CN101240024B - Single-chain antibody scFv of hBAFF and application thereof in producing BAFF human source monoclonal antibody - Google Patents

Abstract

The invention relates to gene engineering field, particularly relates to single-chain antibody scFv of hBAFF, and application thereof in preparation of BAFF human original monoclonal antibody. The aforementioned monoclonal antibody scFv of hBAFF has sequence shown in SEQ ID NO.1. The aforementioned monoclonal antibody scFv of hBAFF is applicable in production of BAFF humanized monoclonal antibody.The invention produce BAFF humanized monoclonal antibody by monoclonal antibody scFv of hBAFF, which has advantages of stable, effective. The invention is reagent for clinical diagnosis aimed at human immune disease (SLE, RA and so on) and gene engineering medicament with potential therapeutic efficacy.

Description

The single-chain antibody scFv of hBAFF and the application in producing BAFF humanized monoclonal antibody thereof

Technical field:

The present invention relates to the genetically engineered field, be specifically related to the single-chain antibody scFv of hBAFF, with and application in BAFF humanized Monoclonal Antibody.

Technical background:

Immune disorder is the main pathogenic factor of numerous disease, can effectively intervene the pathologic immunologic process at the antibody of immunity system important molecule, becomes field very active in the exploitation of therapeutic monoclonal antibody.The human b lymphocyte activator factor (hBAFF) is that newfound a kind of and human immunity in 1999 are regulated and control closely-related cytokine, belongs to tumour necrosis factor (TNF) superfamily.Positive regulating factor as the bone-marrow-derived lymphocyte growth, hBAFF has the differentiation that promotes bone-marrow-derived lymphocyte in vivo, the effect of survival, and too much hBAFF can cause the overactivity of bone-marrow-derived lymphocyte, impel body to produce a large amount of antinuclear antibody, anti-ds-DNA antibody and anti-SSA antibody etc., thereby cause inflammatory reaction, bring out autoimmune disorder.The biologic activity of therefore managing to block BAFF is significant for the treatment of these autoimmune disorders.At present, the preparation of blocking-up hBAFF biological action mainly contains soluble receptors (for the fusion rotein of soluble receptors sTACI and Fc) and anti--hBAFF antibody two classes of hBAFF.The mankind, at SLE, FDA anti--hBAFF antibody the LymphoStat-B of official approval U.S. HGSI company development enters the III phase clinical experiment stage, and from I clinical trial phase data, LymphoStat-B has reduced the CD20 that produces antinuclear antibody in the SLE patient body significantly ⁺The B cell number has greatly improved patient's the state of an illness, and several having no side effect, and simultaneously, LymphoStat-B is also carrying out at the II clinical trial phase of RA.The treatment of autoimmune disease lacks the ideal way so far, cyclosporin A is present widely used immunosuppressor, but its side effect and complication---Toxicity of Kidney, hypertension and hyperlipidemia also allow very headache of hospitalized patients, so LymphoStat-B has shown good commercial application prospect.Preparation also is in the blank stage but China is at present about hBAFF humanized monoclonal antibody.

Prokaryotic expression system lacks glycosylation modified system can't manufacture of therapeutic glycoprotein, cause people's immune response easily and utilize lymphoma to merge the monoclonal antibody of producing, in the eukaryotic cell, Chinese hamster ovary cell (ChineseHamster Ovary Cell, CHO) being the first-selected system of present therapeutic recombinant glycoprotein, is that the U.S. FDA approval is used for one of expression system of pharmaceutical production.Chinese hamster ovary celI has post transcriptional modificaiton function accurately, and the glycosylated protein of expression is approaching the native protein molecule most aspect molecular structure, physicochemical property and the biological function, reduces immunological cross-reaction simultaneously.At present existing increasing pharmaceutical protein has obtained to efficiently express in Chinese hamster ovary celI, and wherein the part medicine is put on market, for example EPO, G-CSF etc.But the output of eukaryotic gene expression is often very low, and the amplification of foreign gene in Chinese hamster ovary celI is to improve one of Critical policies of exogenous gene expression level.Dihydrofolate reductase gene (dhfr) amplification system is the most frequently used.DHFR can be suppressed by the cry of certain animals (MTX) of talking endlessly of folacin ammonia first, improves constantly MTX concentration, and the talk endlessly clone of cry of certain animals of the anti-ammonia first of carrying out property selection, result can cause the coamplification of the foreign gene that is associated in dhfr, and its copy number can increase hundreds of to several thousand times.

