CN107298719A

CN107298719A - Pre-dyed luminescent protein marker preparation method

Info

Publication number: CN107298719A
Application number: CN201710707539.4A
Authority: CN
Inventors: 方卫斌; 张海灵
Original assignee: Tian Yue (beijing) Biotechnology LLC
Current assignee: Tian Yue (beijing) Biotechnology LLC
Priority date: 2017-08-17
Filing date: 2017-08-17
Publication date: 2017-10-27

Abstract

The invention provides a kind of pre-dyed luminescent protein marker preparation method, its step includes：The weight chain constant area gene (CH genes) of proteinA genes, proteinG genes, MBP gene, mouse and rabbit source antibody is obtained, said gene is cut or combined according to each albumen size in pre-dyed luminescent protein marker；Expressing protein, purifying, the albumen of different molecular weight is mixed according to a certain percentage, luminescent protein marker is obtained；The luminescent protein marker mixed up and pre-dyed albumen marker is mixed in different ratios.The pre-dyed luminescent protein marker of the present invention can recognize the primary antibody or secondary antibody of separate sources, add the binding site of albumen, strengthen optical signal, can not only in electrophoresis process and transferring film after indicator protein approximate location, and can on X films indicator protein exact position, without extra increase pre-dyed marker swimming lanes, and extra antibody need not be added, luminescent protein can combining source in the IgG or anti-rabbit and the secondary antibody of mouse of the species such as people, rat, mouse, rabbit.

Description

Pre-dyed luminescent protein marker preparation method

Technical field

The present invention relates to a kind of pre-dyed luminescent protein marker preparation method, belong to molecular biology and led with genetic engineering Domain.

Background technology

Protein marker is widely used in protein electrophorese as a kind of standard of protein molecular weight size.It The mixture being made up of the albumen or polypeptide of known different molecular weight size.Western Blot need one kind to be used for tracking The scale of the positive antigen signals of detection and transferring film efficiency, then generates pre-dyed albumen Marker.Pre-dyed albumen Marker be by Each albumen (or polypeptide) in general proteins marker passes through chemical modification, and the dyestuff with a kind of blueness or other colors is covalent Coupling is formed.Pre-dyed albumen marker appearance brings great convenience to Western Blot, and researcher, can root after transferring film The Position Approximate of purpose band is determined according to film blue (or other colors) albumen marker size, so as to be cut to film The processing such as cut.But, pre-dyed albumen Marker can not be exposed on film, and exposure signal is needed according to the pre-dyed albumen on film Marker judges that in addition mobility changes pre-dyed albumen Marker in SDS-PAGE electrophoresis processes after chemical modification Become, molecular weight information is no longer accurate, often lead to Western Blot results and judge relatively large deviation occur.

Luminous Western Blot Marker are the molecular weight standards exclusively for Western Blot experimental designs. In western blot experiments, marker bands can specifically bind primary antibody or secondary antibody, make protein sample and albumen marker After chemiluminescence detection, it can be shown simultaneously on X-ray.

It can be used for the luminous marker of exposure on the market at present, different company employs different technologies.CST companies Luminous marker be purifying albumen and the covalently bound mixture of biotin, antibiotin HRP antibody is in Western It is used for detecting biotinylated protein ladder in blots.Most of companies are luminous using the albumen composition for being capable of binding antibody IgG Marker composition, such as thermo.

But, all there is certain deficiency in current product, biotinylated albumen marker need to add antibiotin- HRP antibody or streptavidin-HRP, this causes experimental implementation to become cumbersome.In addition, only containing the albumen that can be lighted Marker be unable to the positive antigen signals of tracing detection with transferring film efficiency, it is necessary in addition plus together with pre-dyed marker, it is not only cumbersome and And take swimming lane, reduce can electrophoresis sample size.

The content of the invention

The present invention is intended to provide one kind can be shown with destination protein on X films, so as to more accurately indicate simultaneously The particular location of albumen, and the pre-dyed luminescent protein marker of additional agents preparation method need not be added.

