CN103588874A - Human anti-HBV surface antigen genetic engineering antibody, and preparation method and application thereof - Google Patents
Human anti-HBV surface antigen genetic engineering antibody, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a human anti-HBV surface antigen genetic engineering antibody, and a preparation method and application thereof. According to the invention, the phage surface display technology is adopted, peripheral blood lymphocyte of person having high titer surface-antibody after immunization of hepatitis B vaccine is collected is obtained, a human anti-HBV surface antigen genetic engineering antibody library is established through a genetic engineering means, and a Fab section of the specific anti-HBV surface antigen genetic engineering antibody is obtained through screening. The obtained Fab section of the antibody is named to be HBFab21. The amino acid sequence of a light chain variable region of the HBFab21 is shown in SEQ ID No.1, and the amino acid sequence of a heavy chain variable region of the HBFab21 is shown in SEQ ID No.2. According to the invention, a foundation for hepatitis B virus infection prevention and researches on treatment to hepatitis B virus is laid; through the foundation, an antibody product having an effect of neutralizing hepatitis B virus infection is obtained, and can be applied to preparation of a clinical drug or diagnostic reagent for liver diseases relating to hepatitis B virus.
Description
Technical field
The present invention relates to genetic engineering antibody technology, particularly relate to a kind of people source anti-hepatitis B virus surface antigen genetic engineering antibody; The invention still further relates to the application of this antibody in preparation prevention or treatment hepatitis B virus related liver disease medicine.
Background technology
Hepatitis B virus infection is a worldwide main public health problem, and acute hepatitis B has later the lasting HBV of 5%-10% to infect to develop into chronic hepatopathy and comprises chronic active hepatitis, liver cirrhosis and primary hepatocellular carcinoma.The whole world has 500,000 to 1,000 every year according to estimates, and 000 people dies from the various hepatic diseases relevant with HBV.China is hepatitis big country, and hepatitis B virus carrying rate is population 9.75%.Approximately there are 1.3 hundred million people's Hepatitis B carriers in whole world Patients with Hepatitis B Virus Infection 4.2Yi， China.The circulation way of hepatitis B virus is broadly divided into vertical transmission and the large class of horizontal transmission two.Vertical transmission refers to the propagation of HBV mother carrier to fetus, and the overwhelming majority occurs in last three months of gestation.Once baby infects hepatitis B virus, in them, 85%-90% can develop into chronic band hepatitis B virus state, wherein 25% after growing up, will die from liver cirrhosis and liver cancer.99 years studies show that, China's Patients with Hepatitis B Virus Infection namely has 80%-85% to derive from the infection of father and mother in reproduction growing process in hepatitis B virus carriers and chronic hepatitis B.Horizontal transmission may be summarized to be in perinatal transmission, family propagation, iatrogenic infection and spreads through sex intercourse, and wherein perinatal transmission is more serious.
About the treatment of chronic hepatitis B, going back at present the gratifying method of neither one, for the prejudicial patient of liver function, by the liver protecting therapy overwhelming majority, can make liver function be restored, is very difficult but will remove hepatitis B virus.Chronic carrier is all the more so for hepatitis B virus.Its reason is mainly immunological tolerance, and namely the vivo immuning system of Hepatitis B Virus Infection can not produce effective scavenging(action) to hepatitis B virus.The reason of immunological tolerance be human infection hepatitis B virus time early, generally believed before 3 years old and infect.
Before not finding suitable methods for the treatment of, stop virus infection just to seem very important.At present for the prevention of hepatitis B virus, there are two kinds of active immunity and passive immunizations.Active immunity inject Hepatitis B virus vaccine, occurs first hemogenic vaccine from the end of the seventies, through developing into of decades, had now ripe recombinant vaccine.The good immune effect of Hepatitis B virus vaccine is one of effective means of prevention hepatitis B propagation.Passive immunization is injection hepatitis B immune globulin (HBIG), is mainly used in blocking the passive immunization of mother-to-baby transmission (using with combined with hepatitis B vaccine) and mishap.There are some researches show that the combined with hepatitis B vaccine of employing HBIG blocking-up mother-to-baby transmission effect is better than alone vaccine person and can reduces chronically infected ratio.HBIG all derives from the positive serum of the anti-HbsAg of people at present, and this just makes its a large amount of preparations be restricted, so easily there is the pathophorous infection of haematogenous owing to deriving from serum simultaneously.As haematogenous vaccine originally, to the transformation of recombinant vaccine, be also badly in need of now substituting haematogenous HBIG with genetically engineered hepatitis B surface antibody, the solution that to have a liking for somatic antibody storehouse technology be this problem looks for another way.
