure.Remove BD antibody by absorption and in this mixture, be rich in bioactive anti-N-terminal antibody.By under R1 with 1 * 10 ⁸CbpA defective antibacterial is cultivated 200 μ l serum together and prepared this antibody in 1 hour.Other choline binding protein on this mutant adsorbs anti-CBD antibody, then by centrifugal and remove to degerm and shift out described antibody from antiserum.

For the biological activity of the anti-CbpA antibody that proves absorption, the antiserum blocking-up streptococcus pneumoniae of determining absorption adheres to the ability on the model receptor LNnT.Cultivating the R6x streptococcus pneumoniae with the antiserum of dilution in 1: 600 is added in the hole of using LNnT albumin bag quilt then.

Table 4

The anti-CbpA antiserum blocking-up of absorption adheres to

Antiserum (1: 600)	Pneumococcal number ± the SD in every hole (contrast %)
Antiserum (1: 600)	Pneumococcal number ± the SD in every hole (contrast %)	No antibody	????563±11(100％)
Antiserum before the immunity	????479±11(85％)	No antibody	????563±11(100％)
Antiserum before the immunity	????479±11(85％)	Anti-CbpA antiserum	????294±72(52％)
Remove the anti-CbpA antiserum of CBD antibody through absorption	????175±38(31％)	Anti-CbpA antiserum	????294±72(52％)

These results show that the antibody of the Cbp/AN stub area of its native conformation blocks adhesion strongly.This activity is greater than the activity of intercepting thing among Fig. 5, and the intercepting thing does not have activity when 1: 600 dilution factor.Titration research among Fig. 5 further shows the sero-fast this activity of anti-CbpA of absorption.Show that by triangle streptococcus pneumoniae 4 types and LNnT wrap adherent by the hole baseline.Cultivating streptococcus pneumoniae in advance with the antiserums of various dilution absorption (square) or absorption (rhombus) shows to make and adhere to reduce.Two kinds of antiserums show that in the fact that adheres to similar minimizing the blocking-up activity of the antibody (that is, removing the antibody in choline binding district by absorption) of the terminal CbpA residue of most of N-does not reduce biological activity.Embodiment 3: the generation of the sero-fast passive protection rabbit of anti-R2 immune serum:

(Denver PA) has produced the rabbit immune serum of anti-polypeptide R2 (CbpA intercepts thing) and CbpA at Covance.After collecting preimmune serum, make the New Zealand white rabbit immunity with the R2 that contains amino terminal duplicate block (above preparing 483: 58) of 250 μ g in complete Freund's adjuvant.Strengthened for these rabbit 125 μ g R2 in incomplete Freund's adjuvant at the 21st day and blood drawing in the 31st day.Second rabbit carries out similar immunity with the CbpA of purification.The passive protection of mice

The anti-R2 of rabbit in aseptic PBS of peritoneal injection 100 μ l 1: 2 dilution or preimmune serum (before the immunity and the 31st day immune serum) make C3H/Hej mice (5/group) passive immunity.Give serum after 1 hour, attack mice with 1600CFU streptococcus pneumoniae serotype 6B (bacterial strain SP317).14 days survival condition of monitoring mice.Use the rabbit immune serum mice immunized that produces at polypeptide R2 that 80% survival (Fig. 4) is arranged after attack.Rabbit anteserum mice immunized to all death in 7 days before the useful immunity of institute.

These data show that the antibody that is specific to CbpA has protective effect to system's pneumococcal infection.These data show that further the choline binding district is optional for protection, are enough because be specific to the antibody (the choline binding duplicate block that disappearance is conservative) of the protein and peptide R2 of intercepting for protection.In addition, at the serum of CbpA serotype 4 attack of serotype 6B there is protective effect.Embodiment 4: the sero-fast automatic protective effect of anti-R1

Peritoneal injection CbpA intercepting protein R1 (15 μ g add 50 μ l complete Freund's adjuvants in 50 μ l PBS) makes C3H/HeJ mouse (10/group) immunity.One group of 10 false mice immunized is accepted PBS and adjuvant.Immunity for the second time gave after 4 weeks, and peritoneal injection 15 μ g have the protein (false immunity accept PBS and IFA) of incomplete Freund's adjuvant.Extract blood (posterior orbit is got blood) in the 3rd, 6 and 9 weeks and carry out the immunoreation analysis.The anti-CbpA intercepting of the ELISA terminal point thing titre of the serum of collecting from 10 CbpA mice immunized when 9 weeks is 4,096,000.In the serum of false immune mouse, do not detect antibody.Attack mice in the 10th week with 560CFU streptococcus pneumoniae serotype 6B (bacterial strain SPSJ2p, by P.Flynn, St.Jude Children ' sResearch Hospital, Memphis, TN provides).In 14 days, detect the survival condition of mice.With CbpA intercepting protein R1 mice immunized 80% survival is arranged after attack.All false mice immunized were death (Fig. 7) in the 8th day.

These data show with the generation of CbpA recombinant fragment immune induction can resist systemic pneumococcal infection and dead specific antibody.These data show that further the choline binding district is optional for protective effect, because immunogen is the protein R1 of intercepting.In addition, the result hints that an aminoterminal duplicate block may be enough to induce protective reaction.Also shown cross-protection, because produced reorganization pneumoprotein matter, with having observed protective effect after the attack of serotype 6B separator based on serotype 4 DNA sequence.The nasopharynx bacterium colony cluster of embodiment 5 prevention rats childhood

External, the N-terminal district competitive inhibition streptococcus pneumoniae of CbpA adheres to.In order to prove treatment practicality with this active peptide, give the peptide of rat intercepting childhood, attack and estimate the bacterium colony cluster situation of nasopharynx then with streptococcus pneumoniae.

Contain 0.8 μ g polypeptide R2 or R1 or nonprotein PBS via intranasal application with 10 μ l and handle rat.After 15 minutes, via intranasal application is introduced 3 type streptococcus pneumoniae (bacterial strain SIII), and (10 μ l contain 1 * 10 ⁵Cfer).In order to determine the adhesion of polypeptide competitive inhibition streptococcus pneumoniae and to move living ability, the pneumococcal number that in the time of 72 hours, reclaims in washing nasal cavity and definite each animal, every group of 4 animals.The per 10 μ l of rat that only accept SIII have the individual bacterium colony in 2200,6500,6900 and 8700 (average 6075).The animal of handling with intercepting thing R2 shows maximum minimizing (3600,3500,2500,2100), and average 10 μ l have 2925 antibacterials (contrast 48%).The animal of handling with intercepting thing R1 shows that also the bacterium colony cluster reduces (5000,4800,3500,1600), average out to 3725 (contrast 61%).

This experiment shows that giving streptococcus pneumoniae that specified animal can make this animal resist subsequently in treatment research with peptide of the present invention attacks.Discuss:

Show polypeptide R2:1 by experiment) induce during as the vaccine antigen administration and produce protection antibody and be a kind of preferred composition that is used for bacterin preparation; With 2) as delivery of peptides when respiratory tract and/or nasopharynx are attacked in the body, prevent streptococcus pneumoniae to adhere to competitively and be a kind of preferred composition that moves the effect of giving birth to or affecting conditions control agent that is used to resist.And CbpA intercepting thing plays the agglutinin effect and does not have CBD.Discern two kinds of carbohydrate: LNnT, by the peptide identification and the sialic acid that contain two terminal repetition districts of N-(A and C) among Fig. 1, by the peptide identification that only contains single N-least significant end duplicate block (A).The intercepting thing that contains the terminal repetition district of N-polypeptide R1 and R2 shows that also activity of lectin is arranged in cell culture is measured.

The active key character of polypeptide R2 comprises: 1) polypeptide R2 has relevant fully biological activity with total length CbpA in identification purification glycoconjugate receptor analogs, pneumonocyte and animal model.Antibody for them has also proved dependency; With 2) 4 type derivating agents and in external test, use between other serotype antibacterial of (as, 6B and 2) and have cross-protection, this is important for operable vaccine, prevention and therapeutic modality.

Although this paper, is appreciated that the present invention by mentioning various concrete materials, method and embodiment and describe and the present invention being described and is not limited to the particular combinations of material and method selected for this purpose.One skilled in the art will recognize that the various variations that can imply some details.Similarly, any list of references of quoting in this article from the degree of correlation with the disclosure of invention all be considered be incorporated herein for reference.

Sequence table＜110〉Tuomanen, Elaine I.

Wizemann，Theresa

Masure，H.R.

Johnson，Leslie?S.

Koenig, Scott＜120〉comprise the amino acid whose polypeptide of the terminal intercepting of choline binding protein AN-thing,

By this polypeptide derivative vaccine and application＜130 thereof〉1340-1-017 msc＜140〉09/056,019＜141〉1998-04-07＜160〉39＜170〉PatentIn Ver.2.0＜210〉1＜211〉406＜212〉PRT＜213〉streptococcus pneumonia＜400〉1Glu Asn Glu Gly Ala Thr Gln Val Pro Thr Ser Ser Asn Arg Ala Asn 15 10 15Glu Ser Gln Ala Glu Gln Gly Glu Gln Pro Lys Lys Leu Asp Ser Glu

20??????????????????25??????????????????30Arg?Asp?Lys?Ala?Arg?Lys?Glu?Val?Glu?Glu?Tyr?Val?Lys?Lys?Ile?Val

35??????????????????40??????????????????45Gly?Glu?Ser?Tyr?Ala?Lys?Ser?Thr?Lys?Lys?Arg?His?Thr?Ile?Thr?Val

50??????????????????55??????????????????60Ala?Leu?Val?Asn?Glu?Leu?Asn?Asn?Ile?Lys?Asn?Glu?Tyr?Leu?Asn?Lys?65??????????????????70??????????????????75??????????????????80Ile?Val?Glu?Ser?Thr?Ser?Glu?Ser?Gln?Leu?Gln?Ile?Leu?Met?Met?Glu

85??????????????????90??????????????????95Ser?Arg?Ser?Lys?Val?Asp?Glu?Ala?Val?Ser?Lys?Phe?Glu?Lys?Asp?Ser

100?????????????????105?????????????????110Ser?Ser?Ser?Ser?Ser?Ser?Asp?Ser?Ser?Thr?Lys?Pro?Glu?Ala?Ser?Asp

115?????????????????120?????????????????125Thr?Ala?Lys?Pro?Asn?Lys?Pro?Thr?Glu?Pro?Gly?Glu?Lys?Val?Ala?Glu

130?????????????????135?????????????????140Ala?Lys?Lys?Lys?Val?Glu?Glu?Ala?Glu?Lys?Lys?Ala?Lys?Asp?Gln?Lys145?????????????????150?????????????????155?????????????????160Glu?Glu?Asp?Arg?Arg?Asn?Tyr?Pro?Thr?Ile?Thr?Tyr?Lys?Thr?Leu?Glu

165?????????????????170?????????????????175Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val?Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu

180?????????????????185?????????????????190Leu?Val?Lys?Val?Lys?Ala?Asn?Glu?Pro?Arg?Asp?Glu?Gln?Lys?Ile?Lys

195?????????????????200?????????????????205Gln?Ala?Glu?Ala?Glu?Val?Glu?Ser?Lys?Gln?Ala?Glu?Ala?Thr?Arg?Leu

210?????????????????215?????????????????220Lys?Lys?Ile?Lys?Thr?Asp?Arg?Glu?Glu?Ala?Glu?Glu?Glu?Ala?Lys?Arg225?????????????????230?????????????????235?????????????????240Arg?Ala?Asp?Ala?Lys?Glu?Gln?Gly?Lys?Pro?Lys?Gly?Arg?Ala?Lys?Arg

245?????????????????250?????????????????255Gly?Val?Pro?Gly?Glu?Leu?Ala?Thr?Pro?Asp?Lys?Lys?Glu?Asn?Asp?Ala

260?????????????????265?????????????????270Lys?Ser?Ser?Asp?Ser?Ser?Val?Gly?Glu?Glu?Thr?Leu?Pro?Ser?Pro?Ser

275?????????????????280?????????????????285Leu?Lys?Pro?Glu?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu

290?????????????????295?????????????????300Ala?Lys?Lys?Lys?Ala?Glu?Asp?Gln?Lys?Glu?Glu?Asp?Arg?Arg?Asn?Tyr305?????????????????310?????????????????315?????????????????320Pro?Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp

325?????????????????330?????????????????335Val?Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys

340?????????????????345?????????????????350Glu?Pro?Arg?Asn?Glu?Glu?Lys?Val?Lys?Gln?Ala?Lys?Ala?Glu?Val?Glu

355?????????????????360?????????????????365Ser?Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Lys?Ile?Lys?Thr?Asp?Arg

370?????????????????375?????????????????380Lys?Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala?Ala?Glu?Glu?Asp?Lys385?????????????????390?????????????????395?????????????????400Val?Lys?Glu?Lys?Pro?Ala

