CN1296015A - IgG Fab-BPI fusion protein and DNA sequence thereof - Google Patents
IgG Fab-BPI fusion protein and DNA sequence thereof Download PDFInfo
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Abstract
The present invention discloses IgG Fab-BPI fusion protien, made up by using antibody IgG 1 and bactericial/permeability protein BPI through the process of fusion. It is characterized by that its amino (N) terminal possesses heavy chain and light chain of IgG1, and its carboxyli (C) terminal possesses BPI protein or BPI bio-active fragment, in which IgG1 is broad spectrum antiendotoxin LPS monoclonal antibody (mAb), they are fused and expressed in eukaryotic cell, and the obtained fusion protein expression product possesses broad spectrum and strong action of neutralizing endotoxin and killing bacteria, can be used for curing gram-negative bacterial infection diseases of human body. This invention also discloses DNA sequence of said fusion protein.
Description
The invention belongs to the fused protein that is used in anti-human body infectation of bacteria, i.e. the dna sequence dna of this fused protein of IgG Fab-BPI fused protein, and coding.
Antibody LPS mAb generally all have certain in and LPS effect (Zigler, J infect Disl988; 158 (2): 286-290), still, because species variation and the endogenous heterogeneity of bacterium LPS, most mAb can only neutralize immunity with the LPS of LPS and/or kind bacterium close with it, and rare provide protection (Mine, Km, Infect Immun 1996; 52 (1) 56-62; Mutharia LM, Ingect Immun1984; 45:631-636; Brade L, Infect Immun 1987; 55:462-486).
For one of significant especially report of the present invention is cross reactivity (Heumann D, the J Infect Dis.1991 that identifies the anti-LPS mAb of many strains with LPS-HDL (lipopolysaccharides-high-density lipoprotein (HDL)) mixture; 163:762-768), this studies show that most of anti-LPS mAb lack cross reactivity with it, find through investigation, LPS-HDL is the common product of different bacterium LPS the human body metabolism, the LPS of purifying about 90% forms the LPS-HDL mixture, LPS in the bacterial outer membrane fragment more than 50% forms the LPS-HDL mixture, complete bacterial outer membrane and complete bacterium then have 20% and 10% LPS to form LPS-HDL (JiaoBing Hua respectively, " intramolecularly toxinology ", Shanghai scientific and technical literature press 1995).And LPS-HDL still has complete LPS to cause the biologic activity of shock and DIC, and visible LPS-HDL is one of morbific key factor of intracellular toxin.At present do not find to prepare the research of intracellular toxin antibody with LPS-HDL.
Bactericidal (BPI) is a kind of cationic protein in people's polymorphonuclear granulocyte azurophilic granule, combining the effect that strengthens the Gram-negative bacteria permeability thereby have with bacterium LPS, is highly effective endogenous bacteriocidal substance (Ooi J Biol Chem 1987; 262:14891-94).BPI in conjunction with LPS also have the free LPS of neutralization effect (Weiss, Blood 1987; 69 (2): 652-659).BPI holds at its N-in conjunction with the position of LPS, and speaking by the book is in the 65-99 amino acids.But the binding ability of itself and LPS is limited, promptly rough type LPS there is keying action preferably, the binding ability of the LPS of smooth type is then reduced (Elasbach P et al.Basic Principlesand clinical corelates.1992 along with the lengthening of LPS specific polysaccharide chain; 2:603-636).
BPI N end is because of the about 25Kba of the molecular weight of expression product, and the transformation period lacks, and need adhere to macromolecular substance could play a role preferably (Dahlberg PS, et al.J Surg Ras 1996,63 (1): 44-48).
The LPS that has high homology with BPI combines composition in the blood that the albumen (LBP) that exists in the human body is a kind of solubility, but its effect is the depolymerization of strengthening LPS, strengthens its toxicity (Marra M etal.Gritical care Medicine, 1994; 22 (4): 553-564).Inject BPL simultaneously thereby cause BPI following situation: LPS attack mouse to occur in application, protection efficient reaches 90-95%; LPS attacks mouse and injects BPI after 1 hour, and protection efficient is only injected after 50%, 2 hour, then is 0.As seen BPI is used for clinical practice, still needing overcomes the competitive inhibitory effect of LBP to BPI, uses BPI merely and can not fine inhibition vivotoxin be encroached on by human body.
