CN1609123A - Variable region sequence of hepatitis B virus monoclonal antibody, gene engineering antibody containing the variable region and its use - Google Patents
Variable region sequence of hepatitis B virus monoclonal antibody, gene engineering antibody containing the variable region and its use Download PDFInfo
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Abstract
The present invention relates to new mouse monoclonal antibody variable region sequence to hepatitis B virus preS1, variable region including heavy strand and light strand and its conservative mutant, nucleic acid molecule encoding the variable region amino acid sequence, recombinant expression vector and host cell containing the nucleic acid molecule, genetic engineering antibody assembled with the said variable region sequence, fused protein including the genetic engineering antibody, composition with coupled genetic engineering antibody and hepatitis B treating medicine molecule(s), the use of the genetic engineering antibody in preparing medicine for diagnosing, preventing and/or treating hepatitis B virus, and medicine composition including the genetic engineering antibody, pharmacologically acceptable carrier and/or adjuvant.
Description
Invention field
The present invention relates to new mouse monoclonal antibody variable region sequences at hepatitis B virus preS1, comprise heavy chain and variable region of light chain and conservative property varient thereof, the encode nucleic acid molecule of this change region amino acid sequence, the recombinant expression vector and the host cell that comprise this nucleic acid molecule, the genetic engineering antibody that is assembled into by described variable region sequences, the fusion rotein that comprises this genetic engineering antibody, drug molecule link coupled mixture by this genetic engineering antibody and one or more treatment hepatitis B viruses, this genetic engineering antibody is used for the preparation diagnosis, prevent and/or treat the purposes of the medicine of hepatitis B virus, and the pharmaceutical composition that comprises this genetic engineering antibody and pharmaceutically acceptable carrier and/or adjuvant.
Background technology
On average have every year 2000000 people to suffer from hepatitis in China, wherein 50% is hepatitis B infection, and has an appointment about 100,000,000 people and to be hepatitis B surface antigen(HBsAg) (HBsAg) carrier.The World Health Organization's statistics in 1996 shows, the whole world has at least 30% about 2,000,000,000 people of population to infect hepatitis B virus (Hepatitis B Virus, HBV), wherein about 3.5 hundred million people are Chronic HBV carrier (chronic HBV carriers) (The world health report 1996-Fightingdisease, fostering development).Infecting the chronic hepatopathy and the liver cancer that cause by HBV is one of the most serious human health problem.More distinct issues are, still have at present adult Chronic HBV carrier more than 300,000,000 and tens million of chronic hepatitis patients (people such as Alper, 1989. New England Journal of Medicines (N Engl J Med.) 321 (11): 708-712 in the world; People such as Belloni, 1998. vaccines (Vaccine) .16 (4): 399-402), as can not get effective treatment, will mean has in the period of the following 30-40 tens of millions of people may be dead because of liver cirrhosis or liver cancer.
At present, people have brought up to molecular level to the understanding of hepatitis B virus.Problems such as the genome structure of relevant HBV, proteins encoded, route of synthesis, assembling approach are illustrated substantially.
HBV is one of eukaryotic cell dna virus of known minimum, is a kind of retrovirus, and genome structure is quite accurate, unique, is the little annular DNA of the about 3.2kb of a total length.The HBVDNA double-chain length is asymmetric, the minus strand of total length and virus mRNA complementation; Normal chain only 5 ' end is fixing, the about 1700-2600nt of length.The HBV minus strand has 4 open reading frame: S, C, P and X, the membranin of encoding respectively, nucleocapsid, polysaccharase and HBX albumen.S opens frame encode respectively S gene, preS2 district and preS1 district, and the three respectively has its codon ATG.HBV makes virus can adhere to and invade new liver cell by three kinds of outer membrane protein parcels of big or middle, main albumen virion.Main albumen is HBsAg, is made up of 226 amino acid.Middle albumen is preS2+HBsAg, is made up of 281 amino acid.Large protein is preS1+preS2+HBsAg, forms (people such as Lau, 1993. lancets (Lancet), 342:1335-1340 by 389-400 amino acid; Luo Kangxian. hepatitis B-basis and clinical (first version), Beijing: the People's Health Publisher, 1997, p13-16).It is generally acknowledged that preS1 is relevant with its sterie configuration with main proteic function, preS2 function and its primary structure are closely related.In typical HBV carrier blood the mol ratio of preS1, preS2 and S be about 1: 20: 10000 (Luo Kangxian. hepatitis B-basis and clinical (first version), Beijing: the People's Health Publisher, 1997, p30-32).
HBV infects and has strict host's species specificity and tissue tropism, comparatively definite evidence shows that it only has infectivity to the human and chimpanzee, main infringement hepatic tissue (people such as Barker, 1975. infectious diseases magazines (J Infect Dis), 132 (4): 451; People such as Barker, 1975.Am J Med Sci, 270 (1): 189).Membranin has determined the infection preferendum of HBV, membranin by with liver plasma membrane on the combining mediation virus absorption and enter liver cell (people such as De, 1997. viral hepatitis magazines (J Viral Hepat), 4 (3): 145-153) of acceptor.At present majority think that preS1 is a virus and the combining site of liver plasma membrane acceptor, because synthetic peptide of preS1 21-47 and corresponding antibodies thereof can vitro inhibition HBV and the combining of human liver cancer cell, the variation of preS121-47 can cause HBV to lose infectivity.
PreS albumen has stronger immunogenicity at the T cell levels, also contains the target epi-position of neutralizing antibody in the preS district, and preS antibody also has provide protection to the host.Two from immunity after the polypeptide 12-47 of preS1 and 94-117 and the adjuvant KLH coupling, all makes orangutan be protected (people such as Neurath, 1989. vaccines (Vaccine), 7 (3): 234-236).Clinically, the appearance of preS antibody often is counted as the early sign that HBV infects removing.Studies show that anti-preS1 antibody is that a kind of membrane antibody the earliest appears when removing in acute self limiting hepatitis B patient HBV.Discover in producing the negative crowd of anti-preS1 (21-47) only have 7.6% (5/66) patient successfully to remove virus (people such as Alberti, 1990. hepatology (Hepatology), 12 (2): 199-203).(1999. virological investigations (ResVirol), 141 (5): 563-570) such as Coursaget.(1999. virological investigations (Res Virol) such as Coursaget, 141 (5): 563-570) the preS1 antibody response of different HBV infection populations is studied, found that acute self limiting hepatitis B, chronic viral hepatitis B patient's anti-preS1 (21-47) recall rate is respectively 37% and 1%.In (1999. Chinese Medical Journals (Chin Med J (Engl)), 112 (4): in research 321-324), find that the appearance of anti-preS1 in the acute self limiting hepatitis B patient serum is relevant with the lifting of ALT level such as Mi.
Up to now, the prevention of human virus's disease is mainly still based on virus vaccines, and the treatment of virus disease is except that specific antiviral antibody, and any virus infection does not still have the specific drugs treatment.Because present most of antiviral only can be come the amelioration of inflammation reactivity by suppressing hbv replication, still can't thoroughly remove virus, so in clinical trial, the disappearance of the antiviral therapy terminal point of standard is chronic HBV infection person's serum HbeAg (anti-HBe sun change or do not have all can) and serum HBV dna level are reduced to and can't detect, rather than the removing fully of virus.
The characteristic of antibody molecule is single-minded at certain antigen.Antiviral antibody demonstrates different functions with its specificity in conjunction with the proteic characteristic of different virus.Many antiviral polyclonal antibodies, monoclonal antibody and multi-form genetically engineered recombinant antibodies since its specificity in conjunction with virus surface proteins or viral after birth glycoprotein, can wrap up mechanism such as virion, blocking virus absorption, the interior killer cell function of inductor neutralizes and the blocking virus infection, therefore, can be used for preparing the antiviral antibody medicine.
People such as Morrison linked together mouse monoclonal antibody variable region and human IgG constant region on gene level in 1984, and having successfully constructed first genetic engineering antibody is people-mouse chimeric antibody (human-mouse chimeric antibody).After this, range gene engineered antibody emerge in multitude.1986, the CDR district of the CDR district displacement human IgG of human mouse source monoclonal antibodies such as Jones successfully made up first reshaping antibody (reshaped antibody), also claims CDR grafted antibody (CDR grafting antibody).In addition, comprise the multiple unit price small molecular antibody of Fab, Fv, scFv, diabody, single domain antibody etc. and develop swift and violent bi-specific antibody, multi-specificity antibody successfully constructs successively.
