CN1294263C - Heavy chain and light chain variable region gene of human B lymphocyte stimulation factor monoclonal antibody and its application - Google Patents
Heavy chain and light chain variable region gene of human B lymphocyte stimulation factor monoclonal antibody and its application Download PDFInfo
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Abstract
The present invention relates to genes of variable regions of heavy chains and variable regions of light chains of monoclonal antibodies of human B lymphocyte stimulating factors, the application of polypeptide of gene codes, a carrier containing the genes, the genes and the polypeptide in the preparation of clinical test reagents for BLyS associated autoimmune diseases, and a preparation method thereof. The overall length of the genes of variable regions of heavy chains is 342 bp, the nucleotide sequence is shown in <400>1, and the amino acid sequence is shown in <400>3. The overall length of the genes of variable regions of light chains is 321 bp, the nucleotide sequence is shown in <400>2, and the amino acid sequence is shown in <400>4. Protein produced through the expression of gene engineering reserves the capacity of combination with antibodies, and small scFv molecules can be combined with a quantity of other effector molecules easily through reconstruction by gene engineering technology; thereby, a foundation is laid for the construction of biologic missiles. The present invention also has the advantages of greatly reduced cost and favorable industrialized production.
Description
Technical field
The present invention relates to the heavy chain and the chain variable region gene of human B lymphocyte stimulation factor monoclonal antibody, polypeptide by described genes encoding, the carrier and the described gene and the application of polypeptide in the clinical detection reagent of the relevant autoimmune disorder of preparation BLyS that contain described gene, with and preparation method thereof.Particularly, heavy chain of the present invention and chain variable region gene are from BALB/C mice hybridoma ABL-1.
Background technology
Human B lymphocyte stimulating factor (hBLyS) is people (Science.1999 Jul9 such as Moore PA; 285 (5425): 260-3) newfound a kind of and human immunity in 1999 are regulated and control closely-related cytokine, belong to tumour necrosis factor (TNF) superfamily, be II type transmembrane protein, mainly in tissue such as human peripheral blood monocyte, spleen, lymphoglandula, marrow, express, and can be under the effect of some metalloprotease with born of the same parents outside soluble fractions (hsBLyS) be free in the peripheral blood and play a role.HsBLyS is a kind of lymphocytic costimulating factor, there is intensive to promote the hormesis of propagation and differentiation to activatory B cell: to normal B cell, after with PMA and the pre-activation of Anti-IgM, hsBLyS can induce the various immunoglobulin (Ig) of its a large amount of propagation justacrines, and wherein major part is IgM and IgM.In vivo, hBLyS is to the activation of the outer B cell of capsule and the secretion of antigen-specific IgM; The homotype conversion of immunoglobulin (Ig); Spleen germinal center (GC) is formed with vital role; HBLyS is to CD4 simultaneously
+And CD8
+Hypotype T cell also has the indirect activation effect.The positive regulating factor that hBLyS grows as bone-marrow-derived lymphocyte, has the differentiation that promotes bone-marrow-derived lymphocyte in vivo, the effect of survival, and too much hBLyS can cause the overactivity of bone-marrow-derived lymphocyte and cause autoimmune disorder, evidence is as follows: (1) hBLyS transgenic mice is because overexpression hBLyS, the destruction that has caused self tolerance multiple autoantibody occurs in serum and the tissue, simultaneously with systemic lupus erythematous sample symptom.(2) in the lupoid acne model mice (NZB * NZWF1 and MRL-lpr/lpr), serum hBLyS titre obviously raises, and the level of hBLyS is parallel relation with the progress of disease.(3) all can detect the hBLyS titre in various autoimmune disease (as systemic lupus erythematous, rheumatoid arthritis and systemic sclerosis and myasthenia gravis etc.) patients serum obviously raises.BLyS (M.Petri et al. in 2003, abstract 1712, ACR 2003meeting) thus the development that is used as a kind of Biomarker. BLyS antibody of diagnositc system lupus erythematosus clinically for the diagnosis of the relevant autoimmune disorder of BLyS with treat significant.
