CN100402554C - Preparation and use of testes specificity protein 50 human source antibody - Google Patents

Abstract

The present invention provides a preparation process of a testes specific protein 50 human source single chain antibody. A purified single-chain antibody is obtained by using a phage antibody display technique, using human testis specific protein 50 as a target antigen, using an immune affinity screening process to screen a variable region gene library for recombinant phage human antibodies, separating and cloning TSP 50 specific single-chain antibody genes AI, AII and C8 so as to respectively insert the single-chain antibody genes AI, AII and C8 into a prokaryotic expression vector PelB containing histag, transforming Rosetta bacteria, using IPTG to induce and express foreign protein, and using Ni-NTA column affinity chromatography after cracking thalli; the purified single-chain antibody can be combined with TSP50 holoprotein and 50 peptide specifically via ELISA identification, and the human source single-chain antibody can be massively express by a gene engineering means. The present invention also discloses uses of the testis specific protein 50 human source single-chain antibody in the diagnosis and the treatment of female breast cancer, and other diseases correlative to TSP50.

Description

The preparation and the purposes of testes specificity albumen 50 human antibodies

Technical field

The invention belongs to the humanized genetic engineering antibody field, what relate to is preparation and application with closely-related testes specificity albumen 50 human single chain variable fragments antibodies of tumour such as mammary cancer, this antibody can be used for the diagnosis and the treatment of TSP50 relative disease, belongs to technical field of biological genetic engineering.

Background technology

Mammary cancer is women's common cancer, and sickness rate has the trend that rises year by year, and from the epidemiology development trend, mammary cancer more and more concentrates on the big city, leaps to the first place of women's malignant tumour.At present, mainly be by iconography and biopsy for the diagnosis of mammary cancer, still there is not effective method of early diagnosis.The treatment means of mammary cancer is still based on operation, and radiotherapy, chemotherapy are auxilliary.Along with the development of society, radical operation of mastocarcinoma will be protected breast conserving surgery and be replaced, easier postoperative recurrence and transfer, and the side effect of radiotherapy, chemotherapy and to the patient bring painful obvious to all.Therefore the biology treatment will become one of more promising methods of treatment, and proto-oncogene Antybody therapy method is one of main method wherein, but major part trace expression and bring into play the important physical function in healthy tissues all in the present proto-oncogene, this brings very big inconvenience for application of its antibody.

Testes specificity albumen 50 involved in the present invention (testes specific protein 50, hereinafter to be referred as: TSP50) be a nearest newfound proto-oncogene, its 385 amino acid whose albumen of encoding, and this albumen has similar structure to multiple serine protease, but its catalytic residue Ser is replaced by Thr, so it may be the serine/threonine protein enzyme of a specific type.Under normal circumstances it is expressed in people's the testis tissue, and only is present in the spermatogonium, and not in sperm, points out it to have important effect in the process that sperm produces.And in other healthy tissuess beyond the testis, this gene is not expressed (Yuan, L., Shan, J., De Risi, D., Broome, J., Levecchio, J., Gal, D., Vinciguerra, V., and Xu HP.Isolation of a novel gene, TSP50, by a hypomethylated DNA fragmentin human breast cancer.Cancer Res.1999,59,3215-3221).But nearest studies show that, finds the unusual high expression level of TSP50 in the breast cancer tissue more than 90%, and (Shan, J. in this kind expression epithelial cell of being confined to cancerate, Yuan, L, Xiao, Q., Chiorazzi, N., Budman, D., Teichberg, S., and Xu, HP, TSP50, A possible protease in human testis, is activated in breastcancer epithelial cells.Cancer Res.2002,62,290-294).We infer that TSP50 is a new tumor-testis antigen (cancer testes antigen thus, CTA), play an important role in mammary cancer generation and sperm forming process as the serine/threonine protein enzyme, it will be the important target spot of breast cancer diagnosis and treatment.

But in order to produce the antigenic antibody of targeting specific, usually method this method of employing Kohler and Milstein generally comprises and uses the antigen immune mouse, splenocyte and murine myeloma cell with mouse merges then, selects the hybridoma that can secrete at the monoclonal antibody of specific antigens from the hybridoma that produces.

