CN104628854A - Method for preparing polyclonal antiserum for resisting bombyx mori BmGATA6 as well as polyclonal antiserum and application thereof - Google Patents
Method for preparing polyclonal antiserum for resisting bombyx mori BmGATA6 as well as polyclonal antiserum and application thereof Download PDFInfo
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Abstract
The invention discloses a method for preparing a polyclonal antiserum for resisting bombyx mori BmGATA6 and a polyclonal antiserum prepared from the method and an application thereof. The method comprises the steps of adopting an antigen shown in the sequence SEQ ID NO:1 to obtain the polyclonal antiserum for resisting bombyx mori BmGATA6 through the animal immunization. The antigen is prepared from the following steps: (1) cloning and analyzing the bombyx mori BmGATA6; and (2) performing prokaryotic expression and protein purification on the bombyx mori BmGATA6. The antiserum can be used for the fundamental research on the bombyx mori BmGATA6 protein and the highly conserved Lepidoptera GARA protein function, including the detection on the western and immune fluorescence and the like.
Description
Technical field
The present invention relates to a kind of polyclonal antiserum, be specifically related to a kind of preparation method of anti-silkworm BmGATA6 polyclonal antiserum and polyclonal antiserum prepared therefrom and application.
Background technology
GATA protein family is one group of transcription factor that can be combined with containing conservative (T/A) GATA (A/G) motif promotor, family member has found extensively exist in the biologies such as animal, fungi, plant, in Eukaryotic propagation, Differentiation and development, play important role.Have 6 GATA albumen so that the mankind are identified in Mammals, respectively at hemopoietic system, heart, plays a part key in the Differentiation and development of the histoorgans such as enteron aisle.In insect for fruit bat also identified go out multiple GATA albumen, wherein studied is in hematopoiesis, play the part of the Serpent of commander's regulating effect and in enteron aisle, eyes, mane etc. are grown, play the Panner of regulating effect the most widely.But it is about the research of silkworm GATA albumen rarely has report, also unclear to its effect in silkworm.Although GATA is the protein family comparatively guarded, but the difference between species, the factor such as selection of aminoacid sequence time prepared by antibody, GATA antibody is all caused not share, therefore the function of silkworm BmGATA6 albumen in silkworm will be resolved, carry out follow-up basic experiment smoothly, need the invention of silkworm BmGATA6 proteantigen and antibody.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of anti-silkworm BmGATA6 polyclonal antiserum and polyclonal antiserum prepared therefrom and application.
Particularly, the preparation method of described anti-silkworm BmGATA6 polyclonal antiserum, comprises and adopts the antigen that shown in SEQ ID NO:1, sequence represents to obtain described anti-silkworm BmGATA6 polyclonal antiserum by animal immune.
Particularly, described antigen can be obtained by following step:
1) clone of silkworm BmGATA6 and analysis:
According to the aminoacid sequence of the BmGATA6 of prediction on NCBI, design the amplimer as shown in SEQ ID NO:4-5, carry out pcr amplification, obtain the first amplified production;
Obtain first after described first amplified production is connected with the first connection carrier and connect product, described first connects product conversion first competent cell, obtain the first positive colony, then carry out sequence verification to the first positive colony, the BmGATA6 total length coding gene sequence that order-checking obtains is as shown in SEQ ID NO:3;
2) prokaryotic expression of silkworm BmGATA6 and protein purification:
According to step 1) the BmGATA total length coding gene sequence that obtains, intercept the sequence of N end from 43bp to 986bp, obtain the sequence shown in SEQ ID NO:1, design the prokaryotic expression primer as shown in SEQ ID NO:6-7, carry out pcr amplification, obtain the second amplified production;
Obtain second after described second amplified production is connected with the second connection carrier and connect product, described second connects product conversion second competent cell, carries out protein induced expression;
SDS-PAGE electrophoresis detection is carried out to the albumen of abduction delivering, selects the purer elutriant containing BmGATA6 albumen, repeatedly dialyse, concentrated, its freeze-drying is become protein dry powder, preserves, for subsequent use.
Further, during described abduction delivering, 5 hour respectively carry out BmGATA6 protein induced expression to final concentration for cultivating under cultivate 20h and 37 DEG C condition after being respectively 0.4mM, 0.6mM, 0.8mM, 1.0mM under 16 DEG C of conditions with IPTG.
Further, with IPTG concentration for 0.6mM, the condition of cultivating 20h under 16 DEG C of conditions is final inductive condition.
