CN102649818B - CD4 protein-resistant monoclonal antibody and active fragment and application thereof - Google Patents

Abstract

The invention provides a CD4 protein-resistant monoclonal antibody and an active fragment and an application thereof. The antibody can be used for preventing a human immunodeficiency virus (HIV) from invading cells through combination with a human CD4 protein. The antibody can be used for detecting, diagnosing, preventing and treating diseases in which CD4 cells are taken as target cells, particularly an acquired immunodeficiency syndrome caused by the HIV. The invention further provides a relevant hybrid tumor cell strain, a separated nucleic acid molecule, a small peptide, a medicinal composition comprising the monoclonal antibody, and a kit.

Description

The monoclonal antibody of anti-CD4 albumen and active fragments thereof and purposes

Invention field

The present invention relates to break up bunch 4 (Cluster ofDifferentiation 4 by specific binding people surface antigen, CD4) monoclonal antibody of albumen, and examples of conservative variations or active fragments, the correlative coding sequence of its polypeptide or polypeptide analog, produce the cell strain of described monoclonal antibody, and apply this antibody or fragment for preventing, method and the purposes of immunotherapy and diagnosis; Prevention or treatment primates, comprise the mankind, and the source of infection is with CD4 ⁺the disease that the major objective cell of cell causes: these diseases comprise acquired immune deficiency syndrome (AIDS) (acquired immune deficiency syndrome (AIDS), AIDS), the disease that acquired immune deficiency syndrome (AIDS) is relevant, human immunodeficiency virus (human immunodeficiencyvirus, HIV) infects.

Background technology

Since finding HIV last century, many investigators are devoted to the research of the aspects such as the viral molecular biology, immunity, vaccine of HIV, but still there is many difficulties in the development for HIV vaccine and immunotherapy thereof, the variability that comprises HIV-1, multiple route of transmission/the mode of virus, and the relevant in advance necessary immune response of preventing HIV infection is still disagreed etc.

HIV-1 virus is the enveloped virus of approximately 100 nanometers.Its rna gene group and some activated proteins are wrapped in the taper core being comprised of capsid protein, the inside of viromembrane is capsid protein, coating is positioned at the outermost layer of virion, its main body is phospholipid bilayer, is embedded with furcella shape (spike) structure that gp120 and gp41 by env genes encoding form.After HIV coating furcella shape structure contacts with cell the first acceptor CD4 albumen, occurred conformation changes, then with the second acceptor Chemokine Receptors albumen (chemokine receptors, comprise CCR5, CXCR4 etc.) effect, finally to realize film and merge, the gene of releasing virus is invaded cell.

HIV viral envelope glycoprotein be precursor forms with gp160 at cells out, ripe envelope glycoprotein is cut into approximately 481 amino acid whose gp120 glycoprotein and approximately 345 amino acid whose gp41 glycoprotein (Ratner, L. wait people, Nature, 1985,313:277-284).Gp120 and gp41 exist with trimerical functional unit form on HIV coating, participate in virus and the combination of cell receptor and course of infection (Kwong, the people such as P.D., Nature, 2002, the 420:678-682 of virus and cytogamy; Diskin, the people such as R., Nat Struct Mol Biol, 2010,17:608-613; Buzon, the people such as V., PLoS Pathog, 2010,6 (5): e1000880.).

HIV-1 envelope glycoprotein gp120 by with cell receptor CD4 albumen (Maddon, P.j. wait people, Cell, 1986,47:333-348) and with the interaction host cells infected (Alkhatib of co-receptor (co-receptor is mainly CCR5 or CXCR4 albumen), G. wait people, Science, 1996,272:1955-1958; Deng, the people such as H., Nature, 1996,381:661-666; Dragic, the people such as T., Nature, 1996,381:667-673; Feng, the people such as Y., Science, 1996,272:872-877).Cause viral envelope glycoprotein gp120 conformational change with the combination of cell receptor, and then the gp41 of the cross-film unit structural rearrangement (rearrangements) of induction and envelope glycoprotein makes virus and cytogamy (Wyatt, the people such as R., Science, 1998,280:1884-1888; Pierson, the people such as T.C., Curr Top Microbiol Immunol, 2003,281:1-27).

CD4 full name surface antigen differentiation bunch 4 (Cluster of Differentiation 4), the glycoprotein of expressing comprising the cell surfaces such as helper cell, adjusting T cell, monocyte, scavenger cell and dendritic cell, it is one of surface markers (surface markers) of helper cell, it is its important acceptor of exercising its function, participate in regulating the propagation of T immunocyte, the interaction of the release of lymphokine and participation immunocyte regulates the generation of antibody.That many pathogenic agent (comprising human immunodeficiency virus etc.) infect CD4 simultaneously ⁺the receptor Glycoprotein of cell (Isobe, the people such as M., Proc Natl Acad Sci USA, 1986; 83 (12): 4399-402; Ansari-Lari, the people such as M.A., Genome Res.1996; 6 (4): 314-26; Bofill, the people such as M., Clin Exp Immunol.1992; 88 (2): 243-52).

Similar to a lot of other cell surface receptor/tagged molecule, CD4 albumen is the member of contactin (Immunoglobulin superfamily) also.Complete CD4 is 433 amino acid membrane glycoproteins by CD4 genes encoding, its molecular weight is about 55kDa-62kDa, comprise 4 the cytolemma external structure territories (domain) that formed by 1-375 amino acids, (the people such as Maddon P.J. of afterbody (cytoplasmic tail) in the tenuigenin that the cross-film district that 376-395 amino acids forms and 396-433 amino acids form, Cell, 1985,42:93-104; Littman, the people such as D.R., Cell, 55:541).Wherein extracellular 4 structural domains are respectively the first structural domain (domain 1, is called for short D1, comprises 1-100 amino acids), the second structural domain (domain2, is called for short D2, comprises 101-180 amino acids), (domain 3 for the 3rd structural domain, be called for short D3, comprise 181-290 amino acids) and the 4th structural domain (domain 4, abbreviation D4, comprise 291-375 amino acids) (Maddon, P.J. wait people, Cell, 1985; 42:93-104; Maddon, the people such as P.J., Cell, 1986; 47:333-348; Maddon, the people such as P.J., Cell, 1988; 54:865-874; Wang, the people such as J.H., Nature, 1990,348:411-418); Wherein D1 and D3 are structurally similar to immune globulin variable region structural domain (immunoglobulinvariable domain, IgV domain), D2 and D4 are structurally similar to constant region for immunoglobulin structural domain (immunoglobulin constant domains, IgC domain).

As the receptor protein of HIV cells infected, CD4 albumen in the replicative cycle of virus of AIDS, play an important role (Dalgleish, the people such as A.G., Nature, 1984; 312:763-767; Klatzmann, the people such as D., Science, 1984; 225:59-63; Klatzmann, the people such as D., Nature, 1984,312:767-768).CDR3 sample district (CDR3-like region) in first structural domain (D1) of CD4 and the second immunoglobulin-like complementary determining region (CDR2-like region) of HIV-1 envelope glycoprotein gp120 have been participated in interaction (Arthos directly, J. wait people, Cell, 1989,57:469-481; Clayton, the people such as L.K., Nature, 1989,339:548-551; Mizukami, the people such as T., Proc Natl Acad Sci USA.1988,85:9273-9277; d. wait people, J Biol Chem.1992,267:6664-6671; Camerini, the people such as D., Cell.1990,60:747-754; Kalyanaraman, the people such as V.S., J Immunol.1990,145:4072-4078; Lifson, the people such as J.D., Science.1988,241:712-716; Truneh, the people such as A., J Biol Chem.1991,266:5942-5948; Diskin, R. wait people, NatStruct Mol Biol.2010,17:608-613), the interaction of two protein moleculars causes that CD4-gp120 mixture occurred conformation changes, and makes the gp120 albumen can be further and cell the second acceptor, i.e. chemokine receptor 5 (chemokine receptor-5, CCR5) or CX Chemokine Receptors 4 (CX chemokine receptor-4, CXCR4) combination.Cause glycoprotein tripolymer conformational change with the combination of cell the second acceptor, make the gp41 albumen on HIV surface insert cytolemma, finally cause fusion (Kuritzkes, D.R., Curr opinHIV AIDS.2009, the 4:82-87 of peplos and cytolemma; Briz, the people such as V., J Antimicrob Chemother.2006,57:619-627; Castagna, the people such as A., Drugs.2005,65:879-904).

CD4 ⁺after cell is infected by HIV, the quantity of cell function and cell is all subject to obvious impact, and wherein the minimizing of T cell quantity is because viral cracking type infects on the one hand, simultaneously also with the metainfective CD4 of HIV ⁺cell occurs and CD4 other infection or that do not infect ⁺the fusion phenomenon of+cell relevant (Sodroski, the people such as J., Nature, 1986,322:470-474).CD4 ⁺cell depleting, can cause patient's body to show as immunosuppression, thereby is more and more easily subject to opportunistic infection and malignant tumour occurs.In acquired immunodeficiency syndrome, (acquired immune deficiency syndrome (AIDS) AIDS) extensively exists in patient this immunosuppression phenomenon.Conventionally acquired immune deficiency syndrome (AIDS) clinical manifestation is the compound symptom feature relevant to virus of AIDS, as the enlargement of persistence lymph nodes of body as a whole, and the syndrome of fever and weight loss etc.In some cases, due to HIV (human immunodeficiency virus) infection CD4 ⁺brain cell make acquired immune deficiency syndrome (AIDS) be attended by central nervous system disorder (Popovic, the people such as M., Science, 1984,224:497-500).The object of attack of human immunodeficiency virus is CD4 ⁺cell, causes in the process of acquired immune deficiency syndrome (AIDS) in virus, CD4 in patient body ⁺the quantity of cell and the development of the state of an illness have close relationship, so CD4 ⁺the detection of cell quantity plays an important role to the judgement for the treatment of AIDS effect with to patient immune function's judgement.

Because having by disturbing HIV and CD4 molecule and/or the second acceptor interaction and then affecting HIV, anti-CD 4 antibodies invades recipient cell, performance prevents and/or treats the effect that HIV infects, and some investigators are just in relation and the application of Effect of Anti CD4 antibody blocking virus of AIDS and cell interaction.Report is blocking HIV (human immunodeficiency virus) infection CD4 at present ⁺in recipient cell, effect obviously, have simultaneously and be applied to anti-CD 4 antibodies that acquired immune deficiency syndrome (AIDS) aspect that HIV infect to cause has good prospect as 5A8 and DB-81, the epi-position of its identification is all positioned at D2 structural domain (Song, the people such as R. of CD4 albumen, J Virol.2010,84:6935-6942; Burastero, the people such as S.E., J Transl Med.2009Nov 28; 7:101).

Reported the research about the monoclonal antibody of other epi-positions of anti-CD4 or structural domain, the monoclonal antibody of the specific areas such as monoclonal antibody that peripheral blood T4 antigen detects as can be used for, but in correlation report, also reported the weak point of these antibody on treating AIDS.

For example, the antibody Leu3A of being combined with CD4 molecule D1 structural domain and OKT4A have effect (people such as Sattentau Q.J., Science, 1986, the 234:1120-1123 of the cell Syncytium formation phenomenon that blocking-up aids infection poison causes; The people such as Jameson B.A., Science.1988,240:1335-1339; The people such as Peterson A., Cell.1988,54:65-72).But because the epi-position of the identification epi-position of these antibody and HIV envelope glycoprotein gp120 identification is overlapping, these antibody cannot react with the CD4 protein molecular that combines envelope glycoprotein gp120, therefore there is the major defect (people such as Bates P.A. as the medicine use of HIV (human immunodeficiency virus) infection in these antibody, Protein Eng.1989,3:13-21; The people such as McDougal J.S., J Immunol.1986,137:2937-2944; The people such as Landau N.R., Nature.1988,334:159-162; The people such as Dalgleish A.G., Lancet.1987,2:1047-1050), in body, also can there is due to the immunosuppression of body applied defect people such as (, J Transl Med.2009,7:101) Samuele E.B. simultaneously.

It is reported, the monoclonal antibody OKT4B of the identification CD4 molecule D2 structural domain (people such as T.Kieber-Emmons T., Biochim Biophys Acta.1989,989:281-300) combination for HIV envelope glycoprotein gp120 and CD4 albumen has the obvious interference effect (people such as McDougal J.S., J.Immunol., J Immunol.1986,137:2937-2944; The people such as Lundin K., J Immunol Methods.1987,97:93-100; The people such as Lamarre D., EMBO J., 1989,8:3271-3277), but it stops effect that cell that HIV infects forms synplasm phenomenon obviously than a little less than OKT4A, the more outstanding (people such as SattentauQ.J. of the weakness in treatment, Science.1986,234:1120-1123; The people such as Moore J.P., Science.1990,250:1139-42).

The anti-CD 4 antibodies of other epi-positions also comprises MT151, VIT4, with (the people such as Sattentau Q.J. such as MT321, Science.1986,234:1120-1123), the epi-position that the epi-position of these antibody recognition is combined with HIV envelope glycoprotein gp120 has certain overlapping (people such as Sattentau Q.J., J Exp Med in conformation, 1989,170:1319-1334; The people such as Bates P.A., Protein Eng.1989,3:13-21; The people such as Landau N.R., Nature.1988,334:159-162; The people such as Merkenschlager M., J Immunol.1990,145:2839-2845; The people such as Benkirane M., J Virol.1995,69:6898-6903).

Epi-position on antibody recognition CD4 protein D 1 of the present invention (D1-2) structural domain, after having HIV existence/CD4 albumen to be combined with HIV envelope glycoprotein gp120, this epi-position can show with the present invention's monoclonal antibody better reactive behavior, significantly blocking-up HIV invades cell, virus can not be copied in cell, the cytopathy that blocking-up causes due to HIV infection, thus viral intercellular propagation stoped.Such antibody has using value aspect the new modes for the treatment of correlative diseases such as blocking-up HIV infection and acquired immune deficiency syndrome (AIDS).

Summary of the invention

The invention provides and can break up bunch 4 (Cluster ofDifferentiation 4 by specific binding people surface antigen, CD4) monoclonal antibody of albumen, be that human immunodeficiency virus capable of blocking (human immunodeficiency virus, HIV) is invaded CD4 ⁺cell, suppresses HIV and infects, blocks HIV at CD4 ⁺between propagate, the monoclonal antibody for the treatment of acquired immune deficiency syndrome (AIDS) activity.

Monoclonal antibody of the present invention can be broken up with people's surface antigen the first structural domain (D1 of bunch 4 albumen, 1-113 amino acids) the epi-position combination on, also can with the 1-2 structural domain (D1-2 that comprises this structural domain, 1-180 amino acids) combination, and break up bunch 4 albumen, recombinant expressed CD4, the CD4 protein binding of cell surface with people's surface antigen of the other forms of brachymemma that comprises the first structural domain.The present invention's antibody on human para-immunity defective virus gp120 and the interaction of CD4 albumen is simultaneously without obvious blocking effect, and the present invention's monoclonal antibody HIV virus infected cell capable of blocking, blocks HIV virus copying in recipient cell.

In addition, the invention provides the multiple homology form of anti-CD 4 antibodies: the fragment of monoclonal antibody, immunoglobulin (Ig) (as being at least an immunocompetence section of immunoglobulin molecules), as Fab, Fab ', F (ab ') 2, Fv fragment, single-chain antibody molecule or the multi-specificity antibody that formed by any fragment of the immunoglobulin molecules that contains one or more CDR district.In addition, the present invention also provides the antibody that the one or more CDR district in human normal immunoglobulin such as recombinant antibodies, recombined chimeric antibody, recombinant humanized antibody forms in conjunction with the framework region of one or more different human normal immunoglobulins.And the simulation substance of the CDR functional area of antibody of the present invention.

The invention provides hybridoma cell strain, separated nucleic acid molecule and small peptide, and the pharmaceutical composition that comprises monoclonal antibody of the present invention and medical diagnositc equipment and test kit.The present invention also provides and has utilized that monoclonal antibody of the present invention is surveyed, diagnosis, prevention and treatment Mammals, comprises the mankind, and the source of infection is with CD4 ⁺cell is the disease of major objective cell: these diseases comprise acquired immune deficiency syndrome (AIDS) (acquired immune deficiency syndrome (AIDS), AIDS), the disease that acquired immune deficiency syndrome (AIDS) is relevant, the disease that human immunodeficiency virus (human immunodeficiency virus, HIV) infects.

In one aspect, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof, it has at least one item and is selected from following feature:

(i) specific binding people surface antigen differentiation bunch 4 albumen;

(ii) react with people's surface antigen differentiation bunch 4 polypeptid specificities of the clipped form that contains people's surface antigen differentiation bunch 4 protein D 1 structural domains;

(iii) human immunodeficiency virus's envelope glycoprotein gp120 is had no significant effect with people's surface antigen differentiation bunch reacting of 4 albumen; With

(iv) blocking-up human immunodeficiency virus infection CD4 ⁺cell and infecting again, and/or the disease that causes thus of blocking-up, for example acquired immune deficiency syndrome (AIDS).

In one embodiment, the invention provides monoclonal antibody or its antigen-binding portion thereof, the CDR1 of the light chain of the CDR1 of the heavy chain of wherein said antibody, CDR2 and CDR3 and wherein said antibody, CDR2 and CDR3 are respectively heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and the CDR3 of the monoclonal antibody 15A7 that produces of the hybridoma of CCTCC preserving number C201098.

In one embodiment, the invention provides monoclonal antibody or its antigen-binding portion thereof, the CDR1 of the light chain of the CDR1 of the heavy chain of wherein said antibody, CDR2 and CDR3 and wherein said antibody, CDR2 and CDR3 are respectively heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and the CDR3 that hybridoma produces monoclonal antibody 14G7.

In one embodiment, the invention provides monoclonal antibody or its antigen-binding portion thereof, the variable region of heavy chain of wherein said antibody and variable region of light chain are respectively variable region of heavy chain and the variable region of light chain of the monoclonal antibody 15A7 that produces of the hybridoma of CCTCC preserving number C201098.

In one embodiment, the invention provides monoclonal antibody or its antigen-binding portion thereof, the variable region of heavy chain of wherein said antibody and variable region of light chain are respectively variable region of heavy chain and the variable region of light chain of the monoclonal antibody 14G7 of hybridoma generation.

In one embodiment, the invention provides monoclonal antibody or its antigen-binding portion thereof of specific binding people surface antigen differentiation bunch CD4 albumen, nucleotide sequence coded as shown in SEQ ID NO:2 or by SEQ ID NO:1 of the aminoacid sequence of the variable region of heavy chain of wherein said antibody; Nucleotide sequence coded as shown in SEQ ID NO:4 or by SEQ ID NO:3 of the aminoacid sequence of the variable region of light chain of wherein said antibody.

In one embodiment, the invention provides monoclonal antibody or its antigen-binding portion thereof of specific binding people surface antigen differentiation bunch CD4 albumen, nucleotide sequence coded as shown in SEQ ID NO:18 or by SEQ ID NO:17 of the aminoacid sequence of the variable region of heavy chain of wherein said antibody; Nucleotide sequence coded as shown in SEQ ID NO:20 or by SEQ ID NO:19 of the aminoacid sequence of the variable region of light chain of wherein said antibody.

In one embodiment, the heavy chain CDR that antibody of the present invention comprises the one or more SEQ of being selected from ID NOs:5-7; And/or the light chain CDR that comprises the one or more SEQ of being selected from ID NOs:8-10;

In one embodiment, heavy chain CDR1, the CDR2 of antibody of the present invention and the aminoacid sequence of CDR3 are respectively SEQ ID NO:5-7 or one or more codings in the nucleotide sequence of SEQ ID NO:11-13;

In one embodiment, light chain CDR1, the CDR2 of antibody of the present invention and the aminoacid sequence of CDR3 are respectively SEQ ID NO:8-10 or one or more codings in the nucleotide sequence of SEQ ID NO:14-16;

In one embodiment, monoclonal antibody of the present invention or its antigen-binding portion thereof are Fab, Fab ', F (ab ') ₂, Fv or single-chain antibody.

In one embodiment, the heavy chain CDR that antibody of the present invention comprises the one or more SEQ of being selected from ID NOs:21-23; And the light chain CDR that comprises the one or more SEQ of being selected from ID NOs:24-26;

In one embodiment, heavy chain CDR1, the CDR2 of antibody of the present invention and the aminoacid sequence of CDR3 are respectively SEQ ID NO:21-23 or one or more codings in the nucleotide sequence of SEQ ID NO:27-29;

In one embodiment, light chain CDR1, the CDR2 of antibody of the present invention and the aminoacid sequence of CDR3 are respectively SEQ ID NO:24-26 or one or more codings in the nucleotide sequence of SEQ ID NO:30-32;

In one embodiment, monoclonal antibody of the present invention or its antigen-binding portion thereof are Fab, Fab ', F (ab ') ₂, Fv or single-chain antibody.