Monoclonal antibody is made up of two identical heavy chains and light chain.Each bar heavy chain (VH) and light chain (VL) all are divided into constant region and variable region two parts.Wherein, VH and VL form antigen-binding site jointly.The specificity of antibody is determined by this position just.VH and VL are about 110 amino acid, account for 1/2 and 1/4 of weight chain respectively.Real and antigenic determinant space conformation complementary are 3 amino acid variation zones (CDR) greatly in antigen-binding site.Relative with the variable region is the skeleton district, and its aminoacid sequence is conservative relatively.ScFv is spliced by VH and VL and a flexible amino acid short peptide.
There is report to screen antibody (the ArthritisRheum.2003 Nov such as Baker KP of BLyS from phage display library abroad; 48 (11): 3253-65.), but because this antibody is not cross through immunity, general avidity is supported, and is not suitable as clinical detection reagent.The domestic report (Zhang Zhi side etc., Zhongshan Medical Univ.'s journal, 2002 03 phases) that has the strain of BLyS antibody hybridoma cell to set up, but also do not have the report of genes involved sequence up to now.
Summary of the invention
Purpose of the present invention at first provides the scFv type genetic engineering antibody fragment that a kind of human B lymphocyte stimulation factor monoclonal antibody chain variable region gene and heavy chain variable region gene are assembled, and makes the extensive cheap antibody of producing become possibility.Solve the strain of prior art antibody hybridoma cell with this and be applied in the scale operation sudden change easily, the cell strain instability is lost easily, and many shortcomings such as cost dearly.
Another object of the present invention is that human B lymphocyte stimulation factor monoclonal antibody and scFv antibody fragment thereof are used for the application of the detection reagent of the relevant autoimmune disorder of BLyS in preparation.
According to an aspect of the present invention, the present invention relates to a kind of human B lymphocyte stimulation factor monoclonal antibody chain variable region gene and heavy chain variable region gene, wherein, described heavy chain variable region gene total length is 342bp, its nucleotide sequence is as＜400〉shown in 1, its amino acid sequence coded is as＜400〉shown in 3; Described chain variable region gene total length is 321bp, and its nucleotide sequence is as＜400〉shown in 2, its amino acid sequence coded is as＜400〉shown in 4.
A kind of bone-marrow-derived lymphocyte stimulating factor scFv antibody fragment is between said heavy chain variable region gene and the said chain variable region gene, is connected with one section flexible amino acid short peptide.
A kind of expression vector has described heavy chain variable region gene and chain variable region gene, and said heavy chain variable region gene and chain variable region gene connect by one section flexible amino acid short peptide.Specifically can be pET28a-scFv as shown in Figure 2.
Relate to the expressed polypeptide of above-mentioned expression vector, can with the BLyS effect, can be used to prepare the detection reagent of the relevant autoimmune disorder of BLyS.
Producing the proteic method of described scFv with genetically engineered is: the weight chain variable region gene of isolating antibody from the strain of BLyS monoclonal cell, and assembling scFv, scFv with assembling makes up prokaryotic expression carrier, transformed into escherichia coli, make up genetic engineering bacterium, and abduction delivering, to expression product separate, purifying and renaturation.
