CN107383208A - Luminescent protein marker preparation method - Google Patents

Luminescent protein marker preparation method Download PDF

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Publication number
CN107383208A
CN107383208A CN201710708173.2A CN201710708173A CN107383208A CN 107383208 A CN107383208 A CN 107383208A CN 201710708173 A CN201710708173 A CN 201710708173A CN 107383208 A CN107383208 A CN 107383208A
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genes
luminescent protein
albumen
marker
protein marker
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方卫斌
张海灵
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Tian Yue (beijing) Biotechnology LLC
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Tian Yue (beijing) Biotechnology LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4722—G-proteins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Peptides Or Proteins (AREA)

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 genes, 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, obtains luminescent protein marker.The luminescent protein marker of the present invention can identify the primary antibody or secondary antibody of separate sources, add the binding site of albumen, strengthen optical signal.The luminescent protein marker of the present invention need not add extra antibody, and display can be exposed on X films need to be only incubated with primary antibody secondary antibody simultaneously after；Luminescent protein can combining source in the IgG or anti-rabbit of the species such as people, rat, mouse, rabbit and the secondary antibody of mouse；Compared with the luminous marker of other companies, there is broader indicating range, molecular weight is from 16kDa to 200kDa.

Description

Luminescent protein marker preparation method
Technical field
The present invention relates to a kind of luminescent protein marker preparation method, belong to molecular biology and genetic engineering field.
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.Marker points of albumen is common marker, in advance Contaminate marker and luminous marker.Commonly marker bands can just be seen only after coomassie brilliant blue staining, therefore can only For
In SDS-PAGE electrophoresis.Western Blot need one kind to be used for tracing detection positive antigen signals and transferring film efficiency Scale, then generate pre-dyed albumen Marker.Pre-dyed albumen Marker is by each albumen in general proteins marker (or polypeptide) passes through chemical modification, is formed with the dyestuff covalent coupling of a kind of blueness or other colors.Pre-dyed albumen marker's Occur bringing great convenience to WesternBlot, researcher, can be according to film blue (or other colors) albumen after transferring film Marker size determines the Position Approximate of purpose band, so as to carry out the processing such as shearing to film.But pre-dyed albumen Marker can not expose on film, and exposure signal needs to be judged according to the pre-dyed albumen Marker on film, in addition pre-dyed egg Mobility changes white Marker in SDS-PAGE electrophoresis processes after chemical modification, and 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 blot.Most of companies such as Thermo are using the protein groups for being capable of binding antibody IgG Into luminous marker composition.
But certain deficiency all be present in current product, biotinylated albumen marker need to add antibiotin- HRP antibody or streptavidin-HRP, this causes experimental implementation to become cumbersome.ProteinA and ProteinG resist to different plant species The combination of body is selective, causes to show on X films for some antibody.Moreover, it can expose mostly at present Marker indicating range is limited, and clear and definite indicative function can not be played for the albumen of macromolecule.
The content of the invention
A kind of the present invention is intended to provide preparation method for the luminescent protein marker that need not add additional agents.
The purpose of the present invention is achieved through the following technical solutions：
A kind of luminescent protein marker preparation method, its step include：
A, the heavy chain constant region base of proteinA genes, proteinG genes, MBP genes, mouse and rabbit source antibody is obtained Because of (CH genes), the IgG Binding domain for determining proteinA, proteinG are annotated according to NCBI, are lighted according to pre-dyed In albumen marker each albumen size to proteinA genes, proteinG genes, MBP genes, CH genes are cut or group Close, the Binding domain of albumen are retained in cutting process；
B, proteinA, proteinG, MBP and CH gene after cutting or combination are connected on expression vector respectively Carry out expression and purification, the albumen purified；
C, the albumen of different molecular weight is mixed according to a certain percentage, obtains luminescent protein marker.
Preferably, the expression vector is pET21a or pET28a.
Preferably, the host cell is E.coli.
Preferably, the albumen marker molecular weight is from 16kDa to 200kDa.
The luminescent protein marker of the present invention 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 in marker Albumen can identify the primary antibody or secondary antibody of a variety of separate sources, add the binding site of albumen, strengthen optical signal.This hair Bright luminescent protein marker need not add extra antibody, such as anti-biotin antibodies-HRP or streptavidin-HRP, only Display can be exposed on X films need to be incubated with primary antibody secondary antibody simultaneously after；Luminescent protein can combining source in people, rat, mouse, The IgG or anti-rabbit of the species such as rabbit and the secondary antibody of mouse；Compared with the luminous marker of other companies, there is broader instruction model Enclose, molecular weight is from 16kDa to 200kDa.
Brief description of the drawings
Fig. 1 is the electrophoretogram after luminescent protein marker exposures.
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, staphylococcus aureus immunity Lysozyme associated proteins A (proteinA) and streptococcus immune globulin G is synthesized 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), synthesized by Sangon Biotech (Shanghai) Co., Ltd. and be built into more grams of pET28a-his-MBP Grand carrier.
3rd, different genetic fragment combinations is 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 form
Note：pA:proteinA
pG:proteinG
According to each genetic fragment design primer in upper table, such as table 2：
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, carry out the induced expression of albumen.Specifically Experimental implementation refers to《Molecular cloning handbook》.
6th, the albumen of different molecular weight isolates and purifies
Carrier is that the albumen of pET21a plasmid expression is purified 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.
<110>Zhang Hailing
<120>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 (4)

