CN101215329A - Soluble human program death protein-1-lgV and preparation method thereof - Google Patents

Abstract

The invention discloses soluble programmed dead protein-1-IgV and a preparation process. The protein sequence of is showed as followed: PPTFFPAL LVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQ DSRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAE LRVTERRAEV PTAHPSP. The invention obtains gene of Hpd-1-IgV through applying artificial synthesis according to the structure of human PD-1, target gene is cloned into pQE-30 pronucleus expression carrier to transform colibacillus and is expressed in colibacillus with high efficiency, bacterioclasis shows that target protein is existed in inclusion body form, and purified soluble programmed dead protein-1-IgV is finally obtained through inclusion body scouring, affinity chromatography, gel column chromatography and dialysis renaturation. The soluble programmed dead protein-1-IgV can respectively combine with mPD-L1/Fc and hPD-L1/Fc in extra-corporal shPD-1IgV and on cell level and molecular level, and has certain tumor killing effect in internal shPD-1IgV. Therefore, shPD-1IgV has excellent internal and external biological activity, provides experiment basis for curing tumor, establishes experiment foundation, and has potential application prospect.

Description

Soluble human program death protein-1-lgV and preparation method thereof

Technical field

The invention belongs to the medical biotechnology field, relate to that gene is synthetic, protein expression purifying, external activity detect, press down technology such as knurl detection, particularly a kind of soluble human program death protein-1-lgV (hPD-1-IgV) and preparation method thereof in the animal body.

Background technology

Body immune system can be discerned " oneself/nonego ", " nonego " is carried out immunosurveillance, discerns and replys, so that with its elimination.Yet the immunoprotection system that this body is powerful but seems at a loss what to do in face of tumour.Tumour still can develop, shift under the powerful immunity function of human body, shows that tumour has the protection mechanism of oneself.Tumour cell can be by to the modification of self surface antigen and change immunity identification that microenvironment around the tumor tissues escapes body and attack Immune escape of tumor that Here it is.Tumour exists the mechanism of many escape immune system recognition and attack, comprising: the down-regulated expression of HLA-I class antigen and immune costimulatory molecules or lose; The down-regulated expression of tumour antigen, lose or suddenly change; Tumor cell secretion inhibitive ability of immunity soluble factor; Express the inhibitive ability of immunity molecule on the tumor cell membrane; Induce regulatory T lymphocyte with immune suppression function etc.Wherein, the soluble immunoresponse supressor in tumour source is to influence the function of dendritic cell (DC) and lymph T cell.These factors cause the undesired differentiation of medullary cell, cause the increase of the ripe DC quantity decline of general, the increase of immature DC (iDC) quantity and immature medullary cell (iMCs).Mainly contain: interleukin 10 (IL-10), transforming growth factor-beta (TGF-β), prostaglandin E2 PGE2) and chemokine (as CCL2/MCP-1) etc.In the antineoplastic immune effect, CD8 ⁺The T cell plays a major role, by identification to tumour antigen, can the direct killing tumour cell, but the microenvironment of tumor by local comprises a large amount of cytokines, these cytokines can be individually or are influenced the susceptibility that CTL activation or tumour cell kill and wound CTL synergistically, altogether immune stimulatory inhibition molecule plays an important role in this process, they otherwise the CD8+T cell can not be activated, or make activatory CD8 ⁺Or the forfeiture of T cell function makes CD8 ⁺T cell proliferation, these common immune stimulatory inhibition molecules mainly contain: CTLA4/B7, PD-1/PD-L1, ICOS/B7-H2, BTLA/B7-H4, TIM3 etc.

The PD-1 molecule is named as CD279 in December, 2004 in the 8th the human leukocyte differentiation antigen meeting in the world.Be to clone from hybridoma and hemopoietic progenitor cell system that mouse is in apoptotic state the earliest, be considered to relevant and called after programmed death-1 (programmed death-1) with apoptosis by cutting down hybridization technique.It is the inhibition acceptor of the activation-inducing of immunoglobulin superfamily, and the negativity adjusting function to immunne response is brought into play in two tyrosine residuess by its cytoplasmic domain and the signaling molecule effect in downstream.The PD-1 molecule can be in lymphoid tissue and the lymphocytic activation of organa parenchymatosum's tissue site double inhibition, plays an important role aspect the peripheral tolerance of body keeping.Studies show that PD-1 and its part PD-L1 (B7-H1) interact, form the PD-1/PD-L1 path, and the PD-1/PD-L1 path may participate in the generating process of tumour immunity escape, inflammation, autoimmune disorder, transplantation tolerance and virus infection etc.

The applicant is a goal in research with PD-1 IgV on the basis of analyzing the CCR5 structure, utilizes genetic engineering means and purified technology of protein, obtains the recombinant protein sample of purifying.The outer detection in this fusion rotein body can combine with PD-L1, tumor-bearing mice pressed down the observation of knurl experiment with fusion rotein.The experiment basis research of PD-1 IgV fusion rotein has been carried out in our invention, for further oncotherapy provides experimental basis and laid a good foundation.

1 Programmed death-1 (PD-1) molecular studies progress

1.1 the discovery of PD-1

The PD-1 molecule is named as CD279 in December, 2004 in the 8th the human leukocyte differentiation antigen meeting in the world.Be to clone from hybridoma and hemopoietic progenitor cell system that mouse is in apoptotic state the earliest, be considered to relevant and called after programmed death-1 (programmed death-1) with apoptosis by cutting down hybridization technique.It is the inhibition acceptor of the activation-inducing of immunoglobulin superfamily, and the negativity adjusting function to immunne response is brought into play in two tyrosine residuess by its cytoplasmic domain and the signaling molecule effect in downstream.The PD-1 molecule can be in lymphoid tissue and the lymphocytic activation of organa parenchymatosum's tissue site double inhibition, plays an important role aspect the peripheral tolerance of body keeping.Studies show that PD-1 and its part PD-L1 (B7-H1) interact, form the PD-1/PD-L1 path, and the PD-1/PD-L1 path may participate in the generating process of tumour immunity escape, inflammation, autoimmune disorder, transplantation tolerance and virus infection etc.

1.2 the structure of PD-1

PD-1 molecule relative molecular mass is 55,000 immunoglobulin superfamily I type transmembrane glycoproteins, has 288 amino acid and forms (shown in amino acid is composed as follows).

001 MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFFPA LLVVTEGDNATFTCSFSNTS

061 ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSVVRARRNDSGT

121 YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLVVGVVGGLLGS

181 LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFSVDYGELDFQW REKTPEPPVP

241 CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL

PD-1 molecule extracellular region comprises an IgV spline structure territory, has 4 important N to connect glycosylation site, and by the severe glycosylation.The notable attribute of PD-1 molecule is that the afterbody of cytoplasmic domain contains two tyrosine residuess, and wherein the tyrosine residues of N-terminal and other amino-acid residue have been formed an ITIM (immunity receptor tyrosine suppresses motif) jointly.ITIM is present in many inhibition acceptors, and its common trait is the common sequences that contains one 6 amino-acid residues (Ile/Val/Leu/Ser)-X-Tyr-X-X-(Leu/Val), is playing a significant role aspect the immunoreactive negative regulation.Nearest experiment confirm, the tyrosine residues of its C-terminal also can suppress lymphocytic activation with a series of signal transduction molecule effect in downstream.

1.3 the distribution of PD-1

The PD-1 molecule excites the back inducible expression at T, B cell antigen receptor, and the specified phase that immature T, B cell are grown in thymus gland and marrow also has expression.Some tumor cell lines have been proved the proteic expression of PD-1, as the Jurkat lymphoma, use Buddhist ripple ester and can obviously induce its up-regulated expression.The part tumor cell line has the expression of PD-1mRNA, as Daudi (Burkitt lymphoma), and HL260 (marrow series leukemia before acute) etc.