The people source of containing specific antibody or animal serum immunoglobulin (Ig) are with a long history in order to prevention and treatment transmissible disease.In the extracorporeal antivirus effect of monoclonal antibody and active and endogenous protective human body opposing virus attack obtained many experiment showed, and can be in vivo 100% watch for animals and avoid virus attack as mouse-anti hepatitis A virus (HAV), Hantaan virus, Measles virus, the neutralizing monoclonal antibody such as RSV is viral, CMV is viral.The approach that obtains polyvalent antibody with antigen-immunized animal is the classical way that obtains antibody always, but lacks specificity and homogeneity.Then the B lymphocyte hybridoma technology of setting up makes numerous scientists can directionally prepare in vitro various monoclonal antibodies (monoclonal antibody, McAb) by cell engineering, its high specificity, and character homogeneous, is easy to a large amount of production.Yet McAb mostly is mouse, the reaction of the heterology of mouse source McAb has greatly limited McAb application at human body as treatment preparation.Immunoglobulin (Ig) (Vaccinia immune globulin, VIG) as antibody component mainly from donor (convalescent) immune serum, from obtaining positive serum, to detecting by security, all need to spend a large amount of manpower and financial resources, this just makes its a large amount of preparations be restricted, so easily there is the pathophorous infection of haematogenous owing to deriving from serum simultaneously.Therefore end user source gene engineering product Blood substitute goods can overcome these defects, and deepening continuously of human genetically engineered antibody research brought new hope and bright prospects to the biological products development in this field.Restructuring by antibody molecule gene level can obtain diversified specific murine source and human antibody, and making has had breakthrough and more and more demonstrated its significance and practice prospect the research of monoclonal antibody.
The phage antibody gene pool technology rise of rising the beginning of the nineties at the end of the eighties and the source, development ，Shi people from world today in whole genetic engineering antibody technical study field or the development research of genetic engineering antibody obtain remarkable progress and by phase of basic research, step into substantive applied research and development phase.People's source antivirus genetic engineering antibody, especially the research of people source whole antibody success, brought new hope to specificity prevention and the treatment of various viral infectious, in anti-virus infection biological medicament field, formed gradually the new antiviral drug of a class, i.e. so-called antibody medicine (Antibody Drug).As haematogenous vaccine originally to the transformation of recombinant vaccine; also be badly in need of now substituting haematogenous VIG with genetic engineering antibody; as passed through chimeric antibody technology (Boulianne GL; Hozumi N, Shulman MJ.Production of functional chimaeric mouse/human antibody.Nature.1984 Dec 13-19; 312 (5995): 643-6; Morrison SL, Oi VT.Transfer and expression of immunoglobulin genes.Annu Rev Immunol.1984; 2:239-56.), humanized antibody technology (Jones PT; Dear PH; Foote J; Neuberger MS; Winter G., Replacing the complementarity-determining regions in a human antibody with those from a mouse.Nature.1986 May 29-Jun 4; 321 (6069): 522-5.), the transgcnic mouse techniques (Green of carrier's monoclonal antibody; L.L.et al., Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs.Nat Genet.1994 May; 7 (1): 13-21.), allosome hybridoma technology (James, K.et al.Human monoclonal antibody production.Current status and future prospects.J Immunol Methods.1987 Jun 26; 100 (1-2): 5-40.), phage display technique (Barbas, C.F.et al.Assembly of combinatorial antibody libraries on phage surfaces:the gene III site.Proc Natl Acad Sci U S is Sep 15 A.1991; 88 (18): 7978-82.) etc. produce humanized antibody, now become the great direction of domestic and international research, and progressively led to success.
Summary of the invention
First object of the present invention is to provide a kind of people source anti-hepatitis b surface antigen gene engineered antibody and active fragments thereof.
Second object of the present invention is to provide the gene of the above-mentioned antibody of coding or its active fragments.
The 3rd object of the present invention is to provide above-mentioned antibody and active fragments thereof in preparation prevention or the medicine for the treatment of hepatitis B virus related liver disease or the application in diagnostic reagent.
The present invention uses phage surface to present technology, the peripheral blood lymphocyte after collection hepatitis b vaccine immune with high titre surface antibody person, by genetic engineering means, built anti-hepatitis B virus surface antigen genetic engineering antibody library, people source, and screening obtains special anti-hepatitis B virus surface antigen genetic engineering antibody Fab section.The Fab section antibody called after HBFab21 obtaining.
This strain recombinant antibodies is to be determined by hypervariable region (CDRs) the specific gene sequence being present in light chain of antibody and heavy chain gene variable region, and in prokaryotic cell prokaryocyte, obtains the functional antibodies of the specific binding hepatitis B virus of effective expression.Their specific recognition hepatitis B surface antigen, and all for " a " determinant, have with hepatitis B virus surface antigen that obvious enzyme linked immunological (ELISA) reacts and higher avidity, have stop HBV infect sensitive cells in and function.
The specific light chain of HBFab21 and heavy chain variable region gene derive from the rich long-pending screening of the specificity in anti-hepatitis B virus surface antigen antibody gene storehouse, people source, and the foundation of this antibody library derives from Chinese hepatitis b vaccine immune person peripheral blood lymphocyte gene.Between corresponding three the CDR region sequences combination in its light chain and variable region of heavy chain and CDR district thereof, framework region sequence has formed each antibody variable region sequence signature, and HBFab21 is under the jurisdiction of the VH4， of the heavy chain of antibody family light chain of antibody VK3 of family.Antibody protein function specificity nucleotide sequence and complementary institute thereof in being present in the complementary region CDR1 of decision family, the CDR2 of antibody gene light chain and variable region of heavy chain and CDR3 determine, 6 corresponding CDR region amino acid sequences have formed the specific antigens calmodulin binding domain CaM of antibody, determine that the antigen of antibody in invention is in conjunction with feature and hepatitis B virus resisting functional character.Determine that the light chain of antibody of every strain neutralizing antibody function and variable region of heavy chain amino acid detailed sequence and comparative result thereof are as shown in table 1:
Table 1 light chain of antibody and variable region of heavy chain amino acid detailed sequence and comparative result thereof
The aminoacid sequence of HBFab21 variable region of light chain is as shown in SEQ ID No.1, and the aminoacid sequence of its variable region of heavy chain is as shown in SEQ ID No.2.
