CN101337991A - Preparation and applications of rabbit anti-human IRGMa full-length protein polyclone antibody - Google Patents

Abstract

The invention discloses the preparation and the application of rabbit anti-human IRGMa full-length protein polyclonal antibody, which belong to the field of disease intervention by traditional medicine monomer. A full-length IRGMa protein is selected for preparing the antibody and includes a function region. The preparation method is also provided on the basis of optimal selection, and a primer is designed according to the primer design principle to constitute His and GST fusion protein containing IRGMa full-length gene to prepare the antibody. The antibody prepared by the method has high specificity and sensitivity, which is beneficial to researching expression spectrum analysis and function of IRGM gene and to researching the specific effect of IRGM in the process of pathogenesis of MS, thereby discovering the pathogenesis of MS and finding effective measures for gene immunotherapy. Accordingly, ginsenoside Rg1 has a certain effect of intervening multiple sclerosis, and can remarkably reduce the incidence rate of the multiple sclerosis on an animal model, i.e. EAE mouse.

Description

The preparation of rabbit anti-human IRGMa full-length protein polyclone antibody and application

Technical field

The present invention relates to proteic preparation of IRGMa and application.A kind of preparation of rabbit anti-human IRGMa full-length protein polyclone antibody and application specifically.

Background technology

MS is the cell-mediated autoimmune disease of T, discovers the clinical phenotypes severe patient, and IFN-γ express cell significantly increases, and prompting IFN-γ can make the MS pathology increase the weight of.Mouse Irgm1 (LRG-47) is by IFN-γ inductive albumen, finds that after deliberation Irgm1 is relevant with the pathogenesis of the animal model EAE of MS.

Irgm1 is downstream effect of IFN-γ, and lymphocytic survival is had the downstream regulating effect, plays an important role in immune defense.Human IRGM gene was cloned in 2006, was positioned 5q33.1, the G district of encode an aminoterminal and carboxyl terminal brachymemma.It and mouse Irgm homology are necessary in the cytophagous autophagy process of the mankind, can induce the maturation of BCG phagosome.

The IRGM gene has 5 transcripts because of 3 ' end cut mode difference, is respectively IRGMa, IRGMb, IRGMc, IRGMd and IRGMe.The amino acid number of 5 transcripts is respectively 182,211,192,199,192.182 amino acid of IRGMa are that 5 transcripts are total, and therefore, IRGMa is this proteic major function district.The IRGM antibody of foreign scholar's preparation at present is that the C end is contained the antibody that more than 10 amino acid adds a halfcystine (CQIRENVLENLQKER), method with immunofluorescence or Western blot, this antibody can not detect the cell endogenous signal, because it does not comprise the major function district, limited the scope that antibody uses.And there is not commercial IRGM antibody at present.Therefore, use the IRGMa full-length proteins and prepare the anti-people's polyclonal antibody of rabbit, and be applied to study IRGM expression of gene spectrum analysis and in the MS pathogenic process, have practical function.

Summary of the invention

The objective of the invention is in the IRGM gene, to optimize one section encoding sequence, and prepare IRGMa full-length proteins polyclonal antibody and application thereof.

The technical solution adopted for the present invention to solve the technical problems is: a kind of preparation of rabbit anti-human IRGMa full-length protein polyclone antibody is selected for use, it is characterized in that: at the proteic functional zone of IRGM, select for use IRGMa to prepare antibody.The albumen of selecting for use which section albumen to prepare antibody and how obtaining correct sequence is key of the present invention.IRGM has 5 transcripts, 5 transcript amino acid numbers are respectively IRGMa (182), IRGMb (211), IRGMc (192), IRGMd (199), IRGMe (192), 182 amino acid of IRGMa are that 5 transcripts are total, therefore, 1-182 amino acid should be this proteic major function district.And IRGMa is an exon transcription product, and IRGMb, IRGMc, IRGMd, IRGMe are a plurality of exon transcription products, is both convenient so prepare antibody with IRGMa, has comprised the major function district again, is feasible therefore.

The only synthetic amino acid whose polypeptide of dozens of prepares the anti-people IRGMa of rabbit polyclonal antibody, may cause antibodies specific not high enough; If but at the proteic functional zone of IRGMa, directly polypeptide synthesizes so big fragment and the correct albumen of encoding is very expensive by synthesizing, and is unpractical.So the present invention selects GST and the proteic method of His gene fusion to prepare this target protein fragment.Express GST and His gene fusion albumen, must make up the GST and the His gene fusion carrier that contain IRGMa full-length gene (546bp); According to design of primers principle design primer.

The extracting method of rabbit anti-human IRGMa full-length protein polyclone antibody of the present invention:

1, from people's peripheric venous blood extracting genomic dna.

2, the selection of design of primers and gst gene fusion vector: selected pCMV-myc, pGEX4T-2 and Pet28a (+)-His carrier, and select for use restriction enzyme EcoRI and XhoI as tie point; According to design of primers principle design primer, accomplish that same primer do not have palindrome hairpin structure, 5 ' end is not complementary with 4 bases of 3 ' end, and forward and reverse primer sequence is not complementary, Tm value close (2A+2T+4G+4C), and on Genebank, can not find homologous series; Designed 2 pairs of primers altogether, sequence is as follows:

First pair: be used to reprint pCMV-myc-IRGM and pGEX4T-2-IRGM carrier

1F:5 '-AgCgAATTCCTATggAAgCCATgAATgTTg-3 ' (containing EcoR I point of contact)

1R:5 '-gCgCTCgAgTTAgTATTCACATACCCgCTC-3 ' (containing the XhoI point of contact)

Second pair: be used to reprint pET-28a (+)-IRGM carrier

2F:5 '-AgCgAATTCATggAAgCCATgAATgTTg-3 ' (containing EcoR I point of contact)