In one embodiment, monoclonal antibody of the present invention or its antigen-binding portion thereof are in conjunction with the K of people's surface antigen differentiation bunch 4 albumen _dvalue is less than 1 * 10 ^-5m.

In one embodiment, monoclonal antibody of the present invention comprises FeiCDR district, and Gai Fei CDR district is from the species that are not muroid.

In yet another aspect, the invention provides the monoclonal antibody of specific binding people surface antigen differentiation bunch 4 albumen, it is that preserving number is the hybridoma 15A7 generation of CCTCC NO:C201098, and its corresponding hybridoma is the hybridoma of CCTCC NO:C201098.

In yet another aspect, the invention provides separated nucleic acid molecule, the variable region of heavy chain of its encode monoclonal antibody of the present invention or its antigen-binding portion thereof.In one embodiment, the aminoacid sequence of the variable region of heavy chain of described antibody is SEQ ID NO:2.In another embodiment, described sequence of nucleic acid molecules is SEQ ID NO:1.

In yet another aspect, the invention provides separated nucleic acid molecule, the variable region of light chain of its encode monoclonal antibody of the present invention or its antigen-binding portion thereof.In one embodiment, the aminoacid sequence of the variable region of light chain of described antibody is SEQ ID NO:4.In another embodiment, described sequence of nucleic acid molecules is SEQ ID NO:3.

In yet another aspect, the invention provides separated nucleic acid molecule, the variable region of heavy chain of its encode monoclonal antibody of the present invention or its antigen-binding portion thereof.In one embodiment, the aminoacid sequence of the variable region of heavy chain of described antibody is SEQ ID NO:18.In another embodiment, described sequence of nucleic acid molecules is SEQ ID NO:17.

In yet another aspect, the invention provides separated nucleic acid molecule, the variable region of light chain of its encode monoclonal antibody of the present invention or its antigen-binding portion thereof.In one embodiment, the aminoacid sequence of the variable region of light chain of described antibody is SEQ ID NO:20.In another embodiment, described sequence of nucleic acid molecules is SEQ ID NO:19.

In yet another aspect, the invention provides the expression vector that comprises nucleic acid molecule of the present invention.In yet another aspect, the invention provides the host cell that comprises expression vector of the present invention.

In yet another aspect, the invention provides the method that detects people's surface antigen differentiation bunch 4 albumen in sample, it comprises the steps:

A) described sample is contacted with monoclonal antibody of the present invention or its antigen-binding portion thereof;

B) detect reacting of described monoclonal antibody and albumen in described sample;

C) detect described monoclonal antibody and the albumen in described sample and human immunodeficiency virus's bag

The reaction of membranin.

In one embodiment, described monoclonal antibody is attached on solid phase carrier.In another embodiment, described solid phase carrier is selected from: titer plate, magnetic particle, latex particle and nitrocellulose membrane.In a preferred embodiment, described monoclonal antibody is to be attached in described solid phase by direction like this, and this direction can increase the joint efficiency of described monoclonal antibody and described sample.In another embodiment, described monoclonal antibody is attached in described solid phase by its constant region.In an embodiment, described reaction is developed the color and is measured by enzyme.In another concrete embodiment, described reaction is measured by fluorescence.In another concrete embodiment, described reaction is measured by chemoluminescence.One preferred embodiment in, the monoclonal antibody described in aforesaid method is Fab, Fab ', F (ab ') ₂or Fv.Another preferred embodiment in, described sample comes from birds or the mankind.

In yet another aspect, the invention provides pharmaceutical composition, it comprises monoclonal antibody of the present invention or its antigen-binding portion thereof, and pharmaceutically acceptable carrier.In one embodiment, pharmaceutical composition of the present invention further comprises other antiviral ingredients.In another embodiment, the antibody in pharmaceutical composition of the present invention is Fab, Fab ', F (ab ') ₂or Fv.

In yet another aspect, the invention provides the method for the disease that treatment causes by human immunodeficiency virus infection in experimenter, it comprises to the pharmaceutical composition of the present invention of significant quantity on described experimenter's administering therapeutic.

In yet another aspect, the invention provides monoclonal antibody of the present invention for the preparation for the treatment of or prevent with CD4 ⁺cell is the purposes of medicine of the disease (as acquired immune deficiency syndrome (AIDS)) of target cell.

In yet another aspect, the invention provides monoclonal antibody of the present invention and infect CD4 for the preparation of blocking-up HIV ⁺cell and the again purposes of the medicine of infection.

In yet another aspect, the invention provides monoclonal antibody of the present invention for the purposes of people's surface antigen differentiation bunch 4 albumen mimic epitopes peptide screenings.

In yet another aspect, the invention provides above-mentioned analogue epi-peptide and take the purposes of vaccine of the disease (as acquired immune deficiency syndrome (AIDS)) that CD4+ is target cell for the preparation of prevention.

In yet another aspect, the invention provides above-mentioned analogue epi-peptide for the preparation for the treatment of or diagnose with CD4 ⁺purposes for disease (as the acquired immune deficiency syndrome (AIDS)) test kit of target cell.

Accompanying drawing explanation

Fig. 1 is people CD4 extracellular region aminoacid sequence, marks D1 structural domain in figure, D2 structural domain, D3 structural domain, the start-stop position of four structural domains of D4 structural domain.

Fig. 2 is the SDS-PAGE result of recombinant human CD4 protein expression and purification, and each figure is followed successively by A:CD4 (d1); B:CD4 (d2); C:CD4 (d3); D:CD4 (d4); E:CD4 (d1-2); F:sCD4 (being D1-D4 structural domain); Ge little Tu Ge road sample is followed successively by from left to right: 1: molecular weight of albumen marker; 2: after induction, express thalline; 3:2M urea supernatant; 4:4M urea supernatant; 5:8M urea supernatant; 6: sample after purifying; The unloaded thalline of 7:BL21.

Fig. 3 is the graphic representation that each dilution monoclonal antibody 15A7 blocking-up different subtype HIV experiment strain virus infects Tzm-bl cell, and experiment strain virus comprises HIV _nL4-3(B hypotype), HIV _89.6(B hypotype), HIV _94UG114(D hypotype), HIV _mJ4(C hypotype), HIV _wCML249(D/C hypotype).

Fig. 4 is the graphic representation that each dilution monoclonal antibody 14G7 blocking-up different subtype HIV experiment strain virus infects Tzm-bl cell, and experiment strain virus comprises HIV _nL4-3(B hypotype), HIV _89.6(B hypotype), HIV _94UG114(D hypotype), HIV _mJ4(C hypotype), HIV _wCML249(D/C hypotype).

Fig. 5 is monoclonal antibody 15A7 and Tzm-bl cell, the laser confocal microscope detected result of the immunofluorescence of U87.CD4.CXCR4 cell and U87.CD4.CCR5 Cell binding, the monoclonal antibody H5F4 that use therein contrast is anti-p24.

Fig. 6 is monoclonal antibody 14G7 and Tzm-bl cell, the laser confocal microscope detected result of the immunofluorescence of U87.CD4.CXCR4 cell and U87.CD4.CCR5 Cell binding, the monoclonal antibody H5F4 that use therein contrast is anti-p24.

Fig. 7 is the flow cytometer detected result of recombinant protein c D4 (d1), CD4 (d2), CD4 (d3), CD4 (d4), CD4 (d1-2) and sCD4 blocking-up 15A7 antibody and cell response.

Fig. 8 is the flow cytometer detected result of recombinant protein c D4 (d1), CD4 (d2), CD4 (d3), CD4 (d4), CD4 (d1-2) and sCD4 blocking-up 14G7 antibody and cell response.

Fig. 9 is the single-chain antibody expression plasmid figure of monoclonal antibody 15A7 and 14G7.

Figure 10 is monoclonal antibody purifying SDS-PAGE result, and each road sample is followed successively by from left to right: 1: molecular weight of albumen Marker; 2: monoclonal antibody 15A7 reduces processing sample; 3:15A7 Fab fragments reduction processing sample; 4:15A7 antibody F (ab ') ₂fragment reduction; 5: the non-reduced processing sample of monoclonal antibody 15A7; The non-reduced processing sample of 6:15A7 monoclonal antibody; 7:15A7 antibody F (ab ') ₂non-reduced processing sample.

Figure 11 is monoclonal antibody 15A7 and F (ab ') thereof ₂, Fab antibody fragment and HIV _nL4-3virus reaction, blocks the result that it enters Tzm-bl cell, contrasts as damping fluid.

Figure 12 is the not flow cytometer detected result of isoacceptor of Tzm-bl cell surface, and wherein the little figure of A is to be primary antibodie with monoclonal antibody 15A7, and the rabbit anti-mouse antibody (RAM-FITC, Cat.No.F9006Sigma) of FITC mark is two anti-marks cell to be measured; The little figure of B is the CD4 receptor protein with anti-CD4 (Cat.No.555346, BD Biosciences) the mark cell surface to be measured of FITC mark; The little figure of C is the CXCR4 receptor protein with anti-CXCR4 (Cat.No.555974, BD Biosciences) the mark cell surface to be measured of PE mark; The little figure of D is the CCR5 receptor protein with anti-CCR5 (Cat.No.556889, BD Biosciences) the mark cell surface to be measured of PE-Cy5 mark; The little figure of E is unmarked cell contrast.

Figure 13 is the not flow cytometer detected result of isoacceptor of MT4 cell surface, and wherein the little figure of A is to be primary antibodie with monoclonal antibody 15A7, and the rabbit anti-mouse antibody (RAM-FITC, Cat.No.F9006Sigma) of FITC mark is two anti-marks cell to be measured; The little figure of B is the CD4 receptor protein with anti-CD4 monoclonal antibody (Cat.No.555346, BD Biosciences) the mark cell surface to be measured of FITC mark; The little figure of C is the CXCR4 receptor protein with anti-CXCR4 monoclonal antibody (Cat.No.555974, BD Biosciences) the mark cell surface to be measured of PE mark; The little figure of D is the CCR5 receptor protein with anti-CCR5 monoclonal antibody (Cat.No.556889, BD Biosciences) the mark cell surface to be measured of PE-Cy5 mark; The little figure of E is unmarked cell contrast.

Figure 14 is the not flow cytometer detected result of isoacceptor of H9 cell surface, and wherein the little figure of A is to be primary antibodie with monoclonal antibody 15A7, and the rabbit anti-mouse antibody (RAM-FITC, Cat.No.F9006Sigma) of FITC mark is two anti-marks cell to be measured; The little figure of B is the CD4 receptor protein with anti-CD4 monoclonal antibody (Cat.No.555346, BD Biosciences) the mark cell surface to be measured of FITC mark; The little figure of C is the CXCR4 receptor protein with anti-CXCR4 monoclonal antibody (Cat.No.555974, BD Biosciences) the mark cell surface to be measured of PE mark; The little figure of D is the CCR5 receptor protein with anti-CCR5 monoclonal antibody (Cat.No.556889, BD Biosciences) the mark cell to be measured of PE-Cy5 mark; The little figure of E is unmarked cell contrast.

Figure 15 is the not flow cytometer detected result of isoacceptor of U87.CD4.CXCR4 cell surface, wherein the little figure of A is to be primary antibodie with monoclonal antibody 15A7, the rabbit anti-mouse antibody (RAM-FITC, Cat.No.F9006Sigma) of FITC mark is two anti-marks cell to be measured; The little figure of B is the CD4 receptor protein with anti-CD4 monoclonal antibody (Cat.No.555346, BD Biosciences) the mark cell surface to be measured of FITC mark; The little figure of C is the CXCR4 receptor protein with anti-CXCR4 monoclonal antibody (Cat.No.555974, BD Biosciences) the mark cell surface to be measured of PE mark; The little figure of D is the CCR5 receptor protein with anti-CCR5 monoclonal antibody (Cat.No.556889, BD Biosciences) the mark cell surface to be measured of PE-Cy5 mark; The little figure of E is unmarked cell contrast.

Figure 16 is the not flow cytometer detected result of isoacceptor of U87.CD4.CCR5 cell surface, wherein the little figure of A is to be primary antibodie with monoclonal antibody 15A7, the rabbit anti-mouse antibody (RAM-FITC, Cat.No.F9006Sigma) of FITC mark is two anti-marks cell to be measured; The little figure of B is the CD4 receptor protein with anti-CD4 monoclonal antibody (Cat.No.555346, BD Biosciences) the mark cell surface to be measured of FITC mark; The little figure of C is the CXCR4 receptor protein with anti-CXCR4 monoclonal antibody (Cat.No.555974, BD Biosciences) the mark cell surface to be measured of PE mark; The little figure of D is the CCR5 receptor protein with anti-CCR5 monoclonal antibody (Cat.No.556889, BD Biosciences) the mark cell surface to be measured of PE-Cy5 mark; The little figure of E is unmarked cell contrast.

Figure 17 is the not flow cytometer detected result of isoacceptor of human peripheral blood single nucleus cell (PBMC) cell surface, wherein the little figure of A is to be primary antibodie with monoclonal antibody 15A7, the rabbit anti-mouse antibody (RAM-FITC, Cat.No.F9006Sigma) of FITC mark is two anti-marks cell to be measured; The little figure of B is the CD4 receptor protein with anti-CD4 monoclonal antibody (Cat.No.555346, BDBiosciences) the mark cell surface to be measured of FITC mark; The little figure of C is the CXCR4 receptor protein with anti-CXCR4 monoclonal antibody (Cat.No.555974, BD Biosciences) the mark cell surface to be measured of PE mark; The little figure of D is the CCR5 receptor protein with anti-CCR5 monoclonal antibody (Cat.No.556889, BD Biosciences) the mark cell surface to be measured of PE-Cy5 mark; The little figure of E is unmarked cell contrast.

The situation of the 293FT cell of Figure 18 is the monoclonal antibody 15A7 marker expression that detects of flow cytometer HIV, use therein control cells is blank 293FT cell, the monoclonal antibody H5F4 that control antibodies is anti-p24.

Figure 19 is the laser confocal microscope detected result of the immunofluorescence of monoclonal antibody 15A7 and the 293FT cell of having expressed HIV, and use therein control cells is blank 293FT cell, the monoclonal antibody H5F4 that control antibodies is anti-p24.

Figure 20 is the CD4 albumen of various brachymemmas, comprises recombinant protein c D4 (d1), CD4 (d2), CD4 (d3), CD4 (d4), CD4 (d1-2) and sCD4 blocking-up HIV _nL4-3infect the effect of Tzm-bl cell.

Figure 21 is the immunoblotting reaction result of brachymemma CD4 albumen and the monoclonal antibody 15A7 of purifying.Wherein the little figure of A1 is brachymemma CD4 albumen reduction SDS-PAGE figure, and the little figure of A2 is also virgin rubber immunoblotting reaction figure of brachymemma CD4 albumen; The little figure of B1 is the non-reduced glue figure of brachymemma CD4 albumen, and the little figure of B2 is the non-reduced glue immunoblotting reaction of brachymemma CD4 albumen.Ge little Tu Ge road sample is followed successively by 1: molecular weight of albumen marker; 2:CD4 (d1); 3:CD4 (d2); 4:CD4 (d3); 5:CD4 (d4); 6:CD4 (d1-2); 7:sCD4; 8: reference protein; The unloaded thalline of 9:BL21.

Figure 22 is the flow cytometer detected result with 15A7 mark TZM-bl cell, comprising adding HIV _nL4-3do not add HIV _nL4-3two kinds of situations.

Figure 23 is in recombinant protein c D4 (d1), CD4 (d1-2) and restructuring sCD4 blocking-up 15A7 and HIV _nL4-3infect Tzm-bl cell in and laboratory test results.

Figure 24 uses CD4 (d1), CD4 (d1-d2) and has recombinated sCD4 and transfection the 293FT cell response of pNL4-3, anti-as two with monoclonal antibody 15A7, the result of carrying out flow cytometer detection after the rabbit anti-mouse antibody mark with FITC mark again, wherein the little figure of A is the reaction result of monoclonal antibody 15A7; The little figure of B uses anti-CD4 monoclonal antibody (clone Q4120, the Cat.No.C1805Sigma) reaction result of mark cell surface CD4 to be measured receptor protein in contrast.

Figure 25 is the immunoblotting reaction result of monoclonal antibody 15A7 and point mutation CD4 (d1-2) albumen.Wherein the little figure of A1 is each point mutation CD4 (d1-2) albumen reduction SDS-PAGE figure; The little figure of A2 is the non-reduced sample SDS-PAGE figure of each point mutation CD4 (d1-2) albumen; The little figure of B1 is also former state immunoblotting reaction hybridization figure of each point mutation CD4 (d1-2) albumen; The little figure of B2 is the non-reduced sample immunoblotting reaction hybridization of each point mutation CD4 (d1-2) albumen figure.Ge little Tu Ge road sample is sequentially followed successively by 1: molecular weight of albumen marker; 2:CD4 (d1-2)-N73A; 3:CD4 (d1-2)-R59A; 4:CD4 (d1-2)-K46A; 5:CD4 (d1-2)-F43A; 6: reference protein CD4 (d1-2).

Embodiment

The relational term relating in the present patent application is defined as follows:

" surface antigen differentiation bunch 4 " word in the present invention refers to any surface antigen differentiation bunch 4 albumen by surface antigen differentiation bunch 4 genes encodings.

" CD4 in the present invention ⁺cell " a word phalangeal cell surface has the cell of CD4 glycoprotein, comprising CD4 ⁺t lymphocyte and CD4 ⁺clone, as TZM-bl, H9 and C8166 etc.

" CD4 the first structural domain (D1) " word in the present invention refers to surface antigen differentiation bunch 4 polypeptide of the brachymemma of the 1st to the 98th.

" CD4 the second structural domain (D2) " word in the present invention refers to surface antigen differentiation bunch 4 polypeptide of the brachymemma of the 99th to the 183rd.

" CD4 the first structural domain (D3) " word in the present invention refers to surface antigen differentiation bunch 4 polypeptide of the brachymemma of the 184th to the 291st.

" CD4 the first structural domain (D4) " word in the present invention refers to surface antigen differentiation bunch 4 polypeptide of the brachymemma of the 292nd to the 365th.

" CD4 the first/bis-structural domain (D1-2) " word in the present invention refers to surface antigen differentiation bunch 4 polypeptide of the brachymemma of the 1st to the 183rd.

" recombinant soluble CD4 (sCD4, rsCD4, D1-4) " word in the present invention refers to surface antigen differentiation bunch 4 polypeptide of the brachymemma of the 1st to the 365th.

" anti-CD 4 antibodies " word in the present invention refers to that identification is positioned at the antibody of the epi-position on surface antigen differentiation bunch 4 protein moleculars, the antibody that epi-position on surface antigen differentiation bunch 4 albumen is combined.

" envelope glycoprotein " word in the present invention refers to the envelope glycoprotein of human immunodeficiency virus.Envelope glycoprotein mediates human immunodeficiency virus for the absorption of host cell and enters process.Its precursor forms is gp160, after processing, is gp120 and gp41, on human immunodeficiency virus coating, with polymeric functional unit form, exists.

" antibody " word in the present invention refers to any one immunoglobulin (Ig), comprise can binding specificity monoclonal antibody, many anti-, dual specifics or the multi-specificity antibody of antigen.A complete antibody comprises two heavy chains and two light chains.Every heavy chain contains a variable region and three constant regions of first, second, third, etc.; Every light chain comprises a variable region and a constant region.Antibody is " Y " type, the second and the 3rd constant region that the neck of " Y " type structure contains two heavy chains, and it forms by disulfide-bonded.Every arm of " Y " type structure contains wherein the first constant region and the variable region of a heavy chain, and the variable region of a light chain and constant region.The variable region of light chain and heavy chain determines the combination of antigen; Three hypervariable regions are all contained in the variable region of every chain, and (CDR of light chain (L) comprises LCDR1, LCDR2, LCDR3, and the CDR of heavy chain (H) comprises HCDR1, HCDR2, HCDR3 to be called complementary determining region (CDR).It is named by people such as Kabat, sees Sequences ofProteins of Immunological Interest, Fifth Edition (1991), 1-3 volume, NIHPublication 91-3242, Bethesda MD).Wherein, three CDR are spaced apart by framework region (FR).BiCDR district, framework region is more conservative and form a shelf shape support structure hypervariable region.The constant region of heavy chain and light chain is combined irrelevant with antigen, but has multiple effector function.Antibody can be divided into several classes according to the aminoacid sequence of CH, mainly: IgA, IgD, IgE, IgG and IgM, wherein some class is also further divided into subclass, as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 etc.

