CN101519448B - Epididymal protease inhibitor protein monoclonal antibody and application thereof - Google Patents
Epididymal protease inhibitor protein monoclonal antibody and application thereof Download PDFInfo
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- CN101519448B CN101519448B CN2009100250769A CN200910025076A CN101519448B CN 101519448 B CN101519448 B CN 101519448B CN 2009100250769 A CN2009100250769 A CN 2009100250769A CN 200910025076 A CN200910025076 A CN 200910025076A CN 101519448 B CN101519448 B CN 101519448B
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Abstract
The invention discloses an epididymal protease inhibitor protein monoclonal antibody and application thereof, which belong to the field of biological engineering. The monoclonal antibody is secreted by hybridoma preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C200849. The monoclonal antibody can be combined to the epididymal protease inhibitor protein Eppin protein of specific expression in a testis and epididymis tissue by specificity. The monoclonal antibody can be used for preparing male infertility diagnostic reagents, male contraceptives and anti-fertility study biological products.
Description
Technical field
The invention belongs to biotechnology, cell engineering and immunodiagnosis field, relate to a kind of epididymal protease inhibitor protein monoclonal antibody and uses thereof.More specifically, the present invention relates to a kind of monoclonal antibody, a kind of epididymal protease inhibitor protein of this antibody capable specific recognition, this albumen be a kind of in testis and epididymis tissue specific expressed epididymal protease inhibitor protein, mainly be expressed in sperm surface; The invention still further relates to the application in preparation antifertility postgraduate Tetramune, male sterility diagnostic reagent of this protein-specific MONOCLONAL ANTIBODIES SPECIFIC FOR method and this monoclonal antibody.
Background technology
Epididymis is capacitation and sophisticated place, sperm in the epididymis microenvironment by structure function change obtain the fertilization and fertility.Though still do not have a kind of male contraceptive pill that directly acts on epididymis and can use for clinical trial so far, epididymal function and sperm be studies show that epididymis very likely becomes the optimal target organ of male-contraception in that epididymis is sophisticated.
The Eppin gene finds the mankind first that in calendar year 2001 it is positioned human No. 20 karyomit(e)s, the Eppin albumen of this genetic expression be a kind of in testis and epididymis tissue specific expressed epididymal protease inhibitor protein, mainly be expressed in sperm surface.Eppin albumen the regulation and control seminal fluid solidify and aspects such as the motion of liquefaction, sperm and capacitation play an important role.Be the proteic immunological contraception effect of clear and definite Eppin, O ' Rand studies as the immunological contraception model with male rhesus macaque.Found that have in 9 male monkeys that grow up to have occurred the anti-Eppin antibody of high titre in 7 serum and female monkey is become pregnant, the male monkey fertility of the anti-Eppin antibody of high titre is kept in prompting; There are 5 male monkeys to recover fertility after immunity stops, and autoimmune disorder do not occur.O ' Rand has carried out the Characterization of antigenic epitopes of anti-Eppin antibody simultaneously to the serum of the male monkey of immunity, the epitope of results suggest Eppin protein N terminal 20-39 aminoacid sequence (QGPGLTDWLFPRRCPKIRE) for playing a role.I chamber researchist has carried out deep research to the proteic immunological contraception mechanism of Eppin, studies confirm that: Eppin albumen can be in conjunction with semenogelin (semennogenlin), and then keeps the seminal fluid microenvironment.It is as the infringement of proteinase inhibitor participation protective sperm protein enzyme to epididymal in addition, is connected in the spermioteleosis to work.Anti-Eppin antibody interferes with normal Eppin to the interaction of sperm surface and semenogelin and to the protection of epididymal, influence spermioteleosis and ejaculation after fixing, thus the performance antifertility action.As seen, Eppin albumen is a kind of extremely rising, effective, reversible male contraception vaccine.
Because Eppin albumen plays a significant role in the male fertility process, this monoclonal antibody also can be used for diagnosing the reagent composition of male sterility.
Therefore, preparing specific recognition Eppin albumen and high-affinity bonded monoclonal antibody with it, be used to prepare the reagent composition of diagnosis male reproductive function obstacle, and be used for antifertility research, is present biomedical sector problem demanding prompt solution.
Summary of the invention:
Purpose of the present invention is exactly in order to provide a kind of at the proteic monoclonal antibody of Eppin, overcomes the above problems.
A further object of the invention provides the application of this monoclonal antibody in preparing male sterility diagnostic reagent or male contraceptive pill, antifertility postgraduate Tetramune.
It can be IgG that monoclonal antibody of the present invention is interpreted as, and also can be the Fab segment, F (ab ') of this antibody
2Segment, single-chain antibody etc., its antigen binding fragment is disconnected to carry out specificity with Eppin albumen and combines.
The typical hybridoma cell strain of the present invention is deposited in Chinese typical culture collection center (CCTCC), preserving number C200849, the preservation time is on December 9th, 2008, and the cell strain that will secrete the Eppin monoclonal antibody here is expressed as " people eppin protein monoclonal antibody hybridoma cell strain 2F2F7 ".
Can pass through general in fact chemiluminescent polypeptide synthetic technology, obtain one section polypeptide at epitope place in the people Eppin albumen, be used for the antigen of the immunization of animal and screening, evaluation antibody.