1.4 the part of PD-1

PD-L1 (B7-H1) and PD-L2 (B7-DC) are two parts of PD-1, and the cDNA of its total length obtains from the cDNA library of placenta by the homology screening.These two molecules all are positioned human chromosome 9p24.2, originate in same direction, at interval 42kb.They belong to B7 family together, thereby herewith other members of family are the same, and sophisticated PD-L molecule includes IgV sample district, IgC sample district, strides the cytoplasmic domain afterbody of a film district and a weak point.At extracellular region 4 conserved cysteine residue are arranged, participate in the formation of disulfide linkage in the Ig spline structure.Compare with PD-L2, the cytoplasmic domain of PD-L1 is more conservative Of Mice and Men.PD-L1 and PD-L2mRNA not only can be expressed in organa parenchymatosum's tissue site, as heart, lung and placenta etc., and expression are arranged also on antigen presenting cell, as activated B cell, monocyte and dendritic cell.Nearest research finds that also the expression of PD-L1mRNA is arranged the thymic epithelial cells.PD-L1 is confirmed on activated T, B and monocyte in the expression on the protein level.

1.5 the effect of PD-1/PD-L path

1.5.1 the effect of PD-1 and PD-L is to the influence of external T cell function

The propagation of suppressor T cell suppresses the generation of its pair cell factor IL-2, IFN-γ and IL-10 simultaneously but PD-1 and PD-L1 interact.Discover that PD-L1 and PD-1 are relevant to the intensity of the influence of T cell function and TCR and CD28 signal.Under suboptimal dose TCR strength of signal, PD-L1 is obvious to the inhibited proliferation of T cell; And when the TCR of optimal dose signal, only under the situation that does not have CD28 to stimulate altogether, just to T cell proliferation performance restraining effect.PD-L2 and PD-1 interact also can suppress T cell proliferation and generation IL-4, IL-10 and the IFN-γ that TCR mediates.Under low antigen concentration, PD-L2 and PD-1 effect can suppress the CD4 of the preactivate of TCR and B7-2 mediation ⁺The propagation of T cell, and the generation of minimizing Th1 and Th2 cytokines.But along with the rising of antigen concentration, the inhibition of its on cell proliferation descends, but still can suppress production of cytokines.Studies have shown that further lower concentration antigen can stimulate the PD-1 high level expression, along with the rising of antigen concentration, it expresses decline.Therefore, the weak antigenic stimulation of the interaction partners of PD-L/PD-1 reacts more responsive, and this class antigen mainly results from the generating process of autoimmune disorder and tumour.But other research thinks that under the TCR of suboptimal dose signal, PD-L2 (B72DC) can effectively stimulate the generation of the propagation and the cytokine (mainly being IFN-γ) of external T cell.Someone finds that when the anti-CTLA-4 monoclonal antibody of external application fragment was blocked its effect, can produce the different T cell subsets of same antigen-specific stimulated or retarding effect, and this different effect depends on the state of activation of T cell and the strength of signal of TCR.

1.5.2 the negative regulation effect between PD-1 and the PD-L

The intracellular region of PD-1 contains the tyrosine inhibitory motifs, by raising the negative conditioning signal of the protein factor transmission that contains tyrosine phosphatase enzymic activity and SH2 structural domain.2000, reports such as Freeman, B7-H1 combine with PD-1 can reduce T cell proliferation; 2002, Iwai etc. discovered that B7-H1 combines the immunologic escape that can promote tumour cell with PD-1.Though, all can increase specific for tumour antigen CD8 in the blood circulation by the method for inoculation or adoptive transfer ⁺The quantity of T cell, but along with the continuous progress of tumour, tumor tissues usually can occur CD8 ⁺The destruction or the damage of T cytological effect function.After PD-1 and B7-H1 interact, may mediate the retarding effect of T cell activation reaction by the progress that suppresses the cell cycle.2004, people such as Shengdian Wang found by mutating experiment, play a role (Fig. 2,3) that mutually combine, the IgV sample district of B7-H1 by its IgV sample district and PD-1.Simultaneously, Christian Blank etc. find to intervene the interaction between B7-H1 and PD-1, can strengthen specific for tumour antigen CD8 ⁺The function of T cell in tumor microenvironment.Simultaneously, there are a lot of documents and materials to show, blocking-up B7-H1, degeneration that can induced tumor tissue develops, but not merely relies on the PD-1 approach.

1.5.3 PD-1 and PD-L participate in T, B cell negative regulation mechanism of action

The B lymphoma cell line of in-vitro transfection PD-1 or Fc γ RII-PD-1 antigen-4 fusion protein gene be studies show that PD-1 can suppress the pungency signal of B-cell receptor.The crosslinked Ca that suppresses of BCR and PD-1 ²⁺The phosphorylation of the tyrosine residues of interior stream and downstream signal activating molecules such as Syk, phosphatidylinositol 3-kinase, Phospholipid hydrolase-3 and Vav.But this retarding effect does not need the tyrosine residues in the PD-1 cytoplasmic domain ITIM motif to participate in, but the tyrosine residues by the C end combines realization with the SHP-2 tyrosine phosphatase.In addition, PD-1 also can suppress the propagation of B lymphoma cell line by the tyrosine residues phosphorylation that suppresses mitogen activated protein kinase (MAPK).In addition, to the research of Jurkat clone also show TCR and PD-1 the crosslinked SHP-2 of causing phosphorylation and raise to the cytoplasmic domain of PD-1.The T cell of external application preactivate shows that the PD-L/PD-1 approach does not cause the apoptosis of T cell, and only makes cell cycle arrest in the G0/G1 phase.Yeast-two hybrid technique with improvement finds, can combine with the SH2 structural domain of the carboxyl terminal of tyrosine phosphatase SHP-1 after the ITIM tyrosine residues phosphorylation of the cytoplasmic domain of PD-1.Therefore, can combine performance negativity immunoregulation function after two tyrosine residues phosphorylations of PD-1 cytoplasmic domain respectively with SHP-1 and SHP-2.

2 PD-1 and disease

2.1 PD-1 and tumour

B7-H1 can improve anti-liver cancer cell in the mouse body (H22 tumour cell) immune response with solubility PD-1 blocking-up.Experiment in vitro confirms that B7-H1 can improve the lymphocytic early activation of part with solubility PD-1 blocking-up.PD-L1 is high expression level on the tumour cell of multiple tissue, and the interaction of it and PD-1 has shown the potential mechanism that tumour immunity is escaped.Effect between B7-H1 and the PD-1 has suppressed the effect of activated T cells, may realize that the B7-H1 that tumour is relevant has improved the apoptosis of T cells with antigenic specificity by suppressing the cell cycle, causes tumour to escape immunosurveillance.Experiment shows that the B7-H1 that expresses has promoted the apoptosis of activated tumor response T cell in the body on mouse P815 tumour, and impels in the body and the immune B7-H1 (+) of generation growth of tumor.PD-1 and PD-L1 interact and have reduced the lymphocytic propagation and the production of cytokines of TCR mediation.PD-1 is expressed on the tumour cell of various human and mouse, and is stimulated rise by IFN-γ.Discover,, strengthened the killing activity of tumour-specific CTL with solubility PD-1 blocking-up PD-1/PD-L1 path.And PD-1/PD-L1 interacts with the monoclonal antibody blocking-up, has reversed by specific for tumour antigen CD8 ⁺T cell function table loses the tumour tolerance that causes, has strengthened anti tumor immune response.