" antibody " word in the present invention, ultrawhite except refering in particular to complete immune globulin, also the fragment (as being at least an immunocompetence section of immunoglobulin molecules) that refers to immunoglobulin (Ig), as Fab, Fab ', F (ab ') 2, Fv fragment, single-chain antibody molecule or the multi-specificity antibody that formed by any fragment of the immunoglobulin molecules that contains one or more CDR district.In addition, the antibody the present invention relates to antibody that can be also one or more CDR district in a specific human normal immunoglobulin form in conjunction with the framework region of one or more different human normal immunoglobulins.In Fragment Protein molecule, only comprise 1 CDR in heavy chain CDR, 2 CDR, 3 CDR can realize the recognition reaction of identical epi-position.In Fragment Protein molecule, only comprise 1 CDR in light chain CDR, 2 CDR to 3 CDR section can be realized the recognition reaction of identical epi-position equally.1 or 2 GeCDR districts only retaining mouse monoclonal antibody, all the other are all brand-new aminoacid sequences, the new antibodies of acquisition still has the activity of parent mouse monoclonal antibody.1998, Rader etc. are transplanted to the HCDR3 on mouse monoclonal antibody LM609 variable region and LCDR3 in human antibody sequence, success obtains humanized antibody (the Rader C that only retains mouse HCDR3 and LCDR3, Cheresh DA, Barbas CF 3rd.A phage display approach for rapid antibodyhumanization:designed combinatorial V gene libraries.Proc Natl AcadSci U S A.1998,95 (15): 8910-5).2000, the acquisitions such as Klimka only retain humanized antibody (the Klimka A of the anti-CD-30 of mouse HCDR3, Matthey B, Roovers RC, et al, Human anti-CD30 recombinant antibodies by guided phageantibody selection using cell panning.Br J Cancer.2000,83 (2): 252-60).

" Fab " fragment that antibody is relevant refers to variable region and the variable region of constant region and a heavy chain and a part for the antibody molecule that constant region is got up through disulfide-bonded that contains a light chain.

" Fab ' " fragment refers to the Fab fragment that has comprised part hinge area.

F (ab ') ₂refer to the dimer of Fab '.

" Fc " of antibody refers to a part for the antibody that second, third constant region of the first heavy chain and second, third constant region of the second heavy chain become through disulfide-bonded.The Fc section of antibody has multiple different function, but does not participate in the combination of antigen.

" Fv " section of antibody refers to can be in conjunction with the minimal segment of the antibody of complete antigen binding site.A Fv fragment comprises that the variable region of a light chain is attached to the variable region of a heavy chain.

" single-chain antibody " in the present invention or " scFv " refer to the engineered antibody (Houston 1988) that is directly connected with variable region of heavy chain or is formed by connecting by a peptide chain by variable region of light chain

" single-chain antibody Fv-Fc " in the present invention or " scFv-Fc " also comprise that scFv connects the engineered antibody of the Fc section formation of antibody.

" antigenic determinant " in the present invention (or claim epi-position) refers in antigen molecule part amino acid or the atomic radical with antibodies.

" monoclonal antibody " word in the present invention refers to a fragment from antibody in the antibody molecule of a group height homology or antibody, also except the spontaneous mutation that only may occur under a few cases, and the identical antibody molecule of a group.Monoclonal antibody has high specific to the single epi-position on antigen.How anti-different monoclonal antibody from, and resist is the antibody molecule that has comprised the different epi-positions on identification antigen more.Although traditional monoclonal antibody is secreted by hybridoma, the monoclonal antibody the present invention relates to is not limited in this preparation method.The monoclonal antibody the present invention relates to can adopt the hybridoma technology of the reported first such as Kohler obtain ( g. wait people, Nature, 1975,256:495-497), also can adopt recombinant DNA technology to obtain (as referring to U.S.P 4,816,567).

" chimeric antibody " word in the present invention refers to light chain of antibody or/and a part for heavy chain is the antibody that is derived from a certain Special Thing species or genus or homology identical in the sequence of a certain specific antibodies class or subclass, and light chain of antibody is or/and another part of heavy chain is the antibody that is derived from another species or belongs to the identical or homology of the sequence of another antibody class or subclass.In any case, this antibody fragment still retained to the combination of target antigen active (U.S.P 4,816,567; The people such as Morrison, Proc.Natl.Acad.Sci.USA, 81:6851 6855 (1984)).

In the present invention, antibody or antibody fragment that all or part of CDR district that " humanized antibody " word refers to people source immunoglobulin (Ig) (receptor antibody) obtains after being replaced by non-human antibody (donor antibody) CDR district, donor antibody wherein can be to have expection specificity, affinity and reactive mouse, rat or rabbit antibody.In addition, the aminoacid sequence of the framework region of people source immunoglobulin (Ig) (FR) also can be replaced by the aminoacid sequence of corresponding non-human antibody.And the amino-acid residue of humanized antibody also can be neither derive from receptor antibody, also non-donor antibody CDR district or the framework region sequence of deriving from.These manually modified objects are further to improve or optimization antibody performance.In a word, humanized antibody refer to contain at least one, two complete variable regions almost normally, wherein corresponding all or nearly all CDR district is from non-human antibody, the district of FR all or almost all is wherein from human antibody.Desirable humanized antibody at least contains the part in immunoglobulin (Ig) Fc district, normally immunoglobulin (Ig) Fc district, people source.More detailed contents refer to: the people such as Jones P.T., Nature, 1986,321:522-525; The people such as Reichmann L., Nature.1988,332:323-327; Presta, Curr Opin StructBiol, 1992,2:593-596and Clark M.Immunol Today 2000,21:397-402.

" separated " word the present invention relates to, refers under native state and to obtain through artificial means.If material or the composition of a certain " separated " appear in occurring in nature, may be that its residing natural surroundings has occurred change or from natural surroundings, isolate this material so, or the two situation all have generation.Such as, natural existence certain not separated polynucleotide or polypeptide in a certain living animal body, and highly purified identical polynucleotide or the polypeptide from this native state, separated are referred to as separated.Here " separated " do not got rid of and is mixed with artificial or synthetic material, do not get rid of other impurity that existence does not affect material activity yet.

In the present invention, " carrier " word refers to, and the polynucleotide of certain albumen of coding can be inserted wherein and make albumen obtain a kind of nucleic acid launch vehicle of expressing.Carrier can, by conversion, transduction or transfection host cell, be expressed its genetic material element carrying in host cell.For instance, carrier comprises: plasmid; Phagemid; Coemid; Artificial chromosome is as the artificial chromosome (PAC) in yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1 source; Phage is as lambda particles phage or M13 phage and animal virus etc.Animal virus kind as carrier has retrovirus (comprising slow virus), adenovirus, adeno-associated virus, simplexvirus (as hsv), poxvirus, baculovirus, papilloma virus, papova viruses (as SV40).Carrier may contain the element that various control is expressed, and comprises promoter sequence, transcriptional initiation sequence, enhancer sequence, selectors and reporter gene.In addition, carrier also can contain replication origin.Carrier also likely comprises assists it to enter the composition of cell, as virion, liposome or protein coat, but not only only has these materials.

In the present invention, " host cell " word refers to the cell that imports carrier, comprise following many cell types, as prokaryotic cell prokaryocytes such as intestinal bacteria or withered grass bacterium, as fungal cells such as yeast cell or aspergillus tubigensis, as insect cells such as S2 drosophila cell or Sf9, or as fibroblast, Chinese hamster ovary celI, COS cell, NSO cell, HeLa cell, bhk cell, the zooblast of HEK 293 cells or people's cell.

" neutralizing antibody " word refers to can remove or significantly reduce target viral antigen in conjunction with antibody or the antibody fragment of virulence.

" sequence same percentage " word refer to nucleic acid in candidate sequence or amino acid respectively with corresponding nucleic acid or the per-cent of peptide sequence amplifying nucleic acid or amino acid homogeny.Here the term relevant with nucleotide sequence or peptide sequence " sequence similarity per-cent " relating to be defined as candidate nucleic acid sequence or amino acid residue sequence respectively to the similar per-cent of object nucleotide sequence or aminoacid sequence.For some sequences, are compared in it and aim sequence, can skip sudden change breach if desired, to reach the maximum similar per-cent of gene, and do not go any conservative property of considering similar sequences to suddenly change.The multiple comparison method of this area can be used for determining the similarity of nucleic acid or aminoacid sequence, as available computer software comprises BLAST, and BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) etc.The staff of skilled understands that some algorithms that are included as the maximum comparability of use reach the comparison of full length sequence for the suitable measuring parameter of comparison setting.

" specific binding " word refers to, and two intermolecular nonrandom association reactions, as antibody with produce the reaction between the antigen of this antibody.Herein, the antibody in conjunction with the first antigen is that can't detect or very weak to the binding affinity of the second antigen.In some embodiments, certain antigen-specific antibodies refers to avidity (KD)≤10 ^-5m is (as 10 ^-6m, 10 ^-7m, 10 ^-8m, 10 ^-9m, 10 ^-10m etc.), in conjunction with this antigen, wherein KD refers to the ratio (k of dissociation yield and combination rate _off/ k _on), it can adopt the familiar method of those skilled in the art to measure.

Antibody

Monoclonal antibody of the present invention can specific binding surface antigen differentiation bunch 4 acceptors.One aspect of the present invention relates to monoclonal antibody and the corresponding Fab thereof of energy specific binding surface antigen differentiation bunch 4 acceptors.

In one embodiment, monoclonal antibody of the present invention is secreted by mouse hybridoma cell strain 15A7.The title of these monoclonal antibodies is named with its corresponding hybridoma cell strain.Namely, these monoclonal antibodies 15A7 is produced by hybridoma 15A7, and called after 15A7.Monoclonal antibody 15A7 can specific binding surface antigen differentiation bunch 4 albumen.Mouse hybridoma cell 15A7 is at Chinese Typical Representative culture collection center (CCTCC, Wuhan University, Wuhan, China) carry out preservation, preserving number is CCTCC NO:C201098 (hybridoma 15A7, October 22 2010 preservation time).

Monoclonal antibody of the present invention also comprises the monoclonal antibody that can block monoclonal antibody 15A7 mating surface antigen differentiation bunch 4 albumen.The epi-position that the epi-position of surface antigen differentiation bunch 4 albumen of these monoclonal antibody combinations can be identified with monoclonal antibody 15A7 is identical.The epi-position that the epi-position that these monoclonal antibodies are identified also can be identified with monoclonal antibody 15A7 spatially has overlapping.Such monoclonal antibody can reduce the bonding force at least 30% of monoclonal antibody 15A7 and surface antigen differentiation bunch 4 receptor proteins, or at least 40%, or preferably at least 50%, or more preferably at least 60%, or more preferably at least 70%, or more preferably at least 80%, or more preferably at least 90%, or more preferably 95%, or most preferably 99%.

Monoclonal antibody of the present invention can not significantly suppress HIV envelope glycoprotein gp120 and people's surface antigen differentiation bunch 4 protein binding.These antibody affect HIV envelope glycoprotein gp120 and people's surface antigen differentiation bunch 4 albumen (or people's surface antigen differentiation bunch 4 albumen of recombinant human surface antigen differentiation bunch 4 albumen sCD4 and brachymemma) at the most 30%, or more preferably 20%, or most preferably 0%.

Unaffected in the situation that is combined in HIV envelope glycoprotein gp120 existence of monoclonal antibody of the present invention and people's surface antigen differentiation bunch 4 protein binding (breaking up bunch 4 albumen containing cell surface, people's surface antigen recombinant expressed and brachymemma), such monoclonal antibody is 30% in the bonding force impact of the mixture of HIV envelope glycoprotein gp120 and surface antigen differentiation bunch 4 receptor proteins, or be preferably 20%, or more preferably 5%.

Can adopt ordinary method as Antibodies:A Laboratory Manual, ColdSpring Harbor Laboratory, the method of describing in Ed Harlow and David Lane (1988), measures the ability that a certain unknown monoclonal antibody reduces a certain known monoclonal antibody mating surface antigen differentiation bunch 4 albumen.For example, first that antigen is pre-coated on microwell plate, then the known monoclonal antibody after the mark of the antibody unlabelled to be measured of serial dilution and certain concentration is jointly added in above-mentioned microwell plate after pre-coated and hatched, after washing, measure different dilution antibody known antibodies to be measured and be attached to the quantity on plate.The ability of antibody competition known antibodies conjugated antigen to be measured is stronger, and the ability of known antibodies conjugated antigen is just more weak.Conventionally, antigen is pre-coated on 96 hole microwell plates, and utilizes Radio labeled method or enzyme labelling method to measure the unmarked MAbs blocking ability of mark monoclonal antibody.

Can adopt the hybridoma preparation method of the report in Nature 256:495 (1975) such as Kohler to prepare monoclonal antibody.First by immunogen (adding adjuvant in the time of necessity) immunization mouse or other suitable host animal.The injection system of immunogen or adjuvant is generally subcutaneous multi-point injection or abdominal injection.Immunogen is coupled to some known albumen in advance, on serum albumin or soybean pancreatin inhibitor, may contribute to the immunogenicity of enhancement antigen in host.Adjuvant can utilize freund adjuvant or MPL-TDM etc.Animal is subject to after immunity, and the lymphocyte that has the immunogenic antibody of secretion specific binding in body produces.In addition, lymphocyte also can utilize external immunity to obtain.Collect object lymphocyte and myeloma cell and with suitable fusogen, as PEG, merge to obtain hybridoma (Goding, Monoclonal Antibodies:Principles andPractice, pp.59-103, Academic Press, 1996).

The hybridoma of above-mentioned preparation can be inoculated in suitable nutrient solution grows, and preferably contains material that one or more can suppress not merge, parent myeloma cell growth in nutrient solution.For example, to lacking the parent myeloma cell of xanthoglobulin guanine monophosphate transferring enzyme (HGPRT or HPRT), in nutrient solution, add the growth that the materials such as xanthoglobulin, aminopterin-induced syndrome and thymus pyrimidine (HAT substratum) can suppress HGPRT-deficient cells.

It is high that preferred myeloma cell should have fusion rate, and antibody-secreting ability is stable, to abilities such as HAT nutrient solution sensitivities.Wherein, the first-selected mouse of myeloma cell source myelomatosis, as MOP-21 and derivative strain (the THE Salk Institute Cell Distribution Center of MC-11 mouse tumor, San Diego, Calif.USA), with SP-2/0 or X63-Ag8-653 cell strain (AmericanType Culture Collection, Rockville, Md.USA).Also studies have reported that in addition and utilize human myeloma and people mouse allos myeloma cell strain to prepare people's monoclonal antibody (Kozbor, J.Immunol., 133:3001 (1984); The people such as Brodeur, Monoclonal Antibody ProductionTechniques and Applications, pp.51-63, Marcel Dekker, Inc., New York, 1987).

The nutrient solution of Growth of Hybridoma Cell is for detection of the generation of the monoclonal antibody for specific antigen.The method of the binding specificity of the monoclonal antibody that mensuration hybridoma produces has immunoprecipitation or external combination test, as radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA), flow cytometry (FACS).For example, the Scatchard analytical method that Munson etc. describes at Anal.Biochem.107:220 (1980) can be used to measure the avidity of monoclonal antibody.

After specificity, avidity and the reactivity of the antibody that hybridoma produces are determined, object cell strain can pass through (Goding, Monoclonal Antibodies:Principles and Practice, pp.59-103, Academic Press, 1996) limiting dilution assay of described standard carries out subcloning.Suitable nutrient solution can be DMEM or RPMI-1640 etc.In addition, the form that hybridoma can also ascitic tumor is grown in animal body.

Utilize traditional immunoglobulin purification method, as albumin A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography etc., the monoclonal antibody of subclone emiocytosis can be separated from cell culture fluid, ascites or serum.

Monoclonal antibody of the present invention can also obtain by genetically engineered recombinant technology.Utilize the nucleic acid primer of specific binding monoclonal antibody heavy chain and light chain gene to carry out pcr amplification, the DNA molecular of can be from hybridoma separated obtain encoding monoclonal antibody heavy chain and light chain gene.Gained DNA molecular inserts in expression vector, and transfection host cell then, as E.coli cell, ape and monkey COS, Chinese hamster ovary celI or other do not produce the myeloma cell of immunoglobulin (Ig).Target antibody is cultivated and expressed to host cell after transfection under given conditions.

Antibody on human surface antigen differentiation bunch 4 protein binding of the present invention have high specific and high-affinity.These antibody have cross reactivity to other primates surface antigen differentiation bunch 4 receptor proteins.

Monoclonal antibody of the present invention can be the antibody of traditional " Y " type structure shape of comprising two heavy chains and two light chains.In addition, described antibody can be also Fab fragment, Fab ', the F (ab ') having kept on the antibody of traditional " Y " type structure shape of effects on surface antigen differentiation bunch 4 receptor protein avidity ₂, Fv or other type Partial Fragment, its avidity in conjunction with hemagglutinin can be higher or lower than the antibody of traditional " Y " type structure shape.

Antibody fragment of the present invention can utilize the complete antibody molecule of hydrolysis to obtain (referring to people such as Morimoto, the people such as J.Biochem.Biophys.Methods 24:107-117 (1992) andBrennan, Science 229:81 (1985)).In addition, these antibody fragments also can directly produce (reviewed in Hudson, Curr.Opin.Immunol.11:548-557 (1999) by recombinant host cell; The people such as Little, Immunol.Today, 21:364-370 (2000)).Such as, Fab ' fragment can directly from E.coli cell, obtain or chemical coupling forms F (ab ') ₂fragment (people such as Carter, Bio/Technology, 10:163-167 (1992)).For another example F (ab '), ₂fragment can connect and obtain with leucine zipper GCN4.In addition, Fv, Fab, Fab ' or F (ab ') ₂fragment also can be directly from recombinant host cell nutrient solution separation obtain.Those of ordinary skill in the art knows other technology of Dispersal risk fragment completely.

Antibody nucleotide sequence

The present invention relates to the antibody of specific binding people surface antigen differentiation bunch 4 albumen (containing people's surface antigen differentiation bunch 4 albumen cell surface, recombinant expressed and recombinant expressed brachymemma) or the coding nucleic acid molecule of antibody fragment.The nucleic acid molecule of encoding antibody can obtain from hybridoma in separation.Those of ordinary skill in the art knows the nucleotide sequence that utilizes routine techniques can measure these molecules completely.The antibody nucleic acid molecule the present invention relates to also can utilize traditional genetically engineered recombinant technology or chemical synthesis process to obtain.The sequence of the antibody nucleic acid molecule the present invention relates on the one hand, has comprised the anti-human surface antigen differentiation variable region of heavy chain of bunch 4 protein antibodies or the part nucleotide sequence of antibody molecule.The sequence of the antibody nucleic acid molecule the present invention relates on the other hand, also comprises the anti-human surface antigen differentiation variable region of light chain of bunch 4 protein antibodies or the part nucleotide sequence of antibody molecule.The sequence of the antibody nucleic acid molecule the present invention relates on the other hand, also comprises the CDR sequence of heavy chain or variable region of light chain.Complementary determining region (complementary determinant region, CDR) be the position of being combined with epitope, the CDR sequence in this research is determined by IMGT/V-QUEST (http://imgt.cines.fr/textes/vquest/).But the CDR sequence that different division methods obtains is slightly different.

An aspect of of the present present invention relates to the nucleic acid molecule of coding monoclonal antibody 15A7 heavy chain and light chain variable region sequence.The nucleic acid molecule of monoclonal antibody 15A7 weight chain variabl area sequence is corresponding to SEQ ID NO:1.The nucleic acid molecule of monoclonal antibody 15A7 light chain variable region sequence is corresponding to SEQ ID NO:3.The invention still further relates to the nucleic acid molecule variant or the analog that have comprised monoclonal antibody 15A7 heavy chain and light chain variable region sequence.

On the other hand, the invention still further relates to the variant of the nucleic acid molecule of various separation.Particularly, the identity of the sequence of nucleic acid variant and nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3 at least reaches 70%, preferably at least reaches 75%, more preferably at least reaches 80%, more preferably at least reaches 85%, more preferably at least reaches 90%, most preferably at least reaches 95%.