The Eppin albumen n end 20-39 aminoacid sequence that technical scheme of the present invention at first obtains the male monkey serum Characterization of antigenic epitopes of immunity result according to O ' Rand, in conjunction with bioinformatic analysis, final people Eppin albumen the preceding paragraph epitope that obtains, i.e. 20-33 (QGPGLTDWLFPRRC) (SEQ ID No.1), totally 14 amino acid.Subsequently by 14 amino acid whose polypeptide of this section of chemosynthesis, purity＞90%.Use this peptide vaccination mouse again, after treating to form immunne response in the animal body, in this animal body, extract splenic lymphocyte, and can merge at the murine myeloma cell of external infinite multiplication, set up hybridoma cell line, from this clone, filter out the cell strain that all can produce specific antibody.These cell strains are carried out repeated screening and mono-clonal cultivation, optimize the monoclonal cell strain that affinity of antibody is the strongest, specificity is the highest, the antibody that is produced is monoclonal antibody of the present invention.
The present invention adopts the ELISA method that the hybridoma of secrete monoclonal antibody is screened, antagonist is tired and is measured, and adopts Western blotting, immunohistochemical method, immunocytochemical method and blocking test that the specificity of prepared monoclonal antibody is identified.
Below be concrete technical measures of the present invention:
The monoclonal antibody of a kind of epididymal protease inhibitor protein Eppin, this monoclonal antibody are by being preserved in Chinese typical culture collection center, and preserving number is the hybridoma excretory monoclonal antibody of C200849.
Described monoclonal antibody, its antigen binding fragment is disconnected to carry out specificity with Eppin albumen and combines.
The immortal cell line of secretion said monoclonal antibody.Described immortal cell line is a hybridoma cell line; This hybridoma is to be preserved in Chinese typical culture collection center, and preserving number is the C200849 hybridoma cell line.
The preparation method of described sperm protein monoclonal antibody may further comprise the steps:
A. obtain people Eppin albumen the preceding paragraph epitope by software analysis;
B. use chemical synthesis to synthesize this section polypeptide, purity＞95%;
C. with this section peptide vaccination mouse, get its splenic lymphocyte and can merge, set up hybridoma cell line at the murine myeloma cell of external infinite multiplication;
D. the hybridoma cell strain of this antibody of screening secretion from above-mentioned hybridoma clone is selected the cell strain the most stable, that antibody activity is the highest, and the antibody that they produced is described monoclonal antibody.
The auxiliary material that a kind of pharmaceutical composition that is used for antifertility, said composition contain the described monoclonal antibody of significant quantity and pharmaceutically allow.
The application of described monoclonal antibody in preparing male sterility diagnostic reagent or male contraceptive pill, antifertility postgraduate Tetramune.
Beneficial effect of the present invention:
The invention provides a kind of monoclonal antibody, this antibody can specificity be incorporated into specific expressed epididymal protease inhibitor protein in testis and the epididymis tissue.
The prepared monoclonal antibody of the present invention has high degree of specificity (example 4-7 all illustrates the specificity of this monoclonal antibody, can discern reorganization and natural Eppin albumen), can be used for male antifertility research, preparation male contraceptive pill.
The prepared monoclonal antibody of the present invention is applicable to and detects the immunity male sterility, to help to judge its concrete cause of disease.
Description of drawings:
Fig. 1. software analysis Eppin epitope figure (square frame is selected sequence)
Fig. 2 .RT-PCR amplification Eppin electrophorogram
Wherein: the 1.PT-PCR product; 2. molecular weight standard
Fig. 3. recombinant plasmid and enzyme are cut product
Wherein: 1. molecular weight standard; The product after (2.Pet28a+)/hEppin plasmid process Nco 1 and Xho 1 enzyme are cut
Fig. 4 .Eppin sequencer map
Fig. 5. affinitive layer purification reorganization Eppin albumen, the specificity (2F2F7 strain) of Western checking monoclonal antibody
Wherein: A figure from left to right is followed successively by: albumen marker, before IPTG inductive total protein, dissolved inclusion body purification, cross the Ni-post pass liquid, washings, Ni column purification after, the SDS-PAGE after the renaturation examines and dyes figure.
B figure is from left to right for to scheme with Western through the Eppin albumen behind the Ni column purification, after the renaturation.
Fig. 6 .Western Blot checking Eppin monoclonal antibody is to the proteic specificity of natural sperm Eppin.Three bands are respectively the proteic tetramer of people's sperm Eppin, pentamer and heptamer among the figure.
Fig. 7. people's testis tissue immunohistochemical methods light microscopic figure: the specificity association reaction takes place in the Eppin of spermoblast and mature sperm normal expression in anti-Eppin monoclonal antibody and the people's testis, and cell is dyed tawny.
Fig. 8. people's epididymis histogenic immunity group light microscopic figure: the specificity association reaction takes place in the Eppin of epithelial cell and mature sperm normal expression in anti-Eppin monoclonal antibody and the people's epididymis, and cell is dyed tawny.
Fig. 9. people's testis tissue immunohistochemical methods light microscopic figure: blocked by free Eppin albumen with the reaction of anti-Eppin monoclonal antibody in people's testis tissue, the tawny cell do not occur.
Figure 10. people's epididymis histogenic immunity group light microscopic figure: blocked by free Eppin albumen with the reaction of anti-Eppin monoclonal antibody in people's epididymis tissue, the tawny cell do not occur.
