CN1239999A - Truncated platelet-activating factor acetylhydrolase - Google Patents

Abstract

The present invention provides purified and isolated polynucleotide sequences encoding human plasma platelet-activating factor acetylhydrolase. Also provided are materials and methods for the recombinant production of platelet-activating factor acetylhydrolase products which are expected to be useful in regulating pathological inflammatory events.

Description

The platelet-activating factor acetylhydrolase of brachymemma

Invention field

The present invention relates generally to platelet-activating factor acetylhydrolase, relate more specifically to new coding human plasma platelet-activating factor acetylhydrolase purifying with isolating polynucleotide, relate to by the platelet-activating factor acetylhydrolase product of described polynucleotide encoding, relate to the recombinant production of platelet-activating factor acetylhydrolase product material and method, relate to the specific antibody materials of platelet-activating factor acetylhydrolase.

Background of invention

Platelet activating factor (PAF) is by various cell type synthetic biologic activity phosphatide.In the body and 10 ^-10To 10 ^-9During the normal concentration scope of M, PAF activates the target cell [Venable etc., J.Lipid Res., 34:691-701 (1993)] such as thrombocyte and neutrophilic granulocyte by combining with specificity G albumen coupling cell surface receptor.PAF has 1-O-alkyl-2-ethanoyl-sn-glyceryl-3-phosphorylcholine structure.For reaching best biologic activity, the sn-1 position of PAF glycerol backbone must be and the ehter bond of fatty alcohol that the sn-3 position must have the phosphorylcholine headgroup.

PAF (for example works in normal physiological processes, inflammation, hemostasis and childbirth), and with the pathology inflammatory reaction (for example, asthma, anaphylaxis, septic shock and sacroiliitis) [Venable etc., see above-mentioned, with Lindsberg etc., Ann.Neurol., 30:117-129 (1991)] relevant.PAF relates to the possibility of pathologic reaction and has impelled the activity of attempting regulating PAF, and the principal focal point of these trials is the active antagonists of exploitation PAF, and described antagonist interference PAF combines with cell surface receptor.Referring to, for example, Heuer etc., Clin.Exp.Allergy, 22:980-983 (1992).

It seems that the synthetic and secretion of PAF and degraded thereof and removing be subjected to strict control.The excessive generation of the malfunctioning generation of PAF regulation mechanism, produce inadequately or lack degraded, cause the pathology inflammatory effect of PAF, in this respect, regulate the active alternative method of PAF and will be referred to simulate or increase the natural process that inflammation disappears and takes place.Scavenger cell [Stafforini etc. have been reported, J.Biol.Chem., 265 (17): 9682-9687 (1990)], liver cell and human liver cell oncocyte are HepG2[Satoh etc., J.Clin.Invest, 87:476-481 (1991) and Tarbet etc., J.Biol.Chem., 266 (25): 16667-16673 (1991)] PAF PAF-AH (PAF-AH) enzymic activity of release passivation PAF.Except passivation PAF, PAF-AH is the phosphatide of passive oxidation fragmentation also, such as the product of the arachidonic acid cascade of transmitting inflammation.Referring to, Stremler etc., J.Biol.Chem., 266 (17): 11095-11103 (1991).PAF-AH mainly takes place by the sn-2 ethanoyl of hydrolysis PAF the passivation of PAF, and PAF-AH is by removing sn-2 acyl group metabolism oxidation fragmentation phosphatide.Differentiated two types PAF-AH: tenuigenin form that (in endotheliocyte and red corpuscle) found in various cell types and tissue and the extracellular form of in blood plasma and serum, finding.The phosphatide that not hydrolysis of blood plasma PAF-AH is complete, except PAF, this substrate specificity allows this enzyme to circulate in vivo and have no adverse effect with complete active condition.It seems that blood plasma PAF-AH [Stafforini etc., J.Biol.Chem., 262 (9): 4223-4230 (1987)] are responsible in the degraded of all PAF in the human blood in vitro.

Though the tenuigenin form of PAF-AH it seems to have identical substrate specificity with the blood plasma form, blood plasma PAF-AH has makes it be different from the biochemical characteristics of tenuigenin PAF-AH and other lipase that has characterized.Particularly, blood plasma PAF-AH is associated with hdl particle, is suppressed by diisopropylfluorophosphate, is not subjected to calcium ion effect, and to the protease hydrolysis relative insensitivity, the performance molecular weight is 43,000 dalton.Referring to, Stafforini etc. (1987) see above-mentioned.The same piece of writing article of Stafforini etc. has been described from the program of human plasma fraction's purifying PAF-AH and has been utilized the amino acid of the blood plasma material that this program obtains to form.Stafforini etc., J.Biol.Chem., 268 (6) 3857-3865 (1993) report from red corpuscle purifying cells matter PAF-AH, also described 10 n terminal residues of tenuigenin PAF-AH in this articles.Hattori etc., J.Biol.Chem., 268 (25): 18748-18753 (1993) has described from ox brain purifying cells matter PAF-AH.After its female application is submitted to, Hattori etc., J.Biol.Chem., 269 (237): 23150-23155 (1994) has delivered the nucleotide sequence of ox brain cell matter PAF-AH.On January 5 nineteen ninety-five, during after its female application is submitted to three months, delivered the relevant phospholipase A of lipoprotein among Smithkline Beecham PLC Patent Cooperation Treaty (PCT) the international publication number WO95/00649 ₂(Lp-PLA ₂) nucleotide sequence.Compare Lp-PLA with the nucleotide sequence of PAF-AH of the present invention ₂Nucleotides sequence be listed on the position variant.This nucleotide difference (corresponding to the position 1297 of SEQ ID NO:7) causes by the amino acid difference between the enzyme of described polynucleotide encoding.The amino acid that is positioned at the position 379 of SEQ ID NO:8 is Xie Ansuan, and Lp-PLA ₂The amino acid of correspondence position is L-Ala.In addition, the nucleotide sequence of PAF-AH of the present invention is included in described Lp-PLA ₂20 bases of 124 bases of non-existent 5 ' end and 3 ' end in the sequence.After three months, April 10 nineteen ninety-five, a Lp-PLA ₂Sequence is stored in GenBank with registration number U24577, and this sequence is compared on 11 positions with the nucleotide sequence of PAF-AH of the present invention and had any different.Described nucleotide difference (corresponding to the position 79,81,84,85,86,121,122,904,905,911,983 and 1327 of SEQ ID NO:7) causes by four amino acid differences between the enzyme of described polynucleotide encoding.The position 249,250,274 of SEQ ID NO:8 and 389 amino acid are respectively Methionin, aspartic acid, phenylalanine and leucine, and the amino acid of correspondence position is respectively Isoleucine, arginine, leucine and Serine in the described GenBank sequence.

The recombinant production of PAF-AH will make can be used external source PAF-AH imitation or increase the normal processes that inflammation disappears in the body.Give PAF-AH the physiology advantage that is better than giving paf receptor antagonists is provided, because PAF-AH is the normal product of finding in blood plasma.In addition, because suppress natural PAF-AH activity, stoped the metabolism of needed PAF metabolism and oxidation fragmentation phosphatide thus with the paf receptor antagonists of PAF structurally associated.Therefore, the activity of paf receptor antagonists inhibition PAF-AH has been resisted the competitiveness obstruction of described antagonist to paf receptor.Referring to, Stremler etc. see above-mentioned.In addition, at the acute inflammation position, for example, the release of oxygenant causes the passivation of natural PAF-AH enzyme, and then causes PAF and PAF sample compound local horizontal to raise, and the paf receptor antagonists competition that described compound will give with any external source combines with paf receptor.In contrast, treat the endogenous enzyme that will increase endogenous PAF-AH activity and compensate any passivation with reorganization PAF-AH.

Therefore in this area, need to differentiate material and the method that can be used for the PAF-AH recombinant production with the polynucleotide sequence that separates coding human plasma PAF-AH, needs exploitation, need preparation to detect the reagent of PAF-AH in the blood plasma.

Brief summary of the invention

The invention provides the segmental new purifying of coding human plasma PAF-AH or its enzymic activity with isolating polynucleotide (that is, sense strand and antisence strand dna and RNA).Preferred dna sequence dna of the present invention comprises the dna sequence dna of genome sequence and cDNA sequence and all or part of chemosynthesis.The present invention considered to be set forth in SEQID NO:7 coding PAF-AH dna sequence dna and under the standard stringent condition with the dna sequence dna of its noncoding strand hybridization or if not since the Feng Yu of genetic code will with the dna sequence dna of its hybridization.The present invention has also considered the bioautography thing (that is, in the body or the copy of the DNA isolation sequence of external generation) of dna sequence dna of the present invention.Also provide the DNA of the PAF-AH that such as the plasmid that adds the PAF-AH sequence and viral DNA carrier, especially wherein encodes to may be operably coupled to the self-replicating recombination to construct thing of the carrier of endogenous or heterogenous expression control dna sequence dna and transcription terminator.

According to another aspect of the present invention, so that the mode that the PAF-AH that needs expresses therein stably transforms protokaryon or eukaryotic host cell with dna sequence dna of the present invention.The host cell of expressing the PAF-AH product can be various useful purpose services.This cell has been formed valuable immunogen source with the immunoreactive antibody materials of PAF-AH specifically for exploitation.Host cell of the present invention is useful significantly in the method for scale operation PAF-AH, wherein said cell grows in the suitable medium, separates the polypeptide product that needs by for example immunoaffinity purification from the substratum of described cell or the growth of described cell.

The present invention considers is used for may further comprise the steps from the non-immunological method of blood plasma purifying PAF-AH: (a) separate low-density lipoprotein particle; (b) described low-density lipoprotein particle is dissolved in the damping fluid that comprises 10mM CHAPS, to produce first PAF-AH enzyme solution; (c) with DEAE anion-exchange column on described first PAF-AH enzyme solution; (d) use the about pH7.5 damping fluid that comprises 1mMCHAPS to wash described DEAE anion-exchange column; (e) use comprises about pH7.5 damping fluid of 0-0.5M NaCl gradient from the described DEAE anion-exchange column wash-out PAF-AH of branch enzyme; (f) will merge from the fraction collection liquid with PAF-AH enzymic activity of described DEAE anion-exchange column wash-out; (g) regulate the active fraction collection liquid of merging that obtains from described DEAE anion-exchange column to 10mM CHAPS, to produce second PAF-AH enzyme solution; (h) with sample on described second PAF-AH enzyme solution to blue dyestuff part affinity column; (i) use comprises the damping fluid of 10mM CHAPS and chaotropic salt from described blue dyestuff part affinity column wash-out PAF-AH enzyme; (j) with sample on the elutriant of described blue dyestuff part affinity column to Cu part affinity column; (k) use comprises the damping fluid of 10mM CHAPS and imidazoles from described Cu part affinity column wash-out PAF-AH enzyme; (l) elutriant with described Cu part affinity column carries out SDS-PAGE; (m) separate about 44kDa PAF-AH enzyme from described SDS-polyacrylamide gel.Preferably, the damping fluid of step (b) is 25mM Tris-HCl, 10mM CHAPS, pH7.5; The damping fluid of step (d) is 25mM Tris-HCl, 1mM CHAPS; The post of step (h) is a Blue SepharoseFast Flow post; The damping fluid of step (i) is 25mM Tris-HCl, 10mM CHAPS, 0.5MKSCN, pH7.5; The post of step (j) is a Cu chelating Sepharose post; And the damping fluid of step (k) is 25mM Tris-HCl, 10mM CHAPS, 0.5M NaCl, 50mM imidazoles, and the pH scope is about 7.5-8.0.

The present invention considers is used for may further comprise the steps from the method for the active PAF-AH of intestinal bacteria purifying enzyme that produces PAF-AH: (a) from producing the colibacillary lysate preparative centrifugation supernatant liquor of PAF-AH enzyme; (b) with sample on the described centrifuged supernatant to blue dyestuff part affinity column; (c) use comprises the damping fluid of 10mM CHAPS and chaotropic salt from described blue dyestuff part affinity column wash-out PAF-AH enzyme; (d) with sample on the described elutriant of described blue dyestuff part affinity column to Cu part affinity column; (e) use comprises the damping fluid of 10mM CHAPS and imidazoles from described Cu part affinity column wash-out PAF-AH enzyme.Preferably, the post of step (b) is a Blue Sepharose Fast Flow post; The damping fluid of step (c) is 25mM Tris-HCl, 10mM CHAPS, 0.5M KSCN, pH7.5; The post of step (d) is a Cu chelating Sepharose post; The damping fluid of step (e) is 25mMTris-HCl, 10mM CHAPS, 0.5M NaCl, 100mM imidazoles, pH7.5.

The present invention considers is used for may further comprise the steps from the other method of the active PAF-AH of intestinal bacteria purifying enzyme that produces PAF-AH: (a) from producing the colibacillary lysate preparative centrifugation supernatant liquor of PAF-AH enzyme; (b) the described centrifuged supernatant of dilution in comprising the low pH damping fluid of 10mM CHAPS; (c) with sample on the centrifuged supernatant of described dilution as for about pH 7.5 equilibrated cationic exchange coloums; (d) use 1M salt from described cationic exchange coloum wash-out PAF-AH enzyme; Improve the pH of the described elutriant of described cationic exchange coloum, the salt concn of described elutriant is adjusted to about 0.5M salt; (f) with sample on the described adjusting elutriant of described cationic exchange coloum to blue dyestuff part affinity column; (g) use comprise about 2M extremely the damping fluid of about 3M salt from described blue dyestuff part affinity column wash-out PAF-AH enzyme; (h) use the dialyse described elutriant of described blue dyestuff part affinity column of the damping fluid comprise about 0.1% tween.Preferably, the damping fluid of step (b) is 25mMMES, 10mM CHAPS, 1mM EDTA, pH4.9; The post of step (c) is an equilibrated Ssepharose post in 25mMMES, 10mM CHAPS, 1mM EDTA, 50mM NaCl, pH5.5; Use 1mM NaCl wash-out PAF-AH in the step (d); The pH of elutriant is adjusted to pH7.5 with 2M Tris alkali in the step (e); Post in the step (f) is the sepharose post; The damping fluid of step (g) is 25mM Tris, 10mM CHAPS, 3M NaCl, 1mM EDTA, pH7.5; Damping fluid in the step (h) is 25mM Tris, 0.5M NaCl, 0.1% tween 80, pH7.5.

The present invention considers is used for may further comprise the steps from the method again of the active PAF-AH of intestinal bacteria purifying enzyme: (a) preparation produces the intestinal bacteria extracting solution of dissolved PAF-AH supernatant liquor after containing the damping fluid bacteriolyze of CHAPS; (b) dilute described supernatant liquor, last sample is as for about pH8.0 equilibrated anion-exchange column; (c) from described anion-exchange column wash-out PAF-AH enzyme; (d) with sample on the described adjusting elutriant of described anion-exchange column to blue dyestuff part affinity column; (e) use the described blue dyestuff part affinity column of buffer solution elution that comprises 3.0M salt; (f) described blue dyestuff elutriant is diluted into the suitable buffer that is used to carry out hydroxyapatite (hydroxylapatite) chromatography; (g) carry out hydroxyapatite chromatography, wherein use damping fluid (containing or do not contain CHAPS) to finish washing and wash-out; (h) described hydroxyapatite elutriant is diluted to suitable salt concn to carry out cation-exchange chromatography; (i) with sample on the hydroxyapatite elutriant of described dilution to the cationic exchange coloum of the about 6.0-7.0 of pH scope; (j) use suitable preparation damping fluid from described cationic exchange coloum wash-out PAF-AH; (k) carry out cation-exchange chromatography at low temperature; (l) lacking under the situation of CHAPS with liquid or frozen form preparation PAF-AH.

Preferably, the lysis buffer in the above-mentioned steps (a) is 25mM Tris, 100mMNaCl, 1mM EDTA, 20mM CHAPS, pH8.0; In step (b), the supernatant liquor that will be used for anion-exchange chromatography is 3-4 times of 25mM Tris, 1mM EDTA, 10mM CHAPS, pH8.0 dilution, and described post is an equilibrated Q-Sepharose post in 25mM Tris, 1mM EDTA, 50mM NaCl, 10mM CHAPS, pH8.0; Use 25mMTris, 1mM EDTA, 350mM NaCl, 10mM CHAPS, the described anion-exchange column of pH8.0 wash-out in the step (c); In the step (d) directly with sample on the elutriant of step (c) to blue dyestuff affinity column; Use 3M NaCl, 10mM CHAPS, 25mM Tris, the described post of pH8.0 buffer solution elution in the step (e); In the step (f) by in 10mM sodium phosphate, 100mM NaCl, 10mMCHAPS, pH6.2, diluting, finish the dilution of the described blue dyestuff elutriant that is used for hydroxyapatite chromatography; Use in the step (g) and finish hydroxyapatite chromatography, use 50mM sodium phosphate, 100mMNaCl (contain or do not contain) 10mM CHAPS, pH7.5 to finish wash-out with 10mM sodium phosphate, 100mM NaCl, 10mM CHAPS equilibrated hydroxyapatite column; In the step (h) by at the about 6.0-7.0 of pH scope, comprise in the sodium phosphate damping fluid of (containing or do not contain CHAPS) and dilute, finish the dilution of the described hydroxyapatite elutriant that is used for cation-exchange chromatography; Step (i) is middle with 50mM sodium phosphate, (contain or do not contain) 10mM CHAPS, pH6.8 balance S Sepharose post; Use such as the preparation damping fluid of the 50mM potassiumphosphate that contains 0.01% tween 80,12.5mM aspartic acid, 125mM NaCl, pH7.5 in the step (j) and finish wash-out; Finish cation-exchange chromatography in 2-8 ℃ in the step (k).Being used for step (l) stablizes the example of the suitable preparation damping fluid of PAF-AH and comprises 50mM potassiumphosphate, 12.5mM aspartic acid, 125mM NaCl pH7.4 (about value, contain or not tween 80 and or Pluronic F68) or 25mM potassium phosphate buffer, contain (at least) 125mM NaCl, 25mM arginine and 0.01% tween 80 (containing or do not contain about 0.1 and 0.5% Pluronic F68).

The present invention considers is used for may further comprise the steps from the another method of the active rPAF-AH product of intestinal bacteria purifying enzyme: (a) preparation produces the intestinal bacteria extracting solution of dissolved rPAF-AH product supernatant liquor after containing the damping fluid bacteriolyze of Triton X-100; (b) dilute described supernatant liquor, last sample is as for the affine exchange column of about pH8.0 equilibrated immobilization metal; (c) with comprising the damping fluid of imidazoles from the affine exchange column wash-out of described immobilization metal rPAF-AH product; (d) regulate salt concn, with sample on the described elutriant of the affine exchange column of described immobilization metal to hydrophobic interaction post (HIC#1); (e) by reducing salt concn and/or increasing the described HIC#1 of detergent concentration wash-out; (f) with the titration of described HIC#1 elutriant to about pH6.4; (g) with sample on the HIC#1 elutriant of described adjusting as for about pH6.4 equilibrated cationic exchange coloum (CEX#1); (h) use concentration? the described CEX#1 of sodium-chlor wash-out; (i) with sodium-chlor with described CEX#1 elutriant to about 2.0M concentration; (j) with sample on the CEX#1 elutriant of described adjusting as for about pH8.0 and about 2.0M sodium-chlor equilibrated hydrophobic interaction post (HIC#2); (k) by reducing salt concn and/or increasing the described HIC#2 of detergent concentration wash-out; (l) dilution described HIC#2 elutriant and be adjusted to about pH6.0; (m) with sample on the HIC#2 elutriant of described adjusting as for about pH6.0 equilibrated cationic exchange coloum (CEX#2); (n) with suitable preparation damping fluid from the described rPAF-AH product of described CEX#2 wash-out.

Preferably, the lysis buffer in the above-mentioned steps (a) is 90mM TRIS, 0.125% TritonX-100,0.6M NaCl, pH8.0, carries out bacteriolyze in high-pressure homogenizer; In the step (b) described supernatant liquor is diluted in level pad (20mM TRIS, 0.5M NaCl, 0.1% Triton X-100, pH8.0), with zinc chelate column (Chelating Sepharose Fast Flow, Pharmacia, Uppsala, Sweden) the dress post, with the level pad balance, with sample on the supernatant liquor of described dilution, with 20mM TRIS, 0.5M NaCl, 4M urea, 0.1% Triton X-100, pH8.0 washing, then wash with 20mM TRIS, 0.5M NaCl, 0.02% Triton X-100, pH8.0; Use 20mM Tris, 50mM imidazoles, 0.02% Triton X-100, pH8.0 to finish wash-out in the step (c); In the step (d) elutriant is adjusted to 1mM EDTA and 2M NaCl, with Phenyl Sepharose 6 Fast Flow (Pharmacia) level pad (2.0M NaCl, 25mM Tris, 0.02% Triton X-100, pH8.0) balance, in room temperature with the elutriant of adjusting of step (c) on sample, with the level pad washing, with 30cm/ hour flow velocity 25mMNaPO ₄, 0.02% Triton X-100, pH6.5 the washing; Use 25mM NaPO in the step (e) ₄, 3% Triton X-100, pH6.5 finish wash-out; (Bio-Rad Labs, Richmond is CA) with level pad (20mM NaPO with Macro-Prep High S post in the step (g) ₄, 0.02%Triton X-100, pH6.4) balance, with sample on the elutriant of adjusting of step (f), with the level pad washing, with 25mM Tris, 0.02% Triton X-100, pH8.0 washing; Finish wash-out with 25mM Tris, 0.02% Triton X-100,1.3M NaCl, pH8.0 in the step (h); In the step (j) with Bakerbond Wide Pore Hi-Propyl C ₃(Baker, Phillipsburg, NJ) with level pad (2.0M NaCl, 25mM Tris, 0.02% Triton X-100, pH8.0) balance, with the adjusting elutriant of step (i) on room temperature sample, with the level pad washing, with 30cm/ hour with 25mM Tris, 0.02% Triton X-100, pH8.0 washing; Finish wash-out with 10mMTris, 3.0% Triton X-100, pH8.0 in the step (k); Dilution in level pad (20mM succinate, 0.1% PLURONIC F68, pH6.0) in the step (l); In the step (m) with SP Sepharose Fast Flow (Pharmacia) post with the level pad balance of step (l), with sample on the elutriant of step (l), wash with level pad; Use 50mM NaPO in the step (n) ₄, 0.7M NaCl, 0.1% PLURONIC F68,0.02% tween 80, pH7.5.

The PAF-AH product can obtain as the isolate in n cell source or can chemosynthesis, but preferably produces by the reorganization program that relates to protokaryon of the present invention or eukaryotic host cell.Considered to have part or all PAF-AH product of the aminoacid sequence that is set forth among the SEQ ID NO:8.Considered to lack preceding 12 the amino acid whose fragments of N-terminal that reach most of the one-tenth acquaintance PAF-AH aminoacid sequence that is set forth in SEQ ID NO:8 especially, particularly those have the Met of SEQID NO:8 ₄₆, Ala ₄₇Or Ala ₄₈As the amino acid whose fragment of initial N-terminal.Also considered that it lacks the most nearly 30 amino acid whose fragments of C-terminal of the aminoacid sequence of SEQ ID NO:8, particularly those have Ile ₄₂₉And Leu ₄₃₁Fragment as the C-terminal residue.Considered varient or the PAF-AH of PAF-AH again or in the sequence of SEQ ID NO:8, had to be selected from following amino-acid substitution: S108A, S273A, D286A, D286N, D296A, D304A, D338A, H351A, H395A, H399A, C67S, C229S, C291S, C334S, C407S, D286A, D286N and D304A.As mentioned above, the invention provides this class fragment of coding or the segmental polynucleotide of varient (comprising DNA), and produce the method for this class fragment or varient by the host cell growth that comprises this DNA is recombinated.The PAF-AH product that this paper recommends comprises the amino-acid residue Met of coding SEQ ID NO:8 ₄₆To Asn ₄₄₁The protokaryon expression of polypeptides product (called after rPH.2) of DNA and the amino-acid residue Met of coding SEQID NO:8 ₄₆To Ile ₄₂₉The protokaryon expression of polypeptides product (called after rPH.9) of DNA.For example, compare with the corresponding prokaryotic expression product of the DNA that is positioned at translation initiation codon coding PAF-AH total length mature sequence afterwards, rPH.2 and rPH.9 product all show less N-terminal heterogeneity.In addition, rPH9 shows higher C-terminal homogeneity (consistence).Expection uses mammalian host cell to provide may to need the posttranslational modification (for example, myristoylation (myristolation), glycosylation, brachymemma, fatization (lipidation) and tyrosine, Serine or Threonine phosphorylation) of the best biologic activity of giving recombination expression product of the present invention.PAF-AH product of the present invention can be full-length polypeptide, fragment or varient.Varient can comprise the PAF-AH analogue, wherein one or more regulations (promptly, natural coding) aminoacid deletion or replaced, or wherein added one or more non-regulation amino acid: (1) does not lose distinctive enzymic activity of one or more PAF-AH or immunological characteristic; Or (2) make the particular biological loss of activity of PAF-AH specifically.Can use with PAF-AH bonded albumen or other molecule to regulate its activity.

The present invention also considers the specific antibody materials of PAF-AH (for example, antibody of mono-clonal and polyclonal antibody, single-chain antibody, chimeric antibody, CDR-transplanting or the like) and other conjugated protein.Of the present invention specifically described conjugated protein be the monoclonal antibody that produces with hybridoma 90G11D and 90F2D, described hybridoma is preserved in American type culture collection (ATCC) with preserving number HB 11724 and HB 11725 respectively on September 30th, 1994,12301 ParklawnDrive, Rockville, MD 20852.It is conjugated protein that the present invention also describes is the monoclonal antibody that produces with hybridoma 143A, and this hybridoma is preserved in ATCC June 1 nineteen ninety-five, and preserving number is HB 11900.Use is from the PAF-AH of separating plasma, reorganization PAF-AH, PAF-AH varient or express the cell of this product, can differentiate specifically and PAF-AH bonded albumen or other molecule (for example, lipid or small molecules).Conjugated proteinly can be used for immune composition conversely and be used for purifying PAF-AH, and can be used for detecting or the quantitative PAF-AH of body fluid or tissue samples by known immunology program.Also considered the specific antiidiotypic antibody of PAF-AH specific antibody.

The scientific value of the information of contributing in DNA of the present invention and aminoacid sequence disclosure is tangible.As a series of example, the knowledge of the cDNA sequence of PAF-AH makes can separate the genomic dna sequence of the PAF-AH expression regulating and controlling sequence of coding PAF-AH and specialization (specifying) such as promotor, operator gene or the like by DNA/DNA hybridization.The DNA/DNA hybridization program of under the strict standard condition of this area, using dna sequence dna of the present invention to carry out, might expect other structurally associated protein of making it possible to separate coding PAF-AH allelic variant, one or more biological chemistries of sharing PAF-AH and/or immunological properties and with PAF-AH homologous non-human species protein DNA.Dna sequence dna information provided by the invention makes can be tactful by homologous recombination or " rejectings " [referring to, for example, Kapecchi, Science, 244:1288-1292 (1989)], development can not expressive function PAF-AH enzyme or is expressed the rodent of variation PAF-AH enzyme.Polynucleotide of the present invention can be used for detecting the hybridization assays that cell synthesizes the ability of PAF-AH when suitable mark.Polynucleotide of the present invention can also be to be used for differentiating that the PAF-AH locus constitutes the basis of diagnostic method of hereditary change on the basis of one or more morbid states.The present invention also makes and can obtain expressing relevant antisense polynucleotides with the PAF-AH of those cells of regulating normal expression PAF-AH.

It is particularly human to alleviate the pathology inflammatory condition to have considered to give mammalian subject with PAF-AH preparation of the present invention.Relate to the pathology inflammatory condition based on PAF, give PAF-AH and be suitable for for example treating asthma [Miwa etc., J.Clin.Invest., 82:1983-1991 (1988); Hsieh etc., J.Allergy Clin.Immunol., 91:650-657 (1993); With Yamashita etc., Allergy, 49:60-63 (1994)], anaphylaxis [Venable etc., see above-mentioned], shock [Venable etc., see above-mentioned], reperfusion injury and central nervous system local asphyxia [Lindsberg etc. (1991), see above-mentioned], sacroiliitis [the Zarco etc. of antigen induction, Clin.Exp.Immunol., 88:318-323 (1992)], atheroma forms [Handley etc., Drug Dev.Res., 7:361-375 (1986)], regional ileitis [Denizot etc., Digestive Diseases and Sciences, 37 (3): 432-437 (1992)], ischemic bowel necrosis/necrotizing enterocolitis [Denizot etc., see above-mentioned and Caplan etc., Acta Paediatr., Suppl.396:11-17 (1994)], ulcerative colitis (Denizot etc., see above-mentioned), ischemic stroke [Satoh etc., Stroke, 23:1090-1092 (1992)], ischemic brain injury [Lindsberg etc., Stroke, (1991) such as 21:1452-1457 (1990) and Lindsberg, see above-mentioned], systemic lupus erythematous [Matsuzaki etc., Clinica Chimica Acta, 210:139-144 (1992)], acute pancreatitis [Kald etc., Pancreas, 8 (4): 440-442 (1993)], septicemia (Kald etc., see above-mentioned), suis property glomerulonephritis [Mezzano etc. after the acute infection, J.Am.Soc.Nephrol., 4:235-242 (1993)], pulmonary edema [the Rabinovici etc. that cause by the IL-2 therapeutics, J.Clin.Invest., 89:1669-1673 (1992)], alterative inflammation [Watanabe etc., Br.J.Pharmacol., 111:123-130 (1994)], ischemic renal failure [Grino etc., Annals of InternalMedicine, 121 (5): 345-347 (1994); Premature labor [Hoffman etc., Am.J.ObstetGynecol., 162 (2): 525-528 (1990) and Maki etc., Proc.Natl.Acad.Sci.USA, 85:728-732 (1988)]; Adult respiratory distress syndrome [Rabinovici etc., J.ApplPhysiol., 74 (4): 1791-1802 (1993); Matsumoto etc., Clin.Exp.Pharmacol.Physiol., 19:509-515 (1992); With Rodriguez-Roisin etc., J.Clin.Invest., 93:188-194 (1994)].Also considered to use the human immunodeficiency virus (HIV) of PAF-AH prepared product treatment central nervous system to infect." treatment " used herein comprises preventative and therapeutic treatment.

The animal model of many aforementioned pathologic conditions has been described in this area.The mouse model of asthma and rhinitis for example, has been described among the embodiment 16 of this paper; At Zarco etc., see and described arthritic rabbit model in above-mentioned; At Furukawa etc., Ped.Res., 34, (2): 237-241 (1993) and Caplan etc. see the rabbit model of having described ischemic bowel necrosis/necrotizing enterocolitis in above-mentioned; At Lindsberg etc., (1990), the rabbit model of apoplexy of having seen foregoing description; At Matsuzaki etc., see the mouse model of having described lupus in above-mentioned; At Kald etc., see the rat model of having described acute pancreatitis in above-mentioned; At Rabinovici etc., see the rat model of having described the pulmonary edema that causes by the IL-2 therapeutics in above-mentioned; At Watanabe etc., see above-mentioned) in the rat model of alterative inflammation has been described; In Watson etc., Transplantation, 56 (4): the canine model of having described renal homotransplantation among the 1047-1049 (1993); At Rabinovici etc., see above-mentioned and Lellouch-Tubiana respectively, Am.Rev.Respir. Dis. has described the rat and the guinea pig model of adult respiratory distress syndrome among the 137:948-954 (1988).

PAF-AH composition in the mammiferous method that the present invention has considered to be used for the treatment of the pathologic conditions susceptible that PAF-AH is mediated or to suffer from this illness especially, the amount that comprises to be enough to pathology amount PAF in additional endogenous PAF-AH activity and the described Mammals of passivation gives described Mammals with PAF-AH.

Treatment/pharmaceutical compositions that the present invention considers comprises PAF-AH product and physiology acceptable diluent or carrier, can also comprise the other medicines with anti-inflammation effect.The dosage of indicating will be enough to replenish the PAF of endogenous PAF-AH activity and passivation pathology amount.Consider about general dosage, referring to Remmington ' s Pharmaceutical Sciences, the 18th edition, MackPublishing Co., Easton, PA (1990).Dosage will change between the about 1000 μ g PAF-AH/kg body weight of about 0.1-.Therapeutic composition of the present invention can give by different approaches, depends on the pathologic conditions of desire treatment.For example, can give by intravenously, subcutaneous, oral, suppository and/or lung approach.

For the pathologic conditions of lungs, indicated especially by the lung approach to give PAF-AH.Consider to be used for that lung gives is miscellaneous transfer device, comprise that for example atomizer, metering administration sucker and powder inhalator all are this area standards.Various albumen are passed to the lungs and the recycle system, Adjei etc., Pharm.Res., 7 (6): 565-569 (1990) (leuprorelin acetate) following the description by sucking aerosol preparations; Braquet etc., J.Cardio.Pharm., 13 (Supp.5): s.143-146 (1989) (endothelin-1); Hubbard etc., Annals ofInternal Medicine, III (3), 206-212 (1989) (alpha1-antitrypsin); Smith etc., J.Clin.Invest., 84:1145-1146 (1989) (α-1-proteinase inhibitor); Debs etc., J.Immunol., 140:3482-3488 (1933) (reorganization IFN-and tumor necrosis factor alpha); In disclosed Patent Cooperation Treaty on September 15 (PCT) international publication number WO94/20069 (reorganization Pegylation granulocyte colony-stimulating factor) in 1994.

Brief Description Of Drawings

Considering following detailed description the in detail and reference wherein during accompanying drawing, countless others of the present invention and advantage will be conspicuous:

Fig. 1 is the photo that contains from the pvdf membrane of the PAF-AH of human plasma purifying;

Fig. 2 is the chart that shows recombinant human blood plasma PAF-AH enzymic activity.

Fig. 3 describes to recombinate the diagram diagram of PAF-AH fragment and catalytic activity thereof.

Fig. 4 describe to recombinate mass spectrum result of PAF-AH product rPH.2.

Fig. 5 describe to recombinate mass spectrum result of PAF-AH product rPH.9.

Fig. 6 is the histogram of describing by topical administration reorganization of the present invention PAF-AH blocking-up PAF inductive rat podedema.

Fig. 7 is a histogram of describing to give by intravenously PAF-AH blocking-up PAF inductive rat podedema.

Fig. 8 shows PAF-AH blocking-up PAF inductive oedema and the histogram of not blocking zymosan A inductive oedema.

Fig. 9 A and 9B show the dose response result of the anti-inflammatory activity of PAF-AH in the rat podedema.

The result that Figure 10 A and 10B show shows interior effectiveness of body of the single dose PAF-AH of process in time.