2R:5 '-gCgCTCgAgTTAgTATTCACATACCCgCTC-3 ' (containing the XhoI point of contact)

3, prepare target gene fragment and be connected to myc, on GST and the His gene fusion carrier: with normal people's genomic dna is template, adopts above-mentioned 3 pairs of primers and general T aq enzymatic amplification IRGMa complete encoding sequence respectively; Use TA clone test kit the PCR product is connected respectively on the PCR2.1 carrier, transform and the positive TA clone of picking, shake bacterium (e.colistraindh5, Shanghai Zhuo Kang bio tech ltd), and adopt plasmid extraction purification kit extracting and purifying plasmid; EcoRI and XhoI double digestion confirm that positive TA clone and employing T carrier primer and the order-checking of ABI PRISMR3700DNA sequenator identify the exactness that is connected the target gene fragment sequence on the PCR2.1 carrier; Correct TA clones employing and uses EcoRI and XhoI double digestion, reclaims the myc that target gene fragment also is connected to the recovery of process EcoRI and XhoI double digestion, GST and His gene fusion carrier pCMV-myc, pGEX4T-2, the last and conversion of pET-28a (+)-His; The extracting plasmid, sequence verification.

4, containing the GST of target gene fragment and the expression of His gene fusion carrier identifies: 1 of picking transforms good GST and His gene fusion carrier cloning respectively, shake bacterium (intestinal bacteria origamiDE3 with 5ml 2YT substratum, Novagen company product), centrifugal collection bacterium and preparation GST and His gene fusion albumen in a small amount, adopt PAGE-SDS gel electrophoresis protein isolate fragment, Coomassie brilliant blue dyeing shows GST and His gene fusion protein fragments size.

5, preparation and purifying gst gene fusion rotein and collection target protein: prepare the gst gene fusion rotein in a large number, crossing column purification reclaims also quantitatively, 23 ℃ of enzymes of application Thrombin are cut and are spent the night, or the gst gene fusion rotein is hung the GST pearl earlier, cut with 23 ℃ of enzymes of Thrombin and to spend the night, second day crosses post and collects IRGMa total length target protein.

6, prepare His gene fusion albumen in a large number: because of IRGMa-His gene fusion albumen is insoluble albumen, so with the bacterium of centrifugal collection with sample Buffer cracking on 1 * albumen after employing PAGE-SDS gel electrophoresis protein isolate fragment, Coomassie brilliant blue dyeing, show His gene fusion albumen purpose fragment, it is frozen standby in-80 ℃ of refrigerators to tap rubber.

7, preparation and purifying contain the rabbit anti-human IRGMa full-length protein polyclone antibody of GST-tag and His-tag: subcutaneous multi-point injection immunizing rabbit after the IRGMa albumen that will reclaim after will being cut by the Thrombin enzyme respectively and PAGE glue that contains the His-IRGMa fusion rotein and the incomplete Freund's adjuvant homogenate, get blood 1ml from the auricular vein of rabbit after 1 month, solidify back centrifuging and taking serum, the ELISA method detects the titre of IRGMa polyclonal antibody, and be 1: 2000 this moment; After this tood many or too much for use in every 7-14 days after full freund's adjuvant and albumen homogenate booster immunization 1 time is all got blood 1ml from auricular vein before the per injection, and the ELISA method detects antibody titers; Behind the booster immunization 2 times, the titre that the ELISA method detects antibody has reached more than 1: 10000; Adopt the specificity of western blotting method checking antibody; Get blood from rabbit carotid artery, Immobilized ProteinAColumn (Pierce company) antibody purification is adopted in the packing of centrifugal collection antibody serum, and detectable level and packing are stored in-80 ℃.

8, the application of the anti-people IRGMa of rabbit polyclonal antibody:

(1) immunoblotting: with the 293T cell of pCMV-myc-IRGMa transfection, the cell pyrolysis liquid of collecting is pressed sample electrophoresis on the different concns, change film, 5% skim-milk is as confining liquid, and room temperature closed protein electricity changes 60 fens kinds of film; The anti-people IRGMa of rabbit polyclonal antibody (concentration is 5 μ g/ μ l) is anti-as one, 1: 500-1: 10000 dilutions, and room temperature is shaken and is educated 2 hours, or 4 degree shake to educate and spend the night, and the 1XTBST damping fluid shakes washes 3 times, each 5 minutes kinds; The goat anti-rabbit igg of horseradish peroxidase-labeled is anti-as two, dilution in 1: 5000, and room temperature is shaken and is educated 45 minutes, and the 1XTBST damping fluid shakes washes 3 times, and each 5-15 branch is planted, the colour developing of horseradish peroxidase substrate, compressing tablet and punching.

(2) ELISA: the gst gene fusion rotein (antigen) of purifying is made a gradient by the amount of 1ng, 2.5ng, 5ng, 10ng, 100ng, and with before the immunity and immunity back serum press preimmune serum 1: 500, immunity back serum with 1: 500,1: 1000,1: 2000,1: 5000,1: 10000 work one antibody gradient, preimmune serum is ELISA in contrast.

(3) cellular immunofluorescence dyeing: the Jurkat cell of untransfected is dripped sheet on the cover glass that poly-lysine was handled, 4% Paraformaldehyde 96 fixed cell, 109 lowlenthal serum room temperature closing cells 60 minutes; The anti-people IRGMa of rabbit antibody concentration is 5ug/ul, 1: 100-1: 200 dilutions, and room temperature reaction 2 hours, 1XPBS shake washes 3 times, each 5-10 minute; The goat anti-rabbit igg of band green fluorescence is anti-as two, 1: 1000-1: 2000 dilutions, and room temperature reaction 30-60 minute, two anti-sealings finished preceding 5 minutes, and DAPI dyes nuclear, and 1XPBS shakes and washes 3 times, each 5-10 minute; The mounting mirror is observed down.