As preferred scheme, concrete operations are:
1) from the variable region gene of BLyS monoclonal cell strain ABL-1 separation antibody:
Collect well-grown, can secrete the hybridoma cell strain ABL-1 of BLyS monoclonal antibody, extract total RNA with TRIZOL reagent,
The design degenerate primer, VH employing primer VH Back (5 '-AGGTSMARCTGCAGSAGTCWGG-3 ') and primer VH For (5 '-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3 '),
The VL gene adopts primer VL back (5 '-GACATTGAGCTCACCCAGTCTCCA-3 ') and primer VLFor (5 '-GTTAGATCTCGAGCTTGGTCCC-3 ') according to Promega company single stage method RT-PCR test kit increase respectively VH and VL, adopt the RT-PCR method respectively, angle and get antibody variable region VH and VL gene, the clone enters carrier pMD18-T respectively;
2) VH, the clone of VL gene amplification product:
Design of primers is as follows:
VH P
1(5 '-ACCATG
GAATTCCTGCAGGAGTCTGGTGGCTTG-3 '), contain the EcoRI restriction enzyme site,
VH?P
2(5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTG-3’)，
VL?P
1(5’-CAGGCGGAGGTCGCTCTGGCGGTAGTGACATTGAGCTCAC-3’)，
VL P
2(5 '-GTGGTG
AAGCTTTCACTCGAGCTTGGTACCACCTC-3 '), contain the HindIII restriction enzyme site,
Adopt Over-lap PCR, one section flexible linker (G of design between VH and VL
4S)
3, VH-linker-VL clones in efficient prokaryotic expression carrier pET28a;
3) efficiently express, purifying, renaturation scFv:
PET28a-scFv transforms and enters BL21 (DE3), and the IPTG abduction delivering is collected the inclusion body precipitation, urea dissolving, Ni
2+-IDA His-bind affinitive layer purification albumen, dilution method and dialysis method are to the target protein renaturation, the albumen of purifying is diluted to renaturation buffer I:50mM Tris-HCl, 0.15mM NaCl, 2M urea, 0.5Mm reduced glutathion, 0.1mM Sleep-promoting factor B, pH 8.0, final concentration is 100 μ g/ml, renaturation 24h, renaturation buffer II:50mmol/L Tris-HCl, 0.15mol/LNaCl, pH 8.0 then pack albumen into, dialysis renaturation 12h, ultrafiltration pipe Centricon 30 protein concentrates, 13%SDS-PAGE detects, protein-20 ℃ preservation.
We utilize genetic engineering means at first to obtain the weight chain variable region gene of monoclonal antibody from the hybridoma cell strain of monoclonal antibody of secretion BLyS, and assembled the scFv small molecular antibody, be cloned in the pET28a expression vector, and can in intestinal bacteria, efficiently express, the protein of its generation has kept the ability with antibodies, can with the BLyS effect, and with traditional sense on hybridoma antibody relatively have following advantage: the scFv molecule is little, easy transformation by genetic engineering technique, combine with some other effector molecules, lay the first stone for making up biological missile; Hybridoma cell strain is not easy to preserve, large-scale production cost height, and genetic engineering antibody can adopt prokaryotic expression system, can produce antibody very easily, cost reduces greatly, is beneficial to suitability for industrialized production.Therefore the production of genetic engineering antibody is with the obvious advantage than hybridoma cell strain generation antibody, thereby can replace the conventional hybridization tumor cell strain to produce antibody.In addition, the genetic engineering antibody of the BLyS affinity chromatography medium that can be used for making BLyS is used for the purifying of BLyS.
Description of drawings
Fig. 1 is the RT-PCR product agarose gel electrophoresis analytical results of ABL-1 antibody variable region VH and VL, and wherein swimming lane 1 is a standard protein; Swimming lane 2 is the VH gene, and molecular weight is greatly about 340bp; Swimming lane 3 is the VL gene, and molecular weight is greatly about 330bp; Swimming lane 4 is the VH-linker-VL gene, and size is about 750bp.
Fig. 2 is the assembling of ABL-1 scFv gene and the synoptic diagram of expression vector establishment.
Fig. 3 is the abduction delivering of ABL-1 scFv, the analytical results of purifying and evaluation, wherein (A1): standard protein; (A2): do not induce whole bacterial protein; (A3): induce the back whole bacterial protein; (A4): ultrasonic supernatant; (A5): ultrasound precipitation; (A6): purified product; (B1): Western blotting detects proteic expression.
Fig. 4 is the synoptic diagram that combines that non-competitive ELISA is measured ABL-1 scFv and hsBLyS.
Fig. 5 is that competitive ELISA is measured the synoptic diagram that combines that ABL-1 scFv suppresses ABL-1 monoclonal antibody and hsBLyS.