Summary of the invention

The single-chain antibody scFv that the purpose of this invention is to provide a kind of hBAFF, with and application in producing BAFF humanized monoclonal antibody.

The cDNA of the single-chain antibody scFv of a kind of hBAFF, its sequence is shown in SEQ ID NO.1.

The cDNA of the single-chain antibody scFv of above-mentioned hBAFF can be applied to produce BAFF humanized monoclonal antibody.

The application of the cDNA of the single-chain antibody scFv of said hBAFF in producing BAFF humanized monoclonal antibody specifically may further comprise the steps:

1) BAFF humanized monoclonal antibody Construction of eukaryotic:

The cDNA of the single-chain antibody scFv of hBAFF is connected synthetic scFv-Fc, the dna sequence dna of said scFv-Fc such as SEQ ID NO.3 with human IgG1 Fc (hinge+CH2+CH3) cDNA sequence;

Utilize the primer extension technology that mouse IgG κ chain signal peptide is combined with scFv-Fc, and change the pcDNA3.0 carrier for expression of eukaryon over to, expression plasmid called after pcDNA3.0/scFv-Fc after the sequence enzyme that obtains cut;

2) stably express of the full humanized's genetic engineering antibody of anti-hsBAFF scFv-Fc

Adopt Chinese hamster ovary cell Tetrahydrofolate dehydrogenase defective strain (CHO/dhfr), liposome method is with expression plasmid pcDNA3.0/scFv-Fc and sign plasmid pSV-dhfr, and cotransfection is gone into the CHO/dhfr cell; Utilize the clone of G418 screening transfection success earlier, then utilize the MEM that does not contain xanthoglobulin and thymus pyrimidine (H.T.) to select nutrient solution, the strain of screening dhfr positive cell clone; Ammonia first cry of certain animals (MTX) concentration gradient of talking endlessly increases afterwards, and pressurization amplification foreign gene improves the scFv-Fc expression amount, cultivates through too much wheel pressurization, screens the cell strain that scFv-Fc stablizes, efficiently expresses;

3) expression and purification of scFv-Fc

Change the high expressing cell strain that obtains over to serum free medium and cultivate production scFv-Fc; Collect supernatant, centrifugal, Protein A affinitive layer purification albumen, again with the albumen collected in 4 ℃ to pure water dialysis 16h, after the filtration sterilization ,-70 ℃ of freezing preservations of packing.

In the present invention, we utilize display technique of bacteriophage to screen the single chain antibody fragment scFv that independent intellectual property right has blocking-up hsBAFF biologic activity from total man's endogenous antibody library, though single-chain antibody has advantages such as molecule is little, penetration power is strong, but a little less than its avidity, half life, lack in vivo, and effector function is poor.Therefore by genetic engineering technique people's IgG antibody 1 constant region fc structural domain is introduced single-chain antibody, not only can make single-chain antibody have two valency antigen binding sites, can give the effector function of single-chain antibody mediation ADCC and CDC again, thereby improve the performance of single-chain antibody, the introducing in Fc effect district makes the genetic engineering antibody molecule that makes up more approach the structure of natural antibody molecule, the Fc district can combine with ProteinA or Protein G, and this has also made things convenient for the separation and purification of antibody molecule.

The signal peptide sequence of mouse Ig κ chain is also adopted in this experiment, has successfully made up carrier for expression of eukaryon pcDNA3.0/scFv-Fc, and with expression plasmid pcDNA3.0/scFv-Fc and sign plasmid pSV-dhfr, cotransfection is gone into the CHO/dhfr cell by liposome method.The cell strain of stablizing, efficiently expressing by continuous administration pressurization screening scFv-Fc, and expression product is carried out purifying identify is for the large-scale production of the full humanized's genetic engineering antibody of hsBAFF scFv-Fc lays the foundation.

The present invention takes the mammalian cell strain expression system, and expressed glycosylated protein is approaching the native protein molecule most aspect molecular structure, physicochemical property and the biological function, reduces immunological cross-reaction simultaneously.And adopt MTX to act on the gene amplification system of dhfr, thereby make rotaring redyeing gene obtain very high-caliber amplification and expression, the cell strain that obtains by long-time screening is grown up in serum free medium and can be produced antibody easily, and is easy to purifying.