The source phage antibody library technique of should choosing and antibody engineering platform technology, the research of anti-virus infection curative people source or humanized genetic engineering antibody has obtained remarkable progress.At present, hepatitis B virus (HBV) people source antivirus genetic engineering antibody obtains the laboratory stage success.
The reorganization Fab antibody of hepatitis B virus resisting HBsAg all has the report of success both at home and abroad.
Because the commercialization HBsAg recombinant vaccine and the HBsAg purifying, the recombinant antibodies that obtains people source anti-HBsAg is easy than other antiviral antibodies relatively, obtains Fab antibody and often has the avidity higher with HBsAg.Yet, the research of current HBV genetic engineering antibody, majority all with HBsAg as target antigen.But clinical study shows, the disappearance of preS1 is often indicating the good progress (people such as Delfini of the course of disease, 1989. medical virology magazine (J Med Virol), 28 (3): 169-175), the appearance of preS1 antibody then is counted as the early sign that HBV infect to remove (people such as Budkowska, 1986. hepatology (Hepatology), 6 (3): 360-368).PreS1 may contain liver plasma membrane receptor binding site and viral neutralizing epitope, and preS1 antibody may participate in the removing of virus by two kinds of mechanism: the opsonization of 1.preS1 antibody can be removed free virus in the circulation; 2.preS1 the neutralizing effect of antibody can stop newborn virus to infect hepatocellular again.
Research at the antigenic genetic engineering antibody of preS1 is not achieved success as yet.Have only the superfine people of Zhang Zhi (2002. biological chemistries and biophysics progress (Prog Biochem Biophys) at present, 29 (4): 572-575) carried out the screening study of the human single chain variable fragments antibody of the anti-preS1 of high-affinity (20-47), phage antibody library carry out 3 take turns elutriation after, the antigen antibody reaction result shows, obtained avidity 10 from immune storehouse
-7～10
-8The single-chain antibody of the anti-preS1 of M is higher than the result (10 who obtains from the natural antibody storehouse
-6～10
-7M).People such as Budkowska (1995. Journal of Virologies (J Virol), 69:840-848) reported mouse monoclonal antibody 5a19, the linear epitope of identification preS1 (36-43), people such as Maeng (2000. virusology (Virology), 270:9-16) prove that this monoclonal antibody can stop HBV to enter the human liver cell of cultivation, people such as Pizarro (2001.FEBS Lett, 509 (3): 463-468) further obtain its heavy chain and variable region of light chain, its aspects such as antigen binding kinetics are studied.People such as Choi (1998. hybridomas (Hybridoma), 17:535-540) from natural recombinant antibodies storehouse, filter out people's Fab antibody fragment of recombinating, identification preS1, improved its avidity (people such as Park subsequently, 2000. biological chemistry and biophysical research communication (Biochem Biophys ResCommu), 275:553-557).But the report that does not also have further research and use.
Therefore, in existing antiviral antibody medicine, because human genetically engineered antibody is fully matured not as yet, it is leading that haematogenous immune globulin antibody goods still account for market, for example, the Nabi-HBTM that resisting HBV virus efficient immunoglobulin (Ig) BayHep BTM that Bayer company produces and U.S. Nabi company produce, origin comes from the high donor blood plasma purification of hepatitis B virus resisting HbsAg antibody and is prepared from.
The inventor designs for target spot at hepatitis B virus preS1 and prepares mouse monoclonal antibody.Amplify the heavy chain and the variable region of light chain encoding gene of described monoclonal antibody on this basis with the RT-PCR method, further be assembled into single-chain antibody (scFv), and in intestinal bacteria, obtained good representation.4D11 single-chain antibody after purified kept former mouse only the clonal antibody specificity in conjunction with the ability of preS1 (21-47).Lay a good foundation for successfully developing genetically engineered recombinant antibodies of the present invention.
Summary of the invention
What unless otherwise defined, used here all technology and scientific term were all expressed is in the common implication that those of skill in the art understood that the present invention relates in the field.Here used name and be widely used conventional steps in the corresponding field in cell cultures, molecular genetics, nucleic acid chemistry, Immunology Lab operation steps.
But one aspect of the present invention relates to the variable region sequences of specificity in conjunction with the mouse monoclonal antibody of hepatitis B virus preS1, wherein: 1) the weight chain variable region amino acid sequence shown in SEQ ID NO:1, or the conservative property varient that obtains through one or more aminoacid addition, deletion, replacement, the sudden change of modification conservative property of this sequence; 2) the light chain variable region amino acid sequence is shown in SEQ IDNO:2, or the conservative property varient that obtains through one or more aminoacid addition, deletion, replacement, the sudden change of modification conservative property of this sequence.
Antibody comprises the polypeptide chain aggregate that is connected together by disulfide-bridged, two, and two polypeptide main chains that are called light chain and heavy chain constitute all primary structure classifications (analogs) of antibody.Heavy chain and light chain all are further divided into some subprovinces that are called variable region and constant region.Heavy chain comprises one variable region and three different constant regions, and light chain then comprises single variable region (variable region that is different from heavy chain) and one constant region (constant region that is different from heavy chain).The binding specificity of antibody is responsible in the variable region of heavy chain and light chain.
" variable region of heavy chain " is meant a peptide species, and (1) its length is 110 to 125 amino acid; (2) the heavy chain amino-acid sequence that begins from heavy chain N-terminal amino acid corresponding to monoclonal antibody of the present invention of its amino-acid sequence.Equally, " variable region of light chain " is meant a peptide species, and (1) its length is 95 to 115 amino acid; (2) the light chain amino acid order that begins from light chain N-terminal amino acid corresponding to monoclonal antibody of the present invention of its amino-acid sequence.
Term used herein " the conservative property varient " be meant and kept its maternal characteristic basically, as the varient of basic immunology biological nature, structural performance, control characteristic or biochemical characteristic.General, the aminoacid sequence of the conservative property varient of polypeptide is different from maternal polypeptide.But difference is limited, so that closely similar generally with the sequence and the conservative property varient of maternal polypeptide, and is identical in many zones.Difference on conservative property varient and the maternal polypeptid acid sequence for example can be: the replacement of one or more amino-acid residues and arbitrary combination thereof, interpolation and deletion.The amino-acid residue of replacing or inserting can be encoded by genetic code, also can can't help the genetic code coding.The conservative property varient of polypeptide can produce naturally, and perhaps it can the spontaneous varient of right and wrong.The non-spontaneous conservative property varient of polypeptide can be by induced-mutation technique or directly synthetic the generation.
The invention still further relates to a kind of by variable region of heavy chain part or its examples of conservative variations in the described variable region sequences, and/or the genetic engineering antibody that is assembled into of the aminoacid sequence of variable region of light chain part or its conservative property varient, it still keeps the ability of specific combination hepatitis B virus preS1.
In the preferred embodiment of the invention, described genetic engineering antibody, be weight chain variable region amino acid sequence or its conservative property varient in the variable region sequences of the mouse monoclonal antibody that comprises described preS1, and/or single-chain antibody, the chimeric mAb of light chain variable region amino acid sequence or its conservative property varient, change the monoclonal antibody of shape monoclonal antibody or other humanization forms, or the fragment of described antibody, it still keeps the ability of specific combination hepatitis B virus preS1.
Another aspect of the invention relates to a kind of fusion rotein that two or more states genetic engineering antibody or described antibody fragment that comprises, and it still keeps the ability of specific combination hepatitis B virus preS1.In a preferred embodiment of the invention, described fusion rotein is a bispecific antibody.Concrete, described fusion rotein can be selected from least a single-chain antibody of the present invention or chimeric mAb or change the shape monoclonal antibody or other humanization form monoclonal antibodies for comprising, or keeps the fusion rotein of the described antibody fragment of the ability that the specific combination hepatitis B virus sprays into preS1.
Further aspect of the present invention relates to a kind of mixture, it comprises and is selected from least a above-mentioned genetic engineering antibody, or it keeps the fragment of the ability of specific combination hepatitis B virus preS1, and the drug molecule of one or more treatment hepatitis B of phase link coupled with it.Known medicine with treating hepatitis B effect all can be used in and genetic engineering antibody coupling of the present invention at present, in order to prepare described mixture, to strengthen result of treatment.