The present invention also provide can specific binding the nucleic acid molecule of encoding sequence of antibody fragment of people's surface antigen differentiation bunch 4 albumen.

The present invention further relates to the separated nucleic acid molecule that encoding amino acid sequence is the antibody heavy chain variable region of SEQ ID NO:2.The invention still further relates to the nucleic acid molecule that encoding amino acid sequence is the antibody chain variable region of SEQ IDNO:4.

The present invention relates to the recombinant expression vector that contains described nucleic acid molecule, also relate to the host cell that has transformed these molecules.And, the invention still further relates to and utilize the host cell that comprises described nucleic acid molecule to cultivate under given conditions and the separated method that obtains inventing described antibody.

Antibody polypeptides sequence

Monoclonal antibody 15A7 heavy chain and light chain variable region amino acid sequence can be derived and obtain from corresponding nucleotide sequence.Monoclonal antibody 15A7 weight chain variable region amino acid sequence is SEQ ID NO:2.Monoclonal antibody 15A7 light chain variable region amino acid sequence is SEQ ID NO:4.

On the other hand, the invention provides the aminoacid sequence of variable region of heavy chain and the sequence similarity of SEQ ID NO:2 at least reaches 70%, preferably at least 75%, preferably at least 80%, preferably 85%, more preferably at least 90%, be most preferably at least 95% antibody.

On the other hand, the invention provides the aminoacid sequence of variable region of light chain and the sequence similarity of SEQ ID NO:4 at least reaches 70%, preferably at least 75%, preferably at least 80%, preferably 85%, more preferably at least 90%, be most preferably at least 95% antibody.

The aminoacid sequence of the heavy chain of monoclonal antibody 15A7 and the CDR of variable region of light chain is respectively:

CDR1, the CDR2 of monoclonal antibody 15A7 heavy chain and the aminoacid sequence of CDR3 are respectively SEQ ID NOs:5-7.CDR1, the CDR2 of monoclonal antibody 15A7 light chain and the aminoacid sequence of CDR3 are respectively SEQ ID NOs:8-10.

On the other hand, the invention provides antiCD4 mAb heavy chain or fragment, it contains the one or more CDR that are selected from SEQID NOs:5-7.

Can on SEQ ID NOs:5-7, there are one or more amino acid whose sudden changes or increase or lack in the aminoacid sequence that on the other hand, the heavy chain of antiCD4 mAb or the CDR of fragment contain.Preferably, the amino acid of sudden change or interpolation or disappearance is no more than 3 amino acid.More preferably, the amino acid of sudden change or interpolation or disappearance is no more than 2 amino acid.Most preferably, the amino acid of sudden change or interpolation or disappearance is no more than 1 amino acid.

On the other hand, the invention provides antiCD4 mAb light chain or fragment and contain the one or more CDR that are selected from SEQ IDNOs:8-10.

Can on SEQ ID NOs:8-10, there are one or more amino acid whose sudden changes, increase or lack in the aminoacid sequence that on the other hand, the light chain of anti-human CD4 monoclonal antibody or the CDR of fragment contain.The amino acid of preferably, sudden change, interpolation or disappearance is no more than 3 amino acid.The amino acid of more preferably, sudden change, interpolation or disappearance is no more than 2 amino acid.The amino acid of most preferably, sudden change, interpolation or disappearance is no more than 1 amino acid.

The ability that variant afterwards still retains specific binding CD4 is undergone mutation, adds or lacked to the amino acid of the variable region of above-mentioned antibody or CDR.The present invention also comprises the variant of such Fab.

Monoclonal antibody variant of the present invention can obtain by traditional gene engineering method.Those skilled in the art knows the method for utilizing nucleic acid mutation transformation DNA molecular completely.In addition, the nucleic acid molecule of encoding heavy chain and light chain variant also can obtain by chemosynthesis.

Chimeric antibody, humanized antibody and fusion rotein

On the other hand, the present invention also provides chimeric antibody, and it is comprised of in conjunction with Human monoclonal antibody constant region the variable region heavy chain of mouse source monoclonal antibody 15A7 or its variant and/or intact light chain or part.And the present invention also comprises humanized antibody, they by one or more CDR graftings of mouse source monoclonal antibody 15A7 or its variant to forming on the framework of human antibody.

On the other hand, the present invention's coupling is also provided fusion rotein that contains monoclonal antibody of the present invention wholly or in part of certain molecule.

Chimeric antibody, humanized antibody and fusion rotein can utilize traditional genetic engineering technique to obtain.As, the mouse source sequence that the DNA of coding monoclonal antibody can replace to homology the sequence of the constant region of the heavy chain of human antibody and light chain by the method for sudden change is transformed into (Morrison, Deng people, Proc.Nat.Acad.Sci.81:6851 (1984)), or by immunoglobulin coding sequence and NIg encoding sequence are all or in part carried out to covalent coupling obtain chimeric or humanized antibody or fusion rotein.

Neutralizing antibody

On the other hand, the invention provides can in and human immunodeficiency virus's anti-CD 4 antibodies.In one embodiment, this neutralizing antibody can in and human immunodeficiency virus infection CD4 ⁺cell active at least 60%, or at least 70%, or preferably at least 75%, or preferably at least 80%, or preferably at least 85%, or preferably at least 90%, more preferably at least 95%, most preferably at least 99.99%.In vitro and in HIV Viral experiment, in IC50 and active concentration preferably between 0.0001-10 μ g/mL, or be preferably 50 μ g/mL.

Those of ordinary skills know completely utilize traditional technological method can measure in antibody and human immunodeficiency virus's cells infected active.The neutralization that can be used for measuring the some specific CD4 monoclonal antibody in the present invention as the method for the neutralization test described in embodiments of the invention is active.

Detection method

The present invention also provides a kind of and has utilized monoclonal antibody of the present invention to detect T4 antigen in sample or the method for its stand-in and/or antibody.

On the one hand, the invention provides the detection method of CD4 and CD4 associated treatment, comprise the following steps: (i) antigen in above-mentioned sample is combined and is gone into antibody-antigen or antibody fragment-antigenic compound by monoclonal antibody of the present invention or its fragment; (ii) detect this sample composites to determine whether have T4 antigen or its stand-in in sample.

Detection method can be used Enzyme-linked Immunosorbent Assay (ELISA), enzyme immunodetection, chemiluminescence immunoassay detection, radioimmunity detection, fluorescence immunoassay detection, immunochromatography, competition law and similar detection method.Utilize competition law or sandwich assay mode, above-mentioned detection method can be for detection of target antigen or antibody.

In competition law comparative sample, the competition of the labelled antigen of antigen and a kind of known quantity is in conjunction with the quantitative relation of monoclonal antibody of the present invention.Carry out that immunology detection based on competition law joins the known physics of prior use by the sample of the target antigen that contains unknown number or chemical process is coated with monoclonal antibody of the present invention on solid support and carries out.Add the target antigen after quantitative in advance mark to react simultaneously.After hatching, rinse solid support, detect the activity that is attached to the marker on this upholder.

In sandwich assay, the target antigen in sample is sandwiched between coated monoclonal antibody and mark monoclonal antibody, and then adds marker such as the substrate of enzyme, is detected and judged the existence of antigen by the variation of substrate colors.Carry out the immunology detection based on sandwich assay, for example, first the sample that contains a kind of unknown number target antigen is joined by physics or chemical process and has been coated with in advance on the solid support of monoclonal antibody described in the present invention and reacted.Then, add mark monoclonal antibody of the present invention to react.After hatching, rinse this upholder, then detect being attached to the activity of the marker on this upholder.Marker can be radio isotope if the substrate of 125 iodine, enzyme, enzyme, luminophore are if different luminol,3-aminophthalic acid cyclic hydrazide and acridinium ester, fluorescent substance are if fluorescein and rhodamine, vitamin H and coloring matter are as latex particle and Radioactive colloidal gold etc.The enzyme that mark is used can be peroxidase (as horseradish peroxidase HRP), alkaline phosphatase, beta galactosidase enzyme and glucose oxidase.For suitable substrate in these reactions, have 2; 2 '-azino-bis-(3-ethyl benzo thiophene pyrroline-6 sulfonic acid), luminol,3-aminophthalic acid cyclic hydrazide-hydrogen peroxide, O-Phenylene Diamine-hydrogen peroxide (for peroxidase), para-nitro-pheneye phosphate, 4-methyl acid phosphate umbellate form ketone, 3-(2 '-spiral diamantane)-4-methoxyl group-4-(3 " phosphoryl) phenyl-1,2-diethoxy alkane (for alkaline phosphatase), p-nitrophenyl-β-D-semi-lactosi and methyl umbelliferone-β-D-semi-lactosi (for beta galactosidase enzyme).Other mark comprises quantum dot-labeled, chromophore's mark, enzyme labelling, affinity ligand mark, electromagnetism spin labeling, heavy atom mark, be marked with the probe of nanoparticle scattering of light mark or other nanoparticle, fluorescein isothiocyanate (FITC), TRITC, rhodamine, tetramethyl-rhodamine, R-PE, Cy-3, Cy-5, Cy-7, Texas is red, Phar-Red, different phycoerythrin (APC), Epitope tag is as FLAG or HA epi-position, and enzyme labelling is as alkaline phosphatase, horseradish peroxidase, I ²-tilactase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase and hapten conjugation thing as digoxigenin or dinitrophenol(DNP), maybe can form title complex combination pairing if streptavidin/vitamin H, avidin/biotin or antigen/antibody title complex are as comprised rabbit igg and anti-rabbit igg, fluorophor is as umbelliferose (umbelliferone), fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl-rhodamine, Yihong, green fluorescent protein, algae is red, tonka bean camphor, methylcoumarin, pyrene, Victoria Green WPB, toluylene, fluorescent yellow, Cascade is blue, dichlorotriazine base fluorescein, dansyl chloride, phycoerythrin, fluoresce lanthanide complex compound is as comprised europium and terbium, Cy3, Cy5, molecular beacon (molecular beacons) and its fluorescent derivative, luminescent material is as luminol,3-aminophthalic acid cyclic hydrazide, scattering of light or plasmone group resonance material is as gold or silver-colored particle or quantum mottle (quantum dot): or radio active material as ¹⁴c, ¹²³i, ¹²⁴i, ¹³¹i, Tc99m, ³⁵s or ³h, or ball (spherical shell), and be marked with the probe that any other signal known in the art produces marker.For example, detectable molecule includes but not limited to fluorophor and noted earlier other is known, as the Principles of Fluorescence Spectroscopy being compiled at Joseph R.Lakowicz (Editor), Plenum Pub Corp, the Molecular ProbesHandbook of the sixth version of second edition (July 1999) and Richard P.Hoagland is described.In some embodiments, marker comprises that semiconductor nanocrystals is as quantum mottle (being Qdots), referring to U.S.P 6,207,392.Qdots can buy from Quantum DotCorporation.For semiconductor nanocrystals of the present invention, comprise that the semi-conductive nano microcrystalline of Group II-V is if MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe and its mixture and the semi-conductive nano microcrystalline of Group III-V are as GaAs, InGaAs, InP, InAs and its mixture.Group IV semi-conductor is as the use of germanium or silicon, or organic semi-conductor use, may facilitate under certain conditions feasible.Semiconductor nanocrystals also can comprise alloy, and it contains two or more semi-conductors that is selected from Group III-V compound, Group II-VI compound, Group IV element and its composition.

In some embodiments, fluorescent energy acceptor is connected to the detection probes thing that serves as a mark.In one embodiment, fluorescent energy acceptor can react with singlet oxygen by compound and form fluorescent chemicals and form, or by compound, is reacted and be translated into fluorescent chemicals with an ancillary compound and form.This compounds can be included in the damping fluid in apparatus of the present invention.In other embodiment, fluorescent energy acceptor can be a part that comprises the compound of chemoluminescence agent or group, and for example, fluorescent energy acceptor can comprise that rare earth metal is as metal complexs such as europium, samarium, telluriums.To have sharp-pointed photoluminescent band attractive especially because of it for these materials; And group of the lanthanides marker, as europium (III) can provide effectively signal transmitting for a long time, is difficult for photobleaching, thereby can be so that the test set that contains processing/response sample can be placed longer for some time in the situation that of needs simultaneously.In various allos and homoimmune assay method, used long-life fluorescence europium (III) complex compound nanoparticle thing that serves as a mark, such as can be referring to people Clin.Chem.2004Oct such as Huhtinen; 50 (10): 1935-6.But while using together with the nanoparticle of these inner markers detects with time resolved fluorescence, measure performance and can improve.In allos is measured, the dynamicrange of measuring under lower concentration can be expanded; And, by use, detecting the high specific activity nanoparticle marker of antibody coating, rather than use the detection antibody of conventional mark, the dynamics of mensuration also can improve.In homology is measured, for FRET (fluorescence resonance energy transfer), europium (III) nanoparticle is effectively donor, thereby can carry out simply, screen fast and efficiently.In one embodiment, marker, as the fluorescent marker disclosing, comprises the nanoparticle marker with biomolecules coupling herein.In other words, nanoparticle can be as detecting or capturing probe.For example, in the present invention, can utilize the nanoparticle of europium (the III)-mark that is connected to monoclonal antibody or streptavidin (SA) to detect specific analyte in sample, as the immunoassay of nanoparticle base.Nanoparticle can be used as the substrate that adheres to particular combination agent, and these particular combination agent are for analyte and detection (as marker) or catch composition.The example of associated mark thing can be referring to U.S.P 4,695,554; 4,863,875; 4,373,932; With 4,366,241.U.S.P 4,313, disclosed colloidal metal and dyed particles in 734 and 4,373,932.And disclosed in U.S.P 4,954,452, how to prepare and to use nonmetallic colloid; U.S.P 4,252, disclosed the organic polymer latex particle as marker in 459.

Marker is attached to the method on antigen or antibody, can pass through maleimide method (J.Biochem. (1976), 79,233), vitamin H activation method (J.Am.Chem.Soc. (1978), 100,3585), hydrophobic binding method, ester activation method or isocyanic ester method (Igaku Shoin, " Enzymeimmune assay techniques ", 1987).

If above-mentioned marker is radio isotope, need to make radiation-resistant work top or the liquid-state protective equipment made good use of.If above-mentioned marker is enzyme, need add substrate, the activity of enzyme is measured by colorimetry or photofluorometer.If above-mentioned marker is fluorescent substance, luminophore or coloring matter, measuring method can be used method well known in the art to measure accordingly.

Methods for the treatment of and pharmaceutical composition

Antibody of the present invention and active fragments thereof and homologue thereof provide a kind of CD4 of preventing and/or treating ⁺the virus infection that cell is relevant and relative disease patient's method, especially human immunodeficiency virus (HIV) infection and relevant disease thereof comprise acquired immune deficiency syndrome (AIDS), comprise patient is used to a certain amount of pharmaceutical cpd that has pharmaceutical activity that comprises monoclonal antibody of the present invention.The salt medicine that the present invention also provides a kind of pharmaceutical cpd that contains monoclonal antibody of the present invention or obtained on this basis.

Pharmaceutical composition of the present invention can further comprise other medicament infecting for preventing and/or treating HIV, can with these Anti-virus agents simultaneously, separately or successive administration.For example, antibody of the present invention and active fragments thereof and homologue thereof can with the anti-reverse transcription medicament that can suppress ThermoScript II, as AZT is together used, or together use with the medicament that can suppress hiv protease; In addition, pharmaceutical composition of the present invention further comprises for example Interferon, rabbit of antiviral agent, and immunosuppressor is as Cyclosporin A, but is not limited only to these.

In addition, one or more antibody homologues also can together be used with one or more aforementioned therapies agent.The advantage of this kind of combination treatment is the healing potion that can use compared with low dosage, therefore can avoid toxicity or the side effect that when using single medicament, may have.

Pharmaceutical composition of the present invention comprises the antibody homologue according to one or more immunotherapy significant quantities of the present invention, or derivatives thereof, and pharmaceutically acceptable carrier preferably." immunotherapy significant quantity " refer to enough can prevent because HIV infects or acquired immune deficiency syndrome (AIDS), or be by former target the amount of expressing the immunne response effect that the caused other diseases of infectant of the cell of CD4 causes." pharmaceutically acceptable carrier " refers to can not cause at used patient the carrier of anaphylaxis or other uncomfortable impacts.

Suitable pharmaceutically acceptable carrier comprises, for example, and one or more water, physiological saline, phosphoric acid buffer, sinistrose, glycerine, ethanol and other analogues, and the combination of above-mentioned substance.Pharmaceutically acceptable carrier can further comprise can improve the storage life of antibody or its active fragments or its homologue or the micro-auxiliary substance of effectiveness, for example wetting agent or emulsifying agent, sanitas or damping fluid.

The method of application of pharmaceutical cpd of the present invention can be traditional route of administration, comprise in intravenous drip, intramuscular injection, vagina, oral, oral cavity, hypogloeeis, eyeball, part, parenteral, rectum, leaf sheath, in endoplasm net groove, inguinal region, intravesical, part (as, pulvis, ointment or drops), or nasal, but be not only confined to this.Preferably inject and transfusion form.

Composition of the present invention can have many multi-form.These comprise, for example, and the formulation of solid, semisolid and liquid, for example tablet, pill, powder, solution, dispersion liquid goods suspension, liposome, suppository, injection and transfusion solution.Preferred form is depending on method of application and prevention or treatment application.Preferred composition is the form of the solution of injection and transfusion use.

The pharmaceutical cpd that is applicable to parenteral route injection may contain and meet sterilized water or non-aqueous solution, aerosol, suspension or the emulsion that medicine preparation requires, can be at the sterile powder that faces the injectable solution of resuspended one-tenth of used time or aerosol.As applicable water-based and non-aqueous carrier, instrument and various diluent are as water, ethanol, polyol (as propyleneglycoles, macrogol, glycerol and analogue thereof), suitable mixture, rape oil (as sweet oil), with the alicyclic organic that can be used for injection, as ethane oleic acid, as used Yelkin TTS capsid to maintain the proper flow of medicine, as used aerosol, tensio-active agent to maintain suitable particle size.

Pharmaceutical composition of the present invention also can contain the adjuvant that some play protectiveness, moisturizing, emulsification and aerosolization, also can contain the instant composition that prophylaxis of microbial is polluted, as various antibacterium reagent, antifungal agents; as parabens; chlorobutanol, phenol, Sorbic Acid and analogue.Also can comprise the reagent that maintains osmotic pressure, as sugar, NaCl and analogue thereof.Can extend injectable drug composition adsorption time with the reagent that extends absorption, as Monostearate and gel etc.

Oral solid phase formulation comprises capsule, tablet, pulvis, granule etc.Activeconstituents in these solid phase formulations is at least mixed with a kind of traditional inert pharmaceutical vehicle (or carrier) as Trisodium Citrate, calcium phosphate, or (a) weighting agent or additive as starch, lactose, sucrose, seminose and silicic acid; (b) tackiness agent, as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and Sudan Gum-arabic; (c) wetting agent, as glycerine; (d) cracked dose, as agar, calcium carbonate, potato powder or Tapioca Starch; (e) retardant, as paraffin; (f) short absorption agent, as tetramino mixture; (g) wetting Agent for Printing Inks, as hexadecyl alcohol and glyceryl monostearate; (h) sorbent material, as kaolin and bentonite; (i) lubricant, as talcum, calcium stearate, Magnesium Stearate, solid polyethylene glycol, sulfuric acid Lauryil Sodium, or the mixture of its above-mentioned substance.In Tablet and Capsula formulation, may also contain buffer reagent.

Solid phase formulation can discharge or pulsed release dosage form by making improvement, is in the above-mentioned various direct release excipient of mentioning, to add some can change the vehicle of drug release rate and form, and can be included in the form that also can make coat in formulation.Speed discharges transformation agent and comprises carboxylic propyl methocel, methylcellulose gum, carboxymethyl cellulose sodium, Mierocrystalline cellulose ethane, cellulose acetate, polyethylene oxide, xanthan glycocoll, isopropyl olefin(e) acid ammonia multipolymer, hydrogenation flavor oil, carnauba wax, paraffin, phthalic acid cellulose acetate, phthalic acid carboxylic propyl methocel, the mixture of Sipacril 2739OF or above-mentioned substance.Improvement discharges and pulsed release dosage form may contain a kind of or one group of vehicle with improvement rate of release.