Figure 11 is a graphic representation of representing the pharmacokinetics of PAF-AH in the rat circulation;

Figure 12 is the histogram of the comparison of the PAF antagonist that shows that the antiphlogistic effects of PAF-AH in the rat podedema and effect are relatively poor.

The result that Figure 13 shows shows that the PAF-AH neutralization is from the apoptosis effect of HIV-1 infection and the monocytic conditioned medium of activated.

Detailed Description Of The Invention

Following examples have been described the present invention. Embodiment 1 has showed from human plasma purifying PAF-AH New method. Embodiment 2 has described the little sequence of amino acid of the human plasma PAF-AH of purifying. The clone of the full-length cDNA of encoding human blood plasma PAF-AH has been described among the embodiment 3. At embodiment 4 In the supposition splicing variants of differentiating human plasma PAF-AH gene has been described. In embodiment 5, retouch Stated the clone of the genome sequence of encoding human blood plasma PAF-AH. Embodiment 6 has described and human blood Dog, mouse, ox, chicken, rodent and the macaque cDNA's of slurry PAF-AH cDNA homology The clone. Embodiment 7 has showed the result who detects, this result's proof instantaneous table in the COS7 cell The enzymatic activity of the restructuring PAF-AH that reaches. Embodiment 8 has described at Escherichia coli, brewer's yeast (S. Cerevisiae) and express total length, brachymemma and chimeric people PAF-AH in the mammalian cell DNA. Embodiment 9 has showed from the scheme of Escherichia coli purification of Recombinant PAF-AH and has confirmed its enzyme Active mensuration. Embodiment 10 has described various restructuring PAF-AH products, comprises amino acid replacement Analog and amino and carboxyl brachymemma product, and described and prove natural from plasma separation PAF-AH is glycosylated experiment. In embodiment 11, showed and be used for human plasma PAF-AH The result that the RNA trace that RNA expresses in various tissues and clone is measured is at embodiment 12 In showed in situ hybridization the result. It is special to human plasma PAF-AH that embodiment 13 has described exploitation Monoclonal and the polyclonal antibody of property. Embodiment 14,15,16,17,18 and 19 describes respectively Give restructuring PAF-AH product of the present invention to the acute inflammation in the animal model, pleurisy, Asthma, NEC, ARDS and pancreatitic interior therapeutic Effect. Embodiment 20 has described the restructuring PAF-AH product pair neurotoxicity relevant with the HIV infection External impact. Embodiment 21 has showed the human patients serum that shows shortage PAF-AH activity The result of immunoassays has described and has differentiated significantly to the gene among the responsible patient of described shortage Damage.

Embodiment 1

For the material of amino acid sequencing is provided, from human plasma purifying PAF-AH.A. optimize purification condition

Originally, use Lin Wusuanyan to precipitate low-density lipoprotein (LDL) particle from blood plasma, be dissolved in 0.1% polysorbas20, according to (1987) such as Stafforini, see that above-mentioned method carries out chromatography on DEAE post (Pharmacia, Uppsala, Sweden), but the active inconsistent wash-out of described DEAE post PAF-AH need revalue dissolving and subsequent purification condition.

By centrifugal and gel permeation chromatography, estimate polysorbas20, CHAPS (Pierce ChemicalCo., Rockford, IL) octyl glucoside dissolving LDL particulate ability.CHAPS provides the recovery of 25% lytic activity than polysorbas20 more, provides 300% recovery than octyl glucoside more.To use with 10mM CHAPS dissolved LDL precipitation then and contain the damping fluid of 1mM CHAPS at DEAE Sepharose Fast Flow post (anion-exchange column; Pharmacia) carry out fractional separation on, with big amalgamation liquid (" DEAE amalgamation liquid ") that partially purified PAF-AH is provided to estimate other post.

The DEAE amalgamation liquid is used to be further purified the active various chromatography columns of PAF-AH as starting material with test.The post of test comprises: Blue Sepharose Fast Flow (Pharmacia), dyestuff part affinity column; S-Sepharose Fast Flow (Pharmacia), cationic exchange coloum; Copper chelating Sepharose (Pharmacia), the metal ligand affinity column; Fractogel S (EMSeparations, Gibbstown, NJ), cationic exchange coloum; And Sephacryl-200 (Pharmacia), gel-filtration column.When operating in 1mM CHAPS, these chromatography programs have produced low, unsafty level of purification.Follow-up Sephacryl S-200 gel permeation chromatography in 1mM CHAPS has produced the enzymic activity fraction collection liquid at the 44kDa approximate size wash-out of very among a small circle roomy rather than expection.Take all factors into consideration, these results show that LDL protein condenses in solution.

Therefore by the analysis mode gel permeation chromatography, estimate the active cohesion of PAF-AF of different LDL samples.The last analysis of damping fluid equilibrated Superose 12 (Pharmacia) of containing 1mM CHAPS in usefulness is derived from DEAE amalgamation liquid and the sedimentary sample of fresh dissolved LDL.These two kinds of samples are wash-out in very broad molecular weight ranges all, the most active 150kDa that surpasses of wash-out.When analyzing described sample on 10mM CHAPS equilibrated Superose 12, a large amount of activity is near the 44kDa wash-out, the same with to the active expection of PAF-AH.But described sample contains the PAF-AH activity in some high molecular districts, corresponding to condensation product.

Other sample when then detecting by gel-filtration only in about 44kDa scope wash-out PAF-AH activity.These samples be under having 0.5M NaCl, be dissolved among the 10mM CHAPS LDL precipitation and at the fresh DEAE amalgamation liquid that behind DEAE post wash-out, is adjusted to 10mM CHAPS.These data show, need 10mM CHAPS to keep the PAF-AH of non-cohesion at least.Behind the chromatography on the DEAE but before follow-up chromatographic step, CHAPS concentration is increased to the significant difference that 10mM causes purifying from 1mM.For example, the degree of purification of PAF-AH on S-SepharoseFast Flow adds to 10 times from 2 multiplications.PAF-AH is active in 1mM CHAPS irreversibly combines with Blue Sepharose Fast Flow post, but this post provides the highest level of purification in 10mMCHAPS.Add 10mM CHAPS in advance and do not improve the DEAE chromatography.

Chromatography after Blue Sepharose Fast Flow post on copper chelating Sepharose is with active concentrated 15 times of PAF-AH.Also determined and to have reclaimed the PAF-AH activity from the reductibility sds page, only otherwise sample is boiled.When carrying out sds polyacrylamide gel electrophoresis, the main protein band when dying with this gel silver is consistent from the activity of the material of copper chelating Sepharose post wash-out.The B.PAF-AH purifying procedure

Being used for purifying PAF-AH therefore comprises following 4 ℃ of steps of carrying out with the novel method of carrying out amino acid sequencing.Human plasma is divided into 900ml aliquot sample in the Nalgene bottle, is adjusted to pH8.6.By adding 90ml 3.85% Tungstophosphoric acid, sodium salt, add 23ml 2MMgCl then then ₂Precipitation LDL particle.Then in 3600g with centrifugal 15 minutes of blood plasma.To precipitate resuspending in 800ml 0.2% Trisodium Citrate.By adding 10g NaCl and 24ml 2M MgCl ₂Precipitate LDL once more.By precipitating the LDL particle in centrifugal 15 minutes in 3600g.Repeat this washing secondary.Then precipitation is frozen in-20C °.With the LDL particle resuspending that is derived from 5 liters of blood plasma in 5 liters of buffer A (25mM Tris-HCl, 10mM CHAPS, pH7.5) in and stir and spend the night.With dissolved LDL particle centrifugal 1.5 hours in 3600g.Merge supernatant liquor, remove any residual solid with Whatman 113 filter paper filterings.Sample on the dissolved LDL supernatant liquor is extremely used buffer B (25mM Tris-HCl, 1mM CHAPS, pH7.5) equilibrated DEAE Sepharose FastFlow post (11cm * 10cm; 1 liter of resin volume; 80ml/ minute).Wash this post with buffer B and be back to baseline until absorption.With 8 liters of 0-0.5M NaCl gradient elution albumen, collect 480ml fraction collection liquid.This step is essential for obtaining with following combining of Blue Sepharose Fast Flow post.Basically detect the PAF-AH activity of fraction collection liquid by the method for describing among the embodiment 4.

Merge active fraction collection liquid, it is about 10mMCHAPS that the CHAPS of adding capacity makes amalgamation liquid.With 4ml/ minute this DEAE amalgamation liquid is spent the night last sample to using the buffer A equilibrated Blue Sepharose Fast Flow post (5cm * 10cm that contains 0.5M NaCl; The 200ml bed volume).Washed this post with level pad with 16ml/ minute, be back to baseline until absorption.With 16ml/ minute step wash-out (step elute) PAF-AH activity, be collected in 50ml fraction collection liquid with the buffer A that contains 0.5M KSCN (chaotropic salt).This step has produced and has surpassed 1000 times purifying.Merge active fraction collection liquid, use 1M Tris-HCl pH8.0 that amalgamation liquid is adjusted to pH8.0.With sample on the active amalgamation liquid of Blue Sepharose Fast Flow chromatography at damping fluid C[25mM Tris-HCl, 10mM CHAPS, 0.5M NaCl, pH8.0 (pH7.5 can also)] in equilibrated copper chelating Sepharose post (2.5cm * 2cm; The 10ml bed volume; 4ml/ minute), C washs this post with the 50ml damping fluid.50mM imidazoles wash-out PAF-AH activity with among the 100ml damping fluid C is collected in the 10ml fraction collection liquid.Merge and contain the active fraction collection liquid of PAF-AH, buffer A is dialysed.Except providing that 15 times of PAF-AH are active and concentrating, copper chelating Sepharose column purification effect is very little.Copper chelating Sepharose amalgamation liquid was reduced 15 minutes in 50mM DTT in 37 ℃, last sample is to 0.75mm, 7.5% polyacrylamide gel.Every 0.5cm cutting adhesive tape, adhesive tape is placed the disposable microcentrifuge tube that contains 200 μ l 25mM Tris-HCl, 10mM CHAPS, 150mM NaCl.Adhesive tape is ground, be incubated overnight in 4 ℃.Detect the PAF-AH activity of the supernatant liquor of each adhesive tape then, have the PAF-AH activity to determine which protein band on the SDS-PAGE.In about 44kDa band, find the PAF-AH activity.(Immobilon-P Millipore), uses Coomassie blue stain to pvdf membrane with the albumen electrotransfer on another repetition gel.The photo display of pvdf membrane is in Fig. 1.

As showing in the following table 1, from 5 liters of human plasma purifying 2 * 10 ⁶Doubly produce about 200 μ gPAF-AH.Contrast with it, Stafforini etc. (1987), see foregoing description the active purifying of PAF-AH 3 * 10 ⁴Doubly.

The active total protein ratio of table 1 sample volume is property recovery % purifying multiple while still alive

(ml) (cpm * active concentration (cpm *

10 ⁶) (cmp× (mg/ml) 10 ⁶)

10 ⁹) step accumulative total step accumulative total blood plasma 5,000 23 116 62 0.37 100 100 1 1LDL 4,500 22 97 1.76 12 84 84 33 33DEAE 4,200 49 207 1.08 46 212 178 3.7 124 blue 165 881 14 0.02 54,200 70 126 1,190 1.5 * 10⁵Copper 12 12,700 152 0.15 82,200 104 131 1.5 2.2 * 10 ⁵SDS----------------～10 2.2 * 10 ⁶PAGE

In a word, following steps are unique and crucial to the successful purifying of the blood plasma PAF-AH that is used for microsequencing: dissolve and chromatography at 10mM CHAPS (1), (2) chromatography on such as the blue part affinity column of BlueSepharose Fast Flow; (3) on such as the copper part affinity column of copper chelating Sepharose chromatography and (4) from SDS-PAGE wash-out PAF-AH.

Embodiment 2

For amino acid sequencing, the pvdf membrane of describing from embodiment 1 that contains PAF-AH downcuts about 44kDa protein band, uses the order-checking of Applied Biosystems 473A protein sequencing instrument.N-terminal sequential analysis corresponding to the active about 44kDa protein band of PAF-AH shows that this band contains two chief serieses and two subsequence.The ratio of two chief serieses is 1: 1, therefore is difficult to explain sequencing data.

For distinguishing isolating two proteic sequences of master on sds gel, the pvdf membrane that another multiple is contained the 44kDa band of having an appointment is cut to 2 half, and upper part and the lower part with film checks order respectively.

The N-terminal sequence that lower part of film obtains is:

SEQ?ID?NO：1

FKDLGEENFKALVLIAF

Retrieval to albumen database discloses the fragment that this sequence is a human serum albumin.Also the upper part to same pvdf membrane checks order, and the N-terminal aminoacid sequence of determining is:

SEQ?ID?NO：2

IQVLMAAASFGQTKIP

Any albumen in this sequence and the data retrieved storehouse is all inequality, and is different from following N-terminal aminoacid sequence:

SEQ?ID?NO：3

MKPLVVFVLGG

This sequence is in (1993) such as Stafforini, sees the sequence of the red corpuscle tenuigenin PAF-AH of above-mentioned middle report.Described in following examples 3, utilize this new sequence (SEQ ID NO:2) to carry out the cDNA clone of human plasma PAF-AH.

Embodiment 3

The full-length clone that separates coding human plasma PAF-AH from scavenger cell cDNA library.A. make up scavenger cell cDNA library

Scavenger cell results Poly A from the peripheral blood lymphocytes source ⁺RNA.(San Diego CA) produces double-stranded, flush end cDNA, before the BstXI joint is connected to this cDNA at insertion mammalian expression vector pRc/CMV (Invitrogen) to use the InvitrogenCopy test kit.By electroporation the plasmid that produces is imported coli strain XL-1 indigo plant.With the density of about 3000 bacterium colonies/agarose plate with the bacterium that transforms at 978 dull and stereotyped upper berth flat boards altogether.Independent plasmid DNA from each plate isolation is remained in the independent storehouse, also they are merged into the bigger storehouse of representing 300,000 clones.B. by PCR screening library

By the polymerase chain reaction, utilize degeneracy antisense oligonucleotide PRC primer, screening scavenger cell library based on the new N-terminal aminoacid sequence of in embodiment 2, describing.The sequence of this primer is set forth in down with the IUPAC name, and wherein " I " is inosine.

SEQ?ID?NO：4

5’ACATGAATTCGGIATCYTTIGTYTGICCRAA3’

Use Wada etc., Nuc.Acids Res., the codon option table of 19S:1981-1986 (1991), selection is positioned at the Nucleotide of the 3rd position of this each codon of primer.With this primer with to be positioned at pRc/CMV cloning site flank or SP6 or the specific primer of T7 promoter sequence unite use, to screen 300,000 clones' storehouse, scavenger cell library.All PCR reaction all contains 100ng template cDNA, every kind of each 1 μ g of primer, every kind of each 0.125mM of dNTP, 10mM Tris-HCl pH8.4,50mM MgCl ₂With 2.5 Taq of unit polysaccharases.Initial denaturing step be 94 ℃ 4 minutes, then carry out the amplification in 30 cycles: 94 ℃ 1 minute, 60 ℃ 1 minute and 72 ℃ 2 minutes.With the PCR product cloning that produces go into pBluescript SK-(Stratagene, La Jolla, CA), by its nucleotide sequence of double deoxidating chain termination measuring.This PCR product has the sequence by new peptide chain prediction, corresponding to the Nucleotide 1-331 of SEQ ID NO:7.

Then for differentiating full-length clone, design is set forth in the PCR primer of following PCR fragments specific to above-mentioned clone.

Adopted primer (SEQ ID NO:5) is arranged

5’TATTTCTAGAAGTGTGGTGGAACTCGCTGG3’

Antisense primer (SEQ ID NO:6)

5’CGATGAATTCAGCTTGCAGCAGCCATCAGTAC3’

As the PCR reaction of primer as described in above-mentioned the utilization,, screen the suitable subgroup in about 3000 clones' less storehouse then at first to screen 300,000 clones' cDNA storehouse.Use three 3000 clones' that produce the big or small PCR product of expection storehouse transform bacteria then.C. by the screening by hybridization library

The PCR fragment of using original clone is then screened the DNA of the bacterium that transforms as probe by hybridization.The bacterium colony trace to nitrocellulose, is carried out prehybridization and hybridization in 50% methane amide, 0.75M sodium-chlor, 0.075M Trisodium Citrate, 0.05M sodium phosphate pH6.5,1% polyvinylpyrrolidone, 1%Ficoll, 1% bovine serum albumin and 50ng/ml supersound process salmon sperm DNA.Cause the mark hybridization probe by sexamer at random.After 42 ℃ of hybridization are spent the night, in 0.03M sodium-chlor, 3mM Trisodium Citrate, 0.1% SDS, thoroughly wash trace in 42 ℃.Measure 10 hybridization clones' nucleotide sequence.One of them clone sAH406-3 has by the sequence from the active original peptide sequence prediction of the PAF-AH of human plasma purifying.The dna sequence dna of human plasma PAF-AH and deduced amino acid are set forth in SEQ ID NO:7 and 8 respectively.

The 1.52kb that clone sAH406-3 contains 441 amino acid whose proteic open reading-frame (ORF)s with coded prediction inserts fragment.At N-terminal, differentiated relative hydrophobicity section at N-terminal amino acid (Isoleucine of the position 42 of SEQ ID NO:8) 41 residues before by albumen micrometering preface.Therefore this albumen that is encoded or have long signal sequence or signal sequence adds another peptide of the ripe functional enzyme of digested generation.Having signal peptide is a feature of secretory protein.In addition, comprise total GxSxG primitive (the amino acid 271-275 of SEQ IDNO:8), it is believed that this primitive contains the avtive spot Serine of all known Mammals lipase, microbial lipase and serine protease by clone sAH406-3 encoded protein matter.Referring to Chapus etc., Biochimie, 70:1223-1224 (1988) and Brenner, Nature, 334:528-530 (1988).

Following table 2 is from (1987) such as the human plasma PAF-AH amino acid composition of the present invention of SEQ ID NO:8 supposition and Stafforini, sees the comparison that the above-mentioned amino acid that is known as the material of purifying is formed.

Table 2

Clone sAH406-3 Stafforini etc.

Ala 26 24

Asp and Asn 48 37

Cys 5 14

Glu and Gln 36 42

Phe 22 12

Gly 29 58

His 13 24

Ile 31 17

Lys 26 50

Leu 40 26

Met 10 7

Pro 15 11

Arg 18 16

Ser 27 36

Thr 20 15

Val 13 14

Trp 7 does not determine

Tyr 14 13

The amino acid of human plasma PAF-AH mature form of the present invention is formed with the amino acid that is known as for the material of the previous purifying of human plasma PAF-AH and is formed difference obviously.

When attempting the Nucleotide of the Nucleotide of the ox brain cell matter PAF-AH of (seeing above-mentioned) such as Hattori and deduced amino acid and human plasma PAF-AH of the present invention and aminoacid sequence contrasted, not observing has significant structural similarity in the sequence.

Embodiment 4

When using with the 5 ' non-translational region (the Nucleotide 31-52 of SEQ ID NO:7) of PAF-AH cDNA and crossing over the primer of the district (the Nucleotide 1465-1487 of SEQID NO:7) of translation stop codon of the 3 ' end that is positioned at PAF-AH cDNA hybridizing, when the cDNA of the PBMC of scavenger cell and irriate is carried out PCR, detect the supposition splicing variants of people PAF-AH gene.Described PCR is reflected at and has produced two bands on the gel, and band is corresponding to the expection size of the PAF-AH cDNA of embodiment 3, and another band is then lacked about 100bp.Order-checking to these two bands discloses, and bigger band is the PAF-AH cDNA of embodiment 3, and short band lacks the exon 2 (following embodiment 5) that coding blood plasma PAF-AH infers the PAF-AH sequence of signal and former peptide sequence.Have catalysis triplet and all halfcystines of expection in short clone, therefore the proteinic biochemical activity by this clones coding might be suitable with the biochemical activity of blood plasma enzyme.

Biology dependency for the PAF-AH splicing variants that begins to estimate expection Codocyte matter organized enzyme detects the relative abundance of these two kinds of forms in the scavenger cell of blood mononuclear cell source by the RNA enzyme protection.These two kinds of information all do not exist in the monocyte of fresh separated, find this two kinds of information but become the 2nd day of scavenger cell in the monocyte vitro differentiation, continue to exist at 6 days that cultivate.The quantity of these two kinds of information is roughly the same in the differentiation phase.In contrast, the similar analysis of nervous tissue discloses, and only expresses the total length information of the outer form PAF-AH of expection coding total length born of the same parents.

Embodiment 5

Also separated genome human plasma PAF-AH sequence.By under stringent condition, separating λ and the P1 phage clone that contains the human gene group DNA, measured the structure of this PAF-AH gene by DNA hybridization.Use design to clone among the sAH406-3 with rule annealed primer, the fragment of subclone phage clone and order-checking at interval at cDNA.In addition, use design to be annealed to the new sequencing primer of the intron of exon flank, fall back order-checking to confirm described sequence crossing over exon-intron border.The exon boundary definition is the point of genome and cDNA sequence difference.These analyze announcement, and people PAF-AH is made up of 12 exons.

From the male fetus placenta library that among λ FIX (Stratagene), makes up separate exons 1,2,3,4,5,6 and outside show a part of 7.The plaque trace to nitrocellulose, is carried out prehybridization and hybridization in 50% methane amide, 0.75M sodium-chlor, 75mM Trisodium Citrate, 50mM sodium phosphate (pH6.5), 1% polyvinylpyrrolidone, 1% Ficoll, 1% bovine serum albumin and 50ng/ml supersound process salmon sperm DNA.The hybridization probe that is used to differentiate the phage clone of a part that contains exon 2-6 and 7 is made up of whole cDNA clone sAH 406-3.Use is derived from described cDNA clone's 5 ' end fragment (the Nucleotide 1-312 of SEQ ID NO:7) and differentiates the clone who contains exons 1.These two kinds of probes are all used ³²P causes mark at random by sexamer.After 42 ℃ of hybridization are spent the night, in 30mM sodium-chlor, 3mM Trisodium Citrate, 0.1% SDS, thoroughly wash trace in 42 ℃.Intron sequences is set forth in respectively among the SEQ ID NO:9,10,11,12,13 and 14 around exons 1,2,3,4,5 and 6 dna sequence dna and the part.

From the isolating P1 clone of people P1 genomic library, the remainder of subclone exon 7 and exon 8,9,10,11 and 12.P1 plaque trace to nitrocellulose, is carried out prehybridization and hybridization in 0.75M sodium-chlor, 50mM sodium phosphate (pH7.4), 5mM EDTA, 1% polyvinylpyrrolidone, 1%Ficoll, 1% bovine serum albumin, 0.5% SDS and the total people DNA of 0.1mg/ml.Cause the hybridization probe of mark with 32P at random by sexamer, form by the 2.6kb EcoRI fragment of the genomic dna of the 3 ' end that is derived from above-mentioned isolating λ clone.This fragment contain the exon 6 that is present on the described phage clone and the part of exon 7.After 65 ℃ of hybridization are spent the night, as above-mentioned washing trace.Intron sequences is set forth in respectively among the SEQ ID NO:15,16,17,18,19 and 20 around exon 7,8,9,10,11 and 12 dna sequence dna and the part.

Embodiment 6

Separate total length blood plasma PAF-AH cDNA clone from mouse, dog, ox and chicken spleen cDNA library, separate a part rodents clone from rat chest gland cDNA library.By differentiating described clone (to exon 1-6 description in hybridization conditions and the foregoing description 5 identical, except having used 20% methane amide rather than 50% methane amide) with the low stringency hybridization of people cDNA.End user PAF-AH sAH406-3cDNA clone's 1kb HindIII fragment (the Nucleotide 309-1322 of SEQ ID NO:7) is as probe.In addition, use, separate a part monkey clone from macaque brain cDNA by PCR based on the Nucleotide 285-303 of SEQ ID NO:7 and the primer of 851-867.Mouse, dog, ox, chicken, rat and macaque cDNA clone's nucleotide sequence and deduced amino acid are set forth in SEQ ID NO:21,22,23,24,25 and 26 respectively.

Described cDNA clone's deduced amino acid and people cDNA clone has relatively produced amino acid homogeny per-cent, is set forth in the following table 3.

Table 3

74 69 82 69 55 N of 82 82 64 100 50 chicken 50 50 47 50 100 of people dog mouse ox chicken dog 80 100 64 82 50 mouse 66 64 100 64 47 monkeys, 92 82 69 80 52 rats

About 38% residue is conservative fully in all sequences.The zone of difference maximum is arranged in N-terminal (containing signal sequence) and is shown as the C-terminal not crucial to enzymic activity at embodiment 10.The Gly-Xaa-Ser-Xaa-Gly primitive of finding in neural lipase and other esterase (SEQ ID NO:27) is guarded in ox, dog, mouse, rat and chicken PAF-AH.The Serine at this primitive center works as the avtive spot nucleophile of these enzymes.Die young winter propylhomoserin and the Histidine component of the supposition of avtive spot (embodiment 10A) are also guarded.Therefore human plasma PAF-AH of the present invention seems to have utilized the catalysis triplet, may take the α/β lytic enzyme conformation of neural lipase, although it does not show other sequence homology with described lipase.

In addition, expection human plasma PAF-AH has the low-density lipoprotein and the interactional district of high-density lipoprotein (HDL) particle specificity of a mediation itself and blood plasma.Interacting with these particulate can be by half mediation of N-terminal of described molecule, this half have a big section plant between the amino acid of high conservative, but do not have the catalysis triplet of described enzyme.

Embodiment 7

Has the active protein of PAF-AH, transient expression pRc/CMV expression constructs in the COS7 cell for whether definite human plasma PAF-AH cDNA clone sAH406-3 (embodiment 3) encodes.After 3 days, detect the PAF-AH activity of COS cell culture medium by the transfection of deae dextran method.

Density inoculating cell with every 60mm tissue culture plate 300,000 cells.Next day, described cell was cultivated 2 hours in the DMEM that contains 0.5mg/ml deae dextran, 0.1mM chloroquine and 5-10 μ g plasmid DNA.Then cell was handled 1 minute with the 10%DMSO in the phosphate-buffered saline, with the substratum washing, cultivated in the DMEM that contains 10% foetal calf serum, described foetal calf serum uses diisopropylfluorophosphate (DFP) to handle with the endogenous bovine serum PAF-AH of passivation in advance.Cultivate after 3 days, detect the PAF-AH activity of the substratum of transfectional cell.Exist or do not exist or the situation of 10mM EDTA or 1mM DFP under detect, with determine described recombinase whether be Ca-dependent, whether the previously described serine ester enzyme inhibitors of blood plasma PAF-AH DFP is suppressed by (1987) such as Stafforini (seeing above-mentioned).Negative control comprise with or lack to insert fragment or have reverse sAH406-3 and insert segmental pRc/CMV cells transfected.

Adopt following improvement, by the PAF-AH activity in the method mensuration transfection supernatant liquor of (1990) such as Stafforini (seeing above-mentioned).In brief, by measuring ³H-acetate from [acetyl- ³H] (activity of PAF-AH is measured in hydrolysis MA) to PAF for NewEngland Nuclear, Boston.By (PA) reversed phase column chromatography on is from the substrate separating water-soluble free 3H-acetate of mark for Baker Research Products, Phillipsburg at the 18 alkyl silica gel cylinder.Use transfection supernatant liquor, pH7.2 in the 10 μ l 0.1M Hepes damping fluids, in the reaction volume of 50 μ l, detect.The substrate of 50 picomole is altogether used in each reaction, and ratio is 1: 5 mark: non-marked PAF.Made the reactant incubation 30 minutes in 37 ℃, by adding 40 μ l 10M acetate termination reactions.Wash this solution by described 18 alkyl silica gel post then, this post cleans with the 0.1M sodium acetate then.Collect the water-based elutriant of each sample then, counting is 1 minute in the liquid flashing counting device.With counting/minute expression enzymic activity.

As shown in Figure 2, the substratum with the sAH406-3 cells transfected has the PAF-AH activity higher 4 times than background.There is not the influence of EDTA in this activity, but is eliminated by 1mM DFP.These are observed and confirm clone sAH406-3 coding and the consistent activity of human plasma enzyme PAF-AH.

Embodiment 8

By recombination method, the human plasma PAF-AH DNA and the gomphosis mouse-people PAF-AH DNA that in intestinal bacteria and yeast, express and in mammalian cell, stably express total length and various brachymemmas.A. at expression in escherichia coli

Use the protein coding fragment of PCR from clone sAH406-3 generation human plasma PAF-AH cDNA, it is easy to subclone and goes into coli expression carrier.The section of this subclone starts from 5 ' end coding Ile of described people's gene ₄₂Codon (SEQ ID NO:8), this residue is the N-terminal residue from the enzyme of human plasma purifying.In this construction, comprised the remainder of this gene until natural terminator codon.5 ' of use has adopted PCR primer to be:

SEQ?ID?NO：28

5 ' TATTCTAGAATTATGATACAAGTATTAATGGCTGCTGCAAG3 ' has an XbaI cloning site and a translation initiation codon (underscore).3 ' the antisense primer that uses is:

SEQ?ID?NO：29

5 ' ATTGATATCCTAATTGTATTTCTCTATTCCTG3 ' and comprise the terminator codon of sAH406-3 has an EcoRV cloning site.Basically carry out the PCR reaction as described in example 3 above.With the PCR product that XbaI and EcoRV digestion produce, subclone is gone at the pBR322 carrier that has the Trp promotor near the cloning site upstream [deBoer etc., PNAS, 80:21-25 (1983)].With this expression constructs transformed into escherichia coli strain X L-1 indigo plant, in the L nutrient solution that contains 100 μ g/ml Pyocianils, cultivate.The transformant of precipitation incubated overnight, resuspending is in the lysis buffer that presses down the enzyme peptide that contains 50mM Tris-HCl pH7.5,50mM NaCl, 10mM CHAPS, 1mMEDTA, 100 μ g/ml N,O-Diacetylmuramidases and 0.05 TIU (TIU)/ml.After 2 minutes, detect the PAF-AH activity of lysate in incubation on ice 1 hour and supersound process by the method for describing among the embodiment 4.The intestinal bacteria that transform with this expression constructs (called after trpAH) have produced has the active product of PAF-AH.Referring to the table 6 among the embodiment 9.

Also used and comprised that other three kinds of promotors are tacII promotor (deBoer, see above-mentioned), be derived from pectinose (ara) B promotor [Horwitz etc. of Salmonella typhimurium, Gene, 14:309-319 (1981)], the construction of phage t7 promotor, drive people PAF-AH sequence at expression in escherichia coli.At plasmid pUC19 (New England Biolabs, MA) assembling comprises the construction of Trp promotor (pUC trp AH), tacII promotor (pUC tac AH) and araB promotor (pUCara AH) in, at plasmid pET15B (Novagen, Madison, WI) middle assembling comprises the construction of T7 promotor (pET AH).Also assembling starts the molecular construction pHAB/PH that contains the hybrid promotor by the araB that merges to T7 promoter region ribosome bind site in pET15B.All intestinal bacteria constructions have all produced at 20-50U/ml/OD ₆₀₀PAF-AH activity in the scope.This activity is corresponding to total recombinant protein amount of 〉=1% total cell protein.

Several escherichia coli expression constructions that produce aminoterminal PAF-AH have also been estimated with extension.By amino acid sequencing, the N-terminal of native plasma PAF-AH is differentiated and is Ile ₄₂(embodiment 2).But, Ile ₄₂The sequence that the upstream is right after does not meet with the amino acid of finding at the signal sequence cleavage site [does not promptly follow " 3-1-principle ", because-1 do not find Methionin in the position; Referring to von Heijne, Nuc.Acids Res., 14:4683-4690 (1986)].Infer by classical more signal sequence (M of emiocytosis system identification ₁-A ₁₇Or M ₁-P ₂₁), carry out the endo-protease cutting then.Transformation originates in the whole PAF-AH encoding sequence (the Nucleotide 162-1487 of SEQ IDNO:7) of initial methionine, to use the trp promotor at expression in escherichia coli.As shown in table 4, this construction produces active PAF-AH, but expresses only for originating in Ile ₄₂Original construction level about 1/50.Another starts from Val ₁₈Expression constructs (the Nucleotide 213-1487 of SEQ ID NO:7) produced 1/3 active PAF-AH of original construction level.It is not key or essential that these result's hints, N-terminal are extended the reorganization PAF-AH activity that produces in the intestinal bacteria.

Table 4

PAF-AH activity (U/ml/OD ₆₀₀) construction lysate substratum pUC trp AH (Ile ₄₂N-terminal) 177.7 0.030pUC trp AH Met ₁3.1 0.003pUC tp AH Val ₁₈54.6 0.033

Use the low copy number plasmid and can also in intestinal bacteria, produce the recombinant human PAF-AH product of brachymemma by pectinose being added substratum inductive promotor.A kind of PAF-AH product of this N-terminal brachymemma is the amino-acid residue Met of coding by total length PAF-AH cDNA encoded polypeptides ₄₆-Asn ₄₄₁The recombination expression product of DNA, called after rPH.2.The plasmid that is used at bacterial cell production rPH.2 is pBAR2/PH.2, be based on the plasmid of pBR322, carry the Nucleotide 297-1487 of SEQ IDNO:7 of people PAF-AH that (1) coding originates in the methionine(Met) codon of position 46, (2) be derived from the araB-C promotor and the araC gene of the arabinose operon of Salmonella typhimurium, (3) be derived from the transcription termination sequence of phage t7 and the replication orgin that (4) are derived from phage fl.

Specifically, pBAR2/PH.2 comprises following DNA section: (1) 1994 destructive AatII site is contained replication orgin and is derived from the carrier sequence of the gene of bacterial plasmid pBR322 coding or penbritin or tetracyclin resistance to the EcoRI site that is positioned at Nucleotide 6274 from the position; (2) from the EcoRI site that is positioned at position 6274 to the XbaI site that is positioned at position 131, be derived from Salmonella typhimurium arabinose operon (Genbank preserving number M11045, M11046, M11047, DNA J01797); (3) from the XbaI site that is positioned at position 131 to the NcoI site that is positioned at position 170, contain and be derived from pET-21b (Novagen, Madison, the DNA of ribosome bind site WI); (4) from the NcoI site that is positioned at position 170 to the XhoI site that is positioned at position 1363, people PAF-AH cDNA sequence; (5) from the XhoI site that is positioned at position 1363 to the destructive AatII site that is positioned at position 1993, be derived from pET-21b (Novagen), contain the transcription termination sequence that is derived from phage t7 and be derived from the dna fragmentation of the replication orgin of phage fl.

