CN1164616C - Recombinant hepatitis B virus immune globulin - Google Patents
Recombinant hepatitis B virus immune globulin Download PDFInfo
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Abstract
The present invention relates to a high-technology biological engineering product comprising three kinds of hepatitis b high efficiency immune globulins (hepatitis b antibodies) in different structures, namely anti-hepatitis b virus single-chain antibodies, antibodies Fab and full molecule antibodies. The present invention is mainly characterized in that (1), the antibodies can be specifically combined with hepatitis b virus surface antigens and can perform the functions of preventing and treating hepatitis b in an auxiliary mode by neutralizing hepatitis b viruses; (2), the antibodies belong to human source proteins, and the antibodies can be directly applied to human bodies and can not cause the immunologic rejection of organisms; (3), the antibodies belong to genetic engineering products and not only avoid the easy pathogeny propagation danger of blood source products, but also have the advantages of stable gene and easy large-scale production; (4), the antibodies of the present invention, and the derivatives thereof can be used for preparing reagent boxes used for detecting hepatitis b virus surface antigens for diagnosing hepatitis b.
Description
The invention belongs to the biological gene engineering pharmaceutical field, concrete method is to utilize people's gene engineered antibody storehouse technology, the specific immunoglobulin for preparing a kind of hepatitis B virus, relate to 28kDa human single chain variable fragments antibody FV, the full molecular antibody of 52kDa antibody passage Fab and 150kDa, and their application in preparation treatment, prevention, hepatitis B virus medicine and diagnostic kit.
Hepatitis B is the worldwide disease that is caused by hepatitis B virus (HBV), and the number of hepatitis B surface antigen (HBsAg) is carried above 2.8 hundred million in the whole world according to statistics.China is the high popular area of hepatitis B, has the crowd of 40-60% infected by HBV, has 1.2 hundred million people to carry hepatitis B virus surface antigen approximately.The pregnant and lying-in women that carry hepatitis B virus can pass to the newborn infant with virus.And life more the easy more formation of early infection hepatitis B virus continue the carrier, not only can cause chronic hepatopathy, wherein a part can also develop into liver cirrhosis and liver cancer.
The prevention hepatitis B has two kinds of biological products: a kind of is Hepatitis B virus vaccine, and active immunity can be provided, the prevention before and after being used to contact.Another kind is hepatitis B high titre immunoglobulin (HBIG), and temporary transient passive protection effect is provided, and is used for some contact back crowd's prevention.Single is 50-75% with Hepatitis B virus vaccine to the blocking-up rate of mother-to-baby transmission, and with HBIG and Hepatitis B virus vaccine combined immunization, mother-to-baby transmission blocking-up rate is reached more than 97%.In addition, HBIG can also be used to the transfuse blood prevention of back hepatitis B and the urgent prevention of mishap, also obtains effect preferably with HBIG treatment hepatitis B clinically.In a word, HBIG is a kind of important goods that prevent hepatitis B.It is sure in the effect aspect hepatitis b precaution and the passive immunotherapy.
Both at home and abroad the used HBIG overwhelming majority comes from and gathers high price blood plasma or serum separation and Extraction immunoglobulin (Ig) is made behind the healthy people of hepatitis b vaccine immune for a long time, belongs to blood products.Because passing disease, blood finds that constantly big area uses HBIG to exist certain potentially dangerous in recent years.After ministry of Health of China gate inhibition in 1996 only uses the blood products of HBIG, lack proper prophylactic methods for the prevention of blood transfusion back hepatitis B and the urgent prevention of mishap at present.
The present invention under the situation of this market in urgent need, develops efficient recombination immunoglobulin just.Ziren source hbv antibody was compared with blood products in this complete minute, and its characteristics show the following aspects: (1) does not need immune serum, had solved the problem that is restricted owing to the shortage of immune serum source; (2) avoided blood products HBIG through the blood risk of disease transmission; (3) chemical nature of the present invention belongs to people's source protein matter, can directly apply to human body and does not cause the immunological rejection of body; Be adapted to large-scale industrial production, can improve output, reduce cost; (4) the present invention and derivative thereof can prepare the reagent that detects hepatitis B surface antigen and close, and are used for the diagnosis of hepatitis B.