405＜210〉2＜211〉655＜212〉PRT＜213〉streptococcus pneumoniae＜400〉2Glu Asn Glu Gly Ala Thr Gln Val Pro Thr Ser Ser Asn Arg Ala Asn, 15 10 15Glu Ser Gln Ala Glu Gln Gly Glu Gln Pro Lys Lys Leu Asp Ser Glu

370?????????????????375?????????????????380Lys?Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala?Ala?Glu?Glu?Asp?Lys385?????????????????390?????????????????395?????????????????400Val?Lys?Glu?Lys?Pro?Ala?Glu?Gln?Pro?Gln?Pro?Ala?Pro?Ala?Pro?Lys

405?????????????????410?????????????????415Ala?Glu?Lys?Pro?Ala?Pro?Ala?Pro?Lys?Pro?Glu?Asn?Pro?Ala?Glu?Gln

420?????????????????425?????????????????430Pro?Lys?Ala?Glu?Lys?Pro?Ala?Asp?Gln?Gln?Ala?Glu?Glu?Asp?Tyr?Ala

435?????????????????440?????????????????445Arg?Arg?Ser?Glu?Glu?Glu?Tyr?Asn?Arg?Leu?Thr?Gln?Gln?Gln?Pro?Pro

450?????????????????455?????????????????460Lys?Thr?Glu?Lys?Pro?Ala?Gln?Pro?Ser?Thr?Pro?Lys?Thr?Gly?Trp?Lys465?????????????????470?????????????????475?????????????????480Gln?Glu?Asn?Gly?Met?Trp?Tyr?Phe?Tyr?Asn?Thr?Asp?Gly?Ser?Met?Ala

485?????????????????490?????????????????495Thr?Gly?Trp?Leu?Gln?Asn?Asn?Gly?Ser?Trp?Tyr?Tyr?Leu?Asn?Ser?Asn

500?????????????????505?????????????????510Gly?Ala?Met?Ala?Thr?Gly?Trp?Leu?Gln?Asn?Asn?Gly?Ser?Trp?Tyr?Tyr

515?????????????????520?????????????????525Leu?Asn?Ala?Asn?Gly?Ser?Met?Ala?Thr?Gly?Trp?Leu?Gln?Asn?Asn?Gly

530?????????????????535?????????????????540Ser?Trp?Tyr?Tyr?Leu?Asn?Ala?Asn?Gly?Ser?Met?Ala?Thr?Gly?Trp?Leu545?????????????????550?????????????????555?????????????????560Gln?Tyr?Asn?Gly?Ser?Trp?Tyr?Tyr?Leu?Asn?Ala?Asn?Gly?Ser?Met?Ala

565?????????????????570?????????????????575Thr?Gly?Trp?Leu?Gln?Tyr?Asn?Gly?Ser?Trp?Tyr?Tyr?Leu?Asn?Ala?Asn

580?????????????????585?????????????????590Gly?Asp?Met?Ala?Thr?Gly?Trp?Val?Lys?Asp?Gly?Asp?Thr?Trp?Tyr?Tyr

595?????????????????600?????????????????605Leu?Glu?Ala?Ser?Gly?Ala?Met?Lys?Ala?Ser?Gln?Trp?Phe?Lys?Val?Ser

610?????????????????615?????????????????620Asp?Lys?Trp?Tyr?Tyr?Val?Asn?Gly?Ser?Gly?Ala?Leu?Ala?Val?Asn?Thr625?????????????????630?????????????????635?????????????????640Thr?Val?Asp?Gly?Tyr?Gly?Val?Asn?Ala?Asn?Gly?Glu?Trp?Val?Asn

645 650 655＜210〉3＜211〉284＜212〉PRT＜213〉streptococcus pneumoniae＜400〉3Glu Asn Glu Gly Ala Thr Gln Val Pro Thr Ser Ser Asn Arg Ala Asn, 15 10 15Glu Ser Gln Ala Glu Gln Gly Glu Gln Pro Lys Lys Leu Asp Ser Glu

260?????????????????265?????????????????270Lys?Ser?Ser?Asp?Ser?Ser?Val?Gly?Glu?Glu?Thr?Leu

275 280＜210〉4＜211〉106＜212〉PRT＜213〉streptococcus pneumoniae＜400〉4Lys Pro Glu Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu Ala, 15 10 15Lys Lys Lys Ala Glu Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr Pro

20??????????????????25??????????????????30Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val

35??????????????????40??????????????????45Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Glu

50??????????????????55??????????????????60Pro?Arg?Asn?Glu?Glu?Lys?Val?Lys?Gln?Ala?Lys?Ala?Glu?Val?Glu?Ser?65??????????????????70??????????????????75??????????????????80Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Lys?Ile?Lys?Thr?Asp?Arg?Lys

85???????????????????90??????????????????????95Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala

100 105＜210〉5＜211〉109＜212〉PRT＜213〉streptococcus pneumoniae＜400〉5Thr Glu Pro Gly Glu Lys Val Ala Glu Ala Lys Lys Lys Val Glu Glu, 15 10 15Ala Glu Lys Lys Ala Lys Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr

20??????????????????25??????????????????30Pro?Thr?Ile?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp

35??????????????????40??????????????????45Val?Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Val?Lys?Ala?Asn

50??????????????????55??????????????????60Glu?Pro?Arg?Asp?Glu?Gln?Lys?Ile?Lys?Gln?Ala?Glu?Ala?Glu?Val?Glu?65??????????????????70??????????????????75??????????????????80Ser?Lys?Gln?Ala?Glu?Ala?Thr?Arg?Leu?Lys?Lys?Ile?Lys?Thr?Asp?Arg

85??????????????????90??????????????????95Glu?Glu?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Arg?Ala?ASp?Ala

100 105＜210〉6＜211〉4＜212〉PRT＜213〉streptococcus pneumonia＜400〉6Lys Xaa Xaa Glu 1＜210〉7＜211〉376＜212〉PRT＜213〉streptococcus pneumonia＜400〉7Glu Asn Glu Gly Ser Thr Gln Ala Ala Thr Ser Ser Asn Met Ala Lys 15 10 15Thr Glu His Arg Lys Ala Ala Lys Gln Val Val Asp Glu Tyr Ile Glu

20??????????????????25??????????????????30Lys?Met?Leu?Arg?Glu?Ile?Gln?Leu?Asp?Arg?Arg?Lys?His?Thr?Gln?Asn

35??????????????????40??????????????????45Val?Ala?Leu?Asn?Ile?Lys?Leu?Ser?Ala?Ile?Lys?Thr?Lys?Tyr?Leu?Arg

50??????????????????55??????????????????60Glu?Leu?Asn?Val?Leu?Glu?Glu?Lys?Ser?Lys?Asp?Glu?Leu?Pro?Ser?Glu?65??????????????????70??????????????????75??????????????????80Ile?Lys?Ala?Lys?Leu?Asp?Ala?Ala?Phe?Glu?Lys?Phe?Lys?Lys?Asp?Thr

85??????????????????90??????????????????95Leu?Lys?Pro?Gly?Glu?Lys?Val?Ala?Glu?Ala?Lys?Lys?Lys?Val?Glu?Glu

100?????????????????105?????????????????110Ala?Lys?Lys?Lys?Ala?Glu?Asp?Gln?Lys?Glu?Glu?Asp?Arg?Arg?Asn?Tyr

115?????????????????120?????????????????125Pro?Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Phe?Asp

130?????????????????135?????????????????140Val?Lys?Val?Lys?Glu?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys145?????????????????150?????????????????155?????????????????160Glu?Ser?Arg?Asn?Glu?Gly?Thr?Ile?Lys?Gln?Ala?Lys?Glu?Lys?Val?Glu

165?????????????????170?????????????????175Ser?Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Asn?Ile?Lys?Thr?Asp?Arg

180?????????????????185?????????????????190Lys?Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala?Asp?Ala?Lys?Leu?Lys

195?????????????????200?????????????????205Glu?Ala?Asn?Val?Ala?Thr?Ser?Asp?Gln?Gly?Lys?Pro?Lys?Gly?Arg?Ala

210?????????????????215?????????????????220Lys?Arg?Gly?Val?Pro?Gly?Glu?Leu?Ala?Thr?Pro?Asp?Lys?Lys?Glu?Asn225?????????????????230?????????????????235?????????????????240Asp?Ala?Lys?Ser?Ser?Asp?Ser?Ser?Val?Gly?Glu?Glu?Thr?Leu?Pro?Ser

245?????????????????????250?????????????????255Ser?Ser?Leu?Lys?Ser?Gly?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val

260?????????????????265?????????????????270Glu?Glu?Ala?Glu?Lys?Lys?Ala?Lys?Asp?Gln?Lys?Glu?Glu?Asp?Arg?Arg

275?????????????????280?????????????????285Asn?Tyr?Pro?Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Asp?Leu?Glu?Ile?Ala?Glu

290?????????????????295?????????????????300Ser?Asp?Val?Lys?Val?Lys?Glu?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu305?????????????????310?????????????????315?????????????????320Ala?Lys?Glu?Pro?Arg?Asp?Glu?Glu?Lys?Ile?Lys?Gln?Ala?Lys?Ala?Lys

325?????????????????330?????????????????335Val?Glu?Ser?Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Asn?Ile?Lys?Thr

340?????????????????345?????????????????350Asp?Arg?Lys?Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala?Ala?Glu?Glu

355?????????????????360?????????????????365Asp?Lys?Val?Lys?Glu?Lys?Pro?Ala

370 375＜210〉8＜211〉663＜212〉PRT＜213〉streptococcus pneumoniae＜400〉8Glu Asn Glu Gly Ser Thr Gln Ala Ala Thr Ser Ser Asn Met Ala Lys, 15 10 15Thr Glu His Arg Lys Ala Ala Lys Gln Val Val Asp Glu Tyr Ile Glu

50??????????????????55??????????????????60Glu?Leu?Asn?Val?Leu?Glu?Glu?Lys?Ser?Lys?Asp?Glu?Leu?Pro?Set?Glu?65??????????????????70??????????????????75??????????????????80Ile?Lys?Ala?Lys?Leu?Asp?Ala?Ala?Phe?Glu?Lys?Phe?Lys?Lys?Asp?Thr

260??????????????????265?????????????????270Glu?Glu?Ala?Glu?Lys?Lys?Ala?Lys?Asp?Gln?Lys?Glu?Glu?Asp?Arg?Arg

355?????????????????360?????????????????365Asp?Lys?Val?Lys?Glu?Lys?Pro?Ala?Glu?Gln?Pro?Gln?Pro?Ala?Pro?Ala

370?????????????????375?????????????????380Thr?Gln?Pro?Glu?Lys?Pro?Ala?Pro?Lys?Pro?Glu?Lys?Pro?Ala?Glu?Gln385?????????????????390?????????????????395?????????????????400Pro?Lys?Ala?Glu?Lys?Thr?Asp?Asp?Gln?Gln?Ala?Glu?Glu?Asp?Tyr?Ala

405?????????????????410?????????????????415Arg?Arg?Ser?Glu?Glu?Glu?Tyr?Asn?Arg?Leu?Thr?Gln?Gln?Gln?Pro?Pro

420?????????????????425?????????????????430Lys?Thr?Glu?Lys?Pro?Ala?Gln?Pro?Ser?Thr?Pro?Lys?Thr?Gly?Trp?Lys

435?????????????????440?????????????????445Gln?Glu?Asn?Gly?Met?Trp?Tyr?Phe?Tyr?Asn?Thr?Asp?Gly?Ser?Met?Ala

450?????????????????455?????????????????460Thr?Gly?Trp?Leu?Gln?Asn?Asn?Gly?Ser?Trp?Tyr?Tyr?Leu?Asn?Ala?Asn465?????????????????470?????????????????475?????????????????480Gly?Ala?Met?Ala?Thr?Gly?Trp?Leu?Gln?Asn?Asn?Gly?Ser?Trp?Tyr?Tyr

485?????????????????490?????????????????495Leu?Asn?Ala?Asn?Gly?Ser?Met?Ala?Thr?Gly?Trp?Leu?Gln?Asn?Asn?Gly

500?????????????????505?????????????????510Ser?Trp?Tyr?Tyr?Leu?Asn?Ala?Asn?Gly?Ala?Met?Ala?Thr?Gly?Trp?Leu

515?????????????????520?????????????????525Gln?Tyr?Asn?Gly?Ser?Trp?Tyr?Tyr?Leu?Asn?Ser?Asn?Gly?Ala?Met?Ala

530?????????????????535?????????????????540Thr?Gly?Trp?Leu?Gln?Tyr?Asn?Gly?Ser?Trp?Tyr?Tyr?Leu?Asn?Ala?Asn545?????????????????550?????????????????555?????????????????560Gly?Asp?Met?Ala?Thr?Gly?Trp?Leu?Gln?Asn?Asn?Gly?Ser?Trp?Tyr?Tyr

565?????????????????570?????????????????575Leu?Asn?Ala?Asn?Gly?Asp?Met?Ala?Thr?Gly?Trp?Leu?Gln?Tyr?Asn?Gly