The purpose of the present invention is achieved through the following technical solutions：

A kind of pre-dyed luminescent protein marker preparation method, its step includes：

A, the heavy chain constant region base for obtaining proteinA genes, proteinG genes, MBP genes, mouse and rabbit source antibody Because of (CH genes), the IgG Binding domain for determining proteinA, proteinG are annotated according to NCBI, it is luminous according to pre-dyed Each albumen size is cut or group to proteinA genes, proteinG genes, MBP genes, CH genes in albumen marker Close, the Binding domain of albumen are retained in cutting process；

B, proteinA, proteinG, MBP and CH gene after cutting or combination is connected on expression vector respectively Carry out expression and purification, the albumen purified；

C, the albumen of different molecular weight mixed according to a certain percentage, obtain luminescent protein marker；

D, the luminescent protein marker mixed up and pre-dyed albumen marker mixed in different ratio, by observing transferring film Stripe depth and band brightness selection proper mixture ratio example on X films after development on caudacoria.

It is preferred that, the expression vector is pET21a or pET28a.

It is preferred that, the host cell is E.coli.

It is preferred that, the luminescent protein marker molecular weight is from 16kDa to 200kDa.

The pre-dyed luminescent protein marker of the present invention is in mutually not shadow by luminescent protein marker and pre-dyed albumen marker Mixed according to a certain percentage in the case of sound, so as to realize the molecule figureofmerit after transferring film and molecule on X films after exposure Figureofmerit dual-use function, wherein luminescent protein marker be proteinA, proteinG by being capable of binding antibody IgG or ProteinL, and can be formed by the rabbit source of two anti-bindings or mouse source antibody Fc district gene fusion expression, so that luminescent protein Albumen in marker can recognize the primary antibody or secondary antibody of separate sources, add the binding site of albumen, strengthen optical signal. The present invention pre-dyed luminescent protein marker can not only in electrophoresis process and transferring film after indicator protein approximate location, Er Qieneng The exact position of indicator protein on X films, without extra increase pre-dyed marker swimming lanes, and need not be added extra Antibody, such as anti-biotin antibodies-HRP or streptavidin-HRP, luminescent protein can combining source in people, rat, mouse, rabbit etc. The IgG or anti-rabbit and the secondary antibody of mouse of species.Compared with the luminous marker of other companies, with broader indicating range, point Son amount is from 16kDa to 200kDa.

Brief description of the drawings

Fig. 1 for can luminescent protein marker exposure after electrophoretogram.

Fig. 2 is the electrophoretogram before pre-dyed luminescent protein marker exposes.

Fig. 3 is the electrophoretogram after pre-dyed luminescent protein marker exposes.

Fig. 4 is the architectural feature figure of the polyclonal sequences of MBP+.

The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining The bright present invention, and be not construed as limiting the scope of the present invention.

Embodiment

Embodiment one

1st, synthesis staphylococcus aureus immunity Lysozyme associated proteins A (proteinA) and streptococcus immune globulin G Associated proteins G (proteinG) complete genome sequence；Buy plasmid pFUSEss-CHIg-rG*03 (InvivoGen, Catalog# Pfusess-rchg) resist with pFUSEss-CHIg-mG2b (InvivoGen, Catalog#pfusess-mchg2b) as rabbit source Weight chain constant area gene (mFc) pcr template of body weight chain constant area gene (rFc) and mouse source antibody；From the pET28a- of structure His-MBP carriers are MBP sequence pcr templates.

2nd, pET28a-his-MBP vector constructions

According to pMal-C2T (GenBank:JF795283.1 the MBP nucleotide sequences design polyclonal sequences of his+MBP+ in) (SEQ ID NO.9), is synthesized by Sangon Biotech (Shanghai) Co., Ltd. and is built into many grams of pET28a-his-MBP Grand carrier.