For two of the significant especially report of the present invention is to be re-combined into to comprise that antitumor IgG antigen-binding portion thereof is the hybrid fusion protein matter of the second fusion composition for first composition of fusion with interleukin-(adhesion molecule).Fell HP et al.J Lmmun 1991; 146 (7): narrated the synthetic of IL-2/IgG fusion rotein among the 2446-2452, its main purpose is to be incorporated into the antigen composition with IgG Fab section, attracts to adhere to mononuclear macrophage by IL-2, strengthens mononuclear macrophage engulfing and kill and wound tumour cell.
Have for the present invention special meaning report three, be to be re-combined into that to comprise BPI be first to merge composition and with IgG F in conjunction with LPS
cSection is the hybrid fusion protein (patent No. NO93109046 of the second fusion composition; Dahberg PS, et al.Arch Surg; 1996; 131:1173-1177). at first its composition of selecting IgG for use is a constant region, and purpose is to strengthen mononuclear macrophage etc. to BPI in conjunction with the antigenic phagolysis of LPS rather than in rising and the LPS antigenic action; Secondly, though this IgG is a kind of mAb, its preparation is not to filter out with LPS-HDL.So, its fused protein expression product do not have wide spectrum, strong in and intracellular toxin and germicidal action.
The purpose of this invention is to provide a kind of IgG Fab-BPI fused protein and dna sequence dna thereof, it is on the basis of the monoclonal antibody (mAb) that has prepared wide spectrum antiendotoxin (LPS), with the Fab section (V of this antibody (Immunoglobulin IgG1)
H, Ch
l, V
L, C
kHinge) separate the bactericidal power/permeability that obtains in gene and the white corpuscle and strengthen albumen (BPI) N end 1-629, or the gene fusion of 205-225 position, be expressed in eukaryotic cell, the gained fusion protein expression products have wide spectrum, powerful in and intracellular toxin and germicidal action, can be used for human body gram positive bacterial infection treatment of diseases.
This purpose realizes with following technical proposal: this IgG Fab-BPI fused protein is to be formed by IgG antibody 1 and the gene fusion that strengthens protein B PI (N) end, and the heavy chain and the light chain of IgG antibody 1 arranged at its amino (N) end; At its carboxyl (C) end BPI protein or BPI protein biology active fragments, described IgG antibody 1 being arranged is the monoclonal antibody (mAb) of a kind of wide spectrum antiendotoxin LPS.The heavy chain of wherein said IgG antibody 1 includes variable region of heavy chain and CH; The light chain of IgG antibody 1 includes variable region of light chain and constant region of light chain.The CH of IgG1 is CH1; Constant region of light chain is C
KThe BPI N end group of wherein said fusion is because of mainly referring to 1～629, or 205～225.At BPI protein and IgC
1Between also include hinge region and 15 amino acid whose junction fragments of IgG1.Described BPI protein is made up of 208 amino acid or 55 amino acid.
The dna sequence dna of IgG Fab-BPI fused protein, this dna sequence dna comprises IgG antibody
1Sequence of heavy chain and sequence of light chain; Dna sequence dna also includes the encoding sequence of 68～122 amino-acid residues of the encoding sequence of 1～208 amino-acid residue of PROTEIN B PI and BPI.
IgG Fab-BPI fusion rotein is the combination of two kinds of anti-LPS materials, has remedied shortcoming separately mutually, and has been of value to giving full play to of anti-separately LPS effect advantage.Obviously, what IgG1 of the present invention selected is the Fab section, purpose is to increase the neutralization of fusion rotein to various gram-positive microorganism LPS, so have the effect of the anti-LPS of wide spectrum, remedied the cross reaction limitation of BPI, and improved the competitive inhibitory effect of fusion rotein, prolonged the transformation period LBP: BPI then since its powerful in and the LPS effect, and improved the anti-LPS and the sterilizing ability of fusion rotein.Generally speaking, this fusion rotein will not be subjected to the restriction of disease time, condition, become the newtype drug of the anti-gram positive bacterial infection that positively effect is arranged of energy widespread use.
