CN106119286A - Expression vector and high efficient expression thereof and the method preparing human cystatin C albumen - Google Patents

Expression vector and high efficient expression thereof and the method preparing human cystatin C albumen Download PDF

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Publication number
CN106119286A
CN106119286A CN201610647346.XA CN201610647346A CN106119286A CN 106119286 A CN106119286 A CN 106119286A CN 201610647346 A CN201610647346 A CN 201610647346A CN 106119286 A CN106119286 A CN 106119286A
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China
Prior art keywords
expression vector
human cystatin
recombinant human
albumen
signal peptide
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CN201610647346.XA
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Chinese (zh)
Inventor
崔利兰
刘虹均
由超
丁雨
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WUJIANG NOVOPROTEIN TECHNOLOGY Co Ltd
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WUJIANG NOVOPROTEIN TECHNOLOGY Co Ltd
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Priority to CN201610647346.XA priority Critical patent/CN106119286A/en
Publication of CN106119286A publication Critical patent/CN106119286A/en
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Abstract

The present invention provides a kind of expression vector and high efficient expression thereof and the method preparing human cystatin C albumen, expression vector, including mammalian expression vector and the encoding gene of CD33 protein signal peptide, the sequence of the encoding gene of described CD33 protein signal peptide is the nucleotide sequence shown in SEQ ID NO:1；Said method comprising the steps of: 1) encoding gene of CD33 protein signal peptide is inserted in mammalian expression vector, obtain recombinant human cystatin C expression vector, the encoding gene of described CD33 protein signal peptide is as shown in SEQ ID NO:1；2) again by described recombinant human cystatin C expression vector and host cell infection, transgenic cell is obtained；3) more described transgenic cell is expressed, purification, obtain recombinant human cystatin C albumen.