Pharmaceutical cpd of the present invention also can be comprised of propellant or the solvent that disappears (FDDFs) fast, comprises following composition: aspartyl-phenylalanine methyl ester, sulfanilamide (SN) potassium, citric acid, croscarmellosesodium, crospovidone, xitix, ethyl group acrylate, ethyl group Mierocrystalline cellulose, gelatin, hydroxy propyl methocel, Magnesium Stearate, N.F,USP MANNITOL, methyl second butenoate, seasoning peppermint, polyoxyethylene glycol, silica aerogel, silicon-dioxide, Vivastar P 5000, stearic acid fumaric acid sodium, sorbyl alcohol, Xylitol.Here for describing " atomization and disappear molten " word of FDDFs, depend on the solvability of medicine used, if medicine is insoluble, can be made into nebulizer formulation fast, if medicine is soluble, can be made into solvent-borne type fast.

The solid phase composition of similar type also use such as the polyoxyethylene glycol of lactose or caramel or other high molecular and similarly vehicle make the filling formulation of soft gelatin or glutoid.

Solid dosage such as tablet, sugar-coat agent, capsule and granule etc. can be made by the mode of the outsourcing capsid all known such as casing or other this area ordinary person.Also can be contain opacifying agent, also can be to contain to play slowly, postpone, control the similar composition that active medicine discharges.Also can use the compositions such as polymer and paraffin to carry out embedding.If suitable, also the formulation of the form of micro-capsule made activeconstituents by available above-mentioned one or more vehicle.

For oral liquid dosage form, comprise emulsus agent, solution, suspension, syrup and the elixir etc. that meet medicine requirement.Except activeconstituents, liquid dosage form also can contain some conventional inertia solution of this area, as water or other solvent, soluble reagents and emulsifying agent, as ethyl group alcohol, isopropyl alcohol, ethyl group carbonate, phenyl benzoate, third rare ethylene glycol, 1,3-methyltrimethylene glycol, oil, particularly, Oleum Gossypii semen, Peanut oil, Semen Maydis oil, sweet oil, flavor oil and sesame oil, glycerine, tetrahydrofurfuryl alcohol, polyoxyethylene glycol and fatty acid sorbitol ester, and the mixture of above-mentioned substance or similar material.

Except these inert diluents, pharmaceutical cpd also can comprise the adjuvants such as wetting Agent for Printing Inks, emulsifying agent, suspension agent, saccharifying agent, seasonings and flavouring agent.In addition, pharmaceutical cpd also can comprise the equal PVOH of ethoxylation, polyxyethylated sorbyl alcohol and sorbitanic fat, Microcrystalline Cellulose, an aluminium hydroxide, wilkinite, agar polymkeric substance and tragacanth, or the suspension agent of the mixture of these materials and so on.

Pharmaceutical cpd of the present invention also can be made into the mixture that is applicable to animals for treating, or meet salt for animals, or meet solvent for animals or first patent medicine, and make the dosage of applicable certain particular animals and the dosage forms of approach medicine according to common animal doctor and animal doctor practitioner's requirement.

The preferred pharmaceutical composition of the present invention and those compositions for other antibody of people's passive immunization are similar, for example oncotherapy antibody.Preferred method of application is that non-enteron aisle is used.

One or more monoclonal antibody of the present invention can be in conjunction with other Anti-virus agent for prevention and or treatment CD4 ⁺the disease that cell is relevant, includes but not limited to that human immune deficiency infects and relative disease.Monoclonal antibody can with these Anti-virus agents simultaneously, separately or successive administration.Other Anti-virus agent comprises ribavirin, diamantane, and carboxyl urea, IL-2, IL-12 and five carboxylic chain born of the same parents acid, but be not limited only to these.

These those skilled in the art should be realized that, the immunotherapy significant quantity of antibody homologue of the present invention or its active fragments by the unitary dose of the antibody homologue depending on using program, using, antibody homologue whether together use with other medicaments, the antiviral activity of patient's immunity and health condition, the specific antibodies homologue used.

Embodiment

Below in conjunction with specific embodiment and accompanying drawing, the present invention is further described, but these descriptions are not construed as limiting the invention.

Embodiment 1. can suppress HIV and infect CD4 ⁺the preparation of the anti-CD4 monoclonal antibody of cell

Mouse: 6 week age, female BALB/c Shu Wei Xiamen University school of life and health sciences Experimental Animal Center provided.

The preparation of hybridoma: we use the interior immunization ways of the body of standard and PEG fusion method to obtain monoclonal antibody, detailed method is referring to people such as Ed Harlow, Antibodies A Laboratory Manual, the concise and to the point process of Cold Spring Harbor Laboratory 1988. is as follows:

Mouse immune: will be dissolved in restructuring sCD4 antigenic solution and Freund's complete adjuvant (CFA) the equal-volume mixing and emulsifying of PBS, through limb muscle multi-point injection, the purification of Recombinant sCD4 antigen 5 μ g (cumulative volume 50 μ L) of preparation in every per injection embodiment 2.After first immunisation 15 days and 29 days, with the recombinant antigen solution of same dosage, add Freund's incomplete adjuvant (IFA) respectively and carry out booster immunization.Merge first 72 hours, the antigen 1 that tail vein booster shots 5 μ g do not add adjuvant carries out booster immunization.

Merge: get the mouse spleen cell that serum neutralization tires the highest and merge mutually with murine myeloma cell, first spleen grinding is obtained to splenocyte suspension, cell counting.By 1/6, in the quantity of splenocyte, get the SP2/0 murine myeloma cell of cultivation, centrifugal after mixing, through 50% polyoxyethylene glycol (PEG2000) effect 1 minute, splenocyte and murine myeloma cell SP2/0 are merged.The resuspended fused cell of RPMI1640 substratum that adds 50mL to contain 20%FBS, after cell suspension is mixed with isopyknic feeder cell, be placed in 96 porocyte culture plates (200 μ L/ hole) is in 37 ℃ of cultivations of 5% CO2gas incubator (ESPEC BNA-311).After 3 days, with the HT substratum that contains 20%FBS, (1.361mg xanthine (hypoxanthn, H), 0.388mg thymidine (thymidine, T) add RPMI 1640 (GIBCO company) substratum to 100mL.After dissolving under 45～50 ℃ of conditions, filtration sterilization.Liquid is changed in half reservation.

The screening of hybridoma: after merging, cell is cultivated after 10 days on 96 porocyte plates, draws cell conditioned medium and utilizes following HIV to infect gained hybridoma supernatant nutrient solution in TZM-bl cell and in detection method 96 porocyte culture plates.For the positive cell clone of neutralization, utilize limiting dilution assay to carry out cloning, after 3 time clonings, obtain secreted antibody and can stablize the cell strain of monoclonal antibody (seeing embodiment 3) that suppresses HIV infection TZM-bl cell.

The selection result: obtain two strain monoclonal antibodies, be respectively: 15A7 and 14G7.

The type of antibody and subgroup identification: with the coated elisa plate of purifying sCD4 of preparation in embodiment 2.Every hole adds the cell conditioned medium 100 μ L of monoclonal antibody, 37 ℃ of incubations 30 minutes; On TECAN automatic plate washer, with PBST, wash 5 times, every minor tick 20 seconds, button was dry, adds suitable dilution HRP-goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3 antibody (Serotec company) ELIAS secondary antibody, in 37 ℃ of incubations 30 minutes; On TECAN automatic plate washer, with PBST washing 5 times, every minor tick 20 seconds, detains and does, and adds nitrite ion A (H ₂o ₂) and B (TMB) each 1, in 37 ℃ colour developing 10 minutes, add 1 stop buffer (2M H ₂sO ₄); In TECAN microplate reader, measure OD _450nm(reference wavelength is 620nm), threshold value is the negative average of 2 times, and it is positive that OD value is greater than threshold value, and it is negative that OD value is less than threshold value.Result monoclonal antibody 15A7 is IgG1 type, and 14G7 is IgG2b type.

The cultivation of hybridoma: stable authentic monoclonal antibody cell strain is amplification cultivation in CO2gas incubator first, is transferred to 24 holes after cultivate in 96 holes, transfers to amplification cultivation in 50mL cell bottle.Get the healthy BALB/c mouse in 10 week age, an abdominal injection 0.5mL/ Liquid Paraffin, 1-2 is after week, and every mouse peritoneal injects 1 * 10 ⁶individual hybridoma, collected ascites after 7～10 days.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 packing.

The purifying of monoclonal antibody: by PBS (the 81mL 0.2MNa of 0.02M, pH7.4 for ascites ₂hPO ₄, 19mL 0.2M NaH ₂pO ₄, add physiological saline to 1L) and two-fold dilution, under stirring, dropwise slowly add ammonium sulfate (concentration reaches 50% saturation ratio), 4 ℃ are spent the night.4 ℃, centrifugal 15 minutes of 12000rpm, abandons supernatant, precipitation is dissolved in the PBS of 2 times of former ascites volumes.Under stirring, dropwise slowly add ammonium sulfate, make ammonium sulfate concentrations reach 33% saturation ratio, 4 ℃ of standing over night.4 ℃, centrifugal 15 minutes of 12000rpm, abandons supernatant, precipitation is dissolved in the PBS of 2 times of former ascites volumes.Under stirring, dropwise slowly add ammonium sulfate (concentration reaches 50% saturation ratio), 4 ℃ are spent the night.4 ℃, centrifugal 15 minutes of 12,000rpm, abandons supernatant.Precipitation is dissolved in appropriate PBS, packs in dialysis tubing, put into 50-100 doubly containing 20mM NaCl, desalination about 12 hours under 4 ℃ of stirrings in the 120mM Tris-HCl damping fluid of pH 7.8, during change above dialyzate 3 times.Packing-20 ℃ preservation after taking out.

Embodiment 2. is used as the preparation of the recombinant human CD4 fragment polypeptide of antigen

Be used as the preparation of the people CD4 extracellular region fragment of template: the people CD4 cDNA of take in TZM-bl clone (Cat.No.8129National Institutes of Health AIDS Research and ReferenceReagent Program) is template, CD4 (d1) F (5 '-CGG CATATG AAG AAA GTG GTG CTG GGC-3 ') is forward primer, CD4 (d4) R (5 '-GCGAATTCTTACCATGTGGGCAGAACCTT-3 ') is reverse primer, at PCR thermal cycler (Biometra T3), according to following condition, carries out PCR reaction: 94 ℃ 10 minutes; Be subsequently 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ of 25 circulations of 1 minute, are finally 72 ℃ and extend 10 minutes.Obtain the template DNA fragment of the recombinant human CD4 polypeptide that is used as each brachymemma of preparation of special 1.1kb left and right size.The pMD 18-T carrier that the PCR product of above-mentioned acquisition is sold with business (production of TaKaRa company) is connected, and through Nde I/EcoR I enzyme, cuts evaluation, obtains inserting the positive colony of sCD4 gene.Utilize M13 (+)/(-) primer, the DNA fragmentation conserved sequence of the CD4 extracellular region of preparing through aforesaid method of checking order to obtain, and be used as the template of the recombinant human CD4 polypeptide of each brachymemma of preparation.

The immunoglobulin like domain D1-D4 sequence of N end extracellular region of the people CD4 that as above obtains of take is template, utilizes forward primer (its 5 ' end is introduced restriction enzyme NdeI) and reverse primer (its 5 ' end introducing restricted type restriction endonuclease EcoRI site) in table 1.At PCR thermal cycler (Biometra T3), according to following condition, carry out PCR reaction: 94 ℃ 10 minutes; Be subsequently 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ of 25 circulations of 45 seconds, are finally 72 ℃ and extend 10 minutes.Wherein, CD4 (d1) forward primer used is CD4 (d1) F, and reverse primer is and CD4 (d1) R; CD4 (d2) forward primer used is CD4 (d2) F, and reverse primer is and CD4 (d2) R; CD4 (d3) forward primer used is CD4 (d3) F, and reverse primer is and CD4 (d3) R; CD4 (d4) forward primer used is CD4 (d4) F, and reverse primer is and CD4 (d4) R; CD4 (d1-2) forward primer used is CD4 (d1) F, and reverse primer CD4 (d2) R obtains special DNA fragmentation, is the polynucleotide sequence of the people CD4 of each brachymemma of the present invention of encoding, and its aminoacid sequence as shown in Figure 1.

The recombinant protein clone primer sequence of each structural domain brachymemma of table 1.CD4

Expression vector pTO-T7 according to document build (Luo Wenxin etc., biotechnology journal, 2000,16:53-57).For expressing the concrete steps of the vector construction of polypeptide of the present invention, comprise: the pMD 18-T carrier (production of TaKaRa company) of selling with business as the PCR product of above-mentioned acquisition is connected, through Nde I/EcoR I enzyme, cut evaluation, obtain inserting the positive subclone of each section CD4 gene; Nde I/EcoR I enzyme is cut the CD4 gene fragment that obtains each section, be connected with the pTO-T7 expression vector of cutting through NdeI/EcoR I enzyme, Nde I/EcoR I enzyme is cut and is identified the recombinant expressed clone of polypeptide pTO-T7-CD4 (d1), pTO-T7-CD4 (d2), pTO-T7-CD4 (d3), pTO-T7-CD4 (d4), pTO-T7-CD4 (d1-2) and the pTO-T7-sCD4 that obtains inserting a section CD4 gene again.

The expression of the CD4 recombinant polypeptide of each section brachymemma: competence e. coli bl21 (purchased from New England Biolabs, Inc. (US) Massachusetts, United States of America) prepared by the Calcium Chloride Method of recombinant expression vector plasmid (0.15mg/mL) the conversion 40 μ L of 1 μ L, coat kantlex (final concentration 25mg/mL, the solid LB substratum of resistance down together), 37 ℃ of standing cultivations 10～12 hours, clear and legible to single bacterium colony.Picking list bacterium colony is to the test tube containing the liquid LB substratum of 4mL kalamycin resistance, 37 ℃ of 220 revs/min of shaking culture 10 hours, to cell concentration be OD ₆₀₀≈ 0.8, therefrom gets 1mL and is stored in 4 ℃, and remaining 3mL adds 3.0 μ L 0.8M IPTG (final concentration is 0.8mM), the expression strain of the 37 ℃ 220 revs/min further inducing culture conversions of vibration 4 hours.Get 1.5mL through the expression strain nutrient solution of inducing culture, 12,000 rev/min centrifugal 30 seconds, supernatant discarded, button is dry, with 100 μ L 1 * gel loading buffer (50mM TrisCl (pH6.8), 100mM dithiothreitol (DTT) (DTT), 2%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine) resuspended; Boiling water bath is processed 10 minutes; 12,000 revs/min, centrifugal 10 minutes; Take out 10 μ L and carry out 15% acrylamide SDS-PAGE analysis.Select the conversion bacterial strain that expression amount is higher and carry out great expression long-term conservation.

The triangle of each 1L is cultivated the fresh LB liquid nutrient medium of bottled 500mL, the preservation bacterium liquid of the 200 μ L that transfer respectively, and 37 ℃ of shaking culture 11 hours, to cell concentration OD ₆₀₀≈ 1.0.Then to every culturing bottle, adding 500 μ L 0.8M IPTG is 0.8mM to final concentration, 37 ℃ of vibration inducing culture 4 hours, centrifugal collection thalline.

The washing purifying of CD4 (d1), CD4 (d2), CD4 (d3), CD4 (d4), CD4 (d1-2) and sCD4 brachymemma polypeptide recombinant expressed inclusion body in intestinal bacteria: recombinant bacterial strain is through 4 ℃ 7 of abduction delivering gained nutrient solution, after centrifugal 5 minutes of 000rpm, abandon supernatant; Bacterial sediment adds 20mL lysate (50mM TrisCl, 10mM EDTA, 300mM NaClpH7.2) with every 500mL substratum and calculates, and Eddy diffusion is in lysate.With ultrasonic disruption instrument (the Uilbra-Cell VCX500 type of SONICS & MATERIALS company) smudge cells, take 500mL nutrient solution gained thallus suspension liquid 20mL as example broken condition as: 55% power is set, ice bath, the broken time of every subpulse is 2 seconds, 4 seconds, interval, the altogether broken time is 4 minutes; At 12,000 revs/min 4 ℃ centrifugal 10 minutes, abandon supernatant; Precipitate resuspendedly with the damping fluid I solution (200mM TrisCl, pH8.5,5mM EDTA, 100mM NaCl) of 1/2 thallus suspension liquid volume, add equal-volume to contain the damping fluid I of 4%Triton-X100, wherein the final concentration of Triton-X100 is 2%.At 37 ℃, 200rpm vibration is 30 minutes; Centrifugal 10 minutes of 4 ℃ of 10,000rpm, abandon supernatant; Repeating 2%Triton-X100/ damping fluid I processes once.Precipitation is used with the isopyknic damping fluid I of thallus suspension liquid resuspended, and 37 ℃ of 200rpm vibrate 30 minutes; 4 ℃ 8, centrifugal 10 minutes of 000rpm, abandons supernatant; Repeating damping fluid I processes twice.Precipitation is used with the isopyknic damping fluid I of thallus suspension liquid resuspended; 37 ℃ of 200rpm vibrate 30 minutes; 4 ℃ 10, centrifugal 10 minutes of 000rpm, abandons supernatant; Precipitation is used with the isopyknic 2M urea/damping fluid of thallus suspension liquid I resuspended; 37 ℃ of 200rpm vibrate 30 minutes; 4 ℃ 12, centrifugal 10 minutes of 000rpm, stays supernatant.Sample is numbered respectively CD4 (d1) 2M, CD4 (d2) 2M, CD4 (d3) 2M, CD4 (d4) 2M, CD4 (d1-2) 2M and sCD4 2M; Precipitate resuspended with isopyknic 4M urea/damping fluid I; 37 ℃ of 200rpm vibration 1 hour, 4 ℃ of placements spend the night (10 hours); 4 ℃ 12, centrifugal 10 minutes of 000rpm, stays supernatant, and sample number into spectrum is CD4 (d1) 4M, CD4 (d2) 4M, CD4 (d3) 4M, CD4 (d4) 4M, CD4 (d1-2) 4M and sCD4 4M; Precipitate resuspended with isopyknic 8M urea/damping fluid I; 37 ℃ of 200rpm vibration 1 hour, 4 ℃ of placements spend the night (10 hours); 4 ℃ 12, centrifugal 10 minutes of 000rpm, stays supernatant.Sample number into spectrum is CD4 (d1) 8M, CD4 (d2) 8M, CD4 (d3) 8M, CD4 (d4) 8M, CD4 (d1-2) 8M and sCD48M; Abandon precipitation.Get respectively each 100 μ L of 2M/4M/8M supernatant of CD4 (d1), CD4 (d2), CD4 (d3), CD4 (d4), CD4 (d1-2) and sCD4, add gel loading buffer (50mMTrisCl (pH6.8), 100mM dithiothreitol (DTT) (DTT), 2%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine); Boiling water bath is processed 10 minutes; Centrifugal 10 minutes of 12,000rpm; Take out 10 μ L and carry out 15% acrylamide SDS-PAGE analysis, to judge target protein shared proportion in 2M/4M/8M urea/damping fluid I.

The renaturation of the CD4 recombinant polypeptide of each brachymemma: respectively the 4M of CD4 (d1), CD4 (d2), CD4 (d3), CD4 (d4), CD4 (d1-2) and sCD4 or 8M sample are divided to install to dialysis tubing (U.S. Union Carbide Corp produces, 36DM, molecular weight cut-off 8,000-10,000), in sample volume: dialysate volumes=1: 100 ratio (contains Na in 4 ℃ of gradient dialysis to 1 * PBS in 1L ₂hPO ₄12H ₂o 2.9g, KH ₂pO ₄0.24g, NaCl 8.0g, KCl 0.2g, pH7.4) in (8M urea/damping fluid I-4M urea/damping fluid I-2M urea/damping fluid I-1 * PBS).In theory, when dialysis finishes, the urea content of sample is 4 * 10 ^-6m; Dialyse sample through 4 ℃ 12, centrifugal 10 minutes of 000rpm; Supernatant liquor, by the filtering with microporous membrane of 0.22 μ m, can be used for being further purified.

The HPLC gel filtration chromatography purifying of the CD4 recombinant polypeptide of the brachymemmas such as CD4 (d1), CD4 (d2), CD4 (d3), CD4 (d4), CD4 (d1-2) and sCD4.

To renaturation sample, adopt following HPLC system and method to be further purified.

Instrument system: Agilent Technologies 1200series HPLC, chromatography column: TSKGEL G5000PWXL 7.8mm * 30cm, elutriant: 1 * PBS pH7.4, flow velocity: 0.5mL/min, detector wavelength: 280nm, sample size: 100 μ L.