Therefore, the present invention utilizes the single-chain antibody scFv of hBAFF to produce BAFF humanized monoclonal antibody, have stable, advantage efficiently, and can be at the reagent for clinical diagnosis of mankind itself's immunological disease (SLE, RA etc.) and the genetically engineered drug of potential result of treatment.

Description of drawings

Fig. 1 is the vector construction synoptic diagram

Fig. 2 is the purity of protein behind SDS-PAGE and the Western blotting detection purifying and whether forms homodimer

Fig. 3 detects scFv-Fc by the ELISA method to discern antigenic specificity

Fig. 4 detects scFv-Fc and the affine efficient of antigen bonded by the ELISA method

Fig. 5 is to be target cell with BAFF positive expression cell K-562, and the serum lactic dehydrogenase method for releasing detects the cytotoxicity (antibody-dependent cellular cytotoxicity) of scFv-Fc mediation

Fig. 6 is that fluidic cell method (FACS) detects combining of scFv-Fc and the human lymphocyte of striding film expression BAFF total length

Fig. 7 is the comparison by the tolerance time in serum behind ELISA method detection scFv and the scFv-Fc intravenous injection mouse

Fig. 8 is that mtt assay compares scFv-Fc vitro inhibition hBAFF to the lymphocytic proliferation function of human peripheral blood B

Embodiment:

Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.

Below in conjunction with specific embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.

In following embodiment, various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moiety person indicate when occurring first that all used thereafter identical reagent if no special instructions, and is all identical with the content of indicating first.

In following examples, pcDNA3.0 is available from Invitrogene company; Chinese hamster ovary cell Tetrahydrofolate dehydrogenase defective strain (CHO/dhfr) and plasmid pSV-dhfr are presented by doctor Hu Yunlong of Nanjing Chuanbo Biotechnology Co; The sequence of said human IgG1 Fc (hinge+CH2+CH3) cDNA such as SEQ ID NO.2.

Embodiment one: production, purifying and the evaluation of BAFF humanized monoclonal antibody

1.BAFF humanized's monoclonal antibody Construction of eukaryotic:

The structure of carrier as shown in Figure 1, by of the gene order amalgamation of over-lap round pcr with scFv and Fc; Utilize the primer extension round pcr, mouse Ig κ signal peptide is connected the aminoterminal of scFv-Fc sequence by three PCR; Again goal gene and pcDNA3.0 plasmid being carried out enzyme with Hind III and EcoRV restriction endonuclease respectively cuts; Enzyme is cut product connect, with the plasmid called after pcDNA3.0/scFv-Fc that builds with the T4 ligase enzyme.Specifically:

1.1 cDNA and the following primer of human IgG1 Fc (hinge+CH2+CH3) cDNA sequences Design according to the single-chain antibody scFv of the hBAFF by Phage Display technology screening:

PscFv1：5’GCG?GAG?GTG?CAG?CTG?GTG?3’

PscFv2：5’

TGC?GGC?CGC?3’

PFc1：5’ AAA?ACT?CAC?AC?3’

PFc2：5’GAA?GCT? GAT?ATC?TTA?TTT?ACC?CGG?GGA?C?3’

Wherein PscFv2 and PFc1 italicized item are complementary region, PFc2 contain by EcoRVRestriction enzyme site.Utilize overlapping extension splicing to synthesize scFv-Fc.

1.2 according to mouse IgG κ chain and the following primer of scFv-Fc sequences Design:

HindIII Kozak Ig κ signal peptide

pIgκ1：5’AGG?AAC? AAG?CTT? ACC?ATG?

GTA?CTG?CTG?3’

Ig κ signal peptide

pIgκ2：5’? 3’

Ig κ signal peptide and scFv complementary region

pIgκ3：5’

GCG ?GCG?GAG?GTG?CAG?CTG GTG?3’

Wherein pIg κ 1, and pIg κ 2 and pIg κ 3 italicized items are signal peptide, pIg κ 1 contain by HindIIIRestriction enzyme site and Kozak sequence, pIg κ 3 contain by with the scFv complementary region.Utilize after the primer extension technology composite signal peptide, change the pcDNA3.0 carrier for expression of eukaryon over to, expression plasmid called after pcDNA3.0/scFv-Fc after signal peptide being combined with scFv-Fc and the sequence enzyme that obtains cut by the lap splice technology.