The invention still further relates to a kind of nucleic acid molecule, the variable region of heavy chain of the described variable region amino acid sequence of mouse monoclonal antibody of its coding preS1, it has nucleotide sequence or its degeneracy sequence shown in SEQ ID NO:3; The perhaps variable region of light chain of mouse monoclonal antibody variable region amino acid sequence of its coding preS1, it has nucleotide sequence or its degeneracy sequence shown in SEQ ID NO:4.
The invention still further relates to a kind of recombinant expression vector that comprises above-mentioned nucleic acid molecule.With through above-mentioned recombinant expression vector transformed host cells, it can express genetic engineering antibody of the present invention, or it keeps the fragment of the ability of specific combination hepatitis B virus preS1.
Be applicable to express nucleic acid molecule of the present invention include but not limited to chromosomal, achromosomal and the synthetic dna sequence dna, for example, the derivative of SV40, bacterial plasmid, phage DNA, yeast plasmid, be derived from plasmid and phage DNA bonded carrier, viral DNA, baculovirus, vaccinia virus, adenovirus, fowlpox virus and Pseudorabies virus.But, as long as can duplicate in the host and survive, other carrier also can utilize.
It is worth mentioning that especially the present invention also comprises the recombinant precursor of the encoding sequence that contains the invention described above nucleic acid molecule.This construct comprises carrier, as plasmid or virus vector, has wherein inserted a sequence of the present invention forward or backwards.Of this embodiment preferred aspect, this construct also comprises the adjusting sequence, promotor for example, it is effectively connected on this sequence.To those skilled in the art, many carriers and promotor all are known, and can obtain by commercial sources.Enumerate several carriers below.Bacterium: pQE-70, pQE-60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), pTRC99a, pKK223-3, pDR540, pRIT5 (Pharmacia); Eucaryon: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia).But, so long as can duplicate in the host and survive, other plasmid or carrier also can be used.
The carrier that promoter region can utilize CAT (CAT) carrier or other to have selective marker chooses from the gene of wanting.PKK232-8 and pCM7 are two suitable carriers.The special bacterium promotor that claims comprises LacI, LacZ, T3, T7, gpt, PR, PL and trp.Eukaryotic promoter comprises CMV immediate early promoter, HSV thymidine kinase, early stage and late period SV40, retroviral LTR and mouse metallothionein(MT) I.The selection of suitable carrier and promotor should be within the common state of the art in this area.
Suitable dna sequence dna can be inserted in the carrier by many methods.Usually, dna sequence dna is inserted on the suitable restriction endonuclease sites with method as known in the art.These methods are believed in the ken that is in those skilled in the art.
Dna sequence dna in the expression vector is connected together with a suitable expression control sequenc (promotor) effectively, thereby instructs synthesizing of mRNA.Cited below is other promotor of genetic expression in the PL promotor of the representative of this promotor: LTR or SV40 promotor, colibacillary Lac or trp promotor, phage and known control protokaryon or eukaryotic cell or its virus.Expression vector also comprises needed ribosome bind site of translation initiation and transcription terminator.Carrier can also have the proper sequence that strengthens expression.
In addition, expression vector preferably comprises one or more selectable marker genes that phenotypic characteristic can be provided, so that the screening of transformed host cell, available Tetrahydrofolate dehydrogenase of for example eukaryotic cultivation or neomycin resistance, then available tsiklomitsin of intestinal bacteria or amicillin resistance.
Comprise above-mentioned suitable dna sequence dna, suitable promotor or the carrier of control sequence and can be used to transform appropriate host, allow this albumen of host expresses.
Host cell carries out genetically engineered (transduction, conversion or transfection) with the above-mentioned carrier of the present invention, and carrier can be a cloning vector or expression vector, as plasmid, virus particle, phage or the like.The host cell of through engineering approaches can cultivated for being suitable for activating on traditional nutritional medium that promotor, screening transformant or amplification DRC2 gene change.The original used expression condition of culture condition such as temperature, pH etc. and selecteed cell is consistent, is clearly to those skilled in the art.
Genetic engineering antibody of the present invention all can be expressed by appropriate host cell by engineered method.The present invention can use multiple expression host cell, as prokaryotic host cell, includes but not limited to the bacterial strain of intestinal bacteria, bacillus, streptomyces etc.; Eucaryon host includes but not limited to the bacterial strain of Aspergillus, yeast belong etc., and mammalian cell, vegetable cell etc.The above-mentioned expression of stating target product of the present invention is not limited to concrete expression vector and expressive host, as long as it can express genetic engineering antibody of the present invention, its conservative property varient.From these instruction, the selection of suitable host should be in those skilled in the art's ken.
In another embodiment, the present invention relates to comprise the host cell of above-mentioned construct.Host cell can be a higher eucaryotic cells, such as cells of mamma animals, or eukaryotic cell such as low, such as yeast cell, can also be prokaryotic cell prokaryocyte, such as bacterial cell.The method that construct is imported host cell has: transfection, electroporation or the particle gun method of calcium phosphate transfection, the mediation of DEAE-dextran.
Construct in the host cell can produce the gene product of recombination sequence coding by traditional method.Another approach is that available traditional peptide synthesizer synthesizes polypeptide of the present invention.
Under suitable promotor control, can in cells of mamma animals, yeast, bacterium or other cell, express maturation protein.Utilization also can produce this protein with the cell free translation system by DNA construct derived RNA of the present invention.Being suitable for people such as the clone of protokaryon and eucaryon host and expression vector Sambrook is described in " molecular cloning experiment guide " (second edition, cold spring port press, New York, 1989).
In higher eucaryote, transcribing of the DNA of the variable region sequences of the present invention of encoding can be enhanced by insert an enhancer sequence in carrier.Enhanser is the cis acting factor of DNA, and about 10-300bp is arranged usually, acts on promotor, improves transcribing of it.For example, the enhanser that is positioned at replication orgin rear side 100-270bp place of SV40, the sub-enhanser of cytomegalovirus early promoter, the polyoma enhanser that is positioned at the replication orgin rear side and adenovirus enhanser.
Usually, recombinant expression vector comprises replication orgin and is used for the selective marker of transformed host cell, for example colibacillary ampicillin resistance gene and cereuisiae fermentum TRP1 gene also comprise the promotor of coming from efficiently expressing gene, thereby mediate transcribing of downstream configurations sequence.These promotors can get from the operon of coding glycolytic ferment, such as glycerol 3-phosphate acid kinase (PGK), the factor, acid phosphatase or heat shock protein(HSP).Allogenic structure sequence is assembled together with suitable orientation and translation initiation and terminator sequence and other preferred sequence.Selectable situation is, the allogenic sequence fusion rotein of can encoding, and this albumen contains a N-terminal confirms peptide, shows the feature of expection, the stabilization of for example expressed recombinant products and purify and simplify.
The effective expression carrier that bacterium is suitable for makes up like this: the structural DNA sequence of the target protein of will encoding is inserted in the steerable reading frame that has function in company with suitable translation initiation and termination signal.Carrier should comprise one or more Phenotypic Selection marks and replication orgin, and replication orgin has guaranteed that carrier can remain in the host, and better is to make carrier obtain amplification.The prokaryotic hosts that is suitable for transforming has the various bacteriums of intestinal bacteria, subtilis, Salmonella typhimurium and Pseudomonas, streptomyces and Staphylococcus, and other host also can be selected.
Monoclonal antibody variable region of heavy chain prepared in accordance with the present invention and light chain variable region sequence, and the different variants that produce according to the degeneracy of genetic codon, those skilled in the art can comprise the recombinant expression vector of above-mentioned nucleotide sequence with the several different methods preparation, and by gained recombinant expression vector transformed host cells.The selection of host cell and to be converted into those skilled in the art known, as long as it can express monoclonal antibody of the present invention, its conservative property varient, or the monoclonal antibody or the antibody fragment of single-chain antibody or chimeric antibody or reshaping antibody or other humanization forms, or fusion rotein or bispecific antibody or other forms of polypeptide or polypeptide analog.
Therefore, the invention still further relates to a kind of coding monoclonal antibody of the present invention that comprises, its conservative property varient, or the monoclonal antibody or the antibody fragment of single-chain antibody or chimeric antibody or reshaping antibody or other humanization forms, or the recombinant expression vector of the nucleic acid molecule of fusion rotein or bispecific antibody or other forms of polypeptide or polypeptide analog etc.And by described recombinant expression vector transformed host cells.The present invention can use multiple expression host cell, as prokaryotic host cell, includes but not limited to the bacterial strain of intestinal bacteria, bacillus, streptomyces etc.; Eucaryon host includes but not limited to the bacterial strain of Aspergillus, yeast belong etc., and mammalian cell, insect cell, vegetable cell etc.The expression of the above-mentioned target product of the present invention is not limited to concrete expression vector and expressive host, as long as it can express single-chain antibody of the present invention, its conservative property varient, or the monoclonal antibody or the antibody fragment of single-chain antibody or chimeric antibody or reshaping antibody or other humanization forms, or fusion rotein or bispecific antibody or other forms of polypeptide or polypeptide analog.