3rd, different genetic fragment combinations are designed according to each band molecular size range in luminous marker, specific combination is shown in Table 1：

The luminescent protein marker bands of table 1 are constituted

Note：pA:proteinA

pG:proteinG

According to each genetic fragment design primer, such as table 2 in upper table：

The design of primers table of table 2

4th, using gene described in table 1 as template, corresponding nucleic acid fragment, Ran Houtong are expanded by PCR with the primer in table 2 Cross agarose gel electrophoresis and separate and recover specific purpose fragment；Each fragment is subjected to digestion and connection according to the combination in table 1； Connection product is reclaimed, bacillus coli DH 5 alpha is transferred to after connecting with corresponding carrier enzyme, obtains each expression plasmid.

5th, correct expression plasmid conversion e. coli bl21 (DE3) will be sequenced, the induced expression of albumen is carried out.Specifically Experimental implementation is referred to《Molecular cloning handbook》.

6th, the albumen of different molecular weight is isolated and purified

Carrier is purified for the albumen of pET21a plasmid expression with nickel ion affinity chromatograph；Carrier is pET28a-his-MBP Plasmid expression albumen maltose affinitive layer purification.The operating method and flow of specific various affinity column purifying can To be performed with reference to protein purification laboratory manual.

7th, can luminescent protein marker acquisition

The luminous intensity of each albumen, the i.e. binding ability to antibody are detected with Western Bolt respectively；According to single slice Luminous intensity, the egg of different molecular weight is mixed according to a certain percentage, mixed result is detected by Western Bolt, And mixed proportion is adjusted, finally give the homogeneous mixture of band.Detected by being incubated the different antibodies in different genera source The stability of luminous marker bands.

8th, the luminous marker of pre-dyed acquisition

The luminous marker mixed up and pre-dyed albumen marker (Solarbio, PR1910) is pressed 1:2 ratio mixing, is obtained To pre-dyed luminescent protein marker.

<110>Zhang Hailing

<120>Pre-dyed luminescent protein marker preparation method

<160> 9

<210> 1

<211> 159

<212> PRT

<213>Artificial sequence

<400> 1

MASMTGGQQM GRGSEFAQHD EAQQNAFYQV LNMPNLNADQ RNGFIQSLKD DPSQSANVLG 60

EAQKLNDSQA PKADAQQNNF NKDQQSAFYE ILNMPNLNEA QRNGFIQSLK DDPSQSTNVL 120

GEAKKLNESQ APKADNNFNK EQQNVDKLAA ALEHHHHHH 159

<210> 2

<211> 214

<212> PRT

<213>Artificial sequence

<400> 2

MASMTGGQQM GRGSEFAQHD EAQQNAFYQV LNMPNLNADQ RNGFIQSLKD DPSQSANVLG 60

EAQKLNDSQA PKADAQQNNF NKDQQSAFYE ILNMPNLNEA QRNGFIQSLK DDPSQSTNVL 120

GEAKKLNESQ APKADNNFNK EQQNAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSANLLSE 180