2. the stably express of the full humanized's genetic engineering antibody of anti-hsBAFF scFv-Fc

Adopt Chinese hamster ovary cell Tetrahydrofolate dehydrogenase defective strain (CHO/dhfr).Liposome method is with expression plasmid pcDNA3.0/scFv-Fc and sign plasmid pSV-dhfr, and cotransfection is gone into the CHO/dhfr cell; Utilize the clone of G418 screening transfection success earlier, then utilize the MEM that does not contain xanthoglobulin and thymus pyrimidine (H.T.) to select nutrient solution, the strain of screening dhfr positive cell clone; ELISA detects the cell strain of selecting high expression level, continues to cultivate, and abandons low cell strain of expressing.By the time transfectional cell covers with flat board, be inoculated in new Tissue Culture Dish at 1: 6, add and depress at lower concentration MTX (0.0005 μ M), treat that the resistant gene clone forms, illustrate that intracellular dhff gene has obtained expression, cell covers with flat board by the time, according to being inoculated in new Tissue Culture Dish at 1: 6, improve MTX concentration (0.002 μ M) and carry out next round pressurization screening, so take turns screening, be increased to 20 μ M up to MTX through 7.Obtain the cell strain that scFv-Fc stablizes, efficiently expresses.

3. the purifying of expression product

After the long-time screening of process, change the high expressing cell strain that obtains over to serum free medium (CHO-S-SFM II) and shake production scFv-Fc in the bottle (spinner flask) in trace control.Supernatant after about 1L cultivates is collected back centrifugal (13000rpm, 4 ℃ of 20min) to remove cell debris.Regulate supernatant liquor with 1MNaOH, make the pH value about 7.5; Utilize Biologic LP chromatographic system, supernatant liquor with sample on the 0.5ml/min flow velocity to the rProtein A Sepharose Fast Flow affinity column of binding buffer liquid (1M Tris-HCl, pH 7.5) pre-equilibration (0.8cm, 2cm); Wash with the 0.5ml/min flow velocity with binding buffer liquid, to effluent liquid OD ₂₈₀Value arrives baseline; Receiving sample stays with electrophoresis; With elution buffer (0.05M Gly-HCl, pH 3.0) with 0.5ml/min flow velocity wash-out antibody, to effluent liquid OD ₂₈₀Value arrives baseline, and every 1ml is as a pipe, and substep is collected; The antibody-solutions of using 0.5ml neutralization buffer (1MTris-HCl, pH 8.8) to collect immediately for the solution neutralization; With the target protein collected in 4 ℃ to pure water dialysis 16h, filtration sterilization ,-70 ℃ of freezing preservations of packing.

4.SDS-PAGE and western blot identifies

10 μ l samples are added respectively that equivalent contains or do not contain 2 * sample-loading buffer of beta-mercaptoethanol, constant voltage 100V carries out 12%SDS-PAGE, and coomassie brilliant blue R250 dyeing shows band analysis.

Same sample is behind the 12%SDS-PAGE electrophoresis, be transferred to cellulose nitrate film, after the sealing of 5% skim-milk, add HRP link coupled goat anti-human igg 1Fc, hatch 1h for 37 ℃, TMB develops the color (specifically with reference to Current protocolin immunology unit 8.10). and the result shows that scFv-Fc is about 55kDa at molecular weight under the reductive condition, and forms the dimer (figure .2) that is similar to natural antibody by disulfide linkage under non-reduced condition.