Single-chain antibody of the present invention, its conservative property varient, or the monoclonal antibody or the antibody fragment of single-chain antibody or chimeric antibody or reshaping antibody or other humanization forms, or the application of fusion rotein or bispecific antibody or other forms of polypeptide or polypeptide analog and produce is also known.
But the example of indefiniteness representational as one, the effective expression carrier that bacterium is suitable for comprises the replication orgin of a selective marker and bacterium, and it is derived from that obtain, that have the genetic elements of well-known cloning vector pBR322 (ATCC37017) from commercial channels plasmid.These business-like carriers comprise such as pKK223-3 (Pharmacia) and pGEM1 (Promega) etc." skeleton " part of pBR322 is combined with suitable promotor and the structure sequence of being expressed.
Transforming after appropriate host bacterial strain and host strain grow into appropriate cell density, induce selected promotor with appropriate means (for example temperature transition or pharmaceutical chemicals are induced), and cell is cultivated for some time again.
Usually use the centrifugation method harvested cell, the method smudge cells with physics or chemistry keeps crude extract to be further purified.The microorganism cells that is used for expressing protein can comprise freeze-thaw cycle, ultrasonic wave, Mechanical Crushing with the fragmentation of any method easily, perhaps uses cell lytic agent, and these methods all are well-known to those having ordinary skill in the art.
The culture systems of various mammalian cells also can be used for express recombinant protein.The example of mammals expression system have Gluzman (Cell 23:175,1981) the fibroblastic COS-7 clone of the monkey kidney of describing, and other can express the clone of compatible carrier, for example C127,3T3, CHO, HeLa and bhk cell are.The mammals expression vector comprises replication orgin, suitable promotor, enhanser and any essential ribosome bind site, polyadenous glycosidation site, splicing donor and acceptor site, transcription termination sequence and 5 ' flank non-transcribed sequence.The dna sequence dna and the polyadenous glycosidation site that are derived from SV40 splicing sequence can be used for providing needed non-transcribed genetic elements.
Polypeptide can reclaim from the reconstitution cell culture and purifying comes out, and the method for recovery and purifying has ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and phytohemagglutinin chromatography.Form ripe proteic complete conformation and need proteinic folding step again.High performance liquid chromatography (HPLC) is applied to last purification step.
The present invention also relates to the purposes that fragment that described genetic engineering antibody or its keep the ability of specific combination hepatitis B virus preS1 is used to the medicine for preparing the diagnosis, prevent and/or treat hepatitis B virus on the other hand.
Another aspect of the invention also relates to a kind of hepatitis b virus infected pharmaceutical composition for the treatment of, it comprises the genetic engineering antibody of the present invention for the treatment of significant quantity, or the fragment or the examples of conservative variations of the ability of its reservation specific combination hepatitis B virus preS1, and pharmaceutically acceptable carrier and/or adjuvant.
Among the present invention, " treatment significant quantity " is meant can be to being tried the individual amount that produces effectively protection and neutralization virus.Those skilled in the art know, and described " treatment significant quantity " is with the situation of treatment plan, the course of disease, treatment target and used monoclonal antibody or its segmental difference and difference.In conjunction with document known in the art and instruction and corresponding clinical standard, the clinician should rely on its experience to draw " the treatment significant quantity " of used monoclonal antibody.Preferably, the significant quantity of treatment described in the present invention is every kg body weight 0.001mg-20mg, is preferably 0.01-10mg especially, more preferably 0.1mg-10mg.Described treatment significant quantity is every kg body weight 0.0001mg-0.1mg, is preferably 0.001-0.06mg especially, more preferably 0.01mg-0.04mg.
The present invention further relates to a kind of hepatitis b virus infected method that is used to prevent and/or treat, and it comprises that fragment or conservative property varient with the genetic engineering antibody at least a of the present invention of prevention significant quantity or treatment significant quantity or its reservation specific combination hepatitis B virus preS1 ability are applied to the patient.
" prevention significant quantity " of the present invention is meant the amount that can be enough to cause the immunoprotection reaction in the individuality of inoculation.Those skilled in the art know, and described " prevention significant quantity " is with mode, opportunity, administration object and the used monoclonal antibody of immunization or its segmental difference and difference.In conjunction with document known in the art and instruction and corresponding clinical standard, those skilled in the art should draw " the prevention significant quantity " of used monoclonal antibody by limited test.Preferably, prevention/immune significant quantity is each immune kg body weight 0.0001mg-0.1mg described in the present invention, is preferably 0.001-0.06mg especially, more preferably 0.01mg-0.04mg.
Similarly, " treatment significant quantity " is meant and can produces effectively protection and the viral amount of neutralization to being tried individuality.Those skilled in the art know, and described " treatment significant quantity " is with the situation of treatment plan, the course of disease, treatment target and used monoclonal antibody or its segmental difference and difference.In conjunction with document known in the art and instruction and corresponding clinical standard, the clinician should rely on its experience to draw " the treatment significant quantity " of used monoclonal antibody.Preferably, the significant quantity of treatment described in the present invention is every kg body weight 0.001mg-20mg, is preferably 0.01-10mg especially, more preferably 0.1mg-10mg.Described treatment significant quantity is every kg body weight 0.0001mg-0.1mg, is preferably 0.001-0.06mg especially, more preferably 0.01mg-0.04mg.
The present invention utilizes method well known in the art to obtain the encoding sequence of code book invention monoclonal antibody variable region of heavy chain and variable region of light chain easily from the hybridoma cell line of secretion 4D11 mouse monoclonal antibody.Those skilled in the art can utilize these encoding sequences to analyze for the important amino acid region of monoclonal antibody binding specificity easily in this area, as being present in determinant complementary region (CDR) CDR1, CDR2 and the CDR3 of antibody gene light chain and variable region of heavy chain, its amino acid sequence corresponding has constituted the specific antigens calmodulin binding domain CaM of antibody.Utilize these aminoacid sequences, can make the polypeptide that has with the same or similar specific combination ability of monoclonal antibody, express the single-chain antibody (ScFv) that forms as antibody heavy chain variable region gene and chain variable region gene are linked in sequence with recombination method; Antibody heavy chain variable region gene and chain variable region gene or CDRs gene substitution to the corresponding position of human antibodies, are given expression to the human monoclonal antibody such as chimeric antibody or the reshaping antibody that more approach human antibodies and have former monoclonal antibody binding specificity; Also can be built into the bispecific antibody or the multi-specific antibody of various ways with another or a plurality of monoclonal antibody jointly; Or the like.
Monoclonal antibody is as carrier, carry toxin protein, radionuclide and medicine and in the clinical treatment of tumour, have great application potential, but mouse monoclonal antibody commonly used has many obstacles that limit its curative effect and use in Clinical Application, most importantly the mouse monoclonal antibody has stronger immunogenicity to human body, can produce stronger human antimouse antibody (humananti-mouse antibody, HAMA) reaction.The present invention has been built into the single-chain antibody that keeps the special antigen-binding activity of former mouse monoclonal antibody by the genetic engineering technique transformation, can reduce the mouse source property of antibody largely, reduces the rejection of human body antagonist.The molecule of single-chain antibody is less, and penetration power is strong, the core position of easier arrival focus.According to the needs of diagnosis or treatment, can also further prepare multi-purpose novel antibody.
Description of drawings:
Fig. 1 shows the SDS-PAGE electrophoresis result of recombination, amalgamation and expression and the purified product of hepatitis B virus preS1 and GST.The 1st road is the molecular weight of albumen mark; The 2nd road is a cellular lysate liquid; The 3rd road is the GST-preS1 recombinant antigen behind the Glutathione-Sepharose 4B column purification
Fig. 2 shows the binding ability of preS1 monoclonal antibody and HBV natural antigen.3H5,1G5,6F1,7B6,2A6,7H11,4D11 are the mouse monoclonal antibody of the synthetic peptide of anti-hepatitis B virus preS1 21-47.