Summary of the invention

The invention provides a kind of preparation method of testes specificity albumen 50 human single chain variable fragments antibodies, its purpose is with TSP50 screening human single chain variable fragments antibody storehouse, and with engineered means with this person source single-chain antibody great expression.

The invention also discloses testes specificity albumen 50 human single chain variable fragments antibodies TSP50 diseases related-purposes in the diagnosis of mammary cancer and the treatment.

Below technology of the present invention is elaborated.The present invention utilizes phage antibody surface display system.This system inserts the single-chain antibody variable region gene in the bacteriophage coat protein gene, make single-chain antibody with the formal representation of fusion rotein in the surface of phage, by the screening process of immunoscreening repeatedly-infect-increase-again, obtain the antigen-specific recombinant phages antibody then.

The present invention utilizes phage antibody to present technology, is target antigen with human testicle specificity protein white 50, by the affine sieve method of immunity recombinant phage people antibody variable gene library is screened.Separate and clone TSP50 specific single-chain antibody gene A I, AII and C8.Then single-chain antibody gene AI, AII and C8 are inserted into respectively among the prokaryotic expression carrier PelB that contains the Histidine tail, transform the Rosetta bacterium, by IPTG abduction delivering foreign protein, Ni-NTA post affinity chromatography will be used behind the cellular lysate, obtain the single-chain antibody of purifying, ELISA identify its can with TSP50 whole protein and 50 peptide specific combination.Studies have shown that further it can be inhibited to the human breast carcinoma continuous cell line of vitro culture.

Preparation method's of the present invention concrete technical solution is as follows:

One, the preparation of TSP50 human antibody

According to the aminoacid sequence (seeing accompanying drawing 1 for details) of people TSP50 and with the homology of other serine protease relatively, the amino acid pro pool 156-206 amino acid of choosing in the TSP50 contact reacts functional zone synthesizes, these 50 amino acid whose little peptides are the special peptides of TSP50, with its called after pep-50.According to the cDNA sequence of people TSP50, designed the TSP50 Auele Specific Primer simultaneously,, from people's testis tissue, angled and got the TSP50cDNA fragment, and connect into prokaryotic expression carrier pGEX-4T-3, expressed by the RT-PCR method.

2. use the TSP50 protein screening human antibody library of pep-50 and prokaryotic expression.Concrete grammar is: increased in the library earlier, wrapped and hatched in the culture dish of antigen TSP50, submission is in the human antibody and the antigen mortise of phage surface, to there be the bonded phage to wash away, wash-out and antigen bonded phage are also increased, and the phage after the amplification is dropped into next round screening, 3-4 time so repeatedly again, wash and screen by being increased in the library, making pottery, obtain positive colony.

3. wrap by bacterium vinylbenzene enzyme plate with the phage that screens, add the anti-TSP50 polyclonal antibody of rabbit then, add the anti-rabbit antibody of enzyme mark sheep (or donkey) again, add the substrate colour developing at last, thereby acquired positive colony is further screened.

4. will and check order by the further positive bacteriophage extraction phage DNA that screens of ELISA method, and derive its aminoacid sequence.(seeing Fig. 2, Fig. 3, Fig. 4).

5. the dna fragmentation of expressing human antibody in the positive bacteriophage is cut with restriction enzyme NcoI and NotI enzyme, and connected among the prokaryotic expression carrier PelB, transformed into escherichia coli is again induced the expression of TSP50 human antibody with IPTG.The SDS-PAGE electrophoresis shows that the fusion protein molecule amount is the 31KD (see figure 5).Among the figure

1:A1 does not induce 2:A1 to induce with IPTG with IPTG

3: empty plasmid does not induce 4 with IPTG: empty plasmid IPTG induces

5:Marker 6:C8 does not induce with IPTG

7:C8 induces 8:A11 to induce with IPTG with IPTG

9:A11 induces with IPTG

6. the human antibody with prokaryotic expression carries out purifying by the metal chelating column affinity chromatography.

Two, the purposes of the human antibody of purifying of the present invention

The typical use of human antibody of the present invention is that treatment TSP50 is diseases related, for example mammary cancer etc.