5. the biologic activity of the scFv-Fc of purifying is identified

5.1. use 0.02M, the carbonate bag of pH9.6 is cushioned liquid and induces the outer solvable district segment (hsAPRIL) of propagation part born of the same parents, the outer solvable district segment (msBAFF) of mouse bone-marrow-derived lymphocyte incitant born of the same parents and outer solvable district sheet (hsBAFF) protein series of human b lymphocyte activator factor born of the same parents to be diluted to 10 μ g/ml, 1 μ g/ml, 0.1 μ g/ml and 0.05 μ g/ml bovine serum albumin (BSA), people, in the reacting hole of each polystyrene board, add 100 μ l, each sample repeats three holes, and 4 ℃ of bags are spent the night.Discard solution in the hole next day, washes 3 times with lavation buffer solution, each 3min; Add 200 μ l with the every hole of 5% skimmed milk, 37 ℃ of sealing 2h; Add the goat anti-human igg 1Fc polyclonal antibody of the HRP enzyme labelling of 100 μ l dilution in 1: 1000, put 37 ℃ and hatched 1 hour, then washing; The mouse-anti His that adds the HRP enzyme labelling of dilution in 1: 1000 ₆The goat anti-human igg Fc polyclonal antibody (identification scFv-Fc and human IgG1) of the monoclonal antibody of tag (identification scFv) or HRP enzyme labelling is put 37 ℃ and was hatched 1 hour, then washing; The OPD substrate solution 100 μ l that in each reacting hole, add interim preparation, room temperature reaction 20 minutes; In each reacting hole, add 2M sulfuric acid 50 μ l; 490nm reading on the ELISA detector is surveyed each hole OD value (Fig. 3) with zeroing back, blank hole.Result's demonstration, along with the rising of envelope antigen hsBAFF concentration, the increase of OD value dose-dependently, even antigen concentration reaches 10 μ g/ml, scFv-Fc and BSA, cross reaction still can not take place in hsAPRIL and msBAFF.These results show that scFv-Fc has specific antigen recognition ability.

5.2 be cushioned liquid with the carbonate bag hsBAFF albumen is diluted to two series: series 1 is 10 μ g/ml for fixing hsBAFF protein concentration, and serial 2 are respectively 0.04 μ g/ml, 0.12 μ g/ml, 0.4 μ g/ml, 1.2 μ g/ml, 3.6 μ g/ml, 11 μ g/ml, 33 μ g/ml, 100 μ g/ml for hsBAFF albumen gradient is diluted to protein content.Detect the affine efficient that the identical step of scFv-Fc antigen recognition specificity is identified antibody with the ELISA method.The result is shown in Fig. 4 A, and along with the gradient rising of scFv-Fc antibody concentration, the OD value is the increase of dose-dependently, even but negative control antibody human IgG1 concentration reaches 100 μ g/ml, and hsBAFF reacts also with it and is negative; Wrap simultaneously by a series of antigen concentrations, sessile antibody concentration, the result is shown in Fig.4B, gradient rising along with hsBAFF concentration, the OD value is the increase of dose-dependently also, even but concentration reaches 100ug/ml, and hsBAFF and negative control antibody human IgG1's reaction also is negative.These data show the single-chain antibody than scFv, and scFv-Fc can better recognition hsBAFF antigen.

Contain in the 10% calf serum RPMI-1640 5.3K-562 the human lymphoma cell is resuspended in, transfer cell concn to 1 * 10 ⁵/ ml is standby target cell.Utilize the monocyte action effect cell in the lymphocyte separation medium separation of human peripheral blood.Different concns effector cell and each 100 μ l of target cell suspension are added in the 96 porocyte culture plates, the scFv-Fc solution that adds 50 μ l different concns simultaneously, every increment is originally established 3 repeating holes, establish target cell simultaneously and discharge contrast and maximum release contrast naturally, utilize the serum lactic dehydrogenase method for releasing, select 570nm at EL _XMeasure photoabsorption A (absorbance) value on the 800 type microplate reader, and calculate killing-efficiency.Calculation formula: killing-efficiency (%)=(experimental group A value-release contrasts the A value naturally)/(value-release contrasts the A value to maximum release contrast A naturally) * 100.The result as shown in Figure 5, the Fc segment of recombination fusion protein scFv-Fc has demonstrated the cytotoxicity (antibody-dependent cellular cytotoxicity) that the antibody that mediated by the Fc fragment similarly with natural antibody relies on

5.4 with 5 * 10 ⁵After/ml K-562 cell cleans 2 times with PBS, adding 2 μ g scFv-Fc hatched 1 hour, PBS washs the goat anti-human igg Fc monoclonal antibody that adds the FITC mark after 2 times, by combine (the figure .6) of flow cytometer detection scFv-Fc with the human lymphocyte of striding film expression BAFF total length.The result show scFv-Fc can with the combining of the human lymphocyte of striding film expression BAFF total length.