The gene order of coding HBFab21 variable region of light chain is as shown in SEQ ID No.3, and the gene order of its variable region of heavy chain is as shown in SEQ ID No.4.
Be to be understood that, do not affecting under the prerequisite of Fab antibody activity, those skilled in the art can carry out various replacements, interpolation and/or lack one or several amino acid obtaining the aminoacid sequence with same function to the aminoacid sequence shown in SEQ ID No.1-2, for example in non-hypervariable region, the amino acid with similarity is replaced, as the Gly of the 14th of the light chain VL sequence of HBFab21 is replaced with to Ala.
In addition, consider the degeneracy of codon, for example, can not change under the condition of aminoacid sequence in its coding region, the gene order of the above-mentioned Fab section antibody of encoding is modified, obtain the gene of coding same antibody.Those skilled in the art can be according to the codon-bias of expressing antibody host, and synthetic modifying gene, to improve the expression efficiency of antibody.
Further, the present invention recombinates the variable region of light chain of above-mentioned Fab antibody and variable region of heavy chain, obtains the less single-chain antibody (ScFv) of molecular weight, and this antibody equally can specific recognition hepatitis B virus surface antigen, has the effect of intracellular immunity.Single-chain antibody penetration power is strong, is easy to enter local organization and plays a role.
Can be by the gene of above-mentioned coding Fab antibody, ScFv gene clone in expression vector, and then transform host, by abduction delivering, obtain Fab antibody and single-chain antibody.
In addition, the light chain encoding gene of above-mentioned Fab antibody and heavy Fd fragment gene can be cloned in complete anti-expression vector, and import in host cell, obtain the full anti-immunoglobulin of expressing anti-hepatitis B virus surface antigen.
In an embodiment of the present invention, the light chain of above-mentioned Fab antibody (HBFab21) and heavy Fd fragment gene are cloned into respectively to intact antibody carrier pAC-K-Fc transfection insect Sf 9 cells, utilize baculovirus/insect cell system to realize the secretion type expression of whole antibody, obtain whole antibody HBIgG21.
Utilize ELISA, SDS-PAGE, BIAcore, competition blocking experiment and cell in vitro to learn experiment the whole antibody obtaining is carried out to Function Identification, result shows that this strain humanized IgG whole antibody (HBIgG21) all has specific binding for hepatitis B surface antigen; HBIgG21 demonstrates higher avidity to hepatitis B surface antigen, and affinity constant KD (M) is respectively 3.76 * 10
-9.Competition blocking experiment and cell in vitro are learned experiment and have been confirmed that this strain human antibody (HBIgG21) has the function that stops HBV to infect sensitive cells, has neutralization active.The acquisition of this result is not only for stoping hepatitis B virus infection, simultaneously also for the research of its treatment is laid a good foundation.
The present invention uses phage antibody library technique, has successfully obtained the people source neutrality antibody of 1 strain specificity for hepatitis B virus surface antigen; Utilize the full-antibody gene under people source neutrality anti-hepatitis B virus surface antigen genetic engineering antibody variable region gene, Fab antibody gene and above-mentioned each antibody gene feature of above-mentioned acquisition, can in prokaryotic cell prokaryocyte, yeast cell, eukaryotic cell and any recombination system, express and produce any other gene that contains this antibody gene after this antibody or reconstruction based on this, during acquisition has and the antibody product of hepatitis B virus infection, make clinically the application for medicine or the diagnostic reagent of hepatitis B virus related liver disease.
Accompanying drawing explanation
Fig. 1 is that ELISA detects the specific binding that human Fab's antibody of expressing resists human Fab and HBsAg, and wherein 1-30 is respectively 30 positive colonies;
Fig. 2 is the SDS-PAGE electrophorogram of IgG after purifying, and A is HBIgG21, and B is HBIgG07;
Fig. 3 and Fig. 4 are the results that application surface plasma resonance technology is measured people source anti-hepatitis B virus surface antigen Affinity to MoAbs;
Fig. 5 is the indirect immunofluorescene assay of the HepG2 cell under different condition, and wherein A, D group is green fluorescence, and B, C group is red fluorescence;
Fig. 6 is that in HepG2 cell, HBV Pres1DNA detects, D1 is that mouse monoclonal antibody extent of dilution with HBV is hatched postoperative infection HepG2 at 1: 400, D2 is that mouse monoclonal antibody extent of dilution with HBV is hatched postoperative infection HepG2 at 1: 800, A is HBV infection group and viral infection group, B is that HBIgG21 and HBV are hatched postoperative infection HepG2, and C is that HBIgG07 and HBV are hatched postoperative infection HepG2.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
1. virus, cell, carrier: hepatitis B viruses (HBV) is provided by China Sickness Prevention Control Center Virus Disease Prevention Control Institute's hepatitis chamber.Human hepatoma cell line HepG2 (ATCC); Insect cells Sf9 is from U.S.'s cell cultures center (ATCC).Bacterial strain is XLI-Blue(U.S. Stratagene), carrier is pComb3H(40kb) present of ，You U.S. Scripps institute.Rhabdovirus expression vector is pAC-K-Fc(Germany PROGEN PR3003) (Liang, M.F., Stefan, D., Li, D.X., Queitsch, I., Li, W., and Bautz, E.F.Baculovirusexpression cassette vectors for rapid production of complete human IgG from phage displayselected antibody fragments.Journal of Immunological Methods.247:119-130).Hepatitis B surface antigen(HBsAg) (CHO-HBsAg) is purchased from the safe biological Pharma Inc. in Beijing ten thousand; Mouse-anti hepatitis B surface antigen monoclonal antibody 3E7(Mouse Anti-Hepatitis B Surface Antigen HbsAg, Clone3E7) purchased from DAKO company.Hepatitis b surface antigen positive serum: hepatopathy institute of You Youan hospital is so kind as to give.