580?????????????????585?????????????????590Ser?Trp?Tyr?Tyr?Leu?Asn?Ala?Asn?Gly?Asp?Met?Ala?Thr?Gly?Trp?Val

595?????????????????600?????????????????605Lys?Asp?Gly?Asp?Thr?Trp?Tyr?Tyr?Leu?Glu?Ala?Ser?Gly?Ala?Met?Lys

610?????????????????615?????????????????620Ala?Ser?Gln?Trp?Phe?Lys?Val?Ser?Asp?Lys?Trp?Tyr?Tyr?Val?Asn?Gly625?????????????????630?????????????????635?????????????????640Ser?Gly?Ala?Leu?Ala?Val?Asn?Thr?Thr?Val?Asp?Gly?Tyr?Gly?Val?Asn

645?????????????????650?????????????????655Ala?Asn?Gly?Glu?Trp?Val?Asn

660＜210〉9＜211〉254＜212〉PRT＜213〉streptococcus pneumoniae＜400〉9Glu Asn Glu Gly Ser Thr Gln Ala Ala Thr Ser Ser Asn Met Ala Lys, 15 10 15Thr Glu His Arg Lys Ala Ala Lys Gln Val Val Asp Glu Tyr Ile Glu

210?????????????????215?????????????????220Lys?Arg?Gly?Val?Pro?Gly?Glu?Leu?Ala?Thr?Pro?Asp?Lys?Lys?Glu?Asn225?????????????????230?????????????????235?????????????????240Asp?Ala?Lys?Ser?Ser?Asp?Ser?Ser?Val?Gly?Glu?Glu?Thr?Leu

245 250＜210〉10＜211〉106＜212〉PRT＜213〉streptococcus pneumoniae＜400〉10Lys Ser Gly Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu Ala, 15 10 15Glu Lys Lys Ala Lys Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr Pro

20??????????????????25??????????????????30Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Asp?Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val

35??????????????????40??????????????????45Lys?Val?Lys?Glu?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Glu

50??????????????????55??????????????????60Pro?Arg?Asp?Glu?Glu?Lys?Ile?Lys?Gln?Ala?Lys?Ala?Lys?Val?Glu?Ser?65??????????????????70??????????????????75??????????????????80Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Asn?Ile?Lys?Thr?Asp?Arg?Lys

85??????????????????90??????????????????95Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala

100 105＜210〉11＜211〉107＜212〉PRT＜213〉streptococcus pneumoniae＜400〉11Pro Gly Glu Lys Val Ala Glu Ala Lys Lys Lys Val Glu Glu Ala Lys, 15 10 15Lys Lys Ala Glu Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr Pro Thr

20??????????????????25??????????????????30Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Phe?Asp?Val?Lys

35??????????????????40??????????????????45Val?Lys?Glu?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Glu?Ser

50??????????????????55??????????????????60Arg?Asn?Glu?Gly?Thr?Ile?Lys?Gln?Ala?Lys?Glu?Lys?Val?Glu?Ser?Lys?65??????????????????70??????????????????75??????????????????80Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Asn?Ile?Lys?Thr?Asp?Arg?Lys?Lys

85??????????????????90??????????????????95Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala?Asp?Ala

100 105 <210> 12 <211> 1219 <212> DNA <213> Streptococcus pneumoniae <400> 12 gagaacgagg gagctaccca agtacccact tcttctaata gggcaaatga aagtcaggca 60 gaacaaggag aacaacctaa aaaactcgat tcagaacgag ataaggcaag gaaagaggtc 120 gaggaatatg taaaaaaaat agtgggtgag agctatgcaa aatcaactaa aaagcgacat 180 acaattactg tagctctagt taacgagttg aacaacatta agaacgagta tttgaataaa 240 atagttgaat caacctcaga aagccaacta cagatactga tgatggagag tcgatcaaaa 300 gtagatgaag ctgtgtctaa gtttgaaaag gactcatctt cttcgtcaag ttcagactct 360 tccactaaac cggaagcttc agatacagcg aagccaaaca agccgacaga accaggagaa 420 aaggtagcag aagctaagaa gaaggttgaa gaagctgaga aaaaagccaa ggatcaaaaa 480 gaagaagatc gtcgtaacta cccaaccatt acttacaaaa cgcttgaact tgaaattgct 540 gagtccgatg tggaagttaa aaaagcggag cttgaactag taaaagtgaa agctaacgaa 600 cctcgagacg agcaaaaaat taagcaagca gaagcggaag ttgagagtaa acaagctgag 660 gctacaaggt taaaaaaaat caagacagat cgtgaagaag cagaagaaga agctaaacga 720 agagcagatg ctaaagagca aggtaaacca aaggggcggg caaaacgagg agttcctgga 780 gagctagcaa cacctgataa aaaagaaaat gatgcgaagt cttcagattc tagcgtaggt 840 gaagaaactc ttccaagccc atccctgaaa ccagaaaaaa aggtagcaga agctgagaag 900 aaggttgaag aagctaagaa aaaagccgag gatcaaaaag aagaagatcg ccgtaactac 960 ccaaccaata cttacaaaac gcttgaactt gaaattgctg agtccgatgt ggaagttaaa 1020 aaagcggagc ttgaactagt aaaagaggaa gctaaggaac ctcgaaacga ggaaaaagtt 1080 aagcaagcaa aagcggaagt tgagagtaaa aaagctgagg ctacaaggtt agaaaaaatc 1140 aagacagatc gtaaaaaagc agaagaagaa gctaaacgaa aagcagcaga agaagataaa 1200 gttaaagaaa aaccagctg 1219 <210> 13 <211> 1969 <212> DNA <213> Streptococcus pneumoniae <400> 13 gagaacgagg gagctaccca agtacccact tcttctaata gggcaaatga aagtcaggca 60 gaacaaggag aacaacctaa aaaactcgat tcagaacgag ataaggcaag gaaagaggtc 120 gaggaatatg taaaaaaaat agtgggtgag agctatgcaa aatcaactaa aaagcgacat 180 acaattactg tagctctagt taacgagttg aacaacatta agaacgagta tttgaataaa 240 atagttgaat caacctcaga aagccaacta cagatactga tgatggagag tcgatcaaaa 300 gtagatgaag ctgtgtctaa gtttgaaaag gactcatctt cttcgtcaag ttcagactct 360 tccactaaac cggaagcttc agatacagcg aagccaaaca agccgacaga accaggagaa 420 aaggtagcag aagctaagaa gaaggttgaa gaagctgaga aaaaagccaa ggatcaaaaa 480 gaagaagatc gtcgtaacta cccaaccatt acttacaaaa cgcttgaact tgaaattgct 540 gagtccgatg tggaagttaa aaaagcggag cttgaactag taaaagtgaa agctaacgaa 600 cctcgagacg agcaaaaaat taagcaagca gaagcggaag ttgagagtaa acaagctgag 660 gctacaaggt taaaaaaaat caagacagat cgtgaagaag cagaagaaga agctaaacga 720 agagcagatg ctaaagagca aggtaaacca aaggggcggg caaaacgagg agttcctgga 780 gagctagcaa cacctgataa aaaagaaaat gatgcgaagt cttcagattc tagcgtaggt 840 gaagaaactc ttccaagccc atccctgaaa ccagaaaaaa aggtagcaga agctgagaag 900 aaggttgaag aagctaagaa aaaagccgag gatcaaaaag aagaagatcg ccgtaactac 960 ccaaccaata cttacaaaac gcttgaactt gaaattgctg agtccgatgt ggaagttaaa 1020 aaagcggagg cttgaactag taaaagagga agctaaggaa cctcgaaacg aggaaaaagt 1080 taagcaagca aaagcggaag ttgagagtaa aaaagctgag gctacaaggt tagaaaaaat 1140 caagacagat cgtaaaaaag cagaagaaga agctaaacga aaagcagcag aagaagataa 1200 agttaaagaa aaaccagctg aacaaccaca accagcgccg gctccaaaag cagaaaaacc 1260 agctccagct ccaaaaccag agaatccagc tgaacaacca aaagcagaaa aaccagctga 1320 tcaacaagct gaagaagact atgctcgtag atcagaagaa gaatataatc gcttgactca 1380 acagcaaccg ccaaaaactg aaaaaccagc acaaccatct actccaaaaa caggctggaa 1440 acaagaaaac ggtatgtggt acttctacaa tactgatggt tcaatggcga caggatggct 1500 ccaaaacaat ggctcatggt actacctcaa cagcaatggc gctatggcga caggatggct 1560 ccaaaacaat ggttcatggt actatctaaa cgctaatggt tcaatggcaa caggatggct 1620 ccaaaacaat ggttcatggt actacctaaa cgctaatggt tcaatggcga caggatggct 1680 ccaatacaat ggctcatggt actacctaaa cgctaatggt tcaatggcga caggatggct 1740 ccaatacaat ggctcatggt actacctaaa cgctaatggt gatatggcga caggttgggt 1800 gaaagatgga gatacctggt actatcttga agcatcaggt gctatgaaag caagccaatg 1860 gttcaaagta tcagataaat ggtactatgt caatggctca ggtgcccttg cagtcaacac 1920 aactgtagat ggctatggag tcaatgccaa tggtgaatgg gtaaactaa 1969 <210> 14 <21l> 853 <212> DNA <213> Streptococcus pneumoniae <400> 14 gagaacgagg gagctaccca agtacccact tcttctaata gggcaaatga aagtcaggca 60 gaacaaggag aacaacctaa aaaactcgat tcagaacgag ataaggcaag gaaagaggtc 120 gaggaatatg taaaaaaaat agtgggtgag agctatgcaa aatcaactaa aaagcgacat 180 acaattactg tagctctagt taacgagttg aacaacatta agaacgagta tttgaataaa 240 atagttgaat caacctcaga aagccaacta cagatactga tgatggagag tcgatcaaaa 300 gtagatgaag ctgtgtctaa gtttgaaaag gactcatctt cttcgtcaag ttcagactct 360 tccactaaac cggaagcttc agatacagcg aagccaaaca agccgacaga accaggagaa 420 aaggtagcag aagctaagaa gaaggttgaa gaagctgaga aaaaagccaa ggatcaaaaa 480 gaagaagatc gtcgtaacta cccaaccatt acttacaaaa cgcttgaact tgaaattgct 540 gagtccgatg tggaagttaa aaaagcggag cttgaactag taaaagtgaa agctaacgaa 600 cctcgagacg agcaaaaaat taagcaagca gaagcggaag ttgagagtaa acaagctgag 660 gctacaaggt taaaaaaaat caagacagat cgtgaagaag cagaagaaga agctaaacga 720 agagcagatg ctaaagagca aggtaaacca aaggggcggg caaaacgagg agttcctgga 780 gagctagcaa cacctgataa aaaagaaaat gatgcgaagt cttcagattc tagcgtaggt 840 gaagaaactc ttc 853 <210> 15 <211> 318 <212> DNA <213> Streptococcus pneumoniae <400> 15 aaaccagaaa aaaaggtagc agaagctgag aagaaggttg aagaagctaa gaaaaaagcc 60 gaggatcaaa aagaagaaga tcgccgtaac tacccaacca atacttacaa aacgcttgaa 120 cttgaaattg ctgagtccga tgtggaagtt aaaaaagcgg agcttgaact agtaaaagag 180 gaagctaagg aacctcgaaa cgaggaaaaa gttaagcaag caaaagcgga agttgagagt 240 aaaaaagctg aggctacaag gttagaaaaa atcaagacag atcgtaaaaa agcagaagaa 300 gaagctaaac gaaaagca 318 <2l0> 16 <211> 327 <212> DNA <213> Streptococcus pneumoniae <400> 16 acagaaccag gagaaaaggt agcagaagct aagaagaagg ttgaagaagc tgagaaaaaa 60 gccaaggatc aaaaagaaga agatcgtcgt aactacccaa ccattactta caaaacgctt 120 gaacttgaaa ttgctgagtc cgatgtggaa gttaaaaaag cggagcttga actagtaaaa 180 gtgaaagcta acgaacctcg agacgagcaa aaaattaagc aagcagaagc ggaagttgag 240 agtaaacaag ctgaggctac aaggttaaaa aaaatcaaga cagatcgtga agaagcagaa 300 gaagaagcta aacgaagagc agatgct 327 <210> 17 <211> 1129 <212> DNA <213> Streptococcus pneumoniae <400> 17 gaaaacgaag gaagtaccca agcagccact tcttctaata tggcaaagac agaacatagg 60 aaagctgcta aacaagtcgt cgatgaatat atagaaaaaa tgttgaggga gattcaacta 120 gatagaagaa aacataccca aaatgtcgcc ttaaacataa agttgagcgc aattaaaacg 180 aagtatttgc gtgaattaaa tgttttagaa gagaagtcga aagatgagtt gccgtcagaa 240 ataaaagcaa agttagacgc agcttttgag aagtttaaaa aagatacatt gaaaccagga 300 gaaaaggtag cagaagctaa gaagaaggtt gaagaagcta agaaaaaagc cgaggatcaa 360 aaagaagaag atcgtcgtaa ctacccaacc aatacttaca aaacgcttga acttgaaatt 420 gctgagttcg atgtgaaagt taaagaagcg gagcttgaac tagtaaaaga ggaagctaaa 480 gaatctcgaa acgagggcac aattaagcaa gcaaaagaga aagttgagag taaaaaagct 540 gaggctacaa ggttagaaaa catcaagaca gatcgtaaaa aagcagaaga agaagctaaa 600 cgaaaagcag atgctaagtt gaaggaagct aatgtagcga cttcagatca aggtaaacca 660 aaggggcggg caaaacgagg agttcctgga gagctagcaa cacctgataa aaaagaaaat 720 gatgcgaagt cttcagattc tagcgtaggt gaagaaactc ttccaagctc atccctgaaa 780 tcaggaaaaa aggtagcaga agctgagaag aaggttgaag aagctgagaa aaaagccaag 840 gatcaaaaag aagaagatcg ccgtaactac ccaaccaata cttacaaaac gcttgacctt 900 gaaattgctg agtccgatgt gaaagttaaa gaagcggagc ttgaactagt aaaagaggaa 960 gctaaggaac ctcgagacga ggaaaaaatt aagcaagcaa aagcgaaagt tgagagtaaa 1020 aaagctgagg ctacaaggtt agaaaacatc aagacagatc gtaaaaaagc agaagaagaa 1080 gctaaacgaa aagcagcaga agaagataaa gttaaagaaa aaccagctg 1129 <210> 18 <211> 1992 <212> DNA <213> Streptococcus pneumoniae <400> 18 gaaaacgaag gaagtaccca agcagccact tcttctaata tggcaaagac agaacatagg 60 aaagctgcta aacaagtcgt cgatgaatat atagaaaaaa tgttgaggga gattcaacta 120 gatagaagaa aacataccca aaatgtcgcc ttaaacataa agttgagcgc aattaaaacg 180 aagtatttgc gtgaattaaa tgttttagaa gagaagtcga aagatgagtt gccgtcagaa 240 ataaaagcaa agttagacgc agcttttgag aagtttaaaa aagatacatt gaaaccagga 300 gaaaaggtag cagaagctaa gaagaaggtt gaagaagcta agaaaaaagc cgaggatcaa 360 aaagaagaag atcgtcgtaa ctacccaacc aatacttaca aaacgcttga acttgaaatt 420 gctgagttcg atgtgaaagt taaagaagcg gagcttgaac tagtaaaaga ggaagctaaa 480 gaatctcgaa acgagggcac aattaagcaa gcaaaagaga aagttgagag taaaaaagct 540 gaggctacaa ggttagaaaa catcaagaca gatcgtaaaa aagcagaaga agaagctaaa 600 cgaaaagcag atgctaagtt gaaggaagcc aatgtagcga cttcagatca aggtaaacca 660 aaggggcggg caaaacgagg agttcctgga gagctagcaa cacctgataa aaaagaaaat 720 gatgcgaagt cttcagattc tagcgtaggt gaagaaactc ttccaagctc atccctgaaa 780 tcaggaaaaa aggtagcaga agctgagaag aaggttgaag aagctgagaa aaaagccaag 840 gatcaaaaag aagaagatcg ccgtaactac ccaaccaata cttacaaaac gcttgacctt 900 gaaattgctg agtccgatgt gaaagttaaa gaagcggagc ttgaactagt aaaagaggaa 960 gctaaggaac ctcgagacga ggaaaaaatt aagcaagcaa aagcgaaagt tgagagtaaa 1020 aaagctgagg ctacaaggtt agaaaacatc aagacagatc gtaaaaaagc agaagaagaa 1080 gctaaacgaa aagcagcaga agaagataaa gttaaagaaa aaccagctga acaaccacaa 1140 ccagcgccgg ctactcaacc agaaaaacca gctccaaaac cagagaagcc agctgaacaa 1200 ccaaaagcag aaaaaacaga tgatcaacaa gctgaagaag actatgctcg tagatcagaa 1260 gaagaatata atcgcttgac tcaacagcaa ccgccaaaaa ctgaaaaacc agcacaacca 1320 tctactccaa aaacaggctg gaaacaagaa aacggtatgt ggtacttcta caatactgat 1380 ggttcaatgg caacaggatg gctccaaaac aacggttcat ggtactatct aaacgctaat 1440 ggtgctatgg cgacaggatg gctccaaaac aatggttcat ggtactatct aaacgctaat 1500 ggttcaatgg caacaggatg gctccaaaac aatggttcat ggtactacct aaacgctaat 1560 ggtgctatgg cgacaggatg gctccaatac aatggttcat ggtactacct aaacagcaat 1620 ggcgctatgg cgacaggatg gctccaatac aatggctcat ggtactacct caacgctaat 1680 ggtgatatgg cgacaggatg gctccaaaac aacggttcat ggtactacct caacgctaat 1740 ggtgatatgg cgacaggatg gctccaatac aacggttcat ggtattacct caacgctaat 1800 ggtgatatgg cgacaggttg ggtgaaagat ggagatacct ggtactatct tgaagcatca 1860 ggtgctatga aagcaagcca atggttcaaa gtatcagata aatggtacta tgtcaatggc 1920 tcaggtgccc ttgcagtcaa cacaactgta gatggctatg gagtcaatgc caatggtgaa 1980 tgggtaaact aa 1992 <210> 19 <211> 763 <212> DNA <213> Streptococcus pneumoniae <400> 19 gaaaacgaag gaagtaccca agcagccact tcttctaata tggcaaagac agaacatagg 60 aaagctgcta aacaagtcgt cgatgaatat atagaaaaaa tgttgaggga gattcaacta 120 gatagaagaa aacataccca aaatgtcgcc ttaaacataa agttgagcgc aattaaaacg 180 aagtatttgc gtgaattaaa tgttttagaa gagaagtcga aagatgagtt gccgtcagaa 240 ataaaagcaa agttagacgc agcttttgag aagtttaaaa aagatacatt gaaaccagga 300 gaaaaggtag cagaagctaa gaagaaggtt gaagaagcta agaaaaaagc cgaggatcaa 360 aaagaagaag atcgtcgtaa ctacccaacc aatacttaca aaacgcttga acttgaaatt 420 gctgagttcg atgtgaaagt taaagaagcg gagcttgaac tagtaaaaga ggaagctaaa 480 gaatctcgaa acgagggcac aattaagcaa gcaaaagaga aagttgagag taaaaaagct 540 gaggctacaa ggttagaaaa catcaagaca gatcgtaaaa aagcagaaga agaagctaaa 600 cgaaaagcag atgctaagtt gaaggaagct aatgtagcga cttcagatca aggtaaacca 660 aaggggcggg caaaacgagg agttcctgga gagctagcaa cacctgataa aaaagaaaat 720 gatgcgaagt cttcagattc tagcgtaggt gaagaaactc ttc 763 <210> 20 <211> 318 <212> DNA <213> Streptococcus pneumoniae <400> 20 aaatcaggaa aaaaggtagc agaagctgag aagaaggttg aagaagctga gaaaaaagcc 60 aaggatcaaa aagaagaaga tcgccgtaac tacccaacca atacttacaa aacgcttgac 120 cttgaaattg ctgagtccga tgtgaaagtt aaagaagcgg agcttgaact agtaaaagag 180 gaagctaagg aacctcgaga cgaggaaaaa attaagcaag caaaagcgaa agttgagagt 240 aaaaaagctg aggctacaag gttagaaaac atcaagacag atcgtaaaaa agcagaagaa 300 gaagctaaac gaaaagca 318 <210> 21 <211> 321 <212> DNA <213> Streptococcus pneumoniae <400> 21 ccaggagaaa aggtagcaga agctaagaag aaggttgaag aagctaagaa aaaagccgag 60 gatcaaaaag aagaagatcg tcgtaactac ccaaccaata cttacaaaac gcttgaactt 120 gaaattgctg agttcgatgt gaaagttaaa gaagcggagc ttgaactagt aaaagaggaa 180 gctaaagaat ctcgaaacga gggcacaatt aagcaagcaa aagagaaagt tgagagtaaa 240 aaagctgagg ctacaaggtt agaaaacatc aagacagatc gtaaaaaagc agaagaagaa 300 gctaaacgaa aagcagatgc t 321 <210> 22 <211> 121 <212> PRT <213> Streptococcus pneumoniae <400> 22 Ser Pro Ser Leu Lys Pro Glu Lys Lys Val Ala Glu Ala Glu Lys Lys 151015 Val Glu Glu Ala Lys Lys Lys Ala Glu Asp Gln Lys Glu Glu Asp Arg ...