5.5 16 male ICR mouses are divided into two groups, every group 8 respectively tail vein injection scFv and scFv-Fc, respectively at taking blood sample in 3,6,24,48 and 72 hours, detect the comparison (Fig. 7) of the tolerance time in serum behind scFv and the scFv-Fc intravenous injection mouse by above-described ELISA method.The result shows that scFv just can't be detected in injection in back 24 hours in mice serum, and scFv-Fc still can be detected in injection in back 72 hours.We scFv-Fc of this results suggest has prolonged the tolerance time of antibody in mice serum greatly.

5.6 utilize lymphocyte separation medium separation of human peripheral blood lymphocyte, separate bone-marrow-derived lymphocyte with the magnetic bead that has CD19 antibody afterwards, then with RPMI 1640 diluting cells to 2 * 10 of containing 10% calf serum ⁵/ ml makes cell suspension.Obtained cell suspension adds the every hole 100 μ l of 96 porocyte culture plates, Tissue Culture Plate is put into 37 ℃ subsequently, contains 5%CO ₂Cell culture incubator in adaptability cultivate 4h.The cell that adaptability is cultivated after the 4h is divided into 9 groups, every group 3 hole, and this 8 groups is provided with is as follows:

1 control group

2hsBAFF (2 μ g/ml) group

3anti-IgM (2 μ g/ml) group

4hsBAFF (2 μ g/ml)+anti-IgM (2 μ g/ml) group

5hsBAFF (2 μ g/ml)+anti-IgM (2 μ g/ml)+hIgG 1 (10 μ g/ml) group

6hsBAFF (2 μ g/ml)+anti-IgM (2 μ g/ml)+scFv-Fc (0.5 μ g/ml) group

7hsBAFF (2 μ g/ml)+anti-IgM (2 μ g/ml)+scFv-Fc (1 μ g/ml) group

8hsBAFF (2 μ g/ml)+anti-IgM (2 μ g/ml)+scFv-Fc (5 μ g/ml) group

9hsBAFF (2 μ g/ml)+anti-IgM (2 μ g/ml)+scFv-Fc (10 μ g/ml) group

The hsBAFF and the antibody of corresponding dosage are added in the Tissue Culture Plate, Tissue Culture Plate is put into 37 ℃, contained 5%CO ₂Cell culture incubator in cultivate after the 72h, every hole adds 5mg/ml MTT solution 10 μ l, hatches 4h for 37 ℃ then, every then hole adds DMSO 100 μ l dissolution precipitation things.Select 570nm at EL _XMeasure photoabsorption A value on the 800 type microplate reader.Result such as Fig. 8, hsBAFF can breed by the intense stimulus bone-marrow-derived lymphocyte under the anti-IgM coordinative role, if after adding the scFv-Fc of a series of gradient dilutions simultaneously, being suppressed of bone-marrow-derived lymphocyte propagation gradient dependent form, when especially scFv-Fc reaches 2 μ g/ml, the inhibition degree is the highest, even and human IgG1's concentration can not suppress bone-marrow-derived lymphocyte propagation at 10 μ g/ml.

SEQUENCE?LISTING

＜110〉Nanjing Normal University

＜120〉the single-chain antibody scFv of hBAFF and the application in producing BAFF humanized monoclonal antibody thereof

<160>3

<210>1

<211>723

<212>cDNA

＜213〉people (Homo)

<400>1

gcggcggagg?tgcagctggt?ggagtctggg?ggaggcttgg?tacagcctgg?ggggtccctg 60

agactctcct?gtgcaggctc?tggattcacc?ttcagtagct?atgctatgca?ctgggttcgc?120

caggctccag?gaaaaggtct?ggagtgggta?tcagctattg?gtactggtgg?tggcacatac?180

tatgcagact?ccgtgaaggg?ccgattcacc?acctccagag?acaatgccaa?gaattccttg?240

tatcttcaaa?tgaacagcct?gagagccgag?gacacggccg?tgtattactg?tgcaagaaag?300

atttctgagc?cttggggcca?aggtaccctg?gtcaccgtct?cgagtggtgg?aggcggttca?360

ggcggaggtg?gctctggcgg?tagtgcactt?tcttctgagc?tgactcagga?ccctgctgtg?420

tctgtggcct?tgggacagac?agtcaggatc?acatgccaag?gagacagcct?cagaagctat?480

tatgcaagct?ggtaccagca?gaagccagga?caggcccctg?tacttgtcat?ctatggtaaa?540

aacaaccggc?cctcagggat?cccagaccga?ttctctggct?ccagctcagg?aaacacagct?600

tccttgacca?tcactggggc?tcaggcggaa?gatgaggctg?actattactg?taactcccgg?660

gacagcagtg?ataaccatgt?attcggcgga?gggaccaagc?tgaccgtcct?aggtgcggcc?720

gct 723

<210>2

<211>696

<212>cDNA

＜213〉people (Homo)