The PAF-AH product of another called after rPH.9 is the amino-acid residue Met of coding by total length PAF-AH cDNA (SEQ ID NO:8) encoded polypeptides ₄₆To Ile ₄₂₉The expression product of DNA.The DNA of coding rPH.9 is inserted the identical carrier that is used for producing at bacterial cell rPH.2.This plasmid called after pBAR2/PH.9, comprise following DNA section particularly: (1), is contained replication orgin and is derived from the gene of bacterial plasmid pBR322 coding or penbritin or tetracyclin resistance to the EcoRI site that is positioned at Nucleotide 6239 from the destructive AatII site that is positioned at position 1958 of carrier sequence; (2) from the EcoRI site that is positioned at position 6239 to the XbaI site that is positioned at position 131, be derived from Salmonella typhimurium arabinose operon (Genbank preserving number M11045, M11046, M11047, DNA J01797); (3) from the XbaI site that is positioned at position 131 to the NcoI site that is positioned at position 170, contain and be derived from pET-21b (Novagen, Madison, the DNA of ribosome bind site WI); (4) from the NcoI site that is positioned at position 170 to the XhoI site that is positioned at position 1328, people PAF-AH cDNA sequence; (5) from the XhoI site that is positioned at position 1328 to the destructive AatII site that is positioned at position 1958, be derived from pET-21b (Novagen, Madison, WI), contain the transcription termination sequence that is derived from phage t7 and be derived from the dna fragmentation of the replication orgin of phage fl.

Among pBAR2/PH.2 and the pBAR2/PH 9 under the control that is expressed in the araB promotor of PAF-AH product, this promotor is prevented by strictness when having glucose and lacking pectinose, but works as strong promoter when L-arabinose being added in the substratum that has run out of glucose.By adding to substratum or penbritin (or associated antibiotic) or tsiklomitsin can be finished the screening of the cell that contains this plasmid.Can use the host of various coli strains as recombinant expressed PAF-AH product, include but not limited to the anauxotrophic bacterial strain of pectinose metabolism such as W3110, DH5 α, BL21, C600, JM101 and its derivative to have the bacterial strain for example bacterial strain such as the SB7219 and the MC1061 of CAG629, KY1429 and degraded pectinose capability defect that reduce proteoclastic sudden change.The advantage that use can not be decomposed the bacterial strain of pectinose is that the inductor (pectinose) that produces PAF-AH between inductive phase is not got rid of from substratum, with use can the metabolism pectinose bacterial strain obtain compare the PAF-AH that produces higher level.Can use any suitable medium and culture condition expression activity PAF-AH product in various coli strains.Allow a large amount of rPAF-AH of generation products for example or such as the rich medium prescription of LB, EDM295 (based on replenishing of M9 the minimal medium of yeast extract paste and acid hydrolyzed casein) or " principal component " substratum such as A675, a kind of minimal medium based on A, pH6.75, use glycerine is as carbon source and replenished trace element and VITAMIN.Comprise in the substratum that tsiklomitsin is to keep the described plasmid of screening.

Therefore plasmid pBAR2/PH.2 is transformed into coli strain MC1061 (ATCC53338), and this bacterial strain carries the disappearance of arabinose operon, energy metabolism pectinose not.MC1061 also is a leucine auxotroph, uses the known composition culture medium culturing that contains the casamino acids that replenishes described leucine sudden change by flow addition (batch-fed process) in batches.

The intestinal bacteria M1061 cell that transforms with pBAR2/PH.2 is grown in 30 ℃ of batch culture bases that containing 2gm/L glucose.Glucose plays dual parts the carbon source and the pectinose promoter suppression thing of cell growth.After the batch of material glucose level exhausts (＜50mg/L), the beginning nutritional flow adds (containing 300gm/L glucose).Increase by 16 hours so that the speed that limits sour by product (bi-product) formation is linear.At this moment, nutritional flow is added be converted to the substratum that contains glycerine rather than glucose.Simultaneously, adding the 500gm/L L-arabinose, to make ultimate density be 5gm/L.Keep glycerine stream to add 22 hours with constant stream rate of acceleration.Use tubular fibre to filter harvested cell, about 10 times of concentrated suspension liquid.Cytoplasm is stored in-70 ℃.Obtain the about 80gm/L of final cell amount (OD600=50-60), had the PAF-AH activity of the 65-70U/OD/ml of the about total cell protein 10% of representative.About 75 liters final volume of culture contains 50-60gm PAF-AH.

When expressing pBAR2/PH.2 or PH.9, can reach high level production rPAF-AH product by bacterial strain SB7219 or MC1061.Other bacterial strain of pectinose degraded defective is suitable for high-cell density production.Preferably, cultivate described cell under the following conditions.SB7219 with exponential growth; PBAR2/PH.2 and SB7219; The pBAR2/PH.9 inoculation goes into to be equipped with to contain the fermentor tank of the batch culture base of 2g/L glucose.In case glucose has been consumed, then uses the glycerine solution that contains trace element, VITAMIN, magnesium salts and ammonium salt that jar is carried out stream and add, with the exponential growth of keeping fit.Jar is remained in 30 ℃, provides air with supply oxygen and stirring, surpass about 15% saturation ratio to keep dissolved oxygen level.When the cell density of culture surpasses 110g/L (wet cell weight), carry out the steady flow rate of acceleration, with heavy dose of additive adding culture (final about 0.5%) of L-arabinose.Observe product and form 16-22 hour.Culture reaches 40-50g/L (dry cell weight) usually.By centrifugal cell harvesting, be stored in-70 ℃, purifying rPAF-AH product is analyzed.Usually obtain surpassing 150 units/ml/OD ₆₀₀Specific production rate.B. in yeast cell, express

Also express recombinant people PAF-AH in cereuisiae fermentum.Use yeast ADH2 promoters driven rPAF-AH to express, produce 7U/ml/OD ₆₀₀(seeing the following form 5).

Table 5 construction promotor bacterial strain enzymic activity

(U/ml/OD) pUC tac AH tac intestinal bacteria W3110 30pUC trp AH trp intestinal bacteria W3110 40pUC ara AH araB intestinal bacteria W3110 20pET AH T7 e. coli bl21s (DE3) 50

(Novagen) pHAB/PH araB/T7 intestinal bacteria XL-1 34pBAR2/PH.2 araB MC1061 90pYep ADH2AH ADH2 yeast BJ2.28 7C. expresses PAF-AH in mammalian cell

1. expressing human PAF-AH cDNA construction

Except pSFN/PAFAH.1, structure has used the strong virus promotor of cytomegalovirus, the polyadenylation site of bovine growth hormone gene and the replication orgin of SV40 with the plasmid of expressing PAF-AH, so that described plasmid high copy number in the COS cell duplicates.The plasmid electroporation is entered cell.

Make up the first cover plasmid, wherein with known in mammalian cell with the 5 ' flanking sequence (pDC1/PAFAH.1) of the flanking sequence replacement people PAF-AH cDNA of other gene of high level expression or replace 5 ' or 3 ' flanking sequence (PDC1/PAFAH.2) simultaneously.These plasmid transfections are gone into COS, CHO or 293 cells, cause producing PAF-AH, the level of the transient transfection COS cell rear clone sAH406-3 that proposes among its level and the embodiment 7 basic identical (0.01 unit/ml or exceed background 2-4 doubly).Make up another plasmid, it comprises that the Friend spleen transforming focus that has replaced the cytomegalovirus promotor forms viral promotors.People PAF-AH cDNA is inserted in Friend spleen transforming focus forms plasmid pmH-neo[Hahn under the viral promotors control etc., Gene, 127:267 (1993)].With the plasmid transfection myeloma cell line NS0 of called after pSFN/PAFAH.1, a screening hundreds of clone causes separating two and produces the active transfection body of 0.15-0.5 unit/ml PAF-AH (4B11 and 1C11).Suppose that than work be 5000 unit/milligrams, the productivity of these two NS0 transfection bodies is corresponding to about 0.1mg/ liter.

2. express the chimeric PAF-AH gene constructs of mouse-people

The construction (pRc/MS9) of cDNA with the encoding murine PAF-AH in mammalian expression vector pRc/CMV is after transfection enters the COS cell, and causing the generation level is 5-10 unit/ml excretory PAF-AH of (being higher than 1000 times of backgrounds).Ratio about enzyme with the people alive of supposing mouse PAF-AH is identical, so the expression level of mouse cDNA than the high 500-1000 of people PAF-AH cDNA doubly.

The difference between the expression level in the COS cell for scrutineer and mouse PAF-AH makes up two mouse-people's mosaic genes and expresses in the COS cell tests.First of these constructions, pRc/PH.MHCl contains and merges (the Invitrogen to expression vector pRc/CMV, San Diego, CA) 97 amino acid whose encoding sequences of the N-terminal of 343 amino acid whose mouse PAF-AH polypeptide of the C-terminal of the people PAF-AH in (SEQ IDNO:21).Second mosaic gene in plasmid pRc/PH.MHC2 contains 40 amino acid whose encoding sequences of N-terminal of the mouse PAF-AH polypeptide of 400 residues of C-terminal that merge the people PAF-AH to the pRc/CMV.The PAF-AH activity that causes accumulation 1-2 unit/ml in substratum with the pRc/PH.MHCl rotaring redyeing COS cell.Discovery is derived from the PAF-AH activity that only contains 0.01 unit/ml with the conditioned medium of pRc/PH.MHC2 cells transfected.It seems that from these experiments the difference of expression level is to RNA or the DNA section of small part owing to polypeptide section between residue 40 and 97 or proteic this district of corresponding codes PAF-AH between mouse and the people PAF-AH gene.

3. the first 290bp of recodification PAF-AH encoding sequence

The low-level hypothesis of people PAF-AH synthetic is that the codon that natural gene uses is a suboptimum for effective expression in mammalian cells transfected.But, seem that the codon use can not be responsible to the 500-1000 difference doubly of expression level between mouse and the people's gene, because optimizing codon generally only has 10 times influences at most to expressing.Second hypothesis of explaining mouse and people PAF-AH expression level difference is that the people PAF-AH mRNA in the 5 ' coding region has formed and causes or the relative secondary structure of degrading fast or causing invalid translation initiation or prolongation of described mRNA.

For checking these hypothesis, made up coding from N-terminal to residue 96 real people PAF-AH proteic synthetic fragment, but wherein most codon is not with homotactic but the same amino acid whose codon of encoding is replaced (" recodification ").Second codon becomes GTA from GTG and causes producing the Asp718 site, and it is arranged in a described synthetic segmental end and is present in mouse cDNA.This segmental the other end has the common BamHI site of finding at the codon 97 of people's gene.About 290bp Asp718/BamHI fragment is derived from the PCR fragment, and this PCR fragment is with being described in Sandhu etc., Biotechniques, and two asymmetric PCR methods that being used among the 12:14-16 (1992) makes up synthetic gene produce.The remainder of this synthetic Asp718/BamHI fragment and coding people PAF-AH molecule, the dna fragmentation that originates in the Nucleotide 453 of SEQ ID NO:7 are connected, make the sequence of the people PAF-AH enzyme that coding is real insert mammalian expression vector pRc/CMV (Invitrogen, San Diego), produce plasmid pRc/HRH.4.The complete sequence of the gene of described recodification is set forth in SEQ ID NO:30.Be derived from the flanking sequence (the Nucleotide 1-116 of SEQ ID NO:21) of the mouse cDNA of the coding PAF-AH in pRc/MS9 with the contiguous 5 ' flanking sequence of people PAF-AH encoding sequence among the pRc/HPH.4.

Be the expression of people PAF-AH of check pRc/HPH.4, with pRc/HPH.4 (recodification people's gene), pRc/MS9 (mouse PAF-AH) or pRc/PH.MHCl (mouse-people hybrid 1) transient transfection COS cell.Detect the PAF-AH activity of the conditioned medium of described transfectional cell, find to contain 5.7 units/ml (little musculus cdna), 0.9 unit/ml (mouse-people hybrid) or 2.6 units/ml (recodification people's gene).Therefore, the strategy of the first 290bp encoding sequence of recodification people PAF-AH successfully is increased to about 0.5 microgram/ml with people PAF-AH expression level from several nanogram/ml in instantaneous COS cell transfecting.The recodification PAF-AH gene of pRc/HPH.4 is inserted the mammalian expression vector that contains Tetrahydrofolate dehydrogenase (DHFR) gene, will be with this carrier transfection DHFR-feminine gender Chinese hamster ovary cell.To carry out the methotrexate screening to cells transfected, to obtain producing the clone of high-level people PAF-AH owing to gene amplification.

Embodiment 9

By the whole bag of tricks the recombinant human blood plasma PAF-AH of expression in escherichia coli (is originated in Ile ₄₂) purifying is single coomassie dyeing SDS-PAGE band, detects the activity of natural PAF-AH enzyme performance.A. purification of Recombinant PAF-AH

Similar to natural PAF-AH description among first purifying procedure that uses and the embodiment 1.Carry out following steps at 4 ℃.The precipitation that 50ml is produced the intestinal bacteria (transforming with expression constructs trp AH) of PAF-AH is carried out bacteriolyze as described in example 8 above.By in 10,000g removed solid in centrifugal 20 minutes.With 0.8ml/ minute with sample on the supernatant liquor to damping fluid D (25mMTris-HCl, 10mM CHAPS, 0.5M NaCl, pH7.5) in equilibrated Blue SepharoseFast Flow post (2.5cm * 4cm; The 20ml bed volume).D washs this post with the 100ml damping fluid, with containing the buffer A of 0.5M KSCN with 3.2ml/ minute wash-out.With sample on the active fraction collection liquid of 15ml in damping fluid D equilibrated 1ml copper chelating Sepharose post.D washs this post with the 5ml damping fluid, then with containing the 5ml damping fluid D of 100mM imidazoles with gravity flow velocity wash-out.Contain the active fraction collection liquid of PAF-AH by the SDS-PAGE analysis.

Purifying the results are shown in table 6, one of them unit equal μ mol PAF hydrolysis/hour.On SDS-PAGE, show as the single dark band that is lower than the 43kDa mark in 4 ℃ of purified products that obtain, some diffusion dyeing are arranged down thereon.Compare with the blood plasma PAF-AH prepared product described in the embodiment 1, recombined material is purer significantly and show higher ratio and live.

The active gross activity albumen ratio of table 6 sample volume is property recovery % purifying multiple while still alive

(ml) ((unit * concentration (unit of unit

/ ml) 10 ³) (mg/ml)/mg) step accumulative total step accumulative total lysate 4.5 989 4,451 15.6 63 100 100 11 blue 15 64 960 0.07 914 22 22 14.4 14.4 bronze medals 1 2,128 2,128 0.55 3,869 220 48 4.2 61

When room temperature is carried out same purifying procedure, except the band that is lower than the 43kDa mark, one group of band that is lower than the 29kDa mark is active relevant with the PAF-AH of the gel strips of detection.These lower molecular weight bands may be the proteolytic fragments that keeps the PAF-AH of enzymic activity.

Also carry out different purifying procedures in room temperature.To produce precipitation (100g) resuspending of intestinal bacteria (transforming) of PAF-AH in 200ml lysis buffer (25mM Tris with expression constructs pUC trp AH, 20mM CHAPS, 50mM NaCl, 1mM EDTA, 50 μ g/ml benzamidines, pH7.5), by in 15,000psi three times is by micro-fluidised form instrument (microfluidizer) bacteriolyze.By in 14,300xg removed solid in centrifugal 1 hour.With supernatant liquor at dilution buffer liquid [25mM MES (2-[N-morpholino] ethyl sulfonic acid), 10mM CHAPS, 1mM EDTA, pH4.9] in the dilution 10 times, went up sample extremely at damping fluid E (25mM MES, 10mM CHAPS, 1mMEDTA with 25ml/ minute, 50mM NaCl, pH5.5) middle equilibrated S Sepharose Fast Flow post (200ml) (cationic exchange coloum).Wash this post with 1 liter of damping fluid E,, elutriant is collected with 50ml fraction collection liquid, be adjusted to pH7.5 with 0.5ml 2M Tris alkali with 1M NaCl wash-out.Merge and contain the active fraction collection liquid of PAF-AH, be adjusted to 0.5M NaCl.The S amalgamation liquid was gone up sample extremely at damping fluid F (25mM Tris, 10mM CHAPS, 0.5M NaCl, 1mMEDTA, pH7.5) middle equilibrated Blue Sepharose Fast Flow post (2.5cm * 4cm with 1ml/ minute; 20ml).Wash this post with 4ml/ minute with 100ml damping fluid F, contain the damping fluid F wash-out of 3M NaCl with 100ml.Repeat Blue Sepharose Fast Flow chromatographic step to reduce the level of endotoxin in the sample.Merge and contain the active fraction collection liquid of PAF-AH, damping fluid G (25mM Tris pH7.5,0.5M NaCl, 0.1% tween 80,1mM EDTA) is dialysed.

Purifying the results are shown in table 7, one of them unit equal μ mol PAF hydrolysis/hour.

The active gross activity albumen ratio of table 7 sample volume is property recovery % purifying multiple while still alive

(ml) ((concentration (unit of unit of unit

/ ml) * 10 ³) (mg/ml)/mg) step accumulative total step accumulative total lysate 200 5,640 1,128 57.46 98 100 100 1 1S 111 5,742 637 3.69 1,557 57 56 16 16 blue 100 3,944 394 0.84 4,676 35 62 3 48

The purified product that obtains shows as the single dark band that is lower than the 43kDa mark on SDS-PAGE, some diffusion dyeing are arranged down thereon.Compare with the blood plasma PAF-AH prepared product described in the embodiment 1, recombined material is purer significantly and show higher ratio and live.

The purifying procedure again that the present invention considers relates to following lysis, clarification and the first post step.Lysis buffer (25mM Tris pH7.5,150mM NaCl, 1% tween 80,2mMEDTA) in cell 1: 1 dilution.In the micro-fluidised form instrument of refrigerative with 15,000-20,000psi carries out bacteriolyze by three times to produce＞99% cytoclasis with material.At dilution buffer liquid (25mMTris pH8.5,1mM EDTA) in lysate was diluted 1: 20, last sample is to Q-SepharoseBig Bead chromatography media (Pharmacia) dress post and at 25mM Tris pH8.5,1mM EDTA, equilibrated post in 0.015% tween 80.Elutriant was diluted 1: 10 in 25mM MES pH5.5,1.2M ammonium sulfate, 1mM EDTA, and last sample is to equilibrated butyl Sepharose chromatography media (Pharmacia) in same damping fluid.Wash-out PAF-AH activity in 25mM MES pH5.5,0.1% tween 80,1mM EDTA.

The present invention considers is used for may further comprise the steps from the method again of the active PAF-AH of intestinal bacteria purifying enzyme: (a) preparation produces the intestinal bacteria extracting solution of dissolved PAF-AH supernatant liquor after containing the damping fluid bacteriolyze of CHAPS; (b) dilute described supernatant liquor, last sample is as for about pH8.0 equilibrated anion-exchange column; (c) from described anion-exchange column wash-out PAF-AH enzyme; (d) with sample on the described adjusting elutriant of described anion-exchange column to blue dyestuff part affinity column; (e) use the described blue dyestuff part affinity column of buffer solution elution that comprises 3.0M salt; (f) described blue dyestuff elutriant is diluted at the suitable buffer that is used for hydroxyapatite chromatography; (g) carry out hydroxyapatite chromatography, wherein use damping fluid (containing or do not contain CHAPS) to finish washing and wash-out; (h) described hydroxyapatite elutriant is diluted to suitable salt concn to carry out cation-exchange chromatography; (i) with sample on the hydroxyapatite elutriant of described dilution to the cationic exchange coloum of the about 6.0-7.0 of pH scope; (j) use suitable preparation damping fluid from described cationic exchange coloum wash-out PAF-AH; (k) carry out cation-exchange chromatography at low temperature; (l) lacking under the situation of CHAPS with liquid or frozen form preparation PAF-AH.

Preferably, the lysis buffer in the above-mentioned steps (a) is 25mM Tris, 100mMNaCl, 1mM EDTA, 20mM CHAPS, pH8.0; In the step (b) for carrying out anion-exchange chromatography, described supernatant liquor is diluted 3-4 doubly in 25mM Tris, 1mM EDTA, 10mM CHAPS, pH8.0, this post is an equilibrated Q-Sepharose post in 25mM Tris, 1mM EDTA, 50mM NaCl, 10mM CHAPS, pH8.0; Use 25mMTris, 1mM EDTA, 350mM NaCl, 10mM CHAPS, the described anion-exchange column of pH8.0 wash-out in the step (c); In the step (d) directly with sample on the elutriant of step (c) to blue dyestuff affinity column; Use 3M NaCl, 10mM CHAPS, 25mM Tris, the described post of pH8.0 buffer solution elution in the step (e); By in 10mM sodium phosphate, 100mM NaCl, 10mMCHAPS, pH6.2, diluting, finish the described blue dyestuff elutriant of dilution in the step (f) to carry out hydroxyapatite chromatography; Use in the step (g) and finish hydroxyapatite chromatography, use 50mM sodium phosphate, 100mMNaCl (contain or do not contain) 10mM CHAPS, pH7.5 to finish wash-out with 10mM sodium phosphate, 100mM NaCl, 10mM CHAPS equilibrated hydroxyapatite column; In the step (h) by at the about 6.0-7.0 of pH scope, comprise in the sodium phosphate damping fluid of (containing or do not contain CHAPS) and dilute, finish the dilution of the described hydroxyapatite elutriant that is used for cation-exchange chromatography; Step (i) is middle with 50mM sodium phosphate, (contain or do not contain) 10mM CHAPS, pH6.8 balance S Sepharose; Use in the step (j) such as the suitable preparation damping fluid of the 50mM potassiumphosphate that contains 0.01% tween 80,12.5mM aspartic acid, 125mM NaCl, pH7.5 and finish wash-out; Finish cation-exchange chromatography in 2-8 ℃ in the step (k).Being used for step (l) stablizes the example of the suitable preparation damping fluid of PAF-AH and comprises 50mM potassiumphosphate, 12.5mM aspartic acid, 125mM NaCl pH7.4 (about value, add or do not add tween 80 and or Pluronic F68) or the 25mM potassium phosphate buffer, (at least) 125mM NaCl, 25mM arginine and 0.01% tween 80 (containing or do not contain about 0.1 and 0.5% Pluronic F68) contained.

B. the recombinate activity of PAF-AH

The most significant characteristic of PAF PAF-AH is it has the substrate of short residue to the sn-2 position at substrate a significant specificity.This strict specificity is with PAF PAF-AH and PLA ₂The difference of other form come.Therefore, for whether definite reorganization PAF-AH degrades the phosphatide of longer chain fatty acid is arranged in the sn-2 position, detect the hydrolysis of 1-palmityl-2-arachidonic acyl (arachidonoyl)-sn-glyceryl-3-phosphorylcholine (arachidonic acyl PC (arachidonyolPC)), because this is PLA ₂The preferred substrate of desirable features form identification.The same with the prediction of the previous research of carrying out with natural PAF-AH, this phosphatide is not degraded when with reorganization PAF-AH incubation.In other experiment, with the concentration range of 0-125 μ M arachidonoylPC is included in during standard P AF hydrolysis detects, to determine its PAF-AH hydrolysis that whether suppresses to recombinate to PAF.Even when the PAF-AH of maximum concentration, all not suppressing the PAF hydrolysis, this concentration surpasses 5 times of PAF concentration.Therefore, reorganization PAF-AH shows the substrate selective the same with natural enzyme; Nonrecognition long-chain substrate.In addition, reorganization PAF-AH enzyme is degraded apace and has been carried out sn-2 Fatty Acid Oxidation cracked oxidized phospholipids (glutaryl PC).Native plasma PAF-AH has several other characteristics that itself and other Phospholipid hydrolase is distinguished, and comprises Ca-dependent, to modifying sulfydryl or destroying the resistance of the compound of disulphide.

Natural and reorganization PAF-AH enzyme shows that all to the DFP sensitivity Serine constitutes the part of their avtive spots.The uncommon feature of native plasma PAF PAF-AH be it with circulation in lipoprotein be closely connected, its catalytic efficiency is subjected to the lipoprotein environmental influence.When reorganization PAF-AH of the present invention and human plasma (handling to eliminate endogenous enzyme activity with DFP in advance) insulation, it combines with low density and high-density lipoprotein (HDL) in the mode identical with natural radioactivity.This result is significant, because there is a large amount of evidences to show, the modification of low-density lipoprotein is that oxidation essential, lipid is the initiation factor in this process to observed cholesterol deposits in atheroma.PAF-AH avoids modifying under oxidizing condition at external protection low-density lipoprotein, and this effect may be arranged in vivo.Therefore giving PAF-AH is suitable for suppressing the oxidation of lipoprotein in the atherosclerotic plaque and can diminishing inflammation.

These results confirm that cDNA clone sAH406-3 coding has the active albumen of human plasma PAF-AH.

Embodiment 10

At the various reorganization of expression in escherichia coli PAF-AH product.Product comprises PAF-AH analogue and the PAF-AH fragment with monamino acid sudden change.A.PAF-AH amino-acid substitution product

PAF-AH is a lipase, because its hydrolytic phosphatide PAF.Though significantly not total similarity between PAF-AH and other lipase of having identified has been found conserved residues in the comparison of the lipase of structural characterization.A Serine has been differentiated to be the member of avtive spot.This Serine has formed the catalysis triplet of the avtive spot of representing this lipase with asparagicacid residue and histidine residues.These three residues are non-conterminous in the albumen primary structure, but structural research has confirmed that these three residues are adjacent in three-dimensional structure.Mammals lipase texture ratio is prompting, and this asparagicacid residue is generally apart from 24 amino acid of avtive spot Serine C-terminal.In addition, this Histidine is generally apart from 109-111 amino acid of avtive spot Serine C-terminal.

By site-directed mutagenesis and PCR, modify the single codon of people PAF-AH encoding sequence, with the coding alanine residue and at expression in escherichia coli.As shown in following table 8, for example abbreviation " S108A " shows that the serine residue that is positioned at position 108 is changed to L-Ala, Ser ₂₇₃, Asp ₂₉₆Or His ₃₅₁The point mutation completely destroy PAF-AH activity.Distance between the avtive spot residue is to PAF-AH (Ser to Asp, 23 amino acid; Ser to His, 78 amino acid) and other lipase be similar.These experimental results show that Ser ₂₇₃, Asp ₂₉₆And His ₃₅₁Being to active crucial residue, may be the candidate of catalysis triplet residue therefore.Halfcystine is normally crucial for proteinic functional completeness, because they have the ability that forms disulfide linkage.Blood plasma PAF-AH has 5 halfcystines.For in determining these 5 which is crucial to enzymic activity, each halfcystine is sported Serine individually, the mutant that produces at expression in escherichia coli.Use the preliminary activity of the partial purification prepared product of the mutant that these reorganization produce to the results are shown in the 2nd hurdle of following table 8, use the 3rd hurdle that the results are shown in table 8 of the prepared product of purifying more.Data presentation, all cysteine mutants have roughly the same activity, so all these halfcystines are all optional to the PAF-AH activity.Other point mutation does not also almost have the PAF-AH catalytic activity or not influence.In table 8, " ++ ++ " the about 40-60U/ml/OD of representative ₆₀₀Wild-type PAF-AH activity, the about 20-40U/ml/OD of " +++" representative ₆₀₀Activity, the about 10-20U/ml/OD of " ++ " representative ₆₀₀Activity, "+" represents 1-10U/ml/OD ₆₀₀Activity, "-" shows＜1U/ml/OD ₆₀₀Active.

Table 8

The PAF-of the prepared product of the active purifying of sudden change PAF-AH

AH is than living

Wild-type ++ ++ 6.9mmol/mg/ hour

S108A ++++

S273A -

D286A -

D286N ++

D296A -

D304A ++++

D338A ++++

H351A -

H395A，H399A ++++

C67S +++5.7mmol/mg/ hour

C229S+6.5mmol/mg/ hour

C291S+5.9mmol/mg/ hour

C334S ++ ++ 6.8mmol/mg/ hour

C407S +++6.4mmol/mg/ hour C67S, C334S, C407S 6.8mmol/mg/ hour B.PAF-AH fragment product

By the different time of 3 ' end with exonuclease I II digestion PAF-AH encoding sequence, the plasmid DNA that then encoding sequence that shortens is connected to coding terminator codon in all three frames prepares the C-terminal disappearance.10 different disappearance constructions have been identified by dna sequence analysis, protein expression and PAF-AH living features.Remove 21 to 30 C-terminal amino acid and significantly reduced catalytic activity, the activity of having removed 52 residue completely destroys.Referring to Fig. 3.

N-terminal at PAF-AH has produced similar disappearance.Prepared PAF-AH and escherichia coli thioredoxin fusions, to promote conforming high level expression PAF-AH activity [LaVallie etc., Bio/technology, 11:187-193 (1993)] in N-terminal.Remove the N-terminal (Ile of natural process ₄₂) 19 amino acid reduce active 99%, and the enzymic activity in the fusion rotein of having removed 26 amino acid completely destroys.Referring to Fig. 3.It seems that lack 12 amino acid strengthens about 4 times of enzymic activity.

Afterwards by with embodiment 1 in the similar method described from Freshman blood plasma purifying PAF-AH (using Microcon 30 filters of Amicon to concentrate Blue sepharose elutriant rather than copper post), differentiated two N-terminal except that Ile42, Ser _3sAnd Lys ₅₅This heterogeneity may be the native state of enzyme in the blood plasma, or may take place in purifying.

The material of above-mentioned purifying is carried out the glycosylation analysis.With natural PAF-AH incubation under the situation that has or do not exist the N-glycanase of purifying, this enzyme is removed the carbohydrate that N connects from glycoprotein.The PAF-AH sample of handling is passed through 12% sds polyacrylamide gel electrophoresis, use the rabbit polyclonal antiserum(antisera) to manifest then by Western blot.The protein of handling with the N-glycanase is with the diffusion band migration of 45-50kDa, and the protein of handling with glycanase confirms that with the tight band migration of about 44kDa natural PAF-AH is glycosylated.

Also at reorganization PAF-AH (Ile ₄₂N-terminal) observes the N-terminal heterogeneity in the purifying prepared product.These prepared products are that N-terminal originates in Ala ₄₇, Ile ₄₂Or and Ile ₄₂The artificial initial Met of adjacency ₁The mixture of polypeptide.1.PAF-AH the preliminary comparison of fragment and PAF-AH

The heterogeneity of the PAF-AH that produces in view of observed reorganization prepares other recombinant products and detect uniformity behind recombinant expressed and purifying.Composition at the different time point analysis of the cell fermentation production phase recombination expression product of pBAR2/PH.2 and pBAR2/PH.9 in coli strain MC1061.By the reorganization PH.2 of the cell of a plurality of time points collections between 5-22 hour and the partially purified sample of PH.9 after the expression of matrix auxiliary laser absorption ionization mass spectrometer (MALDIMS) analysis inducible protein.

When using the PH.2 expression vector, in partially purified protein spectrum, be contemplated to the proteic mass value of rPAF-AH place and observe 2 peaks.All observe 2 peaks at all time points, the time point when finishing the fermentation adverse circumstance that shows by accumulation of the acetate in the substratum and/or oxygen consumption is observed bigger heterogeneity.The precision of MALDI-MS is to make an appointment with ± 0.3% in this mass range, approximately is an amino acid whose quality.Observed higher quality peak is consistent with the existence that the total length translation product of the PH.2 carrier of expection deducts the translation initiation methionine(Met) of removing after expection is translated.Lower mass peak lacks 1200 atomic mass units approximately.

When using the PH.9 expression vector, in partially purified protein spectrum, be contemplated to the proteic mass value of rPAF-AH place and observe dominant unimodal.Observe all at all time points that this is unimodal, do not observe heterogeneous increasing at different time points.It is consistent that observed this proteic quality and expection deduct the existence of total length translation product of PH.9 carrier of initial methionine.2. purifying PAF-AH fragment

(Ile encodes for the rPAF-AH with purifying ₄₂-Asn ₄₄₁The expression product of DNA) further relatively, the rPH.2 that purification of Recombinant is expressed (coding Met ₄₆-Asa ₄₄₁The expression product of DNA) and rPH.9 (coding Met ₄₆-Ile ₄₂₉The expression product of DNA) prepared product.RPH.9 is produced by coli strain SB7219, and generally according to above-mentioned zinc chelating purifying procedure purifying, and rPH.2 is produced and as following purifying by coli strain MC1061.By with lysis buffer (the 100mM succinate, 100mM NaCl, 20mM CHAPS, pH6.0) diluting cells precipitation (cell paste) is with the cell transformed bacteriolyze.Mix these slurries and pass through the broken bacteriolyze of high pressure.With the cell centrifugation of bacteriolyze, keep the supernatant liquor that contains rPH.2.Clarifying supernatant liquor is diluted 5 times in containing the 25mM sodium phosphate buffer of 1mM EDTA, 10mMCHAPS, pH7.0.Then with the dilution supernatant liquor on sample to Q Sepharose post.This post washs (washing 1) with the 25mM sodium phosphate buffer that contains 1mM EDTA, 50mM NaCl, 10mM CHAPS, pH7.0 of three times of column volumes earlier, the 25mM Tris damping fluid that contains 1mM EDTA, 10mM CHAPS, pH8.0 with 10 times of column volumes washs (washing 2) then, with the 25mM Tris damping fluid washing (washing 3) that contains 1mm EDTA, 100mM NaCl, 10mM CHAPS, pH8.0 of 10 times of column volumes.Finish wash-out with the 25mM Tris damping fluid that contains 1mM EDTA, 350mM NaCl, 10mM CHAPS, pH8.0.Q Sepharose elutriant is diluted 3 times in 25mM Tris, 1mM EDTA, 10mM CHAPS, pH8.0, go up sample then to Blue Sepharose post.This post at first washs with 25mM Tris, 1mM EDTA, 10mM CHAPS, the pH8.0 of 10 times of column volumes.25mM Tris, 0.5M NaCl, 10mM CHAPS, pH8.0 with 3 times of column volumes washs then.Finish wash-out with 25mM Tris, 3.0M NaCl, 10mM CHAPS, pH8.0.Blue Sepharose elutriant is diluted 5 times in 10mM sodium phosphate, 10mM CHAPS, pH6.2, go up sample then to chromatography column.This post washs with 10mM sodium phosphate, 100mMNaCl, 0.1% Pluronic F68, the pH6.2 of 10 times of column volumes.RPH.2 120mM sodium phosphate, 100mMNaCl, 0.1% Pluronic F68, pH7.5 wash-out.The hydroxyapatite elutriant dilutes 6 times with 10mM sodium phosphate, 0.1% Pluronic F68, pH6.8.Use the 0.5N succsinic acid that the hydroxyapatite elutriant of dilution is adjusted to pH6.8, go up sample then to SP Sepharose post.With 50mM sodium phosphate, 0.1% Pluronic F68, the pH6.8 washing of 10 times of column volumes, use 50mM sodium phosphate, 125mM NaCl, 0.1% Pluronic F68, pH7.5 wash-out then.By being diluted to the rPH.2 that ultimate density among 50mM sodium phosphate, 125mM NaCl, 0.15% Pluronic F68, the pH7.5 is a 4mg/ml preparation wash-out, adding tween 80 to ultimate density is 0.02% tween 80.Product with preparation passes through the 0.2u membrane filtration then, stores before using.3. compare PAF-AH fragment and PAF-AH by order-checking

By using Applied Biosystems 473A type protein sequencing instrument (AppliedBiosystems, Foster City, CA) carry out the N-terminal order-checking, use Hewlett-PackardG1009A type C-terminal protein sequencing instrument to carry out C-terminal order-checking, the relatively rPH.2 of purifying and the rPAF-AH prepared product of rPH.9 prepared product and purifying.Compare with rPAF-AH, the N-terminal heterogeneity of rPH.2 prepared product is lower.The N-terminal analysis of rPH.9 prepared product and rPH.2's is similar, but the C-terminal heterogeneity of observing the rPH.9 prepared product is lower relatively than rPH.2.