The present invention according to the composition structure of this fused protein at preparation process encoded this protein DNA sequence, the i.e. sequence of heavy chain of IgG, sequence of light chain, PROTEIN B PI 1-208 aminoacid sequence, a BPI55 aminoacid sequence.
The anti-LPS mAb of gained of the present invention belongs to Immunoglobulin IgG1 type, Fab section (V
H, CH
1, V
L, C
r) comprise the antigen-binding site and the part constant region of complete antibody, it is small molecular antibody with good antigen binding capacity, have the long transformation period on the one hand, and facilitate penetration of placenta, be difficult for owing to mouse source property causes human body that it is produced immunological rejection on the other hand.IgG1 is the anti-LPS immunoglobulin (Ig) of wide spectrum that filters out with LPS-HDL, selects to comprise V in experimental program
H, CH
1, V
1, C
K, the Hinge gene, and 16 amino acid whose Linker genes of synthetic voluntarily merge composition as first of fusion rotein.
Sterilization, permeability-increasing protein (BPI) are a kind of cationic proteins in people's polymorphonuclear granulocyte azurophilic granule, combine the effect that strengthens the saturating property of Gram-negative bacteria thereby have with bacterium LPS, are highly effective endogenous bacteriocidal substances.Simultaneously, BPI also has the effect of the free LPS of neutralization.BPI holds at its N in conjunction with the position of LPS in the present invention.In its preferred version, the BPI gene is a 1-629 base, has wherein comprised 3 functional zone of BPI, i.e. first structural domain (17-45 amino acids); Second structural domain (65-99 amino acids); The 3rd structural domain (142-169 amino acids).In another preferred version, the BPI gene is the 205-225 base, promptly comprises second functional zone (65-99 amino acids) of BPI.
The present invention also provides each gene composition of clone and expresses the suitable carrier and the host cell of this fusion gene, the light chain gene of IgG can be inserted in the expression vector that contains heavy chain BPI fusion gene, and in host cell, express, finish voluntarily and the amino acid whose combination folding of heavy chain, form complete fused protein.
Itemize out the specific embodiment that this IgGFab-BPI fused protein prepares respectively below in conjunction with first preferred version of the present invention, second preferred version, and the dna sequence dna of the various IgG Fab-BPI of construction expression the present invention fused protein.
Embodiment 1:
1, the preparation of intracellular toxin-high-density lipoprotein (HDL) mixture (LPS-HDL):
Get fresh plasma (containing 20mMEDTA) 3ml, add 100ug LPS, 37 ℃ of I incubation 1h add 3ml KBr (d=1.06), centrifugal 2.5 hours of 65000rpm, get supernatant 2ml, the dialysis tubing of packing into is in 1000ml dialyzate (75Mm Tris, 150Mm NaCL, 0.1EDTA) the middle immersion 24 hours, standby after KBr is removed in dialysis.
2, the preparation of anti-LPS McAb:
Respectively with the dead E.coli J5 thalline 2 * 10 of heat kill
8Mix the subcutaneous multi-point injection in 8 week BALB/c mouse backs in age with LPS 30ug, Fu Shi Freund's complete adjuvant 750ul; Second week rose, weekly the dead thalline 2 * 10 of subcutaneous injection E.coli J5 heat kill
8Mix totally 4 times with LPS 30ug, freund 's incomplete adjuvant 750ul.Put to death mouse, extracting spleen cell 1 * 10
8, with 1 * 10
7Myeloma cell (S/P20) mixes, and adds PEG40001ml and merges, and adds 1%HAT nutrient solution 40ml, is laid on 96 orifice plates and cultivates every hole 100ml, 37 ℃ of CO
2Incubator is cultivated.
Get LPS-HDL, with 50 times of PBS dilutions, and LPS (10ug/ml), wrap respectively by elisa plate, detect cell positive hole supernatant with the ELISA method, 410nm OD value is measured in the ABTS colour developing.Select OD value＞2 * OD
The control wells cellThe cell enlarged culturing.Obtain hybridoma cell strain C3A2.