Collection mode: manually collect.Result shows: sample is through the purity of 15% acrylamide SDS-PAGE analysis purposes protein peak, purity 70～90%.

Fig. 2 has shown that the coli somatic of each expression plasmid conversion is after induction, SDS-PAGE (the little figure of A is that CD4 (d1), the little figure of B are that CD4 (d2), the little figure of C are that CD4 (d3), the little figure of D are that CD4 (d4), the little figure of E are that CD4 (d1-2), the little figure of F are sCD4) shows that CD4 (d1), CD4 (d2), CD4 (d3), CD4 (d4) and CD4 (d1-2), sCD4 are equipped with band of expression in corresponding positions, expression amount is about (the 2nd road of each little figure of Fig. 2 .A-F) between 10～40%, and mainly the form with inclusion body exists.Through 2M, 4M and the washing of 8M urea, visible 2M urea supernatant (the 3rd road of each little figure of Fig. 2 .A～F), 4M urea supernatant (the 4th road of each little figure of Fig. 2 .A～F), and 8M urea supernatant (the 5th road of each little figure of Fig. 2 .A～F); To 4M or 8M supernatant sample with gradient dialysis renaturation after, in HPLC through molecular sieve column chromatography purifying (the 6th road of each little figure of Fig. 2 .A～F).

Embodiment 3. monoclonal antibody blocking-up HIV infect the checking of TZM-bl cytoactive

Select 5 strains to belong to respectively the full gene experiment of the HIV strain virus (HIV of B, C, D, D/C hypotype _nL4-3, HIV _89.6, HIV _94UG114, HIV _mJ4, HIV _wCML249), by cell and measuring monoclonal antibody 15A7,14G7 is to above-mentioned virus infection CD4 ⁺blocking-up/the neutralization of cell.TZM-bl cell is according to 1.5 * 10 ⁴the concentration of individual cells/well is inoculated in 96 porocyte culture plates, after 12 hours for detection of.According to weaker concn as listed in Fig. 3 coordinate dilution monoclonal antibody 15A7 and 14G7, in 96 hole U base plates, every hole adds 100 μ L antibody diluents and each HIV viral suspension (being diluted to 100TCID50) of 50 μ L, 37 ℃ of incubations 1 hour.Add cultured TZM-bl cell on 96 porocyte culture plates, 37 ℃ to cultivate after 48 hours mixed solution 150 μ L, colour developing, Elispot counting, and in calculating and efficiency.Control group replaces 15A7 antibody with PBS.Result is as Fig. 3 and Fig. 4, visible monoclonal antibody 15A7, and 14G7 all has good blocking-up/neutralization to above-mentioned each hypotype HIV, in Table 2:

Table 2. monoclonal antibody 15A7, the cell neutralization of 14G7 to 5 strain HIV viruses

Embodiment 4. monoclonal antibody 15A7, the immunofluorescence that 14G7 labeled cell is expressed CD4 detects

Express the cell strain of CD4: Tzm-bl (Cat.No.8129National Institutes of Health AIDS Research and Reference Reagent Program); U87.CD4.CXCR4 (Cat.No.4036National Institutes of Health AIDSResearch and Reference Reagent Program); U87.CD4.CCR5 (Cat.No.4035National Institutes of Health AIDS Research and ReferenceReagent Program).Above-mentioned 3 strain cells be all can stably express CD4 attached cell.

Prepare cell: test and above-mentioned three strain cells were prepared into single cell suspension with 0.25% tryptic digestion in first 1 day, concentration is 5 * 10 ⁵/ mL, cell suspension is inoculated in 24 orifice plates of completing in advance sterility cover slide according to 100 μ L/ holes, then every hole supplements 400 μ L DMEM perfect mediums (interpolation 10%FBS, 2mM L-glutamine, the nonessential amino acid of 0.1mM and 1% dual anti-) 37 ℃ of 5% CO2gas incubator are cultivated 24 hours.

Cell is fixed: by the substratum sucking-off in 24 orifice plates, every hole adds 500 μ L PBS to wash once, and then every hole adds 500 μ L 4% paraformaldehyde (100mL PBS contains 4g paraformaldehyde) room temperature lucifuges to fix 5 minutes, sucking-off 4% paraformaldehyde stationary liquid.Every hole adds 1mL PBS, and room temperature is placed 5 minutes, and then sucking-off, repeats to wash 2 times with PBS.

Sealing: cover and put a sealed membrane at 24 orifice plates, on 24 orifice plates, the position in each hole drips 25 μ L lowlenthal serums, with ophthalmic tweezers careful from 24 orifice plates, take out cover glass, with thieving paper, suck liquid unnecessary on cover glass, then cover glass is placed in the drop of lowlenthal serum, guarantees that cell faces down.The 24 orifice plate lids that are placed with cover glass are put into wet box (maintenance saturated humidity) incubated at room 1 hour.

Primary antibodie is hatched: carefully take out cover glass, stirring cover slide faces up cell, then puts it into 24 orifice plates, adds 1mL PBS room temperature to place 5 minutes.Get again a new sealed membrane and cover 24 orifice plates and cover, according to the position in hole on 24 orifice plates, drip 25 μ L monoclonal antibody 15A7, the PBS diluent of 14G7 (dilution in 1: 1000).The PBS diluent (dilution in 1: 1000) of monoclonal antibody H5F4 (anti-P24) is dripped in contrast, according to the same operation of sealing, cover glass is positioned in the drop of primary antibodie, guarantees that cell faces down.The 24 orifice plate lids that are placed with cover glass are put into wet box (maintenance saturated humidity) incubated at room 1 hour.

Two anti-hatching: carefully take out cover glass, stirring cover slide faces up cell, then puts it into 24 orifice plates, add 1mL PBS room temperature to place 5 minutes.Then sucking-off PBS, repeats to wash twice with PBS.Get a new sealed membrane and cover 24 orifice plates and cover, according to the position in hole on 24 orifice plates, drip 25 μ L bis-anti-, two resist for PBS dilution in 1: 200 for anti-Mouse IgG (whole molecule)-FITC (Cat.No.F9006, Sigma).According to the same operation of sealing, cover glass is positioned in two anti-drops, guarantee that cell faces down.The 24 orifice plate lids that are placed with cover glass are put into wet box (maintenance saturated humidity) room temperature lucifuge hatches 30 minutes.

DAPI dyes core: carefully take out cover glass, stirring cover slide faces up cell, then puts it into 24 orifice plates, adds 1mL PBS room temperature to place 5 minutes.Then sucking-off PBS, repeats to wash twice with PBS.Getting a new sealed membrane covers 24 orifice plates and covers, according to the position in hole on 24 orifice plates, drip the DAPI (dilution in 1: 2000) that 25 μ L have diluted with PBS, according to the same operation of sealing, cover glass is positioned in the drop of DAPI, guarantees that cell faces down.The 24 orifice plate lids that are placed with cover glass are put into wet box (maintenance saturated humidity) room temperature lucifuge hatches 5 minutes.

Mounting: carefully take out cover glass, stirring cover slide faces up cell, then puts it into 24 orifice plates, adds 1mL PBS room temperature to place 5 minutes.Then sucking-off PBS, repeats to wash twice with PBS.Mark slide glass, and drip mountant in the above.From 24 orifice plates, take out cover glass, with thieving paper, suck liquid unnecessary on cover glass, then cell faces down, and cover glass is placed in to mountant drop.With thieving paper, remove gently the mountant on cover glass, then coat nail varnish at edge, be used for closing cap slide edge.Lucifuge is dry.With common laser scanning co-focusing microscope, observe and take pictures.

Result as shown in Figure 5 and Figure 6, monoclonal antibody 15A7,14G7 can well be combined on the cytolemma of three strain cells.This show monoclonal antibody can with the CD4 protein binding of three strain cell surface expressions.

Monoclonal antibody 15A7 is blocked/suppressed to the CD4 polypeptide of embodiment 5. each brachymemmas, 14G7 mark CD4 ⁺the flow cytometer of cell detects

Tzm-bl is according to 1.5 * 10 ⁴individual cells/well is laid in 96 porocyte culture plates, and 5% 37 ℃ of CO2gas incubator are hatched after 12 hours for experiment.By the CD4 of preliminary purification (d1), CD4 (d2), CD4 (d1-2), CD4 (d3), CD4 (d4), sCD4 albumen is diluted to final concentration 10 μ g/mL with the DMEM substratum of 10%FBS, then adds respectively according to monoclonal antibody 15A7, the 14G7 of dilution, make the final concentration of antibody reach the concentration of mark in Fig. 7 and Fig. 8 axis of abscissa, then hatch 1 hour for 37 ℃.Above-mentioned mixed solution is added to the Tzm-bl cell of completing in advance, hatch 1 hour for 37 ℃.Trypsin digestion cell with 0.25%, proceeds in 4mL centrifuge tube, and 2mLPBS washes once, and 1,500rpm abandons supernatant for centrifugal 5 minutes, and the PBS re-suspended cell that contains RAM-FITC (dilution in 1: 200) with 100 μ L, hatches 30 minutes for 25 ℃.2mL PBS/ pipe is washed once, and 1,500rpm abandons supernatant for centrifugal 5 minutes, and with 500 μ L PBS re-suspended cells, flow cytometer detects antibodies situation.Concrete outcome as shown in Figure 7 and Figure 8.From Fig. 7 and Fig. 8, CD4 (d1-2) has impact significantly (p < 0.01) on monoclonal antibody 15A7,14G7 with reacting of cell; CD4 (d1), sCD4 are to monoclonal antibody 15A7, and 14G7 has impact (p < 0.05) with reacting of cell; And CD4 (d2), CD4 (d3), CD4 (d4) be to monoclonal antibody 15A7,14G7 and cell react impact and PBS difference little (P > 0.05).

The clone of embodiment 6. monoclonal antibody light chain genes and heavy chain gene variable region

Half adherent culture 10 ⁷individual expression antibody 15A7, the hybridoma of 14G7, blowpipe blows afloat attached cell and makes to suspend, transfer in new 4mL centrifuge tube centrifugal 3 minutes of 1,500rpm, the cell of collecting precipitation, is resuspended in the aseptic PBS of 100 μ L (pH7.45), transfers in a new 1.5mL centrifuge tube.Add 800 μ L Trizol (Roche, Germany), put upside down and mix gently, standing 10 minutes.Add 200 μ L chloroforms, thermal agitation 15 seconds, standing 10 minutes, 4 ℃ 12, centrifugal 15 minutes of 000rpm, shifted in the 1.5mL centrifuge tube that supernatant liquid to is new, adds isopyknic Virahol, mixes standing 10 minutes.4 ℃ 12, centrifugal 10 minutes of 000rpm, abandons supernatant, adds 600 μ L 75% washing with alcohol, and 4 ℃ 12, centrifugal 5 minutes of 000rpm, abandons supernatant, is deposited in 60 ℃ of vacuum and drains 5 minutes.Transparent precipitation is dissolved in 70 μ L DEPC H ₂in O, be distributed into two pipes.Every pipe adds 1 μ L reverse transcription primer, the reverse transcription primer that wherein a pipe adds is MVJkR (5 '-CCg TTT (T/g) AT (T/C) TC CAg CTT ggT (g/C) CC-3 '), be used for the chain variable region gene that increases, the reverse transcription primer that another pipe adds is MVDJhR (5 '-C ggT gAC Cg (T/A) ggT (C/g/T) CC TTg (g/A) CCCCA-3 '), for the heavy chain variable region gene that increases.Every pipe adds 1uL dNTP (10mM again, TAKARA), put 72 ℃ of water-baths 10 minutes, be put into immediately ice bath mid-5 minutes, add 10 μ L 5 * reverse transcription damping fluids, 1 μ L AMV (10unit/ μ L, Pormega), 1 μ L RNasin (40unit/ μ L, Promega), becomes cDNA in 42 ℃ by RNA reverse transcription after mixing.

The separation of antibody gene variable region adopts polymerase chain reaction (PCR) method, use is according to the synthetic primer sets of the Ig-Prime kits of Novagen company and synthetic two downstream primer MVJkR, the MVDJhR (Shanghai Bo Ya company is synthetic) of other design, MVJkR is the downstream primer of chain variable region gene amplification, and MVDJhR is the downstream primer of heavy chain variable region gene amplification.Template is above two kinds of synthetic cDNA.PCR condition is: 94 ℃ 5 minutes, 94 ℃ 40 seconds, 53 ℃ of renaturation 1 minute, 72 ℃ 50 seconds, 35 circulations, 72 ℃ are extended 15 minutes.Reclaim object fragment and be also cloned into pMD 18-T carrier (TaKaRa), and order-checking, sequence is determined antibody variable region sequence after Blast analyses and compares, and infers and corresponding aminoacid sequence.

According to aforesaid method, respectively from monoclonal antibody 15A7, in 14G7 hybridoma cell strain, clone its antibody variable gene, and extrapolate corresponding aminoacid sequence.Table 3 is depicted as upstream primer sequence used, monoclonal antibody 15A7 variable region of heavy chain nucleic acid SEQ ID NO:1; Heavy chain amino acid sequence numbering SEQ ID NO:2; Variable region of light chain nucleic acid SEQ ID NO:3; Light-chain amino acid sequence numbering SEQ ID NO:4.Monoclonal antibody 14G7 variable region of heavy chain nucleic acid SEQ ID NO:17; Heavy chain amino acid sequence numbering SEQ ID NO:18; Variable region of light chain nucleic acid SEQ ID NO:19; Light-chain amino acid sequence numbering SEQ ID NO:20.Complementary determining region (complementary determinant region, CDR) by Kabat method, determine (Kabat EA, Wu TT, Perry HM, Gottesman KS, Coeller K.Sequencesof proteins of immunological interest, U.S Department of Health andHuman Services, PHS, NIH, Bethesda, 1991).

Table 3. amplification 15A7, the primer sequence of 14G7 variable region gene

The variable region heavy chain gene sequence input VBASE2 database (http://www.vbase2.org/) obtaining is compared.Result shows, the V fragment of cloning is Igh-VJ558VH1 family, and Vh gene size is respectively 351bp, and the polypeptide that 117 amino-acid residues of coding form has and constitutional features that KABAT structure is completely corresponding.Heavy chain CDR district is as shown in table 4.

The variable region light chain gene sequence input VBASE2 database of acquisition is compared.Result shows, the V fragment of cloning is IGKV21 family, and Vk gene size is respectively 15A7Vk:339bp, the polypeptide that 113 amino-acid residues of coding form, 14G7Vk:324bp, the polypeptide that 108 amino-acid residues of coding form, has the constitutional features completely corresponding with KABAT structure.Light chain CDR district is as shown in table 4.

Table 4.15A7,14G7Vh and Vk cdr amino acid sequence

The expression of embodiment 7. single-chain antibodies and active detection

The heavy chain of antibody gene and variable region of light chain are connected into single-chain antibody DNA fragmentation by (GGGGS) 3 small peptides.Take 15A7VHF as forward primer, and 15A7VHR is that reverse primer amplifies 15A7 variable region of heavy chain fragment, take 15A7VKF as forward primer, and 15A7VKR is that reverse primer amplifies 15A7 variable region of light chain fragment.Primer sequence is in Table 5.Reclaim respectively two antibody gene fragments.Again with variable region of heavy chain and variable region of light chain fragment each other primer and template in new PCR system, carry out overlapping extension, obtain a small amount of complete single chain antibody fragments.Then take complete fragment as template, take 15A7VHF as forward primer, 15A7VKR is that reverse primer carries out the overlapping 15A7 single chain antibody fragments of a large amount of amplifications.Reclaim single chain antibody fragments, with the switchback of NdeI/Xho I enzyme, receive single chain antibody fragments, be cloned in pET-30a-c (+) prokaryotic expression carrier that same enzyme cuts, carrier as shown in Figure 9.Using ER2566 intestinal bacteria as expression strain, adopt ordinary method to express, the protein of results expression is to exist with insoluble inclusion body form.With ordinary method washing purifying inclusion body, result single-chain antibody is mainly dissolved in 8M urea.To be dissolved in single chain antibody protein in 8M urea progressively to 1 * PBS dialysis renaturation, 12,000rpm removes precipitation in centrifugal 10 minutes, and the single-chain antibody solution of the preliminary purification finally obtaining is carried out to determination of activity.

Table 5. single-chain antibody clone primer

The 15A7 single-chain antibody that employing Inhibition ELISA detection preliminary purification goes out and the activity of 14G7 single-chain antibody.Coated T4 antigen in the hole of polystyrene batten, and seal with 1 * ED (adding 2% gelatin, 0.5% casein and 2% sucrose in 1 * PBS solution), treat to add in gaging hole the above-mentioned single-chain antibody solution of 100 μ L, in positive control hole, add respectively 100 μ L mono-clonal 15A7, monoclonal antibody 14G7, in negative control hole, add 100 μ L uncorrelated monoclonal antibody 1F2 diluents or 100 μ L PBS, repeat in three holes.After each hole slightly mixes, in 37 ℃ of reactions, after 1 hour, with 15A7, the 14G7 of biotin mark, react again 1 hour, then add SA-HRP after 30 minutes, to add A, B nitrite ion in 37 ℃ of colour developings 15 minutes as two anti-reactions, after termination reaction, use microplate reader reading.The results are shown in Table 6 and table 7,15A7 single-chain antibody and 14G7 single-chain antibody all show the combination of its corresponding monoclonal antibody of obvious blocking-up and CD4 (D1) albumen.

Table 6.15A7 single-chain antibody detects the activity of the complete anti-blocking-up of 15A7

Table 7.14G7 single-chain antibody detects the activity of the complete anti-blocking-up of 14G7

The 15A7 single-chain antibody, 14G7 single-chain antibody and the CD4 albumen test that adopt ELISA method detection preliminary purification to go out are active.By vitamin H biotin on 15A7 single-chain antibody, 14G7 single-chain antibody mark, and dilute with HCV-ED.Coated T4 antigen CD4 (d1), CD4 (d2), CD4 (d1-2), CD4 (d3), CD4 (d4) and sCD4 in the hole of polystyrene batten, and seal with 1 * ED, the single-chain antibody solution of biotin for the treatment of to have added the above-mentioned mark of 100 μ L in gaging hole, in positive control hole, add 100 μ L marks the monoclonal antibody 15A7 of vitamin H (biotin), mark the monoclonal antibody 14G7 of vitamin H (biotin), in negative control hole, add 100 μ LHCV-ED solution, repeat in three holes.After each hole slightly mixes, in 37 ℃ of reactions, after 1 hour, add SA-HRP after 30 minutes, to add A, B nitrite ion in 37 ℃ of colour developings 15 minutes as two anti-reactions, after termination reaction, use microplate reader reading.The results are shown in Table 8 and table 9, illustrate that the 15A7 single chain antibody protein that preliminary purification goes out has higher activity to CD4 albumen.

The detection of table 8.15A7 single-chain antibody and CD4 albumen test activity

The detection of table 9.14G7 single-chain antibody and CD4 albumen test activity

Embodiment 8. monoclonal antibody Fab fragments and F (ab ') ₂preparation

20mM PB (pH 7.0) damping fluid dialysed overnight for the preparation of monoclonal antibody Fab fragment: the monoclonal antibody 15A7 of purifying (2mg/mL).Papain (purchased from Sigma company) is mixed with to 1mg/mL with same buffer, includes 20mM EDTA, 20mM halfcystine, hatch 30 minutes for 37 ℃, the enzyme buffer liquid of activation is added in the antibody after dialysis to enzyme: antibody weight ratio=1: hatch 15 hours for 1000,37 ℃.Add iodo-acid amide (final concentration 30mM) lucifuge ice bath 1 hour, termination reaction.The monoclonal antibody that enzyme is cut, to 20mM TB (pH8.0) damping fluid dialysed overnight, then goes up DEAE-HPLC purifying.With 20mM TB (pH8.0) balance pillar, with the 15 minutes Fab wash-outs of 20mM TB (pH8.0) linear elution that contain 0.05M NaCl, with the 20mM TB (pH 8.0) that contains 1M NaCl, wash lower Fc and a small amount of do not cut complete anti-.Applied sample amount 10-100mg antibody, flow velocity 1mL/min, detects wavelength 280nm.The FabSDS-PAGE collecting detects after purity concentrated with 10kD specification evaporating pipe, and after 20mMPBS (pH7.4) dialysis, degerming (0.22 μ m millipore filtration) packing, obtains the Fab sterling of monoclonal antibody 15A7.