The rPH.2 prepared product of purifying contains chief series (about 86-89%) with N-terminal Ala47 and the subsequence (about 11-14%) with N-terminal Ala48, and the ratio of these two kinds of N-terminal is quite consistent under the different fermentations condition.The rPH.9 prepared product of purifying also contains and has N-terminal Ala ₄₇Chief series (about 83-90%) and have N-terminal Ala ₄₈Subsequence (about 10-17%).In contrast, attempt in bacterium to produce and originate in Ile ₄₂Polypeptide (rPAF-AH) cause having the Ala of originating in ₄₇(20-53%), Ile ₄₂(8-10%) or and Ile ₄₂The artificial initial Met of adjacency _-1The various mixtures of the polypeptide of methionine(Met) (37-72%).For rPH.2 and rPH.9, removed initial methionine effectively by the N-terminal peptase after synthetic this polypeptide of bacterium, make the L-Ala that is positioned at position 47 L-Ala of position 48 (or be positioned at) as the N-terminal residue.

A collection of rPH.2 is carried out the C-terminal order-checking, observe its C-terminal, with the HOOC-Asn of translation product expection as chief series (about 80%) with HOOC-Asn-Tyr ₄₄₁-Tyr ₄₄₀The C-terminal unanimity, and about 20% be HOOC-Leu.After the rPH.2 prepared product passes through the SDS-PAGE fractional separation, the order-checking again of main band and minor band has produced the C-terminal (AHL of the HOOC-Leu-Met of lower minor band, in following B.5. joint, describe), this has also produced low-level HOOC-His with more consistent than short 10 the amino acid whose products of total length translation product.Further peptide mapping has been presented at the C-terminal that has other in the PH.2 albumen of some batches.The C-terminal of rPH9 by direct order-checking mainly is HOOC-Ile-His (about 78-91% depends on each batch), with the HOOC-Ile of translation product expection ₄₂₉-His ₄₂₈The C-terminal unanimity.Some backgrounds (" noise ") are arranged, so can not get rid of so low-level other sequence in this technology.4. compare PAF-AH fragment and PAF-AH by MALDI-MS

RPH.2 and rPH.9 prepared product to purifying carry out MALDI-MS.Show 2 peaks (referring to Fig. 4) being contemplated to the proteic mass value of rPAF-AH place in the rPH.2 mass spectrum, with similar with the observed pattern of partially purified protein in above-mentioned B.1. joint.Accessory lower molecular weight peak exists with about 20-30% of total amount usually.The mass spectrum of rPH.9 shows the consistent dominant peak (referring to Fig. 5) of total length translation product that deducts the PH.9 carrier of translation initiation methionine(Met) with expection.Also observe the low slightly acromion of little molecular weight of the representative total amount about 5% of rPH.9.5. compare PAF-AH fragment and PAF-AH by SDS-PAGE

RPAF-AH, rPH.2 and rPH.9 prepared product to purifying carry out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Be contemplated to based on the molecular weight of albumen standard around the electrophoretic migration scope of rPAF-AH product, observing the complicated band pattern of rPH.2.In one or some gels, observe two dominant bands, easily observe minor band up and down at master tape.With these upper minor bands, the main band in middle part and lower minor band difference called after AHU, AHM and AHL.All these bands all on Western blot with anti--rPAF-AH monoclonal antibody reactive, therefore differentiated to be the rPAF-AH associated products.The degree of depth of upper minor strips A HU was deepened with the albumen storage time, inferred the modified forms of representing the rPAF-AH product.The SDS-PAGE of rPAF-AH prepared product and rPH.2's is similar.Migration has two master tapes near rPAF-AH expection molecular weight, minor band is arranged on master tape, under the master tape shade is arranged.In contrast, rPH.9 shows the single band of preponderating on SDS-PAGE, not obviously division.Also observe shallow band in two body positions of low slightly position of molecular weight and expection.Do not observe the band of AHU sample.

Also in 2D gel (isoelectrofocusing in urea, then second to carrying out SDS-PAGE), analyze the rPH.2 of purifying and the composition of rPH.9 prepared product.For rPH.9, the 2D gel is presented at isolating 5 main spots on the IEF direction.The electric charge heterogeneity that occurs between each batch of rPH.9 is consistent.In contrast, the 2D gel pattern of rPH.2 is complicated more, because it has isolating about 15 spots on IEF and SDS-PAGE direction.6. the activity that compares PAF-AH fragment and PAF-AH

The rPH.2 of purifying and rPH.9 have and enzymic activity from the endogenous PAF-AH undistinguishable of serum purifying, and rPH.2 and rPH.9 are to combine with lipoprotein with the similar mode of endogenous PAF-AH of purifying.

Embodiment 11

Carried out the initial analysis of human plasma PAF-AH mRNA expression pattern in people's tissue by the RNA blot hybridization.

(Tel-Test " B ", Friendswood TX) prepare RNA from people's brain cortex, heart, kidney, placenta, thymus gland and tonsilla to use RNA Stat 60.In addition, be THP-1 (ATCC TIB 202) preparation RNA from artificial blood precursor like cell, this clone induces differentiation to become the macrophage-like phenotype with phorbol ester acetate Semen Myristicae phorbol (PMA).With organizing RNA and inducing RNA preceding and that induce the back to prepare from promyelocyte THP-1 clone in 1-3 days to carry out electrophoresis, then be transferred to nitrocellulose filter by 1.2% agarose formaldehyde gel.By causing mark total length human plasma PAF-AH cDNA at random, sAH406-3, with embodiment 3 in to the same terms of described those conditions of library screening under with described film hybridization.Initial result shows that this PAF-AH probe and thymus gland, the hybridization of amygdaline 1.8kb band are hybridized with placenta RNA on lesser extent.

PAF is synthetic under normal physiological and pathologic conditions in brain.The short inflammatory matter of known this molecule and potential neurotoxicity character, the synthetic location mechanism of expection PAF or its quick katabolism mechanism are crucial to the health of nervous tissue.It is consistent that the existence of PAF PAF-AH in nervous tissue plays this protective effect with it.What is interesting is, differentiate all that in brain PAF-AH[is in Hattori etc., J.Biol.Chem., 269 (37): the clone who has described it among the 23150-23155 (1994) in the ox heterotrimer born of the same parents] and PAF-AH of the present invention.Whether express for determining these two kinds of enzymes at the similar or different compartment of brain, cloned the people's homologue of PAF-AH cDNA in the ox brain born of the same parents, by the RNA trace, use the essentially identical method of describing in the leading portion, its mRNA in brain is expressed pattern compare with the mRNA expression pattern of PAF-AH of the present invention.The brain zone of detecting by the RNA trace is cerebellum, medullary substance, spinal cord, lenticular nucleus, amygdala (amygdala), caudatum, thalamus and occipital pole, corticocerebral frontal lobe and temporal lobe.In every kind of these tissue, all detect the information of these two kinds of enzymes, although form is than abundant many of secretion form in the heterotrimer born of the same parents.The rna blot analysis of other tissue further discloses the interior form of heterotrimer born of the same parents and is expressing in various tissues and the cell widely, comprises thymus gland, prostate gland, testis, ovary, small intestine, colon, peripheral blood leucocyte, scavenger cell, brain, liver, skeletal muscle, kidney, pancreas and suprarenal gland.This ubiquitous expression prompting, PAF-AH has general house keeper's function in the heterotrimer born of the same parents in cell.

Also detected from the isolating monocyte of human blood and at its PAF-AH when spontaneous differentiation becomes scavenger cell cultivation and expressed.In fresh monocyte, do not detect or seldom detect RNA, induced and keep but during differentiation becomes scavenger cell, express.Have in the substratum of noble cells that PAF-AH is active to follow accumulation.Also in inducing the THP-1 cell RNA of back 1 day rather than 3 days, observe the expression of human plasma PAF-AH transcript.The THP-1 cell is not expressed the mRNA of PAF-AH at base state.

Embodiment 12

The PAF-AH that has detected in people and the mouse tissue by in situ hybridization expresses.

Obtain people's tissue from national disease research and exchange and cooperative people organization network.Collect normal mouse brain and spinal cord and EAE stages 3 mouse spinal cord from the S/JLJ mouse.Collected normal S/JLJ mice embryonic in 7 to 18 days from after fertilization.

Use a spot of OCT compound (Miles, Inc., Elkhart, IN) tissue block is placed the freezing mould of Tissue Tek II (cryomolds) (Miles Laboratories, Inc., Naperville, IL) in.They are placed freezing mould central authorities, be full of freezing mould, place then 2-methylbutane [C is housed with the OCT compound ₂H ₅CH (CH ₃) ₂, Aldrich Chemical Company, Inc., Milwaukee, WI] container in, this container is placed liquid nitrogen.In case tissue in the freezing mould and OCT compound are freezing, then place-80 ℃ of preservations until section with described.It is thick that tissue block is cut into 6 μ m, adhere to Vectabond (Vector Laboratories, Inc., Burlingame, CA) bag is stored in-70 ℃ by on the slide glass, place 50 ℃ to make it warm and remove water of condensation in about 5 minutes, in 4% Paraformaldehyde 96, fix 20 minutes in 4 ℃ then, in each rank in 1 minute (70%, 95%, 100% ethanol) of 4 ℃ of dehydrations, air-dry 30 minutes then in room temperature.To cut into slices in 70 ℃ of sex change 2 minutes in 70% methane amide/2XSSC, in 2XSSC, clean 2 times, dewater air-dry then 30 minutes.With described tissue and radiolabeled strand mRNA in situ hybridization, this strand mRNA mixes by external rna transcription from the DNA of the inside 1 Kb HindIII fragment (the Nucleotide 308-1323 of SEQ ID NO:7) that is derived from the PAF-AH gene ³⁵S-UTP (Amersham) preparation, or from being derived from the DNA preparation by PAF-AH cDNA in the heterotrimer born of the same parents of discriminatings such as Hattori.Use the probe of the 250-500bp of all lengths.In 50 ℃ of hybridization spend the night (12-16 hour); Will ³⁵The ribose probe (riboprobe) (6 * 10 of S mark ⁵Cpm/ section), the water adding hybridization buffer of tRNA (0.5 μ g/ section) and diethylpyrocarbonate (depc) processing, making its ultimate density is 50% methane amide, 0.3M NaCl, 20mM Tris pH7.5,10% dextran sulfate, 1XDenhardt solution, 100mM dithiothreitol (DTT) (DTT) and 5mM EDTA.After the hybridization,, in 60 ℃ of washings 40 minutes in 50% methane amide/1XSSC/10mM DTT, in 2XSSC, washed 30 minutes then, in 0.1XSSC, washed 30 minutes in room temperature in room temperature in room temperature washing slice 1 hour in 4XSSC/10mM DTT.The dehydration of will cutting into slices, air-dry 2 hours, with the embedding of Kodak NTB2 photographic emulsion, air-dry 2 hours, develop (storing the back in the dark fully in 4 ℃) redyed with phenodin/eosin.A. brain

Cerebellum.In mouse and people's brain, in cerebellum Purkinje cell layer, single pericaryon in basket cell and dentate nucleus (one of four dark nuclears of cerebellum), observe the intensive signal.In these cell types, also observe the information of PAF-AH in the heterotrimer born of the same parents.In addition, on the individual cells of the granular layer of grey matter and cellular layer, observe blood plasma PAF-AH signal.

Hippocampus.In people hippocampus section, it seems it is that individual cells in the whole section of pericaryon has shown the intensive signal.These are differentiated to be multiform cell space and granulosa cell.Also in hippocampus, observe PAF-AH information in the heterotrimer born of the same parents.

Brain stem.On the brainstem slice of people and mouse, on the individual cells in grey matter strong signal is arranged.

The degree layer.In people's cortex section of getting from brain, pillow and temporo cortex and on the full brain section of mouse, the individual cells of whole cortex all shows strong signal.These cells are differentiated to be taper, starlike and multiform cell space.In the different layers of cortex, do not express the differentiation that shows.These in situ hybridization result is different with the pallium result who obtains by the RNA trace.This species diversity may be the in situ hybridization result more responsive more than RNA trace.In cerebellum and hippocampus, observe the similar expression pattern of PAF-AH in the heterotrimer born of the same parents.

Hypophysis.Observe more weak signal on the dispersive individual cells in the distal part of people's tissue slice.B. people's colon

Show signal in the lymph aggregation that normal colon and regional colitis's colon all exist in the mucous membrane of section, the signal in the patient's of regional colitis the section is strong slightly.Regional colitis's colon also has strong signal in lamina propria.Similarly, in the section of ill appendix, observe high-caliber signal, and normal appendix shows signal lower but that can detect.The section of patients of ulcerative colitis does not all show tangible signal in the lymph aggregation or in the lamina propria.C. human tonsil and thymus gland

In amygdaline germinal center and among the dispersion group of intrathymic individual cells, observe the intensive signal.D. people's lymphoglandula

Observe the intensive signal at the lymph node section of obtaining from normal donor, the lymphoglandula of cutting into slices, observe more weak signal from the donor of septic shock.E. people's small intestine

Small intestine normal and regional colitis all has weak signal in aggregate lymphatic nodule of cutting into slices and lamina propria, the signal of diseased tissue is slightly high.F. people's spleen and lung

In any spleen (normal section and splenic abscess (abcess) section) or lung (normal section and wind-puff section) tissue, all do not observe signal.G. mouse spinal cord

In the spinal cord in normal and EAE stage 3, strong signal is arranged in gray nucleus, the expression in EAE stages 3 spinal cord is slightly high.In EAE stages 3 spinal cord, the cell in white matter and blood vessel week cover (perivascalar cuff) (may be to soak into scavenger cell and/or other white corpuscle) is presented at unexistent signal in the normal spinal cord.F. mice embryonic

In 11 days embryo, signal is significantly in the central nervous system of fourth ventricle, keeps constant in the time course of this signal when fetal development becomes cerebellum and brain stem.When the embryo was ripe, the signal in the central nervous system in spinal cord (the 12nd day), primary cortex and neuroganglion (the 14th day) and the hypophysis (the 16th day) became obvious.In leaving the supraneural nervus peripheralis system (originating in the 14th or the 15th day) of spinal cord, observe signal,, must occur strong signal on every side the embryo at the 17th day.Also in the 14th day liver and lungs, observe expression, in the aft section (originating in the 16th day) of intestines (originating in the 15th day) and mouth/larynx, observe expression.In the time of the 18th day, express pattern and broken up the signal that becomes in cortex, hindbrain (cerebellum and brain stem), the nerve that leaves the spinal cord lumbar region, mouth/larynx aft section, liver, the kidney, in lung and the intestines signal may be a little less than.G. general introduction

The expression of PAF-AH mRNA in tonsilla, thymus gland, lymphoglandula, aggregate lymphatic nodule, appendix and colon aggregate lymphatic nodule thing is endogenous with the possible dominant body of PAF-AH to be that the conclusion of scavenger cell is consistent, because these are organized a large amount of tissue macrophages are arranged all, it works as engulfing with antigen processing cell.

The expression of PAF-AH and the effect of the scavenger cell in monocyte source are that the hypothesis that diminishes inflammation is consistent in the inflammation tissue.Expection PAF-AH regulates the inflammatory episode cascade that is caused by these media downwards thus with passivation PAF and short scorching phosphatide.

In whole cerebral tissue, detected PAF, the rat cerebellar granule cell secretion PAF in the cultivation.The interior experiment of external and body confirms that PAF is in conjunction with the specific receptors in the nervous tissue, and inducing function and phenotype change, and for example calcium mobilization, transcriptional activation gene are the differentiation of PC12 to adjusted, neural precursor.These observe the physiological action of prompting PAF in brain, and unanimity is with it, uses the experiment of hippocampal tissue slice culture thing and PAF analogue and antagonist to hint in the recent period, and PAF is as the important regression courier of the long-term enhanced of hippocampus.Therefore, except its pathological effect in inflammation, it seems that PAF participates in daily neurone signal generating process.The outer expression of PAF-AH in brain of born of the same parents can play the time span and the intensity of the signal generation of adjusting PAF mediation.

Embodiment 13

Use the intestinal bacteria that produce PAF-AH as immunogen, prepared specific monoclonal antibody reorganization human plasma PAF-AH.

Injected mouse #1342 in the 0th day, the 19th day and the 40th day with reorganization PAF-AH.Strengthen for before merging, inject described mouse with this immunogen among the PBS, put to death mouse after 4 days, its spleen of aseptic taking-up also places 10ml serum-free RPMI1640.By grinding spleen between the freezing end that is soaked in two glass microscope slide among the serum-free RPMI1640, form single-cell suspension liquid, this serum-free RPMI1640 has replenished 2mM L-glutaminate, 1mM Sodium.alpha.-ketopropionate, 100 units/ml penicillin and 100 μ g/ml Streptomycin sulphates (RPMI) (Gibco, Canada).Cell suspending liquid is filtered by aseptic 70 order Nitex cell nutsche filters (Becton Dickinson, Parsippany, New Jersey),, will precipitate resuspending in 20ml serum-free RPMI by in 200g washing in centrifugal 5 minutes 2 times.Prepare 3 thymocytes that are used to the Balb/c mouse of testing first in a similar fashion.To merge preceding 3 days contain 11% foetal calf serum (FBS) (Hyclone Laboratories, Inc., Logan, the NS-1 myeloma cell who remains on logarithmic phase among RPMI Utah) centrifugal 5 minutes in 200g, washing precipitation described in the paragraph is 2 times as described above.

With 1 * 10 ⁸Spleen cell and 2.0 * 10 ⁷The NS-1 cytomixis, centrifugal, inhale and remove supernatant liquor.Make cell precipitation loosening by rapping test tube, in 1 minute time while stirring 37 ℃ of PEG 1500 adding 1ml (among the 75mM Hepes 50%, pH8.0) (BoehringerMannheim), then in 7 minutes time, add 7ml serum-free RPMI.Add 8mlRPMI in addition, with cell centrifugal 10 minutes in 200g.After the abandoning supernatant, will precipitate resuspending in contain 15% FBS, 100 μ M xanthoglobulin sodium, 0.4 μ M aminopterin, 16 μ M thymidines (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 * 10 ⁶Among the 200ml RPMI of thymocyte/ml, be inoculated in 10 flat 96 hole tissue culturing plates of Coming (Coming, Coming New York).

After merging the 2nd, 4 and 6 day taken out the substratum of 100 μ l and replaced with fresh culture from the hole of merging plate.In the time of the 8th day,, detect existence with reorganization PAF-AH bonded mouse IgG by ELISA screening fusions.(Dynatech, Cambridge MA) wrap by 2 hours the reorganization PAF-AH that dilutes in 25mM TRIS, pH7.5 with the 100ng/ hole with Immulon 4 flat boards in 37 ℃.Suction went bag by solution, adds the lock solution [0.5% fishskin gelatin (Sigma) that dilutes in CMF-PBS] in 200ul/ hole, in 37 ℃ of incubations 30 minutes.With the PBS that contains 0.05% polysorbas20 (PBST) wash plate three times, add 50 μ l culture supernatant.In 37 ℃ of incubations after 30 minutes, as above-mentioned washing, be added in the goat anti-mouse IgG (fc) that the 50 μ l horseradish peroxidases that diluted among the PBST 1: 3500 put together (Jackson ImmunoResearch, West Grove, Pennsylvania).As above-mentioned incubation plate,, add by 1mg/ml O-Phenylene Diamine (Sigma) in the 100mM Citrate trianion and 0.1 μ l/ml, 30% H with PBST washing four times ₂O ₂, the 100 μ l substrates formed of pH4.5.By adding 50 μ l 15%H ₂SO ₄, in 5 minutes, stop color reaction.Reading to measure A on the plate instrument ₄₉₀

By in 96 orifice plates, diluting, with fusion hole clone's secondary of selecting, the colony number in the every hole of visual counting after 5 days.Clone's hybridoma is 90D1E, 90E3A, 90E6C, 90G11D (ATCC HB 11724) and 90F2D (ATCC HB 11725).

(IN) monoclonal antibody that hybridoma is produced is carried out common-size analysis for Boehringer Mannheim, Indianapolis to use the Isostrip system.The result shows that the monoclonal antibody that the hybridoma of fusion 90 produces all is IgG ₁

All monoclonal antibodies that the hybridoma of fusion 90 produces function in ELISA detects is good, but can not be in conjunction with PAF-AH in Western blot.For preparing the antibody that to discern PAF-AH by Western blot, with recombinase immune mouse #1958.As preparing hybridoma described to merging 90, but by Western blot rather than ELISA screening, to differentiate the clone that can carry out Western blot.

For Western blot, mix reorganization PAF-AH with the equal-volume sample buffer that contains 125mM Tris pH6.8,4% SDS, 100mM dithiothreitol (DTT) and 0.05% tetrabromophenol sulfonphthalein, boiled before 5 minutes at last sample to 12% sds page (Novex).Behind 40 milliamperes of electrophoresis, in 192mM glycine, 25mM Tris alkali, 20% methyl alcohol and 0.01% SDS in 125V1 hour with the albumen electrotransfer to PVDF membrane (Pierce).(BSA, warm the region between the heart and the diaphragm spends the night among 20mM Tris Sigma), the 100mM NaCl (TBS) containing 5% bovine serum albumin in 4 ℃ with this film.In room temperature and the rabbit polyclonal antiserum(antisera) incubation that is containing dilution 1/8000 among the TBS of 5% BSA 1 hour, then with the TBS washing, the goat anti-mouse IgG of puting together with the alkaline phosphatase in the TBS that contains 5% BSA was in room temperature incubation 1 hour with trace.Wash trace once more with TBS, then with 100mM Tris-HCl pH9.5,100mM NaCl and 5mM MgCl ₂In 0.02% phosphoric acid 5-bromo-4-chloro-3-K-281 and 0.03% nitroblue tetrazolium(NBT) incubation.Carry out water washing repeatedly and stop described reaction.

Fusion hole such as above-mentioned processing the with its supernatant liquor male selection in the western blot analysis.Hybridoma 143A reacts with PAF-AH in Western blot, with its clone (ATCC HB11900).

Pass through immunity in three months by recombinase, in rabbit, produce the specific polyclonal antiserum of human plasma PAF-AH with 100 μ g purifying in the freund's adjuvant.

Embodiment 14

Use rat podedema model [Henriques etc., Br.J.Pharmacol., 106:579-582 (the 1992)] research that experimentizes, to estimate reorganization PAF-AH of the present invention to acutely inflamed curative effect.The result of these researchs confirms that rPAF-AH has blocked PAF inductive oedema.Carried out parallel study, to compare the effectiveness of PAF-AH and two kinds of commercially available PAF antagonists.A. prepare PAF-AH

The intestinal bacteria bacteriolyze that in micro-fluidised form instrument, will transform with PAF-AH expression vector puc trp AH, the centrifugal solid of removing, with sample on the cell conditioned medium liquid to S-Sepharose post (Pharmacia).This post uses the damping fluid of being made up of 50mM NaCl, 10mM CHAPS, 25mM MES and 1mM EDTA, pH5.5 to wash fully.Be increased to 1M wash-out PAF-AH by NaCl concentration with damping fluid.To use the affinity chromatography of Blue Sepharose post (Pharmacia) to use then as extra purification step.Sample is to the Blue Sepharose post on the PAF-AH prepared product, and dilute sample 1: 2 is so that the concentration of NaCl is reduced to 0.5M, with pH regulator to 7.5.After using the damping fluid of forming by 0.5M NaCl, 25mM Tris, 10mM CHAPS and 1mM EDTA, pH7.5 to wash Blue Sepharose post fully, by NaCl concentration being increased to 3.0M wash-out PAF-AH.

Estimate by SDS-PAGE, the purity of the isolating PAF-AH of this mode is generally 95%, and field of activity is 5000-10,000U/ml.Other quality control that each PAF-AH prepared product is carried out comprises measures level of endotoxin and to the erythrocytic hemolytic activity of the fresh rat that obtains.The damping fluid that contains 25mM Tris, 10mM CHAPS, 0.5M NaCl, pH7.5 is as the storage medium of this enzyme and the carrier of administration.The dosage that uses in experiment detects based on the enzymic activity of carrying out before being about to begin in experiment.B. induce oedema

With weight is that (Charles River, Wilmington MA) are used for all experiments for the 6-8 female Long Evans rat in age in week of 180-200 gram.Before experimental implementation, with narcotic ketamine (Fort Dodge Laboratories, Fort Dodge, IA), xylazine (Miles, ShawneeMission, KS) and vetrnquil (Aveco, Fort Dodge, IA) mixture anesthetized animal, about 2.5mg ketamine, 1.6mg xylazine, 0.2mg vetrnquil under every animal skin of every dosage.Give or PAF or zymosan are induced oedema at foot by following.Be each experiment prepared fresh PAF (Sigma #P-1402) from the 19.1mM mother liquor that is stored in chloroform/methanol (9: 1) in-20 ℃.At N ₂Be dried to the volume that needs down, in the damping fluid that contains 150mm NaCl, 10mM Tris pH7.5 and 0.25% BSA, diluted 1: 1000 supersound process 5 minutes.Animal is subcutaneous acceptance 50 μ l PAF (final dose is 0.96nmole) between the metapedes pad, and 1 hour postevaluation oedema was estimated oedema once more after 2 hours in some experiments.Be the zymosan A (Sigma #A-8800) of each experiment prepared fresh as the 10mg/ml suspension among the PBS.Animal is subcutaneous acceptance 50 μ l zymosans (final dose is 500 μ g) between the metapedes pad, 2 hours postevaluation oedema.

By before will giving PAF or zymosan and the fixed time after hitting point with PAF or zymosan merit measure sufficient volume and come quantitative oedema.The milliliter numerical table that increases with sufficient volume shows oedema.Use the zoometry volume displaced measurement of volumeter (plethysmometer) (UGO Basile, #7150 type) of measuring submerged foot metathetical volume of water to anaesthetizing.For guaranteeing that a time point is comparable to the foot immersion of next time point, with indelible ink markings metapedes hair line and the crossing place of heel.Use the same foot of this technology replication to show that tolerance range is within 5%.C.PAF-AH route of administration and dosage

Local injection PAF-AH between the foot pad is perhaps by the injection of injecting systems in the tail cava vein.For topical administration, rat has been accepted 100 μ l PAF-AH (4000-6000U/ml) of subcutaneous transmission between right back foot pad.Left side foot is by giving 100 μ l carriers (buffer salt solution) as contrast.Give PAF-AH for system, rat is received in the PAF-AH that shows unit that 300 μ l carrier medium sized veins give in the tail vein.Accept the carrier intravenous injection of proper volume to impinging upon the tail vein.D. topical administration PAF-AH

Subcutaneous injection 100 μ l PAF-AH (4000-6000U/ml) between the right foot pad of rat (N=4).With 100 μ l carriers (buffer salt solution) injection left side foot.Other four rats are only used vector injection.All rats are all attacked with the PAF by subcutaneous foot injection immediately, attack and estimate sufficient volume in back 1 hour.Fig. 6 has described to have blocked PAF inductive podedema by topical administration PAF-AH, and wherein average increase (the ml) ± SEM with the sufficient volume of each treatment group expresses oedema.The group of having accepted local PAF-AH processing before PAF attacks shows the inflammation that comparison reduces according to the injection group.Compare with 0.63 ± 0.14 (SEM) of the contrast of vehicle treated, in the PAF-AH group, observe the increase of the sufficient volume of 0.08ml ± 008 (SEM).The increase of foot volume is the direct result of PAF injection, because the only animal of injecting in foot with the carrier increase that do not show sufficient volume.E. intravenously gives PAF-AH

Before PAF attacks 15 minutes, with or PAF-AH (2000U in the 300 μ l carriers) or only with carrier intravenous injection rat (every group of N=4) in advance.Fig. 7 has described to give PAF-AH by intravenously and has blocked PAF inductive podedema in the time of 1 and 2 hour in attacking the back, and wherein average increase (the ml) ± SEM with the sufficient volume of each treatment group expresses oedema.The group of accepting 2000U PAF-AH by intravenous route is presented at that inflammation reduces in 2 hours time courses.The average-volume increase of PAF-AH treatment group is 0.10ml ± 0.08 (SEM) in the time of 2 hours, and the average-volume increase of the contrast of vehicle treated is 0.56ml ± 0.11.F. relatively protecting by the PAF-AH in PAF or the zymosan inductive oedema

With or PAF-AH (2000U in the 300 μ l carriers) or only handle rat (every group of N=4) with carrier intravenous injection in advance.After the pre-treatment 15 minutes, each winding was subjected to or PAF or zymosan A, respectively at postevaluation in 1 and 2 hour foot volume.As shown in Figure 8, system gives PAF-AH (2000U) and effectively reduces PAF inductive podedema, but fails to block zymosan inductive oedema, and wherein average increase (the ml) ± SEM with the sufficient volume of each treatment group expresses oedema.The average-volume increase of PAF-AH treatment group is 0.08 ± 0.02, and the average-volume increase of control group is 0.49ml ± 0.03.The effective dose titration of G.PAF-AH protection

In two independent experiments, attacked preceding 15 minutes at PAF, with each the group rat (N=3-4/ group) with 300 μ l volumes or PAF-AH serial dilutions or the pre-treatment of vehicle Control intravenously.Biped is all attacked (as above-mentioned), 1 hour postevaluation oedema with PAF.Fig. 9 has described to have strengthened in the rat with the PAF-AH injection of the dosage that increases and has been protected from PAF inductive oedema, and wherein average increase (the ml) ± SEM with the volume of each treatment group expresses oedema.In these experiments, find the ID of the PAF-AH that gives by intravenous route ₅₀Between the 40-80U/ rat.H. render a service in the body with the PAF-AH that gives the variation of back time

In two independent experiments, 2 groups of rats (N=3-4/ group) are used or PAF-AH (2000U in the 300 μ l carriers) or the pre-treatment of carrier self intravenously.After giving, each is organized and gives back 15 minutes to 47 hours time point in PAF-AH and accept PAF.PAF attacks 1 hour postevaluation oedema.As shown in figure 10, the PAF-AH protection rat that gives 2000U avoids PAF inductive oedema at least 24 hours, and wherein average increase (the ml) ± SEM with the volume of each treatment group expresses oedema.The pharmacokinetics of I.PAF-AH

4 rats are accepted 2000U PAF-AH in the 300 μ l volumes by intravenous injection.Collect blood plasma in each time point, be stored in 4 ℃, measure the plasma concentration of PAF-AH by the ELISA that uses the bispecific monoclonal antibody catch assay.In brief, monoclonal antibody 90G11D (embodiment 13) is diluted to 100ng/ml in 50mM carbonic acid buffer pH9.6, on Immulon 4ELISA plate, fixedly spends the night in 4 ℃.Behind the PBS thorough washing that contains 0.05% polysorbas20, described plate is used in 0.5% fishskin gelatin (Sigma) that dilutes among the PBS sealed 1 hour in room temperature.To contain the serum sample that dilutes among the PBS of 15mM CHAPS and add with double in the ELISA flat board of washing, in room temperature incubation 1 hour.After the washing, the biotin conjugate of monoclonal antibody 90F2D (embodiment 13) concentration with the 5 μ g/ml that dilute in PBS is added in the hand-hole, in room temperature incubation 1 hour.After the washing, 1: 1000 diluent of the ExtraAvidin (Sigma) of 50 μ l is added in the hand-hole, in room temperature incubation 1 hour.After the washing, use OPD to make each hole colour developing, quantitatively as substrate.Calculate enzymic activity from typical curve then.Figure 11 shows that 1 hour blood plasma enzyme level has reached based on the expection concentration that restrains the 5-6ml blood plasma volume of rat for 180-200, and average=374U/ml ± 58.2, wherein data point is represented average ± SEM.Blood plasma level continues to descend after 1 hour, reaches mean plasma concentration 19.3U/ml ± 3.4 in 24 hours, still is significantly higher than the endogenous P of Rats AF-AH level of finding to be about 4U/ml by enzymatic determination.The effectiveness of J.PAF-AH and PAF antagonist relatively

Each is organized a kind of pre-treatment of rat (N=4/ group) with 3 kinds of potential anti-inflammatory agenies: the PAF-AH (2000U) that PAF antagonist Alproazolam (Sigma #A-8800) (2mg in the 200 μ l ethanol) that the PAF antagonist CV3988 (Biomol #L-103) that intraperitoneal gives (2mg in the 200 μ l ethanol), intraperitoneal give or intravenously give.Control rats is with the carrier intravenous injection of 300 μ l volumes.Intraperitoneal gives PAF antagonist, because they are dissolved in ethanol.Give behind the PAF antagonist 30 minutes, attack with PAF and injected or the rat of CV3988 or Alprazolam, so that the PAF antagonist enters circulation, and the rat of PAF-AH and vehicle treated gives to be attacked in back 15 minutes at enzyme.Show with the rat of PAF-AH injection and to surpass the reduction to PAF inductive oedema by the PAF antagonist CV3988 that has established and Alprazolam.Referring to Figure 12, wherein the average increase ± SEM with the volume of each treatment group represents oedema.

Say that briefly rPAF-AH blocks the oedema by the PAF mediation in vivo effectively.Can or topical administration or give PAF-AH product by the intravenous injection systematicness.In dose study, find that the intravenous injection in the 160-2000U/ rat scope reduces the inflammation of PAF mediation significantly, and ID ₅₀Dosage be it seems in the scope of 40-80U/ rat.Based on the calculating of the blood plasma volume of 180-200 gram rat, the plasma concentration in the prediction 25-40U/ml scope should be blocked the oedema that PAF causes.These predictions are subjected to the support of preliminary pharmacokinetic study.The dosage of finding 2000UPAF-AH is effectively blocked the oedema at least 24 hours of PAF mediation.Give behind the PAF-AH 24 hours, the plasma concentration of finding this enzyme is about 25U/ml.Find that PAF-AH more effectively blocks PAF inductive oedema than described 2 kinds of known PAF antagonists of test.