The present invention utilizes antibody gene library and genetic engineering antibody technology, the efficient immunoglobulin (Ig) of developing of multi-form anti-hepatitis B virus surface antigen, i.e. human source gene engineering single-chain antibody, monoclonal antibody and full molecular antibody.These antibody all have different in molecular structure, molecular weight and function aspects.Single-chain antibody is made up of the variable region of heavy chain and the variable region of light chain of antibody, and molecular weight is 28kDa, has the functional zone of a conjugated antigen.Because its molecular weight is little, is convenient to be connected to form bifunctional molecule with other functional molecular, is used for the diagnosis and the treatment of hepatitis B.Monoclonal antibody is made up of the variable region of heavy chain of antibody and first constant region and light chain, and molecular weight is 52kDa, has all features of single-chain antibody.But, to compare with single-chain antibody, monoclonal antibody has higher avidity and stability.Full molecular immune sphaeroprotein is by two heavy chains and two complete antibodies that light chain is formed, and molecular weight is 150kDa, contains two antigen combined function districts and an effector function district.This antibody is compared with above-mentioned two kinds, and its feature shows as (1) avidity and stability is best; (2) not only can specific recognition antigen, and can bring out complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity effect (ADCC); (3) stability of molecule is good, long half time in the body.Yet the common feature of these antibody is that the biochemical property of (1) antibody belongs to people's source protein matter.When they are applied to should not produce untoward reaction to human body when clinical; (2) all have specific recognition also efficiently in conjunction with hepatitis B virus surface antigen, and any cross reaction does not take place with the human body vitals; (3) all belong to gene engineering product, have good stability, be easy to preserve, be adapted to the advantage of large-scale industrial production.In addition, also avoided through the pathophorous potentially dangerous of blood; (4) antibody and derivative thereof and conjugate can both prepare the hepatitis B surface antigen detection reagent and close, and are used for the diagnosis of hepatitis B.
Concrete technical scheme of the present invention is: at first make up hepatitis B virus specific human antibodies storehouse.From the human peripheral lymphocyte of accepting hepatitis b vaccine immune, extract mRNA.Reverse transcription becomes cDNA first chain.Design one cover nucleic acid primer (Antibody Engineering, edited by Carl A.K.Borrebaeck, 1995 New York Oxford) increases with PCR method, and people's antibody is light, the heavy chain variable region gene fragment.By one section coding (Gly
4Ser)
3Dna sequence dna V
HAnd V
LCouple together, constitute V
H-Linker-V
LFusion gene is inserted into this fusion gene in the phasmid expression vector, and with the bacteriophage coat protein gene fusion, ehec infection also makes antibody fragment express the surface that is showed in phage, forms phage people antibody library.(HbsAg) is target antigen with hepatitis B virus surface antigen, adopts immune affine screening antagonist storehouse to carry out multi-turns screen, therefrom obtains the high-affinity single-chain antibody of specific combination HBsAg.
On the basis of single-chain antibody, light, heavy chain variable region gene fragment are cloned into one respectively and are contained in people's antibody constant region expression carrier, transfection mammalian cell (CHO), the engineering cell of screening expressing antibodies in selective medium, and with dilution method engineering cell is further cloned the mono-clonal engineering cell strain of screening high expression level.The expressing antibodies amount is about 10 μ g/ml.With ProteinA-Sepharose affinity chromatography antibody purification from culture supernatant, the purifying rate reaches more than 98%.
The preparation of monoclonal antibody is directly to utilize a cover primer (Antibody Engineering, edited byCarl A.K.Borrebaeck, 1995 New York Oxford), with immune lymphocyte cDNA is template, use pcr amplification light chain of antibody gene and heavy chain Fd (antibody heavy chain variable region and constant region CH1) gene respectively, and two gene clones to the pComb3H expression vector, form the Fab-pCom3H recombinant plasmid.The latter changes intestinal bacteria over to, with containing penbritin selective medium screening reorganization bacterium.Induce the reorganization bacterium to express soluble antibody Fab with IPTG.Separation and purification monoclonal antibody from born of the same parents' pericentral siphon.
Identify the immunocompetence and the biological activity of various antibody with ELISA and immune marking method.Experimental result shows the equal specific combination hepatitis B virus surface antigen of various antibody, and with human normal tissue (after one's own heart, liver, spleen, lung, kidney) any cross reaction does not take place.
The animal toxicity experimental result shows, no matter is single-chain antibody, monoclonal antibody, or full molecular antibody abdominal injection BALB/c mouse, and every injected in mice 1 personal dosage (200 international unit) was observed 7 days continuously, and any abnormal response does not take place mouse.This experimental result points out the experiment of this product safety qualified.