(4) immunohistochemical methods: with tissue paraffin section de-waxing and the rehydration of various human or mouse, 3%H2O2 incubated at room 10 minutes is to eliminate the activity of endogenous peroxydase.Distilled water flushing, PBS soaked 5 minutes, section were put into the container that fills citrate buffer again, put the microwave oven internal heating and made the liquid in container temperature remain between 92 ℃-98 ℃ and to continue to carry out the antigen hot repair in 2 minutes multiple.10% normal goats serum (PBS dilution) sealing, incubated at room 10 minutes.The serum deprivation that inclines is not washed, and drips an anti-or anti-working fluid of suitable proportion dilution, hatches for 37 ℃ to spend the night in 1-2 hour or 4 ℃.The PBS flushing, 5 minutes * 3 times.Drip the biotin labeling two anti-(1%BSA-PBS dilution) of suitable proportion dilution, hatched 30 minutes for 37 ℃; Or drip s-generation biotin labeling two anti-working fluids, 37 ℃ or incubated at room 30 minutes.The PBS flushing, 5 minutes * 3 times.Drip the horseradish enzyme labelling strepto-avidin (PBS dilution) of suitable proportion dilution, hatched 30 minutes for 37 ℃.The PBS flushing, 5 minutes * 3 times.Chromogenic reagent (DAB).Tap water fully washes, and redyes mounting.

(5) histogenic immunity fluorescence: with tissue paraffin section de-waxing and the rehydration of various human or mouse, 3%H2O2 incubated at room 5-10 minute is to eliminate the activity of endogenous peroxydase.Distilled water flushing, PBS soaked 5 minutes, section were put into the container that fills citrate buffer again, put the microwave oven internal heating and made the liquid in container temperature remain between 92 ℃-98 ℃ and to continue to carry out the antigen hot repair in 2 minutes multiple.10% normal goats serum (PBS dilution) sealing, incubated at room 10 minutes.The serum deprivation that inclines is not washed, and dripping the anti-people IRGMa of rabbit antibody concentration is 5ug/ul, 1: 100-1: 200 dilutions, and room temperature reaction 2 hours, 1XPBS washes each 10 minutes 3 times; The goat anti-rabbit igg of band green fluorescence is anti-as two, 1: 100-1: 200 dilutions, and room temperature reaction 45 minutes, two anti-sealings finish preceding 5 minutes, and DAPI dyes nuclear, 1XPBS flushing 3 times, each 5 minutes; The mounting mirror is observed down.

The invention has the beneficial effects as follows: because the present invention design and prepared rabbit anti-human IRGMa full-length protein polyclone antibody, its main functional zone have been contained, thereby guaranteed the high degree of specificity of antibody, help to carry out the research of IRGM protein function and the concrete effect in the MS pathogenic process thereof, thereby to the pathogenesis that discloses MS and seek efficient gene immunotherapy measure positive effect is arranged.

Description of drawings:

Fig. 1 is the photo as a result that antibody of the present invention is used for ELISA.Along with increasing of last sample protein concentration, antigen antibody reaction obviously strengthens, even when the albumen applied sample amount of 1ng, immunity back serum also has 10 times order of magnitude difference with preimmune serum under 1: 10000 extension rate, this result shows, the IRGMa-GST albumen and the antibody of amalgamation and expression have special combination, and tiring of antibody reaches more than 1: 10000.

Fig. 2 antibody of the present invention is used for the photo as a result of immunoblotting.Going up all product is the albumen of 4T-2-IRGMa protokaryon plasmid expression, i.e. IRGMa-GST fusion rotein, size is about 46KDa, between 43 and 66KDa between.4 swimming lane albumen applied sample amount unanimities, but an anti-difference of hybridization usefulness.1: be preimmune serum 1: 500; 2,3,4 are immunity back serum, and dilution is tired and is respectively 1: 2000,1: 5000 and 1: 10000.Immunoblotting is the result show, specific band occurs in the corresponding position of expection size, and prompting IRGMa-GST fusion rotein is able to correct expression, shows that also antibodies specific of the present invention is good.

Fig. 2 antibody of the present invention is used for the photo as a result of immunoblotting.1: be the 293T cell of pCMV-myc-IRGMa transfection, 2: be the 293T cell that does not change.The about 24KDa of IRGMa-myc fusion rotein, between 20.1 and 31KDa between.Immunoblotting is the result show, specific band occurs in the corresponding position of expection size, and prompting IRGMa-myc fusion rotein is able to correct expression, shows that also antibodies specific of the present invention is good.

Fig. 4 antibody of the present invention is used for the photo of Jurkat cellular immunofluorescence.In the endochylema of all Jurkat cells, all see green fluorescence, illustrate that the Jurkat cell has the proteic endogenous expression of IRGM, show that also antibodies specific of the present invention is good.

Fig. 5 antibody of the present invention is used for mouse spinal cord histogenic immunity fluorescence result.In the neurone endochylema of mouse spinal cord, see granular red fluorescence, illustrate in the neurone endochylema of mouse spinal cord, to have the proteic endogenous expression of IRGM, show that also antibodies specific of the present invention is good.

Fig. 6 antibody of the present invention is used for the immunohistochemical methods result of people's pancreatic tissue.In the endochylema of islet cells, can see granular brown signal, in acinous cell, not see, illustrate that there is the proteic endogenous expression of IRGM in the islet cells the people, show that also antibodies specific of the present invention is good.

Fig. 7 antibody of the present invention is used for the immunohistochemical methods result of people's little cerebral tissue.In the endochylema of cerebellum Purkinje's cell and aixs cylinder, all see granular brown signal, and do not see at molecular layer and granular cell layer, illustrate that IRGM albumen has endogenous expression in the Purkinje's cell of people's cerebellum, show that also antibodies specific of the present invention is good.