20??????????????????25??????????????????30Arg?Asn?Tyr?Pro?Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala

35??????????????????40??????????????????45Glu?Ser?Asp?Val?Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu

50??????????????????55??????????????????60Glu?Ala?Lys?Glu?Pro?Arg?Asn?Glu?Glu?Lys?Val?Lys?Gln?Ala?Lys?Ala?65??????????????????70??????????????????75??????????????????80Glu?Val?Glu?Ser?Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Lys?Ile?Lys

85??????????????????90??????????????????95Thr?Asp?Arg?Lys?Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala?Ala?Glu

100?????????????????105?????????????????110Glu?Asp?Lys?Val?Lys?Glu?Lys?Pro?Ala

115 120＜210〉23＜211〉122＜212〉PRT＜213〉streptococcus pneumoniae＜400〉23Pro Ser Ser Ser Leu Lys Ser Gly Lys Lys Val Ala Glu Ala Glu Lys, 15 10 15Lys Val Glu Glu Ala Glu Lys Lys Ala Lys Asp Gln Lys Glu Glu Asp

20??????????????????25??????????????????30Arg?Arg?Asn?Tyr?Pro?Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Asp?Leu?Glu?Ile

35??????????????????40??????????????????45Ala?Glu?Ser?Asp?Val?Lys?Val?Lys?Glu?Ala?Glu?Leu?Glu?Leu?Val?Lys

50??????????????????55??????????????????60Glu?Glu?Ala?Lys?Glu?Pro?Arg?Asp?Glu?Glu?Lys?Ile?Lys?Gln?Ala?Lys?65??????????????????70??????????????????75??????????????????80Ala?Lys?Val?Glu?Ser?Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Asn?Ile

85??????????????????90??????????????????95Lys?Thr?Asp?Arg?Lys?Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala?Ala

100?????????????????105?????????????????110Glu?Glu?Asp?Lys?Val?Lys?Glu?Lys?Arg?Ala

115 120＜210〉24＜211〉428＜212〉PRT＜213〉streptococcus pneumoniae＜400〉24Glu Asn Glu Gly Ala Thr Gln Val Pro Thr Ser Ser Asn Arg Ala Asn, 15 10 15Glu Ser Gln Ala Glu Gln Gly Glu Gln Pro Lys Lys Leu Asp Ser Glu

405?????????????????410?????????????????415Ala?Glu?Lys?Pro?Ala?Pro?Ala?Pro?Lys?Pro?Glu?Asn

20??????????????????25??????????????????30Gln?Thr?Glu?His?Met?Lys?Ala?Ala?Lys?Gln?Val?Asp?Glu?Tyr?Ile?Lys

35??????????????????40??????????????????45Lys?Lys?Leu?Gln?Leu?Asp?Arg?Arg?Lys?His?Thr?Gln?Asn?Val?Gly?Leu

50??????????????????55??????????????????60Leu?Thr?Lys?Leu?Gly?Val?Ile?Lys?Thr?Glu?Tyr?Leu?His?Gly?Leu?Ser?65??????????????????70??????????????????75??????????????????80Val?Ser?Lys?Lys?Lys?Ser?Glu?Ala?Glu?Leu?Pro?Ser?Glu?Ile?Lys?Ala

85??????????????????90??????????????????95Lys?Leu?Asp?Ala?Ala?Phe?Glu?Gln?Phe?Lys?Lys?Asp?Thr?Leu?Pro?Thr

100?????????????????105?????????????????110Glu?Pro?Gly?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu?Ala

115?????????????????120?????????????????125Lys?Lys?Lys?Ala?Glu?Asp?Gln?Lys?Glu?Lys?Asp?Leu?Arg?Asn?Tyr?Pro

130?????????????????135?????????????????140Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Asp?Ile?Ala?Glu?Ser?Asp?Val145?????????????????150?????????????????155?????????????????160Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Glu

165?????????????????170?????????????????175Ser?Arg?Asp?Glu?Lys?Lys?Ile?Asn?Gln?Ala?Lys?Ala?Lys?Val?Glu?Asn

180?????????????????185?????????????????190Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Lys?Asn?Ile?Lys?Thr?Asp?Arg?Glu

195?????????????????200?????????????????205Lys?Ala?Glu?Glu?Ala?Lys?Arg?Arg?Ala?Asp?Ala?Lys?Leu?Gln?Glu?Ala