Fig. 3 shows that the 4D11 single-chain antibody is expressed and the SDS-PAGE electrophoresis result of preliminary purification.The 1st road is the molecular weight of albumen mark; The 2nd road is a cellular lysate liquid; The 3rd road is the ultrasonic supernatant liquor of thalline; The 4th road is the thalline ultrasound precipitation; The 5th road is 2mmol/L urea washing supernatant; The 6th road is 4mmol/L urea washing supernatant; The 7th road is 8mmol/L urea washing supernatant.
Fig. 4 shows 4D11 single-chain antibody pure product each several part collection liquid SDS-PAGE electrophoresis result in His column purification process just.The 1st road was the promptly first pure product of sample before the post; The 2nd road is for penetrating the peak; The 3rd road is the washings first time; The 4th road is the washings second time; The 5th road is the elutriant first time; The 6th road is the elutriant second time; The 7th road is elutriant for the third time; The 8th road is the 4th elutriant; The 9th road is a regenerated liquid.
Below in conjunction with specific embodiment and accompanying drawing, the present invention is further described.Described embodiment is intended to specifically illustrate the present invention with way of example.The value of carrier and host's selection and the concentration of reagent, temperature and its dependent variable just illustrates application of the present invention, and is not construed as limiting the invention.
Embodiment
Embodiment 1: be used as the preparation of antigenic GST-preS1 fusion rotein
The separation of goal gene, clone
Utilize Proteinase K-phenol: chloroform method, the extraction HBV DNA from the chronic viral hepatitis B patients serum sample (numbering 263) that preserve my chamber.With pack into the 1.5mlEppendorf pipe of new sterilization of serum, every pipe 200ul.In every pipe, add STE 156ul, 10%SDS 40ul and 100 * Proteinase K 4ul.In 56 ℃ water-bath 2-3 hour then, jiggle mixing at set intervals once.The phenol that adds 400ul: chloroform: primary isoamyl alcohol, left standstill after shaking up 5-10 minute, unsuitable violent.Centrifugal 10 minutes of 12000g.Carefully supernatant is taken out, add the dehydrated alcohol of two volumes above (780ul) and the NaAc (40ul) that final concentration is 0.3M, placed liquid nitrogen 3 minutes.Centrifugal 10 minutes of 12000g.Discard ethanol rapidly, wash one time with 70% ethanol again.To there not being the ethanol smell, add 20ulddH dry about 5 minutes of thermostatted (55 ℃) again
2The O dissolving.
Design many primers, the PCR segmentation separates the HBV gene, and is cloned into pMD 18-T carrier and checks order.Cloned plasmids pT-preS comprises the preS1 and the preS2 gene of total length.Two special primer S1F of otherwise designed (5 '-AGA TCT CAT ATG AAA AAA TGGTCT TCC AAA CCT CG-3 '), S1R (GGA TCC GGC CTG AGG ATGACT G-3 ') are used for subclone preS1 gene, and 5 ' terminal or 3 ' end of primer has all added 1-2 restriction endonuclease recognition site.With pT-preS is template, and S1F-S1R is a primer amplification 357bppreS1 gene.The PCR product reclaims the back and is connected with pMD 18-T, obtains cloned plasmids pT-preS1.The molecular weight of considering preS1 is less, and we have selected pattern of fusion prokaryotic expression plasmid pGEX-20T as expression plasmid.GST can be used as the immunogenicity that carrier proteins improves hepatitis B antigen.The other benefit of selecting this plasmid for use is that this expression system has advantages such as stable, efficient, and the fusion rotein of expressing also is easy to carry out purifying with affinity chromatography.The product that the expression of recombinant plasmid that makes up with pGEX-20T comes out is the fusion rotein of GST and preS1, and wherein the GST albumen of 26kD is positioned at the aminoterminal of fusion rotein.BamH I/Ec0R I double digestion is handled expression plasmid pGEX-20T, and ethanol precipitation reclaims linear carrier.With Bgl II/EcoR I double digestion pMD 18-T plasmid, glue reclaims test kit and reclaims the preS1 fragment.With the T4 ligase enzyme purpose fragment is connected with pGEX-20T, obtains the pGEX-20T-preS1 plasmid.
The expression of recombinant protein in intestinal bacteria
With pGEX-20T-preS1 plasmid Transformed E .coli ER2566 bacterial strain, picking list colony inoculation is to LB (the containing the Ap microbiotic) liquid nutrient medium of 2ml from flat board, and 37 ℃ of shaking culture are spent the night.Get 200ul incubated overnight liquid and be inoculated in 2ml LB liquid nutrient medium, 37 ℃ of shaking culture 2 hours are to OD
6000.6 about.Add the IPTG final concentration and reach 0.5mM, 28 ℃ or 37 ℃ of inducing culture 4 hours.Get 1ml bacterium liquid, centrifugal collection thalline, button is done.With 60ul aqueous suspension thalline, add 30ul 3 * SDS-PAGE gel loading buffer, boiling water bath 5 minutes.Centrifugal 10 minutes of 12000g.Get sample on the 5ul cellular lysate liquid supernatant, use the 12%SDS-PAGE electrophoretic analysis.The full bacterium lysate of engineering bacteria can be observed a tangible inducible protein band as shown in Figure 1, and expressing protein is about 10% of bacterial protein, and the actual molecular weight size is approximate with theoretical expected value 39kD, and recombinant protein mainly exists with soluble form.
The purifying of soluble antigen
Choose single colony inoculation in 25ml LB (containing the Ap microbiotic), 37 ℃ of shaking culture are spent the night.250ml bacterium liquid is pressed 1: 4 expanding species, to 1000ml.37 ℃ of shaking culture are to OD
6001.0 about.Make the bacterium liquid cooling but, add IPTG to final concentration be 0.2mM, 28 ℃, induced 4 hours.Receive to stop thalline, centrifugal 4 minutes of 4 ℃ of 10000g.After the PBS washing, with an amount of PBS thalline is suspended again.Ice-water bath, ultrasonication.Ultrasound condition is as follows: output control-7, space factor-70%, working hour-35sec.Repeat each 5 minutes at interval 7-9 time.Collect supernatant and cross column purification.Jog medium bottle makes medium Glutathione-Sepharose 4B mixing, draws 1.33ml volume medium homogenate (the medium ratio is 75%, promptly contains the 1ml medium in the 1.33ml volume medium slurry) upper prop.Rap pillar and remove bubble, allow pillar drain off naturally.BVS balance columns with 4 ℃ of precoolings of 10ml.Block down the sample mouth, last sample.Hatched under the room temperature 30 minutes, continuous jog pillar therebetween is to reach with the good binding effect.Open down the sample mouth, allow penetrate under the peak spontaneous current, allow pillar drain off.1 * PBS with 10ml4 ℃ of precooling washes post, repeats 3 times.Stifled letter is the sample mouth down, adds the gsh elution buffer of 1ml, hatches under the room temperature 30 minutes, and continuous jog pillar therebetween is to reach good elute effect.Open down the sample mouth, collect sample with the centrifuge tube of 1.5ml.Repeat wash-out repeatedly, when when firmly shaking sample, not observing bubble till.SDS-PAGE result shows that the purity of the recombinant antigen behind (Fig. 1) purifying is more than 85%.
Embodiment 2: the generation of hybridoma cell line 4D11
The fusion of mouse immune and splenocyte and hybridoma
The Dane particle vein immunity female BALB/c mouse in 6～8 age in week of purifying is mixed back intramuscular injection immune mouse with 50ug recombinant antigen GST-preS1 behind the 2w with CFA.After 30 days, tail vein booster shots 5 μ g do not add the antigen of adjuvant, get eyeball blood examination survey and tire in 72 hours afterwards.Kill mouse, collect blood, get spleen and prepare splenocyte suspension (being suspended from RPMI 1640 substratum of precooling), cell counting count board cell counting.Get the SP2/0 murine myeloma cell of cultivation by 1/6 in the quantity of splenocyte, centrifugal after mixing, utilize polyoxyethylene glycol (PEG 1500) that splenocyte and murine myeloma cell SP2/0 are merged.With after isopyknic feeder cell mix, (200 μ l/ hole) is in 37 ℃ of cultivations of 5% CO2gas incubator (ESPEC BNA-311) in the 96 porocyte culture plates that are placed in cell suspension.After 3 days, ((hypoxanthn, H) (thymidine, T) 0.388mg add RPMI1640 (GIBCO company) substratum to 100mL to the 1.361mg+ thymidine to xanthine with the HT substratum.After dissolving under 45～50 ℃ of conditions, filtration sterilization.) partly keep and change liquid.After 7 days, by plate, utilize as following enzyme-linked immunosorbent assay (ELISA) and detect gained hybridoma supernatant nutrient solution in the 96 porocyte culture plates with 2147 synthetic peptide bags.For the cell clone of ELISA test positive, utilize limiting dilution assay to carry out cloning.