Fig. 8 antibody of the present invention is used for the immunohistochemical methods result of mouse spinal cord tissue.In spinal neuron endochylema and projection, all see granular brown signal, illustrate in the neurone endochylema of mouse spinal cord, to have the proteic endogenous expression of IRGM, show that also antibodies specific of the present invention is good.

The pancreatic tissue immunohistochemical methods result of Fig. 9 mouse.Pancreas islet periphery cell at mice pancreatic can be seen granular brown signal, does not see in acinous cell, illustrates that there is the proteic endogenous expression of IRGM in the islet cells mouse, shows that also antibodies specific of the present invention is good.

Embodiment

Embodiment 1:

A kind of preparation of rabbit anti-human IRGMa full-length protein polyclone antibody is selected for use:

1,, select for use the amino acid whose albumen of the 1-182 of containing IRGMa to prepare antibody at the proteic common function of IRGM district.

2, design two pairs of primers voluntarily according to the design of primers principle, structure contains the GST and the His gene fusion carrier of IRGMa full-length gene (546bp), the amino acid whose target protein fragment of 1-182 of IRGMa is contained in preparation, express GST and His gene fusion albumen, immunizing rabbit, get blood from rabbit carotid artery after 2 months, centrifugal collection antibody serum and antibody purification.

The concrete extracting method of the rabbit anti-human IRGMa full-length protein polyclone antibody of present embodiment:

1, from people's peripheric venous blood extracting genomic dna: because of IRGMa only has an exon, from the genomic dna desired albumen coded sequence that can increase.Therefore, at first from people's peripheric venous blood extracting genomic dna.

2, the selection of design of primers and gst gene fusion vector: selected pCMV-myc, pGEX4T-2 and Pet-28a (+)-His carrier, and select for use restriction enzyme EcoRI and XhoI as tie point; According to design of primers principle design primer, accomplish that same primer do not have palindrome hairpin structure, 5 ' end is not complementary with 4 bases of 3 ' end, and forward and reverse primer sequence is not complementary, Tm value close (2A+2T+4G+4C), and on Genebank, can not find homologous series; Designed 2 pairs of primers altogether, sequence is as follows:

First pair: be used to reprint pCMV-myc-IRGM and pGEX4T-2-IRGM carrier

1F:5 '-AgCgAATTCCTATggAAgCCATgAATgTTg-3 ' (containing EcoR I point of contact)

1R:5 '-gCgCTCgAgTTAgTATTCACATACCCgCTC-3 ' (containing Xho I point of contact)

Second pair: be used to reprint pET-28a (+)-IRGM carrier

2F:5 '-AgCgAATTCATggAAgCCATgAATgTTg-3 ' (containing EcoR I point of contact)

2R:5 '-gCgCTCgAgTTAgTATTCACATACCCgCTC-3 ' (containing the XhoI point of contact)

Above-mentioned 2 pairs of primers are used for the target gene fragment that pcr amplification makes up plasmid.

3, prepare target gene fragment and be connected to myc, on GST and the His gene fusion carrier: with normal people's genomic dna is template, adopts above-mentioned 3 pairs of primers and general T aq enzymatic amplification IRGMa complete encoding sequence respectively; Use TA clone test kit the PCR product is connected respectively on the PCR2.1 carrier, transform and the positive TA clone of picking, shake bacterium (e.colistraindh5, preserve this chamber), and adopt plasmid extraction purification kit extracting and purifying plasmid; EcoRI and XhoI double digestion confirm that positive TA clone and employing T carrier primer and the order-checking of ABI PRISMR3700DNA sequenator identify the exactness that is connected the target gene fragment sequence on the PCR2.1 carrier; Correct TA clones employing and uses EcoRI and XhoI double digestion, reclaims the myc that target gene fragment also is connected to the recovery of process EcoRI and XhoI double digestion, GST and His gene fusion carrier pCMV-myc, pGEX4T-2, the last and conversion of pET-28a (+)-His; The extracting plasmid, sequence verification.

4, containing the GST of target gene fragment and the expression of His gene fusion carrier identifies: 1 of picking transforms good GST and His gene fusion carrier cloning respectively, shake bacterium (intestinal bacteria origamiDE3 with 5ml 2YT substratum, Novagen company product), centrifugal collection bacterium and preparation GST and His gene fusion albumen in a small amount, adopt PAGE-SDS gel electrophoresis protein isolate fragment, Coomassie brilliant blue dyeing shows GST and His gene fusion protein fragments size.

5, preparation and purifying gst gene fusion rotein and collection target protein: prepare the gst gene fusion rotein in a large number, crossing column purification reclaims also quantitatively, 23 ℃ of enzymes of application Thrombin are cut and are spent the night, or the gst gene fusion rotein is hung the GST pearl earlier, cut with 23 ℃ of enzymes of Thrombin and to spend the night, second day crosses post and collects IRGMa total length target protein.

6, prepare His gene fusion albumen in a large number: because of IRGMa-His gene fusion albumen is insoluble albumen, so with the bacterium of centrifugal collection with sample Buffer cracking on 1 * albumen after employing PAGE-SDS gel electrophoresis protein isolate fragment, Coomassie brilliant blue dyeing, show His gene fusion albumen purpose fragment, it is frozen standby in-80 ℃ of refrigerators to tap rubber.