3, the clone of C3A2 monoclonal antibody Fab fragment gene:
Get hybridoma cell strain C3A2 cell 1 * 10
7,, extract RNA with TriZol reagent (GIBCO company) lysing cell.Amplify IgG heavy chain and light chain gene segment respectively through the RT-PCR method.
The primer of amplification heavy chain is (AC) A (AG) CTC CAG (GC) AG TC (AT) GG of back-AT GGA TCC ATG GCC (GC) AG GT (GC), for-TGC TCDT AGA TCA AGG CTT ACTAGT ACA ATC CCT GGG CAC AAT, amplification segment 690bp, amplification condition be 97 ℃ 10 minutes, 94 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 1 minute, after 32 circulations, 72 ℃ were extended 5 minutes.
The primer of amplification light chain is (AT) TG AC (GC) CAG TCT CCA of back-CG AAT TCC ATG GCA GAC ATT (GC), for-C TCG CTC GAG TTA ACA CTC ATT CCT GTT GAA GC, the amplification segment is 720bp, amplification condition be 97 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, after 32 circulations, 72 ℃ were extended 5 minutes.
Respectively heavy chain is connected with PGEM-T easy carrier (Promeg Corporation) with the light chain gene amplified production, transform the JM109 intestinal bacteria, through IPTG and X-ga1 screening, picking white clone, extract plasmid, carry out enzyme with BamH I+Xba I and EcoR I+Xho I respectively and cut evaluation, after the affirmation reorganization is correct, carry out automatic sequencing from 5 ' and 3 ' end respectively with T7 and SSP6 sequencing primer, sequencing reaction is finished by the ABI377 automatic sequencer.Difference called after PGEM-Fd and PGEM-L.
4, people's bactericidal (BPI) N end group because of the clone:
The separation of human peripheral polymorphonuclear leukocytes is extracted total RNA with Trizol, is the synthetic cDNA of primer reverse transcription with oligo (dt) 15.Dna fragmentation with the 25th to 749 base of sleeve type PCR method amplification BPI.First round pcr amplification reaction liquid is formed; 10 * PCR damping fluid 5ul, 25mmol/L MGCL
24ul.10mmol/L dNTP 2ul, each 1ul of 50pmol/L outer primer, the 3UTaq enzyme, 5ulcDNA adds water to 50ul.Amplification condition is 94 ℃ of 1min, 58 ℃ of 1min, and 72 ℃ of 1min, 32 circulations, 72 ℃ are extended 10min then.Second to take turns PCR be template with first round amplified production 5ul, changes with the inner primer amplification, and its reactant of base is formed and the same first round of reaction conditions.(outer primer) back-TTG AGG TTT TGG CAG CTC TGG, for-AAA GGG AGG TGG ATT GTG G; Inner primer: back-AT GGA TCC TGG AGG ATG AGA GAGAAG ATG CT, for-AT TCT AGA CCA TAG TTA GAT TCC AGC CAC).
After the purified recovery of PCR product, be connected to the PGEM-Teasy carrier, transform the JM109 intestinal bacteria, the picking positive colony extracts recombinant plasmid with Wizard plusDNA purification kit, cuts evaluation with Bam HI and Xbal enzyme.After confirming that reorganization is correct, carry out automatic sequencing with T7 SP6 sequencing primer branch from 5 ' and 3 ' end, sequencing reaction is finished by the ABI377 automatic sequencer.Called after PGEM-BPI.
5, BPI-IgG Construction of eukaryotic:
Digest PGEM-FD and pcDNA3 respectively with the EcoR I, through 1% agarose gel electrophoresis, separation and purification Fd and pcDNA3 segment are got 2ulFd and pcDNA3 respectively, add the T4 ligase enzyme, in 16 ℃ of connections of spending the night, transformed into escherichia coli, random choose go out 4 anti-penbritin clones, cultivate in a small amount, extract plasmid, digest evaluation respectively, can cut out the bacterial clone called after pcDNA3-Fd of 690hp with EcoR I and Xba I.