Embodiment one
The development of HBV SCFV
1. gather hepatitis b vaccine immune person peripheral blood lymphocyte, get blood 120ml behind booster immunization 2 HbsAg vaccines of injection (Beijing Biological Product Inst.'s production), the titre of antibody reaches 1: 900.With lymphocyte layering liquid (medical courses in general institute Tianjin blood grind produce) isolated lymphocytes.With quick preparation, purified mRNA test kit (Promega) isolation and purification mRNA from cell.MRNA with purifying is a template, and the first chain cDNA is synthesized in reverse transcription.
2. with cDNA template with PCR method, the amplification antibody variable gene.In the PCR reaction system, add a cover light chain or a variable region of heavy chain primer respectively, 10 * PCR damping fluid, 5 μ l, the dNTP final concentration is 2.5mM, mixing is after 100 ℃ of sex change 5min add 2 Taq of unit archaeal dna polymerases.Total reaction volume is 50 μ l.Add mineral oil behind the mixing.Carry out 30 circulating reactions, each round-robin condition is: 94 ℃ of sex change 30s, and 55 ℃ of annealing 90s, 72 ℃ are extended 90s, and reaction proceeds to last circulation back and be incubated 10min in 72 ℃.After reaction finishes, from heavy, variable region of light chain PCR reaction system, take out 3 μ l respectively and walk 1.5% agarose gel electrophoresis, rest part Sephagles
TMBandprep Kit reclaims.
3. the assembling of single-chain antibody gene and amplification: the V of recovery
HAnd V
LGene fragment be connected primer wait mole or near etc. under the volumetric molar concentration condition, add 2.5mM dNTP and 5 TaqDNA of unit polysaccharases, 10 * PCR damping fluid, 2.5 μ l, 25mM MgCl
22.5 μ l, reaction volume are 25 μ l, with carrying out 7 anneal cycles after the Witco 70 sealing, each round-robin reaction conditions is 94 ℃ of sex change 30 seconds, anneals 4 minutes for 64 ℃.Be assembled into V
H-linker-V
LSingle-chain antibody (scFv) gene, and as template, in above-mentioned reaction volume, add a pair of 5 ' end and contain Sfi I, 3 ' end contains the primer of Not I restriction enzyme site, carries out 30 PCR circulations, each round-robin condition is 94 ℃ of sex change 1 minute, annealed 2 minutes for 55 ℃, 72 ℃ were extended 2 minutes, and last circulation back is incubated 10 minutes at 72 ℃.Take out 3 μ l and walk 1.2% agarose gel electrophoresis from scFv gene PCR amplification reaction system, rest part reclaims with above-mentioned recovery test kit.
4. the foundation in single-chain antibody gene storehouse: the scFv gene after the recovery connects transformed into escherichia coli TG1 competent cell through the plasmid pCANTAB5E of double digestion respectively.In nutrient solution 2 * YT-G (1.7%Bacto-trypone, 1%Bacto-yeast extract, 0.5%NaCl, 2% glucose), cultivate transformed bacteria.Small portion (about 1/10 volume) transformed bacteria is stored in-70 ℃ with 13% glycerine.
5. the expression of recombinant phages antibody and immune affine screening: remaining transformed bacteria dilutes with 10ml2 * YT-G, and is cultured to OD at 37 ℃
600Value is 0.5.Adding penbritin to final concentration then is 100 μ g/ml, M13KO7 to final concentration be 2 * 10
9Pfu/ml cultivated 1 hour for 37 ℃, and is centrifugal.Bacterial sediment is resuspended in 10ml 2 * YT-AK, and (contain in 2 * YT) nutrient solutions of 100 μ g/ml penbritins and 50 μ g/ml kantlex, in 37 ℃ of overnight incubation, centrifugal collection contains the supernatant of recombinant phage.
The supernatant that will contain recombinant phage be coated on HBsAg antigen one on the culture dish and arise from 37 ℃ and hatched 1 hour.With PBST (PBS that contains 0.05%Tween 20) wash dish 20 times, PBS wash dish 20 times.Add the recombinant phages antibody of 100mM triethylamine 1ml elution of bound on plate, use 1M Tris-HCl immediately, the pH9 neutralization is also collected the recombinant phage that elutes.Repeating above-mentioned infection-amplification-screening process 3 takes turns.