Figure 11. the spermatid immunofluorescence: the Eppin of normal expression combines with anti-Eppin monoclonal antibody generation specificity, and sperm head and afterbody send green fluorescence.
Figure 12. the Western of Eppin protein expression identifies figure on the azoospermia patient sperm: compare with left side normal people's sperm Eppin protein expression, the eppin expression amount on the azoospermia patient sperm on the right significantly reduces.Three bands are respectively the proteic tetramer of Eppin, pentamer and heptamer among the figure.
Figure 13. the proteic expression of Eppin Western identifies figure in the smart patient's refining of obstructive nothing: compare the proteic existence of no Eppin in the obstructive azoospermia patient refining with the proteic expression of Eppin in the normal people's refining of the left side.Three bands are respectively the proteic dimer of Eppin, tripolymer and the tetramer among the figure.
Preservation information:
The typical hybridoma cell strain of the present invention is deposited in Chinese typical culture collection center (CCTCC, address: Wuhan, Wuhan University), preserving number C200849, the classification name is people Eppin protein monoclonal antibody hybridoma cell strain 2F2F7, and the preservation time is on December 9th, 2008.
Embodiment:
The invention will be further elaborated by the following examples.
Embodiment 1: anti-Eppin MONOCLONAL ANTIBODIES SPECIFIC FOR
1, software analysis is selected one section proteic epitope of people Eppin (Fig. 1)
The polypeptide of chemosynthesis 14aa (SEQ ID No.1)
2, with Eppin protein immunization inoculation BALB/c mouse
During initial immunity, chemosynthesis Eppin polypeptide (the SEQ ID No.1) 0.5ml (concentration 100 μ g/ml) that gets purifying mixes with equal-volume Fu Shi Freund's complete adjuvant, after in mortar, fully being ground to complete emulsification (being the water-in-oil state), be expelled to (0.1ml/ only) in the mouse peritoneal.Carry out booster immunization the 2nd time after 2 weeks at interval, use the fully emulsified antigen of freund 's incomplete adjuvant instead, injected dose and position are the same.Carry out booster immunization the 3rd time after 2 weeks at interval, directly use antigenic solution, injected dose and position are the same.Measure the antibody titer in the immune serum at interval after 10 days.In cytogamy preceding 3 days, to get a mouse that antibody positive is the strongest and carry out the 4th booster immunization, injected dose and position are the same.
3, measure antibody titer
The 3rd booster immunization be after 10 days, gathers antigen immune and non-immune mouse blood, as sample to be measured and the negative control of ELISA, and separation of serum, 4 ℃ of preservations are to be measured.Is 1 μ g/ml with antigen coated liquid (0.05M carbonate buffer solution, pH 9.6) dilution Eppin polypeptide to final concentration, and bag is by polystyrene board micropore (100 μ l/ hole), and 4 ℃ of bags are spent the night.Next day, PBS-T washes plate 3 times, adds the sealing of 50g/L skim-milk by 200 μ l/ holes, hatches 2 hours for 37 ℃, and PBS-T washes plate 3 times.PBS dilution test serum and negative control sera (dilution in 1: 1000) are blank with PBS, add micropore by 100 μ l/ holes, hatch 1 hour h for 37 ℃.PBS-T washes plate 3 times, adds HRP-sheep anti-mouse igg (dilution in 1: 3000) by 100 μ l/ holes, hatches 1 hour for 37 ℃.PBS-T washes plate 3 times, and every hole adds substrate developer 100ul (article No. 6526, day root biology), hatches 10 minutes for 37 ℃.Add 1mol/L sulfuric acid 50 μ l termination reactions, at wavelength 450nm with the enzyme-linked immunosorbent assay instrument colorimetric and write down the result.
4, mouse boosting cell preparation
Behind the 4th booster immunization 3 days, extract the bloodletting of immune mouse eyes, leave and take the positive control of serum as ELISA, 4 ℃ of preservations are standby.Draw neck to put to death mouse, 75% alcohol-pickled 5 minutes.Cut an osculum at mouse web portion, tear skin, cut off peritonaeum, the tweezer mouse spleen is cut mouse spleen, removes fat and reticular tissue, with the incomplete substratum washing of DMEM mouse spleen.Mouse spleen is cut into 4, at stainless steel mesh (100 orders/cm
2) on, fully grind mouse spleen with the glass syringe nook closing member, squeeze out splenocyte gently.With lapping liquid process stainless steel mesh (200 orders/cm
2) after the filtration, be transferred to centrifuge tube, centrifugal 5 minutes of 1000rpm.Abandon supernatant, the incomplete substratum washed cell of DMEM once, with the abundant re-suspended cell of the incomplete substratum of DMEM, carrying out cell counting and calculating cell concn is 2 * 10 again
10Individual/L, place incubator (37 ℃, 5%CO
2) in stand-by.