Further, the first connection carrier is pMD19-T Simple carrier, and the first competent cell is DH5 α competent cell.
Further, the second connection carrier is pET32a carrier, and the second competent cell is BL21 competent cell.
Further, described animal immune comprises HRP mark mouse, and the antigen coated liquid being 2 μ g/ml with concentration carries out repeatedly multiple spot immunity, calculates tiring of the silkworm BmGATA6 protein antiserum of mouse generation.
Further, described preparation method comprises: in 6 days ages of silkworm 5, the paraffin section of intestines is as sample, carries out Immunofluorescence test to silkworm BmGATA6 protein antiserum.
The present invention also provides the anti-silkworm BmGATA6 polyclonal antiserum prepared according to above-mentioned preparation method.
Can be used in detecting GATA albumen conservative in lepidopterous insects according to anti-silkworm BmGATA6 polyclonal antiserum prepared by above-mentioned preparation method.
Successful clone silkworm BmGATA6 gene of the present invention, Prokaryotic expression, purification goes out activated albumen, polyclonal antiserum is produced by repeatedly immune mouse, the western that can carry out BmGATA6 protein expression level in body detects, and the immunofluorescence after tissue paraffin section de (needing antigen retrieval), the experiments such as the immunofluorescence of cell climbing sheet.The present invention can be further used for the functional study of GATA albumen conservative in silkworm BmGATA6 albumen and lepidopterous insects.This antiserum(antisera) may be used for the fundamental research of silkworm BmGATA6 albumen and the lepidopteran GATA protein function with its high conservative, comprises the detection of the aspect such as western and immunofluorescence.
For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and coordinate accompanying drawing, be described in detail below.
Accompanying drawing explanation
Fig. 1 is silkworm BmGATA6 conservative structural domain prediction view.
Fig. 2 A and Fig. 2 B induces SDS-PAGE electrophoresis detection after BmGATA6 albumen under different concns IPTG and differing temps.Fig. 2 A is 16 DEG C of inductions, and Fig. 2 B is 37 DEG C of inductions.
Fig. 3 be after differing temps induction and ultrasonic disruption in upper cleer and peaceful precipitation the expression of SDS-PAGE electrophoresis detection BmGATA6 albumen, wherein to after the BL21 bacterium liquid supersound process after induction, extract supernatant respectively to carry out examining contaminating detect with the albumen in precipitation, M is marker, 1# is 37 DEG C, 0mM IPTG supernatant; 2# is 37 DEG C, and 0mM IPTG precipitates; 3# is 37 DEG C, and 0.6mM IPTG induces supernatant; 4# is 37 DEG C, 0.6mM IPTG induced precipitation; 5# is 16 DEG C, and 0.6mM IPTG induces supernatant; 6# is 16 DEG C, 0.6mM IPTG induced precipitation.
Fig. 4 is SDS-PAGE electrophoresis detection after different concns imidazoles eluted protein.
Fig. 5 is that the sero-fast western that 3 mouse produce detects.Wherein M represents marker, and be pre-dyed marker in negative serum, antiserum(antisera) ratio is 1: 5000.
Fig. 6 is that Midgut of Silkworm, Bombyx Mori paraffin section detects the sero-fast immunofluorescence effect of BmGATA6.Antibody dilution ratio is 1: 4000.
Embodiment
Test materials
Summary:
Nucleotide sequence shown in SEQ ID NO:2 is cloned and checks order and determine to be connected to correctly BamH I and the Xho I restriction enzyme site of pET32a carrier, after carrying out prokaryotic expression by engineering bacteria BL21, purifying is carried out to BmGATA6 albumen, purification process is as follows: by engineering bacteria IPTG abduction delivering, collected by centrifugation thalline, with containing 20mM Tris respectively after PBS cleaning, 0.5M Nacl, 20mM imidazoles, the lysate of 5% glycerine is resuspended, and carry out liquid nitrogen multigelation, collected by centrifugation supernatant after ultrasonic disruption, suction filtration, then purify with Ni affinity column, with containing 0.2M NaH2PO4 respectively, 0.2M Na2HPO4, 4M Nacl, after the binding buffer liquid of 20mM imidazoles washes away unconjugated albumen, again respectively with containing 200mM, the binding buffer liquid wash-out of 500mM and 1M imidazoles, finally dialyse by PBS solution, obtain BmGATA6 albumen.Be frozen into after protein dry powder packing in-80 DEG C of preservations with freeze drier after the BmGATA6 albumen PEG20000 of acquisition is concentrated.