7, preparation and purifying contain the rabbit anti-human IRGMa full-length protein polyclone antibody of GST-tag and His-tag: subcutaneous multi-point injection immunizing rabbit after the IRGMa albumen that will reclaim after will being cut by the Thrombin enzyme respectively and PAGE glue that contains the His-IRGMa fusion rotein and the incomplete Freund's adjuvant homogenate, get blood 1ml from the auricular vein of rabbit after 1 month, solidify back centrifuging and taking serum, the ELISA method detects the titre of IRGMa polyclonal antibody, and be 1: 2000 this moment; After this tood many or too much for use in per 14 days after full freund's adjuvant and albumen homogenate booster immunization 1 time is all got blood 1ml from auricular vein before the per injection, and the ELISA method detects antibody titers; Behind the booster immunization 2 times, the titre that the ELISA method detects antibody has reached more than 1: 10000; Adopt the specificity of western blotting method checking antibody, the result confirms that this antibody has high degree of specificity; Get blood from rabbit carotid artery, Immobilized ProteinA Column (Pierce company) antibody purification is adopted in the packing of centrifugal collection antibody serum, and detectable level and packing are stored in-80 ℃.

8, the application of the anti-people IRGMa of rabbit polyclonal antibody:

(1) immunoblotting: with the 293T cell of pCMV-myc-IRGMa transfection, the cell pyrolysis liquid of collecting is pressed sample electrophoresis on the different concns, change film, 5% skim-milk is as confining liquid, and room temperature closed protein electricity changes 60 fens kinds of film; The anti-people IRGMa of rabbit polyclonal antibody (concentration is 5 μ g/ μ l) is anti-as one, 1: 500-1: 10000 dilutions, and room temperature is shaken and is educated 2 hours, or 4 degree shake to educate and spend the night, and the 1XTBST damping fluid shakes washes 3 times, each 5 minutes kinds; The goat anti-rabbit igg of horseradish peroxidase-labeled is anti-as two, dilution in 1: 5000, and room temperature is shaken and is educated 45 minutes, and the 1XTBST damping fluid shakes washes 3 times, and each 5-15 branch is planted, the colour developing of horseradish peroxidase substrate, compressing tablet and punching.

(2) ELISA: the gst gene fusion rotein (antigen) of purifying is made a gradient by the amount of 1ng, 2.5ng, 5ng, 10ng, 100ng, and with before the immunity and immunity back serum press preimmune serum 1: 500, immunity back serum with 1: 500,1: 1000,1: 2000,1: 5000,1: 10000 work one antibody gradient, preimmune serum in contrast, be ELISA, result such as Fig. 2 provide.

(3) cellular immunofluorescence dyeing: the Jurkat cell of untransfected is dripped sheet on the cover glass that poly-lysine was handled, 4% Paraformaldehyde 96 fixed cell, 10% lowlenthal serum room temperature closing cell 60 minutes; The anti-people IRGMa of rabbit antibody concentration is 5ug/ul, 1: 100-1: 200 dilutions, and room temperature reaction 2 hours, 1XPBS shake washes 3 times, each 5-10 minute; The goat anti-rabbit igg of band green fluorescence is anti-as two, 1: 1000-1: 2000 dilutions, and room temperature reaction 30-60 minute, two anti-sealings finished preceding 5 minutes, and DAPI dyes nuclear, and 1XPBS shakes and washes 3 times, each 5-10 minute; The mounting mirror is observed down.

(4) immunohistochemical methods: with tissue paraffin section de-waxing and the rehydration of various human or mouse, 3%H2O2 incubated at room 10 minutes is to eliminate the activity of endogenous peroxydase.Distilled water flushing, PBS soaked 5 minutes, section were put into the container that fills citrate buffer (working fluid) again, put the microwave oven internal heating and made the liquid in container temperature remain between 92 ℃-98 ℃ and to continue to carry out the antigen hot repair in 2 minutes multiple.10% normal goats serum (PBS dilution) sealing, incubated at room 10 minutes.The serum deprivation that inclines is not washed, and drips an anti-or anti-working fluid of suitable proportion dilution, hatches for 37 ℃ to spend the night in 1-2 hour or 4 ℃.The PBS flushing, 5 minutes * 3 times.Drip the biotin labeling two anti-(1%BSA-PBS dilution) of suitable proportion dilution, hatched 30 minutes for 37 ℃; Or drip s-generation biotin labeling two anti-working fluids, 37 ℃ or incubated at room 30 minutes.The PBS flushing, 5 minutes * 3 times.Drip the horseradish enzyme labelling strepto-avidin (PBS dilution) of suitable proportion dilution, hatched 30 minutes for 37 ℃.The PBS flushing, 5 minutes * 3 times.Chromogenic reagent (DAB).Tap water fully washes, and redyes mounting.

(5) histogenic immunity fluorescence: with tissue paraffin section de-waxing and the rehydration of various human or mouse, 3%H2O2 incubated at room 5-10 minute is to eliminate the activity of endogenous peroxydase.Distilled water flushing, PBS soaked 5 minutes, section were put into the container that fills citrate buffer (working fluid) again, put the microwave oven internal heating and made the liquid in container temperature remain between 92 ℃-98 ℃ and to continue to carry out the antigen hot repair in 2 minutes multiple.10% normal goats serum (PBS dilution) sealing, incubated at room 10 minutes.The serum deprivation that inclines is not washed, and dripping the anti-people IRGMa of rabbit antibody concentration is 5ug/ul, 1: 100-1: 200 dilutions, and room temperature reaction 2 hours, 1XPBS washes each 10 minutes 3 times; The goat anti-rabbit igg of band green fluorescence is anti-as two, 1: 100-1: 200 dilutions, and room temperature reaction 45 minutes, two anti-sealings finish preceding 5 minutes, and DAPI dyes nuclear, 1XPBS flushing 3 times, each 5 minutes; The mounting mirror is observed down.