2.2 the immunocyte of PD-1 and neoplasm invasiveness

Thompson etc. carry out immunohistochemical staining to RCC (renal cell carcinoma) tumor sample with anti-PD-1 monoclonal antibody, found that the monokaryon immunocyte invades profit and account for 50.9% of patient's sum.The PD-1 immunocyte accounts for 56.6% of total patient's number.And the RCC tumour cell is not expressed PD-1.The PD-1 immunocyte has provide protection for the relevant tumour cell of B7-H1.Therefore, PD-1 (+) immunocyte has strengthened the transfer of tumour, and is relevant with the classification of malignancy of tumor degree.The level of expressing PD-1 on the immunocyte improves on one's body at excessive risk RCC tumour patient, and PD-1/PD-L1 has promoted the generation of RCC tumour.B7-H1 and B7DC express in nonsmall-cell lung cancer, and the quantity of the lymphocyte (TIL) of the neoplasm invasiveness in the B7-H1 male tumor region obviously is less than the tumor area of B7-H1 feminine gender, and the ratio that the TIL of PD-1 is expressed in B7H1 positive tumor cell zone is starkly lower than the negative tumour cell of B7H1 zone.This perhaps is because the PD-1/PD-L1 path causes due to the TIL apoptosis.

2.3 PD-1 and autoimmune disorder

The PD-1 molecule has been brought into play important effect keeping peripheral tolerance, thereby the PD-1 molecule can be used as the pathogenic process that an immunoreactive negativity regulatory molecule participates in autoimmune disorder.This point is confirmed in the PD-1 of different genetic backgrounds knock out mice.C57BL/6-PD-1 ^-/-Slight but gradual splenomegaly takes place in mouse, and the B cell and the medullary cell of spleen increase, and the IgA of serum and IgG2b increase, and IgG3 increases particularly remarkable, and body significantly strengthens the antigenic anti-IgG3 antibody response of TI-2.The proliferative response of the B cell antagonism IgM antibody induction of spleen also strengthens.C57BL/6-PD-1 ^-/-Lupoid acne proliferative glomerulonephritis and sacroiliitis can spontaneously take place in mouse with the growth at age, renal glomerulus has the deposition of over-drastic complement C3 and IgG3, and graininess tubercle and bone injury appear in ankle joint, and littermate control mouse of the same age is then acted normally.When importing the nonsense mutation of Fas gene simultaneously, lupus vegetans sample autoimmune disorder takes place in mouse ahead of time, and the severity of disease also increases greatly.This result shows that PD-1 and Fas have synergy in the generating process of lupoid acne autoimmune disorder.Autoreactivity TCR (2C-TCR) transgenic mouse can spontaneous generation graft versus host sample disease after rejecting the PD-1 gene, and the part mouse is dead before 10 weeks.All the other then show as and lose weight, splenomegaly, skin lesion in various degree: inflammation, hemorrhage and downright bad.The heart of pathology mouse, lung and kidney generation systems inflammatory cell infiltration.The 2C-TCR+T cellular infiltration is to the stratum basale of epidermis.Because 2C-TCR-PD-1 ^-/-The T cell of mouse is uninfluenced in the negative chosen process of thymus gland, and the 2C-TCR of periphery amplification ⁺The T cell presents memory cell phenotype (CDRBlowCD62LlowCD69 ₊), thereby the author thinks that PD-1 may participate in keeping peripheral tolerance.2C-TCR-PD-1 ^{+ /+}Mouse is acted normally, and visible PD-1 genetic flaw is the major reason that graft versus host sample disease takes place.Most BALB/c-PD-1 ^-/-Mouse is dead because of suffering from congestive heart failure when 10 ages in week, but 2C-TCR-PD-1 ^{+ /+}And BALB/c-PD-1 ^-/--RAG-2 mouse is well-grown then.The thoracic cavity ultrasonic examination shows that the ventricular chamber of dead mouse is obviously expanded, and the contractile function of ventricle descends.Spread all over the deposition of IgG and complement C3 between the myocardial cell of the whole expanding ventricular cavity of Microscopic examination showed, but do not have lymphocytic infiltration.Studies show that further autoantibody mainly is to be 33000 albumen at a kind of relative molecular mass that is expressed in the normal myocardium cell surface, with the PD-1 with genetic background ^{+ /+}The contrast mouse is compared BALB/c-PD-1 ^-/-Also can detect this albumen in the serum of mouse.These results show that PD-1 is preventing to have brought into play vital role aspect the BALB/c mouse generation autoimmunity DCM (dilated cardiomyopathy).

The PD-1 that studies confirm that to the PD-1 genetic flaw mouse of different genes background brings into play the negativity immunoregulation effect in vivo.Slight persistence splenomegaly takes place in C57BLP6-PD-1 genetic flaw mouse, and B cell and myeloid cell increase, and blood IgG3 raises and reaches at the antigenic IgG3 increased response of T cell dependent/non-dependent anti-IgM inductive spleen B hyperplasia increased response.C57BLP6-PD-1 genetic flaw mouse carrying out property glomerulonephritis and lupoid acne sacroiliitis can detect PAS positive material and IgG3 deposition in renal glomerulus.DCM (dilated cardiomyopathy) takes place in BALBPC-PD-1 genetic flaw mouse, and these rat hearts of ultrasonic demonstration aroused in interest obviously enlarge, and contractile function is badly damaged, and finds that IgG extensively is deposited between the myocardial cell, and electron microscopic observation finds that there is the IgG deposition on the myocardial cell surface.In addition, occur proteic antibody in the blood, point out people's DCM (dilated cardiomyopathy) of some unknown cause may be relevant with autoimmune response at normal myocardium surface 33 000.PD-1 of blocking-up NOD mouse or PD-L1 can quicken to be in the generation of the female mouse diabetes of each age group of pre-diabetes, and be more obvious in above the average age for marriage mouse.And blocking-up CTLA-4 only lures the morbidity of children mouse in age into, the early stage performance immunoregulation effect that prompting CTLA-4 only takes place at autoimmune response, and PD-1 all has effect in each stage that development takes place autoimmunization.Compare with control group, behind the blocking-up PD1/PD-L1, the scoring of pancreas islet inflammation is obviously increased, and the reactive splenocyte of GAD that produces IFN-γ increases, but anti-insulin antibody does not increase.Express PD-L1 on the islet cells of NOD mouse, do not express PD-L2, the PD-L1 of prompting islet cells oneself expression regulates the active important factor of autoreactive T cell.In by MOG inductive EAE (experimental autoimmune encephalomyelitis) C57BLP6 model research, blocking-up PD-1 can make disease increase the weight of, follow that inflammatory cell infiltration increases in the cerebral tissue, simultaneously, autoimmune response at MOG is strengthened, produce the T cytosis of IFN-γ, the hair style allergy strengthens, and anti-MOG antibody horizontal raises in the blood.Different with the NOD mouse is, blocking-up PD-L2 but not PD-L1 can cause disease to increase the weight of.PD-L1 promptly begins to express in EAE morbidity in early days, and PD-L2 expresses later and expression amount seldom.The experimental asthma Study of model is supported this result, and PD-L1 expresses more than PD-L2 and enriches in lung tissue, and the PD-L2 blocking-up can make disease increase the weight of.So, what meaning the high expression level of PD-L1 in these tissues has still treat further research.Hitachi etc. find in rheumatoid arthritis people's hydrarthrosis, CD4 ⁺T cell PD-1 expresses apparently higher than CD8 ⁺The T cell, CD4 ⁺PD-1 ⁺The T cell is most of expresses CTLA-4 simultaneously, and does not express CD26.CD4 ⁺PD-1 ⁺The T cell produces IL-10, with CD4 ⁺The PD-1-T cell is compared, and the IL-2 of its generation obviously reduces.Rheumatoid arthritis people's hydrarthrosis contains abundant PD-1 ⁺The T cell, the T cell of this subgroup is an anergy.