2. antigen preparation: separated HBV virion:
CsCl
2bed course 4ml, adds 6ml serum specimen above, 30000 revs/min of ultracentrifugations, centrifugal 4 hours; Abandon supernatant, 1ml 1 * PBS will manage end precipitation and hang, and 4 ℃ of placements are spent the night; 12000 revs/min instantaneous centrifugal, and retaining supernatant is HBV viral suspension.
3. the structure of phage antibody library: with lymphocyte separation medium (U.S. Sigma) separated lymphocyte from there is vaccination person's the anticoagulated blood of high titre hepatitis B virus antibody, with RNeasy Mini Kit(Germany QIAGEN) extract total cell RNA, with Oligo-dT primer, adopt the first chain synthetic agent box (SuperScriptTM III First-Strand Synthesis System for RT-PCR.Cat No.18080-051) reverse transcription of Invitrogen company to become cDNA the RNA extracting, with one group of amplification human antibody IgG1 heavy chain Fd and light chain Kappa and Lambda primer, people's endogenous light chain and heavy chain Fab gene are carried out to pcr amplification.PCR condition is: 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 2min, 35 circulations.Banking process carries out (Barbas by document substantially; C.F III.; Kang; A.S.; Lerner; R.A, and Benkovic, S.J.Assembly of combinatorial antibody libraries on phage surface:the gene III site.Proc.Natl.Acad.Sci.USA.1991; 88 (18): 7978-7982).
4. the abduction delivering of the enrichment of phage antibody library screening and Fab section antibody: screening antigen is purifying hepatitis B surface antigen(HBsAg) (CHO-HBsAg).0.1m/LNaHCO during use
3(pH8.6) solution dilution, coated immunity pipe, with 4% skimmed milk-PBS, after 37 ℃ of sealing 2h, add above-mentioned phage antibody library, every pipe 1ml, hatch 2h for 37 ℃, with 5%Tween-20-TBS, repeatedly wash 20 times, finally use glycine-hydrochloric acid elutriant wash-out of every pipe 1ml pH2.2, the Tris liquid neutralization of pH9.6.Phage after wash-out continues to infect the fresh OD of 2ml
600the XLI-Blue bacterium of=1.0 left and right, through helper phage VCSM13(U.S. Stratagene) carry out next round screening after infecting.So repeated screening is 4～5 times.The abduction delivering of concrete enrichment screening method and Fab section carries out (Barbas by document substantially; C.F III.; Kang; A.S.; Lerner; R.A, and Benkovic, S.J.Assembly of combinatorial antibody libraries on phage surface:the gene III site.Proc.Natl.Acad.Sci.USA.1991; 88 (18): 7978-7982).
5. the ELISA of people source anti-hepatitis B virus surface antigen Fab antibody detects
The detection 0.1m/L NaHCO that 5.1Fab expresses
3(pH9.6) in enzyme plate, 4 ℃ are spent the night the coated anti-human Fab antibody of solution (U.S. Sigma, 1 ﹕ 2000 dilutions are used); 4% skimmed milk sealing, 37 ℃ of 1h, add the Fab antibody of expression, 37 ℃ of 1h; Add the anti-human Fab bis-of enzyme mark anti-(U.S. Sigma, 1 ﹕ 2000 dilutions are used), 37 ℃ of 1h; Nitrite ion colour developing, 2M H
2sO
4termination reaction, microplate reader detects absorbance A value.
5.2 Dot-ELISAs detect Fab is combined activity use purifying hepatitis B surface antigen(HBsAg) (CHO-HBsAg) as envelope antigen with hepatitis B surface antigen, step is subsequently the same.
6. the nucleic acid sequence analysis of people source Fab antibody variable gene: with Qiagen Miniprep Kit(Germany QIAGEN) preparing plasmid DNA carries out nucleic acid sequence analysis.The sequencing primer of weight chain is respectively 5 ＇-AAACTAGCTAGTCGCCAAGGA-3 ＇ and 5 ＇-CCGCGGTGGCGGCCGCAAAT-3 ＇.IgG Gene sequence comparison in sequencing result and Internet V-Base gene pool.