AKKLNESQAP KADNKFNKNV DKLAAALEHH HHHH 214

<210> 3

<211> 326

<212> PRT

<213>Artificial sequence

<400> 3

MASMTGGQQM GRGSEFPELL GGPSVFIFPP KPKDTLMISR TPEVTCVVVD VSQDDPEVQF 60

TWYINNEQVR TARPPLREQQ FNSTIRVVST LPIAHQDWLR GKEFKCKVHN KALPAPIEKT 120

ISKARGQPLE PKVYTMGPPR EELSSRSVSL TCMINGFYPS DISVEWEKNG KAEDNYKTTP 180

AVLDSDGSYF LYSKLSVPTS EWQRGDVFTC SVMHEALHNH YTQKSISRSP GKVDPNLEGG 240

PSVFIFPPNI KDVLMISLTP KVTCVVVDVS EDDPDVRISW FVNNVEVHTA QTQTHREDYN 300

STIRVVSALP IQHQDAAALE HHHHHH 326

<210> 4

<211> 368

<212> PRT

<213>Artificial sequence

<400> 4

MASAQHDEAQ QNAFYQVLNM PNLNADQRNG FIQSLKDDPS QSANVLGEAQ KLNDSQAPKA 60

DAQQNNFNKD QQSAFYEILN MPNLNEAQRN GFIQSLKDDP SQSTNVLGEA KKLNESQAPK 120

ADNNFNKEQQ NAFYEILNMP NLNEEQRNGF IQSLKDDPSQ SANLLSEAKK LNESQAPKAD 180

NKFNKEQQNA FYEILHLPNL NEEQRNGFIQ SLKDDPSQSA NLLAEAKKLN DAQAPKADNK 240

FNKEQQNAFY EILHLPNLTE EQRNGFIQSL KDDPSVSKEI LAEAKKLNDA QEFGSSMTTY 300

KLILNGKTLK GETTTEAVDA ATAEKVFKQY ANDNGVDGEW TYDDATKTFT VTEVDKLAAA 360

LEHHHHHH 368

<210> 5

<211> 524

<212> PRT

<213>Artificial sequence

<400> 5

MASAQHDEAQ QNAFYQVLNM PNLNADQRNG FIQSLKDDPS QSANVLGEAQ KLNDSQAPKA 60

DAQQNNFNKD QQSAFYEILN MPNLNEAQRN GFIQSLKDDP SQSTNVLGEA KKLNESQAPK 120

ADNNFNKEQQ NAFYEILNMP NLNEEQRNGF IQSLKDDPSQ SANLLSEAKK LNESQAPKAD 180

NKFNKEQQNA FYEILHLPNL NEEQRNGFIQ SLKDDPSQSA NLLAEAKKLN DAQAPKADNK 240

FNKEQQNAFY EILHLPNLTE EQRNGFIQSL KDDPSVSKEI LAEAKKLNDA QEFPELLGGP 300

SVFIFPPKPK DTLMISRTPE VTCVVVDVSQ DDPEVQFTWY INNEQVRTAR PPLREQQFNS 360

TIRVVSTLPI AHQDWLRGKE FKCKVHNKAL PAPIEKTISK ARGQPLEPKV YTMGPPREEL 420

SSRSVSLTCM INGFYPSDIS VEWEKNGKAE DNYKTTPAVL DSDGSYFLYS KLSVPTSEWQ 480

RGDVFTCSVM HEALHNHYTQ KSISRSPGKV DKLAAALEHH HHHH 524

<210> 6

<211> 738

<212> PRT

<213>Artificial sequence

<400> 6

MASAQHDEAQ QNAFYQVLNM PNLNADQRNG FIQSLKDDPS QSANVLGEAQ KLNDSQAPKA 60

DAQQNNFNKD QQSAFYEILN MPNLNEAQRN GFIQSLKDDP SQSTNVLGEA KKLNESQAPK 120

ADNNFNKEQQ NAFYEILNMP NLNEEQRNGF IQSLKDDPSQ SANLLSEAKK LNESQAPKAD 180

NKFNKEQQNA FYEILHLPNL NEEQRNGFIQ SLKDDPSQSA NLLAEAKKLN DAQAPKADNK 240

FNKEQQNAFY EILHLPNLTE EQRNGFIQSL KDDPSVSKEI LAEAKKLNDA QEFPELLGGP 300

SVFIFPPKPK DTLMISRTPE VTCVVVDVSQ DDPEVQFTWY INNEQVRTAR PPLREQQFNS 360

TIRVVSTLPI AHQDWLRGKE FKCKVHNKAL PAPIEKTISK ARGQPLEPKV YTMGPPREEL 420

SSRSVSLTCM INGFYPSDIS VEWEKNGKAE DNYKTTPAVL DSDGSYFLYS KLSVPTSEWQ 480

RGDVFTCSVM HEALHNHYTQ KSISRSPGKV DPNLEGGPSV FIFPPNIKDV LMISLTPKVT 540

CVVVDVSEDD PDVRISWFVN NVEVHTAQTQ THREDYNSTI RVVSALPIQH QDWMSGKEFK 600