2.3 PD-1 and virus infection

With little gust of analysis and flow cytometry, Barber etc. find that PD-1 exhausts property CD8 in the function of LCMV chronic infection mouse ⁺T cell (exhausted CD8 T cell) is gone up high expression level, and the function memory CD8 of acute infection ⁺Do not express on the T cell.PD-L1 is high expression level on the cell of multiple infection, with barrier monoclonal antibody blocking-up PD-1/PD-L1 path, has strengthened CD8 on chronic LCMV mice infected ⁺The T cell function has activated the CTL activity, causes the generation of IFN and TNF.Day etc. have detected the specific C D8 that HIV infects ⁺The expression of the PD-1 of T cell, the crowd that they select is the HIV patient of not anti-HIV treatment.Found that the obvious high expression level of PD-1 on the special CD8 T cell of HIV, and this expression with to HIV specific C D8 ⁺The positive correlation of T cell function destructiveness, and relevant with the disease generating process.Blocking-up PD-1/PD-L1 path strengthens HIV specific C D4 ⁺, CD8 ⁺The T cell function.

Summary of the invention

The objective of the invention is to, make up human program death protein-1-lgV (hPD-1-IgV) fusion rotein, it can be mutually combined by its corresponding part PD-L1 in vivo and in vitro, thereby PD-1/PD-L1 path in the blocking-up body, breaking tumour immunity escapes, and the killing ability of recovery cytotoxic T lymphocyte, for the development antitumor drug provides new thinking and approach.

In order to realize above-mentioned task, the technical solution adopted in the present invention is:

A kind of soluble human program death protein-1-lgV is characterized in that, its protein sequence is as follows:

PPTFFPAL LVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQDSRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAE LRVTERRAEVPTAHPSP。

The preparation method of above-mentioned soluble human program death protein-1-lgV, it is characterized in that, structure according to people PD-1, using artificial synthetic method has obtained the gene of hPD-1-IgV, goal gene is cloned into the pQE-30 prokaryotic expression carrier, transformed into escherichia coli, in intestinal bacteria, efficiently express, split bacterium and show that target protein exists with the inclusion body form, through inclusion body washing, affinity chromatography, gel filtration chromatography and dialysis renaturation, can obtain the soluble human program death protein-1-lgV of purifying.

Detect combining of shPD-1IgV and PD-L1 with enzyme linked immunological adsorption method (ELISA) and flow cytometry (FCM) respectively with the target protein hPD-1-IgV of the solubility that obtains after purified, and detect shPD-1IgV and compete with anti-PD-L1 monoclonal antibody and combine PD-L1.Mtt assay detects CTL killing and wounding tumour cell.With shPD-1IgV treatment tumor-bearing mice, measure gross tumor volume, observe its tumor killing effect.With CD4 in the peripheral blood in each group of flow cytometry (FCM) detection tumor-bearing mice ⁺CD25 ⁺T (Treg) cell accounts for the ratio of total CD4+T cell.The result shows: shPD-1IgV can combine with commercial mPD-L1/Fc and hPD-L1/Fc respectively, and shPD-1IgV can combine with the PD-L1 that expresses on the tumor cell surface, and shPD-1IgV can combine mPD-L1/Fc with anti-PD-L1 monoclonal antibody competition.Isolating CTL of tumor-bearing mice and control group with the shPD-1IgV treatment relatively have tangible killing tumor cell effect.The tumor-bearing mice and the control group of shPD-1IgV treatment have relatively showed tangible tumor killing effect, has statistical significance, P＜0.05, and the ratio that the CD4+CD25+T cell accounts for total CD4+T cell in the tumor-bearing mice peripheral blood of control group is apparently higher than shPD-1IgV treatment group, for further oncotherapy provides experimental basis and laid a good foundation.Particular content is:

1, synthetic hPD-1-IgV gene order

Analyze the structure of Swiss-Pro Protein Data Bank PD-1, in conjunction with document, the disconnected outward IgV structural domain aminoacid sequence of intercepting PD-1 born of the same parents, obtain PD-1 born of the same parents outward the aminoacid sequence of disconnected IgV (called after: hPD-1-IgV):

PPTFFPAL LVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQDSRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAE LRVTERRAEVPTAHPSP

The gene order of the people PD-1-IgV that announces according to Gene-bank, 5 ends insert the BamHI restriction enzyme site, and 3 ends are introduced and are ended codon TGA and also insert the SalI restriction enzyme site, according to the codon of intestinal bacteria preference, synthetic goal gene.

The pQE-30 prokaryotic expression carrier is from Jieru Meng, and Nan Ma etc. (reference Jieru Meng, Nan Maet a1.NGR Enhanced the Anti-Angiogenic Activity of tum-5.[J] J.Biochem.140, (2006)).Expression vector is gone in the hPD-1-IgV gene clone and be cloned among the carrier pGEX-4T-1 by BamHI and SalI double digestion.

2. construction recombination plasmid pQE-30/hPD-1-IgV

With pGEX-4T-1/hPD-1-IgV carrier transformed into escherichia coli DH5 α, after the extraction plasmid DNA, itself and plasmid pQE-30 are carried out double digestion with BamHI and SalI respectively.The small segment (hPD-1-IgV) of getting above-mentioned pGEX-4T-1/hPD-1-IgV acquisition is connected with the big fragment that enzyme from pQE-30 is cut.Enzyme is cut product and is separated with 2.0% agarose gel electrophoresis, reclaims the small segment of pGEX-4T-1 acquisition and the big fragment among the pQE-30 respectively.With the small segment after reclaiming and greatly fragment be connected at 16 ℃ with the T4 ligase enzyme and spend the night, transform DH5 α competent cell, bed board, cultivation, the picking mono-clonal, extract plasmid, cut evaluation and dna sequencing through enzyme, obtain to contain hPD-1-IgV fusion gene pronucleus cloning vector, name pQE-30/hPD-1-IgV.

3. the abduction delivering of recombinant protein, purifying and evaluation

3.1 the abduction delivering of pQE-30/hPD-1-IgV engineering bacteria

The bacterial strain that will contain the pQE-30-hPD-1IgV recombinant plasmid is inoculated in 10ml and contains in the LB nutrient solution of Amp, and in 37 ℃ of overnight incubation, transferring in 1% ratio next day contains in the LB nutrient solution of Amp in 10ml, in 37 ℃ of shaking culture to logarithmic phase (A _600nm=0.4-0.6) time, add IPTG to final concentration be the 1mmol/L abduction delivering, in 37 ℃ of shaking culture 3-5h.Centrifugal collection thalline, the SDS-PAGE electrophoresis is identified.

3.2 the separation of target protein inclusion body and purifying

Get 10g inductive thalline, resuspended in the ratio of 1g weight in wet base with lysis buffer STE (50mmol/L Tris.Cl pH8.0,50mmol/L NaCl, 1mmol/L EDTA) to 7ml ,-20 ℃ of freeze overnight.Melt next day in room-temperature water bath, splits bacterium with reference to molecular cloning third edition bacteriolyze enzyme process, then adopts the 300W carrying out ultrasonic bacteria breaking again, wherein ultrasonic 5s, and 10s carries out 100 times altogether at interval.After ultrasonic the finishing, 12000rpm/min, 4 ℃ of centrifugal 20min, reject supernatant, precipitation inclusion body washings A (1%TritonX-100,1mmol/L EDTA, 1%DOC, 50mmol/LTris.Cl pH8.5,100mmol/L NaCl) resuspended, 4 ℃ of centrifugal 20min of 12000r/min abandon supernatant.With Ni-NTA post affinitive layer purification institute dissolved precipitation (inclusion body).Adding 10ml in the 1g thalline, to split the ratio of bacterium damping fluid resuspended with thalline, carries out the ultrasonic bacterium of splitting under the condition of ice bath.12000rpm, centrifugal 15min abandons supernatant, and the 1g precipitation adds 10ml6mol/L urea, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris (pH7.5).4 ℃ of stirrings are spent the night, 12000rpm, and centrifugal 15min, centrifugal 2 times altogether, it is stand-by to collect supernatant.Inclusion body through washing is dissolved in 6mol/L urea, 0.1mol/LNaH with the ratio of 1g weight in wet base to 10ml ₂PO ₄, in the damping fluid of 0.01mol/LTris pH 7.5,4 ℃ of stirrings are spent the night, and the centrifugal 30min of 12000r/min collects supernatant, is the target protein crude extract.(gel chromatography of 1.6cm * 80cm) is with working fluid (6mol/L urea, 0.1mol/LNaH for SephacrylS-300 ₂PO ₄, 0.01mol/Ltris pH 7.5) fully after the balance, target protein crude extract 2ml upper prop, with the working fluid wash-out, flow velocity 1ml/min collects the peak that contains target protein, carries out affinitive layer purification then.Use 6mol/L urea, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris (pH 7.5) is balance Ni post fully, earlier with the 6M urea that contains the 10mmol imidazoles, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris (pH 7.5) wash-out foreign protein is again with the 6mol/L urea that contains the 250mmol imidazoles, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris (pH 7.5) wash-out target protein.Collect elution peak, purified product is dialysed to 2M urea, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris (pH 7.5) renaturation, final concentration 1mg/ml.Dialysis is at last gone among the 0.02mol/LPBS (pH 7.2), measures protein concentration with the Lowery method.