7. the structure of whole antibody recombinant expression plasmid: the light chain of the Fab antibody of acquisition is first cut with XbaI/SacI enzyme, with end-filling enzyme (K fragment), fill, be cloned into pAC-K-Fc carrier (German PROGEN PR3003) (Liang, M.F., Stefan, D., Li, D.X., Queitsch, I., Li, W., and Bautz, E.F.Baculovirusexpression cassette vectors for rapid production of complete human IgG from phage displayselected antibody fragments.Journal of Immunological Methods.247:119-130.), utilize XhoI/SpeI site to clone into heavy chain Fd section again, be built into intact antibody carrier.
8. transfection and recombinant virus infection and propagation: the BaculoGo1d cotransfection test kit that adopts U.S. Pharmagen company.Working method outlines as follows: after the recombinant plasmid dna of 5 μ g is mixed with the BaculoGold linear DNA of 0.5 μ g, utilize the Sf9 cell that the transfection reagent transfection stand density in test kit is 50%, cultivate after 4d for 27 ℃, collection supernatant carries out titration and amplification as the seed culture of viruses of recombinant virus.Baculovirus expression vector system handbook (BD Biosciences Pharmagen, USA) is shown in concrete operations.
9. whole antibody IgG secreting, expressing and purifying: the Sf9 cell of recombinant virus infection stand density 70% left and right, 27 ℃ of absorption 1h. use SF-900 II SFM serum-free medium instead, collect supernatant after cultivating 3～5d for 27 ℃.The Protein-A affinity column direct purification expression supernatant of employing Amersham company (Harlow E, Lane D.Antibodies:A Laboratory Manual[M] .New York:Cold Spring Harbor Laboratory Press, 1988.).Utilize ELISA and IFA to identify the IgG antibody function characteristic of obtained purifying.Concrete operations are referring to above-mentioned materials and method 5,6 parts.
10.BIAcore method is measured whole antibody avidity:
The manual tap moving phase of 10.1CM5 sensor chip kinetics buffered soln is used HBS-EP-BUFFER(GE, the U.S.), diluted sample solution is used 10mM NaAc pH4.0, 4.5, 5.0, 5.5, software adopts BIAcore 3000 to control software, with Amine Coupling For 30-50 Immoblization Kit(GE, the U.S.) determine amino coupled HBsAg optimum concn, 10mM NaAc optimal ph, at Microchip flow cell fixing 700RU respectively, 1300RU, 2400RU, with pH 2.0 10mM glycine-hydrochloric acid (Gly-HCl), carrying out respectively the saturation concentration situation of dissociating tests, determine optimal condition of protoplast isolation.
It is sense channel that 10.2 kinetic tests be take respectively HBsAg fixed amount 700RU, 1300RU, 2400RU flow-through cell, coupling HBsAg flow-through cell is not reference channel, HBS is moving phase, flow velocity 30 μ L/min, baseline stability 5min, antibody antigen is in conjunction with 3min, the 5min that dissociates, successively with antibody concentration 32,16,8,4,2,0nM(nmol/L), the empty 8nM3-10 secondary response that runs is to stablize chip in advance, 0nM and 8nM do twice parallel, prevent system drifting.
10.3 data resultss are processed and are adopted Biaevaluation analysis software treatment S PR signal graph, complete each concentration of IgG antibody is expelled to antibody that the collection of illustrative plates after coupling HBsAg flow-through cell deducts injection same concentrations to the resulting collection of illustrative plates of reference flow-through cell, after reference corrected, carry out curve fitting, to describe the dynamic (dynamical) suitable model of antigen antibody reaction, and calculating K A and KD value.
11. anti-human hbsag gene engineered antibody extracorporeal antivirus effect effect researchs: utilize this model of HepG2 cell to evaluate the neutralization activity of the anti-HBsAg antibody in this strain people source of our acquisition.First collect hepatitis B virus positive serum and obtain virion by ultracentrifugation.The HepG2 cell that growth conditions is good is divided into several groups, and A group infects with virion; B group infects after hatching with HBV and human antibody HBIgG21 again; C group infects (positive control) after hatching with HBV and human antibody HBIgG07 again; D group is hatched postoperative infection with HBV and mouse monoclonal antibody; E organizes alone human antibody HBIgG21 and HepG2 cytosis; F organizes alone human antibody HBIgG07 and HepG2 cytosis; G organizes alone mouse monoclonal antibody and HepG2 cytosis.When above-mentioned all kinds of sample and HepG2 cytosis, in substratum, added 10
-4-10
-6the dexamethasone of M.Because dexamethasone can raise copying of HBV by acting on the intragenic glucocorticosteroid element of HBV.Concrete operations as: HepG2 passage is placed in to 96 orifice plates, and the number of cells in every hole is 1 * 10
4; Cell quantity increase puts 5 * 10 two days later
4, the HBV suspension that ultracentrifugation is obtained is pressed 2 times, 4 times, 8 times dilutions with the full substratum of DMEM; Get respectively HBV stoste, each 100 μ l of 2 times, 4 times, 8 times diluents add in 96 orifice plates, hatch 3 hours with 37 ℃, HepG2 cell; Get HBV stoste, each 100 μ l of 2 times, 4 times, 8 times diluents and with human IgG1 00 μ g, 10 μ g, 5 μ g, 2.5 μ g or with 37 ℃ of the mouse monoclonal antibodies of dilution in 1: 100,1: 200,1: 400,1: 800, hatch 1 hour respectively, antigen-antibody mixture is added in the HepG2 cell of 96 orifice plates 37 ℃ to hatch 3 hours (the antigen-antibody mixture of each concentration is done 3 holes); Discard liquid in hole, with 1 * PBS, attached cell is washed three times; In hole, add again the full substratum of DMEM that contains 10-4M dexamethasone and 10-5M Regular Insulin, cultivate one day for 37 ℃; In sucking-off hole, liquid is preserved respectively, with 1 * PBS, attached cell is washed three times, adds the full substratum of DMEM, cultivates one day for 37 ℃; In sucking-off hole, liquid is preserved respectively, adds the full substratum of DMEM that contains 10-6M dexamethasone and 10-6M Regular Insulin, and 37 ℃ of cultivations are changed a nutrient solution for every two days and cultivated 8 days.Utilize respectively ELISA, immunofluorescence (IFA) and PCR to detect the antigen of hepatitis B virus of cell and cell conditioned medium.