SEQUENCE LISTING

＜110〉Huashan Hospital Affiliated To Fudan Univ

＜120〉preparation of rabbit anti-human IRGMa full-length protein polyclone antibody and application

<160>1

<170>PatentIn?version?3.1

<210>1

<211>203

<212>DNA

<213>Helicobacter?pylori

<400>1

ATG?GAA?GCC?ATG?AAT?GTT?GAG?AAA?GCC?TCA?GCA?GAT?GGG?AAC?TTG?CCA?GAG?GTG?ATC?TCT 60

Met?Glu?Ala?Met?Asn?Val?Glu?Lys?Ala?Ser?Ala?Asp?Gly?Asn?Leu?Pro?Glu?Val?Ile?Ser

1 5 10 15 20

AAC?ATC?AAG?GAG?ACT?CTG?AAG?ATA?GTG?TCC?AGG?ACA?CCA?GTT?AAC?ATC?ACT?ATG?GCA?GGG?120

Asn?Ile?Lys?Glu?Thr?Leu?Lys?Ile?Val?Ser?Arg?Thr?Pro?Val?Asn?Ile?Thr?Met?Ala?Gly

25 30 35 40

GAC?TCT?GGC?AAT?GGG?ATG?TCC?ACC?TTC?ATC?AGT?GCC?CTT?CGA?AAC?ACA?GGA?CAT?GAG?GGT?180

Asp?Ser?Gly?Asn?Gly?Met?Ser?Thr?Phe?Ile?Ser?Ala?Leu?Arg?Asn?Thr?Gly?His?Glu?Gly

45 50 55 60

AAG?GCC?TCA?CCT?CCT?ACT?GAG?CTG?GTA?AAA?GCT?ACC?CAA?AGA?TGT?GCC?TCC?TAT?TTC?TCT?240

Lys?Ala?Ser?Pro?Pro?Thr?Glu?Leu?Gta?Lys?Ala?Thr?Gln?Arg?Cys?Ala?Ser?Tyr?Phe?Ser

65 70 75 80

TCC?CAC?TTT?TCA?AAT?GTG?GTG?TTG?TGG?GAC?CTG?CCT?GGC?ACA?GGG?TCT?GCC?ACC?ACA?ACC?300

Ser?His?Phe?Ser?Asn?Val?Val?Leu?Trp?Asp?Leu?Pro?Gly?Thr?Gly?Ser?Ala?Thr?Thr?Thr

85 90 95 100

CTG?GAG?AAC?TAC?CTG?ATG?GAA?ATG?CAG?TTC?AAC?CGG?TAT?GAC?TTC?ATC?ATG?GTT?GCA?TCT?360

Leu?Glu?Asn?Tyr?Leu?Met?Glu?Met?Gln?Phe?Asn?Arg?Tyr?Asp?Phe?Ile?Met?Val?Ala?Ser

105 110 115 120

GCA?CAA?TTC?AGC?ATG?AAT?CAT?GTG?ATG?CTT?GCC?AAA?ACC?GCT?GAG?GAC?ATG?GGA?AAG?AAG?420

Ala?Gln?Phe?Ser?Met?Asn?His?Val?Met?Leu?Ala?Lys?Thr?Ala?Glu?Asp?Met?Gly?Lys?Lys

125 130 135 140

TTC?TAC?ATT?GTC?TGG?ACC?AAG?CTA?GAC?ATG?GAC?CTC?AGC?ACA?GGT?GCC?CTC?CCA?GAA?GTG?480

Phe?Tyr?Ile?Val?Trp?Thr?Lys?Leu?Asp?Met?Asp?Leu?Ser?Thr?Gly?Ala?Leu?Pro?Glu?Val

145 150 155 160

CAG?CTA?CTG?CAG?ATC?AGA?GAA?AAT?GTC?CTG?GAA?AAT?CTC?CAG?AAG?GAG?CGG?GTA?TGT?GAA?540

Gln?Leu?Leu?Gln?Ile?Arg?Glu?Asn?Val?Leu?Glu?Asn?Leu?Gln?Lys?Glu?Arg?Val?Cys?Glu

165 170 175 180

TAC?TAA 546

Tyr?*

182

Claims (5)

1, a kind of preparation of rabbit anti-human IRGMa full-length protein polyclone antibody is selected for use, it is characterized in that: at the proteic functional zone of IRGM, select for use IRGMa to prepare antibody; Select GST and the proteic method of His gene fusion to prepare the target protein fragment.

2, preparation according to claim 1 is selected for use, it is characterized in that: express GST and His gene fusion albumen, must make up the GST and the His gene fusion carrier that contain IRGMa full-length gene 546bp; According to design of primers principle design primer.

3, the extracting method of rabbit anti-human IRGMa full-length protein polyclone antibody of the present invention:

1) from people's peripheric venous blood extracting genomic dna;

2) selection of design of primers and gst gene fusion vector: selected pCMV-myc, pGEX4T-2 and Pet-28a (+)-His carrier, and select for use restriction enzyme EcoRI and XhoI as tie point; According to design of primers principle design primer, accomplish that same primer do not have palindrome hairpin structure, 5 ' end is not complementary with 4 bases of 3 ' end, and forward and reverse primer sequence is not complementary, Tm value close (2A+2T+4G+4C), and on Genebank, can not find homologous series; Designed 2 pairs of primers altogether, sequence is as follows:

First pair: be used to reprint pCMV-myc-IRGM and pGEX4T-2-IRGM carrier

1F:5 '-AgCgAATTCCTATggAAgCCATgAATgTTg-3 ' contains EcoR I point of contact;

1R:5 '-gCgCTCgAgTTAgTATTCACATACCCgCTC-3 ' contains the XhoI point of contact;

Second pair: be used to reprint pET-28a (+)-IRGM carrier

2F:5 '-AgCgAATTCATggAAgCCATgAATgTTg-3 ' contains EcoR I point of contact;

2R:5 '-gCgCTCgAgTTAgTATTCACATACCCgCTC-3 ' contains the XhoI point of contact;