With Pgem-BPI is template, pcr amplification BPI 180bp fragment.The PCR primer is for-CGT CTA GAGTCA GTT ACT GCC CAG CTT CAG ATC-3 ', back-G ACT AGT GGA AGA GTA GTA GGCGGT CGA GCC CTA CTG CGA GCC GTC AAC ATA AGC ATG GTG CCC AAT GTG-3 '.Amplification condition is: 97 ℃ of sex change 5 minutes, 94 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 1 minute, after 32 circulations, 72 ℃ were extended 5 minutes.Through 2% agarose gel electrophoresis, separation and purification 180bp dna fragmentation.
Digest above-mentioned PCR product with Spe I and Xba I, be connected transformed into escherichia coli with the pcDNA3-Fd of same processing.Random choose goes out 4 anti-penbritin clones, cultivates in a small amount, extracts plasmid, digests evaluation respectively with Spe I and xba I, can cut out the bacterial clone called after pcFd-BPI180 of 180bp.
Digest PGEM-L and pcDNA3 plasmid respectively with EcoR and Xho I, through 1% agarose gel electrophoresis, separation and purification L and pcDNA3 segment are got 2ul L and pcDNA3 respectively, add the T4 ligase enzyme, in 16 ℃ of connections of spending the night, transformed into escherichia coli, random choose go out 4 anti-penbritin clones, cultivate in a small amount, extract plasmid, digest evaluation respectively, can cut out the bacterial clone called after pcDNA3-L of 670bp with EcoR I and Xho I.
Pcr amplification pcDNA3-L 1.7kb fragment (comprising the CMN promotor, L chain, BGH poly a-signal).Amplimer is back-GAA CGT TTA GAT CTG CTT CGC G-3 and for-GAG CCC CAA TTG GTTCTT TCC GC-3 ', and amplification condition is: 97 ℃ of sex change minute, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 2 minutes, after 32 circulations, 72 ℃ were extended 10 minutes.Through 1% agarose gel electrophoresis, separation and purification 1.7kbDNA segment, with Bg1 II and Mun I respectively enzyme cut above-mentioned PCR product and pcFd-BPI180, the 1.7kbDNA segment is inserted among the pcFd-BPI180, and transformed into escherichia coli, random choose go out 4 anti-penbritin clones, cultivate in a small amount, extract plasmid, digest evaluation respectively, can cut out the bacterial clone called after pcFBPI180 of 1.7kp with Bg1 II and Mun I.
6, pcFBPI transfection CHO cell:
Get 6 well culture plates, every hole adds 1 * 10
5Cell/2ml perfect medium is put CO
2Cultivated 24 hours in the incubator, cell is merged mutually.
Get one 12 * 75mm sterile tube, add the 10ml serum free medium, dissolving 2ug pcFBPI180 plasmid DNA is designated as the D pipe; Get another sterile tube, add the 100ul serum free medium, molten angle 25ul refers to plastid, is designated as the L pipe.Two pipe solution are mixed, and room temperature is put 40min, to form the DNA-Liposome mixture, is designated as the D-L pipe.
Wash cell with the 2ml serum free medium.Add the 800ul serum free medium in the D-L pipe, mixing is spread to cellular layer gently, puts CO
2Incubator 24 hours adds the 1ml substratum; After 24 hours, change fresh perfect medium; After 72 hours,, put and select to grow in the substratum 10 times of dilutions of cell.
Embodiment 2:
Dna segment with the 645bp of digestion of Hinc II and Xba I and PGEM-BPI cutting-out is inserted into pcDNA3-Fd, transformed into escherichia coli.Random choose goes out 4 anti-penbritin clones, cultivates in a small amount, extracts plasmid, digests evaluation respectively with Hinc II and Xba I, can cut out the bacterial clone called after pcFd-bpiI645 of 645bp.
Pcr amplification pcDNA3-L 1.7kb segment (comprising the CMV promotor, L chain, BGH poly a-signal).Amplimer is 5-agg cg tta gat ctg ctt cgc g-3 and 5-gag ccc caa ttg gtt ctt tccgc-3 ', amplification condition is: 97 ℃ of sex change 5 minutes, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 2 minutes, 32 circulations were extended 10 minutes for back 72 ℃.Through 1% agarose gel electrophoresis, separation and purification 1.7kb dna segment, with Bg1 II and Mun I respectively enzyme cut above-mentioned PCR product and pcFd-BPI645, the 1.7KB fragment is inserted among the psFd-BPI645, and transformed into escherichia coli, random choose go out 4 anti-penbritin clones, cultivate in a small amount, extract plasmid, digest evaluation respectively, can cut out the bacterial clone called after pcFBPI645 of 1.7kp with Bg1 II and Mun I.