5. the preparation of recombinant phages antibody and evaluation: get the recombinant phages antibody 100 μ l that third round elutes and the TG1 cell 200 μ l of logarithmic phase, cultivated 30 minutes in 37 ℃ of shaking tables behind the mixing, cells infected was done doubling dilution (1: 10 with 2 * YT, 1: 100,1: 1000) after, be coated on SOBAG (2%Bacto-trypone, 0.5%Bacto-yeast extract, 0.05%NaCl, 10mMMgCl
2, 2% glucose, 100 μ g/ml penbritins, 1.5%Bacto-Agar) on the solid medium in 30 ℃ of overnight incubation.From flat board, choose 72 single bacterium colonies at random, be inoculated into respectively 100 μ l2 * YT-AG (contain 100 μ g/ml penbritins, in 2 * YT) nutrient solutions of 2% glucose, 30 ℃ of overnight incubation.Get the 20 μ l bacterium that spends the night next day respectively and be transferred in the new 1.5ml centrifuge tube, contain 200 μ l2 * YT-AG and 5 * 10
8Pfu/ml M13KO7.Cultivated 2 hours for 37 ℃, centrifugal, with suspend respectively sedimentation cell in each centrifuge tube of 200 μ l2 * YT-AK (containing penbritin and kantlex) nutrient solution, in 37 ℃ of overnight incubation.Centrifugal and collect supernatant.
By 96 hole elisa plates, coating buffer is 0.05M Na with antigen HBsAg bag
2CO
3(pH9.6), antigen concentration is 10 μ g/ml, and every hole adds coating buffer 100 μ l, and 4 ℃ are spent the night.Add confining liquid (1.5%BSA is dissolved among the PBS), in room temperature sealing 1 hour.100 μ l recombinant phages antibody supernatants are mixed with the confining liquid equal-volume, and room temperature is placed and to be added the people after 10 minutes to the elisa plate of bag quilt, 37 ℃ of incubations 1 hour.With the negative contrast of M13 phage, wash plate 3 times with PBST (PBS that contains 0.05%Tween 20), PBS washes plate 3 times.Add the anti-M13 phage of 100 μ l also with the IgG-HRP (1: 5000) of horseradish peroxidase-labeled, the positive control hole adds 100 μ l sheep anti-mouse igg-HRP, and 37 ℃ were reacted 1 hour, washed plate 3 times with PBST, and PBS washes plate 3 times.Add substrate OPD-H
2O
2100 μ l, room temperature effect 20 minutes adds 50 μ l 2M H
2SO
4Termination reaction, the absorbance value in the every hole of detection, 490nm place.Therefrom select the active higher clone of several strains, establish parallel hole and repeat The above results, determine the strongest positive colony of a strain activity at last.
6. the evaluation of positive colony recombinant plasmid and dna sequence analysis thereof: extract recombinant plasmid, use Sfi I and NotI digestion with restriction enzyme respectively.Enzyme is cut product through 1.2% agarose gel electrophoresis analysis.With T7 DNA Sequence
TMKit measures the dna sequence dna of scFv gene on this recombinant plasmid.The result shows that the scFv gene is made up of 807 nucleic acids, and is as follows according to the anti-hepatis B immunoglobulin aminoacid sequence that dna sequence dna is derived.