Get 3 kunming mices, cut tail respectively and get blood, be separated and obtain negative serum, in-80 DEG C of preservations.Get appropriate BmGATA6 protein dry powder, dissolve with distilled water, protein concentration is surveyed with BCA test kit, get the albumen of every mouse 70 μ g, volume dilution is 250 μ l, subcutaneous and many places, the abdominal cavity immunity Kunming mouse three with the complete Freund's adjuvant of 250 μ l fully emulsified rear first time, 10 days afterwards with the fully emulsified rear second time immunity Kunming mouse of the incomplete Freund's adjuvant of same protein content and same volume, use the same method after 10 days and carry out third time immunity, the 4th immunity is carried out after 15 days, within 15 days, directly impact immunity with 70 μ g BmGATA6 albumen afterwards, carry out excision eyeball after 3 days and get blood.Be separated and obtain anti-BmGATA6 serum, after mixing with equal-volume glycerine, packing is stored in-80 DEG C.
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.
embodiment 1the clone of silkworm BmGATA6 and analysis
Download the aminoacid sequence of the BmGATA6 of prediction from NCBI, its accession number is XM_004924410.1.According to the special primer of the mRNA sequences Design amplification BmGATA6 gene of its prediction, forward primer SEQ ID NO:5, reverse primer SEQ ID NO:6, to make silkworm greatly three days five ages that intestines cDNA[in three days ages of silkworm five buys from domestic silkworm gene group biology National Key Laboratory, the method of synthesizing according to extraction and the cDNA of " molecular biosciences experimental technique and the skill " of being edited by Shen Huangxuan (press of Zhongshan University in June, 2010 publish) chapter 8 RNA is obtained] increase for template, PCR reaction conditions is: 95 DEG C of denaturations 5 minutes, then 95 DEG C of sex change 40 seconds, anneal 40 seconds for 60 DEG C, 72 DEG C extend 2 minutes, totally 28 circulations, last 72 DEG C extend 10 minutes, PCR primer reclaims about 2000bp band through agarose gel electrophoresis, then be connected with pMD19-T Simple carrier, ligation system is carried out in strict accordance with Solution I ligase enzyme operation instruction, condition of contact connects 2 hours under 16 DEG C of conditions, product conversion DH5 α competent cell will be connected, sequence verification after acquisition positive colony pMD19-T Simple/BmGATA6, sequencing result is as shown in SEQ ID NO:3, the CDS of the BmGATA6 gene of cloning by analysis is 1971bp, to encode 657 amino acid (as shown in SEQ ID NO:4), with online software SMART (http://smart.embl-heidelberg.de/), structural analysis is carried out to its aminoacid sequence, as shown in Figure 1, BmGATA6 aminoacid sequence comprises a conservative GATA structural domain.
embodiment 2the prokaryotic expression of silkworm BmGATA6 and protein purification
According to the BmGATA6 total length CDS sequence (as shown in SEQ ID NO:3) that clone obtains, (gene order is as shown in SEQ ID NO:2 to intercept the sequence of N end from 43bp to 986bp, its corresponding aminoacid sequence is as shown in SEQ ID NO:1), design prokaryotic expression primer, forward primer as shown in SEQ ID NO:7,5 ' CG
gGATCCcGAGAGGAAGAAAACAGCCA 3 ', reverse primer is as shown in SEQ ID NO:8, and underlined sequences is BmH I restriction enzyme site, reverse primer 5 ' CCG
cTCGAGaAGAGCTCCTTGTTACTGCCTC 3 ', underlined sequences is Xho I restriction enzyme site, then with intestines cDNA in three days ages of silkworm five for template increases, PCR reaction conditions is: 95 DEG C of denaturations 5 minutes, then 95 DEG C of sex change, 40 seconds, 58 DEG C annealing 40 seconds, 72 DEG C extend 1 minute, totally 28 circulations, last 72 DEG C extend 10 minutes, PCR primer is identified through agarose gel electrophoresis and is reclaimed, reclaim product BamH I and Xho I double digestion, reclaim BmGATA6 gene fragment, pET32a carrier BamH I and Xho I is carried out double digestion simultaneously, reclaim carrier framework, the BmGATA6 endonuclease bamhi of recovery is carried out being connected conversion with pET32a skeleton fragment, obtain pET32a/BmGATA6 recombinant plasmid, then the order-checking of Hua Da genome company is sent to, order-checking is confirmed that correct recombinant plasmid transformed expresses recipient cell e. coli bl21, add IPTG to final concentration for being respectively 0.4mM, 0.6mM, 0.8mM, cultivate cultivate 20h and 37 DEG C condition after 1.0mM under 16 DEG C of conditions under and carry out the protein induced expression of BmGATA6 respectively in 5 hours, SDS-PAGE electrophoresis detection, result as shown in Figure 2 A and 2 B, result is presented at different IPTG concentration, at different temperature, BmGATA6 albumen all has expression, then choose IPTG be 0.6 concentration it is carried out respectively under 16 DEG C of conditions and 37 DEG C of conditions induction, and carry out ultrasonication and be separated, SDS-PAGE electrophoresis detection, result as shown in Figure 3 BmGATA6 albumen all has expression in supernatant and the precipitation of 16 DEG C and 37 DEG C.In order to ensure the activity of the recombinant protein of abduction delivering, finally have chosen with IPTG concentration for 0.6mM, the condition of cultivating 20h under 16 DEG C of conditions is final inductive condition.