<400>2

cccaaatctt?gtgacaaaac?tcacacatgc?ccaccgtgcc?cagcacctga?actcctgggg 60

ggaccgtcag?tcttcctctt?ccccccaaaa?cccaaggaca?ccctcatgat?ctcccggacc?120

cctgaggtca?catgcgtggt?ggtggacgtg?agccacgaag?accctgaggt?caagttcaac?180

tggtacgtgg?acggcgtgga?ggtgcataat?gccaagacaa?agccgcggga?ggagcagtac?240

aacagcacgt?accgtgtggt?cagcgtcctc?accgtcctgc?accaggactg?gctgaatggc?300

aaggagtaca?agtgcaaggt?ctccaacaaa?gccctcccag?cccccatcga?gaaaaccatc?360

tccaaagcca?aagggcagcc?ccgagaacca?caggtgtaca?ccctgccccc?atcccgggag?420

gagatgacca?agaaccaggt?cagcctgacc?tgcctggtca?aaggcttcta?tcccagcgac?480

atcgccgtgg?agtgggagag?caatgggcag?ccggagaaca?actacaagac?cacgcctccc?540

gtgctggact?ccgacggctc?cttcttcctc?tatagcaagc?tcaccatgga?caagagcagg?600

tggcagcagg?ggaacgtctt?ctcatgctcc?gtgatgcatg?aggctctgca?caaccactac?660

acgcagaaga?gcctctccct?gtctccaggt?aaataa 696

<210>3

<211>1419

<212>cDNA

＜213〉artificial sequence

<400>3

gcggcggagg?tgcagctggt?ggagtctggg?ggaggcttgg?tacagcctgg?ggggtccctg 60

agactctcct?gtgcaggctc?tggattcacc?ttcagtagct?atgctatgca?ctgggttcgc 120

caggctccag?gaaaaggtct?ggagtgggta?tcagctattg?gtactggtgg?tggcacatac 180

tatgcagact?ccgtgaaggg?ccgattcacc?acctccagag?acaatgccaa?gaattccttg 240

tatcttcaaa?tgaacagcct?gagagccgag?gacacggccg?tgtattactg?tgcaagaaag 300

atttctgagc?cttggggcca?aggtaccctg?gtcaccgtct?cgagtggtgg?aggcggttca 360

ggcggaggtg?gctctggcgg?tagtgcactt?tcttctgagc?tgactcagga?ccctgctgtg 420

tctgtggcct?tgggacagac?agtcaggatc?acatgccaag?gagacagcct?cagaagctat 480

tatgcaagct?ggtaccagca?gaagccagga?caggcccctg?tacttgtcat?ctatggtaaa 540

aacaaccggc?cctcagggat?cccagaccga?ttctctggct?ccagctcagg?aaacacagct 600

tccttgacca?tcactggggc?tcaggcggaa?gatgaggctg?actattactg?taactcccgg 660

gacagcagtg?ataaccatgt?attcggcgga?gggaccaagc?tgaccgtcct?aggtgcggcc 720

gctcccaaat?cttgtgacaa?aactcacaca?tgcccaccgt?gcccagcacc?tgaactcctg 780

gggggaccgt?cagtcttcct?cttcccccca?aaacccaagg?acaccctcat?gatctcccgg 840

acccctgagg?tcacatgcgt?ggtggtggac?gtgagccacg?aagaccctga?ggtcaagttc 900

aactggtacg?tggacggcgt?ggaggtgcat?aatgccaaga?caaagccgcg?ggaggagcag 960

tacaacagca?cgtaccgtgt?ggtcagcgtc?ctcaccgtcc?tgcaccagga?ctggctgaat?1020

ggcaaggagt?acaagtgcaa?ggtctccaac?aaagccctcc?cagcccccat?cgagaaaacc?1080

atctccaaag?ccaaagggca?gccccgagaa?ccacaggtgt?acaccctgcc?cccatcccgg?1140

gaggagatga?ccaagaacca?ggtcagcctg?acctgcctgg?tcaaaggctt?ctatcccagc?1200

gacatcgccg?tggagtggga?gagcaatggg?cagccggaga?acaactacaa?gaccacgcct?1260

cccgtgctgg?actccgacgg?ctccttcttc?ctctatagca?agctcaccat?ggacaagagc?1320

aggtggcagc?aggggaacgt?cttctcatgc?tccgtgatgc?atgaggctct?gcacaaccac?1380

tacacgcaga?agagcctctc?cctgtctcca?ggtaaataa 1419

Claims (4)

1. the cDNA of the single-chain antibody scFv of a human b lymphocyte activator factor is characterized in that, its sequence is shown in SEQ ID NO.1.