1 Glu?Leu?Thr?Gln?Ser?Pro?Ser?Phe?Leu?Ser?Ala?Ser?Ile?Gly?Asp?Arg 16
17 Val?Thr?Ile?Thr?Cys?Arg?Thr?Ser?Gln?Asp?Ile?Asp?Asn?Tyr?Leu?Ala 32
33 Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Val?Tyr?Ser 48
49 Ala?Ser?Ala?Leu?Gln?Gly?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly 64
65 Ser?Arg?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Asn?Ser?Leu?Gln?Pro?Glu?Asp 80
81 Ser?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Leu?Gly?Ser?Tyr?Pro?Leu?Thr?Phe 96
97 Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Ser?Arg?Arg?Gly?Gly?Gly 112
113 Ser?Arg?Gly?Gly?Gly?Pro?Gly?Gly?Gly?Gly?Ser?Glu?Val?Gln?Leu?Leu 128
129 Asp?Gln?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Ala?Gly?Ser?Leu?Thr?Leu 144
145 Ser?Cys?Ala?Ile?Ser?Gly?Phe?Thr?Tyr?Ser?Glu?Tyr?Ala?Val?Ser?Trp 160
161 Gly?Arg?His?Ala?Leu?Gly?Lys?Gly?Pro?Glu?Trp?Val?Ser?Thr?Ile?Ile 176
177 Gly?Ser?Ala?Gly?Asn?Thr?Tyr?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Leu 192
193 Ile?Ile?Ser?Arg?Asp?Thr?Ser?Arg?Lys?Met?Leu?Phe?Leu?Gln?Met?Asn 208
209 Ser?Leu?Arg?Ala?Gly?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Lys?Val?Lys 224
225 Met?Val?Arg?Gly?Gly?Tyr?Trp?Phe?Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln 240
241 Gly?Thr?Ala?Val?Thr?Val?Ser?Ser?Thr?Ser?Glu?Gln?Lys?Leu?Ile?Ser 256
257 Glu?Glu?Asp?Leu?Gly?Ser?His?His?His?His?His?His 276
With the single-chain antibody gene subclone to the pComB carrier, transformed into escherichia coli carries out the expression of soluble single-chain antibody.Induced 3 hours centrifugal collection supernatant with the nutrient solution that contains 1mM IPTG.The tniema pericentral siphon.Detect soluble single-chain antibody with anti-His-Tag antibody (Phamacia).ELISA and Westem Blot experiment proves that all solubility scFv antibody can combine with HbsAg, and not with other protein bound.
The present invention can be in the application in preparation treatment and prevention hepatitis B virus medicine and the diagnostic kit.
Embodiment two
The preparation of hbv antibody Fab
The basic skills of preparation hbv antibody Fab is as above-mentioned single-chain antibody.Also be that cDNA with immune peripheral blood lymphocyte is a template, with pcr amplification light chain of antibody (κ) and variable region of heavy chain and first constant region (Fd).Different is adds the different primer of a cover in the PCR reaction system, pcr amplification obtains κ and Fd gene, and these gene clones to phage expression vector, by the method for immunity-screening-amplification, acquisition high-affinity antibody Fab.Nucleotide sequence analysis obtains the gene order of light chain of antibody κ and heavy chain Fd, and is as follows according to the aminoacid sequence that this gene is derived:
Hepatitis B virus antibody κ chain amino acid sequence:
1 Asp?Ile?Thr?Gln?Ser?Pro?Ser?Phe?Leu?Ser?Ala?Ser?Ile?Gly?Asp?Arg 16
17 Val?Thr?Ile?Thr?Cys?Arg?Thr?Ser?Gln?Asp?Ile?Asp?Asn?Tyr?Leu?Ala 32
33 Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Val?Tyr?Ser 48
49 Ala?Ser?Ala?Leu?Gln?Gly?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly 64
65 Ser?Arg?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Asn?Ser?Leu?Gln?Pro?Glu?Asp 80
81 Ser?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Leu?Gly?Ser?Tyr?Pro?Leu?Thr?Phe 96
97 Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Gly?Ala?Ser?Val?Phe?Ile 112
113 Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val 128
129 Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys 144
145 Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu 160
161 Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu 176
177 Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr 192
193 His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu 208
209 Cys 227
Hepatitis B virus heavy chain of antibody Fd aminoacid sequence:
Glu?Phe?Glu?Val?Gln?Leu?Leu?Glu?Gln?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly
Gly?Ser?Leu?Thr?Leu?Ser?Cys?Ala?Ile?Ser?Gly?Phe?Thr?Phe?Ser?Glu?Tyr?Ala
Val?Ser?Trp?Gly?Arg?His?Ala?Leu?Gly?Lys?Gly?Pro?Glu?Trp?Val?Ser?Thr?Ile
Ile?Gly?Ser?Ala?Gly?Asn?Thr?Tyr?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Leu?Ile
Ile?Ser?Arg?Asp?Thr?Ser?Arg?Lys?Met?Leu?Phe?Leu?Gln?Met?Asn?Ser?Leu?Arg
Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Lys?Val?Lys?Met?Val?Arg?Gly?Gly
Tyr?Trp?Phe?Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Ala?Val?Thr?Val?Ser
Ser?Gly?Thr?Pro?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe
Pro?Leu?Ala?Pro?Cys?Ser?Arg?Ser?Thr?Ser?Glu?Ser?Thr?Ala?Ala?Leu?Gly?Cys
Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Ser?Ala
Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr
Ser?Leu?Thr?Ala?Trp?Tyr?Pro?Cys?Pro?Pro?Thr?Ala?Trp?Ala?Arg?Arg?Pro?Thr
Separation and purification monoclonal antibody from born of the same parents' pericentral siphon.Detect the immunocompetence and the cross reaction of full molecular antibody by the ELISA and the immune marking.The result shows this antibody specific combination hepatitis B virus surface antigen, not with human normal tissue generation cross reaction.