The ELISA detection method
The synthetic peptide of 21-47 is dissolved in 0.05mol/L carbonic acid bag and is cushioned liquid (20.02g Na2CO3,2.52g NaHCO3 adds deionized water to 1 liter, pH is 9.5) in, concentration is about 3 μ g/ml, 2 hours (37 ℃) of absorption place 4 ℃ to spend the night again on each hole surface of 96 hole polyethylene microtiter plates.With PBST washings (8.0g NaCl, 0.2g KH
2PO
4, 2.9gNa
2HPO
412H
2O, 0.2g KCl and 0.5ml polysorbas20 add deionized water to 1 liter, and pH is 7.4) the washing titer plate to be to remove unconjugated antigen protein.Use then in the confining liquid (1 * PBS solution, form the same) in 200 μ l/ holes and add 2% gelatin, 0.2% casein and 2% sucrose) 37 ℃ of sealings 2 hours.Get rid of clean, pat dry final vacuum sealing, 4 ℃ of preservations.
During detection, in every hole, add 100 μ l cell culture fluid supernatants; Every 96 hole microtiter plate is established a positive control, adds 100 μ l mouse anti GST-preS1 polyvalent antibodies (dilution in 1: 100 is used); Other establishes a negative control, adds 100 μ l HT cell culture fluids.37 ℃ of temperature were bathed 30 minutes, wash 5 times with the PBST washings, pat dry the back and add peroxidase-conjugated sheep anti mouse immunoglobulin (Ig) (HRP-GAM Ig, DAKO company), 37 ℃ of temperature were bathed 30 minutes, take out the back and wash 5 times with the PBST washings, (substrate solution A composition is: 13.42g Na successively to add each 50 μ l of substrate solution A, B after patting dry
2HPO
412H
2O, 4.2g citric acid H
2O and 0.3g hydrogen peroxide are 700ml with adjusted volume in the deionized water; Colour developing liquid B composition is: 0.2g tetramethyl benzidine, 20ml dimethyl formamide are 700ml with adjusted volume in the deionized water), 37 ℃ were developed the color 10 minutes, add 50 μ l stop buffer (2M H2SO4) termination reactions, and on microplate reader, detect the OD450 value in each hole, usually with the OD450 value be higher than negative control more than 2 times the person be considered as the positive.
The acquisition of monoclonal antibody ascites and purifying
Get the healthy Balb/c mouse in 10 ages in week, abdominal injection freund 's incomplete adjuvant, every 0.5ml.After 2～7 days, collect the hybridoma of cloning, the centrifugal supernatant that goes adds the substratum that does not contain serum, regulates cell density to 2 * 10
5～2 * 10
6Individual/ml, every injected in mice 0.5ml.Mouse web portion increases after 7～10 days, begins to collect ascites.Centrifugal 15 minutes of 3000rpm, the liquid of clarification part in the middle of drawing, the filtering with microporous membrane degerming of 0.45 μ m ,-20 ℃ of preservations after the packing.
The ascites of handling well is used PBS (the 81ml 0.2mol/LNa of 0.02mol/L, pH7.4
2HPO
419ml 0.2mol/L NaH
2PO
4, add physiological saline to 100ml) and the two-fold dilution, dropwise slowly add ammonium sulfate (concentration reaches 50% saturation ratio) under stirring, 4 ℃ are spent the night.4 ℃, centrifugal 15 minutes of 12000rpm abandons supernatant, precipitation is dissolved among the PBS of 2 times of former ascites volumes.Dropwise slowly add ammonium sulfate under stirring, make ammonium sulfate concentrations reach 33% saturation ratio, 4 ℃ of standing over night.4 ℃, centrifugal 15 minutes of 12000rpm abandons supernatant, precipitation is dissolved among the PBS of 2 times of former ascites volumes.Dropwise slowly add ammonium sulfate under stirring, (concentration reaches 50% saturation ratio), 4 ℃ are spent the night.4 ℃, centrifugal 15 minutes of 12000rpm abandons supernatant.Precipitation is dissolved among an amount of PBS, in packing into the dialysis band, puts into 50～100 times and contain 20mmol/L NaCl, desalination about 12 hours under 4 ℃ of stirrings in the 120mmol/L Tris-HCl damping fluid of pH7.8, during change dialyzate more than 3 times.Take out back packing-20 ℃ preservation.
With aforesaid method, filtered out 7 strains and secreted the hybridoma cell strain (3H5,1G5,6F1,7B6,2A6,7H11,4D11) of anti-HBV preS1 (21-47) monoclonal antibody.
By 10
9Vge/ hole bag is by Dane particle (contain a spot of tubular particle and tubular particle, be used as the HBV natural antigen and use), and 37 incubations 4 spent the night after 2 hours.PBST washes plate once, confining liquid 37 sealings 2 hours.Add the monoclonal antibody purification after the dilution, all samples is diplopore and repeats) 0.1ml in above-mentioned wrapped by reacting hole in, put 37 and hatched 30 minutes, PBST washing 5 times.In reacting hole, add sheep anti mouse enzyme labelled antibody 0.1ml.37 hatched 30 minutes, PBST washing 5 times.Add substrate solution colour developing termination reaction after 15 minutes, and on microplate reader, detect the OD450 value in each hole.Result (Fig. 2) shows that all 7 kinds of preS1 monoclonal antibodies all can be discerned the HBV natural antigen.As negative control, monoclonal antibody E1, the E2 of hepatitis E virus (HEV) can not discern viral natural antigen.Under the same concentrations (20ug/ml), in 7 kinds of preS1 monoclonal antibodies, the binding ability of 4D11 and HBV natural antigen is the strongest.
Separating of embodiment 4,4D11 monoclonal antibody light chain gene and heavy chain gene variable region
Half adherent culture 10
7Individual 4D11 mouse source hybridoma, blowpipe blows afloat attached cell and makes suspension, transfers in the new 4ml centrifuge tube, the centrifugal 3min of 1500rpm, the cell of collecting precipitation is resuspended among the aseptic PBS of 100 μ l (pH7.45), transfers in the new 1.5ml centrifuge tube.(Roche Germany), puts upside down mixing gently, leaves standstill 10min to add 800 μ l Trizol.Add 200 μ l chloroforms, thermal agitation 15s leaves standstill 10min, and 4 ℃ of centrifugal 15min of 12000rpm shift in the new 1.5ml centrifuge tube of supernatant liquid to, add isopyknic Virahol, and mixing leaves standstill 10min.4 ℃ of centrifugal 10min of 12000rpm abandon supernatant, add 600 μ l, 75% washing with alcohol, and 4 ℃ of centrifugal 5min of 12000rpm abandon supernatant, are deposited in 60 ℃ of vacuum and drain 5min.Transparent precipitation is dissolved in 70 μ l DEPC H
2Among the O, be distributed into two pipes, every pipe 35 μ l.Wherein 1 pipe adds each 0.5 μ l reverse transcription primer MFdR1 (5 '-ACT AGT ACA ATC CCT GGG CAC AAT-3 ')/MFdR2 (5 '-ACT AGT CTT GGG TAT TCT AGG CTC-3 '), and another pipe adds each 0.5 μ l reverse transcription primer MKCR1 (5 '-TCT AGA ATT AAC ACT CAT TCCTGT TGA A-3 ')/MVkR (5 '-CCC AAG CTT ACT GGA TGG TGGGAA GAT GGA-3 ').Every pipe adds 1 μ l dNTP (worker is given birth in Shanghai) again, put 72 ℃ of water-bath 10min, be put into the mid-5min of ice bath immediately, add 10 μ l 5x reverse transcription damping fluids, 1 μ l AMV (10u/ μ l, Pormega), 1 μ l Rnasin (40u/ μ l, Promega), behind the mixing in 42 ℃ of cDNA that the RNA reverse transcription become heavy chain Fd and light chain.
Antibody gene separation of polymeric polymerase chain reaction (PCR) method, use the Ig-Prime kits of Novagen company and design 5 upstreams of synthetic variable region of light chain primer in addition and 5 downstream weight strand primers (Bo Ya company in Shanghai is synthetic), template is the cDNA of above synthetic 4D11 heavy chain Fd and light chain.