Monoclonal antibody F (ab ') ₂the preparation of fragment: the monoclonal antibody 15A7 of purifying (2mg/mL) uses 6mM Na ₂hPO ₄1 ₂h ₂o and 7mM citric acid cocktail buffer dialysed overnight.Stomach en-(purchased from Sigma company) is mixed with to 1mg/mL with same buffer and adds in the antibody after dialysis, enzyme: antibody weight ratio=1: hatch 12 hours for 100,37 ℃.The monoclonal antibody that enzyme is cut, to 20mMTB (pH 8.0) damping fluid dialysed overnight, then goes up DEAE-HPLC purifying.With 20mM TB (pH 8.0) balance pillar, 20mM TB (pH 8.0) linear elution that use contains 1M NaCl 30 minutes, washes respectively lower F (ab ') ₂with a small amount of do not cut complete anti-.Applied sample amount 10-100mg antibody, flow velocity 1mL/min, detects wavelength 280nm.The F (ab ') collecting ₂concentrated with 10kD specification evaporating pipe after SDS-PAGE detects purity, after 20mM PBS (pH7.4) dialysis, degerming (0.22 μ m millipore filtration) packing, obtains the F (ab ') of monoclonal antibody 15A7 ₂sterling.

Monoclonal antibody 15A7 after purifying, monoclonal antibody 15A7Fab fragment and monoclonal antibody F (ab ') ₂fragment SDS-PAGE result is as shown in figure 10: wherein the 1st road is molecular weight of albumen marker; The 2nd road is the monoclonal antibody 15A7 that boiling water bath is processed for 10 minutes after reductive agent beta-mercaptoethanol; The 3rd road is the monoclonal antibody 15A7Fab fragment that boiling water bath is processed for 10 minutes after reductive agent beta-mercaptoethanol; The 4th road is the monoclonal antibody 15A7F (ab ') that boiling water bath is processed for 10 minutes after reductive agent beta-mercaptoethanol ₂fragment; The 5th road is monoclonal antibody 15A7; The 6th road is 15A7Fab fragment; The 7th road is monoclonal antibody 15A7F (ab ') ₂fragment.

In embodiment 9. antibody 15A7 and active fragments thereof and the mensuration of active IC50

Select B subtype virus strain HIV _nL4-3, with detecting in antibody 15A7 and active fragments thereof and active IC50 with experiment in cell.TZM-bl cell is according to 1.5 * 10 ⁴the concentration of individual cells/well is inoculated in 96 porocyte culture plates, after 12 hours for detection of.According to 2 times of dilution antibody 15A7 of the weaker concn of 100 μ g/ml and Fab, F (ab ') ₂fragment, dilutes 20 gradients, and in 96 hole U base plates, every hole adds 100 μ L antibody diluents and 50 μ L HIV viral suspensions (being diluted to 100TCID50), 37 ℃ of incubations 1 hour.Mixed solution 150 μ L are added to cultured TZM-bl cell on 96 porocyte culture plates, cultivate after 48 hours for 37 ℃, after colour developing with Elispot counting, in calculating according to following formula and efficiency, more than 50% neutralization is active is decided to be the positive, lower than in 50% and active negative.Control group replaces 15A7 antibody with PBS.Figure 11 has shown monoclonal antibody 15A7 and its Fab and F (ab ') ₂in fragment and HIV _nL4-3effect, F (ab ') wherein ₂neutralization and whole antibody are suitable.The method of calculation of IC50 are as follows:

lgIC50＝Xm-I(P-(3-Pm-Pn)/4)

Wherein: Xm is lg maximal dose; I is lg (maximal dose/face mutually dosage); The positive reactivity sum of P; Pm is maximum positive reaction rate; Pn is minimum positive reaction rate.Result is summed up in Table 10.

Table 10. monoclonal antibody 15A7 and fragment thereof the cell neutralization to 5 strain HIV viruses

^*nT: do not detect

Embodiment 10. monoclonal antibody 15A7 mark CD4 ⁺the flow cytometer of cell detects

This tests the clone of expression CD4 used: Tzm-bl (Cat.No.8129NationalInstitutes of Health AIDS Research and Reference Reagent Program); U87.CD4.CXCR4 (Cat.No.4036National Institutes of Health AIDSResearch and Reference Reagent Program); U87.CD4.CCR5 (Cat.No.4035National Institutes of Health AIDS Research and ReferenceReagent Program); MT4 (Cat.No.120National Institutes of HealthAIDS Research and Reference Reagent Program); H9 (Cat.No.87National Institutes of Health AIDS Research and Reference ReagentProgram).Above-mentioned 5 strain cells all can stably express CD4, and wherein Tzm-blU87.CD4.CxCR4, U87.CD4.CCR53 strain are attached cell, and MT4, H9 are suspension cell.

PBMC extracts healthy volunteer's venous blood separation, and step is as follows: venous blood samples 20mL is added in heparin (5-10IU/mL) anticoagulant tube, adds 20mL PBS suspension cell.20mL cell suspension is carefully added in to 10mL Ficoll-Paque PLUS (Cat.No.17-1440-03GE Healthcare Bio-Sciences AB) upper, room temperature horizontal centrifugal 500g 20 minutes.Now in centrifuge tube, forming layering, is topmost blood plasma, between plasma layer and lymphocyte separation medium, is PBMC, sucks the blood plasma of the part the superiors, collects the PBMC of plasma layer and lymphocyte separation medium interface in 50mL centrifuge tube.Add 40mL PBS, mix rear 200g centrifugal 10 minutes, discard supernatant, collecting cell precipitation, adds 40mL PBS resuspended, mixes rear 500g centrifugal 10 minutes, then repeats 1 time.With 1% Trypan Blue, detect cell viability (answering > 95%) counting cells, by AIM-V Medium (Cat.No.12055Invitrogen) culture medium culturing.

Test antibody used: the anti-CD4 (Cat.No.555346 of FITC mark, BDBiosciences), PE mark CXCR4 (Cat.No.555974, BD Biosciences), the above antibody of PE-Cy5 mark CCR5 (Cat.No.556889, BD Biosciences) all uses PBS dilution in 1: 200 to use.

Monoclonal antibody 15A7 (dilution in 1: 1000) is as primary antibodie, and Anti-Mouse IgG (whole molecule)-FITC (Cat.No.F9006Sigma) is anti-as two with PBS dilution in 1: 200.

Prepare cell: the 3 strain attached cells such as Tzm-bl, U87.CD4.CXCR4, U87.CD4.CCR5 are prepared into single cell suspension with 0.25% tryptic digestion, MT4, H9 and PBMC are suspension cell, with transfer pipet, blow and beat into single cell suspension, cell concn is all adjusted into 1 * 10 ⁶/ mL, gets 4mL cell suspending liquid equivalent packing 8 pipe, and is divided at random tetra-groups of A, B, C, D, every group two pipe, and a pipe is for antibody labeling, and one manages negative contrast.Every tube cell adds 1.5mL PBS to put upside down to mix, and centrifugal 5 minutes of 1500rpm, discards supernatant, collecting cell precipitation.

Traget antibody: with the antibody-solutions 200 μ L re-suspended cells that diluted and mix.A, B, C group add respectively FITC-anti-CD4, PE-anti-CXCR4, PE-Cy5-anti-CCR5 antibody, and room temperature lucifuge is hatched 30 minutes.Add 2mL PBS to mix, centrifugal 5 minutes of 1,500rpm, discards supernatant, collecting cell precipitation, and resuspended with 500 μ L PBS.Flow cytometer detects antibody labeling situation.

Monoclonal antibody 15A7 solution re-suspended cell for D group primary antibodie, incubated at room 1 hour, adds 2mL PBS to mix, and centrifugal 5 minutes of 1500rpm, discards supernatant, repeats aforesaid operations 1 time, collecting cell.The anti-anti-Mouse IgG of 200 μ L bis-(whole molecule)-FITC solution re-suspended cell also mixes, and room temperature lucifuge is hatched 30 minutes.Add 2mL PBS, centrifugal 5 minutes of 1,500rpm, discards supernatant, collecting cell precipitation, and resuspended with 500 μ L PBS.Flow cytometer detects antibody labeling situation: Figure 12 is the flow cytometer test result after Tzm-bl cell marking; Figure 13 is the flow cytometer test result after MT4 cell marking; Figure 14 is the flow cytometer test result after H9 cell marking; Figure 15 is the flow cytometer test result after U87.CD4.CXCR4 cell marking; Figure 16 is the flow cytometer test result after U87.CD4.CCR5 cell marking; Figure 17 is the flow cytometer test result after PBMC cell marking.Positive rate result after each cell marking is added up in table 11, and combination situation and anti-CD4 antibody that flow cytometer detection result is set in 1%, 15A7 antibody and each clone surface receptor by negative control value have obvious dependency.

Table 11. monoclonal antibody 15A7 is to each clone surface receptor flow cytometer detection result

Embodiment 11. monoclonal antibody 15A7 detect with the flow cytometer of expressing the Cell binding situation of HIV

293-FT cell is according to 5 * 10 ⁴the concentration of individual cells/well is inoculated in 24 porocyte culture plates, and 5% 37 ℃ of CO2gas incubator are hatched 12 hours, with 0.6 μ g pNL4-3 plasmid (HIV _nL4-3the full gene plasmid of infections clone) transfectional cell, transfection reagent is shuttle China (Sofast, Xiamen sun horse biotechnology company limited), transfection method is referring to the operational guidance of this reagent; With the cell of untransfected in contrast.After transfection 48 hours, by the substratum sucking-off in 24 orifice plates, with 0.25% tryptic digestion, prepare single cell suspension, cell is transferred in 4mL centrifuge tube, the corresponding pipe in every hole.Every tube cell suspension adds 1.5mL PBS to mix, and centrifugal 5 minutes of 1,500rpm, discards supernatant, collecting cell precipitation.With 200 μ L monoclonal antibody 15A7 working fluids (dilution in 1: 1000) re-suspended cell; Control group PBS re-suspended cell, as the reference of pNL4-3 transfection effect, mixes rear incubated at room 1 hour with the anti-p24 monoclonal antibody H5F4 of dilution in 1: 100, and every pipe adds 2mL PBS to mix, and centrifugal 5 minutes of 1500rpm, discards supernatant, collecting cell precipitation.Anti-as two with 1: 200 dilution anti-Mouse IgG (whole molecule)-FITC (Cat.No.F9006, Sigma) of PBS, every pipe adds the anti-re-suspended cell of 200 μ L bis-and mixes, and room temperature lucifuge is hatched 30 minutes.Then add 2mL PBS to mix, centrifugal 5 minutes of 1,500rpm, discards supernatant, and collecting cell precipitation is resuspended with 500 μ L PBS.The situation of the cell after hatching with flow cytometer detection antibody labeling and albumen.The results are shown in Figure 18, positive rate the results are summarized in table 12, visible: p24 expresses at 293FT-HIV ⁺in cell, have expression, positive rate is 13.4%, illustrates after pNL4-3 plasmid transfection cell and reaches in time multiplexed cell tabulation.Monoclonal antibody 15A7 is combined effect efficiency with 293FT-HIV be 1%, and known 15A7 is not combined with HIV.

Table 12.15A7 and 293FT and transfection the 293FT of pNL4-3 be combined situation

The immunofluorescence of the cell of embodiment 12. monoclonal antibody 15A7 marker expression HIV detects

Prepare cell: the trypsinase of use 0.25% is by 293FT cell dissociation and be prepared into single cell suspension.By cell according to 5 * 10 ⁴the concentration hole of individual cells/well is inoculated in 24 orifice plates of completing in advance sterility cover slide, then every hole supplements 400 μ L DMEM perfect mediums (interpolation 10%FBS, 2mM L-glutamine, the nonessential amino acid of 0.1mM and 1% dual anti-) 37 ℃ of 5% CO2gas incubator are cultivated 12 hours.With 0.6 μ g pNL4-3 plasmid (HIV _nL4-3the full gene plasmid of infections clone) transfectional cell, transfection reagent is shuttle China (Sofast, Xiamen sun horse biotechnology company limited), transfection method is referring to the operational guidance of this reagent; With the cell of untransfected in contrast.Transfection is carried out cell and is fixed after 48 hours.

Cell is fixing penetrating: by the substratum sucking-off in 24 orifice plates, every hole adds 500 μ L PBS to wash once, then every hole adds 500 μ L 4% paraformaldehyde (100mL PBS contains 4g paraformaldehyde) room temperature lucifuges to fix 5 minutes, sucking-off 4% paraformaldehyde stationary liquid.Every hole adds 1mLPBS, and room temperature is placed 5 minutes, and then sucking-off, repeats to wash 2 times with PBS.By PBS sucking-off, every hole adds 500 μ L 0.3%Triton-X100 (100mL PBS contains 300 μ LTriton-X100) room temperature penetrating 5 minutes, sucking-off 0.3%Triton-X100.Every hole adds 1mLPBS, and room temperature is placed 5 minutes, and then sucking-off, repeats to wash 2 times with PBS.

Sealing: cover and put a sealed membrane at 24 orifice plates, on 24 orifice plates, the position in each hole drips 25 μ L lowlenthal serums, with ophthalmic tweezers careful from 24 orifice plates, take out cover glass, with thieving paper, suck liquid unnecessary on cover glass, then cover glass is placed in the drop of lowlenthal serum, guarantees that cell faces down.The 24 orifice plate lids that are placed with cover glass are put into wet box (maintenance saturated humidity) incubated at room 1 hour.

Primary antibodie is hatched: carefully take out cover glass, stirring cover slide faces up cell, then puts it into 24 orifice plates, adds 1mL PBS room temperature to place 5 minutes.Get again a new sealed membrane and cover 24 orifice plates and cover, according to the position in hole on 24 orifice plates, drip the PBS diluent (dilution in 1: 1000) of 25 μ L monoclonal antibody 15A7.The PBS diluent (dilution in 1: 1000) of monoclonal antibody H5F4 (anti-P24) is dripped in contrast, according to the same operation of sealing, cover glass is positioned in the drop of primary antibodie, guarantees that cell faces down.The 24 orifice plate lids that are placed with cover glass are put into wet box (maintenance saturated humidity) incubated at room 1 hour.

Two anti-hatching: carefully take out cover glass, stirring cover slide faces up cell, then puts it into 24 orifice plates, add 1mL PBS room temperature to place 5 minutes.Then sucking-off PBS, repeats to wash twice with PBS.Get a new sealed membrane and cover 24 orifice plates and cover, according to the position in hole on 24 orifice plates, drip 25 μ L bis-anti-, two resist for PBS dilution in 1: 200 for anti-Mouse IgG (whole molecule)-FITC (Cat.No.F9006, Sigma).According to the same operation of sealing, cover glass is positioned in two anti-drops, guarantee that cell faces down.The 24 orifice plate lids that are placed with cover glass are put into wet box (maintenance saturated humidity) room temperature lucifuge hatches 30 minutes.

DAPI dyes core: carefully take out cover glass, stirring cover slide faces up cell, then puts it into 24 orifice plates, adds 1mL PBS room temperature to place 5 minutes.Then sucking-off PBS, repeats to wash twice with PBS.Getting a new sealed membrane covers 24 orifice plates and covers, according to the position in hole on 24 orifice plates, drip the DAPI (dilution in 1: 2000) that 25 μ L have diluted with PBS, according to the same operation of sealing, cover glass is positioned in the drop of DAPI, guarantees that cell faces down.The 24 orifice plate lids that are placed with cover glass are put into wet box (maintenance saturated humidity) room temperature lucifuge hatches 5 minutes.

Mounting: carefully take out cover glass, stirring cover slide faces up cell, then puts it into 24 orifice plates, adds 1mL PBS room temperature to place 5 minutes.Then sucking-off PBS, repeats to wash twice with PBS.Mark slide glass, and drip mountant in the above.From 24 orifice plates, take out cover glass, with thieving paper, suck liquid unnecessary on cover glass, then cell faces down, and cover glass is placed in to mountant drop.With thieving paper, remove gently the mountant on cover glass, then coat nail varnish at edge, be used for closing cap slide edge.Lucifuge is dry.With laser scanning co-focusing microscope, observe and take pictures.

As shown in figure 19, monoclonal antibody 15A7 is not combined in 293FT and transfection is crossed on the 293FT cell of pNL4-3 plasmid for result.Show that monoclonal antibody is not combined with HIV.

The competition neutralization experiment of the CD4 polypeptide blocking-up HIV cells infected of embodiment 13. brachymemmas

Tzm-bl cell is according to 1.5 * 10 ⁴in individual cells/well paving and 96 orifice plates, 5% 37 ℃ of CO2gas incubator are hatched after 12 hours for neutralizing experiment.By the CD4 of preliminary purification (d1), CD4 (d1-2), CD4 (d2), CD4 (d3), after CD4 (d4) gradient dilution according to the HIV of 100 μ L/ holes and 100TCID50 _nL4-3mix latter 37 ℃ and hatch 1 hour, above-mentioned mixed solution is added to the Tzm-bl cell of completing in advance, 5% 37 ℃ of CO2gas incubator are cultivated 48 hours, after colour developing with Elispot counting, and in calculating and efficiency.Result as shown in figure 20, CD4 (d1), CD4 (d1-2) and sCD4 have the activity of good blocking-up HIV virus infected cell: wherein the IC50 of CD4 (d1) is 0.143 μ g/mL, the IC50 of CD4 (d1-2) is 0.250 μ g/mL, and the IC50 of sCD4 is 1.815 μ g/mL.

The reactivity of the CD4 polypeptide of embodiment 14. monoclonal antibody 15A7 and each brachymemma

Indirect elisa method detects: the CD4 of purifying (d1), CD4 (d2), CD4 (d3), CD4 (d4), CD4 (d1-d2) and sCD4 albumen are diluted to respectively to final concentration 0.5 μ g/mL with the coated damping fluid of 1 * PBS, on each hole surface of 96 hole polyethylene microtiter plates, 37 ℃ of absorption is 2 hours, then is placed in 4 ℃ and spends the night.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) wash titer plate to remove unconjugated antigen protein once.Then use 37 ℃ of confining liquids (adding 2% gelatin, 0.5% casein and 2% sucrose in the 1 * PBS solution) sealing 2 hours in 200 μ L/ holes.Get rid of clean, pat dry final vacuum sealing, 4 ℃ of preservations.

During detection, in every hole, add every hole to add the every secondary response of 15A7 antibody of 100 μ L dilutions that the negative control of one uncorrelated monoclonal antibody is set.37 ℃ of incubations 1 hour, with PBST washings, wash 5 times, sheep anti mouse immunoglobulin (Ig) (the HRP-GAM Ig that adds the peroxidase labelling of dilution in 1: 5000 after patting dry, DAKO company), 37 ℃ of incubations 30 minutes, with PBST washings, wash 5 times, pat dry rear priority and add each 50 μ L of substrate solution A, B (substrate solution A composition is: 13.42gNa ₂hPO ₄12H ₂o, 4.2g citric acid H ₂o and 0.3g hydrogen peroxide are 700mL by adjusted volume in deionized water; Nitrite ion B composition is: 0.2g tetramethyl benzidine, 20mL dimethyl formamide are 700mL by adjusted volume in deionized water), 37 ℃ are developed the color 15 minutes, add 50 μ L stop buffer (2M H ₂sO ₄) termination reaction, and in microplate reader, detect the OD450 value in each hole.Table 13 result is known, and CD4 (d1), CD4 (d1-2) and sCD4 albumen and antibody 15A7 respond.

Western method detects: also former state and non-reduced sample (containing DTT) are respectively with 15% SDS-PAGE electrophoretic separation for each CD4 recombinant protein after HPLC purifying, and electrotransfer, to nitrocellulose filter, adds 5% skimmed milk in room temperature sealing 1.5 hours; The film cutting of sealing with 5% skimmed milk, into about the wide bar of 0.8cm, is added respectively to monoclonal antibody, room temperature reaction 1 hour; TNT (8.765g NaCl, 1.21g Tris Base and 0.5mL polysorbas20, add deionized water to 1L, and adjusting pH is 8.0) washing three times, every minor tick 5 minutes; With AP mark goat anti-mouse (AP-GAM, the H+L) ELIAS secondary antibody (Protos company product) of 5% skimmed milk dilution in 1: 5000, room temperature reaction 1 hour; TNT washing three times, every minor tick 5 minutes; To 10mLAP damping fluid (100mM NaCl, 5mM MgCl ₂100mM Tris-HCl, adjusting pH is 9.5) in add 66 μ L NBT solution (0.5g NBT is dissolved in the dimethyl formamide of 10mL 70%) and 33 μ L BCIP solution (0.5g BCIP is dissolved in the dimethyl formamide of 10mL 70%), room temperature reaction 15 minutes.Western trace the results are shown in Figure 21: visible monoclonal antibody 15A7 only has good reactivity with CD4 (d1-2) and the sCD4 of reduction components; And have good reactivity with CD4 (d1-2) and the sCD4 of non-reduced component, there is weak reactivity with the CD4 (d1) of non-reduced component.