3) preparation target gene fragment and be connected to myc, on GST and the His gene fusion carrier: with normal people's genomic dna is template, adopts above-mentioned 2 pairs of primers and general T aq enzymatic amplification IRGMa complete encoding sequence respectively; Use TA clone test kit the PCR product is connected respectively on the PCR2.1 carrier, transform and the positive TA clone of picking, shake bacterium, and adopt plasmid extraction purification kit extracting and purifying plasmid; EcoRI and XhoI double digestion confirm that positive TA clone and employing T carrier primer and the order-checking of ABI PRISMR3700 dna sequence analysis instrument identify the exactness that is connected the target gene fragment sequence on the PCR2.1 carrier; Correct TA clones employing and uses EcoRI and XhoI double digestion, reclaims the myc that target gene fragment also is connected to the recovery of process EcoRI and XhoI double digestion, GST and His gene fusion carrier pCMV-myc, pGEX4T-2, the last and conversion of pET-28a (+)-His; The extracting plasmid, sequence verification;

4) containing the GST of target gene fragment and the expression of His gene fusion carrier identifies: 1 of picking transforms good GST and His gene fusion carrier cloning respectively, shake bacterium with 5ml 2YT substratum, centrifugal collection bacterium and preparation GST and His gene fusion albumen in a small amount, adopt PAGE-SDS gel electrophoresis protein isolate fragment, Coomassie brilliant blue dyeing shows GST and His gene fusion protein fragments size;

5) preparation and purifying gst gene fusion rotein and collection target protein: prepare the gst gene fusion rotein in a large number, crossing column purification reclaims also quantitatively, 23 ℃ of enzymes of application Thrombin are cut and are spent the night, or the gst gene fusion rotein is hung the GST pearl earlier, cut with 23 ℃ of enzymes of Thrombin and to spend the night, second day crosses post and collects IRGMa total length target protein;

6) prepare His gene fusion albumen in a large number: because of IRGMa-His gene fusion albumen is insoluble albumen, the bacterium of centrifugal collection is adopted PAGE-SDS gel electrophoresis protein isolate fragment after with sample Buffer cracking on 1 * albumen, Coomassie brilliant blue dyeing, show His gene fusion albumen purpose fragment, it is frozen standby in-80 ℃ of refrigerators to tap rubber;

7) preparation and purifying contain the rabbit anti-human IRGMa full-length protein polyclone antibody of GST-tag and His-tag: subcutaneous multi-point injection immunizing rabbit after the IRGMa albumen that will reclaim after will being cut by the Thrombin enzyme respectively and PAGE glue that contains the His-IRGMa fusion rotein and the incomplete Freund's adjuvant homogenate, get blood 1ml from the auricular vein of rabbit after 1 month, solidify back centrifuging and taking serum, the ELISA method detects the titre of IRGMa polyclonal antibody, is 1: 2000; Booster immunization is 1 time after back too many or too much for use full freund's adjuvant and the albumen homogenate in every 7-14 days, all gets blood 1ml from auricular vein before the per injection, and the ELISA method detects antibody titers; Behind the booster immunization 2 times, the titre that the ELISA method detects antibody has reached more than 1: 10000; Adopt the specificity of western blotting method checking antibody; Get blood from rabbit carotid artery, Immobilized ProteinA Column antibody purification is adopted in the packing of centrifugal collection antibody serum, and detectable level and packing are stored in-80 ℃.

4, the application of the anti-people IRGMa of rabbit polyclonal antibody:

1) immunoblotting: with the 293T cell of pCMV-myc-IRGMa transfection, the cell pyrolysis liquid of collecting is pressed sample electrophoresis on the different concns, change film, 5% skim-milk is as confining liquid, and room temperature closed protein electricity changes 60 fens kinds of film; The anti-people IRGMa of rabbit polyclonal antibody concentration is 5 μ g/ μ l as one anti-, 1: 500-1: 10000 dilutions, and room temperature is shaken and is educated 2 hours, or 4 degree shake to educate and spend the night, and the 1XTBST damping fluid shakes washes 3 times, each 5 minutes kinds; The goat anti-rabbit igg of horseradish peroxidase-labeled is anti-as two, dilution in 1: 5000, and room temperature is shaken and is educated 45 minutes, and the 1XTBST damping fluid shakes washes 3 times, and each 5-15 branch is planted, the colour developing of horseradish peroxidase substrate, compressing tablet and punching;

2) ELISA: the gst gene fusion rotein antigen of purifying is made a gradient by the amount of 1ng, 2.5ng, 5ng, 10ng, 100ng, and with before the immunity and immunity back serum press preimmune serum 1: 500, immunity back serum with 1: 500,1: 1000,1: 2000,1: 5000,1: 10000 work one antibody gradient, preimmune serum is ELISA in contrast;

3) cellular immunofluorescence dyeing: the Jurkat cell of untransfected is dripped sheet on the cover glass that poly-lysine was handled, 4% Paraformaldehyde 96 fixed cell, 10% lowlenthal serum room temperature closing cell 60 minutes; The anti-people IRGMa of rabbit antibody concentration is 5ug/ul, 1: 100-1: 200 dilutions, and room temperature reaction 2 hours, 1XPBS shake washes 3 times, each 5-10 minute; The goat anti-rabbit igg of band green fluorescence is anti-as two, 1: 1000-1: 2000 dilutions, and room temperature reaction 30-60 minute, two anti-sealings finished preceding 5 minutes, and DAPI dyes nuclear, and 1XPBS shakes and washes 3 times, each 5-10 minute; The mounting mirror is observed down;