11.1ELISA detects cells and supernatant and uses purifying hepatitis B surface antigen(HBsAg) (CHO-HBsAg) as envelope antigen, detect respectively through the cells and supernatant that HBV or immune complex are hatched one day after, two days, four days, six days, eight days gather, step the same 5.2 subsequently.
11.2 indirect immunofluorescences (IFA) detected the HepG2 cell cultures after HBV or HBV+ antibody incubation is made to antigen sheet after 8 days, and acetone is fixed, and fixes 30 minutes for-20 ℃; Add 37 ℃ of mouse monoclonal antibodies (1: 1000) to hatch 30 minutes, clean, dry up; Add the anti-mouse Fc fluorescence antibody with the Evans blue solution dilution of 1:30000 dilution according to the ratio of 1:30, hatch 30min for 37 ℃, clean, dry up.Fluorescent microscope detects and takes pictures.
11.3PCR detection kit specification sheets extracts the external DNA of cell dyeing, gets 5 μ l and makes template, is half nest-PCR, and primer is as follows:
Primer 1:HBsAg upstream: 5 ' GAC TGG GGA CCC TGC ACC GAA C-3 '
Primer 2: Pres1 upstream: 5 '-GGA AGA TCT CCA TGG GAG GTT GGT C-3 '
Primer 3:Pres1 downstream: 5 '-CCG GAA TTC TTA GGC CTG AGG ATG AC-3 '
First with No. 1 and No. 3 primers, as external primer amplification, go out Pres1+Pres2+S fragment gene;
With above-mentioned amplified production, do that template be take No. 2 again and No. 3 primers amplify Pres1 fragment as inner primer.
The research of antibody after 12. non-hypervariable region sudden changes to hepatitis B virus resistance
Based on HBIgG21 weight chain variable region amino acid sequence, the Gly of the 116th of aminoacid sequence shown in SEQ ID No.2 is replaced with to Ser, the Leu of HBIgG21 variable region of light chain (shown in SEQID No.1) the 9th is replaced with to His.Synthesize respectively heavy chain nucleic acid sequence encoding (codon ggc being replaced with to agc in corresponding position) and the light chain nucleic acid sequence encoding (codon ctg being replaced with to cac in corresponding position) of HBIgG21.Method according to above-mentioned 8～11, is cloned into light chain gene and heavy chain Fd section in pAC-K-Fc, and transfection insect Sf 9 cells, utilizes baculovirus/insect cell system to realize the secretion type expression of whole antibody, and this mutant is carried out to immunology detection.
1. the screening of people source anti-hepatitis B virus surface antigen antibody library
Success builds 4 Kappa storehouses and 4 Lambda storehouses, and storage capacity is 1 * 10
7above, weight chain insertion rate is more than 95%.Respectively Kappa word bank and Lambda word bank are packed, by purifying hepatitis B surface antigen, through three-wheel screening discovery Kappa word bank three-wheel there is enrichment phenomenon in screening, wash-out storage capacity increases gradually, and Lambda word bank does not occur, 100 Kappa word bank clones of picking and 100 lambda word bank clones, ELISA result shows: for HBsAg positive colony, have 30, all from Kappa word bank, the different antibody cloning of 10 strain sequence is found in order-checking altogether, as shown in table 2:
Table 2 antibody library packing and enrichment screening quantum of output
2. the sequential analysis of people source anti-hepatitis B virus surface antigen antibody
With DNAstar Lasergene software analysis, and compare with the IgG sequence in Internet V-Base gene pool, in the different Fab antibody of the 10 strain sequences that filter out, there are 9 strains overlapping with the partial results in hepatitis B gene engineered antibody research in the past, and this part is applied for a patent (application number is 201210167834.2), remaining 1 strain is the newfound antibody gene of the present invention, variable region of heavy chain be sorted in VH4, variable region of light chain be sorted in VK3 called after HBFab21.
3. the specific binding of people source hepatitis B virus resisting Fab antibody to hepatitis B surface antigen
Anti-human Fab is coated shows (see figure 1) with the coated ELISA detected result of HBsAg, and most of screening and cloning Fab antibody induction secretory volume is high, and titre reaches 1:6400; Having 10 strain antibodies (antiHBsAg-Fab4,7,10,13,16,17,20,21,24 and 29) and HBsAg is 1:1600 in conjunction with titre, illustrate that energy specific binding HBsAg and avidity are higher, wherein antiHBsAg-Fab21 is new discovery antibody cloning of the present invention, called after HBFab21, therefore selects this strain to build IgG whole antibody.