In a word, these results confirm that PAF-AH blocks PAF inductive inflammation effectively, may have therapeutic value in PAF is the disease of main medium.

Embodiment 15

At the second individual inner model is test reorganization PAF-AH of the present invention in the PAF inductive pleuritis.When importing pleural space, PAF has shown that induction of vascular leaks [Henriques etc. see above-mentioned].Had 300 μ l reorganization PAF-AH (1500 μ mol/ml/ hours with 200 μ l, preparation as described in example 14 above) in the 0.9% contrast damping fluid or the blue dyestuff of 1% Evans in the equal-volume contrast damping fluid the tail vein injection female rats (Charles River, 180-200g).After 15 minutes, described rat accepts to be injected into 100 μ l PAF (2.0nmol) of pleural space.PAF attacked after 1 hour, put to death rat, collected Pleural fluid by clean this chamber with 3ml heparinization phosphate-buffered saline.Determine the degree of vessel leakage by the amount (absorption value by 620nm is quantitative) of the blue dyestuff of Evans in the pleural space.Find with the vessel leakage few (represent inflammation alleviate surpass 80%) of the pretreated rat comparison of PAF-AH according to animal.

Above result supports to suffer from pleuritic curee with reorganization PAF-AH enzyme treatment of the present invention.

Embodiment 16

Also raise and tested reorganization PAF-AH enzyme of the present invention in the model at the eosinophilic granulocyte of antigen induction.In the air flue gathering of eosinophilic granulocyte be take place in asthma, rhinitis and the eczema late period immunne response feature.With the interval in 2 weeks, by 2 peritoneal injections with BALB/c mouse (Charles River) sensitization, injection liquid by 4mg aluminium hydroxide (Imject alum, PierceLaboratories, Rockford, 1 μ g ovalbumin (OVA) in IL) is formed.Immunity for the second time is after 14 days, with or aerosolized OVA or attack the mouse of sensitization as the salt solution of contrast.

Before the attack, mouse is divided into 4 groups at random, 4 mouse/groups.The mouse of group 1 and 3 uses the 140 μ l that are made up of 25mM Tris, 0.5M NaCl, 1mM EDTA and 0.1% tween 80 that give by intravenous injection to contrast the damping fluid pre-treatment.(activity that gives in the 140 μ l PAF-AH damping fluids is 5,500 units/ml) pre-treatment to group 2 and 4 mouse with the PAF-AH of 750 units.Give after PAF-AH or the damping fluid 30 minutes, be exposed to aerosolized PBS, be exposed to aerosolized OVA and organize 3 and 4 mouse as the following mouse that will organize 1 and 2 group.After 24 hours, by intravenous injection with or 140 μ l damping fluids (group 1 and 3) or 140 μ l damping fluids in the PAF-AH (group 2 and 4) of 750 units handle mouse for the second time.

By animal being exposed to aerosolized OVA, in the mouse of sensitization, induce eosinophilic granulocyte to soak into tracheae.The mouse of sensitization is placed 50ml taper centrifuge tube (Coring), and (Somerset PA) forces and breathed the aerosolized OVA (50mg/ml) that is dissolved in 0.9% salt solution 20 minutes for 646 types, DeVilbiss Corp. to use atomizer.Handle control mice in a similar manner, but what use is 0.9% salt solution in atomizer.Be exposed to aerosolized OVA or salt solution after 48 hours, put to death mouse, cut out tracheae.The tracheae of each group is embedded among the OCT, before section, is stored in-70 ℃.

Eosinophilic granulocyte that be to estimate tracheae soaks into, with or Luna solution and hematoxylin-eosin solution or with the tissue slicies of 4 groups of mouse of peroxidase stain.Cut out 12 the 6 thick sections of μ m from every group of mouse, and corresponding numbering.As the following Luna dyeing of section with odd-numbered.To cut into slices and in formaldehyde acetal (formal alcohol), fix 5 minutes, clean with three times 2 minutes tap water in room temperature in room temperature, then in room temperature at two times 5 minutes dH ₂O cleans.In room temperature with the Luna dyestuff with 5 minutes (the Luan dyestuff is made up of 90ml Weigert iron haematoxylin and 10ml1% Biebrich Scarlet) of tissue section strain.Painted slide glass is dipped in 1% acid alcohol 6 times, in tap water, cleaned 1 minute, in 0.5% Quilonum Retard solution, dip in 5 times, in the mobile tap water, cleaned 2 minutes in room temperature in room temperature.By 70%-95%-100% ethanol (each 1 minute) with slide glass in room temperature dehydration, transparent in two times 1 min Xylene in room temperature then, sealing in Cytoseal 60.

For peroxidase stain, the section of even-numbered is fixed 10 minutes in 4 ℃ of acetone, air-dry.Add 200 μ l DAB solution to each section, left standstill 5 minutes in room temperature.Slide glass was cleaned in tap water 5 minutes in room temperature, 2 1% osmic acids are used for each cut into slices 3-5 second.In room temperature slide glass was cleaned in tap water 5 minutes, be skillful in room temperature with 25 ℃ Mayers bushes and redye.In the mobile tap water, cleaned slide glass 5 minutes then, by 70%-95%-100% ethanol (each is 1 minute) with slide glass in room temperature dehydration, transparent in two times 1 min Xylene in room temperature then, sealing in Cytoseal 60.

Estimate the eosinophilic granulocyte number in the tela submucosa tracheae.The tracheae of discovery group 1 and 2 mouse has the few eosinophilic granulocyte of dispersive in whole telasubmucosa.(this group mouse is with the damping fluid pre-treatment and be exposed to atomizing OVA) finds a large amount of eosinophilic granulocytes in whole telasubmucosa as desired to the tracheae of organizing 3 mouse.In contrast, with the PAF-AH pre-treatment and be exposed in the tracheae of group 4 mouse of atomizing OVA and in telasubmucosa, found and promptly to have organized observed suitable few eosinophilic granulocyte in 1 and 2 with two control groups.

Therefore, shown with the PAF-AH therapeutic treatment curee who shows the immunne response in late period, immunne response comprised such as the eosinophilic granulocyte that takes place in asthma and rhinitis and assembling in air flue described late period.

Embodiment 17

Also in the rat model of two different treatment necrotizing enterocolitiss (NEC) (the acute hemorrhagic bowel necrosis that promptly in LBWI, takes place and cause remarkable M ﹠ M), tested PAF-AH product of the present invention.Previous experiment confirms, handles with glucocorticosteroid and reduced in the animal and the incidence of NEC among the premature infant, and the activity of having pointed out glucocorticosteroid is that blood plasma PAF-AH is active to be taken place by increasing.A. by the activity in the rat of PAF attack inductive NEC

1. prevent NEC

To recombinate PAF-AH product rPH.2 (among the 0.3ml 25,500 units, group 2 and 4) or carrier/damping fluid itself (25mM Tris, 0.5M NaCl, 1mM EDTA and 0.1% tween 80) (group 1 and 3) to give weight be the tail vein that 180-220 restrains female Wistar rats (n=3).As described in [J.Pediatr.Res.34:237-241 (1993)] such as previous Furukawa, behind rPH.2 or vector injection 15 minutes the time, will or BSA (0.25%)-salt solution (group 1 and 2) or the PAF (0.2 μ g/100g) that is suspended in the BSA salt solution go into aorta abdominalis at the horizontal injection of superior mesenteric artery.Ligament from Trietz to caecum after 2 hours takes out small intestine, thoroughly washs with cool brine, checks roughly.Obtain sample from the microscopy of the upper, middle and lower of small intestine.Fixing in being organized in buffered formalin, by handling sample to carry out microscopy with phenodin and eosin dyeing.Repeated experiments three times.

Finding substantially to show, is the intestines of normal appearance in the group with BSA brinish vehicle treated.The rPH.2 that injects when similarly, lacking PAF is to checking not influence substantially.In contrast, PAF be injected into serosa surface that descending aorta causes described intestines fast, serious discoloration and hemorrhage.Notice similarly hemorrhagely when at mucosal surface inspection one joint small intestine, described small intestine be it seems suitable necrosis.When described intestines be it seems normally when the tail vein injection rPH.2 in 15 minutes before giving aorta with PAF.

When carrying out microscopy, from organizing normal fluff structures and the normal populations in 1, the 2 and 4 small intestines confirmation lamina proprias that obtain.In contrast, the full thickness that is presented in the whole mucous membrane of the group of only handling with PAF is downright bad and hemorrhage.

Also measured the activity of blood plasma PAF-AH in the rat of in above-mentioned experiment, using.As following mensuration PAF-AH activity.Before tail vein injection, obtain blood sample.Just obtain blood sample from vena cava before injection PAF and when putting to death rat subsequently.With the blood collecting of about 50 μ l in the heparinization kapillary.Obtain blood plasma (980xg 5 minutes) after centrifugal.As Yasuda and the previous described detection enzyme of Johnston (Endocrinology, 130:708-716 (1992)).

Find before the injection average blood plasma PAF-AH activity of all rats be 75.5 ± 2.5 units (1 unit equal 1nmole * minute ^-1* ml ^-1Blood plasma).15 minutes average blood plasma PAF-AH activity is that 75.2 ± 2.6 units, group 3 are 76.7 ± 3.5 units for group 1 behind the injection carrier.After 15 minutes, with the blood plasma PAF-AH of the animal of 25,500 rPH.2 of unit injection active for group 2 be that 2249 ± 341 units and group 4 are 2494 ± 623 units.The activity of group 2 and 4 keeps higher (1855 ± 257 unit) (rPH.2 injected back 2.25 hours) (group 2=1771 ± 308 when putting to death; Group 4=1939 ± 478).These results show, only use the blood plasma PAF-AH activity (group 1 and 3) of the rat of vector injection not change in experimentation.All animals of only accepting the PAF injection all have the NEC development, are subjected to protecting fully and inject the rat that connects the PAF injection with rPH.2.

The dose-dependently of 2 NEC prevention

For whether the protection of determining antagonism NEC in the rat is dose-dependent, before giving, PAF handled animal with the rPH.2 that increases dosage in 15 minutes.Originally, with 25.5-25, the rPH.2 of 500 unit scopes gives rat tail vein.Then PAF (0.4 μ g in the 0.2ml BSA salt solution) was injected into aorta abdominalis in 15 minutes after giving rPH.2.PAF gives to take out small intestine after 2 hours, checks the development of NEC.Before giving this enzyme, external source gives to measure after back 2.25 hours blood plasma PAF-AH activity with rPH.2.The result is the mean value of 2-5 animal in every group.

The discovery of Jian Chaing shows substantially, and all acceptance have developed NEC less than the rat of 2,000 these enzymes of unit.Accept in the animal of minimal protection amount (2040 unit) enzyme that blood plasma PAF-AH activity is every milliliter of blood plasma 363 units after 15 minutes, representative exceeds 5 times of substrate levels.When being lower than 1,020 total unit and giving rPH.2, the plasmin activity that obtains on average is about 160 or lower, and all animal persons have been developed NEC.

3. resist the time span of the protection of NEC

For determining that external source PAF-AH product provides the time span to the protection of NEC development,, then attack with PAF at different time points by tail vein this enzyme to a fixed amount of rat injection.With rPH.2 (8,500 units among the 0.3ml) or only give in the rat tail vein, give the back different time with PAF (0.36 μ g in the 0.2ml BSA salt solution) in this enzyme and be injected into aorta abdominalis with carrier.PAF injects and took out small intestine in back 2 hours, checks substantially with histological examination to develop to estimate NEC.Different time and PAF give back 2 hours mensuration blood plasma PAF-AH activity after enzyme gives.Determine the enzymic activity mean+SD of each group.

The result shows there is not rat development NEC in preceding 8 hours behind injection rPH.2, but the animal 100% ground development NEC that behind 24 and 48 hours of this enzyme of injection, attacks with PAF.

4.NEC reverse

Whether can reverse development for determining to give the PAF-AH product, this enzymes of 25,500 units is given by being injected into vena cava giving PAF (0.4 μ g) 2 minutes afterwards by PAF injection inductive NEC.There is not animal development NEC.But when giving rPH.2 by this approach in 15 minutes behind injection PAF, all animals are all developed NEC, are consistent with the quick time course that passes through to give PAF inductive NEC development of [seeing above-mentioned] previous report such as Furukawa.

The summary of these observations shows that the active less relatively increase of blood plasma PAF-AH (5 times) can prevent NEC.These are observed and had before confirmed tire rabbit [Maki, Deng, Proc.Natl.Acad.Sci. (USA) 85:728-732 (1988)] and premature infant [Caplan, Deng, J.Pediatr.116:908-964 (1990)] in the active relatively low report of blood plasma PAF-AH combine, prompting can be used for the treatment of NEC to the preventative administration of human reorganization of low birth wt baby PAF-AH product.

Activity in the B.NEC newborn dummy

As the following effectiveness of in the NEC model, estimating PAF-AH product rPH.2, feed by prescription in this model and the common risks and assumptions association of these two kinds of human diseasess that suffocate compels neonate rat.In this model, the 3rd day of life, the animal of about 70-80% developed with newborn infant NEC similarly substantially with damage of intestines microscopic level.From using CO ₂(Harlan Sprague-Dawley, Indianapolis IN) obtain neonate rat to the conceived Sprague-Dawley rat of anaesthetizing and delivering a child by abdominal incision.Collect new born animal, drying keeps in newborn infant's incubator in whole experiment.

At first, use animal groups independently to estimate giving of rPH.2 and absorb feature.Give one of rPH.2 (3 λ, 15 λ or 75 λ) of young three kinds of different intestines of normal newborn rat or intraperitoneal dosage in 0 of time, after 1 hour, 6 hours or 24 hours, collect blood to estimate blood plasma PAF-AH activity.Use the substrate incubation to detect the ELISA of the anti-people rPAF-AH monoclonal antibody (90F2D and 90G11D are described among the embodiment 13) of [Gray etc., Nature, 374:549 (1995)] and each sample of use, measure the PAF-AH activity.For the sample of selecting, use at two kinds of different monoclonal antibodies (90F2D and 90G11D are described among the embodiment 13) of people rPAF-AH exploitation and carry out immunohistochemical analysis.Adopt the standard technique of the antibody that uses dilution in 1: 100 and the incubation that spends the night to carry out immunohistochemistry.

After the normal newborn rat enteron aisle gave rPH.2, use or substrate incubation detection at any time or elisa technique did not all have detectable blood plasma PAF-AH activity.When using intraperitoneal to give rPH.2, after administration 1 hour, use these two kinds of methods that detectable significant cycle P AF-AH activity is all arranged, this activity peaked at 6 hours.(from 3 to 75 λ 10-250U) cause higher blood plasma PAF-AH activity to the rPH.2 of higher dosage.Immunohistochemical analysis discloses, and has the rPAF-AH product in the epithelial cell at intestinal mucosa behind the enteral administration.This reactivity is mainly assembled in intestinal villus, and dyeing is minimum in the pit cell.Dyeing in the ileum is more than jejunum, and immunochemistry has been differentiated some rPAF-AH products in the part of colon.In any check sample or in the sample that from animal, obtains by the intraperitoneal administration without any verifiable dyeing.Therefore, intestines give the local mucous membrane epithelium accumulation that the rPAF-AH product causes this enzyme, absorb without any detectable systematicness, and in contrast, intraperitoneal give the rPAF-AH product and cause high cyclophorase level, but do not have the local mucous membrane accumulation.

In described NEC model, according to Caplan etc., Pediatr.Pathol., 14:1017-1028 (1994) induces NEC in neonate rat.In brief, use from the young prescription of the newborn rat of powder (Esbiliac, Borden Inc) reconstruct and fed animal by the pipe of feeding every 3 hours.The volume of feeding is fed initial with 0.1ml/, be increased to the 4th day of this scheme according to tolerance.All animals by every day in airtight 50 seconds of plastic box internal respiration secondary 100% nitrogen, and be exposed to freezing (4 ℃) 10 minutes, attack with the invasion that suffocates (asphyxial insalt).After feeding, stimulate enteron aisle and bladder function with soft manipulation at every turn.Show painful symptom with animal maintenance 96 hours or until their.Animal abdominal distension, bloody stool, respiratory distress, cyanosis and the lethargic sleep of morbidity cause death by detruncation is painless.After the execution, check the downright bad sign of every rat intestine substantially, formalin fixed is to carry out later histologic analysis then.With sample with paraffin embedding, with slicing machine section, with phenodin and eosin dyeing, by two viewers with blind method inspection.With damage of intestines marking, 1+ is that epithelial cell lifting or separation, 2+ be that epithelial cell sloughs to medium fine hair (mid-villous) level, 3+ are that whole fine hair necrosis, 4+ are the wall necrosis.

Be to estimate the effectiveness of rPH.2, with this compound by intestines transmission, intraperitoneal transmission or organize with 3 different rats of this two kinds of methods processing simultaneously.This rPH.2 prepared product has 0.8mg/ml albumen and about 4000 units/mg PAF-AH activity,＜0.5EU/mg intracellular toxin/albumen ratio.The through port stomach tube is with the dilution animal of rPH.2 enterally administering of 25 λ (80U) of (every 3 hours) that goes into to feed at every turn.The peritoneal injection of secondary gives animal 75 λ of intraperitoneal administration by every day.Control animals received do not contain damping fluid (the 20mM NaPO of the proper volume of rPH.2 ₄, pH7.4), study simultaneously with each experimental group.Each treatment group is estimated mortality ratio and NEC symptom, use the FischerExact detection statistics to analyze difference.＜0.05 p-value is considered to remarkable.The results are shown in following table 9.

Table 9

Four different experiments, intestinal canal administration four that the dead contrast of NEC (giving in the peritonaeum) 7/10 8/10rPH.2 (240U every day 2 peritonaeums in) 6,/11 8/11 contrast (enteron aisle gives) 19/26 21/26rPH.2 (giving 80 6/26 7/26U every 3 hours enteron aisles) contrast (peritonaeum in+enteron aisle give) 10/17 12/17rPH.2 (give 240 3/14 7/14U every day in 2 peritonaeums and give 80U every 3 hours enteron aisles) data represent administration in the peritonaeum test and peritonaeum in+accumulation results of three experiments of intestinal canal administration.

Compare with control animal, enteron aisle rPH.2 has given to reduce significantly NEC and dead incidence.The result of experiment of four different enterally administerings shows, with the rPH.2 pre-treatment NEC is reduced to 6/26 (p＜0.001) from 19/26 (contrast).Handle and control animal between damage of intestines be variable, but in most cases be characterized as medium fine hair necrosis, the whole fine hair necrosis in other zone in some joints, have wall necrotic area, remainder to have normal small intestine sometimes to learn.The worst degree with NEC of the processing animal of damage of intestines and control animal is similarly (the average marking of contrast is 2.8, and the average marking of the rat that rPH.2 handles is 2.4, p＞0.05).

Intraperitoneal administration rPH.2 does not make significant difference to the NEC or the death of this model.Occurring between this group and the contrast of symptom is similarly (contrast is 40 ± 5 hours, and the rat that rPH.2 handles is 36 ± 7 hours), and the degree of NEC is similarly (the average marking of contrast is 2.6, and the average marking that rPH.2 handles rat is 2.5) in these two groups.

Carried out other experiment, wherein rat has been used the dosage identical with single treatment group, promptly carried out enteron aisle and also carry out intraperitoneal administration rPH.2 (, adding that every day, secondary passed through peritoneal injection 75 λ) every 3 hours 25 λ rPH.2 in feeding at every turn.The results are shown in the above-mentioned table 9.Although treatment group and control group do not have significant difference aspect mortality ratio, rPH.2 handles the incidence reduced NEC significantly, and (in the contrast 10/17, rPH.2 handles in the rat 3/14, p=0.04).It should be noted that the intestinal bacteria positive in 6 blood cultivation things that before death, obtain in 7 dead in rPH.2 treatment group animals.

These results further support the provide protection of PAF-AH product in the newborn dummy of non-PAF inductive NEC.Carry out the enteron aisle processing with the rPAF-AH product and prevented NEC, and the processing of the intraperitoneal of this dosage does not have verifiable effect.These find prompting, are used to have the prematurity newborn infant of the formula feeding of NEC risk to replenish the PAF-AH product and can reduce this disease incidence rate.

Embodiment 18

Detected the effectiveness of PAF-AH product in acute respiratory distress syndrome (ARDS) guinea pig model.

The platelet activating factor (PAF) that cavy is gone in intravenous injection has produced the remarkable lung inflammation of early stage ARDS and has recalled in the mankind.Intravenously gives in the PAF several minutes, pulmonary parenchyma hyperemia, and segmental bronchus and bronchiole shrink [Lellouch-Tubiana etc. see above-mentioned].Thrombocyte and polymorph neutrophile leucocytes begin to stand like a wall, along the arteriole of lung identification of cell aggregation [Lellouch-Tubiana, Br.J.Exp.Path., 66:345-355 (1985)] easily.The PAF infusion also destroys bronchial epithelial cell, and it separates and assemble the air flue chamber from airway walls.This destruction to the airway epithelia cell is consistent with the transparent film formation that takes place in the mankind of ARDS development.Neutrophil and thrombocyte stand like a wall, and and then these cell hemocytes ooze out interalveolar septum and the alveolar space that enters lung.The cellular infiltration that is caused by PAF is accompanied by the significant vessel leakage [Kirsch, Exp.Lung Res., 18:447-459 (1992)] that causes the air flue oedema.The evidence of oedema is subjected to the further support of in vitro study, and wherein PAF has induced in dabbling Guinea pig lung ¹²⁵The dose-dependently of the fibrinogen of I mark (10-1000ng/ml) exosmose [Basran, Br.J.Pharmacol., 77:437 (1982)].

Based on above-mentioned observation, developed cavy ARDS model.Intubate is placed the jugular vein of the male Hartly cavy (about 350-400 gram) of anesthesia, will 500 μ l volumes as the phosphate-buffered saline that contains 0.25% bovine serum albumin (PBS-BSA) of carrier in the PAF of dilution, with the total dose of 100-400ng/kg scope infusion in 15 fens clock times.Various time points behind the PAF infusion are put to death animal, collect lung tissue.In the cavy with the PAF infusion, dose-dependently injury of lung and inflammation are very tangible during by 15 minutes, exist in the time of 60 minutes.Neutrophil and red blood cell are present in the alveolar space of the cavy of PAF processing, but do not exist in contrast or false infusion animal.Epithelial cell destructive evidence also is significantly, is the transparent film formed memory of people ARDS patient.From the protein determination of bronchoalveolar lavage (BAL) sample obtained with the cavy of PAF infusion, show proteic remarkable accumulation in the inflammation lung, be the clear evidence of vessel leakage.

Find that rPH.2 is protected from the injury of lung of PAF mediation fully in the ARDS guinea pig model.With or rPH.2 (2000 units among the 500 μ l) or 500 μ l PAF-AH damping fluid pre-treatment cavys group only.After 15 minutes with these cavys of 400ng/kg PAF infusion in the 500 μ l volumes, infusion in 15 fens clock times.In addition, with the false group of 500 μ l PBS-BSA infusion cavys.After the PAF infusion is finished, put to death animal, by using the 10ml salt solution lavation lung secondary that contains 2 μ/ml heparin, in case hemostasis-coagulation is collected the BAL fluid.For measuring the protein concentration among the BAL, sample was diluted in salt solution 1: 10, measure OD280.Find that the false BAL fluidic protein concentration of handling cavy is 2.10 ± 1.3mg/ml.Completely contradict with it, discovery is 12.55 ± 1.65mg/ml with the BAL fluidic protein concentration of the animal of PAF infusion.In with the pretreated cavy of rPH.2, find that BAL fluidic protein concentration is 1.13 ± 0.25mg/ml, this and vacation are handled contrast quite, and confirmation PAF-AH product has been blocked the pulmonary edema that PAF is replied fully.

Embodiment 19

In two different acute pancreatitic models, estimated the effectiveness of PAF-AH product rPH.2.A. the activity in the scorching model of pancreas in rat

(Wilmngton MA) buys male Wistar rat (200-250g) from Charles River Laboratories.Be placed on 23 ± 2 ℃, had for 12 little time/dark round-robin controls environment in the room, with standard laboratory food (chew) and the random feeding animals of water.With the animal random assignment to or control group or experimental group.With 50mg/kg vetanarcol intraperitoneal anesthetized rat, by the incision carotid artery lay polyethylene catheter (the V3 size, Biolab products, Lake Havasu, AZ).With the subcutaneous extremely low-necked dorsal part zone of interting of conduit, make animal from recovery from anesthesia.Make rat obtain water, but overnight fasting.Clear-headed animal was experimentized next day.At off period, keep catheter patency by continuing infusion of saline (0.2ml/h).In experiment day, with rPH.2 or vehicle Control intravenous injection animal, then infusion or (1) 5 μ g/kg caerulin 3.5 hours per hour, perhaps (2) 10 μ g/kg caerulin 5 hours per hour, (Research Plus, Bayonne, NJ).After infusion is finished, use the vetanarcol anesthetized animal immediately, cut its belly, draw 5ml blood to carry out subsequent detection from postcava.Then by the sacrificed by exsanguination animal.Measure serum amylase, serolipase and serum bilirubin, collect pancreas.With pancreas piece or fixing to carry out histology in 4% phosphoric acid buffer formaldehyde solution, perhaps freeze deeply immediately in-80 ℃ to measure activity of myeloperoxidase.The pancreas water content of following other pancreas piece of commentary valency and pancreas amylase and trypsinase.As the following activity of myeloperoxidase that compiles the mensuration of (sequestration) as neutrophil of in pancreas and lung, estimating.Also following commentary valency pulmonary vascular permeability.Use and not match Si Shi t and inspected the statistical analysis of data.Average ± the S.E.M. of data represented at least three different experiments of report.When p＜0.05, the difference among the result is thought significant.

1. pancreas water content

The pancreas piece is blotted, weighs (weight in wet base), then in 120 ℃ of dryings 34 hours, weigh again (dry weight).Difference between weight in wet base and the dry weight is calculated as the pancreas water content and with the percentage expression of pancreas weight in wet base.Think that it is the development of oedema that the pancreas water content raises.

2. serum and pancreas amylase

Use 4,6-ethylidene (G ₇)-p-nitrophenyl (G ₁)-α ₁D-maltoplaside (ET-G ₇PNP) (Sigma Chemical Co., St.Louis, MO) as substrate, according to Pierre etc., Clin.Chem., 22:1219 (1976) measures amylase activity in the serum.The amylase activity of the pancreas tissue of homogenate in 10mM phosphoric acid buffer pH7.4 is measured in use with quadrat method.

3. pancreas trypsinase

Use Boc-Gin-Ala-Arg-MCA as substrate, the fluorometric assay tryptic activity.That in brief, mixes the sample of 200 μ l and 2.7ml in cup contains 150mM NaCl, 1mMCaCl ₂50mM Tris damping fluid (pH8.0) with 0.1% bovine serum albumin.After 20 seconds of preincubation, 100 μ l substrates are added sample begin reaction.Read fluorescence reading (exciting 380nm, emission 440nm), express with slope.For merging the data of different experiments, the tryptic activity in a plurality of parts is expressed as the per-cent of total tryptic activity.

4. histology and morphometry

For optical microscopy, that the complete transverse section at random of the head of pancreas, body and tail is fixing in 10% neutral phosphoric acid buffered formalin.With paraffin-embedded 5 μ m section hematoxylin-eosin (H﹠amp; E) dyeing, by skilled morphologist with blind formula inspection.Acinous cell damage/necrosis is defined as or (a) exists the whole or portion of tissue structure deteriorate of acinous cell ghost or (b) formation of acinous cell cavity and expansion, acinus, these two is all relevant with inflammatory reaction.Use is equipped with computerize planimeter image analysis video unit (the CCD-72 type of NIH-1200 image analysis software, Dage-MTI, Michigan city, IN), it is quantitative that the total area that the quantity and the acinar tissue of acinous cell damage/necrosis occupied carries out norphometry.Each tissue samples is checked 10 fields of microscope of selecting at random (125x).With degree by the percentage expression acinous cell damage/necrosis of the occupied total acinar tissue of the area that meets damage/downright bad standard.

5. pancreas and lung myeloperoxidase (MPO) determination of activity

Organize peroxidase activity to estimate that neutrophil compiles in pancreas and the lung by mensuration.The tissue samples of collecting during with execution is stored in-70 ℃ until detection.Sample (50mg) thawed and in 1ml 20mM phosphoric acid buffer (pH7.4) homogenate and centrifugal (10,000xg, 10 minutes 4 ℃).(Sigma, St Louis in the 50mM phosphoric acid buffer (pH6.0) MO), carry out four round-robin freeze-thaws in containing 0.5% cetrimonium bromide with the precipitation resuspending that produces.Then by supersound process further broken suspension in 40 seconds and centrifugal (10,000xg, 5 minutes 4 ℃).Will be by enzyme, 1.6mM tetramethyl-benzidine (the SigmaChemical Co. of described extraction, St.Louis, MO), the reaction mixture of 80mM sodium phosphate buffer (pH5.4) and 0.3mM hydrogen peroxide is 37 ℃ of 110 seconds of incubation, measures the absorption of 655nm in the CobasBio automatic analyser.Proofread and correct this absorption value at the part dry weight of tissue samples then.

6. mensuration pulmonary vascular permeability

Pancreas is choledoch-to block that also cause usually can be by the relevant injury of lung of the quantitative serious pancreatitis of pulmonary vascular permeability and tissue examination.

Put to death animal preceding 2 hours, give intravenously large bolus injection 5mg/kg fluorescein isothiocyanate albumin (the FITC-albumin, Sigma Chemical Co., St.Louis, MO).Be bled into the bronchovesicular chamber by quantitative FITC-albumin from lumen of vessels, estimate the lung microvascular permeability.In brief, after just having put to death, use pliers to block right segmental bronchus, expose tracheae.Then, by using the intubate lavation right lung that inserts tracheae.Merge three salt solution (60ml irrigating solution) washing lotion, under exciting 494nm and emission 520nm, measure the FITC fluorescence in serum and the irrigating solution.Calculate the fluorescence ratio of irrigating solution and blood, as the mensuration of microvascular permeability in the lung.Also use H﹠amp; E is with lung dyeing and carry out histological examination.

7. the influence that gives of caerulin and rPH.2

Only use caerulin with 5 μ g/kg/ hour infusions 3.5 hours, cause in the rat typical slight secretogogue inductive pancreatitis, it is characterized in that hyperamylasemia, by the pancreas oedema of pancreas moisture determination with comprise that the histology of significant acinous cell vacuolation and pancreas oedema changes.Saline infusion in the control animal can not cause these biological chemistries or histology to change.Before beginning caerulin infusion 30 minutes.Intravenously gives 5,10 or the rPH.2 of 20mg/kg, can significantly not change the degree that is changed by only infusion caerulin inductive pancreas oedema (water content) and histology.Giving rPH.2 does not also influence the activation or the amylase content of caerulin inductive pancreas trypsinogen.

With the caerulin of higher metering promptly 10 μ g/kg/ hours to rat infusion 5 hours, cause more serious pancreatitis, it is characterized in that compared with the control trypsinogen in the active remarkable increase of more significant increase, pancreas MPO of serum amyloid enzymic activity and pancreas oedema and the pancreas activates and the remarkable increase of amylase activity.The pancreas histology shows that pancreas oedema and acinous cell vacuolation are not only arranged, and the downright bad and a small amount of cell that soaks into of some sheets is also arranged.

Give rPH.2 (5 or 10mg/kg intravenously) in 30 minutes before in beginning infusion caerulin (10 μ g/kg/ hours), alleviated the degree that changes by many pancreases of infusion caerulin inductive only.The results are shown in the following table 10.Dosage is that the rPH.2 processing of 5mg/kg causes the serum amyloid enzymic activity to reduce (from 10984 ± 1412 to 6763 ± 1256).Higher 10mg/kg rPH.2 dosage does not cause the further improvement of hyperamylasemia.With or 5 or 10mg/kg rPH.2 handle, also cause some go down (only caerulin is 90.61 ± 0.27, and caerulin+5mg/kg rPH.2 is 88.21 ± 0.61) by the caerulin inductive pancreas oedema development of moisture determination.The rPH.2 of 5mg/kg dosage provides that pancreas MPO is active and has significantly alleviated (only caerulin is for surpassing contrast 2.92+0.32 doubly, and caerulin and rPH.2 are 1.19+0.21, p＜0.05).The rPH.2 of higher dosage does not cause the active further raising of MPO.The rPH.2 of these two kinds of dosage does not change trypsinogen activation or amylase content in the pancreas significantly.The pancreas histology shows after the rPH.2 pre-treatment some improvement are being arranged aspect micronecrosis and the infiltration.

Clinically with in pancreatitic several models, observed the relevant injury of lung of pancreatitis.With 5 μ g/kg/ hour infusion caerulin 3.5 hours, cause the pancreatitis of light form, do not cause the remarkable damage of lung.But, with 10 μ g/kg/ hour infusion caerulin 5 hours, cause more serious pancreatitis, also cause soaking into quantitative injury of lung by the pulmonary vascular permeability (0.31 ± 0.04 to 0.79 ± 0.09), lung MPO activity (showing that neutrophil compiles) and the neutrophil of histological examination that increase.

The 30 minutes dosage with 5mg/kg gives rPH.2 before the infusion caerulin, has alleviated significantly by the only active rising of infusion caerulin inductive lung MPO (only caerulin is 3.55 ± 0.93, and caerulin and rPH.2 are 1.51 ± 0.26).RPH.2 handles and to have reduced behind the caerulin infusion seriousness of observed microscopic change in lung tissue significantly.RPH.2 handles and has reduced the increase of caerulin inductive pulmonary vascular permeability, although there is not statistical significance.Aspect the seriousness that reduces caerulin inductive injury of lung, the dosage that higher 10mg/kgrPH.2 dosage is not lower is more effective.