The TSP50 human antibody that purifying is obtained joins among the MCF-7 MCF7 of vitro culture, after 72 hours, detects the vigor of MCF7 cell by mtt assay.The result shows that the TSP50 single-chain antibody can obviously suppress the propagation (see figure 6) of cell.

Term among the present invention " treatment " comprises " prevention ", " inhibition " or " treatment " these several situations to disease." prevention " is the composition that uses protectiveness before disease is brought out." inhibition " is after disease is brought out, but uses this composition before clinical manifestation is arranged." treatment " is after disease shows clinical symptom, the protective composite of using.

Human antibody of the present invention can with other antibody combined use, especially can with the antibody to other sign reaction on this and relevant people's cell, sign c-erbB-2 (the Bacus SS of mammary cancer for example, Gudkov AV, Esteva FJ, Yarden Y.Expression of erbB Receptors and their Ligands inBreast Cancer:Implications to Biological Behavior and TherapeuticResponse.Breast Dis.1999; 11:63-75.)

Usually, the form that human antibody of the present invention can purifying is used with appropriate carriers pharmaceutically.Generally, these carriers comprise water or alcohol/aqueous solution, emulsion or suspension, comprise salt solution and buffering medium.Non-enteron aisle carrier comprises sodium chloride solution, Lin Ge (Ringer ' s) glucose, glucose and sodium-chlor and Ru Suanlingeshi solution.Keep the complex body in the suspension if necessary, the thickening material of the optional carboxymethyl cellulose freely of the acceptable adjuvant of then suitable physiology, polyvinylpyrrolidone, gelatin and alginate.

Intravenous vehicles comprises liquid and nutritious supplementary and electrolyte replenisher.

Human antibody of the present invention can be used as the composition of using separately, or is used as and other medicine and co-administered composition, and these comprise the panimmunity medicine, as S-Neoral, methotrexate, Zorubicin or Platinol and immunotoxin.Pharmaceutical composition can comprise " the cocktail agent " that various kinds of cell toxic agent or other reagent and human antibody of the present invention are linked together.

According to the route of administration of pharmaceutical composition of the present invention can be those of ordinary skills generally know any.

Be not limited to for comprising for the methods of treatment of immunotherapy, can human antibody of the present invention be used to any patient according to standard technique.Administered in any suitable way be can pass through, non-enteron aisle, intravenously, intramuscular, intraperitoneal administration comprised, perhaps also can be by directly pouring into administration with conduit.The dosage of administration and frequency according to age, sex, patient condition, take the other factors that other medicines, untoward reaction and clinicist consider simultaneously and decide.

Except therepic use, this construct also can be used for studying purposes.TSP50 human antibody of the present invention can be used for the research of TSP50 26S Proteasome Structure and Function, and these antibody can be used as the instrument of conformation research to illustrate the native configurations of TSP50 different sites.In addition, the TSP50 human antibody can be used as diagnostic probe.Can be according to technical mark TSP50 human antibody known in the art.This construct also is suitable for purpose in other body.Whether for example they can be used for the location or the targeted therapy of TSP50 related neoplasms, have mammary cancer to take place as diagnosis, and radioactive substance are taken to the part of tumour by the TSP50 human antibody.

Positively effect of the present invention is: (1) does not influence normal physiological function so the application of this antibody can not resemble the antibody of other proto-oncogenes because TSP50 does not express in women's healthy tissues.(2) because single-chain antibody is to be formed by connecting by a micromolecular connection chain by antibody heavy chain variable region and variable region of light chain, its molecular weight is the sixth of immunoglobulin IgG molecular weight approximately.Therefore its perviousness is better, helps the in-vivo diagnostic and the treatment of solid tumor.(3) it is low to produce the antibody cost with genetic engineering means, is suitable for scale operation.(4) because antibody is the humanized, and do not contain the stable region of antibody, so its immunogenicity is minimum, can obviously reduce the adverse reaction to patient when being used for the treatment of.

Description of drawings

Fig. 1 has shown the aminoacid sequence of people TSP50, has underscore partly to be synthetic 50 peptide positions

Fig. 2 has shown the sequence of the human antibody AI that screens from phage antibody library.

Fig. 3 has shown the sequence of the human antibody AII that screens from phage antibody library.

Fig. 4 has shown the sequence of the human antibody C8 that screens from phage antibody library.