Thalline is collected by containing after the e. coli bl21 enlarged culturing of pET32a/BmGATA6 recombinant plasmid and abduction delivering centrifugal 10 minutes under 7000rpm condition, PBS cleans and uses for 3 times the lysate respectively containing 20mM Tris, 0.5M Nacl, 20mM imidazoles, 5% glycerine resuspended afterwards, and carrying out liquid nitrogen multigelation 3 times, ultrasonic disruption is 16000rpm centrifugal 30 minutes collection supernatants after 30 minutes; Supernatant is purified with carrying out Ni affinity column after 0.45 μm of filter membrane suction filtration.With balance liquid (8.5ml0.2M NaH
2pO
4, 91.5ml 0.2M Na
2hPO
4, 125ml 4M NaCl, 775ml H
2o) Ni affinity column is balanced, after slow for egg white mixture hanging column, pillar is got express developed to wash away unconjugated albumen with binding buffer liquid (balance liquid+20mM imidazoles), 40mM, 60mM, 80mM is contained respectively respectively again with 50ml, 100mM, the balance liquid (elutriant) of 200mM, 500mM imidazoles slowly rinses pillar and collects elutriant, and the elutriant collected carries out SDS-PAGE electrophoresis detection (Fig. 4); Select the purer elutriant containing BmGATA6 albumen, dialyse 36 hours by PBS solution, within every 6 hours, change dialyzate once; Purifying and the BmGATA6 albumen after dialysing are contained in dialysis tubing, concentrate with PEG20000 under 4 DEG C of conditions, be dispensed into after being concentrated to appropriate volume in the centrifuge tube of 2ml, with the freezing concentrating instrument of high speed, its freeze-drying become protein dry powder, be stored in-80 DEG C of refrigerators for subsequent use.
embodiment 3silkworm BmGATA6 protein immunization mouse and antiserum(antisera) are collected
Get 3 male mouse of kunming in 6 week ages (purchased from Chongqing Teng Xin Bioisystech Co., Ltd), cut off a little tail respectively and collect blood, after the number of finishing, room temperature leaves standstill 2 hours, room temperature 5000g is after centrifugal 5 minutes, centrifugal 1 minute of 10000g, collects supernatant in new centrifuge tube, the red corpuscle got below of trying not, supernatant and glycerine with 1: 1 ratio mix after preserve, as negative serum in-80 DEG C;
Get 3 BmGATA6 protein dry powder, the measurement that rear BCA test kit carries out protein concentration is fully dissolved respectively with 300ml deionized water, get 750 μ l about 210 μ g BmGATA6 albumen and 750 μ l complete Freund's adjuvants altogether, blow and beat until albumen is fully emulsified back and forth with two 5ml syringes, respectively the subcutaneous of many places is carried out to 3 Kunming mouses and abdominal injection immune, every only about inject 70 μ g albumen.Second time and third time immunity are carried out in interval after 10 days respectively, and except changing into except incomplete Freund's adjuvant by complete Freund's adjuvant, method is with first time; The 4th immunity is carried out at interval after 15 days, method is with second time; Apart from the 4th immunity within latter 15 days, respectively get 70 μ g BmGATA6 albumen volume is added to 500 μ l after direct injection mouse carry out last immunity and impact, obtain mouse blood respectively by the method for extracing eyeball after 3 days.