Embodiment 15. monoclonal antibody 15A7 are having or are having mark CD4 in situation without HIV virus ⁺the detection of cell

Prepare cell: TZM-bl cell is according to 1 * 10 ⁵the concentration of individual cells/well is inoculated in 24 porocyte culture plates, after 12 hours for experiment.In 24 orifice plates, add virus (being diluted to 100TCID50), contrast replaces with DMEM substratum.Hatch after 2 hours for 37 ℃, abandon containing virulent supernatant liquor, and wash 3 times with PBS.The diluent that adds monoclonal antibody 15A7, hatches 1 hour for 37 ℃.Trypsin digestion cell with 0.25%, proceeds in 4mL centrifuge tube, and 2mLPBS washes once, and 1,500rpm abandons supernatant for centrifugal 5 minutes, and the PBS re-suspended cell that contains RAM-FITC (dilution in 1: 200) with 100 μ L, hatches 30 minutes for 25 ℃.2mL PBS/ pipe is washed once, and 1,500rpm abandons supernatant for centrifugal 5 minutes, and with 500 μ LPBS re-suspended cells, flow cytometer detects antibodies situation.Result is as shown in figure 22: in the situation that HIV exists, monoclonal antibody 15A7 still can mark TZM-b1, and the positive rate of mark has obvious enhancing (p < 0.05).

Embodiment 16. monoclonal antibody 15A7 detect the toxicity of cell

TZM-bl cell is according to 1.5 * 10 ⁴the concentration in/hole is inoculated in 96 porocyte culture plates, 5% 37 ℃ of CO2gas incubator hatch 12 hours for detection of.By the substratum sucking-off in 96 orifice plates, add 100 μ L without phenol red DMEM substratum, 5% 37 ℃ of CO2gas incubator are hatched 2 hours, according to Cell Counting Kit-8 test kit (green skies biotechnology research institute) the every hole of specification sheets, add 10 μ L CCK-8,37 ℃, 5% CO2gas incubator are hatched, and sample by microplate reader and read plate respectively after hatching 20,40,60 minutes, detect its light absorption value.This experiment replaces monoclonal antibody 15A7 in contrast with PBS, without phenol red DMEM substratum, does not add cell as a setting.Specifically light absorption value is in Table the light absorption value of 14,15A7, PBS and DMEM substratum without significant difference (P > 0.05), and this shows that monoclonal antibody 15A7 does not have toxicity to Tzm-bl cell.

Table 14.15A7 monoclonal antibody cytotoxicity detects

The CD4 polypeptide of embodiment 17.15A7 and brachymemma is hatched the neutralization experiment that rear blocking-up HIV infects Tzm-b1 cell

Select the full gene experiment of the HIV strain virus HIV of B hypotype _nL4-3utilize in cell and hatch the rear infection conditions to Tzm-b1 cell with the CD4 polypeptide of experiment detection monoclonal antibody 15A7 and brachymemma.Tzm-bl cell is according to 1.5 * 10 ⁴in/hole paving and 96 orifice plates, after 12 hours for neutralizing experiment.According to the weaker concn gradient dilution monoclonal antibody 15A7 of 100 μ g/mL, 20 gradients of doubling dilution, in the substratum of dilution, add respectively corresponding CD4 (d1), CD4 (d1-2), sCD4 polypeptide, contrast is used sCD4 polypeptide for PBS, experiment according to the concentration of 25pM, and diluent volume is 100 μ L/ holes.With PBS, replace 15A7 antibody gradient dilution antibody simultaneously, in the substratum of dilution, also add respectively corresponding CD4 (d1), CD4 (d1-2), sCD4 polypeptide, thereby the situation that checking sCD4 polypeptide self blocking-up HIV infects Tzm-b1.HIV consumption is 100TCID50/ hole, volume is 50 μ L/ holes, after mixing with above-mentioned diluent, in 37 ℃ of constant incubators, hatch 1 hour, above-mentioned mixed solution is added in the Tzm-bl cell of completing in advance, then after 37 ℃ of 5% CO2gas incubator cultivated 48 hours, fix penetrating and colour developing.In counting and calculate with Elispot and efficiency.Experimental result is shown in accompanying drawing 23: from experimental result, can find out, and CD4 (d1), CD4 (d1-2), sCD4 polypeptide infects TZM-bl to monoclonal antibody 15A7 blocking-up HIV obvious impact (p < 0.01).

Embodiment 18.CD4 (d1), CD4 (d1-2), sCD4 and transfection the cell flow cytometer of HIV infective cloned plasmids detect

293-FT cell is according to 5 * 10 ⁴the concentration of individual cells/well is inoculated in 24 porocyte culture plates, and 5% 37 ℃ of CO2gas incubator are hatched 12 hours, with 0.6 μ g pNL4-3 plasmid (HIV _nL4-3the full gene plasmid of infections clone) transfectional cell, transfection reagent is shuttle China (Sofast, Xiamen sun horse biotechnology company limited), transfection method is referring to the operational guidance of this reagent; With the cell of untransfected in contrast.After transfection 48 hours, by the substratum sucking-off in 24 orifice plates, with 0.25% tryptic digestion, prepare single cell suspension, cell is transferred in 4mL centrifuge tube, the corresponding pipe in every hole.Every tube cell suspension adds 1.5mL PBS to mix, and centrifugal 5 minutes of 1,500rpm, discards supernatant, collecting cell precipitation.With 200 μ L PBS re-suspended cells, adding final concentration is CD4 (d1), CD4 (d1-2), the sCD4 albumen of 0.5 μ M; Control group substitutes protein solution, incubated at room 1 hour with PBS.Every pipe adds 2mL PBS to mix, and centrifugal 5 minutes of 1,500rpm, discards supernatant, collecting cell precipitation.With 200 μ L monoclonal antibody 15A7 working fluids (dilution in 1: 1000) re-suspended cell; Anti-Human CD4 (the CloneQ4120 of 200 μ L dilution in 1: 100 for control group, Sigma, identification epi-position is positioned at CD4D1 structural domain) re-suspended cell, with the anti-p24 monoclonal antibody H5F4 of dilution in 1: 100 as the reference of pNL4-3 transfection effect, mix rear incubated at room 1 hour, every pipe adds 2mL PBS to mix, centrifugal 5 minutes of 1500rpm, discard supernatant, collecting cell precipitation.Anti-as two with 1: 200 dilution anti-Mouse IgG (wholemolecule)-FITC (Cat.No.F9006, Sigma) of PBS, every pipe adds the anti-re-suspended cell of 200 μ L bis-and mixes, and room temperature lucifuge is hatched 30 minutes.Then add 2mL PBS to mix, centrifugal 5 minutes of 1,500rpm, discards supernatant, and collecting cell precipitation is resuspended with 500 μ L PBS.The situation of the cell after hatching with flow cytometer detection antibody labeling and albumen.The results are shown in Figure 24: wherein the little figure of A be take 293FT cell, the 293FT-HIV that monoclonal antibody antibody 15A7 hatches with CD4 (d1), CD4 (d1-2), sCD4 albumen respectively as first antibody mark ⁺the result of cell; The little figure of B 293FT cell, 293FT-HIV that to be anti-Human CD4 (Clone Q4120) hatch with CD4 (d1), CD4 (d1-2), sCD4 albumen respectively for first antibody mark ⁺the result of cell.Positive rate the results are summarized in table 15, visible: p24 expresses at 293FT-HIV ⁺in cell, have expression, positive rate is 7.2%, illustrates after pNL4-3 plasmid transfection cell and reaches in time multiplexed cell tabulation, but because p24 albumen is mainly present in cell interior, is not therefore having penetrating in the situation that, and Positive rate is on the low side; The known CD4 of positive rate result (d1) by control antibodies anti-Human CD4 (Clone Q4120) labeled cell, CD4 (d1-2), sCD4 are all and 293FT-HIV ⁺cell has combination, and wherein sCD4 binding ability is the strongest; Monoclonal antibody 15A7 and the 293FT-HIV that combines above-mentioned CD4 (d1), CD4 (d1-2), sCD4 albumen ⁺the mark situation of cell is more effective with the mark of corresponding cell than positive control antibody anti-Human CD4 (Clone Q4120).

Table 15.15A7 detect CD4 albumen with transfection the 293FT of pNL4-3 be combined situation

The mensuration of embodiment 19. monoclonal antibody 15A7 and CD4 (d1-2), sCD4 avidity

By vitamin H on the CD4 (d1-2) of purifying in embodiment 2, sCD4 protein labeling, concrete operations are as follows: it is 10 μ M that above-mentioned albumen is diluted to 4mL final concentration with PBS, add vitamin H (the EZ-Link Sulfo-NHS-LC-Biotin that 100 μ L concentration are 4mM, Cat.No.21335, Thermo Scientific), mix rear incubated at room 2 hours, then pack in dialysis tubing, put under 4 ℃ of stirrings of 1L PBS and dialyse, within 3 hours, change a dialyzate, repeat 4 times.

Using above-mentioned CD4 (d1-2), the sCD4 that is marked with vitamin H as antigen, in conjunction with Streptavidin (SA) Biosensors probe (Cat.No.185019, ForteBio), according to operational manual, point out, avidity with ForteBio Octet RED protein-interacting instrument analysis list clonal antibody 15A7 and CD4 (d1-2), sCD4 albumen: monoclonal antibody 15A7 with PBS be diluted to 400nM, 100nM, tri-concentration of 50nM detect and calculate avidity, negative control antibody is anti-P24 monoclonal antibody H4F5.The results are shown in Table 16: the avidity of monoclonal antibody 15A7 and two kinds of albumen approaches 1.20 * 10 ^-9m.

The avidity of the CD4 albumen of table 16. monoclonal antibody 15A7 and brachymemma

The expression of CD4 (d1-2) polypeptide of embodiment 20. point mutation

Section CD4 gene polypeptide recombinant expressed clone pTO-T7-CD4 (d1-2) is carried out to rite-directed mutagenesis, is L-Ala by target site amino acid mutation, and primer sequence is in Table 17.With PCR thermal cycler (Biometra T3), according to following condition, carry out PCR reaction: 94 ℃ of 10min; 94 ℃ of 2min subsequently, 56 ℃ of 50s, 17 circulations of 72 ℃ of 7min, are finally 72 ℃ and extend 10min.After PCR finishes, add restriction endonuclease Dpn I to digest template plasmid more than 37 ℃ of reaction 3h, using ER2566 intestinal bacteria as expression strain, adopt ordinary method to express, the albumen of results expression is to exist with insoluble inclusion body form.With ordinary method purifying inclusion body, target protein is mainly dissolved in 8M urea.By being dissolved in albumen in 8M urea, progressively carrying out dialysis and in 1 * PBS, complete renaturation, 12,000rpm removes precipitation in centrifugal 10 minutes, obtains rite-directed mutagenesis PROTEIN C D4 (d1-2)-F43A, CD4 (d1-2)-K46A, CD4 (d1-2)-R59A, CD4 (the d1-2)-N73A of preliminary purification.

Each rite-directed mutagenesis of table 17.CD4 (d1-2) fragment clone's primer sequence

Embodiment 21. indirect elisa methods detect the reactivity of monoclonal antibody 15A7 and point mutation CD4 (d1-2) polypeptide

The CD4 of preliminary purification (d1-2)-F43A, CD4 (d1-2)-K46A, CD4 (d1-2)-R59A, CD4 (d1-2)-N73A and CD4 (d1-d2) albumen are diluted to respectively to final concentration 0.5 μ g/mL with the coated damping fluid of 1 * PBS, according to 100 μ L/ holes, add in 96 hole polyethylene microtiter plates 37 ℃ to hatch 2 hours, then be placed in 4 ℃ and spend the night.With PBST washings (8.0gNaCl, 0.2g KH ₂pO ₄, 2.9g Na ₂hPO ₄12H ₂o, 0.2g KCl and 0.5ml polysorbas20, add deionized water to 1 liter, and pH is 7.4) wash titer plate once to remove unconjugated antigen protein.Then use 37 ℃ of confining liquids (adding 2% gelatin, 0.5% casein and 2% sucrose in the 1 * PBS solution) sealing 2 hours in 200 μ L/ holes.Get rid of clean, pat dry final vacuum sealing, 4 ℃ of preservations.

During detection, every hole adds the 15A7 antibody of 100 μ L dilutions.Hatch 1 hour for 37 ℃, with PBST washings, wash 5 times, sheep anti mouse immunoglobulin (Ig) (the HRP-GAM Ig that adds the peroxidase labelling of dilution in 1: 5000 after patting dry, DAKO company), hatch 30 minutes for 37 ℃, with PBST washings, wash 5 times, pat dry rear priority and add each 50 μ L of substrate solution A, B (substrate solution A composition is: 13.42g Na ₂hPO ₄12H ₂o, 4.2g citric acid H ₂o and 0.3g hydrogen peroxide are 700mL by adjusted volume in deionized water; Nitrite ion B composition is: 0.2g tetramethyl benzidine, 20mL dimethyl formamide are 700mL by adjusted volume in deionized water), 37 ℃ are developed the color 15 minutes, add 50 μ L stop buffer (2M H ₂sO ₄) termination reaction, and in microplate reader, detect the OD450 value in each hole.The results are shown in Table 18 results, wherein F43, K46, R59, the N73 of CD4 (d1-2) are mutated into after L-Ala, and the binding ability of 15A7 is significantly declined.

Embodiment 22.Western marking hybrid method detects the reactivity of monoclonal antibody 15A7 and point mutation CD4 (d1-2) polypeptide

By the CD4 of preliminary purification (d1-2)-F43A, CD4 (d1-2)-K46A, CD4 (d1-2)-R59A, CD4 (d1-2)-N73A and CD4 (d1-d2) albumen also former state and non-reduced sample (containing DTT) respectively with 13.5% SDS-PAGE electrophoretic separation, electrotransfer, to nitrocellulose filter, adds 5% skimmed milk in room temperature sealing 1.5 hours; Film after sealing adds in primary antibodie 15A7 (with the skimmed milk of 1: 200 dilution monoclonal antibody), and 4 ℃ of reactions are spent the night; TNT (8.765g NaCl, 1.21g Tris Base and 0.5ml Tween-20, add deionized water to 1L, and adjusting pH is 8.0) washing three times, every minor tick 5 minutes; AP mark goat anti-mouse (GAM with the dilution in 1: 5000 of 5% skimmed milk _h+L-AP) be ELIAS secondary antibody (Protos company product), room temperature reaction 1 hour; TNT washing three times, every minor tick 5 minutes; To 10mL AP damping fluid (100mM NaCl, 5mM MgCl ₂100mM Tris-HCl, pH is 9.5) in add 66 μ L NBT solution (0.5g NBT is dissolved in the dimethyl formamide of 10mL 70%) and 33 μ LBCIP solution (0.5g BCIP is dissolved in the dimethyl formamide of 10mL 70%), room temperature reaction 15 minutes.Western trace result is referring to Figure 25: wherein F43, K46, R59, the N73 of CD4 (d1-2) are mutated into after L-Ala, and monoclonal antibody 15A7 significantly declines to the binding ability of albumen.

Claims (15)

1. monoclonal antibody or its antigen-binding portion thereof, it has at least one item and is selected from following feature:

(i) specific binding people surface antigen differentiation bunch 4 albumen;

(ii) react with people's surface antigen differentiation bunch 4 polypeptid specificities of the clipped form that contains people's surface antigen differentiation bunch 4 protein D 1 structural domains;

(iii) human immunodeficiency virus's envelope glycoprotein gp120 is had no significant effect with people's surface antigen differentiation bunch reacting of 4 albumen; With

(iv) blocking-up human immunodeficiency virus infection CD4 ⁺cell and infecting again, and/or the acquired immune deficiency syndrome (AIDS) that causes thus of blocking-up;

Heavy chain CDR1, the CDR2 of wherein said antibody and the aminoacid sequence of CDR3 are respectively SEQ ID NOs:5-7 or nucleotide sequence coded by SEQ ID NOs:11-13, and light chain CDR1, the CDR2 of described antibody and the aminoacid sequence of CDR3 are respectively SEQ ID NOs:8-10 or nucleotide sequence coded by SEQ ID NOs:14-16; Or, heavy chain CDR1, the CDR2 of described antibody and the aminoacid sequence of CDR3 are respectively SEQ ID NOs:21-23 or nucleotide sequence coded by SEQ ID NOs:27-29, and light chain CDR1, the CDR2 of described antibody and the aminoacid sequence of CDR3 are respectively SEQ ID NOs:24-26 or nucleotide sequence coded by SEQ IDNOs:30-32.

2. the monoclonal antibody of claim 1 or its antigen-binding portion thereof, the aminoacid sequence of the variable region of heavy chain of wherein said antibody and the aminoacid sequence of variable region of light chain are respectively as shown in SEQ IDNO:2 and SEQ ID NO:4; Or the aminoacid sequence of the variable region of heavy chain of described antibody and the aminoacid sequence of variable region of light chain are respectively as shown in SEQ ID NO:18 and SEQ ID NO:20.

3. monoclonal antibody or its antigen-binding portion thereof as described in claim 1-2 any one, it is Fab, Fab', F (ab') ₂, Fv or single-chain antibody.

4. monoclonal antibody or its antigen-binding portion thereof as described in claim 1-2 any one, wherein, described monoclonal antibody is in conjunction with the K of people's surface antigen differentiation bunch 4 albumen _dvalue is less than 1 * 10 ^-5m.

5. monoclonal antibody or its antigen-binding portion thereof as described in claim 1-2 any one, wherein, described monoclonal antibody comprises FeiCDR district, Gai Fei CDR district is the species from non-muroid.

6. specific binding people surface antigen breaks up a monoclonal antibody for bunch 4 albumen, and it is the antibody 15A7 of the hybridoma generation of CCTCC preserving number C201098.

7. a hybridoma cell strain, it is the hybridoma of CCTCC preserving number C201098.

8. the purposes of the monoclonal antibody of the monoclonal antibody of claim 1-5 any one or its antigen-binding portion thereof or claim 6 in the reagent for the preparation of people's surface antigen differentiation bunch 4 albumen in detecting sample, described detection comprises the steps:

A) described sample is contacted with the monoclonal antibody of claim 1-5 any one or the monoclonal antibody of its antigen-binding portion thereof or claim 6;

B) detect reacting of described monoclonal antibody or its antigen-binding portion thereof and albumen in described sample;

C) detect reacting of described monoclonal antibody or its antigen-binding portion thereof and albumen in described sample and human immunodeficiency virus's envelope protein;

Wherein said monoclonal antibody or its antigen-binding portion thereof are attached on solid phase carrier; Wherein said solid phase carrier is selected from: titer plate, magnetic particle, latex particle and nitrocellulose membrane.

9. the purposes of claim 8, wherein said monoclonal antibody or its antigen-binding portion thereof are to be attached in described solid phase by direction like this, this direction can increase the joint efficiency of described monoclonal antibody and described sample.

10. the purposes of claim 8, wherein said monoclonal antibody or its antigen-binding portion thereof are attached in described solid phase by its constant region.

Monoclonal antibody described in 11. claim 1-2 or 4-6 any one or antigen-binding portion thereof claimed in claim 3 purposes in the reagent of the disease being caused by human immunodeficiency virus infection in for the preparation for the treatment of experimenter.

Monoclonal antibody described in 12. claim 1-2 or 4-6 any one or antigen-binding portion thereof claimed in claim 3 are for the preparation of the purposes that is used for the treatment of or prevents take the medicine of the disease that CD4+ cell is target cell, and wherein said disease is acquired immune deficiency syndrome (AIDS).

Monoclonal antibody described in 13. claim 1-2 or 4-6 any one or antigen-binding portion thereof claimed in claim 3 are for the preparation of the purposes of the medicine for blocking HIV infection CD4+ cell and infecting.

14. 1 kinds of pharmaceutical compositions, it comprises monoclonal antibody as described in claim 1-5 any one or monoclonal antibody and the pharmaceutically acceptable carrier of its antigen-binding portion thereof or claim 6.

The pharmaceutical composition of 15. claims 14, wherein said monoclonal antibody or its antigen-binding portion thereof are Fab, Fab', F (ab') ₂or Fv.