Fig. 5 has shown the electrophoretic analysis result of prokaryotic expression single-chain antibody.

Fig. 6 has shown that the human antibody of artificial expression can obviously suppress the proliferation activity of MCF-7 MCF7 cell.

Embodiment:

The following example will more specifically be described the present invention, not be regarded as limiting of the invention

Embodiment 1:

50 peptides and full-length proteins screening phage antibody library with TSP50

Phage antibody library: the human antibody library that is implemented in the pCOMB3 plasmid is so kind as to give by U.S. Scripps institute.

1. first round screening: 50 peptides and full-length proteins (50 μ g/ml) with TSP50 are wrapped by polystyrene culture dish, with antibody library (5.6 * 10 ¹²Pfu) add culture dish, hatched 2 hours, the sucking-off supernatant is used PBST wash dish 10 times, plate is buckled on aseptic filter paper done, with freshly prepared triethylamine 1ml wash bottle ware; Get 2 aseptic EP pipes, add 300 μ lTris-HCl respectively, add 500 μ l triethylamine elutriants more respectively, be used for infecting TG1 after the neutralization.

2. the amplification of first round screening back antibody library: among every 0.8ml and after eluted product infect the fresh e. coli tg1 of 14ml, 37 ℃ of incubators infected 30 minutes; 37 ℃ of shaking culture 1 hour are taken out 150 μ l and are inoculated among the 100ml2YT/AG1, and 37 ℃ of joltings were cultivated 1 hour, added helper phage M ₁₃K ₀₇, 37 ℃ were infected 30 minutes, added penbritin to final concentration 50 μ g/ml, and 37 ℃ of joltings are spent the night.Next day, centrifugal collection nutrient solution supernatant adds the PEG8000/NaCl of 1/5 volume, and mixing is placed more than 2 hours for 4 ℃, and the centrifugation phage is dissolved in throw out among the 1mlPBS, gets 1 μ l and surveys titre, and all the other 4 ℃ preservations are standby.

3. repetition screening process, and with one times of antigen coated amount reduction, so triplicate.Be that the specificity clone obtains enrichment.

Embodiment 2:

The preparation of mono-clonal phage antibody and active the detection

To eluriate product that enrichment obtains through three-wheel is laid on the 2YT solid medium and grows, 37 ℃ of incubated overnight, next day, 100 single spot inputs of picking are equipped with in the sterilization test tube of 2ml2YT/AG 37 ℃ of overnight incubation at random. and the adding of collection phage has been coated with in the proteic enzyme plate of the TSP50 hole hatched 2 hours.With conventional ELISA method screening, obtain the strong positive clone.

Embodiment 3:

TSP50 human antibody sequencing

Extract ELISA strong positive clone's DNA, entrust Shanghai associating genome company to carry out sequential analysis, and derive its aminoacid sequence, result such as Fig. 2 are shown in 3,4.

Embodiment 4:

The prokaryotic expression of human antibody

1. the structure of recombinant vectors: the DNA and the prokaryotic expression carrier PelB of phage antibody positive colony are cut with NcoI and NotI enzyme respectively, connect with the T4 ligase enzyme then. transform the Rosetta competence bacteria, the single bacterium colony amplification of picking next day is also extracted plasmid, cuts the evaluation recombinant plasmid by PCR and enzyme.

2. the abduction delivering of human antibody: the single positive bacterium colony of picking, be inoculated among the 12ml 2YT, 37 ℃ of joltings are taken out 8ml and are put in the 50ml Erlenmeyer flask when being cultured to OD600 and being 0.5 left and right sides, continue 37 ℃ of shaking culture, until OD600 is to be divided into two parts at 0.8～1.2 o'clock, and the 800mmol/LIPTG that a copy of it is added 5 μ l induces, and final concentration is 1mmol/L, 30 ℃, 140rpm spends the night.

3. the electrophoretic analysis of recombinant protein: got 4 hours and 6 hours IPTG inductive bacteriums centrifugal, to precipitate (by the centrifugal gained of 100ml bacterium) and freeze in-20 ℃, and take out after 1 hour and add PBS12ml, add PMSF (100mM) 8 μ l, 37 ℃ thaw, ultrasonication, centrifugal 30 minutes of 4 ℃ of 12000rpm collect supernatant, 4 ℃ of preservations, get a little and use 12% separation gel, the 5% SDS-PAGE glue that concentrates glue carries out the sex change electrophoresis, and the result is as shown in Figure 5.