Fig. 6 be Western blotting detect ABL-1 scFv and hsBLyS combine wherein (A1): standard protein; (A2): purifying hsBLyS albumen; (B): ABL-1mAb and hsBLyS combine combining of (C): ABL-1scFv and hsBLyS; (D): combine (as the negative control) of irrelevant IgG and hsBLyS.
Embodiment
The present invention is further described below in conjunction with drawings and Examples.
Embodiment 1: collect well-grown from the variable region gene of BLyS monoclonal cell strain ABL-1 separation antibody, can secrete the hybridoma cell strain ABL-110 of BLyS monoclonal antibody
7Individual (this research department makes up, according to " Current protocol in immunology " Production of MonoclonalAntibodies) extracts total RNA with TRIZOL reagent.RT-PCR angles and gets the VH gene:
The method that provides with reference to (the Access RT-PCR System and Access RT-PCRIntroductory System) of Promega company: the single stage method RT-PCR test kit VH gene that increases respectively:
VH gene employing primer VH Back (5 '-AGGTSMARCTGCAGSAGTCWGG-3 ') and primer VH For (5 '-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3 '),
The RT-PCR reaction is total up to 50 μ l systems: total RNA of 1 μ g, and 5 * AMV/Tf1 reaction buffer of 10 μ l, concentration is primer VH Back and each 5 μ l of VH For of 10pmol, concentration is the dNTP1 μ l of 500uM, the MgSO of 3mM
42 μ l; 5u AMV reversed transcriptive enzyme 1 μ l and 5u Tf1 archaeal dna polymerase 1 μ.Reactions steps: 48 ℃ of reverse transcription reactions 45 minutes; 94 ℃ of sex change 30 seconds; Annealed 1 minute for 55 ℃; 68 ℃ were extended totally 35 circulations 1 minute.
RT-PCR angles and gets the VL gene:
VL gene employing primer VL back (5 '-GACATTGAGCTCACCCAGTCTCCA-3 ') and VL For (5 '-GTTAGATCTCGAGCTTGGTCCC-3 ').According to the single stage method RT-PCR of Promega company test kit amplification VL.50 μ l reaction systems are as follows: total RNA of l μ g, and 5 * AMV/Tf1 reaction buffer of 10 μ l, concentration is primer VL back and each 5 μ l of VL For of 10pmol, concentration is the dNTP1 μ l of 500uM, the MgSO of 3mM
42 μ l; 5u AMV reversed transcriptive enzyme 1 μ l and 5u Tf1 archaeal dna polymerase 1 μ.Reactions steps: 48 ℃ of reverse transcription reactions 45 minutes; 94 ℃ of sex change 30 seconds; Annealed 1 minute for 60 ℃; 68 ℃ were extended totally 35 circulations 1 minute.After reaction finished, 1% agarose gel electrophoresis detected.Result such as Fig. 1.The clone of VH and VL gene amplification product:
Design of primers is as follows:
VH P
1(5 '-ACCATG
GAATTCCTGCAGGAGTCTGGTGGCTTG-3 '), contain the EcoR1 restriction enzyme site,
VH?P
2(5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTG-3’)，
VL?P
1(5’-CAGGCGGAGGTGGCTCTGGCGGTAGTGACATTGAGCTCAC-3’)，
VL P
2(5 '-GTGGTG
AAGCTTCACTCGAGCTTGGTACCACCTC-3 '), contain the HindIII restriction enzyme site,
The PCR product uses glue to reclaim the test kit purifying.Replace body pMD18-T 1 μ l, PCR product 4 μ l, T4 ligase enzyme 1 μ l, 5 μ l T4 ligase enzyme damping fluids (2 times), 16 degree connect 18 hours.Connect product 10 μ l transformed competence colibacillus bacillus coli DH 5 alphas.Bed board, second day picking mono-clonal adopts bacterium colony PCR to identify, and delivers the order-checking of Shanghai Bo Ya company.Sequencing result is as sequence table＜400〉shown in 1 and＜400〉2.