The MuIgkV of K light chain is used in the separation of chain variable region gene
L7 groups of upstream primers of 5 '-A to G design 5 upstream primers of synthetic with other, be VkF1 (5 '-CCA CCA TggAgA CAg ACA CAC-3 ')/VkF2 (5 '-TTT TCA AgT gCA gAT TTTCAg-3 ')/VkF3 (5 '-(A/T) CT CAg gTC-3 ' of ATg gAg (A/T) CA CA (g/T))/VkF4 (5 '-(A/g) CT CAg-3 ' of TCC ACC ATg (g/T) CC CC (A/T))/VkF5 (5 '-ACC ATg AAg TTg CCT gTT Agg-3 '), downstream primer is MVJkR (5 '-CCg TTT (T/g) AT (T/C) TC CAg CTT ggT (g/C) CC-3 ').It is right to use these upstream primers and MVJkR to form primer respectively, is that template is carried out pcr amplification with the 4D11 light chain cdna.The PCR condition is: 94 ℃ of 5min, 72 ℃ of 40s35 circulations of 52 ℃ of 1min of 94 ℃ of 30s, 72 ℃ of 15min.The result has 5 upstream primers and MVJkR combination can amplify with expecting fragment of the same size to reclaim and be cloned into pMD 18-T carrier respectively, deliver to the order-checking of Shanghai Bo Ya company, and sequence is definite by MuIgkV through blast comparison back
L5 '-G2 (5 '-CGA CAT GGT (C/T) CT (C/T) AT (A/C/G) TC CTT GCT GTTCTG G-3 ')/MVJkR and two pairs of primers of VkF5/MVJkR can both amplify 4D11 light chain variable region sequence, (seeing SEQ ID NO:4), the aminoacid sequence of its supposition is seen SEQ IDNO:2.
The MuIgGV of IgG is used in the separation of heavy chain variable region gene
H6 groups of upstream primers of 5 '-A to F, downstream primer are MVDJhR (5 '-C ggT gAC Cg (T/A) ggT (C/g/T) CCTTg (g/A) CC CCA-3 '), and template is the cDNA of 4D11 heavy chain Fd, carry out the grads PCR amplification.The pcr amplification condition is 94 ℃ of 5min, 72 ℃ of 40s of 55 ℃ of 40s of 94 ℃ of 30s, 35 circulations, 72 ℃ of 15min.The result has 5 upstream primers and MVDJhR combination can amplify about 400bp band, reclaims and be cloned into pMD 18-T carrier respectively, delivers to the order-checking of Shanghai Bo Ya company, and sequence is determined MuIgGV after the blast comparison
H5 '-C2 (5 '-CGA CATGG (A/C/G) TTG G (C/G) T GTG GA (A/C) CTT GC (C/T) ATTCCT-3 ')/MVDJhR, MuIgGV
H5 '-C2 (5 '-CGA CAT GG (A/C/G) TTGG (C/G) T GTG GA (A/C) CTT GC (C/T) ATT CCT-3 ')/the MVDJhR primer is to amplifying 4D11 weight chain variabl area sequence (seeing SEQ ID NO:3), and the aminoacid sequence of its supposition is seen SEQ ID NO:1.
The clone of embodiment 5,4D11 single-chain antibody, expression, purifying and determination of activity
The heavy chain and the variable region of light chain of 4D11 antibody gene are passed through (GGGGS)
3Small peptide connects into the single-chain antibody dna fragmentation.With 4D11HF1 (5 '-ggA TCC CAG ATT CAGCTG CAG CAG TC-3 ')/4D11HR1 (5 '-gCT ACC ACC CCC TCC AgATCC gCC ACC TCC AgA AgA TTC CgT gAC CgA g-3 ') is primer to amplifying 4D11 heavy chain gene variable region fragment, with 4D11KF1 (5 '-A TCT ggA ggg ggTggT AgC ggT ggA ggC ggg AgT gAT gTT gTg ATg ACC C-3 ')/4D11KR1 (5 '-gTC gAC CCgTTT gAT TTC CAg CTT gg-3 ') primer to amplifying 4D11 light chain gene variable region fragment.Reclaim two fragments respectively, primer and template are carried out overlapping extension in new PCR system each other with these two fragments again, obtain a small amount of complete single chain antibody fragments, be template with complete fragment then, with 4D11HF1/4D11KR1 is that primer increases in a large number, reclaim single chain antibody fragments, be cloned in the pMD 18-T carrier.From the plasmid that obtains, receive single chain antibody fragments again, be cloned in the pTO-T7 prokaryotic expression carrier that same enzyme cuts with the switchback of BamH I/Sal I enzyme.With the recombinant plasmid pTO-T7-4D11 Transformed E R2566 coli strain that obtains, choose single bacterium colony in 500ml LB (containing Kan100ug/ml) substratum, 37 ℃ of shaking culture are spent the night, bacterium liquid OD600 value reaches about 0.8, and the IPTG of 0.2mmol/L induces, and gathers in the crops thalline behind 30 ℃ of expression 6hr, ultrasonication, the centrifugal 10min of 15000rpm, precipitation be dissolved in the isopyknic 20mmol/L Tris-HCl of supernatant (pH7.6) in, get that cleer and peaceful precipitation solution carries out the SDS-PAGE electrophoresis on the equivalent.Spacer gel concentration is 5%, and resolving gel concentration is 12%.Find that marking protein reaches about 15% of bacterial protein amount, mainly exist with insoluble inclusion body form.After ultrasound precipitation was used the 2%Triton washed twice, with 2M, 4M, the dissolving of 8M urea, the each several part lysate found that through the 12%SDS-PAGE electrophoresis single-chain antibody mainly is dissolved in (Fig. 3) in the 8M urea respectively.The single chain antibody protein that is dissolved in the 8M urea is diluted to 50-100ng/ml with 8M urea, progressively to 1x PBS dialysis renaturation, reconcentration is 6 times afterwards, and 12000rpm removed precipitation in centrifugal 10 minutes, and the single-chain antibody solution that finally obtains is carried out determination of activity with indirect elisa method.The antigen of bag quilt is the synthetic peptide of the 21-47 of preS1, and single-chain antibody just pure product is respectively got 100ul to the hole behind the multiple proportions gradient dilution, react 1 hour after scouring, add the goat-anti Histag enzyme labelled antibody of 1: 2000 dilution HRP mark again, react after 30 minutes and developed the color 15 minutes, read plate.The result just OD value of pure product stoste is 3.210, is 0.687 (table 1) after diluting 8 times.The 4D11 single-chain antibody that shows expression has the activity of excellent specificity conjugated antigen.
Table 1. indirect ELISA detects the antigen-binding activity of 4D11 scFv
The first pure product of 4D11 scFv further through the His column purification, are carried out affinity constant mensuration to improve purity.The His post is given a baby a bath on the third day after its birth time with deionized water, uses 1x extracting Buffer (pH7.0PBS) to wash then one time.Suspension Talon resin is inhaled 1ml in pillar, removes ethanol, and the PBS of usefulness 20ml is the balance pillar at twice.Remove PBS, add good 4D11-scFv (0.283mg/ml) and the 2ml PBS of 2.5ml renaturation, mixing is put 4 ℃ of 2h.Collection penetrates the peak, washes 3 times each 2 minutes then with PBS.(PBS contains the 5mM imidazoles, and pH7.0) 1.2ml is to pillar, and mixing leaves standstill 5min, and the collection washings repeats this step 3 time, all collects elutriant to add 1x washing Buffer.(PBS contains the 150mM imidazoles, and pH7.0) 1.2ml is to pillar, and mixing leaves standstill 5min, collects elutriant, repeats this step 3 time, all collects elutriant to add 1x wash-out Buffer.Add 5ml MES regeneration.Collected solution is carried out the SDS-PAGE electrophoresis, silver dye result (Fig. 4) as can be known the purity of 4D11 scFv behind the His column purification reach more than 95%, each several part in the purge process is collected liquid carry out the indirect ELISA detection, the results are shown in Table 2, find for the first time and elutriant active higher for the second time OD
420Be respectively 1.403 and 1.008.