Embodiment 5:

The affinity purification of recombination human source antibody

Get the fresh reorganization of inductive bacterium bacterium liquid 300ml, collect thalline behind the centrifugal 30min of 7000rpm/min, add 5ml buffer A (6M urea, 100mM Na by every gram thalline weight in wet base ₂PO ₄2H ₂O, 10mMTris-HCl, pH8.0), the 5mg N,O-Diacetylmuramidase, fully mixing adds PMSF to whole mass concentration 100mg/L, and 4 ℃ of stirrings are spent the night.Split bacterium liquid through the centrifugal 20min of 14000r/min (4 ℃), get supernatant liquor and cross Ni2+-NTA metal-chelating layer suction post, use 20ml buffer A, buffer B (6M urea, 100mM Na successively ₂PO ₄2H ₂O, 10mMTris-HCl, 1M NaCl pH6.3), damping fluid C (pH5.9, the same buffer B of its composition), damping fluid C+ imidazoles (100mM) wash-out, collects the elution peak of damping fluid C+ imidazoles (100mM), and measures protein content.

Embodiment 6:

Use horseradish peroxidase-labeled antibody

Horseradish peroxidase 8mg is dissolved in the 1ml distilled water, adds 200 μ l, 0.1M metaperiodic acid sodium solution at room temperature left standstill mixed solution 30 minutes.Utilize 1mM acetate buffer (pH4.5), make enzyme solution 4 ℃ of following dialysed overnight.Add 100 μ l, 0.2M carbonate buffer solution (pH9.5) is 9.5 to regulate the pH value.

On the other hand, 8mg is dissolved in 0.1M phosphate buffered saline buffer (pH7.4) with the anti-people TSP50 of purifying human antibody, in, utilize 0.01M sodium carbonate buffer (pH9.5) 4 ℃ of following dialysed overnight. the peroxidase that obtains is mixed with antibody, at room temperature static 2.5 hours, add Sodium Borohydride, descended static 2 hours at 4 ℃.With resulting peroxidase labeled antibodies, utilize phosphate buffered saline buffer, promptly obtain the enzyme labelled antibody binding substances 4 ℃ of dialysed overnight, add equal-volume glycerine-20 ℃ preservation, select suitable extent of dilution during use.

Practical example 1:

Immunohistochemical methods detects the expression of TSP50 in the mammary cancer

Get mammary cancer and cancer beside organism's paraffin section, 0.3%H is used in conventional dewaxing entry ₂O ₂-methyl alcohol soaked 30-40 minute, and PBS washing three times is sealed 30-60 minute with 0.1% gel again, discard unnecessary confining liquid, add the TSP50 human antibody of HRP mark, 37 ℃ 1 hour, it is inferior to give a baby a bath on the third day after its birth with PBS, each 5 minutes, adds freshly prepared DAB (0.5mg/ml)+H ₂O ₂(1 μ l/ml), 3-5 minute, conventional dehydration, mounting, microscopy.

Embodiment 2

With human antibody TSP50 is carried out Position Research in the cell

To cultivate good MCF-7 MCF-7 cell inoculation in 12 well culture plates that are placed with cover glass, every hole 5 * 10 ⁵Individual cell, 37 ℃, CO2 incubator overnight incubation is abandoned supernatant, washes secondary with PBS, fix 30 minutes with 4% Paraformaldehyde 96 room temperature then, give a baby a bath on the third day after its birth time with PBS, add the methyl alcohol effect 20 minutes of ice bath again, add the human antibody of FITC mark with the sealing of 1%BSA room temperature after 1 hour, 4 ℃ of effects 2 hours are sealed to the slide glass the back with cover glass and are observed TSP50 in intracellular location situation with the burnt phase microscope of copolymerization.

Practical example 3:

The medicinal radiative nuclear element of TSP50 human antibody mediation ¹³¹I is to the level diagnosis effect of mammary cancer

Get female nude mice, respectively inject the well-grown human breast carcinoma continuous cell line of 0.2ml MCF-7 cell suspension (cell count 8 * 10 at its belly mammary gland both sides fat pad ⁶Individual), preparation mice with tumor model.