Embodiment three
Hepatitis B virus is divided Ziren antibody entirely
On the basis of single-chain antibody, antibody is light, heavy chain variable region gene is cloned into one respectively and is contained in people's antibody constant region expression carrier, transfection mammalian cell (CHO), the engineering cell of screening expressing antibodies in selective medium, and with dilution method engineering cell is further cloned the mono-clonal engineering cell strain of screening high expression level.Specific implementation process is as follows:
With the primer amplification V that contains XbaI and BamHI restriction enzyme site
LGene, same with the primer amplification V that contains XhoI and HindIII restriction enzyme site
HGene.Respectively V
LAnd V
HGene is connected with expression vector pdHL2.4, at first makes up pdHl2.4-V
LRecombinant plasmid reinstalls V
HGene builds up the pdHL-IgG recombinant plasmid.Identify through double digestion and PCR, prove V
LAnd V
HGene all has been cloned on the complete antibody carrier for expression of eukaryon.Expression vector pdHl2.4 has the partial sequence from pBR322, with convenient genetic manipulation, the insertion antibody gene of carrying out in intestinal bacteria.In addition, pdhl2.4 has two eukaryotic transcription units, be responsible for transcribing of heavy chain of antibody and light chain gene respectively, comprise the 5 ' non-translated sequence of mouse metallothionein(MT)-1 (MT-1) promotor, mouse IgG heavy chain enhancer, MT-1, the transcription termination signal and the polyA tailing signal of antibody gene, and Tetrahydrofolate dehydrogenase (dhfr) selectable marker gene.Except that the functional element that possesses above general eukaryotic expression vector, also comprise the constant region gene of heavy chain of antibody IgG1 and light chain κ among the pdHl2.4, intron is arranged between the gene.
Recombinant plasmid transformed is increased in intestinal bacteria, and the separation and purification recombinant plasmid.The recombinant plasmid that will contain antibody gene with lipofectin imports among the mammalian cell CHO, with MTX selective medium screening transformant, obtains to change the full molecular gene cell strain of hbv antibody.With the situation of ELISA evaluation engineering cell secretory antibody, the cell strain of screening expression activity height and expression stability.With the further cloning screening of these cell strains, obtain the high expression level strain, the expressing antibodies amount is about 10 μ g/ml.With Protein A-Sepharose affinity chromatography antibody purification from culture supernatant, the purifying rate reaches more than 98%.
Set up the seed cell storehouse, the full molecular gene engineered antibody of engineering cell stably express carries out aseptic experiment by " biological products aseptic experiment rules ".The result shows that engineering cell does not have bacterium and mycoplasma contamination.
Detect the immunocompetence and the cross reaction of full molecular antibody by the ELISA and the immune marking.The result shows this antibody specific combination hepatitis B virus surface antigen, avidity
。With human normal tissue (after one's own heart, liver, spleen, lung, kidney) any cross reaction does not take place.
The animal toxicity experimental result shows, no matter is single-chain antibody or full molecular antibody abdominal injection BALB/c mouse, and every injected in mice 1 personal dosage (200 international unit) was observed 7 days continuously, and any abnormal response does not take place mouse.This experimental result points out the experiment of this product safety qualified.