All the other methods and step are with embodiment 1.
Dna sequence dna:
1 ( IgG1 ) TAT GGA TCC ATG GCC CAG GTG AAG CTC CAG CAG TCT GGA CCT GAGCTG GAG AAG CCT GGC GCT TCA GTG AAG ATA TCC TGC AAG GCT TCTGGT TAC TCA TTC ACT GGC TAC AAC ATG AAC TGG GTG AAG CAG AGCAAT GGA AAG AGC CTT GAG TGG ATT GGA AAT ATT GAT CCT TAC TATGGT GGT ACT AGC TAC AAC CAG AAG TTC AAG GGC AAG GCC ACA TTGACT GTA GAC AAA TCC TCC AGC ACA GCC TAC ATG CAG CTC AAG AGCCTG ACA TCT GAG GAC TCT GCA GTC TAT TAC TGT GCA AGA TCC GGG GGTAAC TAC GGG GGA GCC TGG TTT GCT TAC TGG GGC CAA GGG ACC ACGGTC ACC GCC AAA ACG ACA CCC CCA TCT GTC TAT CCA CTG GCC CCT GGATCT GCT GCC CAA ACT AAC TCC ATG GTG ACT CTG GGA TGC CTG GTC AAGGGC TAT TTC CCT GAG CCA GTG ACA GTG ACC TGG AAC TCT GGA TCC CTGTCC AGC GGT GTG CAC ACC TTC CCA GCT GTC CTG CAG TCT GAC CTC TACACT CTG AGC AGC TCA GTG ACT GTC CCC TCC AGC ACC TGG CCC AGCGAG ACC GTC ACC TGC AAC GTT GCC CAC CCG GCC AGC AGC ACC AAGGTG GAC AAG AAA ATT GTG CCC AGG GAT TGT ACT AGT AAG CCT TGATCT AGA GCA
2 ( IgG1 ) TCG AAT TCC ATG GCA GAC ATT CTG TTG ACG CAG TCT CCA CTC ACTTTG TCG GTT ACC ATT GGA CAA CCA GCC TCC ATC TCT TGC AAG TCAAGT CAG AGC CTC TTA GAT AGT GAT GGA AAG ACA TAT TTG AAT TGGTTG TTA CAG AGG CCA GGC CAG TCT CCA AAG CGC CTA ATC TAT CTGGTG TCT AAA CTG GAC TCT GGA GTC CCT GAC AGG TTC ACT GGC AGTGGA TCA GGG ACA GAT TTC ACA CTG AAA ATC AGC AGA GTG GAG GCTGAG GAT TTG GGA GTT TAT TAT TGC TGG CAA GGT ACA CAT TTT CCATTC ACG TTC GGC TCG GGG ACA AAG TTG GAA ATA AAA CGG GCT GATGCT GCA CCA ACT GTA TCC ATC TTC CCA CCA TCC AGT GAG CAG TTAACA TCT GGA GGT GCC TCA GTC GTG TGC TTC TTG AAC AAC TTC TACCCC AAA GAC ATC AAT GTC AAG TGG AAG ATT GAT GGC AGT GAA CGACAA AAT GGC GTC CTG AAC AGT TGG ACT GAT CAG GAC AGC AAA GACAGC ACC TAC AGC ATG AGC AGC ACC CTC ACG TTG ACC AAG GAC GAGTAT GAA CGA CAT AAC AGC TAT ACC TGT GAG GCC ACT CAC AAG ACATCA ACT TCA CCC ATT GTC AAG AGC TTC AAC AGG AAT GAG TGT TAACTC GAG CGA G
3 ( BPⅠ1～208 ) GGC ACC GCC GTG ACA GCG GCC GTC AAC CCT GGC GTC GTG GTC AGGATC TCC CAG AAG GGC CTG GAC TAC GTC AGC CAG CAG GGG ACG GCCGCT CTG CAG AAG GAG CTG AAG AGG ATC AAG ATT CCT GAC TAC TCAGAC AGC TTT AAG ATC AAG CAT CTT GGG AAG GGG CAT TAT AGC TTCTAC AGC ATG GAC ATC CGT GAA TTC CAG CTT CCC AGT TCC CAG ATAAGC ATG GTG CCC AAT GTG GGC CTT AAG TTC TCC ATC AGC AAC GCCAAT ATC AAG ATC AGC GGG AAA TGG AAG GCA CAA AAG AGA TTC TTAAAA ATG AGC GGC AAT TTT GAC CTG AGC ATA GAA GGC ATG TCC ATTTCG GCT GAT CTG AAG CTG GGC AGT AAC CCC ACG TCA GGC AAG CCCACC ATC ACC TGC TCC AGC TGC AGC AGC CAC ATC AAC AGT GTC CACGTG CAC ATC TCA AAG AGC AAA GTG GGG TGG CTG ATC CAA CTC TTCCAC AAA AAA ATT GAG TCT GTG CTT CGA AAC AAG ATG AAC AGC CAGGTC TGC GAG AAA GTG ACC AAT TCT GTA TCC TCC GAG CTG CAA CCTTAT TTC CAG ACT CTG CCA GTA ATG ACC AAA ATA GAT TCT GTG GCT GGAATC AAC TAT GAG GTA