3.3 the renaturation of target protein

Use the sample after solubilization of inclusion bodies liquid dilutes affinity chromatography, make protein concentration be about 0.1mg/ml, the gradient dialysis, extracellular fluid dialysis adds CuSO ₄With beta-mercaptoethanol as redox system, 4 ℃ of dialysis were changed liquid once in per 6 hours, gradient is, 4M urea, 2M urea, 1M urea carries out dialysis distilled water at last, the centrifugal supernatant that stays, freeze-drying.HPLC detects purity of protein.

Description of drawings

Fig. 1 is the structure of recombinant expression plasmid.Wherein, figure (A) is that the enzyme of recombinant expression plasmid pQE-30-rhPD-1IgV is cut evaluation, the mark 1:DNA marker (DL2000) among the figure, 2:pQE-30-rhPD-1IgV digested by BamHI and SalI; Figure (B) is clone's strategy in rhPD-1IgV district;

Enzyme is cut carrier pGEX-4T-1-rhPD-1IgV and pQE-30, obtain recombination, amalgamation and expression carrier pQE-30-rhPD-1IgV through connecting, with carrier pQE-30-rhPD-1IgV transformed competence colibacillus cell DH5 α, plasmid is extracted in the amplification back, carry out double digestion evaluation figure (A) with BamHI and SalI, obtain the insertion fragment of consistent size with expected results.Order-checking confirms through magnificent promise biotechnology Services Co., Ltd, and it is in full accord with expection that this inserts segmental gene order figure (B).

Fig. 2 is protein expression purifying and evaluation.Wherein, being labeled as among the figure (A): 1: molecular weight protein matter standard, 2:pQE-30 empty plasmid, 3: the rhPD-1IgV after inducing, 4: split the bacterium supernatant, 5: split the bacterium precipitation.

Behind the recombinant plasmid transformed intestinal bacteria, through abduction delivering, stable screening makes up engineering bacteria.After fermentation, collect a large amount of thalline, split bacterium and show that there be (figure A) in target protein with the inclusion body form, through inclusion body is washed, SephacrylS-300 gel filtration chromatography (figure B) affinity chromatography (figure C), SDS-PAGE shows the single band of albumen (figure D), and Western-blot identifies that demonstration can combine (figure E) with anti-His monoclonal antibody specificity.Its purity of HPLC analysis revealed is greater than 95%.

Fig. 3 is that the external molecular level of fusion rotein hPD-1IgV combines detection with mouse or people B7-H1/Fc (mB7-H1/Fc or hB7-H1/Fc).In conjunction with ELISA show shPD-1IgV can combine with hB7-H1/Fc and mB7-H1/Fc respectively (figure A, B).ShPD-1IgV can with bag by every hole 100nghB7-H1/Fc with mB7-H1/Fc is effective combines, in conjunction with presenting dose-dependently, show that bright shPD-1IgV can combine with hB7-H1/Fc and mB7-H1/Fc, and, under the dosage of 1000ngshPD-1IgV, shPD-1IgV combine with hB7-H1/Fc or mB7-H1/Fc present saturated.The competitive ELISA result shows that shPD-1IgV can block combine (the figure C) of hB7-H1/Fc and anti-hB7-H1 monoclonal antibody.After anti-hB7-H1 monoclonal antibody of 100ng and 100nghB7-H1/Fc were hatched jointly, the dosage that increases shPD-1IgV can reduce combining of anti-hB7-H1 monoclonal antibody and hB7-H1/Fc.Wherein, after shPD-1IgV was increased to 800ng, blocking effect tended towards stability.

Fig. 4 is that applying flow cytometry detects on the fusion rotein hPD-1IgV cell levels and the combining of B7-H1.(A) negative control wherein, (B) SP2/0 cell, (C) HT-29 cell; ShPD-1IgV can combine (Fig. 4) with the B7-H1 on the cell surface.With compare, shPD-1IgV can combine with the B7-H1 that SP2/0 cell (murine myeloma cell) is expressed, combination rate is 12%, and the B7-H1 of the shPD-1IgV HT-29 cell expressing that can stimulate with IFN-γ combines, combination rate is 15.6%.

Fig. 5 is that mtt assay detects the CTL killing activity that fusion rotein hPD-1IgV evokes.With to physiological saline relatively according to group, treatment group (dosage I group, dosage II group, dosage III group) has tangible CTL killing activity, is respectively 52.3%, 55.4% and 56.1%.The CTL killing activity of CTX group still is starkly lower than the treatment group apparently higher than control group.

Table 1 is respectively to organize the CTL killing activity relatively.

Table 1. is respectively organized the CTL killing activity relatively

	12.5∶1	25∶1	50∶1
				Dosage I group	0.311±0.015	0.461±0.022	1.231±0.134
Dosage II group	0.240±0.043	0.447±0.034	1.225±0.162
				Dosage III group	0.329±0.031	0.488±0.030	1.296±0.104
The physiological saline control group	0.190±0.065	0.247±0.034	0.392±0.043

Fig. 6 is the ratio that the CD4+CD25+Treg cell accounts for the CD4+T cell in the Flow cytometry tumor-bearing mice peripheral blood.Wherein, figure (A) is the physiological saline control group, and figure (B) is a dosage I group, and figure (C) is a dosage II group, and figure (D) is a dosage dosage III group, and figure (E) is the contrast histogram; Compare CD4 in the treatment group peripheral blood with the physiological saline control group ⁺CD25 ⁺The Treg cell accounts for CD4 ⁺The ratio of T cell obviously reduce (figure A, B, C, D, E), table 2 is a CD4CD25Treg per-cent.

Table 2

CD4CD25 Treg per-cent
		Dosage I group	24.8±2.876
Dosage II group	19.7±2.934
		Dosage III group	18.2±2.673
Control group	28.3±3.002
		The CTX group	10.3±3.231

CD4 in the peripheral blood in each treatment group ⁺CD25 ⁺The Treg cell accounts for CD4 ⁺The ratio of T cell is respectively 24.8%, 19.7%, 18.2%, is starkly lower than 28.3% of control group.Statistical analysis shows that each group of treatment group relatively has notable difference, P＜0.05 with control group.Simultaneously, compare with treatment II group and treatment III group, treatment I group has notable difference, P＜0.05.But treatment treatment II group and treatment III group do not have significant difference, P＞0.05.

Fig. 7 is the tumor-inhibiting action of hPD-1IgV fusion rotein to the mouse of SP2/0 lotus knurl.Among the figure, ^aCompare with the physiological saline group P＜0.05; ^cCompare with the CTX group P＜0.05; Use shPD-1IgV to BALB/c mice with tumor treatment 10 days, the result shows, compare with the physiological saline group, the treatment group has significant difference (P＜0.05), but, do not have statistical significance between 5mg/Kg treatment group and the 10mg/Kg treatment group, but statistical significance (P＜0.05) is all arranged with 15mg/Kg treatment group.Simultaneously, treatment group and endoxan (CTX) group more also has significant difference (P＜0.05).