210?????????????????215?????????????????220Asn?Val?Ala?Thr?Ser?Glu?Gln?Asp?Lys?Ser?Lys?Arg?Arg?Ala?Lys?Arg225?????????????????230?????????????????235?????????????????240Glu?Val?Xaa?Gly?Glu?Leu?Ala?Thr?Pro?Asp?Lys?Lys?Glu?Asn?Asp?Ala

245?????????????????250?????????????????255Lys?Ser?Ser?Asp?Ser?Ser?Val?Gly?Glu?Glu?Thr?Leu?Thr?Ser?Pro?Ser

260?????????????????265?????????????????270Leu?Lys?Pro?Glu?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu

275?????????????????280?????????????????285Ala?Lys?Lys?Lys?Ala?Glu?Asp?Gln?Lys?Glu?Glu?Asp?Arg?Arg?Asn?Tyr

290?????????????????295?????????????????300Pro?Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp305?????????????????310?????????????????315?????????????????320Val?Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys

325?????????????????330?????????????????335Glu?Ser?Arg?Asn?Glu?Glu?Lys?Ile?Lys?Gln?Val?Lys?Ala?Lys?Val?Glu

340?????????????????345?????????????????350Ser?Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Asn?Ile?Lys?Thr?Asp?Arg

355?????????????????360?????????????????365Lys?Lys?Ala?Glu?Glu?Glu?Glu?Ala?Lys?Arg?Arg?Ala?Ala?Glu?Glu?Asp

370?????????????????375?????????????????380Lys?Val?Lys?Glu?Lys?Pro?Ala?Glu?Gln?Pro?Gln?Pro?Ala?Pro?Ala?Pro385?????????????????390?????????????????395?????????????????400Gln?Pro?Glu?Lys?Pro?Thr?Glu?Glu?Pro?Glu?Asn?Pro?Ala?Pro?Ala?Pro

405?????????????????410?????????????????415Ala?Pro?Lys?Pro?Glu?Asn?Pro?Ala?Glu?Lys?Pro?Lys?Ala?Glu?Lys?Pro

420?????????????????425?????????????????430Ala?Asp?Gln?Gln?Ala?Glu?Glu

435＜210〉29＜211〉437＜212〉PRT＜213〉streptococcus pneumoniae＜400〉29Ala Val Ala Ser Leu Phe Met Gly Ser Val Val His Ala Thr Glu Lys, 15 10 15Glu Val Thr Thr Gln Val Ala Thr Ser Ser Asn Arg Ala Asn Lys Ser

210?????????????????215?????????????????220Asn?Val?Ala?Thr?Ser?Glu?Gln?Asp?Lys?Ser?Lys?Arg?Arg?Ala?Lys?Arg225?????????????????230?????????????????235?????????????????240Glu?Val?Leu?Gly?Glu?Leu?Ala?Thr?Pro?Asp?Lys?Lys?Glu?Asn?Asp?Ala

420?????????????????425?????????????????430Ala?Asp?Gln?Gln?Ala

435＜210〉30＜2ll〉439＜212〉PRT＜213〉streptococcus pneumoniae＜400〉30Val Ala Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu, 15 10 15Lys Glu Val Thr Thr Gln Val Ala Thr Ser Ser Asn Arg Ala Asn Glu

20??????????????????25??????????????????30Ser?Gln?Ala?Gly?His?Arg?Lys?Ala?Ala?Glu?Gln?Phe?Asp?Glu?Tyr?Ile

35??????????????????40??????????????????45Lys?Thr?Met?Ile?Gln?Leu?Asp?Arg?Arg?Lys?His?Thr?Gln?Asn?Phe?Ala

50??????????????????55??????????????????60Leu?Asn?Ile?Lys?Leu?Ser?Arg?Ile?Lys?Thr?Glu?Tyr?Leu?Arg?Lys?Leu?65??????????????????70??????????????????75??????????????????80Asn?Val?Leu?Glu?Glu?Lys?Ser?Lys?Ala?Glu?Leu?Pro?Ser?Glu?Thr?Lys

85??????????????????90??????????????????95Lys?Glu?Ile?Asp?Ala?Ala?Phe?Glu?Gln?Phe?Lys?Lys?Asp?Thr?Asn?Arg

100?????????????????105?????????????????110Thr?Lys?Lys?Thr?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu?Ala?Lys

115?????????????????120?????????????????125Lys?Lys?Ala?Lys?Ala?Gln?Lys?Glu?Glu?Asp?His?Arg?Asn?Tyr?Pro?Thr

130?????????????????135?????????????????140Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val?Glu145?????????????????150?????????????????155?????????????????160Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Glu?Ser

165?????????????????170?????????????????175Arg?Asp?Asp?Glu?Lys?Ile?Lys?Gln?Ala?Glu?Ala?Lys?Val?Glu?Ser?Lys

180?????????????????185?????????????????190Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Asn?Ile?Lys?Thr?Asp?Arg?Glu?Lys

195?????????????????200?????????????????205Ala?Glu?Glu?Glu?Ala?Lys?Arg?Arg?Ala?Glu?Ala?Lys?Leu?Lys?Glu?Ala

210?????????????????215?????????????????220Val?Glu?Lys?Asn?Va1?Ala?Thr?Ser?Glu?Gln?Asp?Lys?Pro?Lys?Gly?Arg225?????????????????230?????????????????235?????????????????240Arg?Lys?Arg?Gly?Val?Pro?Gly?Glu?Gln?Ala?Thr?Pro?Asp?Lys?Lys?Glu

245?????????????????250?????????????????255Asn?Asp?Ala?Lys?Ser?Ser?Asp?Ser?Ser?Val?Gly?Glu?Glu?Ala?Leu?Pro

260??????????????????265?????????????????270Ser?Pro?Ser?Leu?Lys?Pro?Glu?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys

275?????????????????280?????????????????285Val?Ala?Glu?Ala?Glu?Lys?Lys?Ala?Lys?Ala?Gln?Lys?Glu?Glu?Asp?Arg

290?????????????????295?????????????????300Arg?Asn?Tyr?Pro?Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala305?????????????????310?????????????????315?????????????????320Glu?Ser?Asp?Val?Lys?Val?Lys?Glu?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu

325?????????????????330?????????????????335Glu?Ala?Lys?Glu?Ser?Arg?Asn?Glu?Glu?Lys?Val?Asn?Gln?Ala?Lys?Ala

340?????????????????345?????????????????350Lys?Val?Glu?Ser?Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Lys?Ile?Lys

355?????????????????360?????????????????365Thr?Asp?Arg?Lys?Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala?Ala?Glu

370?????????????????375?????????????????380Glu?Asp?Lys?Val?Lys?Glu?Lys?Pro?Ala?Glu?Gln?Pro?Gln?Pro?Ala?Pro385?????????????????390?????????????????395?????????????????400Ala?Pro?Gln?Pro?Glu?Lys?Pro?Thr?Glu?Glu?Pro?Glu?Asn?Pro?Ala?Pro

405?????????????????410?????????????????415Ala?Pro?Lys?Pro?Glu?Lys?Pro?Ala?Glu?Gln?Pro?Lys?Ala?Glu?Lys?Thr

420?????????????????425?????????????????430Asp?Asp?Gln?Gln?Ala?Glu?Glu

435＜210〉31＜211〉419＜212〉PRT＜213〉streptococcus pneumoniae＜400〉31Ala Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu Asn, 15 10 15Glu Gly Thr Thr Gln Ala Pro Thr Ser Ser Asn Arg Gly Asn Glu Ser

20??????????????????25??????????????????30Gln?Ala?Glu?His?Met?Lys?Ala?Ala?Lys?Gln?Val?Asp?Glu?Tyr?Ile?Glu

35??????????????????40??????????????????45Lys?Met?Leu?Gln?Leu?Asp?Arg?Arg?Lys?His?Thr?Gln?Asn?Val?Gly?Leu

50??????????????????55??????????????????60Leu?Thr?Lys?Leu?Gly?Ala?Ile?Lys?Thr?Glu?Tyr?Leu?Arg?Gly?Leu?Ser?65??????????????????70??????????????????75??????????????????80Val?Ser?Lys?Glu?Lys?Ser?Thr?Ala?Glu?Leu?Pro?Ser?Glu?Ile?Lys?Glu

85??????????????????90??????????????????95Lys?Leu?Thr?Ala?Ala?Phe?Lys?Gln?Phe?Lys?Lys?Asp?Thr?Leu?Lys?Pro

100?????????????????105?????????????????110Glu?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Ala?Glu?Ala?Lys?Lys

115?????????????????120?????????????????125Lys?Ala?Glu?Asp?Gln?Lys?Glu?Glu?Asp?Arg?Arg?Asn?Tyr?Pro?Thr?Ile

130?????????????????135?????????????????140Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val?Glu?Val145?????????????????150?????????????????155?????????????????160Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Val?Lys?Ala?Asn?Glu?Pro?Arg

165?????????????????170?????????????????175Asp?Glu?Glu?Lys?Ile?Lys?Gln?Ala?Glu?Ala?Glu?Val?Glu?Ser?Lys?Lys

180?????????????????185?????????????????190Ala?Glu?Ala?Thr?Arg?Leu?Lys?Lys?Ile?Lys?Thr?Asp?Arg?Glu?Lys?Ala

195?????????????????200?????????????????205Glu?Glu?Glu?Ala?Lys?Arg?Arg?Val?Asp?Ala?Lys?Glu?Gln?Asp?Glu?Ser

210?????????????????215?????????????????220Ser?Lys?Arg?Arg?Lys?Ser?Arg?Val?Lys?Arg?Gly?Asp?Val?Gly?Glu?Gln225?????????????????230?????????????????235?????????????????240Ala?Thr?Pro?Asp?Lys?Lys?Glu?Asn?Asp?Ala?Lys?Ser?Ser?Asp?Ser?Ser

245?????????????????250?????????????????255Val?Gly?Glu?Glu?Thr?Leu?Pro?Ser?Pro?Ser?Leu?Lys?Pro?Gly?Lys?Lys

260?????????????????265?????????????????270Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu?Ala?Asp?Lys?Lys?Ala?Lys

275?????????????????280?????????????????285Ala?Gln?Lys?Glu?Glu?Asp?Arg?Arg?Asn?Tyr?Pro?Thr?Asn?Thr?Tyr?Lys

290?????????????????295?????????????????300Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val?Glu?Val?Lys?Lys?Ala305?????????????????310?????????????????315?????????????????320Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Glu?Pro?Arg?Asn?Glu?Glu

325?????????????????330?????????????????335Lys?Val?Lys?Gln?Ala?Lys?Ala?Glu?Val?Glu?Ser?Lys?Lys?Ala?Glu?Ala

340?????????????????345?????????????????350Thr?Arg?Leu?Glu?Lys?Ile?Lys?Thr?Asp?Arg?Lys?Lys?Ala?Glu?Glu?Glu

355?????????????????360?????????????????365Ala?Lys?Arg?Lys?Ala?Ala?Glu?Glu?Asp?Lys?Val?Lys?Glu?Lys?Pro?Ala

370?????????????????375?????????????????380Glu?Gln?Pro?Lys?Pro?Ala?Pro?Ala?Pro?Gln?Pro?Glu?Lys?Pro?Ala?Pro385?????????????????390?????????????????395?????????????????400Lys?Pro?Glu?Asn?Pro?Ala?Glu?Gln?Pro?Lys?Ala?Glu?Lys?Pro?Ala?Asp

405 410 415Gln Gln Ala＜210〉32＜211〉437＜212〉PRT＜213〉streptococcus pneumoniae＜400〉32Val Ala Ser Leu Phe Met Gly Ser Val Val His Ala Thr Glu Lys Glu, 15 10 15Val Thr Thr Gln Val Ala Thr Ser Ser Asn Lys Ala Asn Lys Ser Gln

20??????????????????25??????????????????30Thr?Glu?His?Met?Lys?Ala?Ala?Lys?Gln?Val?Asp?Glu?Tyr?Ile?Lys?Lys

35??????????????????40??????????????????45Lys?Leu?Gln?Leu?Asp?Arg?Arg?Lys?His?Thr?Gln?Asn?Val?Gly?Leu?Leu

50??????????????????55??????????????????60Thr?Lys?Leu?Gly?Val?Ile?Lys?Thr?Glu?Tyr?Leu?His?Gly?Leu?Ser?Val?65??????????????????70??????????????????75??????????????????80Ser?Lys?Lys?Lys?Ser?Glu?Ala?Glu?Leu?Pro?Ser?Glu?Ile?Lys?Ala?Lys

85??????????????????90??????????????????95Leu?Asp?Ala?Ala?Phe?Glu?Gln?Phe?Lys?Lys?Asp?Thr?Leu?Pro?Thr?Glu

100?????????????????105?????????????????110Pro?Gly?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu?Ala?Lys