5, cytogamy
Abundant mixing immune spleen cell (10
8Individual) and murine myeloma cell SP2/0 (10
7Individual) suspension, centrifugal 5 minutes of 1000rpm.Abandon supernatant, remove residual liquid as far as possible, finger flicks centrifuge tube makes cell precipitation comparatively loosening.Centrifuge tube is placed 37 ℃ of water-baths, add 1ml 50%PEG 4000 in 30 seconds, edged appropriateness in limit stirs, and adds the back and continues to stir 15 seconds, leaves standstill 90 seconds.Add the incomplete substratum of DMEM, liquid feeding speed from slow to fast, concrete scheme: add 1ml, 2ml, 3ml, 4ml, 5ml and 10ml respectively in per 2 minutes continuously, stop fusion.Centrifugal 5 minutes of 800rpm abandons supernatant,, washed cell resuspended gently with the incomplete substratum of DMEM 1 time.Add 50ml DMEM perfect medium, re-suspended cell joins 96 well culture plates by 100 μ l/ holes gently, places cell incubator (37 ℃, 5%CO
2) the middle cultivation.
6, detection of antibodies
With synthetic Eppin polypeptide as envelope antigen, getting in the micropore to be measured culture supernatant 0.1ml is used for measuring,, as negative control, anti-as two with immune mouse periphery serum with HRP-sheep anti mouse (dilution in 1: 3000) with the culture supernatant of murine myeloma cell SP2/0 as positive control.
7, hybridoma cloning is cultivated
Identify the male subclone after getting 10 fusions, add 10ml HT substratum and abundant mixing, join in 96 well culture plates by the 0.1ml/ hole.Detect antibody after 10 days, filter out 6 the strongest monoclonal cell strains of antibody positive, be respectively 1E5G5,1G5G2,2F2F7,3H2B11,5F7D3 and 6B10D5.Then, use DMEM perfect medium (containing 10% foetal calf serum) instead, repeatedly carry out subclone and cultivate, up to all contain cell clone be antibody positive till.So obtain 1E5G5,1G5G2,2F2F7,3H2B11,5F7D3 and 6B10D5 cell strain.
8, the foundation of hybridoma cell line
With 1E5G5,1G5G2,2F2F7,3H2B11,5F7D3 and the 6B10D5 monoclonal cell enlarged culturing of antibody positive, set up hybridoma cell line, carry out an antibody test week about.When hybridoma cell line reached certain quantity, freeze-stored cell was left and taken supernatant and is used to measure tiring of its monoclonal antibody.Hybridoma cell strain in-vitro cultivation liquid nitrogen cryopreservation after 1 month still can stably excreting Eppin monoclonal antibody after the recovery.
Embodiment 2: the titration of anti-Eppin monoclonal antibody
Adopt the ELISA method.By polystyrene board micropore (100 μ l/ hole), 4 ℃ are spent the night with Eppin polypeptide (1mg/L) bag.Next day, PBS-T washes plate 3 times, adds 37 ℃ of 50g/L skim-milk sealings 2 hours by 200 μ l/ holes, and PBS-T washes plate 3 times.Add sample to be tested (culture supernatant of hybridoma cell strain, or the mouse ascites monoclonal antibody of serial dilution), hatched 1 hour for 37 ℃.PBS-T washes plate 3 times, adds HRP-sheep anti-mouse igg (dilution in 1: 3000), hatches 1 hour for 37 ℃.PBS-T washes plate 3 times, and every hole adds TMB-H
2O
2Solution was hatched 10 minutes for 37 ℃.Add 2 1mol/L H
2SO
4Termination reaction is in wavelength 450nm enzyme-linked immunosorbent assay instrument colorimetric.The result: the culture supernatant antibody titer of hybridoma cell strain 1E5G5,1G5G2,2F2F7,3H2B11,5F7D3 and 6B10D5 was respectively 1: 800,1: 1600,1: 1600,1: 1600,1: 800 and 1: 800; The antibody titer of ascites is 1: 2.7 * 10
4Get the highest 2F2F7 cell strain of titre and carry out preservation.
The highest 2F2F7 cell strain carries out preservation.
The table 1. six strain monoclonal cell strain ELISA detection of tiring
Embodiment 3:Eppin clone and prokaryotic expression
In order to verify the specificity of monoclonal antibody, at first to carry out the clone and the prokaryotic expression of Eppin gene.We utilize the RT-PCR method, and clone Eppin gene from the normal fertile men testis tissue after PCR, double digestion evaluation, carries out dna sequencing (SEQ ID No.2).Then, make up recombinant expression vector pET-28a (+)/Eppin, after PCR and double digestion evaluation, transformed into escherichia coli BL21 (DE3), IPTG abduction delivering and SDS-PAGE detect reorganization Sp17, nickel ion affinity chromatograph purification of recombinant proteins.
1.1 experimental specimen: people's testis tissue
1.2 the double-stranded cDNA fragment of preparation Eppin
1.2.1 design of primers
With the design of DNAMAN analysis software, the PCR primer sequence is as follows:
Upstream primer (primer 1):
5 ' GTTC
CCATGGTCCAGGGACCTGGTCTGA-3 ' (SEQ ID No.3) (line place is a Nco I restriction enzyme site)
Downstream primer (primer 2):
5 '-GTTC
CTCGAGTCACACCACCACGGGAAAGCGTTTATTCTTGCAG ' (SEQ IDNo.4) (line place is an Xho I restriction enzyme site).