CKVNNKDLPS PIERTISKIK GLVRAPQVYI LPPPAEQLSR KDVSLTCLVV GFNPGDISVE 660

WTSNGHTEEN YKDTAPVLDS DGSYFIYSKL DIKTSKWEKT DSFSCNVRHE GLKNYYLKKT 720

ISRSPGKAAA LEHHHHHH 738

<210> 7

<211> 955

<212> PRT

<213>Artificial sequence

<400> 7

MGSSHHHHHH GSSMKIEEGK LVIWINGDKG YNGLAEVGKK FEKDTGIKVT VEHPDKLEEK 60

FPQVAATGDG PDIIFWAHDR FGGYAQSGLL AEITPDKAFQ DKLYPFTWDA VRYNGKLIAY 120

PIAVEALSLI YNKDLLPNPP KTWEEIPALD KELKAKGKSA LMFNLQEPYF TWPLIAADGG 180

YAFKYENGKY DIKDVGVDNA GAKAGLTFLV DLIKNKHMNA DTDYSIAEAA FNKGETAMTI 240

NGPWAWSNID TSKVNYGVTV LPTFKGQPSK PFVGVLSAGI NAASPNKELA KEFLENYLLT 300

DEGLEAVNKD KPLGAVALKS YEEELAKDPR IAATMENAQK GEIMPNIPQM SAFWYAVRTA 360

VINAASGRQT VDEALKDAQT NSSSNNNNNN NNNNLGIEEN LEVLFQGPVP GSPGSAQHDE 420

AQQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP KADAQQNNFN 480

KDQQSAFYEI LNMPNLNEAQ RNGFIQSLKD DPSQSTNVLG EAKKLNESQA PKADNNFNKE 540

QQNAFYEILN MPNLNEEQRN GFIQSLKDDP SQSANLLSEA KKLNESQAPK ADNKFNKEQQ 600

NAFYEILHLP NLNEEQRNGF IQSLKDDPSQ SANLLAEAKK LNDAQAPKAD NKFNKEQQNA 660

FYEILHLPNL TEEQRNGFIQ SLKDDPSVSK EILAEAKKLN DAQEFPELLG GPSVFIFPPK 720

PKDTLMISRT PEVTCVVVDV SQDDPEVQFT WYINNEQVRT ARPPLREQQF NSTIRVVSTL 780

PIAHQDWLRG KEFKCKVHNK ALPAPIEKTI SKARGQPLEP KVYTMGPPRE ELSSRSVSLT 840

CMINGFYPSD ISVEWEKNGK AEDNYKTTPA VLDSDGSYFL YSKLSVPTSE WQRGDVFTCS 900

VMHEALHNHY TQKSISRSPG KVDTSVDLQL EIKRASQPEL APEDPEDVEH HHHHH 955

<210> 8

<211> 1684

<212> PRT

<213>Artificial sequence

<400> 8

MGSSHHHHHH GSSMKIEEGK LVIWINGDKG YNGLAEVGKK FEKDTGIKVT VEHPDKLEEK 60

FPQVAATGDG PDIIFWAHDR FGGYAQSGLL AEITPDKAFQ DKLYPFTWDA VRYNGKLIAY 120

PIAVEALSLI YNKDLLPNPP KTWEEIPALD KELKAKGKSA LMFNLQEPYF TWPLIAADGG 180

YAFKYENGKY DIKDVGVDNA GAKAGLTFLV DLIKNKHMNA DTDYSIAEAA FNKGETAMTI 240

NGPWAWSNID TSKVNYGVTV LPTFKGQPSK PFVGVLSAGI NAASPNKELA KEFLENYLLT 300

DEGLEAVNKD KPLGAVALKS YEEELAKDPR IAATMENAQK GEIMPNIPQM SAFWYAVRTA 360

VINAASGRQT VDEALKDAQT NSSSNNNNNN NNNNLGIEEN LEVLFQGPVP GSMKIEEGKL 420

VIWINGDKGY NGLAEVGKKF EKDTGIKVTV EHPDKLEEKF PQVAATGDGP DIIFWAHDRF 480

GGYAQSGLLA EITPDKAFQD KLYPFTWDAV RYNGKLIAYP IAVEALSLIY NKDLLPNPPK 540

TWEEIPALDK ELKAKGKSAL MFNLQEPYFT WPLIAADGGY AFKYENGKYD IKDVGVDNAG 600

AKAGLTFLVD LIKNKHMNAD TDYSIAEAAF NKGETAMTIN GPWAWSNIDT SKVNYGVTVL 660

PTFKGQPSKP FVGVLSAGIN AASPNKELAK EFLENYLLTD EGLEAVNKDK PLGAVALKSY 720

EEELAKDPRI AATMENAQKG EIMPNIPQMS AFWYAVRTAV INAASGRQTV DEALKDAQTE 780

FMKIEEGKLV IWINGDKGYN GLAEVGKKFE KDTGIKVTVE HPDKLEEKFP QVAATGDGPD 840

IIFWAHDRFG GYAQSGLLAE ITPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN 900