Will with known Idogen or chloramine-t method mark medicinal radiative nuclear element ¹³¹I (but also mark ⁹⁹Tc ^m, ¹²³I reaches ¹¹¹In) human antibody AI injects through mouse tail blood vessel.After 24,48,60,72,84,96 hours, detect with the radio immuno imaging method ¹³¹The I traget antibody is the video picture situation in the tumor bearing nude mice body, and the result shows after artery injects radiolabeled human antibody, and the video picture rate is to rise in the cancer district in 86.9%, 36 hour to gather, and 60 hours the most clear, and T/NT is 5.2.

Practical example 4

Detect the proteic expression of TSP50 with the ELISA method

Test kit is formed:

1. standard substance: mammary cancer marker molecule TSP50 protein solution 400 μ l, its concentration is 0.1mg/ml

2. human antibody: mouse anti TSP50 human antibody 50 μ l, antibody concentration is 0.1mg/ml, it is 1: 1000 that ELISA tires

3. polyclonal antibody: the anti-TSP50 polyclonal antibody 50 μ l of rabbit, antibody concentration is 1mg/ml, and it is 1: 1000 that ELISA tires, and is this chamber self-control, and its preparation method is a known technology.

4. enzyme labelled antibody: it is 1: 1000 that goat anti-rabbit igg antibody 50 μ l (available from promega company), antibody ELISA tire

Test kit comprises mentioned reagent at least, for convenience simultaneously, and the enzyme plate of also can packing into, concentrated cleaning solution, concentrated confining liquid, developer A, developer B, stop buffer, semi-logarithmic coordinate paper etc.

Described concentrated cleaning solution is a phosphoric acid buffer, and confining liquid is that BSA-phosphate buffered saline buffer, developer A are 1% phosphorus phenylenediamine (OPD), and developer B is a hydrogen peroxide, and stop buffer is a 2M sulfuric acid.

Using method:

1. various concentrated solutions are diluted to application concentration, are about to 100 times (concentration is 0.01M during detection) of concentrated cleaning solutions dilution specifically; 10 times (BSA concentration is 1% during detection) of confining liquid dilution

2. prepare different dilution standard substance: the washings with dilution is diluted to 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 100ng/ml, 10ng/ml, 1ng/ml with standard substance.

3. with the anti-TSP50 human antibody of purifying, be diluted to 0.4 μ g/ml, wrap by 96 hole enzyme plates with 0.01M PBS damping fluid (pH7.4), 100 μ l/ holes, 4 ℃ are spent the night.

4. discard antibody sandwich liquid, with 1%BSA sealing, 43 ℃, 30 minutes.

5. abandon confining liquid, use the 0.1%Tween20-PBS washed twice, each 5 minutes.

6. add different dilution TSP50 albumen, add testing liquid simultaneously, 100 μ l/ holes, 43 ℃, 30 minutes.

7. with 0.1%Tween20-PBS washing three times, each 5 minutes.

8. add the anti-TSP50 polyclonal antibody of rabbit, 100 μ l/ holes, 43 ℃, 30 minutes.

9. with 0.1%Tween20-PBS washing three times, each 5 minutes.

10. adding goat anti-rabbit igg, 100 μ l/ holes, 43 ℃, 30 minutes.

11. with 0.1%Tween20-PBS washing three times, each 5 minutes.

12. add the OPD chromogenic substrate, room temperature reaction 5 minutes.

13. every hole adds 50 μ l 2M H ₂SO ₄Termination reaction, microplate reader is surveyed the OD492 value.

Make typical curve according to TSP50 standard substance measurement result, from then on all samples to be checked can find its corresponding value on the typical curve according to its OD492 value, and this numerical value is the content of TSP50 in this sample.