4. whole antibody IgG expresses and purifying
By the light chain of HBFab21 antibody and heavy chain Fd fragment gene, be cloned into respectively transfection insect Sf 9 cells after intact antibody carrier pAC-K-Fc, utilize baculovirus/insect cell system to realize the secretion type expression of whole antibody, called after HBIgG21.Adopt the Protein-A affinity column direct purification of Amersham company to express supernatant, by SDS-PAGE, check the Expression and purification situation of whole antibody IgG, result confirms to obtain compared with pure protein, can clearly observe that antibody after unwinding is light, heavy chain, lays respectively at about 28kD, 55kD place.As shown in Figure 2.
5. avidity is measured
Here we utilize biosensor BIAcoreTM 3000 to measure the avidity of Human monoclonal antibody, it is exactly to utilize the resonance angle that the variations in refractive index of surface plasma resonance (surface plasmon resonance) phenomenon monitoring vane surface dielectric causes to change that the principle of biosensor work and primary process and principle of work sum up, and this change is directly proportional to the amount of the biomacromolecule of vane surface knot platform, in solution, free molecule does not affect the size of resonance angle, therefore very special, responsive.With biosensor, detecting interaction that any biomacromolecule asks all will be through several like this step: combination, dissociate, and regeneration.The present invention uses the hepatitis B surface antigen(HBsAg) (CHO-HBsAg) of purifying to be coupled to as antigen the avidity that bio-sensing chip is measured 2 strain Human monoclonal antibodies, and affinity costant is calculated with BIAcore bundled software BIAevaluation 3.0, as shown in Figures 3 and 4.HBIgG21 reaches nmole level, i.e. KD=3.76 * 10 for the avidity of hepatitis B surface antigen(HBsAg) (CHO-HBsAg)
-9m.In addition, control antibodies HBIgG07 conjugated antigen avidity reaches nmole level, i.e. KD=9.53 * 10
-9m, this strain people source anti-hepatitis b surface antigen monoclonal antibody all has very high avidity (table 3) as can be seen here.
Table 3 surface plasma resonance technology is measured affinity of antibody
6. anti-human hbsag gene engineered antibody extracorporeal antivirus effect effect research
6.1 the HBsAg ELISA of cells and supernatant detects
Collect respectively the cells and supernatant of respectively organizing the 2nd day, the 4th day, the 8th day, coated HBsAg, ELISA detects supernatant, the detection that found that in A group (infection group and viral infection group) three days is all positive, wherein the OD value of second day is 0.5 left and right, the OD value of the 4th day, the 8th day does not have difference, is 0.8 left and right.In B group, the HBIgG21 of different concns and HBV are hatched in the postoperative infection HepG2 culture supernatant of tri-days HBsAg and are detected all negative.In C group, the HBIgG07 of different concns and HBV are hatched in the postoperative infection HepG2 culture supernatant of tri-days HBsAg and are detected all negative.In D group when mouse monoclonal antibody extent of dilution is 1: 800, the supernatant test positive of collecting the 4th day and the 8th day, all the other are negative.The monoclonal antibody interpolation group separately that E, F, G are tri-groups detects also all negative.Result is summarized as follows:
A group:
| 2 |
4 |
8 days |
| HBsAg（＋） | HBsAg（＋） | HBsAg（＋） |
B group:
C group:
D group:
6.2 cellmediated immunity fluorescence (IFA) detect
It is full after 8 days that each organizes cell cultures, and its point is done to immunofluorescence detection to slide, and as shown in Figure 5, A group cell Microscopic observation is to green fluorescence; Under B group and C group cell mirror, be red; D group cell is that 1: 800 o'clock Microscopic observation is to green fluorescence at mouse monoclonal antibody extent of dilution; Under E, F, G group cell mirror, be redness, negative findings figure slightly.
The PCR that 6.3HBV infects Pres1 in positive cell detects
Collect the HepG2 cell of cultivating 8 days, extract the nonchromosomal DNA in cell, with half nest-PCR method amplification Pres1, as shown in Figure 6, result shows that A group PCR detects the positive band that has occurred about 350bp size; B group and C group all do not detect positive band; D group cell is less than at 1: 800 o'clock at mouse monoclonal antibody extent of dilution and positive band (in Fig. 6, D1, M-antibody 1: 400) do not detected, and is that 1: 800 o'clock pcr amplification is to positive band (D2, M-antibody 1:400) at mouse monoclonal antibody extent of dilution.
Comprehensive above-mentioned three aspects: hepatitis B surface antigen (HBsAg) detected result, find A group test positive, B group and C group detect all negative, and in C group when antibody is greatest dilution, in culture supernatant, HBsAg can be detected, may be because our concentration of added human antibody is higher, block the infection of HBV completely, and mouse monoclonal antibody its concentration when greatest dilution is not enough to block, the infection of HBV causes.Take HepG2 cell as model, with HBV or HBV and antibody, jointly hatch HepG2 separately respectively.Detect culture supernatant after one week, find that there is the HepG2 that in the HepG2 culture supernatant that antibody intervenes, HBsAg titre infects well below alone HBV.The antibody that proof the present invention obtains has the function that stops HBV to infect sensitive cells, is neutralizing antibody.