Table 10

Contrast caerulin (CER) CER+ CER+

( CER ) 10μg/kg/h 5mg/kg rPH.2 10mg/kg rPH.2 ( U/l ) 961±174 10984±1412 6763±1256 8576±1024 ( ％ ) 72.71±0.64 90.61±0.27 88.21±0.61 89.00±0.94MPO ( 1.0 2.92±0.32 1.19±0.21 1.42±0.19 ) ( 1000x/ 0.12±0.06 9.70±2.50 8.33±1.75 9.15±1.28μgDNA ) ( U/μgDNA ) 0.28±0.06 0.42±0.07 0.45±0.04 0.46±0.044 ( /％ ) 0.31±0.04 0.79±0.09 0.70±0.09 0.70±0.07MPO ( 1.0 3.55±0.93 1.51±0.26 1.64±0.22 ) B. ( opossum )

Obtain the health of two kinds of sexes, the America didelphid of catching at random (Didelphis virginiana) (2.0kg-4.0kg) from Scott-Hass, be placed on 23 ± 2 ℃, had for 12 little time/dark round-robin controls environment in the room, arbitrarily feeds with standard laboratory food and water.Behind the overnight fasting, with 50mg/kg vetanarcol (Veterinary Laboratories Inc., Lenexa, KS) intraperitoneal anesthetized rat.Under aseptic condition, carry out laparotomy by midline incision, with the total tubal ligations of courage pancreas of all animals to induce acute necrotizing pancreatitis.In addition, the ligation cystic duct is to prevent that gall-bladder is as the bile storehouse.With the animal random assignment to or contrast or experimental group.From second day of ligation ductus pancreaticus, the rPH.2 (solution with 4mg/ml provide) of experimental group by accepting 5mg/kg body weight every day in the tail cava vein, and control group is only accepted the intravenous injection of the placebo carrier of same volume.Handle back 1 day and 2 days (after the pancreatic ligation the 3rd day and the 4th day), use excessive vetanarcol to cause death animal is painless.Extract blood sample to measure serum amylase, serolipase and serum bilirubin from heart, collect pancreas.With pancreas piece or fixing to carry out histology in 4% phosphoric acid buffer formaldehyde solution, perhaps freeze deeply immediately in-80 ℃ to measure activity of myeloperoxidase.As the A joint of above-mentioned present embodiment, estimate the pancreas water content and the pancreas amylase of other pancreas piece.As the above-mentioned activity of myeloperoxidase of in pancreas, estimating the mensuration of compiling as neutrophil.Also as above commentary valency pulmonary vascular permeability.

Average ± average poor (SEM) that data represented a plurality of mensuration from three or more independent experiments of report obtain.When data are only formed by two groups, use Si Shi t-check, maybe when relatively three groups or when more organizing by analysis of variance (ANOVA), the significance of evaluation variation.If ANOVA shows that there were significant differences, then use the Tukey method as post hoc check, the difference of data between analysis bank.When p value＜0.05, think to show significant difference.

The results are shown in table 11.The pancreas obstruction of common bile duct causes with hyperamylasemia, hemolipase is too much and the necrosis of the pancreas degree of depth is the serious necrotizing pancreatitis of feature.In addition, the pancreas obstruction of common bile duct is relevant with the remarkable increase of abnormal level of serum total bilirubin.The beginning intravenously gave rPH.2 (5mg/kg/ days) after the pancreatic ligation second day, had alleviated by pipe to block and the degree that changes of the many pancreases of placebo treatment inductive only.Compare with the animal of placebo treatment, rPH.2 handles and to reduce a serum-amylase level in, although this difference is not remarkable statistically, compares with placebo, and rPH.2 handles two days (after the pancreatic ligation the 4th day) and reduced serum-amylase level significantly.Compared with the control, rPH.2 handles and reduced the serolipase level in one day or two days, although this difference is not remarkable statistically.Compared with the control, rPH.2 handles and reduced pancreas amylase content in two days, causes pancreas amylase to increase in one day although handle.Do not observe with the rPH.2 processing and influence abnormal level of serum total bilirubin, pancreas activity of myeloperoxidase or pancreas water content.

Learn variation by courage pancreatemphraxis inductive principal character sex organization, comprise the remarkable expansion of remarkable necrosis, inflammatory cell infiltration, acinous cell vacuolation and alveolar lumen.Norphometry inspection to the damage of pancreas acinous cell shows.RPH.2 is to the main protective effect of pancreas after rPH.2 handles one day and two days.RPH.2 handled after one day, and the acinous cell damage is reduced to the about 23% of total acinous cell tissue, formed with 48% damage of the animal of placebo treatment to contrast.Handle after two days, this reduction of acinous cell damage is more remarkable, and this moment, rPH.2 handled about 35% damage that causes total acinous cell tissue, forms with about 60% damage of the animal of placebo treatment to contrast.

Injecting quantitative pulmonary vascular permeability by FITC shows.Compare with placebo, rPH.2 handles the highly significant difference after one day and two days.In the animal of all placebo treatment of Microscopic examination showed of lung serious injury of lung is arranged all.Being characterized as of injury of lung have main scavenger cell, lymphocyte and neutrophil between the degree of depth inflammatory reaction of soaking in matter and the alveolar, and sheet but significant interstitial edema and alveolar membrane thicken.Give remarkable minimizing that rPH.2 causes inflammatory cell infiltration and free interstitial edema reduce.

In a word, these results show, beginning in 48 hours gives rPH.2 with 5mg/kg/ days dosage intravenously after the pancreatic ligation, cause that amylase and lipase blood levels rise significantly alleviate and by H ﹠amp; Significantly alleviating of the quantitative acinous cell of the MORPHOMETRIC ANALYSIS OF EXFOLIATED of E stained damage and significantly alleviating of the seriousness of pancreatitis inductive injury of lung.In pancreatitic this clinical correlation model, give the rPAF-AH product and shown the useful effect that reduces pancreatitis seriousness.

Table 11

Handle (to put to death) after one day to handle after two days and (put to death) in the 4th day in the 3rd day

Placebo rPH2.5 placebo rPH.25mg/kg

Mg/kg serum bilirubin (mg/dl) 5.49 ± 0.96 7.10 ± 0.60 6.54 ± 0.55 4.91 ± 0.79 serum amylases (U/ liter) 5618 ± 899 4288 ± 675 6538 ± 1,355 3106 ± 467 ^*Serolipase, (U/ liter) 2226 ± 554 1241 ± 263 1424 ± 257 1023 ± 295 pancreas water content, (%) 81.10 ± 0.56 81.52 ± 0.79 80.05 ± 1.07 79.32 ± 0.49 pancreas MPO 1345 ± 286 1142 ± 83 1149 ± 232 1033 ± 130, (OD/ part dry weight) pancreas amylase 706 ± 92 1101 ± 105 950 ± 85 712 ± 131, (U/ μ g DNA) pulmonary vascular permeability 0.76 ± 0.09 0.21 ± 0.04 ^*0.57 ± 0.13 0.23 ± 0.04 ^*(FITC irrigating solution/serum %) acinous cell damages 48% 23% 60% 35% (total acinar tissue %) ^*P=0.02 compares with placebo ^*P＜0.001 is compared with placebo

Embodiment 20

Study to estimate rPH.2 pair of PAF-AH product and the relevant neurovirulent influence of HIV infection.People's 1 type immunodeficiency virus (HIV-1) infects central nervous system and causes the neurone loss that caused by apoptosis.Stimulate the monocyte that comprises that the HIV-1 with the neurocyte contact activation infects by various antigenicities, the neurotoxicity pro-inflammatory cytokine of secreting high levels comprises PAF.Estimated rPH.2 to neurovirulent influence by HIV infection and the monocytic conditioned medium of activated.

Infect and the activated mononuclear cell with HIV as following.Reclaim monocyte from the peripheral bone myelocyte (PBMC) of HIV and hepatitis B seronegativity donor afterwards in white corpuscle extraction method (leukopheresis), as Genis etc., described in the J.Exp.Med.176:1703-1718 (1992) by adverse current centrifugal elutriation purifying (＞98%).Contain recombination of human macrophage colony stimulating factor (MSCF) (Genetics Institute, Inc Cambridge, DMEM MA) (Sigma, St.Louis, MO) in as adherent single-layer culturing cell (in the T-75 culturing bottle 1 * 10 ⁴Cell/ml).Under these conditions, monocyte differentiation becoming scavenger cell.Cultivate after 7-10 days, scavenger cell is exposed to HIV-1 with the infectious virus particle/target cell of infection multiplicity (MOI) 0.01 _ADA(preservation number M60472).Under these conditions, the monocyte of 20-50% HIV-1 inoculate back 7 days infected, can determine [Kalter etc., J.Immunol., 146:298-306 (1991)] by immunofluorescence and hybridization in situ technique.Every 2-3 days, with fresh culture again stream add all cultures.HIV-1 infected the back 5-7 days and according to Kalter etc., saw the reverse transcriptase activity peak period (10 of above-mentioned evaluation ⁷Cpm/ml), the culture that HIV-1 is infected and do not infect monocytic parallel culture and stimulated 30 minutes in 37 ℃ with LPS (10ng/ml) or carrier, then in-80 ℃ of quick freezing until being used for the neurotoxicity detection.

As following people's pallium neuronal cell culture of setting up.According to the Banker and the Cowan that revise, Brain Res., the program of 126:397-425 (1977) obtains human fetal brain tissue from the human fetal brain tissue akrencephalon in second three months (pregnant 13-16 week).In brief, collect cerebral tissue, (contain Ca at the cold Hank BSS of 30ml ^{+ 2}And Mg ^{+ 2}+ 25mM HEPES and 5X gentamicin) middle washing, from adherent meninges and blood separation, be cut into 2mm ³Piece.The tissue compressing is ground 10-15 time by 230 μ M Nitex bags and by the Pasteru suction pipe that flame is polished.To be organized in 4 ℃ in 550rpm centrifugal 5 minutes, the precipitation resuspending is contained N1 component (Regular Insulin, 5mg/l in 5-10ml; Transferrin, 5mg/l; Selenite, 5 μ g/l; Progesterone 20nM; Putrescine, 100 μ MM) and 10% foetal calf serum (FCS), PSN antibiotic cocktail (penicillin, 50mg/l; Streptomycin sulphate, 50mg/l; Xin Meisu, 100mg/l) and MEM-hipp (D-glucose, the 5g/l of amphotericin (2.5mg/l); L-glutaminate, 2mM; HEPES, 10mM; Sodium.alpha.-ketopropionate, 1mM; KCl, 20mM) in.By measuring cell counting and survival rate with 0.4% Trypan Blue dilution Hank BSS (1: 1 v/v) and with the hematimeter counting.With the soft grinding cell of 10ml suction pipe 5 times, with 10 ⁵The density of cell/12mm glass cover slide is planted plate, and (St.Louis MO) wraps quilt to described cover glass in advance, places 24 hole culture dish for 70K-150K MW, Sigma with poly-l-lysine.The 1ml substratum is sucked each culture hole.At 5% CO ₂In 37 ℃ of culturing cell 10-28 days, changed substratum every 3 days in the wet air of/95% air.Under these conditions, culture is＞neurone of 60-70% homogeneous, the 20-30% astroglia cell is arranged,＜1% little colloid and～10% scavenger cell and the dyeing of little colloid.Cultivate after 14-28 days, the neurone culture is expressed the N-methyl-D-aspartate (NMDA) or the non--nmda receptor of enough levels, and is dead after giving NMDA or alpha-amino group-3-hydroxy-5-methyl base-4 isoxazole propionic acid (AMPA) exitotoxicity (excitotoxic) dosage.

Detect as the following neurotoxicity of carrying out.Specimen was used for the neuronal cell culture 24 hours with 1: 10 v/v concentration, specimen is the monocytic conditioned medium that the HIV-1 of (a) LPS stimulation infects, (b) control medium (c) contains the conditioned medium of the rPH.2 that adds with 51 μ g/ml or (d) added the conditioned medium of the carrier of rPH.2.Use commercially available medicine box (Apop Tag; ONCOR, Gaithersburg, MD), measure neurotoxicity by original position discriminating apoptotic nucleus on the fixed neurone cover glass in 4% Paraformaldehyde 96, this medicine box uses terminal deoxynucleotidyl transferase (TdT) to cut free 3 '-OH end (TUNEL dyeing) of DNA digoxigenin-dUPT is bonded to new enzyme.Using a computer norphometry instrument (MCID, Imaging Research, St.Catherine, Ontario, Canada), analyze TUNEL staining cell nuclear volume/total neuron number in each 50X visual field of the painted neuronic digitized picture of TUNEL in 〉=15 fields of microscope of selecting at random.Neuronal cell nuclear per-cent ± SEM expression data with the TUNEL stained positive is shown among Figure 13.By ANOVA or the definite significance,statistical check that contrasts and test between handling of pairing t-check, significance is p≤0.05.These cultures quantitative confirmed that HIV infects and the monocytic conditioned medium of activated, in the neuronic total group of pallium near 25% in induced neuronal cell death, rPH.2 can be reduced to this toxicity less than total neuronic 5%.RPH.2 self impassivity toxicity is because 50 μ g/mlrPH.2 compare the dead not influence of neuronal cell with the culture of handling with control medium.These results clearly illustrate that, by the neurovirulent main component of monocytic conditioned medium inductive of using activated HIV to infect must be because PAF, because can almost completely eliminate neurotoxicity by being total to incubation with the PAF-AH product, this enzyme is responsible for the metabolism of PAF in the central nervous system.These find prompting.Infect potential treatment intervention in the relevant CNS sacred disease in treatment with HIV-1.

Embodiment 21

The blood plasma PAFAH activity level of the Japanese population near 4% is low maybe can not to be detected.This defective is relevant with the serious respiratory syndrome of asthmatic children, and [82:1983-1991 (1988) 1 for Miwa etc., J.Clin.Invest., and this children it seems with the heredity of autosomal recessive mode this defective.

For determine whether this defective by non-activity but the enzyme that exists or cause by not synthesizing PAF-AH detects the PAF-AH activity (by the method for 10 pairs of transfection volume descriptions of embodiment) and the following existence of using monoclonal antibody 90G11D and 90F2D (embodiment 13) detection PAF-AH in sandwich ELISA of patient's blood plasma of the active defectives of a plurality of PAF-AH.With the monoclonal antibody 90G11D in 100ng/ hole bag by Immulon 4 flat undersides (Dynatech, Chantilly, VA) and store overnight.Be used in 0.5% fishskin gelatin (Sigma) that dilutes among the CMF-PBS and sealed described plate 1 hour, wash then 3 times in room temperature.Dilution patient blood plasma in the PBS that contains 15mM CHAPS adds in each hole of entering plate (50 μ l/ hole).In these plates of room temperature incubation 1 hour, wash 4 times.To add in each hole by standard method biotinylation and the 50 μ l that in PBST, dilute, 5 μ g/ml monoclonal antibody 90F2D,, wash then 3 times in these plates of room temperature incubation 1 hour.Then will in CMF-PBST, dilute 1/1000 50 μ l ExtraAvidin (Sigma) and add in each hole, with these plates before colour developing in room temperature incubation 1 hour.

Observe active directly related with enzyme level of PAF-AH.Lacking activity among the patients serum is reflected by lacking detectable enzyme.Similarly, the plasma sample with half normal activity contains the PAF-AH of half normal level.These observe prompting, and the active defective of PAF-AH is to cause owing to the non-activity enzyme that can not synthesize this enzyme or can not discern owing to described monoclonal antibody.

Further experiment discloses, and this defective is because the genetic damage of human plasma PAF-AH gene.The genomic dna that has separated PAF-AH defective individuality is used as the template of the PCR reaction of PAF-AH gene-specific primer.At first increasing one by one, each encoding sequence exon of body also checks order.The single Nucleotide of observing in the exon 9 changes (G of the position 996 of SEQ ID NO:7 becomes T).This Nucleotide changes the Xie Ansuan of the position 279 that causes PAF-AH sequence (V279F) and is replaced by phenylalanine.The genomic dna amplification exon 9 that has other 11 PAF-AH defective individualities of same point mutation from discovery.

Whether weakened this enzyme for testing this sudden change, by with embodiment 10 in the similar method preparation described contain the escherichia coli expression construction of this sudden change.When importing intestinal bacteria, this expression constructs does not produce the PAF-AH activity, and the contrast construction of this sudden change does not have complete activity.This amino-acid substitution probably causes structural modification, and this modification has caused the immunoreactivity of observed active defective and shortage and PAF-AH antibody of the present invention.

Therefore PAF-AH specific antibody of the present invention can be used for detecting the diagnostic method of progress of the treatment pathologic conditions of serum PAF-AH abnormal level (normal level be about 1-5U/ml) and tracking usefulness PAF-AH.In addition, the gene infringement in the discriminating PAF-AH gene makes and can screen the PAF-AH defective that Japanese patients shows.This sudden change causes increases restriction endonuclease sites (Mae II), therefore makes to distinguish activity and mutation allele with the simple method that restricted length polymorphism (RFLP) is analyzed.Referring to Lewin, the 136-141 page or leaf is stated from Genes V, Oxford University Press, New York, New York (1994).

By with MaeII dna digestion, southern blotting technique and with exon 9 probes (the Nucleotide 1-396 of SEQ ID NO:17) hybridization, screen 12 PAF-AH defective patients' genomic dna.Find that all patients all have the RFLP consistent with described mutation allele.

Though in specific embodiments, described the present invention, should understand and those skilled in the art will envision that multiple variation and modification.Therefore, have only this restriction that occurs in claims of appendix could limit the present invention.Sequence table (1) physical data:

(i) applicant: ICOS CORPORATION

(ii) denomination of invention: platelet-activating factor acetylhydrolase

(iii) sequence number: 30

(iv) contact address:

(A) addressee: Marshall, O ' Toole, Gerstein, Murray ﹠amp; Borun

(B) street: 6300 Sears Tower, 233 South Wacker Drive

(C) city: Chicago

(D) state: Illinois

(E) country: the U.S.

(F) postcode: 60606-6402

(v) computer-reader form:

(A) medium type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release #1.0, version #1.25

(vi) current request for data:

(A) application number:

(B) applying date:

(C) classification:

(viii) attorney/proxy's data:

(A) name: Rin-Laures, Li-Hsien

(B) number of registration: 33,547

(C) reference/file number: 27866/34026

(ix) telecommunications data:

(A) phone: (312) 474-6300

(B) fax: (312) 474-0448

(C) information of fax: 25-3658 (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 17 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:1:

Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu?Ile?Ala

1 5 10 15

The information of Phe (2) SEQ ID NO:2:

(i) sequence signature:

(A) length: 16 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:2:

Ile?Gln?Val?Leu?Met?Ala?Ala?Ala?Ser?Phe?Gly?Gln?Thr?Lys?Ile?Pro

The information of 15 10 15 (2) SEQ ID NO:3:

(i) sequence signature:

(A) length: 11 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:3:

Met?Lys?Pro?Leu?Val?Val?Phe?Val?Leu?Gly?Gly

The information of 15 10 (2) SEQ ID NO:4:

(i) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA

(ix) feature:

(A) title/keyword: decorating site

(B) position: group (13,21,27)

(C) out of Memory :/note=" Nucleotide in each these position is inosine.″

(xi) sequence description: the information of SEQ ID NO:4:ACATGAATTC GGNATCYTTG NGTYTGNCCR AA 32 (2) SEQ ID NO:5:

(i) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:5:TATTTCTAGA AGTGTGGTGG AACTCGCTGG 30 (2) SEQ ID NO:6:

(i) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:6:CGATGAATTC AGCTTGCAGC AGCCATCAGT AC 32 (2) SEQ ID NO:7:

(i) sequence signature:

(A) length: 1520 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: eDNA

(ix) feature:

(A) title/keyword: CDS

(B) position: 162..1484

(xi) sequence description: SEQ ID NO:7:GCTGGTCGGA GGCTCGCAGT GCTGTCGGCG AGAAGCAGTC GGGTTTGGAG CGCTTGGGTC 60GCGTTGGTGC GCGGTGGAAC GCGCCCAGGG ACCCCAGTTC CCGCGAGCAG CTCCGCGCCG 120CGCCTGAGAG ACTAAGCTGA AACTGCTGCT CAGCTCCCAA G ATG GTG CCA CCC 173

Met?Val?Pro?Pro

1AAA?TTG?CAT?GTG?CTT?TTC?TGC?CTC?TGC?GGC?TGC?CTG?GCT?GTG?GTT?TAT 221Lys?Leu?His?Val?Leu?Phe?Cys?Leu?Cys?Gly?Cys?Leu?Ala?Val?Val?Tyr 5 10 15 20CCT?TTT?GAC?TGG?CAA?TAC?ATA?AAT?CCT?GTT?GCC?CAT?ATG?AAA?TCA?TCA 269Pro?Phe?Asp?Trp?Gln?Tyr?Ile?Asn?Pro?Val?Ala?His?Met?Lys?Ser?Ser

25 30 35GCA?TGG?GTC?AAC?AAA?ATA?CAA?GTA?CTG?ATG?GCT?GCT?GCA?AGC?TTT?GGC 317Ala?Trp?Val?Asn?Lys?Ile?Gln?Val?Leu?Met?Ala?Ala?Ala?Ser?Phe?Gly

40 45 50CAA?ACT?AAA?ATC?CCC?CGG?GGA?AAT?GGG?CCT?TAT?TCC?GTT?GGT?TGT?ACA 365Gln?Thr?Lys?Ile?Pro?Arg?Gly?Asn?Gly?Pro?Tyr?Ser?Val?Gly?Cys?Thr

55 60 65GAC?TTA?ATG?TTT?GAT?CAC?ACT?AAT?AAG?GGC?ACC?TTC?TTG?CGT?TTA?TAT 413Asp?Leu?Met?Phe?Asp?His?Thr?Asn?Lys?Gly?Thr?Phe?Leu?Arg?Leu?Tyr

70 75 80TAT?CCA?TCC?CAA?GAT?AAT?GAT?CGC?CTT?GAC?ACC?CTT?TGG?ATC?CCA?AAT 461Tyr?Pro?Ser?Gln?Asp?Asn?Asp?Arg?Leu?Asp?Thr?Leu?Trp?Ile?Pro?Asn?85 90 95 100AAA?GAA?TAT?TTT?TGG?GGT?CTT?AGC?AAA?TTT?CTT?GGA?ACA?CAC?TGG?CTT 509Lys?Glu?Tyr?Phe?Trp?Gly?Leu?Ser?Lys?Phe?Leu?Gly?Thr?His?Trp?Leu

105 110 115ATG?GGC?AAC?ATT?TTG?AGG?TTA?CTC?TTT?GGT?TCA?ATG?ACA?ACT?CCT?GCA 557Met?Gly?Asn?Ile?Leu?Arg?Leu?Leu?Phe?Gly?Ser?Met?Thr?Thr?Pro?Ala

120 125 130AAC?TGG?AAT?TCC?CCT?CTG?AGG?CCT?GGT?GAA?AAA?TAT?CCA?CTT?GTT?GTT 605Asn?Trp?Asn?Ser?Pro?Leu?Arg?Pro?Gly?Glu?Lys?Tyr?Pro?Leu?Val?Val

135 140 145TTT?TCT?CAT?GGT?CTT?GGG?GCA?TTC?AGG?ACA?CTT?TAT?TCT?GCT?ATT?GGC 653Phe?Ser?His?Gly?Leu?Gly?Ala?Phe?Arg?Thr?Leu?Tyr?Ser?Ala?Ile?Gly

150 155 160ATT?GAC?CTG?GCA?TCT?CAT?GGG?TTT?ATA?GTT?GCT?GCT?GTA?GAA?CAC?AGA 701Ile?Asp?Leu?Ala?Ser?His?Gly?Phe?Ile?Val?Ala?Ala?Val?Glu?His?Arg165 170 175 180GAT?AGA?TCT?GCA?TCT?GCA?ACT?TAC?TAT?TTC?AAG?GAC?CAA?TCT?GCT?GCA 749Asp?Arg?Ser?Ala?Ser?Ala?Thr?Tyr?Tyr?Phe?Lys?Asp?Gln?Ser?Ala?Ala

185 190 195GAA?ATA?GGG?GAC?AAG?TCT?TGG?CTC?TAC?CTT?AGA?ACC?CTG?AAA?CAA?GAG 797Glu?Ile?Gly?Asp?Lys?Ser?Trp?Leu?Tyr?Leu?Arg?Thr?Leu?Lys?Gln?Glu

200 205 210GAG?GAG?ACA?CAT?ATA?CGA?AAT?GAG?CAG?GTA?CGG?CAA?AGA?GCA?AAA?GAA 845Glu?Glu?Thr?His?Ile?Arg?Asn?Glu?Gln?Val?Arg?Gln?Arg?Ala?Lys?Glu

215 220 225TGT?TCC?CAA?GCT?CTC?AGT?CTG?ATT?CTT?GAC?ATT?GAT?CAT?GGA?AAG?CCA 893Cys?Ser?Gln?Ala?Leu?Ser?Leu?Ile?Leu?Asp?Ile?Asp?His?Gly?Lys?Pro

230 235 240GTG?AAG?AAT?GCA?TTA?GAT?TTA?AAG?TTT?GAT?ATG?GAA?CAA?CTG?AAG?GAC 941Val?Lys?Asn?Ala?Leu?Asp?Leu?Lys?Phe?Asp?Met?Glu?Gln?Leu?Lys?Asp245 250 255 260TCT?ATT?GAT?AGG?GAA?AAA?ATA?GCA?GTA?ATT?GGA?CAT?TCT?TTT?GGT?GGA 989Ser?Ile?Asp?Arg?Glu?Lys?Ile?Ala?Val?Ile?Gly?His?Ser?Phe?Gly?Gly

265 270 275GCA?ACG?GTT?ATT?CAG?ACT?CTT?AGT?GAA?GAT?CAG?AGA?TTC?AGA?TGT?GGT 1037Ala?Thr?Val?Ile?Gln?Thr?Leu?Ser?Glu?Asp?Gln?Arg?Phe?Arg?Cys?Gly

280 285 290ATT?GCC?CTG?GAT?GCA?TGG?ATG?TTT?CCA?CTG?GGT?GAT?GAA?GTA?TAT?TCC 1085Ile?Ala?Leu?Asp?Ala?Trp?Met?Phe?Pro?Leu?Gly?Asp?Glu?Val?Tyr?Ser

295 300 305AGA?ATT?CCT?CAG?CCC?CTC?TTT?TTT?ATC?AAC?TCT?GAA?TAT?TTC?CAA?TAT 1133Arg?Ile?Pro?Gln?Pro?Leu?Phe?Phe?Ile?Asn?Ser?Glu?Tyr?Phe?Gln?Tyr

310 315 320CCT?GCT?AAT?ATC?ATA?AAA?ATG?AAA?AAA?TGC?TAC?TCA?CCT?GAT?AAA?GAA 1181Pro?Ala?Asn?Ile?Ile?Lys?Met?Lys?Lys?Cys?Tyr?Ser?Pro?Asp?Lys?Glu325 330 335 340AGA?AAG?ATG?ATT?ACA?ATC?AGG?GGT?TCA?GTC?CAC?CAG?AAT?TTT?GCT?GAC 1229Arg?Lys?Met?Ile?Thr?Ile?Arg?Gly?Ser?Val?His?Gln?Asn?Phe?Ala?Asp

345 350 355TTC?ACT?TTT?GCA?ACT?GGC?AAA?ATA?ATT?GGA?CAC?ATG?CTC?AAA?TTA?AAG 1277Phe?Thr?Phe?Ala?Thr?Gly?Lys?Ile?Ile?Gly?His?Met?Leu?Lys?Leu?Lys

360 365 370GGA?GAC?ATA?GAT?TCA?AAT?GTA?GCT?ATT?GAT?CTT?AGC?AAC?AAA?GCT?TCA 1325Gly?Asp?Ile?Asp?Ser?Asn?Val?Ala?Ile?Asp?Leu?Ser?Asn?Lys?Ala?Ser

375 380 385TTA?GCA?TTC?TTA?CAA?AAG?CAT?TTA?GGA?CTT?CAT?AAA?GAT?TTT?GAT?CAG 1373Leu?Ala?Phe?Leu?Gln?Lys?His?Leu?Gly?Leu?His?Lys?Asp?Phe?Asp?Gln

390 395 400TGG?GAC?TGC?TTG?ATT?GAA?GGA?GAT?GAT?GAG?AAT?CTT?ATT?CCA?GGG?ACC 1421Trp?Asp?Cys?Leu?Ile?Glu?Gly?Asp?Asp?Glu?Asn?Leu?Ile?Pro?Gly?Thr405 410 415 420AAC?ATT?AAC?ACA?ACC?AAT?CAA?CAC?ATC?ATG?TTA?CAG?AAC?TCT?TCA?GGA 1469Asn?Ile?Asn?Thr?Thr?Asn?Gln?His?Ile?Met?Leu?Gln?Asn?Ser?Ser?Gly

425 430 435ATA?GAG?AAA?TAC?AAT?TAGGATTAAA?ATAGGTTTTT?TAAAAAAAAA?AAAAAA 1520Ile?Glu?Lys?Tyr?Asn

The information of 440 (2) SEQ ID NO:8:

(i) sequence signature:

(A) length: 441 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:8:Met Val Pro Pro Lys Leu His Val Leu Phe Cys Leu Cys Gly Cys Leu 15 10 15Ala Val Val Tyr Pro Phe Asp Trp Gln Tyr Ile Asn Pro Val Ala His

20 25 30Met?Lys?Ser?Ser?Ala?Trp?Val?Asn?Lys?Ile?Gln?Val?Leu?Met?Ala?Ala

35 40 45Ala?Ser?Phe?Gly?Gln?Thr?Lys?Ile?Pro?Arg?Gly?Asn?Gly?Pro?Tyr?Ser

50 55 60Val?Gly?Cys?Thr?Asp?Leu?Met?Phe?Asp?His?Thr?Asn?Lys?Gly?Thr?Phe?65 70 75 80Leu?Arg?Leu?Tyr?Tyr?Pro?Ser?Gln?Asp?Asn?Asp?Arg?Leu?Asp?Thr?Leu

85 90 95Trp?Ile?Pro?Asn?Lys?Glu?Tyr?Phe?Trp?Gly?Leu?Ser?Lys?Phe?Leu?Gly

100 105 110Thr?His?Trp?Leu?Met?Gly?Asn?Ile?Leu?Arg?Leu?Leu?Phe?Gly?Ser?Met

115 120 125Thr?Thr?Pro?Ala?Asn?Trp?Asn?Ser?Pro?Leu?Arg?Pro?Gly?Glu?Lys?Tyr

130 135 140Pro?Leu?Val?ValPhe?Ser?His?Gly?Leu?Gly?Ala?Phe?Arg?Thr?Leu?Tyr145 150 155 160Ser?Ala?Ile?Gly?Ile?Asp?Leu?Ala?Ser?His?Gly?Phe?Ile?Val?Ala?Ala

165 170 175Val?Glu?His?Arg?Asp?Arg?Ser?Ala?Ser?Ala?Thr?Tyr?Tyr?Phe?Lys?Asp

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

195 200 205Leu?Lys?Gln?Glu?Glu?Glu?Thr?His?Ile?Arg?Asn?Glu?Gln?Val?Arg?Gln

210 215 220Arg?Ala?Lys?Glu?Cys?Ser?Gln?Ala?Leu?Ser?Leu?Ile?Leu?Asp?Ile?Asp225 230 235 240His?Gly?Lys?Pro?Val?Lys?Asn?Ala?Leu?Asp?Leu?Lys?Phe?Asp?Met?Glu

245 250 255Gln?Leu?Lys?Asp?Ser?Ile?Asp?Arg?Glu?Lys?Ile?Ala?Val?Ile?Gly?His

260 265 270Ser?Phe?Gly?Gly?Ala?Thr?Val?Ile?Gln?Thr?Leu?Ser?Glu?Asp?Gln?Arg

275 280 285Phe?Arg?Cys?Gly?Ile?Ala?Leu?Asp?Ala?Trp?Met?Phe?Pro?Leu?Gly?Asp

290 295 300Glu?Val?Tyr?Ser?Arg?Ile?Pro?Gln?Pro?Leu?Phe?Phe?Ile?Asn?Ser?Glu305 310 315 320Tyr?Phe?Gln?Tyr?Pro?Ala?Asn?Ile?Ile?Lys?Met?Lys?Lys?Cys?Tyr?Ser

325 330 335Pro?Asp?Lys?Glu?Arg?Lys?Met?Ile?Thr?Ile?Arg?Gly?Ser?Val?His?Gln

340 345 350Asn?Phe?Ala?Asp?Phe?Thr?Phe?Ala?Thr?Gly?Lys?Ile?Ile?Gly?His?Met

355 360 365Leu?Lys?Leu?Lys?Gly?Asp?Ile?Asp?Ser?Asn?Val?Ala?Ile?Asp?Leu?Ser

370 375 380Asn?Lys?Ala?Ser?Leu?Ala?Phe?Leu?Gln?Lys?His?Leu?Gly?Leu?His?Lys385 390 395 400Asp?Phe?Asp?Gln?Trp?Asp?Cys?Leu?Ile?Glu?Gly?Asp?Asp?Glu?Asn?Leu

405 410 415Ile?Pro?Gly?Thr?Asn?Ile?Asn?Thr?Thr?Asn?Gln?His?Ile?Met?Leu?Gln

420 425 430Asn?Ser?Ser?Gly?Ile?Glu?Lys?Tyr?Asn

The information of 435 440 (2) SEQ ID N0:9:

(i) sequence signature:

(A) length: 1123 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: do not determine

( xi ) ：SEQ ID NO：9：AAATATAAAT TTTAATAACA CCACACATAA ATTTCAAACT ACTTTCCCTA AGTTTCTAGC 60TGAAGTTTTA AATGAGTGTG TTTTTAATTT ATTAGAAAGT GGATTGAAGA GAAAACATTG 120GAAGATGAAG GAAGGCGTTT CAGTTAAACC CCAAATAACT CTGTGTTACA CTGAGCTATG 180AAACGGCTCC TTCTAGCTCC ATTTCTCCTC AGACCTAAGT GCTATTCCTG ATTGTCCTTC 240ATTGTCATTT CCAGGGAGAA ATGACACCAG CACAGTGGCA GGCCTTCCAA TCTGGAGCAC 300GGTCCACACA ACTTCCGAAT TGGTGTTCAG TGTAAAGTGT ATCGGAGTGC GGAAAATGCG 360CAGGGCATTG CCAACTATAG ATGCTCGGAG TAATTCAGTG TATTCAGAGA ACACGGTGAA 420ACAAGGAAAA CCGGCCTGAC TGGGGGGTGA ATTCAGCAGG GAGTAAATCT GATCGGCATC 480AGGTCTGCGG AAAGGAGCTG GTGAGCACGA CACCACCAGG CATTGCCTGG CTCTCTCCGC 540GGCGGGCTAA GTTAACCTCG GGTCCAGGTG CGGGCCATGG TCTTGGGGAG GGTGCTGGGT 600GCGCTCGAGC AGGCTACGTC GGGAGCCGCC GCTGCTAGTG AGAGCCGGGC CACACACGCT 660CCTCCCCGGT ACCTCCTCCA GCATCACCAG GGGAGGAGAG GGTCGGGCAC AAGGCGCGCT 720AGGCGGACCC AGACACAGCC GCGCGCAGCC CACCCGCCCG CCGCCTGCCA GAGCTGCTCG 780GCCCGCAGCC AGGGGGACAG CGGCTGGTCG GAGGCTCGCA GTGCTGTCGG CGAGAAGCAG 840TCGGGTTTGG AGCGCTTGGG TCGCGTTGGT GCGCGGTGGA ACCCCCCAGG GACCCCAGTT 900CCCGCGAGCA GCTCCGCGCC GCGCCTGAGT GAGGAGGGGC CCCGGGGGCG AGGCGGGAGT 960GGGAGGAAGG GCACGGTCGC CGCGCTGGAG GTCGGGACCC CGGAGCGGCG ACCGGCCGGG 1020GTGGGCTCGC TGAGTCGCAC CCGCTCTGCT GGCCGGTCCT GGGCTCACAG TCCCTGCAGC 1080CCTCGGAAAC AGCGCTAGGA TCCTTCGGGA GAGGAGAGAT GAC 1123 ( 2 ) SEQ ID N0：10：