The present invention is described in further detail below in conjunction with drawings and Examples.

Embodiment

4. the structure of fusion rotein PD-1-IgV, expression and purifying

4.1 the design of PD-1-IgV gene

Analyze the structure of Swiss-Pro Protein Data Bank PD-1, in conjunction with document, the disconnected outward IgV structural domain aminoacid sequence of intercepting PD-1 born of the same parents obtains PD-1 born of the same parents' aminoacid sequence (called after: hPD-1-IgV) of disconnected IgV outward.The gene order of the people PD-1-IgV that announces according to Gene-bank, 5 ends insert the BamHI restriction enzyme site, and 3 ends are introduced and are ended codon TGA and also insert the SalI restriction enzyme site, according to the codon of intestinal bacteria preference, synthetic goal gene.The synthetic gene order,

GGA TCC CAG TAT ATA AAA GCA AAT TCT AAA TTT ATA GGT ATA ACT GAAGCA GCA GCA GAA TTC GAC CTA TAT GTG GTA GAG TAT GGT AGC AAT ATG ACAATT GAA TGC AAA TTC CCA GTA GAA AAA CAA TTA GAC CTG GCT GCA CTA ATTGTC TAT TGG GAA ATG GAG GAT AAG AAC ATT ATT CAA TTT GTG CAT GGA GAGGAA GAC CTG AAG GTT CAG CAT AGT AGC TAC AGA CAG CGT GCC CGG CTGTTG AAG GAC CAG CTC TCC CTG GGA AAT GCT GCA CTT CAG ATC ACA GAT GTGAAA TTG CAG GAT GCA GGG GTG TAC CGC TGC ATG ATC AGC TAT GGT GGT GCCGAC TAC AAG CGA ATT ACT GTG AAA GTC AAT TGA GTC GAC

Its protein sequence is:

PPTFFPAL LVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQDSRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAE LRVTERRAEVPTAHPSP。

PGEX-4T-1 carrier (the living worker in Shanghai biotech company synthesizes and provides) is provided goal gene.

4.1.1 the preparation of competent cell

Get in the LB substratum that DH5 α glycerine bacterial classification inoculates in 1: 100 ratio, 37 ℃ of shaking culture are spent the night, and transfer once next day, continues to be cultured to OD ₆₀₀About about 0.4.(aseptic technique) with bacterium liquid ice bath 10min, centrifugal (3000rpm * 5min, 4 ℃) are supernatant discarded afterwards, adds the 100mmol/L CaCl of 1/2 volume precooling ₂, blow afloat precipitation gently, ice bath 40min, centrifugal (3000rpm * 5min, 4 ℃) abandon the 100mmol/L CaCl that contains 25% glycerine that adds 1/25 volume behind the supernatant ₂, blow afloat precipitation, divide in the Eppendorf pipe of packing into ,-70 ℃ of preservations are standby.

4.1.2 the conversion of competent cell, cultivation

Competent escherichia coli cell is taken out from-70 ℃, and ice bath melted 5-10 minute, added the pUC57 contain goal gene, and slight mixing continued ice bath 30 minutes, bed board then, 37 ℃ of overnight incubation.

4.1.3 the extraction of plasmid and evaluation

Transform picking neat in edge on the culture dish of back 37 ℃ of incubated overnight at plasmid, the clone that growth conditions is good, being inoculated into 10ml contains in the LB substratum of Amp, 37 ℃, 200rpm were cultivated 8 hours, extract plasmid with plasmid extraction kit, behind BamHI and double digestion, whether whether row agarose gel electrophoresis is observed has the segment of inserting segment and insertion consistent with the length of expection, enzyme is cut identified that the male clone serves Hai Boya company and carries out dna sequencing.

4.2 the structure of recombinant plasmid pQE-30/hPD-1-IgV, expression, purifying and renaturation

4.2.1 the structure of recombinant plasmid pQE-30/hPD-1-IgV

With pGEX-4T-1/hPD-1-IgV carrier transformed into escherichia coli DH5 α, after the extraction plasmid DNA, itself and plasmid pQE-30 are carried out double digestion with BamHI and SalI respectively.Getting small segment that pGEX-4T-1 described in 3.1 obtains is connected with big fragment from pQE-30.Enzyme is cut product and is separated with 2.0% agarose gel electrophoresis, reclaims the small segment of pGEX-4T-1 acquisition and the big fragment among the pQE-30 respectively.With the small segment after reclaiming and greatly fragment be connected at 16 ℃ with the T4 ligase enzyme and spend the night, transform DH5 α competent cell, bed board, cultivation, the picking mono-clonal, extract plasmid, cut evaluation and dna sequencing through enzyme, obtain to contain hPD-1-IgV fusion gene pronucleus cloning vector, name pQE-30/hPD-1-IgV.

4.2.2 the abduction delivering of recombinant protein, purifying and evaluation

4.2.2.1 the abduction delivering of pQE-30/hPD-1-IgV engineering bacteria

The bacterial strain that will contain the pQE-30-hPD-1IgV recombinant plasmid is inoculated in 10ml and contains in the LB nutrient solution of Amp, and in 37 ℃ of overnight incubation, transferring in 1% ratio next day contains in the LB nutrient solution of Amp in 10ml, in 37 ℃ of shaking culture to logarithmic phase (A _600nm=0.4～0.6) time, add IPTG to final concentration be the 1mmol/L abduction delivering, in 37 ℃ of shaking culture 3-5h.Centrifugal collection thalline, the SDS-PAGE electrophoresis is identified.

4.2.2.2 the separation of target protein inclusion body and purifying

Get 10g inductive thalline, resuspended in the ratio of 1g weight in wet base with lysis buffer STE (50mmol/L Tris.Cl pH8.0,50mmol/L NaCl, 1mmol/L EDTA) to 7ml ,-20 ℃ of freeze overnight.Melt next day in room-temperature water bath, splits bacterium with reference to molecular cloning third edition bacteriolyze enzyme process, then adopts the 300W carrying out ultrasonic bacteria breaking again, wherein ultrasonic 5s, and 10s carries out 100 times altogether at interval.After ultrasonic the finishing, 12000rpm/min, 4 ℃, centrifugal 20min, reject supernatant, precipitation inclusion body washings A (1%TritonX-100,1mmol/L EDTA, 1%DOC, 50mmol/LTris.Cl pH8.5 is 100mmol/LNaCl) resuspended, 4 ℃ of centrifugal 20min of 12000r/min abandon supernatant.With Ni-NTA post affinitive layer purification institute dissolved precipitation (inclusion body).Adding 10ml in the 1g thalline, to split the ratio of bacterium damping fluid resuspended with thalline, carries out the ultrasonic bacterium of splitting under the condition of ice bath.12000rpm, centrifugal 15min abandons supernatant, and the 1g precipitation adds 10ml6mol/L urea, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris (pH 7.5).4 ℃ of stirrings are spent the night, 12000rpm, and centrifugal 15min, centrifugal 2 times altogether, it is stand-by to collect supernatant.Inclusion body through washing is dissolved in 6mol/L urea, 0.1mol/LNaH with the ratio of 1g weight in wet base to 10ml ₂PO ₄, in the damping fluid of 0.01mol/LTris pH 7.5,4 ℃ of stirrings are spent the night, and the centrifugal 30min of 12000r/min collects supernatant, is the target protein crude extract.(gel chromatography of 1.6cm * 80cm) is with working fluid (6mol/L urea, 0.1mol/LNaH for SephacrylS-300 ₂PO ₄, 0.01mol/LTris pH 7.5) fully after the balance, target protein crude extract 2ml upper prop, with the working fluid wash-out, flow velocity 1ml/min collects the peak that contains target protein, carries out affinitive layer purification then.Use 6mol/L urea, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris (pH 7.5) is balance Ni post fully, earlier with the 6M urea that contains the 10mmol imidazoles, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris (pH 7.5) wash-out foreign protein is again with the 6mol/L urea that contains the 250mmol imidazoles, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris (pH 7.5) wash-out target protein.Collect elution peak, purified product is dialysed to 2M urea, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris (pH 7.5) renaturation, final concentration 1mg/ml.Dialysis is at last gone among the 0.02mol/LPBS (pH 7.2), measures protein concentration with the Lowery method.