The assembling of embodiment 2:scFv gene
For obtaining scFv, between VH and VL, add a flexible linker (G
4S)
3, for prevent the sudden change that induces one in amplification procedure, we adopt high-fidelity enzyme Pyrobest (Dalian is precious biological).Whole assembling synoptic diagram is Fig. 2, result such as Fig. 1.
First round PCR reaction amplification VH:
50 μ l PCR reaction systems are as follows: 10 * reaction buffer of 5 μ l; Concentration is the primer VHP of 10 μ M
1With VH P
22.5 the dNTP of μ l:4 μ l; 35 μ l H
2O; High-fidelity Pyrobest enzyme 0.5 μ l.95 ℃ of sex change 5min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 26 circulations; 72 ℃, extend 5min.
Second takes turns PCR reaction amplification VL:
50 μ l PCR reaction systems are as follows: 10 * reaction buffer of 5 μ l; Concentration is the primer VL P of 10 μ M
1With VL P
22.5 the dNTP of μ l:4 μ l; 35 μ l H
2O; High-fidelity Pyrobest enzyme 0.5 μ l.95 ℃ of sex change 5min; 95 ℃ of sex change 30s, 52 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 26 circulations; 72 ℃, extend the 5min. rubber tapping and reclaim the PCR product.
Third round PCR reaction amplification VH-linker-VL:
50 μ l PCR reaction systems are as follows: 10 * reaction buffer of 5 μ l; Concentration is the primer VHP of 10 μ M
1With VL P
22.5 the dlTP of μ l:4 μ l; 35 μ l H
2O; High-fidelity Pyrobest enzyme 0.5 μ l, each 1 μ l of last two-wheeled PCR product is as template.95 ℃ of sex change 5min; 95 ℃ of sex change 30s, 52 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 26 circulations; 72 ℃, extend the 5min. reaction.1% agarose gel electrophoresis, product is reclaimed in rubber tapping.
The enzyme of embodiment 3:scFv gene is cut digestion and is connected
Double digestion pET2 8a:
EcoRI and HindIII are the Takara product.10 * M reaction buffer of 2 μ l; The carrier pET28a of 1O μ l; Each 1 μ l of EcoRI and HindIII; 7 μ l H
2O.37 ℃ were reacted 6 hours.: the third round amplified production among the double digestion embodiment 2:
10 * M reaction buffer of 2 μ l; The third round PCR product of 10 μ l; Each 1 μ l of EcoRI and HindIII; 7 μ l H
2O.37 ℃ were reacted 6 hours.
1% agarose gel electrophoresis, product is reclaimed in rubber tapping.
Ligation:
The T4 ligase enzyme is the Takara product.The ligation damping fluid H of 2 μ l
2O; Last wheel enzyme is cut each 1 μ l of product; 16 μ l H
2O.16 ℃ were reacted 12 hours.
To get 10 μ l products and transform transformed competence colibacillus DH5 α. bed board, second day picking mono-clonal adopts bacterium colony PCR to identify, and delivers the order-checking of Shanghai Bo Ya company.
The abduction delivering of embodiment 4:scFv single-chain antibody in intestinal bacteria.
Clone's transformed competence colibacillus BL21 (DE3) that the picking order-checking is correct.The single colony inoculation of picking is in 3ml LB nutrient solution (containing 50 μ g/ml kantlex), cultivated 10 hours for 37 ℃, collect bacterium, being inoculated into 50ml by 1: 100 contains among the same concentrations microbiotic LB, 37 ℃ when being cultured to OD600=0.6, add IPTG (1mmol/L) and induce, cultivated 5 hours for 37 ℃, 13%SDS-PAGE detects expression.Result such as Fig. 3 clearly show that the expressed proteins molecular weight is about 27Kd.Mainly be present in the inclusion body.