1.2.2 the extraction of the total RNA of people's testis tissue
Cut the 50mg tissue block and put into mortar, add an amount of liquid nitrogen immediately, be fully grinding under the frozen state at tissue, make it to become Powdered, and then be transferred to and contain 1ml Trizol (article No.: 15596-018, Ying Jun company) in the centrifuge tube of RNA extracting solution, put upside down mixing, room temperature was placed 15 minutes.Add the 0.2ml chloroform, abundant mixing, centrifugal 15 minutes of 4 ℃ of 12000rpm.Shift the colourless water in upper strata to new centrifuge tube, add the 0.5ml Virahol, abundant mixing, room temperature was placed 10 minutes.Centrifugal 10 minutes of 4 ℃ of 12000rpm abandon supernatant, add 1ml 75% ethanol, vortex vibration, centrifugal 5 minutes of 4 ℃ of 7500rpm.Abandon supernatant, with 50 μ l DEPC (diethylpyrocarbonate) treating water dissolving RNA precipitation, the 8g/L agarose gel electrophoresis detects the effect that RNA extracts, and sample RNA solution is frozen stand-by at-70 ℃ of refrigerators.
1.2.3 reverse transcription reaction
(20 μ l) is composed as follows for the total reaction system:
10 * RT-damping fluid, 2 μ l
25mmol/L MgCl2 4μl
10mmol/L dNTP 2μl
RNase inhibitor 0.5 μ l
Total rna solution 3 μ l
AMV reversed transcriptive enzyme 0.75 μ l (15U)
Nuclease free ultrapure water (nuclease free water) is supplied reaction volume to 20.0 μ l
Carry out reverse transcription reaction by the test kit specification sheets: hatched RNA solution 10 minutes for 70 ℃, ice bath is 5 minutes immediately; Add other reacted constituents, the vibration mixing, instantaneous centrifugal, incubated at room 10 minutes; 42 ℃ of reverse transcription reactions 30 minutes, 95 ℃ of heating 5 minutes, deactivation AMV reversed transcriptive enzyme ,-70 ℃ of refrigerators are preserved standby.
1.2.4PCR amplification
(25 μ l) is composed as follows for the total reaction system:
10 * PCR-damping fluid, 2.5 μ l
25mmol/L MgCl
2 1.5μl
10mmol/L dNTP 0.5μl
5 μ mol/L Eppin upstream primers, 1.0 μ l
5 μ mol/L Eppin downstream primers, 1.0 μ l
Total cDNA single-stranded template 1.0 μ l
Sterile purified water 16.5 μ l
Taq enzyme (0.5U/ μ l) 1.0 μ l
The PCR parameter is: 94 ℃ of pre-sex change 5 minutes, 95 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 30 seconds, 35 circulations, 72 ℃ were extended 7 minutes.17g/L agarose gel electrophoresis check and analysis PCR product, result such as Fig. 2.
1.2.5PCR the purifying of product and quantitative
Utilize commercially available purification kit, to specifications the PCR product is carried out purifying, with the concentration of the quantitative dna solution of ultraviolet spectrophotometer.
1.3 the structure of recombinant cloning vector Pet28a (+)/hEppin and evaluation
The Eppin dna fragmentation after 1.3.1 the rubber tapping purifying enzyme is cut
1.3.2 test kit extracts plasmid pET-28a (+)
1.3.3Nco I, Xho I double digestion plasmid pET-28a (+)
(100 μ l) is composed as follows for the total reaction system:
Sterile purified water 50.0 μ l
10 * K damping fluid, 10.0 μ l
Plasmid pET-28a (+) is 30.0 μ l (0.16mg/ml)
Nco I(10U/μl) 5.0μl
Xho I(10U/μl) 5.0μl
Vibration mixing reaction system is hatched 1h for 37 ℃, adds 10 μ l, 10 * sample-loading buffer and stops endonuclease reaction, and 10g/L agarose gel electrophoresis enzyme analysis is cut product.
1.3.5 rubber tapping purifying linearization plasmid pET-28a (+).
1.3.6 ligation (reaction system is composed as follows):
10 * ligase enzyme damping fluid, 1.0 μ l
Eppin DNA 2.0 μ l (43 μ g/ml) behind the purifying
Linearization plasmid pET-28a (+) 1.0 μ l (177 μ g/ml) behind the purifying
T4DNA ligase enzyme 1.0 μ l
Nuclease free water 5.0 μ l
Vibration mixing reaction system, 4 ℃ of ligations are spent the night.After reaction finished, 65 ℃ were incubated 10 minutes, deactivation T4DNA ligase enzyme.
1.3.7 recombinant plasmid transformed intestinal bacteria TOP10 competent cell
10 μ l ligation liquid are joined in the 100 μ l TOP10 competent cells, the tube wall several of flapping gently, fully the mixing content placed ice bath 30 minutes.Centrifuge tube is placed 42 ℃ of water-baths 90 seconds, go to ice bath immediately, left standstill 2 minutes.Slowly add LB substratum 0.5ml, 37 ℃ of 100rpm joltings were cultivated 45 minutes, bacterium liquid were coated on the LB flat board that contains kantlex (0.1g/L) 37 ℃ of overnight incubation.
1.3.8 screening and amplification cultivation positive colony
5 single bacterium colonies of white of picking are inoculated in 5 culture test tubes (the LB substratum that contains the 0.1g/L kantlex) 37 ℃ of 250rpm jolting overnight incubation respectively at random.
1.3.9DNA recombinant plasmid is identified in order-checking
To cut the recombinant plasmid (Fig. 3) of evaluation through PCR and enzyme, and send Nanjing Jin Site company to carry out dna sequencing, the sequence in sequencing result and Genbank and other bibliographical informations will be compared result such as Fig. 4.