Claims (4)
1, a kind of hepatitis B virus immune globulin is characterized in that variable region Fv is the immunoglobulin (Ig) with 28kDa, is made up of the variable region of heavy chain and the variable region of light chain of antibody, and the aminoacid sequence that this antibody gene is derived is as follows:
1 Glu?Leu?Thr?Gln?Ser?Pro?Ser?Phe?Leu?Ser?Ala?Ser?Ile?Gly?Asp?Arg 16
17 Val?Thr?Ile?Thr?Cys?Arg?Thr?Ser?Gln?Asp?Ile?Asp?Asn?Tyr?Leu?Ala 32
33 Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Val?Tyr?Ser 48
49 Ala?Ser?Ala?Leu?Gln?Gly?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly 64
65 Ser?Arg?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Asn?Ser?Leu?Gln?Pro?Glu?Asp 80
81 Ser?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Leu?Gly?Ser?Tyr?Pro?Leu?Thr?Phe 96
97 Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Ser?Arg?Arg?Gly?Gly?Gly 112
113?Ser?Arg?Gly?Gly?Gly?Pro?Gly?Gly?Gly?Gly?Ser?Glu?Val?Gln?Leu?Leu 128
129?Asp?Gln?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Ala?Gly?Ser?Leu?Thr?Leu 144
145?Ser?Cys?Ala?Ile?Ser?Gly?Phe?Thr?Tyr?Ser?Glu?Tyr?Ala?Val?Ser?Trp 160
161?Gly?Arg?His?Ala?Leu?Gly?Lys?Gly?Pro?Glu?Trp?Val?Ser?Thr?Ile?Ile 176
177?Gly?Ser?Ala?Gly?Asn?Thr?Tyr?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Leu 192
193?Ile?Ile?Ser?Arg?Asp?Thr?Ser?Arg?Lys?Met?Leu?Phe?Leu?Gln?Met?Asn 208
209?Ser?Leu?Arg?Ala?Gly?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Lys?Val?Lys 224
225?Met?Val?Arg?Gly?Gly?Tyr?Trp?Phe?Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln 240
241?Gly?Thr?Ala?Val?Thr?Val?Ser?Ser?Thr?Ser?Glu?Gln?Lys?Leu?Ile?Ser 256
257?Glu?Glu?Asp?Leu?Gly?Ser?His?His?His?His?His?His。276
2, a kind of hepatitis B virus immune globulin is characterized in that Fab is the immunoglobulin (Ig) with 52kDa, has aminoacid sequence as follows,
Hepatitis B virus antibody κ chain amino acid sequence:
1 Asp?Ile?Thr?Gln?Ser?Pro?Ser?Phe?Leu?Ser?Ala?Ser?Ile?Gly?Asp?Arg 16
17 Val?Thr?Ile?Thr?Cys?Arg?Thr?Ser?Gln?Asp?Ile?Asp?Asn?Tyr?Leu?Ala 32
33 Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Val?Tyr?Ser 48
49 Ala?Ser?Ala?Leu?Gln?Gly?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly 64
65 Ser?Arg?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Asn?Ser?Leu?Gln?Pro?Glu?Asp 80
81 Ser?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Leu?Gly?Ser?Tyr?Pro?Leu?Thr?Phe 96
97 Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Gly?Ala?Ser?Val?Phe?Ile 112
113?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val 128
129?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys 144
145?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu 160
161?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu 176
177?Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr 192
193 His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu 208
209 Cys 227
Hepatitis B virus heavy chain of antibody Fd aminoacid sequence:
Glu?Phe?Glu?Val?Gln?Leu?Leu?Glu?Gln?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly
Gly?Ser?Leu?Thr?Leu?Ser?Cys?Ala?Ile?Ser?Gly?Phe?Thr?Phe?Ser?Glu?Tyr?Ala
Val?Ser?Trp?Gly?Arg?His?Ala?Leu?Gly?Lys?Gly?Pro?Glu?Trp?Val?Ser?Thr?Ile
Ile?Gly?Ser?Ala?Gly?Asn?Thr?Tyr?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Leu?Ile
Ile?Ser?Arg?Asp?Thr?Ser?Arg?Lys?Met?Leu?Phe?Leu?Gln?Met?Asn?Ser?Leu?Arg
Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Lys?Val?Lys?Met?Val?Arg?Gly?Gly
Tyr?Trp?Phe?Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Ala?Val?Thr?Val?Ser
Ser?Gly?Thr?Pro?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe
Pro?Leu?Ala?Pro?Cys?Ser?Arg?Ser?Thr?Ser?Glu?Ser?Thr?Ala?Ala?Leu?Gly?Cys
Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Ser?Ala
Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr
Ser?Leu?Thr?Ala?Trp?Tyr?Pro?Cys?Pro?Pro?Thr?Ala?Trp?Ala?Arg?Arg?Pro?Thr。
3, a kind of hepatitis B virus immune globulin is characterized in that full molecular antibody is the immunoglobulin (Ig) with 150kDa, contains claim 1 or 2 described aminoacid sequences.
4, according to claim 1,2, the application of 3 described arbitrary immunoglobulin (Ig)s in preparation treatment, prevention hepatitis B virus medicine or preparation hepatitis B virus surface antigen diagnostic kit.
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