(i) sequence signature:

(A) length: 417 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: 145..287

(xi) sequence description: the information of SEQ ID NO:10:GTACCAATCT AAAACCCAGC ACAGAAAAAT ACATGTTTTA TTTTTTCCAA GTGTTACTAG 60TACCTCAGCC TTTCTTGATT TGTCAGCTTA TTTAAGGCCT CTTCATTGCA TACTTCTTTT 120TTCTTTTAAT CATCTGCTTC GAAGGAGACT AAGCTGAAAC TGCTGCTCAG CTCCCAAGAT 180GGTGCCACCC AAATTGCATG TGCTTTTCTG CCTCTGCGGC TGCCTGGCTG TGGTTTATCC 240TTTTGACTGG CAATACATAA ATCCTGTTGC CCATATGAAA TCATCAGGTA AGAGGTGTAT 300TTGTTCAAGG TCTTGAGCAA CTGATCTGTC GCCATACTTC AAGTGGGCCC CAAGAAGTTG 360CACATCTGCA CATCTAAACA AGTCCTATTT AAAGGCTTAT GGAGATCCTG TATTCTC 417 (2) SEQ ID NO:11:

(i) sequence signature:

(A) length: 498 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: 251..372

( xi ) ：SEQ ID NO：11：CATTAGGAGG TAACAGTCCA AGGCAGCTGA GAGAAAGGCT ATGTCTACTT TCATCTCTTT 60ACCCTCCAAA ACCCCTACAC AGTGTTTCAA ACAGAGAGAC CCTCAATAAT TGCATATCTT 120ACTTGTTAGG TTGAGAAAGA AAGAAGGCCA GAAACTATGG GAAGTAACTT GATTCCGTTG 180GAATTCTTTT GCATAATAAA ATCTGATATG TAATGGATGA CAAATGAGAT AATATTTACC 240TGTTTTTCAG CATGGGTCAA CAAAATACAA GTACTGATGG CTGCTGCAAC GTTTGGCCAA 300ACTAAAATCC CCCGGGGAAA TGGGCCTTAT TCCGTTGGTT GTACAGACTT AATGTTTGAT 360CACACTAATA AGGTAATGCT TTGATTTATA CAACTTATCC TGATACTCTA ATATTGTCTG 420TCGCTATGGA CCACTAGAAG GTGTTCAAAT GTGACCTTGC CCTCACCTGA GAATGACTCA 480TTTTCGAATT TGTATTGT 498 ( 2 ) SEQ ID NO：12：

(i) sequence signature:

(A) length: 433 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: 130..274

(xi) sequence is retouched also: the information of SEQ ID NO:12:CAGCAGCCTA AAGTCTTAGA CTTTGTGAAC ACAGAGGTAT TGAGTCCCAC TAATTAATAT 60CGAAAATAGC TGCTGGAATA TGTTTGAGAC ACAACTTCTC TAAAAGTGCA TTAATTTCTT 120TCTTAACAGG GCACCTTCTT GCGTTTATAT TATCCATCCC AAGATAATGA TCACCTTGAC 180ACCCTTTGGA TCCCAAATAA AGAATATTTT TGGGGTCTTA GCAAATTTCT TGGAACACAC 240TGGCTTATGG GCAACATTTT GAGGTTACTC TTTGGTAAGA TTTCTGTTGA TCCTTCTTTG 300TAGGCTCTTG CATGTATGAA AACCTTGAAA ACAACAAGAA CTTCAAGTAG TTAAGACCAA 360AGTAGATTTT TCTTCAGTCC AAATAGCTCC TAAAATGATA AGGAAAGTAT TTCTTTAAAG 420CCCAGGCAAC TAC 433 (2) SEQ ID NO:13:

(i) sequence signature:

(A) length: 486 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: 164..257

( xi ) ：SEQ ID NO：13：TTGGTGGGTA TCTAGTAGCA GTCTTTTTAA TGAATCTACT ATTCATCCAT AAAAAAGTAG 60ATATAAATCA GATGGGTCTG CATTTTATGC TAATGAGATA TGAATTAAAT TCACTAGCAA 120CACTCAGAGA AAACCTTAAC TATAACCTTC CATTGTTGTC TAGGTTCAAT GACAACTCCT 180GCAAACTGGA ATTCCCCTCT GAGGCCTGGT GAAAAATATC CACTTGTTGT TTTTTCTCAT 240GGTCTTGGGG CATTCAGGTA ATGTTTGAGA GGTTGAACAA TTTTGGCTTC CAGGAATAAA 300TGACAATTTT TTTATTCAAG AAAGAAATAG CAGAGTTTGG AATGTCATGC AGGCCCTTGT 360CTGGAGGAGT TGGGGTTCCT CAATAATTGG CTGTGGGTCT ATTGATCAGT CCTAGACCTG 420TCTGGTCAAG TAGTTTTTTC CCTACTATCA GCTCATTGGG ATTAGCCTCA CAGCAGAGAA 480GAAAGG 486 ( 2 ) SEQ ID NO：14：

(i) sequence signature:

(A) length: 363 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: 113..181

(xi) sequence description: the information of SEQ ID NO:14:CCCCAGGCTC TACTACAGGG TGTAATGGCC TCCATGTTCC CAGTTTTATT AGTGACTCAG 60CCTTGTAATT CATGACTGGT AGTTGTAATT CTTCCCTCTT TTTGTTTTGA AGGACACTTT 120ATTCTGCTAT TGGCATTGAC CTGGCATCTC ATGGGTTTAT AGTTGCTGCT GTAGAACACA 180GGTATGTTAC CTGATATAAT TGGGCTCTTT GGCCAACTAC AGGGAATGTC AATGCTCATA 240ACTATGTTTC TAATTTTCAT AAAAGTTTAT TTAAAATGTT GATGGAACTT TCAAGTATGG 300TAACATCATG AGCAAAAAAG GAGATTGAGT TTTATCGACT TAAAAGACTT AAAAGCACCT 360AAC 363 (2) SEQ ID NO:15:

(i) sequence signature:

(A) length: 441 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: 68..191

(xi) sequence description: the information of SEQ ID NO:15:GAACTGAGAA ACATGGTCAG ATGAGGAAGG GAAGGAGCAT GCATAAATAA TTTTGCTTGT 60ATTATAGAGA TAGATCTGCA TCTGCAACTT ACTATTTCAA GGACCAATCT GCTGCAGAAA 120TAGGGGACAA GTCTTGGCTC TACCTTAGAA CCCTGAAACA AGAGGAGGAG ACACATATAC 180GAAATGAGCA GGTACATTGC AGTGAAAGGA GAGGTGGTTG GTGACCTAAA AGCATGTACA 240AAAGGATGAC ATTTGTTAAT TTAATTTTAC ACCTGGCAAG TTATGCTCCT AGCTCTCCTA 300TTTCCCATTC CCAAAAGATC TGTCAATAGA TTCCTGGAGC AGTAAAATTC CCTTAATGGA 360ATATCTAGTT CATAGTAAAA ACAAAGGCAA ATACAAAAAT TTGGGAGATG ACAGTGAATA 420TTCAGAATTC CTCGAGCCGG G 441 (2) SEQ ID NO:16:

(i) sequence signature:

(A) length: 577 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: 245..358

( xi ) ：SEQ ID NO：16：GGTTAAGTAA ATCGTCTGAA GTCACATAGT AGGTAAGGCA AAACAGAGCC AGGATTTGGA 60CTAAGGCTAT ACCTATGTGC AAAGCTGGGG CCTGTGTCAT TATGGTAGCA AGTAATAGTC 120ACTAATCAGA TTTCCAGTTT ATAACTGACC AACGATTTTT CCCAAATACA GCTTCTACCT 180AAACTTTAAA ATAAGTGTTA TAACTTTTTA CTTTGTCATT TCCTTCTTCT AATAATTATA 240TTAGGTACGG CAAAGAGCAA AAGAATGTTC CCAAGCTCTC AGTCTGATTC TTGACATTGA 300TCATGGAAAG CCAGTGAAGA ATGCATTAGA TTTAAAGTTT GATATGGAAC AACTGAAGGT 360AAGCTATAAA AAGTAATTTT TCTCTTGTCC TACAGTTCTT TATTGTTTTT TGTCATTTAA 420TTTTCTGCCT ATATTGCAAG GTACAATATG ATAAAGGGCT GCAACCAGCC CCTCCCCAAT 480GCGCACACAC AGACACACAA AGCAGTACAG GTAAAGTATT GCAGCAATGA AGAATGCATT 540ATCTTGGACT AGATATGAAT GCAAAGTTAG TCAGTTT 577 ( 2 ) SEQ ID NO：17：

(i) sequence signature:

(A) length: 396 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: 108.199

(xi) sequence description: the information of SEQ ID NO:17:ATCAATGTAT TTACCATCCC CATGAAATGA ACAATTATAT GATTGACAAA TCATTTCTTC 60TAACACCACG AAATAGCTAT AAATTTATAT CATGCTTTTT CAAATAGGAC TCTATTGATA 120GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC AACGGTTATT CAGACTCTTA 180GTGAAGATCA GAGATTCAGG TAAGAAAATA AGATAGTAAA GCAAGAGAAT AGTAAATTAT 240TGGAAGAAAT TATATTGTGA GATATAATTT TTATTCAAAT TCTTAGTGAA GGAAGGGGAT 300CTCTTGGAGT TTATAAGGCT ATTCTTTTGC CCCCATAAAA TACTCTATAT ACATTTTCCT 360AGGCTAAAAC ATCTCCTCTC CTGCTATTAA AATCTC 396 (2) SEQ ID NO:18:

(i) sequence signature:

(A) length: 519 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: 181..351

( xi ) ：SEQ ID N0：18：CTTACAAAGT TAATCATATC CCTTTCCCAC ATTGAAGTAT GATACCTCTT TATTCCAATC 60AGATAACCCA TAATAAACTG GTATGGTGCG TGTCCACCAA TCCTAGCATT ATTAGGATGT 120CCTCAATGTT GGCTAGTATG TAACCAGTTT AATTTCATCA TTGTCAACAA ATATCTACAG 180ATGTGGTATT GCCCTGGATG CATGGATGTT TCCACTGGGT GATGAAGTAT ATTCCAGAAT 240TCCTCAGCCC CTCTTTTTTA TCAACTCTGA ATATTTCCAA TATCCTGCTA ATATCATAAA 300AATGAAAAAA TGCTACTCAC CTGATAAAGA AAGAAAGATG ATTACAATCA GGTAAGTATT 360AGTGACTTAT TTCATTATGT GAAACAAACT TGAAGCTTGG GTAAATATCA ATCGATATCA 420TTTGGTAACT ATTAAAGAAT TGCTGAATTG GTTGTTTAGA CTTTCAATAA GGAGAGAATT 480AGATAATCTC AGTTTCTAAG TACATTTAGT CTACTCTTT 519 ( 2 ) SEQ ID NO：19：

(i) sequence signature:

(A) length: 569 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: 156..304

( xi ) ：SEQ ID NO：19：TGAAACACAT CTAAGTAGAT CAAATTACAA GTTTTATTTC TTCTTTGGTT TTCAGTAAAC 60AGACCAACAA GACCAGTACC TTTCCTTACA CTCTAACTAA AAAAATAATA ATTTTATCAA 120ACAATGTGAC TTTTAAATGT CTTGTTCTCT TTTAGGGGTT CAGTCCACCA GAATTTTGCT 180GACTTCACTT TTGCAACTGG CAAAATAATT GGACACATGC TCAAATTAAA GGGAGACATA 240GATTCAAATG TAGCTATTGA TCTTAGCAAC AAAGCTTCAT TAGCATTCTT ACAAAAGCAT 300TTAGGTAAGA AACTATTTTT TTCATGACCT AAACCGAGAT GAATCTCGAG GACAAAGCTG 360TCTATCTTAA TACAGCTTTA GTACTATTTA AACTATTTCC AGTTGGTTTA CAATGGAACA 420AAGCAGTATA TCAATTTGAA AACAGAAATT TGAGAAAGTC AATTTTGCTG CTTTACATCT 480CTATATCATA GAAAGCAAAT CAACTGTTAA AGGTAATATT CTTTGTATGA GCTAGAGTGA 540CTCATGTGAG GATATCGAAC GACGGTGCT 569 ( 2 ) SEQ ID NO：20：

(i) sequence signature:

(A) length: 469 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(ix) feature:

(A) title/keyword: exon

(B) position: 137..253

( xi ) ：SEQ ID NO：20：GATACAGAGG CACATCGTCT CTACCATCCT AACGGAACTT GTGTAATTTG TAAATCTTTA 60TTGCCACCTA GGGGCATCCA AACTGTTTAA TGCTCTCAAA AGTTTAATAT GTTGATTAAC 120ACTTTATATT TTATAGGACT TCATAAAGAT TTTGATCAGT GGGACTGCTT GATTGAAGGA 180GATGATGAGA ATCTTATTCC AGGGACCAAC ATTAACACAA CCAATCAACA CATCATGTTA 240CAGAACTCTT CAGGAATAGA GAAATACAAT TAGGATTAAA ATAGGTTTTT TAAAAGTCTT 300GTTTCAAAAC TGTCTAAAAT TATGTGTGTG TGTGTGTGTG TGTGTGTGTG AGAGAGAGAG 360AGAGAGAGAG AGAGAGAATT TTAATGTATT TTCCCAAAGG ACTCATATTT TAAAATGTAG 420GCTATACTGT AATCGTGATT GAAGCTTGGA CTAAGAATTT TTTCCCTTT 469 ( 2 ) SEQ ID NO：21：

(i) sequence signature:

(A) length: 1494 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(ix) feature:

(A) title/keyword: CDS

(B) position: 117..1436

(xi) sequence description: SEQ ID NO:21:GGCACGAGCT AGGATCTGAC TCGCTCTGGT GGCATTGCTG CGCTCAGGGT TCTGGGTATC 60CGGGAGTCAG TGCAGTGACC AGAACATCAA ACTGAAGCCA CTGCTCAGCT CCTAAG 116ATG GTA CCA CTC AAA CTG CAG GCG CTT TTC TGC CTC CTC TGC TGC CTC 164Met Val Pro Leu Lys Leu Gln Ala Leu Phe Cys Leu Leu Cys Cys Leu 15 10 15CCA TGG GTC CAT CCT TTT CAC TGG CAA GAC ACA TCT TCT TTT GAC TTC 212Pro Trp Val His Pro Phe His Trp Gln Asp Thr Ser Ser Phe Asp Phe

20 25 30AGG?CCG?TCA?GTA?ATG?TTT?CAC?AAG?CTC?CAA?TCG?GTG?ATG?TCT?GCT?GCC 260Arg?Pro?Ser?Val?Met?Phe?His?Lys?Leu?Gln?Ser?Val?Met?Ser?Ala?Ala

35 40 45GGC?TCT?GGC?CAT?AGT?AAA?ATC?CCC?AAA?GGA?AAT?GGA?TCG?TAC?CCC?GTC 308Gly?Ser?Gly?His?Ser?Lys?Ile?Pro?Lys?Gly?Asn?Gly?Ser?Tyr?Pro?Val

50 55 60GGT?TGT?ACA?GAT?CTG?ATG?TTC?GGT?TAT?GGG?AAT?GAG?AGC?GTC?TTC?GTG 356Gly?Cys?Thr?Asp?Leu?Met?Phe?Gly?Tyr?Gly?Asn?Glu?Ser?Val?Phe?Val?65 70 75 80CGT?TTG?TAC?TAC?CCA?GCT?CAA?GAT?CAA?GGT?CGC?CTC?GAC?ACT?GTT?TGG 404Arg?Leu?Tyr?Tyr?Pro?Ala?Gln?Asp?Gln?Gly?Arg?Leu?Asp?Thr?Val?Trp

85 90 95ATC?CCA?AAC?AAA?GAA?TAT?TTT?TTG?GGT?CTT?AGT?ATA?TTT?CTT?GGA?ACA 452Ile?Pro?Asn?Lys?Glu?Tyr?Phe?Leu?Gly?Leu?Ser?Ile?Phe?Leu?Gly?Thr

100 105 110CCC?AGT?ATT?GTA?GGC?AAT?ATT?TTA?CAC?CTC?TTA?TAT?GGT?TCT?CTG?ACA 500Pro?Ser?Ile?Val?Gly?Asn?Ile?Leu?His?Leu?Leu?Tyr?Gly?Ser?Leu?Thr

115 120 125ACT?CCT?GCA?AGC?TGG?AAT?TCT?CCT?TTA?AGG?ACT?GGA?GAA?AAA?TAC?CCG 548Thr?Pro?Ala?Ser?Trp?Asn?Ser?Pro?Leu?Arg?Thr?Gly?Glu?Lys?Tyr?Pro

130 135 140CTC?ATT?GTC?TTT?TCT?CAT?GGT?CTC?GGA?GCC?TTC?AGG?ACG?ATT?TAT?TCT 596Leu?Ile?Val?Phe?Ser?His?Gly?Leu?Gly?Ala?Phe?Arg?Thr?Ile?Tyr?Ser145 150 155 160GCT?ATT?GGC?ATT?GGC?TTG?GCA?TCT?AAT?GGG?TTT?ATA?GTG?GCC?ACT?GTC 644Ala?Ile?Gly?Ile?Gly?Leu?Ala?Ser?Asn?Gly?Phe?Ile?Val?Ala?Thr?Val

165 170 175GAA?CAC?AGA?GAC?AGA?TCT?GCA?TCG?GCA?ACT?TAC?TTT?TTT?GAA?GAC?CAG 692Glu?His?Arg?Asp?Arg?Ser?Ala?Ser?Ala?Thr?Tyr?Phe?Phe?Glu?Asp?Gln

180 185 190GTG?GCT?GCA?AAA?GTG?GAA?AAC?AGG?TCT?TGG?CTT?TAC?CTG?AGA?AAA?GTA 740Val?Ala?Ala?Lys?Val?Glu?Asn?Arg?Ser?Trp?Leu?Tyr?Leu?Arg?Lys?Val

195 200 205AAA?CAA?GAG?GAG?TCG?GAA?AGT?GTC?CGG?AAA?GAA?CAG?GTT?CAG?CAA?AGA 788Lys?Gln?Glu?Glu?Ser?Glu?Ser?Val?Arg?Lys?Glu?Gln?Val?Gln?Gln?Arg

210 215 220GCA?ATA?GAA?TGT?TCC?CGG?GCT?CTC?AGT?GCG?ATT?CTT?GAC?ATT?GAA?CAT 836Ala?Ile?Glu?Cys?Ser?Arg?Ala?Leu?Ser?Ala?Ile?Leu?Asp?Ile?Glu?His225 230 235 240GGA?GAC?CCA?AAA?GAG?AAT?GTA?CTA?GGT?TCA?GCT?TTT?GAC?ATG?AAA?CAG 884Gly?Asp?Pro?Lys?Glu?Asn?Val?Leu?Gly?Ser?Ala?Phe?Asp?Met?Lys?Gln

245 250 255CTG?AAG?GAT?GCT?ATT?GAT?GAG?ACT?AAA?ATA?GCT?TTG?ATG?GGA?CAT?TCT 932Leu?Lys?Asp?Ala?Ile?Asp?Glu?Thr?Lys?Ile?Ala?Leu?Met?Gly?His?Ser

260 265 270TTT?GGA?GGA?GCA?ACA?GTT?CTT?CAA?GCC?CTT?AGT?GAG?GAC?CAG?AGA?TTC 980Phe?Gly?Gly?Ala?Thr?Val?Leu?Gln?Ala?Leu?Ser?Glu?Asp?Gln?Arg?Phe

275 280 285AGA?TGT?GGA?GTT?GCT?CTT?GAT?CCA?TGG?ATG?TAT?CCG?GTG?AAC?GAA?GAG 1028Arg?Cys?Gly?Val?Ala?Leu?Asp?Pro?Trp?Met?Tyr?Pro?Val?Asn?Glu?Glu

290 295 300CTG?TAC?TCC?AGA?ACC?CTC?CAG?CCT?CTC?CTC?TTT?ATC?AAC?TCT?GCC?AAA 1076Leu?Tyr?Ser?Arg?Thr?Leu?Gln?Pro?Leu?Leu?Phe?Ile?Asn?Ser?Ala?Lys305 310 315 320TTC?CAG?ACT?CCA?AAG?GAC?ATC?GCA?AAA?ATG?AAA?AAG?TTC?TAC?CAG?CCT 1124Phe?Gln?Thr?Pro?Lys?Asp?Ile?Ala?Lys?Met?Lys?Lys?Phe?Tyr?Gln?Pro

325 330 335GAC?AAG?GAA?AGG?AAA?AAT?GAT?TAC?AAT?CAA?GGG?CTC?AGG?CAC?CAG?AAC 1172Asp?Lys?Glu?Arg?Lys?Asn?Asp?Tyr?Asn?Gln?Gly?Leu?Arg?His?Gln?Asn

340 345 350TTT?GAC?GAC?TTT?ACT?TTT?GTA?ACT?GGC?AAA?ATA?ATT?GGA?AAC?AAG?CTG 1220Phe?Asp?Asp?Phe?Thr?Phe?Val?Thr?Gly?Lys?Ile?Ile?Gly?Asn?Lys?Leu

355 360 365ACA?CTG?AAA?GGA?GAA?ATC?GAT?TCC?AGA?GTA?GCC?ATC?GAC?CTC?ACC?AAC 1268Thr?Leu?Lys?Gly?Glu?Ile?Asp?Ser?Arg?Val?Ala?Ile?Asp?Leu?Thr?Asn

370 375 380AAA?GCT?TCG?ATG?GCT?TTC?TTA?CAA?AAG?CAT?TTA?GGG?CTT?CAG?AAA?GAC 1316Lys?Ala?Ser?Met?Ala?Phe?Leu?Gln?Lys?His?Leu?Gly?Leu?Gln?Lys?Asp385 390 395 400TTT?GAT?CAG?TGG?GAC?CCT?CTG?GTG?GAA?GGA?GAT?GAT?GAG?AAC?CTG?ATT 1364Phe?Asp?Gln?Trp?Asp?Pro?Leu?Val?Glu?Gly?Asp?Asp?Glu?Asn?Leu?Ile

405 410 415CCT?GGG?TCA?CCC?TTT?GAC?GCA?GTC?ACC?CAG?GCC?CCG?GCT?CAG?CAA?CAC 1412Pro?Gly?Ser?Pro?Phe?Asp?Ala?Val?Thr?Gln?Ala?Pro?Ala?Gln?Gln?His

420 425 430TCT?CCA?GGA?TCA?CAG?ACC?CAG?AAT?TAGAAGAACT?TGCTTGTTAC?ACAGTTGCCT 1466Ser?Pro?Gly?Ser?Gln?Thr?Gln?Asn

The information of 435 440TTTAAAAGTA GAGTGACATG AGAGAGAG, 1494 (2) SEQ ID NO:22:

(i) sequence signature:

(A) length: 2191 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(ix) feature:

(A) title/keyword: CDS

(B) position: 92..1423

(xi) sequence description: SEQ ID NO:22:CCGCGCGCTC CGGCCGGGGG ACCCTGGTTC CGGCGAGCGG CTCAGCGCGG CGCCCGGAAG 60TTTAAGCTGA AACCACTGCT CAGCTTCCAA G ATG TTG CCA CCC AAA CTG CAT 112

Met?Leu?Pro?Pro?Lys?Leu?His

1 5GCG?CTT?TTC?TGC?CTC?TGC?AGC?TGC?CTC?ACA?CTG?GTT?CAT?CCT?ATT?GAC 160Ala?Leu?Phe?Cys?Leu?Cys?Ser?Cys?Leu?Thr?Leu?Val?His?Pro?Ile?Asp

10 15 20TGG?CAA?GAC?CTA?AAT?CCT?GTT?GCC?CAT?ATT?AGA?TCA?TCA?GCA?TGG?GCC 208Trp?Gln?Asp?Leu?Asn?Pro?Val?Ala?His?Ile?Arg?Ser?Ser?Ala?Trp?Ala

25 30 35AAT?AAA?ATA?CAA?GCT?CTG?ATG?GCT?GCT?GCA?AGT?ATT?AGG?CAA?AGT?AGA 256Asn?Lys?Ile?Gln?Ala?Leu?Met?Ala?Ala?Ala?Ser?Ile?Arg?Gln?Ser?Arg?40 45 50 55ATT?CCC?AAA?GGA?AAT?GGA?TCT?TAT?TCT?GTC?GGT?TGT?ACA?GAT?TTG?ATG 304Ile?Pro?Lys?Gly?Asn?Gly?Ser?Tyr?Ser?Val?Gly?Cys?Thr?Asp?Leu?Met

60 65 70TTT?GAT?TAT?ACT?AAT?AAG?GGC?ACC?TTT?TTG?CGT?TTG?TAT?TAT?CCA?TCG 352Phe?Asp?Tyr?Thr?Asn?Lys?Gly?Thr?Phe?Leu?Arg?Leu?Tyr?Tyr?Pro?Ser

75 80 85CAA?GAG?GAT?GAC?CAC?TCT?GAC?ACG?CTT?TGG?ATC?CCA?AAC?AAA?GAA?TAT 400Gln?Glu?Asp?Asp?His?Ser?Asp?Thr?Leu?Trp?Ile?Pro?Asn?Lys?Glu?Tyr

90 95 100TTT?TTT?GGT?CTT?AGT?AAA?TAT?CTT?GGA?ACA?CCC?TGG?CTT?ATG?GGC?AAA 448Phe?Phe?Gly?Leu?Ser?Lys?Tyr?Leu?Gly?Thr?Pro?Trp?Leu?Met?Gly?Lys

105 110 115ATA?TTG?AGC?TTC?TTT?TTT?GGT?TCA?GTG?ACA?ACT?CCT?GCG?AAC?TGG?AAT 496Ile?Leu?Ser?Phe?Phe?Phe?Gly?Ser?Val?Thr?Thr?Pro?Ala?Asn?Trp?Asn120 125 130 135TCC?CCT?CTG?AGG?ACT?GGT?GAA?AAA?TAT?CCA?CTG?ATT?GTT?TTT?TCT?CAT 544Ser?Pro?Leu?Arg?Thr?Gly?Glu?Lys?Tyr?Pro?Leu?Ile?Val?Phe?Ser?His

140 145 150GGT?CTT?GGA?GCA?TTC?CGG?ACA?ATT?TAT?TCT?GCT?ATT?GGC?ATT?GAT?CTA 592Gly?Leu?Gly?Ala?Phe?Arg?Thr?Ile?Tyr?Ser?Ala?Ile?Gly?Ile?Asp?Leu

155 160 165GCA?TCA?CAT?GGG?TTC?ATC?GTT?GCT?GCT?ATA?GAA?CAC?AGA?GAT?GGA?TCC 640Ala?Ser?His?Gly?Phe?Ile?Val?Ala?Ala?Ile?Glu?His?Arg?Asp?Gly?Ser

170 175 180GCC?TCT?GCG?ACT?TAC?TAT?TTC?AAG?GAC?CAG?TCT?GCT?GCA?GAA?ATA?GGG 688Ala?Ser?Ala?Thr?Tyr?Tyr?Phe?Lys?Asp?Gln?Ser?Ala?Ala?Glu?Ile?Gly

185 190 195AAC?AAA?TCT?TGG?TCT?TAT?CTT?CAA?GAA?CTA?AAA?CCA?GGG?GAT?GAG?GAG 736Asn?Lys?Ser?Trp?Ser?Tyr?Leu?Gln?Glu?Leu?Lys?Pro?Gly?Asp?Glu?Glu200 205 210 215ATA?CAT?GTT?CGA?AAT?GAG?CAG?GTA?CAG?AAA?AGG?GCA?AAG?GAG?TGC?TCC 784Ile?His?Val?Arg?Asn?Glu?Gln?Val?Gln?Lys?Arg?Ala?Lys?Glu?Cys?Ser

220 225 230CAA?GCT?CTC?AAC?TTG?ATT?CTG?GAC?ATT?GAT?CAT?GGA?AGG?CCA?ATT?AAG 832Gln?Ala?Leu?Asn?Leu?Ile?Leu?Asp?Ile?Asp?His?Gly?Arg?Pro?Ile?Lys

235 240 245AAT?GTA?CTA?GAC?TTA?GAG?TTT?GAT?GTG?GAA?CAA?CTG?AAG?GAC?TCT?ATT 880Asn?Val?Leu?Asp?Leu?Glu?Phe?Asp?ValGlu?Gln?Leu?Lys?Asp?Ser?Ile

250 255 260GAC?AGG?GAT?AAA?ATA?GCA?GTA?ATT?GGA?CAT?TCT?TTT?GGT?GGA?GCC?ACA 928Asp?Arg?Asp?Lys?Ile?Ala?Val?Ile?Gly?His?Ser?Phe?Gly?Gly?Ala?Thr

265 270 275GTT?CTT?CAG?GCT?CTT?AGT?GAA?GAC?CAG?AGA?TTT?AGG?TGC?GGG?ATT?GCC 976Val?Leu?Gln?Ala?Leu?Ser?Glu?Asp?Gln?Arg?Phe?Arg?Cys?Gly?Ile?Ala280 285 290 295TTG?GAT?GCA?TGG?ATG?CTT?CCA?CTG?GAT?GAT?GCA?ATA?TAT?TCC?AGA?ATC 1024Leu?Asp?Ala?Trp?Met?Leu?Pro?Leu?Asp?Asp?Ala?Ile?Tyr?Ser?Arg?Ile

300 305 310CCT?CAG?CCC?CTC?TTT?TTT?ATT?AAC?TCG?GAA?CGG?TTC?CAA?TTT?CCT?GAG 1072Pro?Gln?Pro?Leu?Phe?Phe?Ile?Asn?Ser?Glu?Arg?Phe?Gln?Phe?Pro?Glu

315 320 325AAT?ATC?AAA?AAA?ATG?AAA?AAA?TGC?TAC?TCA?CCT?GAC?AAA?GAA?AGA?AAA 1120Asn?Ile?Lys?Lys?Met?Lys?Lys?Cys?Tyr?Ser?Pro?Asp?Lys?Glu?Arg?Lys

330 335 340ATG?ATT?ACA?ATC?AGG?GGT?TCA?GTC?CAT?CAG?AAC?TTT?GCT?GAT?TTC?ACT 1168Met?Ile?Thr?Ile?Arg?Gly?Ser?Val?His?Gln?Asn?Phe?Ala?Asp?Phe?Thr

345 350 355TTT?ACA?ACT?GGC?AAA?ATA?GTT?GGA?TAC?ATA?TTC?ACA?TTA?AAA?GGA?GAT 1216Phe?Thr?Thr?Gly?Lys?Ile?Val?Gly?Tyr?Ile?Phe?Thr?Leu?Lys?Gly?Asp360 365 370 375ATA?GAT?TCA?AAT?GTA?GCA?ATT?GAT?CTT?TGC?AAC?AAA?GCT?TCA?TTG?GCA 1264Ile?Asp?Ser?Asn?Val?Ala?Ile?Asp?Leu?Cys?Asn?Lys?Ala?Ser?Leu?Ala

380 385 390TTT?TTA?CAA?AAG?CAT?TTA?GGA?CTG?CGG?AAA?GAT?TTT?GAT?CAG?TGG?GAT 1312Phe?Leu?Gln?Lys?His?Leu?Gly?Leu?Arg?Lys?Asp?Phe?Asp?Gln?Trp?Asp

395 400 405TCT?TTG?ATT?GAA?GGA?AAA?GAC?GAA?AAT?CTT?ATG?CCA?GGG?ACC?AAC?ATT 1360Ser?Leu?Ile?Glu?Gly?Lys?Asp?Glu?Asn?Leu?Met?Pro?Gly?Thr?Asn?Ile

410 415 420AAC?ATC?ACC?AAC?GAA?CAT?GAC?ACT?CTA?CAG?AAC?TCT?CCA?GAA?GCA?GAG 1408Asn?Ile?Thr?Asn?Glu?His?Asp?Thr?Leu?Gln?Asn?Ser?Pro?Glu?Ala?Glu

425 430 435AAA TCG AAT TTA GAT TAAAAGCACT TTTTTAAAGA TCTTGTTTAA AAACTGTCAA 1463Lys Ser Asn Leu Asp440AAAATGTGTG TATGACTTTT AATATATTTT CTCAAATAAC TCATATTGGA AAATGTAGGC 1523TATCCCATAA AAGTGATTGA AGCTTGGACT AGGAGGTTTT TTTCTTTAAA GAAAGATTGG 1583TGTCTATCGA AATCATGCCA GCCTAAATTT TAATTTTACT AAAATGATGC TGTGTCAAAA 1643TTAATAACTA CTTTTACATT CTTTAATGGA CAAGTATAAC AGGCACAAGG CTAATGAAAA 1703CGTGTTGCAA TGACATAACA ATGCCTAAAA ATACAGATGT TCTTGCCTCT TTTTTCTATT 1763ATAATTGAGT TTTAGCAACA TGTTATGCTA GGTAGAATTT GGAAGCACTT CCCTTTGACT 1823TTTGGTCATG ATAAGAAAAA TTAGATCAAG CAAATGATAA AAGCAGTGTT TTACCAAGGA 1883TTAGGGATAC TGAACAATTT CACTATGGTA ACTGAATGGG GAGTGACCAA GGGTAAAAAT 1943ATTAAAGCCA AGGCAAAGGC AGCAGATTAG AATGGATTAA AGAGAGTTTA TAATTTGTTT 2003GCATTTACTT GATGGTTTAT CTCATGGATT CATGAGTCAA GAAAGGTGCG TAGGACAGGC 2063CAGGGATTCC AGTTATAACA CATTATTCAC CCAAAGGGTT CTTTAATTCT GTATGAGTAT 2123TGGGAGTGGA TTAGCACAAT AGAGGCATAT GTTGCTTTAA AAAAAAAAAA AAAAAAAAAA 2183AAAAAAAA 2191 ( 2 ) SEQ ID NO：23：