115?????????????????120?????????????????125Lys?Lys?Ala?Glu?Asp?Gln?Lys?Glu?Lys?Asp?Leu?Arg?Asn?Tyr?Pro?Thr

130?????????????????135?????????????????140Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Asp?Ile?Ala?Glu?Ser?Asp?Val?Glu145?????????????????150?????????????????155?????????????????160Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Glu?Ser

165?????????????????170?????????????????175Arg?Asp?Glu?Lys?Lys?Ile?Asn?Gln?Ala?Lys?Ala?Lys?Val?Glu?Asn?Lys

180?????????????????185?????????????????190Lys?Ala?Glu?Ala?Thr?Arg?Leu?Lys?Asn?Ile?Lys?Thr?Asp?Arg?Glu?Lys

195?????????????????200?????????????????205Ala?Glu?Glu?Ala?Lys?Arg?Arg?Ala?Asp?Ala?Lys?Leu?Gln?Glu?Ala?Asn

210?????????????????215?????????????????220Val?Ala?Thr?Ser?Glu?Gln?Asp?Lys?Ser?Lys?Arg?Arg?Ala?Lys?Arg?Glu225?????????????????230?????????????????235?????????????????240Val?Phe?Gly?Glu?Leu?Ala?Thr?Pro?Asp?Lys?Lys?Glu?Asn?Asp?Ala?Lys

245?????????????????250?????????????????255Ser?Ser?Asp?Ser?Ser?Val?Gly?Glu?Glu?Thr?Leu?Thr?Ser?Pro?Ser?Leu

260?????????????????265?????????????????270Lys?Pro?Glu?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu?Ala

275?????????????????280?????????????????285Lys?Lys?Lys?Ala?Glu?Asp?Gln?Lys?Glu?Glu?Asp?Arg?Arg?Asn?Tyr?Pro

290?????????????????295?????????????????300Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val305?????????????????310?????????????????315?????????????????320Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Glu

325?????????????????330?????????????????335Ser?Arg?Asn?Glu?Glu?Lys?Ile?Lys?Gln?Val?Lys?Ala?Lys?Val?Glu?Ser

340?????????????????345?????????????????350Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Asn?Ile?Lys?Thr?Asp?Arg?Lys

355?????????????????360?????????????????365Lys?Ala?Glu?Glu?Glu?Glu?Ala?Lys?Arg?Arg?Ala?Ala?Glu?Glu?Asp?Lys

370?????????????????375?????????????????380Val?Lys?Glu?Lys?Pro?Ala?Glu?Gln?Pro?Gln?Pro?Ala?Pro?Ala?Pro?Gln385?????????????????390?????????????????395?????????????????400Pro?Glu?Lys?Pro?Thr?Glu?Glu?Pro?Glu?Asn?Pro?Ala?Pro?Ala?Pro?Ala

405?????????????????410?????????????????415Pro?Lys?Pro?Glu?Asn?Pro?Ala?Glu?Lys?Pro?Lys?Ala?Glu?Lys?Pro?Ala

420????????????????425????????????????430Asp?Gln?Gln?Ala?Glu

435＜210〉33＜211〉433＜212〉PRT＜213〉streptococcus pneumoniae＜400〉33Cys Thr Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu, 15 10 15Asn Glu Arg Thr Thr Gln Val Pro Thr Ser Ser Asn Arg Gly Lys Pro

20??????????????????25??????????????????30Glu?Arg?Arg?Lys?Ala?Ala?Glu?Gln?Phe?Asp?Glu?Tyr?Ile?Asn?Lys?Met

35??????????????????40??????????????????45Ile?Gln?Leu?Asp?Lys?Arg?Lys?His?Thr?Gln?Asn?Leu?Ala?Phe?Asn?Ile

50??????????????????55??????????????????60Gln?Leu?Ser?Arg?Ile?Lys?Thr?Glu?Tyr?Leu?Asn?Gly?Leu?Lys?Glu?Lys?65??????????????????70??????????????????75??????????????????80Ser?Glu?Ala?Glu?Leu?Pro?Ser?Lys?Ile?Lys?Ala?Glu?Leu?Asp?Ala?Ala

85??????????????????90??????????????????95Phe?Lys?Gln?Phe?Lys?Lys?Asp?Thr?Leu?Pro?Thr?Glu?Pro?Glu?Lys?Lys

100?????????????????105?????????????????110Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu?Ala?Glu?Lys?Lys?Val?Ala

115?????????????????120?????????????????125Glu?Ala?Lys?Lys?Lys?Ala?Lys?Ala?Gln?Lys?Glu?Glu?Asp?His?Arg?Asn

130?????????????????135?????????????????140Tyr?Pro?Thr?Ile?Thr?Tyr?Lys?Thr?Leu?Asp?Leu?Glu?Ile?Ala?Glu?Phe145?????????????????150?????????????????155?????????????????160Asp?Val?Lys?Val?Lys?Glu?Ala?Glu?Leu?Glu?Leu?Val?Lys?Lys?Glu?Ala

165?????????????????170?????????????????175Asp?Glu?Ser?Arg?Asn?Glu?Gly?Thr?Ile?Asn?Gln?Ala?Lys?Ala?Lys?Val

180?????????????????185?????????????????190Glu?Ser?Glu?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Lys?Lys?Ile?Lys?Thr?Asp

195?????????????????200?????????????????205Arg?Glu?Lys?Ala?Glu?Glu?Glu?Glu?Ala?Lys?Arg?Arg?Ala?Asp?Ala?Lys

210?????????????????215?????????????????220Glu?Gln?Asp?Glu?Ser?Lys?Arg?Arg?Lys?Ser?Arg?Gly?Lys?Arg?Gly?Ala225?????????????????230?????????????????235?????????????????240Leu?Gly?Glu?Gln?Ala?Thr?Pro?Asp?Lys?Lys?Glu?Asn?Asp?Ala?Lys?Ser

245?????????????????250?????????????????255Ser?Asp?Ser?Ser?Val?Gly?Glu?Glu?Thr?Leu?Pro?Ser?Pro?Ser?Leu?Lys

260?????????????????265?????????????????270Pro?Gly?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu?Ala?Asp

275?????????????????280?????????????????285Lys?Lys?Ala?Lys?Ala?Gln?Lys?Glu?Glu?Asp?Arg?Arg?Asn?Tyr?Pro?Thr

290?????????????????295?????????????????300Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val?Lys305?????????????????310?????????????????315?????????????????320Val?Lys?Glu?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Glu?Ser

325?????????????????330?????????????????335Arg?Asn?Glu?Glu?Lys?Ile?Lys?Gln?Ala?Lys?Ala?Lys?Val?Glu?Ser?Lys

340?????????????????345?????????????????350Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Lys?Ile?Lys?Thr?Asp?Arg?Lys?Lys

355?????????????????360?????????????????365Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala?Ala?Glu?Glu?Asp?Lys?Val?Lys

370?????????????????375?????????????????380Glu?Lys?Pro?Ala?Glu?Gln?Pro?Gln?Pro?Ala?Pro?Ala?Pro?Gln?Pro?Glu385?????????????????390?????????????????395?????????????????400Lys?Pro?Ala?Glu?Glu?Pro?Glu?Asn?Pro?Val?Pro?Ala?Pro?Lys?Pro?Glu

405?????????????????410?????????????????415Asn?Pro?Ala?Glu?Gln?Pro?Lys?Ala?Glu?Lys?Pro?Ala?Asp?Gln?Gln?Ala

420 425 430Glu＜210〉34＜211〉427＜212〉PRT＜213〉streptococcus pneumoniae＜400〉34Val Ala Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu, 15 10 15Lys Glu Val Thr Thr Gln Val Pro Thr Tyr Ser Asn Met Ala Lys Thr

20??????????????????25??????????????????30Glu?His?Arg?Lys?Ala?Ala?Lys?Gln?Val?Val?Asp?Glu?Tyr?Ile?Glu?Lys

35??????????????????40??????????????????45Met?Leu?Arg?Glu?Ile?Gln?Leu?Asp?Arg?Arg?Lys?His?Thr?Gln?Asn?Phe

50??????????????????55??????????????????60Ala?Phe?Asn?Met?Lys?Leu?Ser?Ala?Ile?Lys?Thr?Glu?Tyr?Leu?Tyr?Gly?65??????????????????70??????????????????75??????????????????80Leu?Lys?Glu?Lys?Ser?Glu?Ala?Glu?Leu?Pro?Ser?Glu?Val?Lys?Ala?Lys

85??????????????????90??????????????????95Leu?Asp?Ala?Ala?Phe?Glu?Gln?Phe?Lys?Lys?Asp?Thr?Leu?Lys?Leu?Gly

100?????????????????105?????????????????110Glu?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys

115?????????????????120?????????????????125Ala?Lys?Ala?Gln?Lys?Glu?Glu?Asp?Arg?Arg?Asn?Tyr?Pro?Thr?Asn?Thr

130?????????????????135?????????????????140Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val?Glu?Val?Lys145?????????????????150?????????????????155?????????????????160Lys?Ala?Glu?Leu?Glu?Leu?Leu?Lys?Glu?Glu?Ala?Lys?Thr?Arg?Asn?Glu

165?????????????????170?????????????????175Asp?Thr?Ile?Asn?Gln?Ala?Lys?Ala?Lys?Val?Glu?Ser?Lys?Lys?Ala?Glu

180?????????????????185?????????????????190Ala?Thr?Lys?Leu?Glu?Glu?Ile?Lys?Thr?Asp?Arg?Lys?Lys?Ala?Glu?Glu

195?????????????????200?????????????????205Glu?Ala?Lys?Arg?Lys?Ala?Glu?Ala?Glu?Glu?Asp?Lys?Val?Lys?Asp?Lys

210?????????????????215?????????????????220Leu?Lys?Arg?Arg?Thr?Lys?Arg?Ala?Val?Pro?Gly?Glu?Pro?Ala?Thr?Pro225?????????????????230?????????????????235?????????????????240Asp?Lys?Lys?Glu?Asn?Asp?Ala?Lys?Ser?Ser?Asp?Ser?Ser?Val?Gly?Glu

245?????????????????250?????????????????255Glu?Thr?Leu?Pro?Ser?Pro?Ser?Leu?Lys?Ser?Gly?Lys?Lys?Val?Ala?Glu

260?????????????????265?????????????????270Ala?Glu?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Ala?Lys?Asp?Gln?Lys

275?????????????????280?????????????????285Glu?Glu?Asp?Arg?Arg?Asn?Tyr?Pro?Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Asp

290?????????????????295?????????????????300Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val?Lys?Val?Lys?Glu?Ala?Glu?Leu?Glu305?????????????????310?????????????????315?????????????????320Leu?Val?Lys?Glu?Glu?Ala?Lys?Gly?Ser?Arg?Asn?Glu?Glu?Lys?Ile?Asn

325?????????????????330?????????????????335Gln?Ala?Lys?Ala?Glu?Val?Glu?Ser?Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu

340?????????????????345?????????????????350Glu?Lys?Ile?Lys?Thr?Asp?Arg?Lys?Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg

355?????????????????360?????????????????365Lys?Ala?Ala?Glu?Glu?Asp?Lys?Val?Lys?Glu?Lys?Pro?Ala?Glu?Gln?Pro

370?????????????????375?????????????????380Gln?Pro?Ala?Pro?Ala?Pro?Gln?Pro?Glu?Lys?Pro?Thr?Glu?Glu?Pro?Glu385?????????????????390?????????????????395?????????????????400Asn?Pro?Ala?Pro?Ala?Pro?Lys?Pro?Glu?Lys?Pro?Ala?Glu?Gln?Pro?Lys

405?????????????????410?????????????????415Ala?Glu?Lys?Thr?Asp?Asp?Gln?Gln?Ala?Glu?Glu

420 425＜210〉35＜211〉413＜212〉PRT＜213〉streptococcus pneumoniae＜400〉35Glu Asn Glu Gly Ser Thr Gln Ala Ala Thr Ser Ser Asn Met Ala Lys, 15 10 15Thr Glu His Arg Lys Ala Ala Lys Gln Val Val Asp Glu Tyr Ile Glu

245?????????????????250?????????????????255Ser?Ser?Leu?Lys?Ser?Gly?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val

370?????????????????375?????????????????380Thr?Gln?Pro?Glu?Lys?Pro?Ala?Pro?Lys?Pro?Glu?Lys?Pro?Ala?Glu?Gln385?????????????????390?????????????????395?????????????????400Pro?Lys?Ala?Glu?Lys?Thr?Asp?Asp?Gln?Gln?Ala?Glu?Glu

405 410＜210〉36＜211〉425＜212〉PRT＜213〉streptococcus pneumoniae＜400〉36Tyr Ile Ala Ser Leu Phe Leu Gly Gly Val Val His Ala Glu Gly Val, 15 10 15Arg Ser Glu Asn Asn Pro Thr Val Thr Ser Ser Gly Gln Asp Ile Ser