Room temperature after the serum number of finishing of the anti-BmGATA6 albumen collected is left standstill 2 hours, and the mode with process negative serum processes, packing and preservation.
embodiment 4the detection of anti-silkworm BmGATA6 albumen serum
Join the coating buffer (Na that antigen concentration is 2 μ g/ml
2cO
30.17g, NaHCO
30.28g is settled to 100ml), in enzyme plate, every hole adds 100 μ l, goes to 4 DEG C refrigerator overnight after leaving standstill 1 hour after sealing in 37 DEG C, take out enzyme plate, with PBST (KH
2pO
40.2g, KCl 0.2g, NaCl 8.0g, NaHPO
412H
2o 2.95, Tween-20 0.5ml, be settled to 1L) wash 3 times, each 5 minutes, by the antiserum(antisera) (1# of 3 mouse, 2#, 3#) respectively with 1: 500, 1: 1000, 1: 2000, 1: 4000, 1: 8000, 1: 16000, 1: 32000, 1: 64000, the ratio of 1: 128000 and 1: 256000 is got 100 μ l respectively and is added bag by the hole of antigen after diluting, respectively stay one to wrap equally in addition and added 100 μ l respectively with the negative serum of 1: 8000 dilution proportion by the hole of antigen, the HRP of 1: 30000 dilution proportion marks mouse IgG and PBST, 37 DEG C hatch 1 as a child PBST wash 3 times, each 5 minutes, add respectively and mark mouse IgG 100 μ l with the dilution proportion HRP of 1: 30000,37 DEG C hatch 1 hour after PBST wash 3 times, each 5 minutes, develop the color with TMB colouring reagents box, and measure the OD value of A450 and calculate tiring of BmGATA6 protein antiserum that 3 mouse produce, result is as shown in table 1, and the antiserum titre of 3 mouse is all up to 1: 128000.
Table l
| 1∶500 | 1∶1000 | 1∶2000 | 1∶4000 | 1∶8000 | 1∶16000 | 1∶32000 | 1∶64000 | 1∶128000 | 1∶256000 | Negative serum | Blank | |
| First | 1.500406 | 1.460213 | 1.461861 | 1.430188 | 1.45121 | 1.396746 | 1.379622 | 1.02275 | 1.136666 | 0.1432329 | 0.138952 | 0.1182988 |
| Second | 1.483028 | 1.46698 | 1.479357 | 1.432371 | 1.395874 | 1.441161 | 1.406163 | 1.269189 | 1.233514 | 0.09170054 | 0.1712278 | 0.1119433 |
| 3rd | 1.464646 | 1.450806 | 1.377811 | 1.501604 | 1.438726 | 1.388982 | 1.394825 | 1.360386 | 1.140426 | 0.1333108 | 0.1278683 | 0.1169995 |
Be sample with silkworm 5 Midgut protein in 7 days ages, antagonism silkworm BmGATA6 albumen serum carries out western blot detection, albumen is after SDS-PAGE electrophoresis and transferring film, with the TBST (NaCl8.8g containing 5%BSA, 1M Tris-HCl (ph 8.0) 20ml, 0.5ml Tween20, constant volume is to 1L) close in 4 DEG C and spend the night, the antiserum(antisera) respectively 3 mouse produced with after 1: 5000 dilution in incubated at room 2 hours, TBST washes 3 times, each 10 minutes, the HRP of the dilution proportion with 1: 30000 marked mouse IgG incubated at room after 1 hour, again wash 3 times with TBST, each 10 minutes, carry out developing the color and exposing with imager with ECL nitrite ion, as shown in Figure 5, the antiserum(antisera) that 3 mouse produce can be special display 5 Midgut of Silkworm, Bombyx Mori in 3 days ages in BmGATA6 protein band, size is about 80kD.
Also Immunofluorescence test is carried out to silkworm BmGATA6 protein antiserum.In 6 days ages of silkworm 5, the paraffin section of intestines is as sample, after antigen retrieval, develop a film son after 3 times each 5 minutes with PBS, by the PBS incubated at room containing 1%Triton-X 100 15 minutes, 5 minutes are washed again with PBS, after washing 3 times, with the PBS confining liquid containing 3%BSA and 10% lowlenthal serum in 4 DEG C close spend the night, with confining liquid with 1: 1000,1: 2000,1: 4000 dilution proportion 2# antiserum(antisera), and with 1: 2000 dilution proportion negative serum, respectively at incubated at room sample after 2 hours, with PBS wash 3 times each 10 minutes, then use Alexa
594Donkey Anti-Mouse IgG (H+L) antibody at room temperature hatches 1 hour, with PBS wash 3 times each 10 minutes; By the DAPI incubated at room of suitable proportion after 40 minutes, PBS washes 10 minutes, add anti-fluorescence quenching and then use fluorescent microscope imaging with after nail varnish edge sealing, as shown in Figure 6, compared with negative serum, BmGATA6 antiserum(antisera) can be special mark Midgut of Silkworm, Bombyx Mori in BmGATA6 albumen, and show it and be expressed in the nucleus of Midgut of Silkworm, Bombyx Mori cell.