(i) sequence signature:

(A) length: 1533 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: protein

(ix) feature:

(A) title/keyword: CDS

(B) position: 62..1394

(xi) sequence description: SEQ ID NO:23:CCGCGAGCAG TTCACCGCGG CGTCCGGAAG GTTAAGCTGA AACGGCAGCT CAGCTTCGGA 60G ATG TTA CCG TCC AAA TTG CAT GCG CTT TTC TGC CTC TGC ACC TGC 106 Met Leu Pro Ser Lys Leu His Ala Leu Phe Cys Leu Cys Thr Cys

1 5 10 15CTT?GCA?CTG?GTT?TAT?CCT?TTT?GAC?TGG?CAA?GAC?CTG?AAT?CCA?GTT?GCC 154Leu?Ala?Leu?Val?Tyr?Pro?Phe?Asp?Trp?Gln?Asp?Leu?Asn?Pro?Val?Ala

20 25 30TAT?ATT?GAA?TCA?CCA?GCA?TGG?GTC?AGT?AAG?ATA?CAA?GCT?CTG?ATG?GCT 202Tyr?Ile?Glu?Ser?Pro?Ala?Trp?Val?Ser?Lys?Ile?Gln?Ala?Leu?Met?Ala

35 40 45GCT?GCA?AAC?ATT?GGT?CAA?TCT?AAA?ATC?CCC?AGA?GGA?AAT?GGA?TCT?TAT 250Ala?Ala?Asn?Ile?Gly?Gln?Ser?Lys?Ile?Pro?Arg?Gly?Asn?Gly?Ser?Tyr

50 55 60TCC?GTC?GGT?TGT?ACA?GAC?TTG?ATG?TTT?GAT?TAC?ACT?AAT?AAG?GGC?ACC 298Ser?Val?Gly?Cys?Thr?Asp?Leu?Met?Phe?Asp?Tyr?Thr?Asn?Lys?Gly?Thr

65 70 75TTC?TTG?CGT?TTG?TAT?TAT?CCA?TCT?CAA?GAT?GAT?GAT?CAC?TCC?GAC?ACC 346Phe?Leu?Arg?Leu?Tyr?Tyr?Pro?Ser?Gln?Asp?Asp?Asp?His?Ser?Asp?Thr?80 85 90 95CTT?TGG?ATC?CCA?AAC?AAA?GAA?TAT?TTT?TTG?GGT?CTT?AGT?AAA?TTT?CTT 394Leu?Trp?Ile?Pro?Asn?Lys?Glu?Tyr?Phe?Leu?Gly?Leu?Ser?Lys?Phe?Leu

100 105 110GGA?ACA?CAC?TGG?CTT?GTG?GGC?AAA?ATT?ATG?GGC?TTA?TTC?TTC?GGT?TCA 442Gly?Thr?His?Trp?Leu?Val?Gly?Lys?Ile?Met?Gly?Leu?Phe?Phe?Gly?Ser

115 120 125ATG?ACA?ACT?CCT?GCA?GCC?TGG?AAT?GCA?CAT?CTG?AGG?ACT?GGG?GAA?AAA 490Met?Thr?Thr?Pro?Ala?Ala?Trp?Asn?Ala?His?Leu?Arg?Thr?Gly?Glu?Lys

130 135 140TAC?CCA?CTA?ATT?ATT?TTT?TCT?CAT?GGT?CTT?GGA?GCA?TTC?AGG?ACG?ATT 538Tyr?Pro?Leu?Ile?Ile?Phe?Ser?His?Gly?Leu?Gly?Ala?Phe?Arg?Thr?Ile

145 150 155TAT?TCT?GCT?ATT?GGC?ATT?GAT?CTG?GCA?TCC?CAC?GGG?TTT?ATA?GTT?GCT 586Tyr?Ser?Ala?Ile?Gly?Ile?Asp?Leu?Ala?Ser?His?Gly?Phe?Ile?Val?Ala160 165 170 175GCT?GTA?GAA?CAC?AGG?GAT?GGC?TCT?GCA?TCC?TCG?ACA?TAC?TAT?TTC?AAG 634Ala?Val?Glu?His?Arg?Asp?Gly?Ser?Ala?Ser?Ser?Thr?Tyr?Tyr?Phe?Lys

180 185 190GAC?CAG?TCT?GCT?GTA?GAA?ATA?GGC?AAC?AAG?TCT?TGG?CTC?TAT?CTC?AGA 682Asp?Gln?Ser?Ala?Va1?G1u?Ile?Gly?Asn?Lys?Ser?Trp?Leu?Tyr?Leu?Arg

195 200 205ACC?CTG?AAG?CGA?GGA?GAG?GAG?GAG?TTT?CCT?TTA?CGA?AAT?GAG?CAG?TTA 730Thr?Leu?Lys?Arg?Gly?Glu?Glu?Glu?Phe?Pro?Leu?Arg?Asn?Glu?Gln?Leu

210 215 220CGG?CAA?CGA?GCA?AAG?GAA?TGT?TCT?CAA?GCT?CTC?AGT?TTG?ATT?CTG?GAC 778Arg?Gln?Arg?Ala?Lys?Glu?Cys?Ser?Gln?Ala?Leu?Ser?Leu?Ile?Leu?Asp

225 230 235ATT?GAT?CAC?GGG?AGG?CCA?GTG?ACG?AAT?GTA?CTA?GAT?TTA?GAG?TTT?GAT 826Ile?Asp?His?Gly?Arg?Pro?Val?Thr?Asn?Val?Leu?Asp?Leu?Glu?Phe?Asp240 245 250 255GTG?GAA?CAG?CTG?AAG?GAC?TCT?ATT?GAT?AGG?GAT?AAA?ATA?GCC?ATT?ATT 874Val?Glu?Gln?Leu?Lys?Asp?Ser?Ile?Asp?Arg?Asp?Lys?Ile?Ala?Ile?Ile

260 265 270GGA?CAT?TCT?TTT?GGT?GGA?GCC?ACA?GTT?ATT?CAG?ACT?CTT?AGT?GAA?GAC 922Gly?His?Ser?Phe?Gly?Gly?Ala?Thr?Val?Ile?Gln?Thr?Leu?Ser?Glu?Asp

275 280 285CAG?AGA?TTC?AGG?TGT?GGC?ATT?GCT?CTG?GAT?GCA?TGG?ATG?TTT?CCC?GTG 970Gln?Arg?Phe?Arg?Cys?Gly?Ile?Ala?Leu?Asp?Ala?Trp?Met?Phe?Pro?Val

290 295 300GGT?GAT?GAA?GTA?TAT?TCC?AGA?ATT?CCT?CAA?CCC?CTC?TTT?TTT?ATC?AAC 1018Gly?Asp?Glu?Val?Tyr?Ser?Arg?Ile?Pro?Gln?Pro?Leu?Phe?Phe?Ile?Asn

305 310 315TCG?GAA?CGA?TTC?CAA?TAC?CCT?TCT?AAT?ATC?ATA?AGA?ATG?AAA?AAA?TGC 1066Ser?Glu?Arg?Phe?Gln?Tyr?Pro?Ser?Asn?Ile?Ile?Arg?Met?Lys?Lys?Cys320 325 330 335TTC?TTA?CCT?GAT?AGA?GAA?CGA?AAA?ATG?ATT?ACA?ATC?AGG?GGT?TCG?GTC 1114Phe?Leu?Pro?Asp?Arg?Glu?Arg?Lys?Met?Ile?Thr?Ile?Arg?Gly?Ser?Val

340 345 350CAT?CAG?AAT?TTT?GTT?GAC?TTC?ACT?TTT?GCC?ACT?AGC?AAA?ATA?ATT?GGC 1162His?Gln?Asn?Phe?Val?Asp?Phe?Thr?Phe?Ala?Thr?Ser?Lys?Ile?Ile?Gly

355 360 365TAC?CTA?TTC?ACA?CTG?AAA?GGA?GAC?ATC?GAT?TCC?AAT?GTA?GCC?ATC?AGC 1210Tyr?Leu?Phe?Thr?Leu?Lys?Gly?Asp?Ile?Asp?Ser?Asn?Val?Ala?Ile?Ser

370 375 380CTT?AGC?AAC?AAA?GCT?TCC?TTA?GCG?TTC?TTA?CAA?AAA?CAT?TTA?GGA?CTT 1258Leu?Ser?Asn?Lys?Ala?Ser?Leu?Ala?Phe?Leu?Gln?Lys?His?Leu?Gly?Leu

385 390 395CAG?AAA?GAT?TTT?GAT?CAG?TGG?GAT?TCT?TTA?GTT?GAA?GGC?GAA?GAT?CAC 1306Gln?Lys?Asp?Phe?Asp?Gln?Trp?Asp?Ser?Leu?Val?Glu?Gly?Glu?Asp?His400 405 410 415AAT?CTT?ATT?CCA?GGG?ACC?AAC?ATT?AAC?ACA?ACC?AAC?CAC?CAA?GCC?ATT 1354Asn?Leu?Ile?Pro?Gly?Thr?Asn?Ile?Asn?Thr?Thr?Asn?His?Gln?Ala?Ile

420 425 430CTG?CAG?AAC?TCC?ACA?GGA?ATA?GAG?AGA?CCA?AAT?TTA?GAT?T?AAAAGAGCTT 1404Leu?Gln?Asn?Ser?Thr?Gly?Ile?Glu?Arg?Pro?Asn?Leu?Asp

The information of 435 440TTTAAAAAGT TTTGTTTACG AACTTGTCTA AAAGTGTGTG TGTGTATGAT TTAAATGTAT 1464TTTCTCAAAT AGCTCATATT AAAAAATGTA GGCTATAGCA CAAAAAAAAA AAAAAAAAAA 1524AAAAAAAAA, 1533 (2) SEQ ID NO:24:

(i) sequence signature:

(A) length: 1876 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: protein

(ix) feature:

(A) title/keyword: CDS

(B) position: 468..1734

(xi) sequence description: SEQ ID N0:24:CGGCGGGCTG CTGGCCCTTC CCGGCTGTTC GTAGAGCCGG ATCCTGCAGC GCCCCTGAGA 60CGAACCGCCC CGATGCGGTG CTCCTCAGCG CCACGGGACG CAGCCGGGGC CGGCCGTGTT 120GGCGCAGCTC CCACGACGTA CGCTTCCTTT CCAGGCTCGA GGAAAGCCTC TCCCACAAAC 180ACCGTCCCAG CTGGGAAGTG AGGCGGAGTT TTGGTCCCTC CCCTCCGGCA GCGCCCGGCA 240TTCCGTCCGT CCGTCCGTCC GTCCGTGCGG CGCACGGCGC CCTGCAGAGC CGGGACACCG 300CAGCAGGGTA GGAGGACCCG GAGGTGGTGT GCAGCCACAG GTTTCCATCC TGCCCCCACC 360TCCCGGGGAG CAGCCCTGTG CTATACCCAA CCCCCCGCAC AGAGCACTGA GCCGGCTGCT 420GCCTGCCTGC ACCCCGCCGT GGGACCTTCT GCTCTTCCCA ACAAGTG ATG GCA TCG 476

Met?Ala?Ser

1CTG?TGG?GTG?AGA?GCC?AGG?AGG?GTG?TTC?ATG?AAA?AGT?CGT?GCT?TCA?GGT 524Leu?Trp?Val?Arg?Ala?Arg?Arg?Val?Phe?Met?Lys?Ser?Arg?Ala?Ser?Gly

5 10 15TTC?TCG?GCG?AAG?GCG?GCG?ACG?GAG?ATG?GGG?AGC?GGC?GGC?GCG?GAG?AAG 572Phe?Ser?Ala?Lys?Ala?Ala?Thr?Glu?Met?Gly?Ser?Gly?Gly?Ala?Glu?Lys?20 25 30 35GGC?TAT?CGG?ATC?CCC?GCC?GGG?AAG?GGC?CCG?CAC?GCC?GTG?GGC?TGC?ACG 620Gly?Tyr?Arg?Ile?Pro?Ala?Gly?Lys?Gly?Pro?His?Ala?Val?Gly?Cys?Thr

40 45 50GAT?CTG?ATG?ACC?GGC?GAC?GCG?GCC?GAG?GGA?AGC?TTT?TTG?CGC?CTG?TAT 668Asp?Leu?Met?Thr?Gly?Asp?Ala?Ala?Glu?Gly?Ser?Phe?Leu?Arg?Leu?Tyr

55 60 65TAC?CTA?TCG?TGT?GAC?GAC?ACA?GAT?ACT?GAA?GAG?ACA?CCC?TGG?ATT?CCA 716Tyr?Leu?Ser?Cys?Asp?Asp?Thr?Asp?Thr?Glu?Glu?Thr?Pro?Trp?Ile?Pro

70 75 80GAT?AAA?GAG?TAC?TAC?CAG?GGG?CTG?TCT?GAC?TTC?CTC?AAC?GTG?TAC?CGG 764Asp?Lys?Glu?Tyr?Tyr?Gln?Gly?Leu?Ser?Asp?Phe?Leu?Asn?Val?Tyr?Arg

85 90 95GCC?CTG?GGA?GAA?AGG?CTT?TTC?CAG?TAC?TAC?GTT?GGC?TCA?GTG?ACC?TGT 812Ala?Leu?Gly?Glu?Arg?Leu?Phe?Gln?Tyr?Tyr?Val?Gly?Ser?Val?Thr?Cys100 105 110 115CCT?GCA?AAA?TCA?AAC?GCT?GCT?TTT?AAG?CCA?GGA?GAG?AAA?TAC?CCA?CTG 860Pro?Ala?Lys?Ser?Asn?Ala?Ala?Phe?Lys?Pro?Gly?Glu?Lys?Tyr?Pro?Leu

120 125 130CTC?GTT?TTT?TCC?CAT?GGA?CTT?GGA?GCT?TTT?CGG?ACC?ATC?TAT?TCT?GCT 908Leu?Val?Phe?Ser?His?Gly?Leu?Gly?Ala?Phe?Arg?Thr?Ile?Tyr?Ser?Ala

135 140 145ATC?TGC?ATA?GAG?ATG?GCT?TCT?CAA?GGC?TTT?CTA?GTG?GCA?GCT?GTG?GAG 956Ile?Cys?Ile?Glu?Met?Ala?Ser?Gln?Gly?Phe?Leu?Val?Ala?Ala?Val?Glu

150 155 160CAC?AGA?GAT?GAA?TCG?GCT?TCA?GCA?ACG?TAT?TTC?TGT?AAA?AAG?AAG?GCT 1004His?Arg?Asp?Glu?Ser?Ala?Ser?Ala?Thr?Tyr?Phe?Cys?Lys?Lys?Lys?Ala

165 170 175GAT?TCT?GAG?CCA?GAG?GAG?GAT?CAA?ACA?TCA?GGC?GTG?GAG?AAG?GAG?TGG 1052Asp?Ser?Glu?Pro?Glu?Glu?Asp?Gln?Thr?Ser?Gly?Val?Glu?Lys?Glu?Trp180 185 190 195ATC?TAC?TAC?AGG?AAG?CTC?AGA?GCA?GGA?GAG?GAG?GAG?CGC?TGT?CTG?CGT 1100Ile?Tyr?Tyr?Arg?Lys?Leu?Arg?Ala?Gly?Glu?Glu?Glu?Arg?Cys?Leu?Arg

200 205 210CAC?AAG?CAG?GTA?CAG?CAG?AGA?GCA?CAG?GAG?TGC?ATC?AAA?GCG?CTC?AAC 1148His?Lys?Gln?Val?Gln?Gln?Arg?Ala?Gln?Glu?Cys?Ile?Lys?Ala?Leu?Asn

215 220 225CTC?ATT?CTT?AAG?ATC?AGT?TCA?GGA?GAG?GAA?GTG?ATG?AAT?GTG?CTG?AAC 1196Leu?Ile?Leu?Lys?Ile?Ser?Ser?Gly?Glu?Glu?Val?Met?Asn?Val?Leu?Asn

230 235 240TCA?GAC?TTT?GAC?TGG?AAC?CAC?CTG?AAG?GAT?TCT?GTT?GAT?ACT?AGC?AGA 1244Ser?Asp?Phe?Asp?Trp?Asn?His?Leu?Lys?Asp?Ser?Val?Asp?Thr?Ser?Arg

245 250 255ATA?GCT?GTG?ATG?GGA?CAC?TCT?TTT?GGT?GGT?GCT?ACA?GTT?ATT?GAG?AGC 1292Ile?AIa?Val?Met?Gly?His?Ser?Phe?Gly?Gly?Ala?Thr?Val?Ile?Glu?Ser260 265 270 275CTC?AGC?AAA?GAA?ATT?AGA?TTT?AGG?TGT?GGC?ATT?GCC?CTT?GAT?GCG?TGG 1340Leu?Ser?Lys?Glu?Ile?Arg?Phe?Arg?Cys?Gly?Ile?Ala?Leu?Asp?Ala?Trp

280 285 290ATG?CTC?CCG?GTA?GGC?GAT?GAC?ACT?TAC?CAA?AGC?AGT?GTG?CAG?CAA?CCA 1388Met?Leu?Pro?Val?Gly?Asp?Asp?Thr?Tyr?Gln?Ser?Ser?Val?Gln?Gln?Pro

295 300 305CTG?CTC?TTT?ATT?AAT?TCC?GAA?AAA?TTC?CAG?TGG?GCT?GCC?AAT?ATC?TTA 1436Leu?Leu?Phe?Ile?Asn?Ser?Glu?Lys?Phe?Gln?Trp?Ala?Ala?Asn?Ile?Leu

310 315 320AAG?ATG?AAG?AAG?CTT?AGC?TCC?AAT?GAT?ACC?AAC?AAG?AAA?ATG?ATC?ACC 1484Lys?Met?Lys?Lys?Leu?Ser?Ser?Asn?Asp?Thr?Asn?Lys?Lys?Met?Ile?Thr

325 330 335ATC?AAA?GGA?TCG?GTA?CAT?CAG?AGC?TTT?CCT?GAT?TTT?ACT?TTT?GTG?AGT 1532Ile?Lys?Gly?Ser?Val?His?Gln?Ser?Phe?Pro?Asp?Phe?Thr?Phe?Val?Ser340 345 350 355GGA?GAA?ATC?ATT?GGA?AAG?TTT?TTC?AAG?TTA?AAA?GGA?GAA?ATA?GAC?CCA 1580Gly?Glu?Ile?Ile?Gly?Lys?Phe?Phe?Lys?Leu?Lys?Gly?Glu?Ile?Asp?Pro

360 365 370AAT?GAA?GCT?ATT?GAT?ATA?TGC?AAC?CAC?GCT?TCA?TTG?GCC?TTC?CTG?CAG 1628Asn?Glu?Ala?Ile?Asp?Ile?Cys?Asn?His?Ala?Ser?Leu?Ala?Phe?Leu?Gln

375 380 385AAA?CAT?CTG?AGT?CTT?AAG?AGA?GAT?TTT?GAT?AAG?TGG?GAT?TCA?CTC?GTG 1676Lys?His?Leu?Ser?Leu?Lys?Arg?Asp?Phe?Asp?Lys?Trp?Asp?Ser?Leu?Val

390 395 400GAT?GGC?ATA?GGA?CCC?AAT?GTT?ATT?TCT?GGT?ACC?AAT?ATC?GAC?TTA?TCT 1724Asp?Gly?Ile?Gly?Pro?Asn?Val?Ile?Ser?Gly?Thr?Asn?Ile?Asp?Leu?Ser

The information of 405 410 415CCA ACT GAG T AAGGAGTACA AGAAGTACTG CAAAGGCCAC CAGCAGCAGG 1774Pro Thr G1u420ACACCAACGT TGGCCACACA TTGCTTGGAG CTGAGATAGC ACTGGCCTCC CACACAGCTT 1834TTGGAGTGTG AAACAACAAA AAAAAAAATC ACAGGGGAGC CG, 1876 (2) SEQ ID NO:25:

(i) sequence signature:

(A) length: 517 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: eDNA

(ix) feature:

(A) title/keyword: CDS

(B) position: 2..514

(xi) sequence description: SEQ ID NO:25:GGG GGG CAT TCT TTT GGA GGA GCA ACA GTT TTT CAA GCC CTA AGT GAA 46 Gly His Ser Phe Gly Gly Ala Thr Val Phe Gln Ala Leu Ser Glu

1 5 10 15GAC?CAG?AGA?TTC?AGA?TGT?GGG?ATT?GCC?CTT?GAT?CCG?TGG?ATG?TTT?CCC 94Asp?Gln?Arg?Phe?Arg?Cys?Gly?Ile?Ala?Leu?Asp?Pro?Trp?Met?Phe?Pro

20 25 30GTG?AGT?GAG?GAG?CTG?TAC?TCC?AGA?GTT?CCT?CAG?CCT?CTC?TTC?TTT?ATC 142Val?Ser?Glu?Glu?Leu?Tyr?Ser?Arg?Val?Pro?Gln?Pro?Leu?Phe?Phe?Ile

35 40 45AAC?TCT?GCC?GAA?TTC?CAG?ACT?CCA?AAG?GAC?ATT?GCA?AAA?ATG?AAA?AAC 190Asn?Ser?Ala?Glu?Phe?Gln?Thr?Pro?Lys?Asp?Ile?Ala?Lys?Met?Lys?Asn

50 55 60TTC?TAC?CAG?CCT?GAC?AAG?GAA?AGG?AAA?ATG?ATT?ACG?ATC?AAG?GGC?TCA 238Phe?Tyr?Gln?Pro?Asp?Lys?Glu?Arg?Lys?Met?Ile?Thr?Ile?Lys?Gly?Ser

65 70 75GTG?CAC?CAG?AAT?TTT?GCT?GAC?GGG?ACT?TTT?GTA?ACT?GGC?AAA?ATA?ATT 286Val?His?Gln?Asn?Phe?Ala?Asp?Gly?Thr?Phe?Val?Thr?Gly?Lys?Ile?Ile?80 85 90 95GGA?AAC?AAG?CTG?TCA?CTG?AAA?GGA?GAC?ATA?GAC?TCC?AGA?GTT?GCC?ATA 334Gly?Asn?Lys?Leu?Ser?Leu?Lys?Gly?Asp?Ile?Asp?Ser?Arg?Val?Ala?Ile

100 105 110GAC?CTC?ACC?AAC?AAG?GCT?TCC?TTG?GCT?TTC?TTA?CAA?AAA?CAT?TTA?GGA 382Asp?Leu?Thr?Asn?Lys?Ala?Ser?Leu?Ala?Phe?Leu?Gln?Lys?His?Leu?Gly

115 120 125CTT?CAT?AAA?GAC?TTT?GAT?CAG?TGG?GAC?TGT?CTG?GTG?GAG?GGA?GAG?AAC 430Leu?His?Lys?Asp?Phe?Asp?Gln?Trp?Asp?Cys?Leu?Val?Glu?Gly?Glu?Asn

130 135 140GAG?AAC?CTC?ATC?CCG?GGG?TCA?CCC?TTT?GAT?GTA?GTC?ACC?CAG?TCC?CCG 478Glu?Asn?Leu?Ile?Pro?Gly?Ser?Pro?Phe?Asp?Val?Val?Thr?Gln?Ser?Pro

The information of 145 150 155GCT CTG CAG AGT TCT CCC GGA TCA CAC AAC CAG AAT TAG 517Ala Leu Gln Ser Ser Pro Gly Ser His Asn Gln Asn160,165 170 (2) SEQ ID NO:26:

(i) sequence signature:

(A) length: 580 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..580

(xi) sequence description: SEQ ID NO:26:CAA GTA CTG ATG GCT GCT GCA AGC TTT GGC GAA CGT AAA ATC CCT AAG 48Gln Val Leu Met Ala Ala Ala Ser Phe Gly Glu Arg Lys Ile Pro Lys 15 10 15GGA AAT GGG CCT TAT TCC GTT GGT TGT ACA GAC TTA ATG TTT GAT TAG 96Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr Asp Leu Met Phe Asp Tyr

20 25 30ACT?AAA?AAG?GGC?ACC?TTC?TTG?CGT?TTA?TAT?TAT?CCA?TCC?CAA?GAT?GAT 144Thr?Lys?Lys?Gly?Thr?Phe?Leu?Arg?Leu?Tyr?Tyr?Pro?Ser?Gln?Asp?Asp

35 40 45GAT?CGC?CTT?GAC?ACC?CTT?TGG?ATC?CCA?AAT?AAG?GAG?TAT?TTT?TGG?GGT 192Asp?Arg?Leu?Asp?Thr?Leu?Trp?Ile?Pro?Asn?Lys?Glu?Tyr?Phe?Trp?Gly

50 55 60CTT?AGC?AAG?TAT?CTT?GGA?AAA?CAC?TGG?CTT?ATG?GGC?AAC?ATT?TTG?AGT 240Leu?Ser?Lys?Tyr?Leu?Gly?Lys?His?Trp?Leu?Met?Gly?Asn?Ile?Leu?Ser?65 70 75 80TTA?CTC?TTT?GGT?TCA?GTG?ACA?ACT?CCT?GCA?AAC?TGG?AAT?TCC?CCT?CTG 288Leu?Leu?Phe?Gly?Ser?Val?Thr?Thr?Pro?Ala?Asn?Trp?Asn?Ser?Pro?Leu

85 90 95AGG?CCT?GGT?GAA?AAA?TAC?CCA?CTT?GTT?GTT?TTT?TCT?CAT?GGT?CTT?GGA 336Arg?Pro?Gly?Glu?Lys?Tyr?Pro?Leu?Va1?Val?Phe?Ser?His?Gly?Leu?Gly

100 105 110GCA?TTC?AGG?ACA?ATT?TAT?TCT?GCT?ATT?GGC?ATT?GAC?CTG?GCA?TCT?CAT 384Ala?Phe?Arg?Thr?Ile?Tyr?Ser?Ala?Ile?Gly?Ile?Asp?Leu?Ala?Ser?His

115 120 125GGG?TTT?ATA?GTT?GCT?GCT?GTA?GAA?CAC?AGA?GAT?AGA?TCT?GCA?TCT?GCA 432Gly?Phe?Ile?Val?Ala?Ala?Val?Glu?His?Arg?Asp?Arg?Ser?Ala?Ser?Ala

130 135 140ACT?TAC?TAT?TTC?AAG?AAC?CAA?TCT?GCT?GCA?GAA?ATA?GGG?AAA?AAG?TCT 480Thr?Tyr?Tyr?Phe?Lys?Asn?Gln?Ser?Ala?Ala?Glu?Ile?Gly?Lys?Lys?Ser145 150 155 160TGG?CTC?TAC?CTT?AGA?ACC?CTG?AAA?GAA?GAG?GAG?GAG?ATA?CAT?ATA?CGA 528Trp?Leu?Tyr?Leu?Arg?Thr?Leu?Lys?Glu?Glu?Glu?Glu?Ile?His?Ile?Arg

165 170 175AAT?AAG?CAG?GTA?CGA?CAA?AGA?GCA?AAA?GAA?TGT?TCC?CAA?GCT?CTC?AGT 576Asn?Lys?Gln?Val?Arg?Gln?Arg?Ala?Lys?Glu?Cys?Ser?Gln?Ala?Leu?Ser

The information of 180 185 190CTG A 580Leu (2) SEQ ID NO:27:

(i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: peptide

(xi) sequence description: the information of SEQ ID NO:27:Gly Xaa Ser Xaa Gly1 5 (2) SEQ ID N0:28:

(i) sequence signature:

(A) length: 41 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:28:TATTCTAGAA TTATGATACA AGTATTAATG GCTGCTGCAA G 41 (2) SEQ ID NO:29:

(i) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:29:ATTGATATCC TAATTGTATT TCTCTATTCC TG 32 (2) SEQ ID NO:30:

(i) sequence signature:

(A) length: 1335 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

( xi ) ：SEQ ID NO：30：ATGGTACCCC CAAAGCTGCA CGTCCTGTTT TGTCTGTGTG GATGTCTCGC CGTCGTGTAC 60CCCTTCGATT GGCAGTATAT CAACCCCGTG GCTCACATGA AGAGCAGCGC CTGGGTGAAT 120AAGATCCAGG TGCTCATGGC CGCACCAAGC TTCGGTCAGA CCAAGATTCC TAGAGGCAAC 180GGCCCCTACA GCGTGGGCTG CACCGATCTG ATGTTCGACC ATACCAACAA AGGAACTTTT 240CTGAGACTGT ACTACCCCAG CCAGGACAAC GACAGACTGG ATACTCTGTG GATCCCAAAT 300AAAGAATATT TTTGGGGTCT TAGCAAATTT CTTGGAACAC ACTGGCTTAT GGGCAACATT 360TTGAGGTTAC TCTTTGGTTC AATGACAACT CCTGCAAACT GGAATTCCCC TCTGAGGCCT 420GGTGAAAAAT ATCCACTTGT TGTTTTTTCT CATGGTCTTG GGGCATTCAG GACACTTTAT 480TCTGCTATTG GCATTGACCT GGCATCTCAT GGGTTTATAG TTGCTGCTGT AGAACACAGA 540GATAGATCTG CATCTGCAAC TTACTATTTC AAGGACCAAT CTGCTGCAGA AATAGGGGAC 600AAGTCTTGGC TCTACCTTAG AACCCTGAAA CAAGAGGAGG AGACACATAT ACGAAATGAG 660CAGGTACGGC AAAGAGCAAA AGAATGTTCC CAAGCTCTCA GTCTGATTCT TGACATTGAT 720CATGGAAAGC CAGTGAAGAA TGCATTAGAT TTAAAGTTTG ATATGGAACA ACTGAAGGAC 780TCTATTGATA GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC AACGGTTATT 840CAGACTCTTA GTGAAGATCA GAGATTCAGA TGTGGTATTG CCCTGGATGC ATGGATGTTT 900CCACTGGGTG ATGAAGTATA TTCCAGAATT CCTCAGCCCC TCTTTTTTAT CAACTCTGAA 960TATTTCCAAT ATCCTGCTAA TATCATAAAA ATGAAAAAAT GCTACTCACC TGATAAAGAA 1020AGAAAGATGA TTACAATCAG GGGTTCAGTC CACCAGAATT TTGCTGACTT CACTTTTGCA 1080ACTGGCAAAA TAATTGGACA CATGCTCAAA TTAAAGGGAG ACATAGATTC AAATGTAGCT 1140ATTGATCTTA GCAACAAAGC TTCATTAGCA TTCTTACAAA AGCATTTAGG ACTTCATAAA 1200GATTTTGATC AGTGGGACTG CTTGATTGAA GGAGATGATG AGAATCTTAT TCCAGGGACC 1260AACATTAACA CAACCAATCA ACACATCATG TTACAGAACT CTTCAGGAAT AGAGAAATAC 1320AATTAGGATT CTAGA 1335

Claims (16)

1. purifying and isolating human plasma platelet-activating factor acetylhydrolase (PAF-AH) polypeptide fragment, its disappearance are set forth in preceding 12 the N-terminal amino acid that reach most of one-tenth acquaintance PAF-AH aminoacid sequence among the SEQ ID NO:8.

2. be selected from the PAF-AH polypeptide fragment of following claim 1:

(a) has the Met of SEQ ID NO:8 ₄₆As the amino acid whose polypeptide of initial N-terminal;

(b) has the Ala of SEQ ID NO:8 ₄₇As the amino acid whose polypeptide of initial N-terminal;

(c) has the Ala of SEQ ID NO:8 ₄₈As the amino acid whose polypeptide of initial N-terminal;

3. claim 1 or 2 any one PAF-AH polypeptide fragment, the most nearly 30 C-terminal amino acid of the aminoacid sequence of its disappearance SEQ IDNO:8.

4. have the residue that is selected from following SEQ ID NO:8 any one PAF-AH polypeptide fragment as the claim 1 of its C-terminal residue or 2:

(a) Ile ₄₂₉，

(b) Leu ₄₃₁And

(c) Asn ₄₄₁。

5. the varient of the PAF-AH polypeptide fragment of claim 1, it has one and is selected from following amino-acid substitution in the sequence of SEQ ID NO:8:

(a) S108A，

(b) S273A，

(c) D286A，

(d) D286N，

(e) D296A，

(f) D304A，

(g) D338A，

(h) H351A，

(i) H395A，

(j) H399A，

(k) C67S，

(l) C229S，

(m) C291S，

(n) C334S and

(o) C407S。

6. people PAF-AH polypeptide variants, it has one and is selected from following amino-acid substitution in the sequence of SEQ ID NO:8:

(a) D286A

(b) D286N

(c) D304A。

7. coding is according to any one segmental isolating polynucleotide of PAF-AH polypeptide fragment, varient or varient of claim 1 or 6.

8. coding has the Met of SEQ ID NO:8 ₄₆As N-terminal residue and Ile ₄₂₉Or Asn ₄₄₁People PAF-AH fragment or the segmental isolating polynucleotide of varient as the C-terminal residue.

9. be the claim 7 of DNA or 8 any one polynucleotide.

10. the dna vector that comprises the DNA of claim 9.

11. use according to the DNA of claim 9 with the mode stable conversion in described PAF-AH polypeptide fragment, varient or the segmental host cell of varient, expressed or the host cell of transfection.

12. produce the segmental method of PAF-AH polypeptide fragment, varient or varient of blood plasma PAF-AH, be included in to make in the suitable nutrition and separate described PAF-AH fragment, varient or varient fragment according to the host cell growth of claim 11 and from the substratum of described cell or its growth.

PAF-AH polypeptide fragment, varient or varient fragment that 13 methods of passing through claim 12 produce.

14. pharmaceutical compositions comprises claim 1,6 or 13 any one PAF-AH fragment, varient or varient fragment and pharmacy acceptable diluent, adjuvant or carrier.

15. treatment is to the pathologic conditions susceptible of PAF mediation or suffer from the mammiferous method of the pathologic conditions of PAF mediation, is included in the described Mammals to be enough to replenish PAF-AH amount active and passivation PAF pathologic effect and gives pharmaceutical compositions according to claim 14.

16. according to the method for claim 15, wherein pathologic conditions is pleuritis, asthma, rhinitis, necrotizing enterocolitis, acute respiratory distress syndrome, acute pancreatitis or infects relevant sacred disease with HIV.

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