Sequence 4 (aminoacid sequences of 55 Chinese han populations of BP I) CTC GAG ATA AGC ATG GTG CCC AAT GTG GGC CTT AAG TTC TCC ATC AGCAAC GCC AAT ATC AAG ATC AGC GGG AAA TGG AAG GCA CAA AAG AGATTC TTA AAA ATG AGC GGC AAT TTT GAC CTG AGC ATA GAA GGC ATGTCC ATT TCG GCT GAT CTG AAG CTG GGC AGT AACTCT AGA
Claims (8)
1, a kind of IgG Fab-BPI fused protein, it is to be formed by the gene fusion of IgG antibody 1 with bactericidal BPI (N) end for its feature, and the heavy chain and the light chain of IgG antibody 1 arranged at its amino (N) end; At its carboxyl (C) end BPI protein or BPI protein biology active fragments, described IgG antibody 1 being arranged is the monoclonal antibody (mAb) of a kind of wide spectrum antiendotoxin LPS.
2, IgG Fab-BPI fused protein according to claim 1 is characterized in that the heavy chain of described IgG antibody 1 includes variable region of heavy chain and CH; The light chain of described IgG antibody 1 includes variable region of light chain and constant region of light chain.
3, IgG Fab-BPI fused protein according to claim 1 is characterized in that PROTEIN B PI (N) end group because of mainly referring to 1～629, or 205～225.
4, IgG Fab-BPI fused protein according to claim 2 is characterized in that described IgG1 CH is CH
1Described IgG1 constant region of light chain is C
k
5, IgG Fab-BPI fused protein according to claim 1 is characterized in that also including hinge region and 15 amino acid whose junction fragments of IgG1 between PROTEIN B PI and IgG antibody 1.
6, IgG Fab-BPI fused protein according to claim 4 is characterized in that PROTEIN B PI is made up of 208 amino acid or 55 amino acid.
7, a kind of dna sequence dna of IgG Fab-BPI fused protein is characterized in that this dna sequence dna comprises the sequence of heavy chain and the sequence of light chain of IgG antibody 1.
8, the dna sequence dna of IgG Fab-BPI fused protein according to claim 7 is characterized in that this dna sequence dna also includes the encoding sequence of 68～122 amino-acid residues of the encoding sequence of 1～208 amino-acid residue of PROTEIN B PI and PROTEIN B PI.
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|---|---|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101344600B (en) * | 2007-07-13 | 2011-01-26 | 鸿富锦精密工业（深圳）有限公司 | Production method of plated film lens |
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2000
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101344600B (en) * | 2007-07-13 | 2011-01-26 | 鸿富锦精密工业（深圳）有限公司 | Production method of plated film lens |
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