Table 2. indirect ELISA detects the activity that each several part is collected liquid in the His column purification process
Wash secondary again and again and give a baby a bath on the third day after its birth that time to wash elutriant right
Stoste penetrates peak regeneration
Washing Tuo Tuotuo shines
OD
420 3.275 0.109 0.033 1.403 1.008 0.183 0.055 0.044
Sequence table
＜110〉Xiamen University
Yangshengtang Co., Ltd
＜120〉hepatitis B virus monoclonal antibody variable region sequences and contain genetic engineering antibody of described variable region and uses thereof
<130>IDC030094
<160>4
<170>PatentIn?version?3.1
<210>1
<211>119
<212>PRT
<213>artificial
<400>1
Gln?Ile?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Glu?Leu?Val?Lys?Pro?Gly?Thr
1 5 10 15
Ser?Val?Lys?Ile?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Asp?Tyr
20 25 30
Tyr?Ile?Asn?Trp?Val?Lys?Gln?Lys?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile
35 40 45
Gly?Trp?Ile?Phe?Pro?Gly?Ser?Gly?Asn?Ile?Lys?Tyr?Asn?Glu?Asn?Phe
50 55 60
Lys?Gly?Lys?Ala?Thr?Leu?Thr?Val?Asp?Thr?Ser?Ser?Asn?Thr?Ala?Tyr
65 70 75 80
Met?Gln?Leu?Ser?Ser?Leu?Thr?Phe?Glu?Asp?Thr?Ala?Val?Tyr?Phe?Cys
85 90 95
Thr?Arg?Pro?Ala?Asn?Met?Ile?Thr?Thr?Leu?Ala?Tyr?Trp?Gly?Gln?Gly
100 105 110
Thr?Ser?Val?Thr?Glu?Ser?Ser
115
<210>2
<211>113
<212>PRT
<213>artificial
<400>2
Asp?Val?Val?Met?Thr?Gln?Thr?Pro?Leu?Ser?Leu?Pro?Val?Ser?Leu?Gly
1 5 10 15
Asp?Gln?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Leu?Val?Tyr?Ser
20 25 30
Asn?Gly?Asn?Thr?Tyr?Leu?His?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser
35 40 45
Pro?Lys?Leu?Leu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro
50 55 60
Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile
65 70 75 80
Ser?Arg?Leu?Glu?Ala?Glu?Asp?Leu?Gly?Val?Tyr?Phe?Cys?Ser?Gln?Thr
85 90 95
Thr?His?Val?Pro?Pro?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys
100 105 110
Arg
<210>3
<211>357
<212>DNA
<213>genome
<400>3
cagatccagc?tgcagcagtc?tggacctgag?ctggtgaagc?ctgggacttc?agtgaagata 60
tcctgcaagg?cttctggcta?caccttcact?gactactata?taaactgggt?gaagcagaag 120
cctggacagg?gacttgagtg?gattggatgg?atttttcctg?gaagcggtaa?tattaagtac 180
aatgagaact?tcaagggcaa?ggccacattg?actgtagaca?catcctccaa?cacagcctac 240
atgcaactca?gcagcctgac?atttgaggac?actgctgtct?atttctgtac?aagacccgct 300
aatatgatta?cgacgcttgc?ttactggggc?caaggaacct?cggtcaccga?atcttct 357
<210>4
<211>339
<212>DNA
<213>genome
<400>4
gatgttgtga?tgacccaaac?tccactctcc?ctgcctgtca?gtcttggaga?tcaagcctcc 60
atctcttgca?gatctagtca?gagccttgta?tacagtaatg?gaaacaccta?tttacactgg 120
tacctgcaga?agccaggcca?gtctccaaag?ctcctgatct?acaaagtttc?caaccgattt 180
tctggggtcc?cagacaggtt?cagtggcagt?ggatcaggga?cagatttcac?actcaagatc 240
agcagactgg?aggctgagga?tctgggagtt?tatttctgct?cteaaactac?acatgttcct 300
ccgacgttcg?gtggaggcac?caagctggaa?atcaaacgg 339
Claims (11)
1. but specificity is in conjunction with the variable region sequences of the mouse monoclonal antibody of hepatitis B virus preS1, wherein: 1) the weight chain variable region amino acid sequence shown in SEQ ID NO:1, or the conservative property varient that obtains through one or more aminoacid addition, deletion, replacement, the sudden change of modification conservative property of this sequence; 2) the light chain variable region amino acid sequence is shown in SEQ ID NO:2, or the conservative property varient that obtains through one or more aminoacid addition, deletion, replacement, the sudden change of modification conservative property of this sequence.
2. one kind by variable region of heavy chain part in the described variable region sequences of claim 1 or its examples of conservative variations, and/or the genetic engineering antibody that is assembled into of the aminoacid sequence of variable region of light chain part or its conservative property varient, it still keeps the ability of specific combination hepatitis B virus preS1.
3. the described genetic engineering antibody of claim 2, it is for comprising weight chain variable region amino acid sequence or its conservative property varient in the described variable region sequences of claim 1, and/or single-chain antibody, the chimeric mAb of light chain variable region amino acid sequence or its conservative property varient, change the monoclonal antibody of shape monoclonal antibody or other humanization forms, or the fragment of described antibody, it still keeps the ability of specific combination hepatitis B virus preS1.
4. one kind comprises at least a fusion rotein that is selected from described genetic engineering antibody of claim 2 or described antibody fragment, and it still keeps the ability of specific combination hepatitis B virus preS1.
5. the described fusion rotein of claim 4, it is a bispecific antibody.
6. mixture, it comprises and is selected from the described genetic engineering antibody of at least a claim 2 or it keeps the fragment of the ability of specific combination hepatitis B virus preS1, and the drug molecule of one or more treatment hepatitis B of phase link coupled with it.
7. nucleic acid molecule, the variable region of heavy chain of the described variable region amino acid sequence of its coding claim 1, it has nucleotide sequence or its degeneracy sequence shown in SEQ ID NO:3; Perhaps variable region of light chain of its coding claim 1 described variable region amino acid sequence, it has nucleotide sequence or its degeneracy sequence shown in SEQ ID NO:4.
8. recombinant expression vector that comprises the described nucleic acid molecule of claim 7.
9. one kind through the described recombinant expression vector transformed host cells of claim 8, and it can express the described genetic engineering antibody of claim 2 or it keeps the fragment of the ability of specific combination hepatitis B virus preS1.
10. the described genetic engineering antibody of claim 2 or its fragment that keeps the ability of specific combination hepatitis B virus preS1 is used to the purposes of the medicine for preparing the diagnosis, prevent and/or treat hepatitis B virus.
11. a pharmaceutical composition, it comprises the described genetic engineering antibody of the claim 2 for the treatment of significant quantity or it keeps fragment or its examples of conservative variations of the ability of specific combination hepatitis B virus preS1, and pharmaceutically acceptable carrier and/or adjuvant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310101630 CN1609123A (en) | 2003-10-23 | 2003-10-23 | Variable region sequence of hepatitis B virus monoclonal antibody, gene engineering antibody containing the variable region and its use |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310101630 CN1609123A (en) | 2003-10-23 | 2003-10-23 | Variable region sequence of hepatitis B virus monoclonal antibody, gene engineering antibody containing the variable region and its use |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810178602A Division CN101538326A (en) | 2003-10-23 | 2003-10-23 | Variable region sequence of hepatitis B virus monoclonal antibody, gene engineering antibody containing variable region and application thereof |
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| CN1609123A true CN1609123A (en) | 2005-04-27 |
Family
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|---|---|---|---|
| CN 200310101630 Pending CN1609123A (en) | 2003-10-23 | 2003-10-23 | Variable region sequence of hepatitis B virus monoclonal antibody, gene engineering antibody containing the variable region and its use |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100341890C (en) * | 2004-07-12 | 2007-10-10 | 中国科学院上海生命科学研究院 | Peptide specifically associated with front surface antigen zone of large protein of human hepatitis B virus surface antigen and uses thereof |
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2003
- 2003-10-23 CN CN 200310101630 patent/CN1609123A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100341890C (en) * | 2004-07-12 | 2007-10-10 | 中国科学院上海生命科学研究院 | Peptide specifically associated with front surface antigen zone of large protein of human hepatitis B virus surface antigen and uses thereof |
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Effective date of registration: 20071228 Address after: Postal code No. 422, Siming South Road, Siming District, Xiamen, Fujian, China: 361005 Applicant after: Xiamen University Co-applicant after: Beijing Wantai Biological Pharmaceutical Co., Ltd. Address before: Postal code No. 422, Siming South Road, Siming District, Xiamen, Fujian, China: 361005 Applicant before: Xiamen University Co-applicant before: Yangshengtang Co., Ltd |
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