Embodiment 5: the separation and purification of expression product and renaturation
The centrifugal 10min of 4 ℃ of 5000g/min collect thalline, with 5ml 50mmol/L Tris-HCl, and the resuspended precipitation of 0.15mol/L NaCl, ultrasonic on ice then (ultrasonic 5s, gap 5s, 160 times repeatedly) are to cell suspension thickness no longer.The centrifugal 10min of room temperature 12000g/min collects respectively and goes up cleer and peaceful precipitation.Precipitation adding 2.5ml Binding damping fluid (50mmol/L Tris-HCl, 0.15mol/L NaCl, 8M urea, the 5mM imidazoles, pH 8.0), room temperature dissolving 2 hours. the centrifugal 10min of room temperature 12000g/min, collect supernatant.Get Ni
2+-IDA His-bind resin 1ml adorns post, the operational manual purifying protein that provides according to Novagen company.
Collect the albumen of Elution buffer solution elution, measure protein concentration, with renaturation buffer 1 (50mmol/L Tris-HCl, 0.15mol/L NaCl, 2M urea, 0.5mmol/L reduced glutathion, 0.1mmol/L Sleep-promoting factor B pH 8.0) diluted protein to final concentration is 100 μ g/Ml.Renaturation 24h then adorns people's dialysis tubing with albumen, with renaturation buffer II (pH 8.0 for 50mmol/L Tris-HCl, 0.15mol/L NaCl) dialysis renaturation 12h.Ultrafiltration pipe Centricon 30 (Millipore, USA) protein concentrates.13%SDS-PAGE detects.Protein-20 ℃ preservation.
Embodiment 6:scFv single-chain antibody combines determination of activity with hsBLyS
1) non-competing ELISA method is measured:
10 μ g/ml hsBLyS (NaHCO
3, pH9.6) 4 ℃ of wrapper sheets spend the night.Second day with 1% skimmed milk sealing (37 ℃, 2h).The scFv of serial dilution hatch with hsBLyS respectively (37 ℃, 2h).(Novagen is one anti-USA), and HRP link coupled goat anti-mouse igg is two anti-, and conventional ELISA detects (with reference to Current protocol in immunology unit2.1-2.3) with anti-His monoclonal antibody.Result such as Fig. 4
2) the competitive ELISA method is measured:
10 μ g/ml hsBLyS (NaHCO
3, pH9.6) 4 ℃ of wrapper sheets spend the night.The scFv of serial dilution mixes with ABL-1mAb respectively, hatch with hsBLyS (37 ℃, 2h).Detect scFv with HRP link coupled goat anti-mouse igg and suppress combining of ABL-1mAb and hsBLyS.Result such as Fig. 5, result confirm that ABL-1 scFv and ABL-1 monoclonal antibody are that same epi-position combines hsBLyS.
3) Western blotting measures:
Get hsBLyS 20 μ l, the 13%SDS-PAGE electrophoresis is transferred to cellulose nitrate film, after the 1%BSA sealing, adds scFv and hatches 2h for 37 ℃, and (Novagen is one anti-USA) to anti-His monoclonal antibody, and HRP link coupled goat anti-mouse igg is two anti-, the TMB colour developing.(specifically with reference to Current protocol in immunology unit8.10).Result such as Fig. 6, result show the linear epitope of ABL-1 scFv and ABL-1 monoclonal antibody identification hsBLyS.