1.4 make up recombinant expression vector pET-28a (+)/Eppin
1.4.1 recombinant plasmid transformed intestinal bacteria competence BL21 (DE)
Picking is transformed into BL21 (DE) and experiences polypeptide cell through the positive colony of PCR and double digestion evaluation, bacterium liquid is coated on the LB flat board that contains kantlex (0.1g/L), and the back-off plate is placed on overnight incubation in 37 ℃ of incubators.
1.5 the abduction delivering of recombinant plasmid pET-28a (+)/Eppin
Select a mono-clonal next day and be inoculated in the LB substratum that contains kantlex (0.1g/L), 37 ℃ of 250rpm overnight incubation.Next day, in the LB substratum that contains kantlex (0.1g/L), 37 ℃ of 300rpm thermal agitations were cultivated with 1: 50 dilution transferred species.When bacterium liquid OD600 ≈ 0.6, take out 1ml bacterium liquid and do not add inductor as negative control, in bacterium liquid, add inductor isopropylthio-(IPTG, final concentration are 1mmol/L), 37 ℃ inducing culture 3h hour.Simultaneously, with containing unloaded plasmid pET-28a's (+) and not containing the BL21 bacterium of plasmid as blank.Get 1ml and induce bacterium liquid, centrifugal 5 minutes of 7500rpm, abandon supernatant, the abundant resuspended bacterial precipitation of 50 μ l, 1 * sds gel sample-loading buffer, water proof boiled 5 minutes, and centrifugal 5 minutes of 12000rpm gets 10 μ l supernatants and carries out SDS-PAGE (concentrated glue 50g/L, separation gel 150g/L) detects and analyzes expression product, result such as Fig. 4.
1.6 nickel ion affinity chromatograph purification of Recombinant Eppin
Buffer A (pH 8.0 for 50mmol/L Tris-HCl, 300mmol/L NaCl) is soaked balance 2gNi-NTAHis-Bind resin 1h, abandons supernatant after the resin natural sedimentation, centrifugal supernatant of mixing cracking and balance resin, 4 ℃ of 1h that vibrate gently.Shift suspension to chromatography column, allow the liquid in the post bed drain off naturally, and the buffer B of 10 column volumes (50mmol/LTris-HCl, 300mmol/LNaCl, the 20mmol/L imidazoles, pH 8.0) the thorough washing resin.At last, with 4ml imidazoles elution buffer (50mmol/LTris-HCl, 300mmol/LNaCl, 250mmol/L imidazoles, pH8.0) wash-out target protein.In 4 ℃ of refrigerators, PBS dialysis elutriant 48h changes liquid therebetween 8 times.The centrifugal cleer and peaceful purified product of cracking carries out SDS-PAGE (concentrating glue 50g/L, separation gel 150g/L) simultaneously, and the comparative analysis purification effect is seen Fig. 5 .A.Xylene Brilliant Cyanine G G-250 method is measured the concentration of recombinant protein.
The test of embodiment 4:Western Blot trace
With express recombinant Eppin e. coli total protein trace to nitrocellulose filter.The PBS-T sealing that contains the 50g/L skim-milk is spent the night, PBS-T rinsing 3 times, the hybridoma cell strain excretory monoclonal antibody that adds preserving number respectively and be C200849 was hatched PBS-T rinsing 3 times 1 hour, add HRP-sheep anti-mouse igg (1: 2500) and hatched PBS-T rinsing 3 times 1 hour.At last, ECL chemoluminescence method exposure.Only band occurs on recombinant protein band position, other tropina bands are not seen (seeing Fig. 5 .B).
Embodiment 5:Western Blot identifies that the specific reaction of this monoclonal antibody and natural Eppin will extract people's sperm total protein, and with Western blot to nitrocellulose filter.The PBS-T sealing that contains the 50g/L skim-milk is spent the night, PBS-T rinsing 3 times, the hybridoma cell strain excretory monoclonal antibody that adds preserving number respectively and be C200849 was hatched PBS-T rinsing 3 times 1 hour, add HRP-sheep anti-mouse igg (1: 2500) and hatched PBS-T rinsing 3 times 1 hour.At last, ECL chemoluminescence method exposure.Results suggest only band occurs on Eppin protein band position, other sperm protein bands are not seen (see figure 6).
Embodiment 6: the specific reaction of this monoclonal antibody and natural Eppin is identified in the immunohistochemical methods test
A. people's testis paraffin section preparation, 0.03% methyl alcohol-H
2O
2Hatched 30 minutes, boiled in the microwave oven 1 minute.10% sheep blood serum sealing 30 minutes, PBS-T washes 3 times, one anti-for preserving number of the present invention be the hybridoma cell strain excretory monoclonal antibody of C200849, the not negative contrast of immune serum, 4 ℃ of overnight incubation, rinsing 3 times adds HRP-sheep anti-mouse igg (1: 3000), hatched 1 hour for 37 ℃, rinsing 3 times, DAB substrate solution colour developing 10 minutes, Hematorylin was redyed 1 minute, dehydration, transparent, mounting is observed under opticmicroscope and is taken pictures, observe the reactivity of various types of cells in monoclonal antibody and the tissue, Eppin albumen mainly is present in spermatids at different levels from circular sperm to mature sperm and the sustentacular cell of testis (Fig. 7).