4.2.3 the renaturation of target protein

Use the sample after solubilization of inclusion bodies liquid dilutes affinity chromatography, make protein concentration be about 0.1mg/ml, the gradient dialysis, extracellular fluid dialysis adds CuSO ₄With beta-mercaptoethanol as redox system, 4 ℃ of dialysis were changed liquid once in per 6 hours, gradient is, 4M urea, 2M urea, 1M urea carries out dialysis distilled water at last, the centrifugal supernatant that stays, freeze-drying.HPLC detects purity of protein.

5. fusion rotein hPD-1-IgV external activity detects

5.1 the detection on the external molecular level of fusion rotein hPD-1-IgV

5.1.1 ELISA detects shPD-1IgV and combines activity with hB7H1/Fc

Bag is by 96 orifice plates after getting the shPD-1IgV doubling dilution after the renaturation, and each concentration is established 3 multiple holes, and 4 ℃ of bags are spent the night, sealing 2h wash 6 * 2 minutes/time, every hole adding 100ng hB7-H1/Fc, room temperature 1h washed 6 * 2 minutes/time, added anti-hB7-H1 monoclonal antibody, room temperature 1h washs each 2 minutes 6 times, add anti-mouse two and resist, room temperature 30min washs 6 times, each 2 minutes/time, A was detected in OPD colour developing back _490nmValue is contrast with irrelevant his fusion rotein in the experiment.Method steps is as follows:

The bag quilt: get 96 hole elisa plates, the shPD-1IgV fusion rotein behind the doubling dilution is dissolved in bag is cushioned in the liquid, 4 ℃ of bags are spent the night;

Outwell coating buffer, add confining liquid, room temperature sealing 2h;

Discard confining liquid, wash 6 times, each 2min with washings;

Difference mouse anti human B7-H1 monoclonal antibody (500 μ g/mL) 100 μ L/ holes, incubated at room 1h;

Discard monoclonal antibody, wash 6 times, each 3min with washings;

The anti-mouse two that adds the HRP mark is anti-, 100 μ L/ holes, incubated at room 1h;

Discard two and resist, wash 6 times, each 3min with washings;

Add substrate colour developing liquid, 100 μ L/ holes, room temperature, colour developing 10-20min;

Add stop buffer, 50 μ L/ holes, termination reaction;

The microplate reader reading is measured the light absorption value of every hole at 490nm.

5.1.2 ELISA detects shPD-1IgV and combines activity with mB7H1/Fc

96 orifice plate bags are by the mB7-H1/Fc of different concns, and each concentration is established 3 multiple holes, and 4 ℃ of bags are spent the night, sealing 2h wash 6 * 2 minutes/time, every hole adding 500ugshPD-1IgV, room temperature 1h washed 6 * 2 minutes/time, added anti-his monoclonal antibody, room temperature 1h, washed 6 * 2 minutes/time, it is anti-to add anti-mouse two, room temperature 30min, washed 6 * 2 minutes/time, A is detected in OPD colour developing back _490nmValue is contrast with irrelevant his fusion rotein in the experiment.

5.1.3 ELISA detects the competition activity of shPD-1IgV to hB7-H1/Fc and anti-hB7H1 monoclonal antibody

96 orifice plate bags are by 100ng hB7-H1/Fc, if 3 multiple holes, 4 ℃ of bags are spent the night, sealing 2h, washed 6 * 2 minutes/time, every hole adds the shPD-1IgV and the anti-hB7-H1 monoclonal antibody of 100ng of doubling dilution, and room temperature 1h washed 6 * 2 minutes/time, adding anti-mouse two resists, room temperature 30min washed 6 * 2 minutes/time, and A is detected in OPD colour developing back _490nmValue is contrast with irrelevant his fusion rotein in the experiment.

Combine activity 5.2 detect fusion rotein hPD-1IgV on the Flow cytometry cell levels with B7H1

Behind the people HT-29 cell attachment, adding final concentration is the IFN-γ of 20ng/mL, stimulates HT-29 cell high expression level B7-H1 molecule after 2 days; The low B7-H1 albumen of expressing of mouse SP2/0 cell (murine myeloma cell).B7-H1 with HT-29 cell and SP2/0 cell expressing combines with shPD-1IgV fusion rotein Flow cytometry.Method steps is as follows:

Prepare HT-29 cell and SP2/0 cell suspension respectively, adjusting cell concn with 10%FCS RPMI1640 is 5 * 10 ⁶-1 * 10 ⁷/ ml.Get cuvette or plastic centrifuge tube that 40 μ l cell suspensions add the anti-His mAb of specificity (5-50 μ l) in advance, add 50 μ l (diluting) deactivation in 1: 20 normal rabbit serum again, 4 ℃ of 30min with DPBS.With washings washing 2 times, add about washings 2ml (1000rpm * 5min). at every turnAbandon supernatant, add the sheep anti mouse fluorescent marker of 50 μ l working concentrations, shake well, 4 ℃ of 30min.With washings washing 2 times, about each liquid feeding 2ml (1000rpm*5min).Add 500 μ l stationary liquids.

Reagent is formulated as follows:

DPBS (10 *, stock solution):

NaCl:80g, KCl:2g, Na ₂HPO4:11.5g, KH ₂PO4:2g, distilled water adds to 1000mL, faces and uses preceding dilution.

Washings: DPBS * 1:900mL, FCS:50mL, 4%NaN ₃: 50mL.

Stationary liquid: DPBS * 1:1000mL, Glucose:20g, Formaldehyde:10mL, NaN ₃: 0.2g.

6. fusion rotein hPD-1-IgV activity in vivo is observed

6.1 inoculated tumour cell

BALB/c mouse, totally 40, body weight: 18-22g, male.The take the logarithm SP2/0 tumour cell of state in vegetative period is according to 1 * 10 ⁷Individual cell/only be seeded to mouse back is subcutaneous.Observe the mouse interior tumor volume change every day, wait to observe and divide into groups after forming tumour.

6.2 tumor-bearing mice grouping

Choose the uniform tumor-bearing mice of gross tumor volume, reject the too big or too little tumor-bearing mice of gross tumor volume.Divide 5 groups at random with it, 6 every group.Physiological saline group control group; Dosage I group: the heavy 5mg shPD-1IgV of every kg mouse fusion rotein; Dosage II group: the heavy 10mg shPD-1IgV of every kg mouse fusion rotein; Dosage III group: the heavy 15mgshPD-1IgV fusion rotein of every kg mouse; If endoxan positive controls.

6.3 administering mode and dosage

Abdominal injection, the physiological saline group was given physiological saline once in per 12 hours, and treatment group (dosage I group, dosage II group, dosage III group) was administered once according to dosage in per 12 hours, and endoxan (CTX) group was administered once in per 24 hours.Continuous 10 days, measure the major diameter and the minor axis of tumour every day, calculate gross tumor volume TV (mm by following formula ³)=a * b ²* π/6, wherein, a is the tumour major diameter, b is and the vertical minor axis of a.