Practical example 5

The anti-tumor activity of the human antibody of prokaryotic expression is identified

Cultivate going down to posterity and well-grown MCF-7 MCF7 cell with 0.25% trysinization after, the accent cell concn is 1 * 10 ⁵After join in 96 orifice plates every hole 100 μ l, 37 ℃, 5% CO ₂Incubator spends the night, and three kinds of TSP50 human antibodies of prokaryotic expression are joined in each hole with different concns, and its final concentration is respectively 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0 μ g/ml, each concentration is established three multiple holes, simultaneously with the negative contrast of PBS, 37 ℃, 5% CO ₂Cultivate after 68 hours in the incubator, every hole adds 10 μ l MTT (5mg/ml), continues to cultivate 4 hours, inhales and goes cell conditioned medium, every hole to add 100 μ l DMSO, treats that the cell endoparticle dissolves the back fully and surveys the vigor that the OD570 value is judged cell with microplate reader.The result shows that three strain TSP50 single-chain antibodies all can obviously suppress the propagation (see figure 6) of breast cancer cell.

Practical example 6

The medicinal radiative nuclear element of TSP50 human antibody mediation ¹³¹I is to the therapeutic action of mammary cancer

Get female nude mice, respectively inject the well-grown human breast carcinoma continuous cell line of 0.2ml MCF-7 cell suspension (cell count 8 * 10 at its belly mammary gland both sides fat pad ⁶Individual), preparation mice with tumor model.

Will with known Idogen or chloramine-t method mark medicinal radiative nuclear element ¹³¹I (but also mark ⁹⁹Tc ^m, ¹²³I reaches ¹¹¹In) human antibody AII injects through mouse tail blood vessel.After 1,3,5,7,14 day, observe with the calculated value of CT ¹³¹The I traget antibody is to the influence of tumor bearing nude mice tumor growth, and the result shows that the tumour of 89.3% mice with tumor is dwindled, and wherein its tumour of 78.1% mice with tumor is dwindled more than 50%.

Practical example 7

With the TSP50 human antibody TSP50 is carried out the epi-position structural analysis

Human antibody screening phage 15 peptide storehouses (can buy commercialization peptide storehouse) with TSP50, its concrete grammar is: increased in the peptide storehouse earlier, join then to have wrapped and hatched in the culture dish of anti-TSP50 human antibody, submission phage surface can with the little peptide of antibodies will with the antibody mortise, to there be the bonded phage to wash away, the phage of wash-out and antibodies is also increased, phage after the amplification is dropped into the next round screening again, 3-4 time so repeatedly, wash and screen by being increased in the peptide storehouse, making pottery, obtain positive colony.The gained positive colony is further screened with the competitive ELISA method, find with TSP50 competition and human antibody bonded clone, concrete grammar is: personnel selection source antibody sandwich enzyme plate, BSA sealing back adds TSP50 albumen and gained phage, be control group with independent adding TSP50 albumen hole simultaneously, the TSP50 polyclonal antibody that adds horseradish peroxidase-labeled then, the OPD colour developing, measure the OD492 value, find and with the phage of TSP50 competition and antibodies and to extract its DNA and to check order, find out its conserved sequence, this conserved sequence is the epi-position of the TSP50 that infers in theory, further determines the epi-position structure of TSP50 again by computer simulation.

Claims (3)

1. three kinds of testes specificity albumen 50 human single chain variable fragments antibodies, it is characterized in that: they all are to utilize phage antibody to present technology, with human testicle specificity protein white 50 is target antigen, the screening human antibody library, and gained antibody gene sequence clone expressed to the prokaryotic expression carrier PelB and prepare, this three-type-person source antibody has following sequence general formula:

MAEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV 50

SAISGSGGSTYYADSVKGRFTISRDN-①-KNTLYLQMNSLREDTAVYYCAR- 100

②-WGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSVALG 150

QTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSS 200

SGNTASLTITGAQAEDEADYYCNSRDSSG-③-VFGGGTKLTVVLGAA 245

Wherein, 1. be S, 2. be RQRLRHYANN, when 3. being F, this sequence is AI;

1. being P, 2. is RQRKSGRK, and when 3. being K, this sequence is AII;

1. being S, 2. is VRHRASRANL, and when 3. being RV, this sequence is C8.

2. testes specificity albumen 50 human single chain variable fragments antibodies of claim 1 are used to prepare the diseases related diagnosis of testes specificity albumen 50 and the purposes of medicine.

3. pharmaceutical composition, it contains any one human antibody and medicinal acceptable vehicle in the with good grounds claim 1.

Images