B. immunohistochemical analysis is carried out in people's epididymis paraffin section preparation equally, the reactivity of various types of cells in observing monoclonal antibody and organizing, and Eppin albumen mainly is expressed in (Fig. 8) in the epididymal cell.
C. blocking test is in order further to identify the specificity of monoclonal antibody of the present invention, carried out blocking test, the Eppin albumen (100mg/L) that promptly in the monoclonal anti liquid solution, adds purifying, in 37 ℃ hatch 30min after, carry out immunohistochemical staining with this monoclonal anti liquid solution again, the result shows that the positive reaction of monoclonal antibody of the present invention and testis epididymis tissue all can be blocked (Fig. 9, Figure 10), thereby the albumen in the proof monoclonal antibody of the present invention institute bonded tissue is Eppin albumen.
Embodiment 7: immunofluorescent test identify the specific reaction of this monoclonal antibody and natural Eppin leave and take the row conventional semen efamination people's semen sample, centrifugal 5 minutes of liquefaction back 1000rpm, PBS-T washes 3 times, sperm concentration is adjusted to 5 * 106/ml, smear, seasoning overdoes on the spirit lamp and fixes for 3 times.10% sheep blood serum sealing 30 minutes, wash 3 times, adding preserving number is the hybridoma cell strain excretory monoclonal antibody of C200849, the not negative contrast of immune serum, hatched 2 hours for 37 ℃, rinsing 3 times adds FITC-sheep anti-mouse igg (1: 100), hatches 1 hour for 37 ℃, rinsing 3 times, (Vector Laboratories, USA) mounting are observed and take pictures (Figure 11) under high power lens to add the anti-short agent of going out of fluorescence.
Embodiment 8: detect azoospermia for example
Collect 10 routine normal male semen samples and 10 routine azoospermia patient samples, through the percoll separated sperm, with phosphate buffered saline buffer (137mM sodium-chlor, 2.7mM Repone K, 1.5mM SODIUM PHOSPHATE, MONOBASIC, pH 7.4) the washing secondary, be resuspended in the protein lysate (7M urea, 2M thiocarbamide) ultrasonic and centrifugal (15000g, 5 minutes) crack protein, the Bradford method is measured concentration.With Western blot to nitrocellulose filter.The PBS-T sealing that contains the 50g/L skim-milk is spent the night, PBS-T rinsing 3 times, the hybridoma cell strain excretory monoclonal antibody that adds preserving number respectively and be C200849 was hatched PBS-T rinsing 3 times 1 hour, add HRP-sheep anti-mouse igg (1: 2500) and hatched PBS-T rinsing 3 times 1 hour.At last, ECL chemoluminescence method exposure.Results suggest, the proteic expression amount of Eppin is than normal people low (seeing Figure 12) in the azoospermia patient sperm.
Embodiment 9: detect complete Treatment of azoospermia for example
Collect fully Treatment of azoospermia patient semen samples of 5 routine normal male semen samples and 5 examples, 3000g got refining after centrifugal, and added sample damping fluid (62.5mmol/L Tris-HCl in 10 minutes; 10% glycerine; 0.05% tetrabromophenol sulfonphthalein) carries out Western after boiling.The PBS-T sealing that contains the 50g/L skim-milk is spent the night, PBS-T rinsing 3 times, the hybridoma cell strain excretory monoclonal antibody that adds preserving number respectively and be C200849 was hatched PBS-T rinsing 3 times 1 hour, add HRP-sheep anti-mouse igg (dilution in 1: 2500) and hatched PBS-T rinsing 3 times 1 hour.At last, ECL chemoluminescence method exposure.Results suggest, the proteic expression of no Eppin (seeing Figure 13) in the complete obstructive azoospermia patient refining.
Reference:
1.Naz RK，Gupta SK，Gupta JC，Vyas HK，Talwar AG.Recent advances in contraceptivevaccine development：a mini-review.Hum Reprod，2005，20(12)：3271-83.
2.Naz RK.Vaccine for contraception targeting sperm.Immunol Rev，1999，171：193-202.
3.Herr JC，Flickinger CJ，Homyk M，Klotz K，John E.Biochemical and morphologicalcharacterization of intra-acrosomal antigen SP-10 from human sperm，Biol Repord，1990，42(1)：181-93.
4.Lea IA，van Lierop MJ，Widgren EE，Grootenhuis A，Wen Y，van Duin M，O′Rand MG.Achimeric sperm peptide induces antibodies and strain-specific reversible infertility in mice.Biol Reprod，1998，59(3)：527-36.
5.Santhanam R，Naz RK.Novel human testis-specific cDNA：molecular cloning，expression and immunobiological effects of the recombinant protein.Mol Reprod Dev，2001，60(1)：1-12.
6.Naz RK，Zhu X，Kadam AL.Cloning and sequencing of cDNA encoding for a novelhuman testis-specific contraceptive vaccinogen：role in immunocontraception.Mol ReprodDev，2001，60(1)：116-27.
7.Miki K，Willis WD，Brown PR，Goulding EH，Fulcher KD，Eddy EM.Targeteddisruption of the Akap4 gene causes defects in sperm flagellum and motility.Dev Biol，2002，248(2)：331-42.
8.Shankar S，Mohapatra B，Verma S，Selvi R，Jagadish N，Suri A.Isolation andcharacterization of a haploid germ cell specific sperm associated antigen 9(SPAG9)from thebaboon.Mol Reprod Dev，2004，69(2)：186-93.