20??????????????????25??????????????????30Lys?Lys?Tyr?Ala?Asp?Glu?Val?Lys?Ser?His?Leu?Glu?Lys?Ile?Leu?Ser

35??????????????????40??????????????????45Glu?Ile?Gln?Thr?Asn?Leu?Asp?Arg?Ser?Lys?His?Ile?Lys?Thr?Val?Asn

50??????????????????55??????????????????60Leu?Ile?Asn?Lys?Leu?Gln?Asp?Ile?Lys?Arg?Thr?Tyr?Leu?Tyr?Glu?Leu?65??????????????????70??????????????????75??????????????????80Asn?Val?Leu?Glu?Asp?Lys?Ser?Lys?Ala?Glu?Leu?Pro?Ser?Lys?Ile?Lys

85??????????????????90??????????????????95Ala?Glu?Leu?Asp?Ala?Ala?Phe?Glu?Gln?Phe?Lys?Lys?Asp?Thr?Leu?Pro

100?????????????????105?????????????????110Thr?Glu?Pro?Gly?Lys?Lys?Val?Ala?Glu?Ala?Lys?Lys?Lys?Val?Glu?Glu

115?????????????????120?????????????????125Ala?Glu?Lys?Lys?Ala?Lys?Ala?Gln?Iys?Glu?Glu?Asp?Tyr?Arg?Asn?Tyr

130?????????????????135?????????????????140Pro?Thr?Ile?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp145?????????????????150?????????????????155?????????????????160Val?Lys?Val?Lys?Glu?Ala?Glu?Leu?Glu?Leu?Val?Lys?Lys?Glu?Ala?Asp

165?????????????????170?????????????????175Glu?Ser?Arg?Asn?Glu?Gly?Thr?Ile?Asn?Gln?Ala?Lys?Ala?Lys?Val?Glu

180?????????????????185?????????????????190Ser?Glu?Gln?Ala?Glu?Ala?Thr?Arg?Leu?Lys?Lys?Ile?Lys?Thr?Asp?Arg

195?????????????????200?????????????????205Glu?Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Arg?Ala?Asp?Ala?Lys?Glu?Gln

210?????????????????215?????????????????220Asp?Glu?Ser?Lys?Arg?Arg?Lys?Ser?Arg?Val?Lys?Arg?Gly?Asp?Phe?Gly225?????????????????230?????????????????235?????????????????240Glu?Pro?Ala?Thr?Pro?Asp?Lys?Lys?Glu?Asn?Asp?Ala?Lys?Ser?Ser?Asp

245?????????????????250?????????????????255Ser?Ser?Val?Gly?Glu?Glu?Thr?Leu?Pro?Ser?Pro?Ser?Leu?Lys?Pro?Gly

260?????????????????265?????????????????270Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu?Ala?Glu?Lys?Lys

275?????????????????280?????????????????285Ala?Lys?Asp?Gln?Lys?Glu?Glu?Asp?His?Arg?Asn?Tyr?Pro?Thr?Ile?Thr

290?????????????????295?????????????????300Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val?Glu?Val?Lys305?????????????????310?????????????????315?????????????????320Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Gly?Ser?Arg?Asn

325?????????????????330?????????????????335Glu?Glu?Lys?Val?Lys?Gln?Ala?Lys?Ala?Glu?Val?Glu?Ser?Lys?Lys?Ala

340?????????????????345?????????????????350Glu?Ala?Thr?Arg?Leu?Glu?Lys?Ile?Lys?Thr?Asp?Arg?Lys?Lys?Ala?Glu

355?????????????????360?????????????????365Glu?Glu?Ala?Lys?Arg?Lys?Ala?Ala?Glu?Glu?Asp?Lys?Val?Lys?Glu?Lys

370?????????????????375?????????????????380Pro?Ala?Glu?Gln?Pro?Gln?Pro?Ala?Pro?Ala?Pro?Gln?Pro?Glu?Lys?Pro385?????????????????390?????????????????395?????????????????400Ala?Pro?Ala?Pro?Lys?Pro?Glu?Asn?Pro?Ala?Glu?Gln?Pro?Lys?Ala?Glu

405?????????????????410?????????????????415Lys?Pro?Ala?Asp?Gln?Gln?Ala?Glu?Glu

420 425＜210〉37＜211〉439＜212〉PRT＜213〉streptococcus pneumoniae＜400〉37Ala Ser Leu Phe Leu Gly Gly Val Val His Ala Glu Gly Val Arg Ser, 15 10 15Gly Asn Asn Ser Thr Val Thr Ser Ser Gly Gln Asp Ile Ser Lys Lys

20??????????????????25??????????????????30Tyr?Ala?Asp?Glu?Val?Glu?Ser?His?Leu?Gln?Ser?Ile?Leu?Lys?Asp?Val

35??????????????????40??????????????????45Asn?Lys?Asn?Leu?Lys?Lys?Val?Gln?His?Thr?Gln?Asn?Ala?Asp?Phe?Asn

50??????????????????55??????????????????60Lys?Lys?Leu?Ser?Lys?Ile?Lys?Thr?Lys?Tyr?Leu?Tyr?Glu?Leu?Asn?Val?65??????????????????70??????????????????75??????????????????80Leu?Glu?Glu?Lys?Ser?Glu?Ala?Glu?Leu?Thr?Ser?Lys?Thr?Lys?Glu?Thr

85??????????????????90??????????????????95Lys?Glu?Glu?Leu?Thr?Ala?Ala?Phe?Glu?Gln?Phe?Lys?Lys?Asp?Thr?Leu

100?????????????????105?????????????????110Ser?Thr?Glu?Pro?Glu?Lys?Lys?Val?Ala?Glu?Ala?Lys?Lys?Lys?Val?Glu

115?????????????????120?????????????????125Glu?Ala?Lys?Lys?Lys?Ala?Glu?Asp?Gln?Lys?Glu?Lys?Asp?Arg?Arg?Asn

130?????????????????135?????????????????140Tyr?Pro?Thr?Ile?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser145?????????????????150?????????????????155?????????????????160Asp?Val?Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Val?Lys?Ala

165?????????????????170?????????????????175Asn?Glu?Pro?Arg?Asp?Glu?Glu?Lys?Ile?Lys?Gln?Ala?Glu?Ala?Lys?Val

180?????????????????185?????????????????190Glu?Ser?Lys?Gln?Ala?Glu?Ala?Thr?Arg?Leu?Lys?Lys?Ile?Lys?Thr?Asp

195?????????????????200?????????????????205Arg?Glu?Gln?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Asn?Ile?Lys?Thr?Asp?Arg

210?????????????????215?????????????????220Glu?Gln?Ala?Glu?Glu?Glu?Ala?Lys?Val?Lys?Asp?Glu?Pro?Lys?Lys?Arg225?????????????????230?????????????????235?????????????????240Thr?Lys?Arg?Gly?Val?Leu?Gly?Glu?Pro?Ala?Thr?Pro?Asp?Lys?Lys?Glu

245?????????????????250?????????????????255Asn?Asp?Ala?Lys?Ser?Ser?Asp?Ser?Ser?Val?Gly?Glu?Glu?Thr?Leu?Pro

260?????????????????265?????????????????270Ser?Pro?Ser?Leu?Lys?Pro?Glu?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys

275?????????????????280?????????????????285Val?Glu?Glu?Ala?Lys?Lys?Lys?Ala?Glu?Asp?Gln?Lys?Glu?Glu?Asp?Arg

290?????????????????295?????????????????300Arg?Asn?Tyr?Pro?Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala305?????????????????310?????????????????315?????????????????320Glu?Ser?Asp?Val?Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu

325?????????????????330?????????????????335Glu?Ala?Lys?Glu?Pro?Arg?Asn?Glu?Glu?Lys?Val?Lys?Gln?Ala?Lys?Ala

340?????????????????345?????????????????350Glu?Val?Glu?Ser?Lys?Gln?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Asn?Ile?Lys

370?????????????????375?????????????????380Glu?Asp?Lys?Val?Lys?Glu?Lys?Pro?Ala?Glu?Gln?Pro?Gln?Pro?Ala?Pro385?????????????????390??????????????????395????????????????400Ala?Pro?Gln?Pro?Glu?Lys?Pro?Ala?Pro?Lys?Pro?Glu?Lys?Pro?Ala?Pro

405?????????????????410?????????????????415Ala?Pro?Lys?Pro?Glu?Asn?Pro?Ala?Glu?Gln?Pro?Lys?Ala?Glu?Lys?Pro

420?????????????????425?????????????????430Ala?Asp?Gln?Gln?Ala?Glu?Glu

435＜210〉38＜211〉460＜212〉PRT＜213〉streptococcus pneumoniae＜400〉38Cys Ile Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu, 15 10 15Asn Glu Gly Ala Thr Gln Val Pro Thr Ser Ser Asn Arg Ala Asn Glu

20??????????????????25??????????????????30Ser?Gln?Ala?Glu?Gln?Gly?Glu?Gln?Pro?Lys?Lys?Leu?Asp?Ser?Glu?Arg

35??????????????????40???????????????????45Asp?Lys?Ala?Arg?Lys?Glu?Val?Glu?Glu?Tyr?Val?Lys?Lys?Ile?Val?Gly

50??????????????????55??????????????????60Glu?Ser?Tyr?Ala?Lys?Ser?Thr?Lys?Lys?Arg?His?Thr?Ile?Thr?Val?Ala?65??????????????????70??????????????????75?????????????????80Leu?Val?Asn?Glu?Leu?Asn?Asn?Ile?Lys?Asn?Glu?Tyr?Leu?Asn?Lys?Ile

85??????????????????90??????????????????95Val?Glu?Ser?Thr?Ser?Glu?Ser?Gln?Leu?Gln?Ile?Leu?Met?Met?Glu?Ser

100?????????????????105?????????????????110Arg?Ser?Lys?Val?Asp?Glu?Ala?Val?Ser?Lys?Phe?Glu?Lys?Asp?Ser?Ser

115?????????????????120?????????????????125Ser?Ser?Ser?Ser?Ser?Asp?Ser?Ser?Thr?Lys?Pro?Glu?Ala?Ser?Asp?Thr

130?????????????????135?????????????????140Ala?Lys?Pro?Asn?Lys?Pro?Thr?Glu?Pro?Gly?Glu?Lys?Val?Ala?Glu?Ala145?????????????????150?????????????????155?????????????????160Lys?Lys?Lys?Val?Glu?Glu?Ala?Glu?Lys?Lys?Ala?Lys?Asp?Gln?Lys?Glu

165?????????????????170?????????????????175Glu?Asp?Arg?Arg?Asn?Tyr?Pro?Thr?Ile?Thr?Tyr?Lys?Thr?Leu?Glu?Leu

180?????????????????185?????????????????190Glu?Ile?Ala?Glu?Ser?Asp?Val?Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu

195?????????????????200?????????????????205Val?Lys?Val?Lys?Ala?Asn?Glu?Pro?Arg?Asp?Glu?Gln?Lys?Ile?Lys?Gln

210?????????????????215?????????????????220Ala?Glu?Ala?Glu?Val?Glu?Ser?Lys?Gln?Ala?Glu?Ala?Thr?Arg?Leu?Lys225?????????????????230?????????????????235?????????????????240Lys?Ile?Lys?Thr?Asp?Arg?Glu?Glu?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Arg

245?????????????????250?????????????????255Ala?Asp?Ala?Lys?Glu?Gln?Gly?Lys?Pro?Lys?Gly?Arg?Ala?Lys?Arg?Gly

260?????????????????265?????????????????270Val?Pro?Gly?Glu?Leu?Ala?Thr?Pro?Asp?Lys?Lys?Glu?Asn?Asp?Ala?Lys

275?????????????????280?????????????????285Ser?Ser?Asp?Ser?Ser?Val?Gly?Glu?Glu?Thr?Leu?Pro?Ser?Pro?Ser?Leu

290?????????????????295?????????????????300Lys?Pro?Glu?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu?Ala305?????????????????310?????????????????315?????????????????320Lys?Lys?Lys?Ala?Glu?Asp?Gln?Lys?Glu?Glu?Asp?Arg?Arg?Asn?Tyr?Pro

325?????????????????330?????????????????335Thr?Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val

340?????????????????345?????????????????350Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Glu

355?????????????????360?????????????????365Pro?Arg?Asn?Glu?Glu?Lys?Val?Lys?Gln?Ala?Lys?Ala?Glu?Val?Glu?Ser

370?????????????????375?????????????????380Lys?Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Lys?Ile?Lys?Thr?Asp?Arg?Lys385?????????????????390?????????????????395?????????????????400Lys?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala?Ala?Glu?Glu?Asp?Lys?Val

405?????????????????410?????????????????415Lys?Glu?Lys?Pro?Ala?Glu?Gln?Pro?Gln?Pro?Ala?Pro?Ala?Pro?Lys?Ala

420?????????????????425?????????????????430Glu?Lys?Pro?Ala?Pro?Ala?Pro?Lys?Pro?Glu?Asn?Pro?Ala?Glu?Gln?Pro