KDLLPNPPKT WEEIPALDKE LKAKGKSALM FNLQEPYFTW PLIAADGGYA FKYENGKYDI 960

KDVGVDNAGA KAGLTFLVDL IKNKHMNADT DYSIAEAAFN KGETAMTING PWAWSNIDTS 1020

KVNYGVTVLP TFKGQPSKPF VGVLSAGINA ASPNKELAKE FLENYLLTDE GLEAVNKDKP 1080

LGAVALKSYE EELAKDPRIA ATMENAQKGE IMPNIPQMSA FWYAVRTAVI NAASGRQTVD 1140

EALKDAQTVD AQHDEAQQNA FYQVLNMPNL NADQRNGFIQ SLKDDPSQSA NVLGEAQKLN 1200

DSQAPKADAQ QNNFNKDQQS AFYEILNMPN LNEAQRNGFI QSLKDDPSQS TNVLGEAKKL 1260

NESQAPKADN NFNKEQQNAF YEILNMPNLN EEQRNGFIQS LKDDPSQSAN LLSEAKKLNE 1320

SQAPKADNKF NKEQQNAFYE ILHLPNLNEE QRNGFIQSLK DDPSQSANLL AEAKKLNDAQ 1380

APKADNKFNK EQQNAFYEIL HLPNLTEEQR NGFIQSLKDD PSVSKEILAE AKKLNDAQEF 1440

PELLGGPSVF IFPPKPKDTL MISRTPEVTC VVVDVSQDDP EVQFTWYINN EQVRTARPPL 1500

REQQFNSTIR VVSTLPIAHQ DWLRGKEFKC KVHNKALPAP IEKTISKARG QPLEPKVYTM 1560

GPPREELSSR SVSLTCMING FYPSDISVEW EKNGKAEDNY KTTPAVLDSD GSYFLYSKLS 1620

VPTSEWQRGD VFTCSVMHEA LHNHYTQKSI SRSPGKLQLE IKRASQPELA PEDPEDVEHH 1680

HHHH 1684

<210> 9

<211> 1350

<212> PRT

<213>Artificial sequence

<400> 9

ATGGGTTCTT CTCACCATCA CCATCACCAT GGTTCTTCTA TGAAAATCGA AGAAGGTAAA 60

CTGGTAATCT GGATTAACGG CGATAAAGGC TATAACGGTC TCGCTGAAGT CGGTAAGAAA 120

TTCGAGAAAG ATACCGGAAT TAAAGTCACC GTTGAGCATC CGGATAAACT GGAAGAGAAA 180

TTCCCACAGG TTGCGGCAAC TGGCGATGGC CCTGACATTA TCTTCTGGGC ACACGACCGC 240

TTTGGTGGCT ACGCTCAATC TGGCCTGTTG GCTGAAATCA CCCCGGACAA AGCGTTCCAG 300

GACAAGCTGT ATCCGTTTAC CTGGGATGCC GTACGTTACA ACGGCAAGCT GATTGCTTAC 360

CCGATCGCTG TTGAAGCGTT ATCGCTGATT TATAACAAAG ATCTGCTGCC GAACCCGCCA 420

AAAACCTGGG AAGAGATCCC GGCGCTGGAT AAAGAACTGA AAGCGAAAGG TAAGAGCGCG 480

CTGATGTTCA ACCTGCAAGA ACCGTACTTC ACCTGGCCGC TGATTGCTGC TGACGGGGGT 540

TATGCGTTCA AGTATGAAAA CGGCAAGTAC GACATTAAAG ACGTGGGCGT GGATAACGCT 600

GGCGCGAAAG CGGGTCTGAC CTTCCTGGTT GACCTGATTA AAAACAAACA CATGAATGCA 660

GACACCGATT ACTCCATCGC AGAAGCTGCC TTTAATAAAG GCGAAACAGC GATGACCATC 720

AACGGCCCGT GGGCATGGTC CAACATCGAC ACCAGCAAAG TGAATTATGG TGTAACGGTA 780

CTGCCGACCT TCAAGGGTCA ACCATCCAAA CCGTTCGTTG GCGTGCTGAG CGCAGGTATT 840