Sequence table
＜110〉Nanjing Normal University
＜120〉heavy chain of human B lymphocyte stimulation factor monoclonal antibody and chain variable region gene and application thereof
<160>4
<210>1
<211>342
<212>DNA
＜213〉mouse (Mus)
<400>1
gtgcaactgc?aggagtcagg?aggaggcttg?gtacagcctg?ggggttctct?gagactctcc?60
tgtgcaactt?ctgggttcac?cttcactgat?tactacatga?gctgggtccg?ccagcctcca?120
ggaaaggcac?ttgagtggtt?gggttttatt?agaaacaaag?ctaatggtta?cacaacagag?180
tacagtgcat?ctgtgaaggg?tcggttcacc?atctccagag?ataattccca?aagcatcctc?240
tatcttcaaa?tgaacaccct?gagagctgag?gacagtgcca?cttattactg?tgcaagagat?300
atcacccttc?tgggccaagg?gaccacggtc?accgtctcct?ca?342
<210>2
<211>321
<212>DNA
＜213〉mouse (Mus)
<400>2
gacattgagc?tcacccagtc?tccagcaatc?atgtctgcat?ctctagggga?acgggtcacc?60
atgacctgca?ctgccagctc?aagtgtaagt?tccagttact?tgcactggta?ccagcagaag?120
ccaggatcct?cccccaaact?ctggatttat?agcacatcca?acctggcttc?tggagtccca?180
gctcgcttca?gtggcagtgg?gtctgggacc?tcttactctc?tcacaatcag?cagcatggag?240
gctgaagatg?ctgccactta?ttactgccac?cagtatcatc?gttccccgta?cacgttcgga?300
ggggggacca?agctcgag?318
<210>3
<211>114
<212>PRT
＜213〉mouse (Mus)
<400>3
Val?Gln?Leu?Gln?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Thr?Ser?Gly?Phe?Thr?Phe?Thr?Asp
20 25 30
Tyr?Tyr?Met?Ser?Trp?Val?Arg?Gln?Pro?Pro?Gly?Lys?Ala?Leu?Glu
35 40 45
Trp?Leu?Gly?Phe?Ile?Arg?Asn?Lys?Ala?Asn?Gly?Tyr?Thr?Thr?Glu
50 55 60
Tyr?Ser?Ala?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn
65 70 75
Ser?Gln?Ser?Ile?Leu?Tyr?Leu?Gln?Met?Asn?Thr?Leu?Arg?Ala?Glu
80 85 90
Asp?Ser?Ala?Thr?Tyr?Tyr?Cys?Ala?Arg?Asp?Ile?Thr?Leu?Leu?Gly
95 100 105
Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser
110 114
<210>4
<211>106
<212>PRT
＜213〉mouse (Mus)
<400>4
Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro?Ala?Ile?Met?Ser?Ala?Ser?Leu
1 5 10 15
Gly?Glu?Arg?Val?Thr?Met?Thr?Cys?Thr?Ala?Ser?Ser?Ser?Val?Ser
20 25 30
Ser?Ser?Tyr?Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Ser?Ser?Pro
35 40 45
Lys?Leu?Trp?Ile?Tyr?Ser?Thr?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro
50 55 60
Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr
65 70 75
Ile?Ser?Ser?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?His
80 85 90
Gln?Tyr?His?Arg?Ser?Pro?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu
95 100 105
Claims (5)
1, a kind of bone-marrow-derived lymphocyte stimulating factor scFv antibody fragment is characterized in that, between the heavy chain variable region gene and chain variable region gene of human B lymphocyte stimulation factor monoclonal antibody, is connected with one section flexible amino acid short peptide; Said heavy chain variable region gene has＜400〉1 sequence; Said chain variable region gene has＜400〉2 sequence.
2, a kind of expression vector has the said scFv antibody fragment of claim 1.
3, expression vector as claimed in claim 2 specifically is pET28a-scFv.
4, the polypeptide expressed as the expression vector of claim 2 or 3.
5, the described polypeptide of claim 4 is used for the application of the detection reagent of the relevant autoimmune disorder of BLyS in preparation.
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| CN101851291B (en) * | 2010-02-09 | 2011-10-26 | 中国人民解放军第四军医大学 | Heavy chain and light chain variable regions of anti-human BAFF monoclonal antibody |
| CN104045713B (en) * | 2013-03-13 | 2019-02-12 | 江苏诺迈博生物医药科技有限公司 | The monoclonal antibody of anti-Blys a kind of and pharmaceutical composition containing the antibody |
| CN105061596B (en) * | 2015-08-05 | 2019-01-29 | 江苏诺迈博生物医药科技有限公司 | The monoclonal antibody and its application of human B lymphocyte stimulating factor |
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| CN1308127A (en) * | 2000-11-02 | 2001-08-15 | 南京师范大学 | Expression carrier for recombinant human B lymphocyte stimulating factor and its construction process |
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2005
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308127A (en) * | 2000-11-02 | 2001-08-15 | 南京师范大学 | Expression carrier for recombinant human B lymphocyte stimulating factor and its construction process |
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