435?????????????????440?????????????????445Lys?Ala?Glu?Lys?Pro?Ala?Asp?Gln?Gln?Ala?Glu?Glu

450 455 460＜210〉39＜211〉459＜212〉PRT＜213〉streptococcus pneumoniae＜400〉39Ile Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu Asn, 15 10 15Glu Gly Ala Thr Gln Val Pro Thr Ser Ser Asn Arg Ala Asn Glu Ser

20??????????????????25??????????????????30Gln?Ala?Glu?Gln?Gly?Glu?Gln?Pro?Lys?Lys?Leu?Asp?Ser?Glu?Arg?Asp

35??????????????????40??????????????????45Lys?Ala?Arg?Lys?Glu?Val?Glu?Glu?Tyr?Val?Lys?Lys?Ile?Val?Gly?Glu

50??????????????????55??????????????????60Ser?Tyr?Ala?Lys?Ser?Thr?Lys?Lys?Arg?His?Thr?Ile?Thr?Val?Ala?Leu?65??????????????????70??????????????????75??????????????????80Val?Asn?Glu?Leu?Asn?Asn?Ile?Lys?Asn?Glu?Tyr?Leu?Asn?Lys?Ile?Val

85??????????????????90??????????????????95Glu?Ser?Thr?Ser?Glu?Ser?Gln?Leu?Gln?Ile?Leu?Met?Met?Glu?Ser?Arg

100?????????????????105?????????????????110Ser?Lys?Val?Asp?Glu?Ala?Val?Ser?Lys?Phe?Glu?Lys?Asp?Ser?Ser?Ser

115?????????????????120?????????????????125Ser?Ser?Ser?Ser?Asp?Ser?Ser?Thr?Lys?Pro?Glu?Ala?Ser?Asp?Thr?Ala

130?????????????????135?????????????????140Lys?Pro?Asn?Lys?Pro?Thr?Glu?Pro?Gly?Glu?Lys?Val?Ala?Glu?Ala?Lys145?????????????????150?????????????????155?????????????????160Lys?Lys?Val?Glu?Glu?Val?Glu?Lys?Lys?Ala?Lys?Asp?Gln?Lys?Glu?Glu

165?????????????????170?????????????????175Asp?Arg?Arg?Asn?Tyr?Pro?Thr?Ile?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu

180?????????????????185?????????????????190Ile?Ala?Glu?Ser?Asp?Val?Glu?Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val

195?????????????????200?????????????????205Lys?Val?Lys?Ala?Asn?Glu?Pro?Arg?Asp?Lys?Gln?Lys?Ile?Lys?Gln?Ala

210?????????????????215?????????????????220Glu?Ala?Glu?Val?Glu?Ser?Lys?Gln?Ala?Glu?Ala?Thr?Arg?Leu?Lys?Lys225?????????????????230?????????????????235?????????????????240Ile?Lys?Thr?Asp?Arg?Glu?Glu?Ala?Glu?Glu?Glu?Ala?Lys?Arg?Arg?Ala

245?????????????????250?????????????????255Asp?Ala?Lys?Glu?Gln?Gly?Lys?Pro?Lys?Gly?Arg?Pro?Lys?Arg?Gly?Val

260?????????????????265?????????????????270Pro?Gly?Glu?Leu?Ala?Thr?Pro?Asp?Lys?Lys?Glu?Asn?Asp?Ala?Lys?Ser

275?????????????????280?????????????????285Ser?Asp?Ser?Ser?Val?Gly?Glu?Glu?Thr?Leu?Pro?Ser?Pro?Ser?Leu?Lys

290?????????????????295?????????????????300Pro?Glu?Lys?Lys?Val?Ala?Glu?Ala?Glu?Lys?Lys?Val?Glu?Glu?Ala?Lys305?????????????????310?????????????????315?????????????????320Lys?Lys?Ala?Glu?Asp?Gln?Lys?Glu?Glu?Asp?Arg?Arg?Asn?Tyr?Pro?Thr

325?????????????????330?????????????????335Asn?Thr?Tyr?Lys?Thr?Leu?Glu?Leu?Glu?Ile?Ala?Glu?Ser?Asp?Val?Glu

340?????????????????345?????????????????350Val?Lys?Lys?Ala?Glu?Leu?Glu?Leu?Val?Lys?Glu?Glu?Ala?Lys?Glu?Pro

355?????????????????360?????????????????365Arg?Asn?Glu?Glu?Lys?Val?Lys?Gln?Ala?Lys?Ala?Glu?Val?Glu?Ser?Lys

370?????????????????375?????????????????380Lys?Ala?Glu?Ala?Thr?Arg?Leu?Glu?Lys?Ile?Lys?Thr?Asp?Arg?Lys?Lys385?????????????????390?????????????????395?????????????????400Ala?Glu?Glu?Glu?Ala?Lys?Arg?Lys?Ala?Ala?Glu?Glu?Asp?Lys?Val?Lys

405?????????????????410?????????????????415Glu?Lys?Pro?Ala?Glu?Gln?Pro?Gln?Pro?Ala?Pro?Ala?Pro?Lys?Thr?Glu

420?????????????????425?????????????????430Lys?Pro?Ala?Pro?Ala?Pro?Lys?Pro?Glu?Asn?Pro?Ala?Glu?Gln?Pro?Lys

435?????????????????440?????????????????445Ala?Glu?Lys?Pro?Ala?Asp?Gln?Gln?Ala?Glu?Glu

450?????????????????455

Claims

1, a kind of isolating polypeptide comprises the terminal aminoacid sequence that intercepts thing of choline binding protein AN-.

2, the isolated polypeptide of claim 1, wherein aminoacid sequence is to describe among the SEQ ID NO 1, comprises its fragment, mutant, variant, analog or derivant.

3, the isolated polypeptide of claim 1, wherein aminoacid sequence is to describe among the SEQ ID NO 3, comprises its fragment, mutant, variant, analog or derivant.

4, the isolated polypeptide of claim 1, wherein aminoacid sequence is to describe among the SEQ ID NO 6.

5, the isolated polypeptide of claim 1, wherein aminoacid sequence is to describe among the SEQ ID NO 7, comprises its fragment, mutant, variant, analog or derivant.

6, the isolated polypeptide of claim 1, wherein aminoacid sequence is to describe among the SEQ ID NO 9, comprises its fragment, mutant, variant, analog or derivant.

7, a kind of isolating polypeptide comprises the terminal aminoacid sequence that intercepts thing of choline binding protein AN-, has SEQ ID NO 24 described aminoacid, and wherein this polypeptide shows its tertiary structure.

8, the isolated polypeptide of claim 7, wherein said tertiary structure is corresponding with the structure in being present in native protein.

9, the isolated polypeptide of claim 7, wherein said polypeptide are by preparing with hydroxylamine cleavage total length choline binding protein A, and wherein azanol produces the terminal intercepting of the N-thing of choline binding protein A thus at aminoacid 475 places cracking choline binding protein A.

10, the separating analogous thing of the polypeptide of claim 1.

11, the isolated polypeptide of claim 10, wherein analog comprises the aminoacid sequence with N-terminal methionine or the terminal polyhistidyl of N-.

12, the isolated polypeptide of claim 1, wherein said fragment are the proteolytic digestion products of polypeptide.

13, a kind of isolating polypeptide comprises the aminoacid sequence of the terminal intercepting of choline binding protein AN-thing, and wherein polypeptide has activity of lectin and not in conjunction with choline.

14, a kind of isolating immunogenic polypeptide comprises the terminal aminoacid sequence that intercepts thing of choline binding protein AN-.

15, the immunogenic polypeptide of claim 14, wherein aminoacid sequence is to describe among the SEQ ID NO 1, comprises its fragment, mutant, variant, analog or derivant.

16, the immunogenic polypeptide of claim 14, wherein aminoacid sequence is to describe among the SEQ ID NO 3, comprises its fragment, mutant, variant, analog or derivant.

17, the immunogenic polypeptide of claim 14, wherein aminoacid sequence is to describe among the SEQ ID NO 7, comprises its fragment, mutant, variant, analog or derivant.

18, the immunogenic polypeptide of claim 14, wherein aminoacid sequence is to describe among the SEQ ID NO 9, comprises its fragment, mutant, variant, analog or derivant.

19, a kind of isolating nucleic acid of coded polypeptide, described polypeptide comprise the aminoacid sequence of the terminal intercepting of choline binding protein AN-thing.

20, the isolating nucleic acid of claim 19, its amplifying nucleic acid are to describe among the SEQ ID NO 12, comprise its fragment, mutant, variant, analog or derivant.

21, the isolating nucleic acid of claim 19, its amplifying nucleic acid are to describe among the SEQ ID NO 14, comprise its fragment, mutant, variant, analog or derivant.

22, the isolating nucleic acid of claim 19, its amplifying nucleic acid are to describe among the SEQ ID NO 17, comprise its fragment, mutant, variant, analog or derivant.

23, the isolating nucleic acid of claim 19, its amplifying nucleic acid are to describe among the SEQ ID NO 19, comprise its fragment, mutant, variant, analog or derivant.

24, the isolating nucleic acid of claim 19, its amplifying nucleic acid is DNA.

25, the isolating nucleic acid of claim 19, its amplifying nucleic acid is cDNA.

26, the isolating nucleic acid of claim 19, its amplifying nucleic acid is a genomic DNA.

27, the isolating nucleic acid of claim 19, its amplifying nucleic acid is RNA.

28, the isolating nucleic acid of claim 19, it can be connected on the transcribe rna promoter effectively.

29, a kind of carrier comprises the nucleic acid molecules of claim 19.

30, the carrier of claim 29, wherein promoter comprises antibacterial, yeast, insecticide or mammalian promoter.

31, the carrier of claim 30, wherein said carrier are plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage or eucaryon viral DNA.

32, a kind of host carrier system of producing polypeptide, it comprises the carrier of claim 30 in proper host cell.

33, the host carrier system of claim 32, wherein proper host cell comprises protokaryon or eukaryotic cell.

34, a kind of cell line, it comprises the nucleic acid of claim 19.

35, a kind of method that obtains the purified form polypeptide, this method comprises:

(a) carrier of claim 19 is introduced in the proper host cell;

(b) cultivate the gained host cell so that produce polypeptide;

(c) polypeptide that produces in the recycling step (b); With

(d) polypeptide that reclaims in the purification step (c).

36, a kind of can specificity in conjunction with the antibody of the polypeptide of claim 1 or 7.

37, the antibody of claim 36, wherein antibody is monoclonal antibody.

38, the antibody of claim 36, wherein antibody is polyclonal antibody.

39, the antibody of claim 36, wherein antibody is chimeric (bispecific) antibody.

40, a kind of pharmaceutical composition, it contains polypeptide and the pharmaceutically suitable carrier or the diluent of a certain amount of claim 1.

41, a kind of method of induction of immunity reaction in being exposed to or having infected pneumococcal individuality comprises the pharmaceutical composition that gives a certain amount of claim 40 of described individuality, induction of immunity reaction thus.

42, a kind of individuality that prevents is comprised giving the pharmaceutical composition that described individuality effectively prevents the claim 40 of streptococcus pneumoniae adhesion amount by the method for pneumococcal infection, prevents pneumococcal infection thus.

43, the method for claim 42, wherein pharmaceutical composition is released in respiratory tract or the nasopharynx.

44, a kind of individuality that prevents is comprised giving described individuality a certain amount of antibody of claim 36 and pharmaceutical composition of pharmaceutically suitable carrier or diluent of containing by the method for pneumococcal infection, prevents pneumococcal infection thus.

45, a kind of vaccine, it comprises polypeptide and the pharmaceutically acceptable adjuvant or the carrier of claim 1.

46, the vaccine of claim 45, wherein polypeptide has SEQ ID NOS:1,3-7,9-11,22 and 23 described arbitrary aminoacid sequences.

47, the vaccine of claim 45, wherein polypeptide comprises the aminoacid sequence of the terminal intercepting of the described choline binding protein AN-of Fig. 2 thing.

48, a kind of vaccine, it comprises polypeptide and pharmaceutically acceptable adjuvant or the carrier with the aminoacid sequence that contains the described conserved region of Fig. 2.

49, the vaccine of claim 48, conserved region is selected from the aminoacid sequence of 158-172,300-321,331-339,355-365,367-374,379-389,409-427 and 430-447.

50, a kind of vaccine, it comprises isolating nucleic acid and the pharmaceutically acceptable adjuvant or the carrier of coding claim 1 polypeptide.

51, a kind of vaccine, it comprises isolating nucleic acid and the pharmaceutically acceptable adjuvant or the carrier of claim 19.

52, a kind of vaccine, it comprises carrier and the pharmaceutically acceptable adjuvant or the carrier of claim 29.

53, a kind of method that has infected or be exposed to pneumococcal individuality for the treatment of comprises the claim 45-51 or 52 vaccine, the treatment individuality thus that give described individual treatment effective dose.