7. the antibody after the sudden change of non-hypervariable region affects hepatitis B virus resistance
Method according to above-mentioned 7～11, to be cloned in pAC-K-Fc based on the amended light chain gene of HBIgG21 and heavy chain gene, and transfection insect Sf 9 cells, utilize baculovirus/insect cell system to realize the secretion type expression of whole antibody, obtain mutant HBIgG21 '.This mutant is carried out to immunology detection, ELISA experiment shows that RVFab21 ' energy specificity is for hepatitis B virus surface antigen, and by competition blocking experiment and cell in vitro, learn experiment and confirmed that its neutralization is active, result show its character and HBIgG21 basic identical.
As can be seen from the above embodiments, the present invention uses phage antibody library technique, has successfully obtained the people source neutrality antibody HBFab21 of 1 strain specificity for hepatitis B virus surface antigen; And its have in vitro stop HBV infect sensitive cells in and function, in host, expression amount is high.Utilize the full-antibody gene under people source neutrality anti-hepatitis B virus surface antigen genetic engineering antibody variable region gene, Fab antibody gene and above-mentioned each antibody gene feature of above-mentioned acquisition, can in prokaryotic cell prokaryocyte, yeast cell, eukaryotic cell and any recombination system, express and produce any other gene that contains this antibody gene after this antibody or reconstruction based on this, during acquisition has and the antibody product of hepatitis B virus infection, make clinically for preventing and treat the specific antibody medicine of hepatitis B virus related liver disease.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a people source anti-hepatitis b surface antigen gene engineered antibody and active fragments thereof, it is characterized in that, this people source anti-hepatitis b surface antigen gene engineered antibody Fab section called after HBFab21, the CDR1 of HBFab21 variable region of light chain is that QSVSSN, CDR2 are that GAS, CDR3 are QQYNNWPQT, and the CDR1 of HBFab21 variable region of heavy chain is that SITGSSYY, CDR2 are that IYSTSGTT, CDR3 are ARPTAHRNVRMVPGQDAFEV; Preferably, the aminoacid sequence of HBFab21 variable region of light chain is as shown in SEQ ID No.1, and the aminoacid sequence of its variable region of heavy chain is as shown in SEQ ID No.2.
2. antibody as claimed in claim 1 and active fragments thereof, is characterized in that, its aminoacid sequence carries out various replacements, interpolation and/or lack one or several amino acid obtaining the aminoacid sequence with same function.
3. antibody as claimed in claim 2 and active fragments thereof, is characterized in that, in non-hypervariable region, the amino acid with similarity replaced, as the Gly of the 14th of the light chain VL sequence of HBFab21 is replaced with to Ala.
4. antibody as claimed in claim 1 and active fragments thereof, is characterized in that, recombinated in the variable region of light chain of described Fab section and variable region of heavy chain, the less single-chain antibody of the molecular weight that obtains.
5. the antibody as described in claim 1-4 any one and the encoding gene of active fragments thereof.
6. encoding gene as claimed in claim 5, is characterized in that, the gene order of coding HBFab21 variable region of light chain is as shown in SEQ ID No.3, and the gene order of its variable region of heavy chain is as shown in SEQ ID No.4.
7. the carrier that comprises the encoding gene described in claim 5 or 6.
8. the host cell that comprises the encoding gene described in claim 5 or 6.
9. encoding gene, carrier claimed in claim 7 or the host cell claimed in claim 8 described in the antibody described in claim 1-4 any one and active fragments thereof, claim 5 or 6 is in preparation prevention or the medicine for the treatment of hepatitis B virus related liver disease or the application in diagnostic reagent.
10. the antibody described in preparation claim 1-4 any one and the method for active fragments thereof.
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| WO2018184593A1 (en) * | 2017-04-07 | 2018-10-11 | 厦门大学 | Antibody for treating hepatitis b infection and related disease |
| WO2019229699A1 (en) * | 2018-05-31 | 2019-12-05 | Novartis Ag | Hepatitis b antibodies |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105001325A (en) * | 2015-07-31 | 2015-10-28 | 北京泰诺迪生物科技有限公司 | Total-humanized anti-hepatitis B virus neutralizing antibody and preparing method and application thereof |
| WO2018184593A1 (en) * | 2017-04-07 | 2018-10-11 | 厦门大学 | Antibody for treating hepatitis b infection and related disease |
| RU2765878C2 (en) * | 2017-04-07 | 2022-02-04 | Сямэнь Юниверсити | Antibodies for treatment of hepatitis b infection and related diseases |
| KR20220126788A (en) * | 2017-04-07 | 2022-09-16 | 시아먼 유니버시티 | Antibodies for treatment of hepatitis B infection and related diseases |
| KR102561555B1 (en) | 2017-04-07 | 2023-07-31 | 시아먼 유니버시티 | Antibodies for treatment of hepatitis B infection and related diseases |
| WO2019229699A1 (en) * | 2018-05-31 | 2019-12-05 | Novartis Ag | Hepatitis b antibodies |
| CN112165974A (en) * | 2018-05-31 | 2021-01-01 | 诺华股份有限公司 | Hepatitis B antibodies |
| US11932681B2 (en) | 2018-05-31 | 2024-03-19 | Novartis Ag | Hepatitis B antibodies |
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