6.4 CD4 in the Flow cytometry peripheral blood ⁺CD25 ⁺The Treg cell accounts for CD4 ⁺The ratio of T cell

Each organizes tumor-bearing mice, and eyeball is got blood, adds the anti-hemostasis-coagulation of 1.5-2.4mgEDTA in every milliliter of blood.Get 100 μ l anticoagulations, add the straight labeling antibody of anti-CD4 and CD25 respectively, room temperature lucifuge 20-30min.Add 2ml erythrocyte cracked liquid (82.9g/LNH ₄Cl, 10g/LKHCO ₃, 370mg/LEDTA), mixing gently, room temperature is placed 10min.4 ℃, the centrifugal 5min of 1000r/min abandons supernatant.Add 2ml washings (8g/LNaCl, 0.2g/LKCl, 1.15g/LNa ₂HPO ₄, 0.2g/LKH ₂PO ₄, 5%FCS, 4%NaN ₃), mixing gently, 4 ℃, the centrifugal 5min of 1000r/min abandons supernatant.Repeat once.Add 500 μ l stationary liquid (8g/LNaCl, 0.2g/LKCl, 1.15g/LNa ₂HPO ₄, 0.2g/LKH ₂PO ₄, 2% glucose, 1% formaldehyde, 0.02%NaN ₃).FCM detects CD4 in the peripheral blood ⁺CD25 ⁺The Treg cell accounts for CD4 ⁺The ratio of T cell.

6.5 mtt assay detects the CTL killing activity

A. the separation of splenocyte: after measuring gross tumor volume for the last time, disconnected neck method is put to death mouse, opens the abdominal cavity under the aseptic condition and gets spleen.Spleen is immersed in the physiological saline, on 80 order steel meshes, grind gently, collect splenocyte.The centrifugal 15min of 1500r/min abandons supernatant.Add erythrocyte cracked liquid (Tris-NH ₄Cl, the NH of 0.16mol/L ₄The Tris of Cl and 0.17mol/L presses 9: 1 mixed, transfers pH=7.2), effect 1min adds physiological saline rapidly, washes 2 times.With containing 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, 10%FBS, the RPMI1640 of 50U/mlIL-2 is resuspended, and trypan blue counting back is stand-by.

The activation of b.CTL: tumour cell is cultivated with splenocyte altogether through 50 μ g/ml ametycin pre-treatment, 10%FCS, RPMI substratum, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates.The 0th, 2,4 days adding IL-2 (2units/ml).After 5 days, collect non-adherent cell, be used for measuring killing activity.

C.MTT detects killing activity: the concentration of tumour cell with 5000 cells in every hole is joined in 96 orifice plates.After treating cell attachment, the splenocyte of the preparation ratio in 12.5: 1 and 25: 1 and 50: 1 is joined in above-mentioned 96 orifice plates.Each concentration is established 3 multiple holes.37 ℃, 5%CO ₂Incubator is cultivated 72h.Discard substratum, every hole adds 50 μ lMTT reaction solutions (0.5mg/ml serum-free 1640 training bases).37 ℃, 5%CO ₂Incubator is cultivated 4h.Discard the MTT reaction solution, every hole adds 100 μ lDMSO, room temperature vibration 10min.Microplate reader is surveyed the light absorption value (A value) at 490nm place.Calculate kill rate.

6.6 the SPSS statistical study is carried out t check analysis, variance analysis to statistic data.

The protein sequence of soluble human program death protein-1-lgV

PPTFFPAL LVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQDSRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAE LRVTERRAEVPTAHPSP。

Claims (3)

1. a soluble human program death protein-1-lgV is characterized in that, its protein sequence is as follows:

PPTFFPAL LVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQDSRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAE LRVTERRAEVPTAHPSP。

2. the preparation method of the described soluble human program death protein-1-lgV of claim 1, it is characterized in that, structure according to people PD-1, using artificial synthetic method has obtained the gene of hPD-1-IgV, goal gene is cloned into the pQE-30 prokaryotic expression carrier, transformed into escherichia coli, in intestinal bacteria, efficiently express, split bacterium and show that target protein exists with the inclusion body form, through inclusion body washing, affinity chromatography, gel filtration chromatography and dialysis renaturation, can obtain the soluble human program death protein-1-lgV of purifying.

3. method as claimed in claim 2 is characterized in that, the step of described expression and purifying is as follows:

1) abduction delivering of pQE-30/hPD-1-IgV engineering bacteria

The bacterial strain that will contain the pQE-30-hPD-1IgV recombinant plasmid is inoculated in 10ml and contains in the LB nutrient solution of Amp, and in 37 ℃ of overnight incubation, transferring in 1% ratio next day contains in the LB nutrient solution of Amp in 10ml, in 37 ℃ of shaking culture to logarithmic phase A _600nmDuring=0.4-0.6, add IPTG to final concentration be the 1mmol/L abduction delivering, in 37 ℃ of shaking culture 3-5h, centrifugal collection thalline, the SDS-PAGE electrophoresis is identified;

2) separation of target protein inclusion body and purifying

Get 10g inductive thalline, in the ratio of 1g weight in wet base to 7ml, resuspended with lysis buffer STE, lysis buffer is:

50mmol/L Tris.Cl, 50mmol/L NaCl, 1mmol/L EDTA, resuspended back the freezing in-20 ℃ of lysis buffer STE spends the night, melt next day in room-temperature water bath, splits bacterium with the bacteriolyze enzyme process, then adopts the 300W carrying out ultrasonic bacteria breaking again, wherein ultrasonic 5s, 10s carries out 100 times altogether at interval; After ultrasonic the finishing, 12000rpm/min, 4 ℃ of centrifugal 20min, the reject supernatant, precipitation is resuspended with inclusion body washings A, and inclusion body washings A is: 1%TritonX-100,1mmol/L EDTA, 1%DOC, 50mmol/LTris.Cl pH8.5,100mmol/L NaCl, 12000r/min, 4 ℃ of centrifugal 20min abandon supernatant; With Ni-NTA post affinitive layer purification institute dissolved precipitation, adding 10ml in the 1g thalline, to split the ratio of bacterium damping fluid resuspended with thalline, carries out the ultrasonic bacterium of splitting under the condition of ice bath, 12000rpm, and centrifugal 15min abandons supernatant, and the 1g precipitation adds 10ml6mol/L urea, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris, 4 ℃ of stirrings are spent the night, 12000rpm, centrifugal 15min, centrifugal 2 times altogether, it is stand-by to collect supernatant; Inclusion body through washing is dissolved in 6mol/L urea, 0.1mol/LNaH with the ratio of 1g weight in wet base to 10ml ₂PO ₄, in the damping fluid of 0.01mol/L Tris, 4 ℃ of stirrings are spent the night, and 12000r/min, centrifugal 30min collect supernatant, are the target protein crude extract; With the SephacrylS-300 gel chromatography, after the abundant balance of working fluid, described working fluid is: 6mol/L urea, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris; Target protein crude extract 2ml upper prop, with the working fluid wash-out, flow velocity 1ml/min collects the peak that contains target protein, carries out affinitive layer purification then;

Use 6mol/L urea, 0.1mol/LNaH ₂PO ₄, the abundant balance Ni post of 0.01mol/LTris is earlier with the 6 M urea that contain the 10mmol imidazoles, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris wash-out foreign protein is again with the 6mol/L urea that contains the 250mmol imidazoles, 0.1mol/LNaH ₂PO ₄, 0.01mol/LTris wash-out target protein is collected elution peak, and purified product is dialysed to 2M urea, with 0.1mol/LNaH ₂PO ₄, the 0.01mol/LTris renaturation, final concentration 1mg/ml, dialysis is at last gone among the 0.02mol/LPBS, measures protein concentration with the Lowery method;

3) renaturation of target protein

Use the sample after solubilization of inclusion bodies liquid dilutes affinity chromatography, make protein concentration be about 0.1mg/ml, the gradient dialysis, extracellular fluid dialysis adds CuSO ₄With beta-mercaptoethanol as redox system, 4 ℃ of dialysis were changed liquid once in per 6 hours, gradient is, 4M urea, 2M urea, 1M urea carries out dialysis distilled water at last, the centrifugal supernatant that stays, freeze-drying, HPLC detects purity of protein.

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