4) immunohistochemical methods: with tissue paraffin section de-waxing and the rehydration of various human or mouse, 3%H2O2 incubated at room 10 minutes; Distilled water flushing, PBS soaked 5 minutes, section were put into the container that fills citrate buffer again, put the microwave oven internal heating and made the liquid in container temperature remain between 92 ℃-98 ℃ and to continue to carry out the antigen hot repair in 2 minutes multiple; 10% normal goats blood-serum P BS dilutes sealing, incubated at room 10 minutes; The serum deprivation that inclines is not washed, and drips an anti-or anti-working fluid of suitable proportion dilution, hatches for 37 ℃ to spend the night in 1-2 hour or 4 ℃; The PBS flushing, 5 minutes * 3 times; Drip the biotin labeling two anti-1%BSA-PBS dilutions of suitable proportion dilution, hatched 30 minutes for 37 ℃; Or drip s-generation biotin labeling two anti-working fluids, 37 ℃ or incubated at room 30 minutes; The PBS flushing, 5 minutes * 3 times; Drip the horseradish enzyme labelling strepto-avidin PBS dilution of suitable proportion dilution, hatched 30 minutes for 37 ℃; The PBS flushing, 5 minutes * 3 times; Chromogenic reagent DAB; Tap water fully washes, and redyes mounting;

5) histogenic immunity fluorescence: with tissue paraffin section de-waxing and the rehydration of various human or mouse, 3%H2O2 incubated at room 5-10 minute; Distilled water flushing, PBS soaked 5 minutes, section were put into the container that fills citrate buffer again, put the microwave oven internal heating and made the liquid in container temperature remain between 92 ℃-98 ℃ and to continue to carry out the antigen hot repair in 2 minutes multiple; 10% normal goats blood-serum P BS dilutes sealing, incubated at room 10 minutes; The serum deprivation that inclines is not washed, and drips; The anti-people IRGMa of rabbit antibody concentration is 5ug/ul, 1: 100-1: 200 dilutions, and room temperature reaction 2 hours, 1XPBS washes each 10 minutes 3 times; The goat anti-rabbit igg of band green fluorescence is anti-as two, 1: 100-1: 200 dilutions, and room temperature reaction 45 minutes, two anti-sealings finish preceding 5 minutes, and DAPI dyes nuclear, 1XPBS flushing 3 times, each 5 minutes; The mounting mirror is observed down.

5, optimize one section encoding sequence in the IRGM gene, SEQUENCE LISTING:

ATG?GAA?GCC?ATG?AAT?GTT?GAG?AAA?GCC?TCA?GCA?GAT?GGG?AAC?TTG?CCA?GAG?GTG?ATC?TCT?60

Met?Glu?Ala?Met?Asn?Val?Glu?Lys?Ala?Ser?Ala?Asp?Gly?Asn?Leu?Pro?Glu?Val?Ile?Ser

1 5 10 15 20

AAC?ATC?AAG?GAG?ACT?CTG?AAG?ATA?GTG?TCC?AGG?ACA?CCA?GTT?AAC?ATC?ACT?ATG?GCA?GGG?120

Asn?Ile?Lys?Glu?Thr?Leu?Lys?Ile?Val?Ser?Arg?Thr?Pro?Val?Asn?Ile?Thr?Met?Ala?Gly

25 30 35 40

GAC?TCT?GGC?AAT?GGG?ATG?TCC?ACC?TTC?ATC?AGT?GCC?CTT?CGA?AAC?ACA?GGA?CAT?GAG?GGT?180

Asp?Ser?Gly?Asn?Gly?Met?Ser?Thr?Phe?Ile?Ser?Ala?Leu?Arg?Asn?Thr?Gly?His?Glu?Gly

45 50 55 60

AAG?GCC?TCA?CCT?CCT?ACT?GAG?CTG?GTA?AAA?GCT?ACC?CAA?AGA?TGT?GCC?TCC?TAT?TTC?TCT?240

Lys?Ala?Ser?Pro?Pro?Thr?Glu?Leu?Gta?Lys?Ala?Thr?Gln?Arg?Cys?Ala?Ser?Tyr?Phe?Ser

65 70 75 80

TCC?CAC?TTT?TCA?AAT?GTG?GTG?TTG?TGG?GAC?CTG?CCT?GGC?ACA?GGG?TCT?GCC?ACC?ACA?ACC?300

Ser?His?Phe?Ser?Asn?Val?Val?Leu?Trp?Asp?Leu?Pro?Gly?Thr?Gly?Ser?Ala?Thr?Thr?Thr

85 90 95 100

CTG?GAG?AAC?TAC?CTG?ATG?GAA?ATG?CAG?TTC?AAC?CGG?TAT?GAC?TTC?ATC?ATG?GTT?GCA?TCT?360

Leu?Glu?Asn?Tyr?Leu?Met?Glu?Met?Gln?Phe?Asn?Arg?Tyr?Asp?Phe?Ile?Met?Val?Ala?Ser

105 110 115 120

GCA?CAA?TTC?AGC?ATG?AAT?CAT?GTG?ATG?CTT?GCC?AAA?ACC?GCT?GAG?GAC?ATG?GGA?AAG?AAG?420

Ala?Gln?Phe?Ser?Met?Asn?His?Val?Met?Leu?Ala?Lys?Thr?Ala?Glu?Asp?Met?Gly?Lys?Lys

125 130 135 140

TTC?TAC?ATT?GTC?TGG?ACC?AAG?CTA?GAC?ATG?GAC?CTC?AGC?ACA?GGT?GCC?CTC?CCA?GAA?GTG?480

Phe?Tyr?Ile?Val?Trp?Thr?Lys?Leu?Asp?Met?Asp?Leu?Ser?Thr?Gly?Ala?Leu?Pro?Glu?Val

145 150 155 160

CAG?CTA?CTG?CAG?ATC?AGA?GAA?AAT?GTC?CTG?GAA?AAT?CTC?CAG?AAG?GAG?CGG?GTA?TGT?GAA?540

Gln?Leu?Leu?Gln?Ile?Arg?Glu?Asn?Val?Leu?Glu?Asn?Leu?Gln?Lys?Glu?Arg?Val?Cys?Glu

165 170 175 180

TAC?TAA 546

Tyr?*

182

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