1. a kind of luminescent protein marker preparation method, its step include：
A, the weight chain constant area gene (CH of proteinA genes, proteinG genes, MBP genes, mouse and rabbit source antibody is obtained Gene), the IgG Binding domain for determining proteinA, proteinG are annotated according to NCBI, according to pre-dyed luminescent protein Each albumen size is cut or combined to proteinA genes, proteinG genes, MBP genes, CH genes in marker, cuts Retain the Binding domain of albumen during cutting；
B, proteinA, proteinG, MBP and CH gene after cutting or combination are connected on expression vector and carried out respectively Expression and purification, the albumen purified；
C, the albumen of different molecular weight is mixed according to a certain percentage, obtains luminescent protein marker.
2. luminescent protein marker according to claim 1 preparation method, it is characterised in that：The expression vector is PET21a or pET28a.
3. luminescent protein marker according to claim 1 preparation method, it is characterised in that：The host cell is E.coli。
4. luminescent protein marker according to claim 1 preparation method, it is characterised in that：The albumen marker points Son amount is from 16kDa to 200kDa.
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CN110408639A (en) * 2019-08-01 2019-11-05 天德悦（北京）生物科技有限责任公司 Pre-dyed albumen marker and preparation method thereof, application can be exposed
CN111257557A (en) * 2020-01-23 2020-06-09 武汉爱博泰克生物科技有限公司 Immunoblotting one-step method
CN113461796A (en) * 2021-08-04 2021-10-01 深圳晶蛋生物医药科技有限公司 Recombinant SGLT2 protein and application thereof
CN113970644A (en) * 2021-12-24 2022-01-25 天德瑞(北京)生物科技有限公司 Working concentration detection method based on HRP (horse radish peroxidase) labeled protein

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CN101421619A (en) * 2004-03-25 2009-04-29 邹松 Reagents, methods and kits for the universal rapid immuno-detection
CN101556287A (en) * 2009-02-26 2009-10-14 杭州松华生物科技有限公司 Novel protein molecular weight standard and preparation method thereof
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CN110408639A (en) * 2019-08-01 2019-11-05 天德悦（北京）生物科技有限责任公司 Pre-dyed albumen marker and preparation method thereof, application can be exposed
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CN113970644A (en) * 2021-12-24 2022-01-25 天德瑞(北京)生物科技有限公司 Working concentration detection method based on HRP (horse radish peroxidase) labeled protein

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