Although the present invention discloses as above with preferred embodiment; so itself and be not used to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the present invention; when doing a little change and improvement, therefore protection scope of the present invention is when being as the criterion depending on the claim person of defining.
Claims (10)
1. a preparation method for anti-silkworm BmGATA6 polyclonal antiserum, is characterized in that, described method comprises the antigen that shown in employing SEQ ID NO:1, sequence represents and obtains described anti-silkworm BmGATA6 polyclonal antiserum by animal immune.
2. preparation method according to claim 1, is characterized in that, described antigen is obtained by following step:
1) clone of silkworm BmGATA6 and analysis:
According to the aminoacid sequence of the BmGATA of prediction on NCBI, design the amplimer as shown in SEQ ID NO:4-5, carry out pcr amplification, obtain the first amplified production;
Obtain first after described first amplified production is connected with the first connection carrier and connect product, described first connects product conversion first competent cell, obtain the first positive colony, then carry out sequence verification to the first positive colony, the BmGATA6 total length coding gene sequence that order-checking obtains is as shown in SEQ ID NO:3;
2) prokaryotic expression of silkworm BmGATA6 and protein purification:
According to step 1) the BmGATA total length coding gene sequence that obtains, intercept the sequence of N end from 43bp to 986bp, obtain the sequence shown in SEQ ID NO:1, design the prokaryotic expression primer as shown in SEQ ID NO:6-7, carry out pcr amplification, obtain the second amplified production;
Obtain second after described second amplified production is connected with the second connection carrier and connect product, described second connects product conversion second competent cell, carries out protein induced expression;
SDS-PAGE electrophoresis detection is carried out to the albumen of abduction delivering, selects the purer elutriant containing BmGATA6 albumen, repeatedly dialyse, concentrated, its freeze-drying is become protein dry powder, preserves, for subsequent use.
3. preparation method according to claim 2, it is characterized in that, during described abduction delivering, 5 hour respectively carry out BmGATA6 protein induced expression to final concentration for cultivating under cultivate 20h and 37 DEG C condition after being respectively 0.4mM, 0.6mM, 0.8mM, 1.0mM under 16 DEG C of conditions with IPTG.
4. preparation method according to claim 3, is characterized in that, with IPTG concentration for 0.6mM, the condition of cultivating 20h under 16 DEG C of conditions is final inductive condition.
5. the preparation method according to any one of claim 2 to 4, is characterized in that, the first connection carrier is pMD19-T Simple carrier, and the first competent cell is DH5 α competent cell.
6. the preparation method according to any one of claim 2 to 4, is characterized in that, the second connection carrier is pET32a carrier, and the second competent cell is BL21 competent cell.
7. the preparation method according to above-mentioned arbitrary claim, it is characterized in that, described animal immune comprises HRP mark mouse, and the antigen coated liquid being 2 μ g/ml with concentration carries out repeatedly multiple spot immunity, calculates tiring of the silkworm BmGATA6 protein antiserum of mouse generation.
8. preparation method according to claim 7, is characterized in that, comprises further: in 6 days ages of silkworm 5, the paraffin section of intestines is as sample, carries out Immunofluorescence test to silkworm BmGATA6 protein antiserum.
9. anti-silkworm BmGATA6 polyclonal antiserum prepared by the preparation method any one of claim 1-8.
10. anti-silkworm BmGATA6 polyclonal antiserum prepared by the preparation method any one of claim 1-8 is detecting the application in GATA albumen conservative in lepidopterous insects.
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| CN101050459A (en) * | 2007-03-16 | 2007-10-10 | 中国农业科学院兰州兽医研究所 | Mature Doppel of Chinese yellow cattle, and method for preparing polyclonal antibody |
| CN103214574A (en) * | 2013-03-18 | 2013-07-24 | 河南师范大学 | Method for preparing carp small peptide transporter protein PepT1 rabbit antiserum |
| CN103509107A (en) * | 2013-08-07 | 2014-01-15 | 浙江大学 | Method of preparing bombyx mori silk fibroin specific antibody by utilizing characteristic polypeptide |
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