2. the application of the cDNA of the single-chain antibody scFv of the said human b lymphocyte activator factor of claim 1 in producing BAFF humanized monoclonal antibody.

3. according to the said application of claim 2, it is characterized in that, may further comprise the steps:

(1) BAFF humanized monoclonal antibody Construction of eukaryotic:

The cDNA of the single-chain antibody scFv of hBAFF is connected synthetic scFv-Fc, the dna sequence dna of said scFv-Fc such as SEQ ID NO.3 with human IgG1 Fc (hinge+CH2+CH3) cDNA sequence;

Utilize the primer extension technology that mouse IgG κ chain signal peptide is combined with scFv-Fc, and change the pcDNA3.0 carrier for expression of eukaryon over to, expression plasmid called after pcDNA3.0/scFv-Fc after the sequence enzyme that obtains cut;

(2) stably express of the full humanized's genetic engineering antibody of anti-hsBAFF scFv-Fc

Adopt Chinese hamster ovary cell Tetrahydrofolate dehydrogenase defective strain CHO/dhfr, liposome method is with expression plasmid pcDNA3.0/scFv-Fc and sign plasmid pSV-dhfr, and cotransfection is gone into the CHO/dhfr cell; Utilize the clone of G418 screening transfection success earlier, then utilize the MEM that does not contain xanthoglobulin and thymus pyrimidine to select nutrient solution, the strain of screening dhfr positive cell clone; The ammonia first cry of certain animals concentration gradient of talking endlessly increases afterwards, and pressurization amplification foreign gene improves the scFv-Fc expression amount, cultivates through too much wheel pressurization, screens the cell strain that scFv-Fc stablizes, efficiently expresses;

(3) expression and purification of scFv-Fc

Change the high expressing cell strain that obtains over to serum free medium and cultivate production scFv-Fc; Collect supernatant, centrifugal, Protein A affinitive layer purification albumen, again with the albumen collected in 4 ℃ to pure water dialysis 16h, after the filtration sterilization ,-70 ℃ of freezing preservations of packing.

4. according to the said application of claim 3, it is characterized in that, said step (1) specifically:

CDNA and the following primer of human IgG1 Fc (hinge+CH2+CH3) cDNA sequences Design according to single-chain antibody scFv:

PscFv1：5’GCG?GAG?GTG?CAG?CTG?GTG?3’

PscFv2：5’

TGC?GGC?CGC?3’

PFc1：5’

AAAACT?CACAC?3’

PFc2：5’GAA?GCT? GAT?ATC?TTA?TTT?ACC?CGG?GGAC?3’

Wherein PscFv2 and PFc1 italicized item are complementary region, PFc2 contain by EcoRVRestriction enzyme site utilizes overlapping extension splicing to synthesize scFv-Fc;

According to mouse IgG κ chain and the following primer of scFv-Fc sequences Design:

HindIII Kozak Ig κ signal peptide

pIgκ1：5’AGG? AAC?AAG?CTT?ACC?ATG

Ig κ signal peptide

pIgκ2：5’

Ig κ signal peptide and scFv complementary region

pIgκ3：5’

GCG?GCG?GAG?GTG?CAG?CTG GTG?3’

Wherein pIg κ 1, and pIg κ 2 and pIg κ 3 italicized items are signal peptide, pIg κ 1 contain by HindIIIRestriction enzyme site and Kozak sequence, pIg κ 3 contain by with the scFv complementary region; Utilize after the primer extension technology composite signal peptide, change the pcDNA3.0 carrier for expression of eukaryon over to, expression plasmid called after pcDNA3.0/scFv-Fc after signal peptide being combined with scFv-Fc and the sequence enzyme that obtains cut by the lap splice technology.

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