9.Richardson RT，Sivashanmugam P，Hall SH，Hamil KG，Moore PA，Ruben SM，FrenchFS，O′Rand M.Cloning and sequencing of human Eppin：a novel family of protease inhibitorsexpressed in the epididymis and testis.Gene，2001，270(1-2)：93-102.
10.O′rand MG，Widgren EE，Sivashanmugam P，Richardson RT，Hall SH，French FS，VandeVoort CA，Ramachandra SG，Ramesh V，Jagannadha Rao A.Reversibleimmunocontraception in male monkeys immunized with eppin.Science，2004，306(5699)：1189-90.
11.Wang Z，Widgren EE，Sivashanmugam P，O′Rand MG，Richardson RT.Association of eppin with semenogelin on human spermatozoa.Biol Reprod，2005，72(5)：1064-70.
12.Wang Z，Widgren EE，Richardson RT，O’Rand MG.Characterization of an eppin proteincomplex from human semen and spermatozoa.Biol Reprod 2007；77：476-484.
13.O’Rand MG，Widgren EE，Stan Beyler，Richardson RT.Inhibition of Human SpermMotility by Contraceptive Anti-Eppin Antibodies from Infertile Male Monkeys：Effect onCyclic Adenosine Monophosphate.Biol Reprod.2008；Oct 22，[Epub ahead of print].
Sequence table
＜110〉Nanjing Medical University
＜120〉a kind of epididymal protease inhibitor protein monoclonal antibody and uses thereof
<160>4
<210>1
<211>14
<212>PRT
＜213〉artificial sequence
<220>
＜223〉Eppin proteantigen epi-position
<400>1
Gln Gly Pro Gly Leu Thr Asp Trp Leu Phe Pro Arg Arg Cys
1 5 10
<210>2
<211>348
<212>DNA
＜213〉native sequences derives from people (Human) testis tissue
<220>
＜223〉Eppin gene
<400>2
gtccagggac ctggtctgac tgattggtta tttcccagga gatgtcccaa aaatcagaga 60
agaatgtgaa ttccaagaaa ggatgtgtgt acaaaggaca gacaatgcca ggacaacaag 120
aagtgttgtg tcttcagctg cggaaaaaaa tgtttagatc tcaaacaaga tgtatgcgaa 180
atgccaaaag aaactggccc ctgcctggct tattttcttc attggtggta tgacaagaaa 240
aataatactt gctccatgtt tgtctatggt ggctgccagg gaaacaataa caacttccaa 300
tccaaagcca actgcctgaa cacctgcaag aataaacgct ttccctga 348
<210>3
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉upstream sequence
<400>3
gttcccatgg tccagggacc tggtctga 28
<210>4
<211>44
<212>DNA
＜213〉artificial sequence
<220>
＜223〉downstream sequence
<400>4
gttcctcgag tcacaccacc acgggaaagc gtttattctt gcag 44
Claims (4)
1. the monoclonal antibody of an epididymal protease inhibitor protein Eppin, this monoclonal antibody are by being preserved in Chinese typical culture collection center, and preserving number is the hybridoma excretory monoclonal antibody of CCTCC NO:C200849.
2. secrete the clone of the described monoclonal antibody of claim 1, it is characterized in that this cell is that to be preserved in Chinese typical culture collection center preserving number be CCTCC NO:C200849, the classification name is the hybridoma cell line of people Eppin protein monoclonal antibody hybridoma cell strain 2F2F7.
3. the auxiliary material that pharmaceutical composition that is used for antifertility, said composition contain the described monoclonal antibody of claim 1 of significant quantity and pharmaceutically allow.
4. the application of the described monoclonal antibody of claim 1 in preparing male sterility diagnostic reagent or male contraceptive pill, antifertility postgraduate Tetramune.
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| CN102262159B (en) | 2010-05-27 | 2014-03-26 | 李建远 | Method for detecting 305 kinds of semen positioning protein of human testicle and epididymis expression related to procreation |
| CN103819563B (en) * | 2013-12-31 | 2016-03-23 | 中国人民解放军第三军医大学第一附属医院 | Fusion rotein MBP-Eppin, albumen Eppin and preparation method thereof |
| CN113045663B (en) * | 2021-04-18 | 2022-04-01 | 深圳市国创纳米抗体技术有限公司 | anti-HE 4 nano antibody 1A8 and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101362795A (en) * | 2008-09-11 | 2009-02-11 | 中国人民解放军第三军医大学 | B cell epitope of epididymal protease inhibitor and antigen peptides containing thereof |
| CN101362796A (en) * | 2008-09-11 | 2009-02-11 | 中国人民解放军第三军医大学 | B cell epitope of epididymal protease inhibitor and multiple antigenic peptide containing thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101362795A (en) * | 2008-09-11 | 2009-02-11 | 中国人民解放军第三军医大学 | B cell epitope of epididymal protease inhibitor and antigen peptides containing thereof |
| CN101362796A (en) * | 2008-09-11 | 2009-02-11 | 中国人民解放军第三军医大学 | B cell epitope of epididymal protease inhibitor and multiple antigenic peptide containing thereof |
Non-Patent Citations (1)
| Title |
|---|
| 王增军等.精子表面Eppin蛋白分子结合网络通路的研究.《第十五届全国泌尿外科学术会议论文集》.2008,1280. * |
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