AACGCCGCCA GTCCGAACAA AGAGCTGGCA AAAGAGTTCC TCGAAAACTA TCTGCTGACT 900

GATGAAGGTC TGGAAGCGGT TAATAAAGAC AAACCGCTGG GTGCCGTAGC GCTGAAGTCT 960

TACGAGGAAG AGTTGGCGAA AGATCCACGT ATTGCCGCCA CTATGGAAAA CGCCCAGAAA 1020

GGTGAAATCA TGCCGAACAT CCCGCAGATG TCCGCTTTCT GGTATGCCGT GCGTACTGCG 1080

GTGATCAACG CCGCCAGCGG TCGTCAGACT GTCGATGAAG CCCTGAAAGA CGCGCAGACT 1140

AATTCGAGCT CGAACAACAA CAACAATAAC AATAACAACA ACCTCGGGAT CGAGGAAAAC 1200

CTGGAAGTTC TGTTCCAGGG GCCGGTACCG GGATCCCCGG AATTCAAGCT TACTAGTGTC 1260

GACCTGCAGC TCGAGATCAA ACGGGCTAGC CAGCCAGAAC TCGCCCCGGA AGACCCCGAG 1320

GATGTCGAGC ACCACCACCA CCACCACTGA 1350

Claims

1. a kind of pre-dyed luminescent protein marker preparation method, its step includes：

A, the CH genes for obtaining proteinA genes, proteinG genes, MBP genes, mouse and rabbit source antibody, are noted according to NCBI The IgG Binding domain for determining proteinA, proteinG are released, it is big according to each albumen in pre-dyed luminescent protein marker It is small that proteinA genes, proteinG genes, MBP genes, CH genes are cut or combined, albumen is retained in cutting process Binding domain；

B, proteinA, proteinG, MBP and CH gene after cutting or combination are connected on expression vector carried out respectively Expression and purification, the albumen purified；

D, the luminescent protein marker mixed up and pre-dyed albumen marker mixed in different ratio, by observing transferring film caudacoria Band brightness selection proper mixture ratio example on X films after upper stripe depth and development.

2. pre-dyed luminescent protein marker according to claim 1 preparation method, it is characterised in that：The expression vector For pET21a or pET28a.

3. pre-dyed luminescent protein marker according to claim 1 preparation method, it is characterised in that：The host cell For E.coli.

4. pre-dyed luminescent protein marker according to claim 1 preparation method, it is characterised in that：The luminescent protein Marker molecular weight is from 16kDa to 200kDa.