Description

Expression vector and high efficient expression thereof and the method preparing human cystatin C albumen
Technical field
The invention belongs to biological technical field, be specifically related to a kind of expression vector and high efficient expression thereof and prepare human cystatin The method of C protein.
Background technology
Bladder chalone C (CysC), also known as cysteine proteinase inhibitor C, is a kind of cystatin. CysC is widely used in the monitoring of the renal function of diabetes, pediatric disease, renal transplantation and Patients of Spinal.Additionally, it The aspects such as cerebrovascular disease, heart disease, tumor and hepatic fibrosis the most also embody the most wide application prospect.In early days Diagnosis kidney injury aspect, CysC has higher sensitivity, is the most substantially to disclosure satisfy that glomerular filtration rate (GFR) The material that preferable endogenous mark requires, thus, combining other diagnosis indexs has important facing to the early diagnosis of kidney disease Bed meaning.But due to some technical shortage, cause CysC diagnostic reagent and the standard substance used on domestic market Mainly provided by offshore company, expensive, make patient and society's medical treatment cost higher, current clinical practice is less.
The preparation method of some bladder chalone C albumen is had been disclosed at present, but is mostly to utilize prokaryotic expression, lack Incapable persons' bladder chalone C albumen post-treatment and modification.Additionally, the protein yield ratio again utilizing eukaryotic expression system to prepare is relatively low, about 3mg/L, relatively costly, it is unfavorable for producing on a large scale and applying.
Summary of the invention
Therefore, it is an object of the invention to provide a kind of expression vector and high efficient expression thereof and preparation natural human bladder chalone C egg White method, such that it is able to make output increased 7-8 times of human cystatin C albumen, and method is simple, low cost.
On the one hand, the present invention provides a kind of recombinant human cystatin C expression vector, including mammalian expression vector and CD33 The encoding gene of protein signal peptide, the sequence of the encoding gene of described CD33 protein signal peptide is the core shown in SEQ ID NO:1 Nucleotide sequence.
Preferably, described mammalian expression vector is pcDNATM3.3-TOPO, PTT5 or pcDNA4/His A.
Preferably, the sequence of described CD33 protein signal peptide is the aminoacid sequence shown in SEQ ID NO:2.
Preferably, described expression vector also includes Kozac sequence, the nucleotide sequence such as SEQ of described Kozac sequence Nucleotide sequence shown in ID NO:5, the introducing of this Kozac sequence can strengthen eukaryotic gene translation efficiency.
On the other hand, the present invention also provides for a kind of new expression vector pcDNATM3.3-TOPO-CD33, including above-mentioned recombined human Bladder chalone C expression vector.
Preferably, described cell is HEK293 cell, NSO cell or Chinese hamster ovary celI.
Further aspect, the present invention also provides for a kind of recombinant human cystatin C expression vector for preparing recombinant human cystatin C Application in albumen.
Another aspect, the present invention also provides for a kind of cell for preparing the application in recombinant human cystatin C albumen.
Another further aspect, the present invention also provides for the preparation method of a kind of recombinant human cystatin C albumen, comprises the following steps:
1) encoding gene of CD33 protein signal peptide is inserted in mammalian expression vector, obtain recombinant human cystatin C Expression vector, the encoding gene of described CD33 protein signal peptide is as shown in SEQ ID NO:1；
3) more described recombiant plasmid is expressed in cell, purification, obtain recombinant human cystatin C albumen.
Preferably, in step 1) in, the encoding gene of described CD33 protein signal peptide is by becoming according to human cystatin C genes Ripe peptide primers clone obtain, the gene order of forward primer as shown in SEQ ID NO:3, the gene of downstream primer Sequence is as shown in SEQ ID NO:4.
The present invention utilizes mammalian cell expression vector, by guide people bladder chalone C protein excretion expression signal peptide Optimize, have employed the signal peptide of CD33 albumen so that the expression in HEK293 cell of the recombinant human cystatin C albumen reaches The expression of 23mg/L, improves 7-8 times than commonsense method of the prior art, overcomes prokaryotic expression system cannot complete translation On the basis of the shortcoming of post-treatment and modification, suckling expression system is utilized to obtain the recombinant human cystatin C albumen of high yield；Logical Cross nickel post one step purification, analyze through SDS-PAGE and obtain purity the recombinant human cystatin C albumen of 95%, divide through Sec-HPLC Analysis, purity reaches more than 99.9%.
Accompanying drawing explanation
Hereinafter, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:
Fig. 1 is mammalian cell expression vector pcDNATM3.3-TOPO plasmid map.
Fig. 2 is the SDS-PAGE reduction of the albumen of recombinant human cystatin C after purification in the present invention and non-reduced electrophoretogram, its Middle R represents the electrophoresis result under reducing condition, and NR represents the electrophoresis result under non-reduced state, and MK represents albumen marker.
Fig. 3 is the Sec-HPLC chromatogram of the recombinant human cystatin C albumen in the present invention.
Detailed description of the invention
Unless specifically stated otherwise, reagent used in following example and medicine all can from regular distributor available from.
The signal peptide optimization of embodiment 1 human cystatin C albumen
CD33 albumen has hypersecretion native signal peptide sequence, and this method uses online signal peptide prediction website SignalP 4.1Server http://www.cbs.dtu.dk/services/SignalP/ presses down with CD33 protein signal peptide replacement people's Guang The signal peptide cutting site of the secretion of human cystatin C mature peptide is predicted by element C native signal peptide, result display cleavage site Between the alanine 16 and methionine 17 of CD33 signal peptide, illustrate that CD33 protein signal peptide can be with guide people bladder chalone C The secretion of mature peptide.
The structure of embodiment 2 human cystatin C mammalian expression vector, expresses and purification
1. the transformation of human cystatin C mammalian expression vector
Mammalian expression vector pcDNATM3.3-TOPO (Invitrogen) is engineered introduces Kozac at TOPO cloning site Sequence (SEQ ID NO:5:GCCGCCACC is used for strengthening eukaryotic gene translation efficiency) and CD33 protein signal peptide (nucleotides sequence Row: SEQ ID NO:1；Aminoacid sequence: SEQ ID NO:2), after signal peptide, introduce Xho I and EcoR I enzyme action simultaneously Site, in order to the insertion of gene, the named pcDNA of improved carrierTM3.3-TOPO-CD33, pcDNATM3.3-TOPO- Vector linearization is processed by CD33 vector plasmid with Xho I and EcoR I double digestion, and glue reclaims.
2. the structure of human cystatin C mammalian expression vector
According to pcDNATM3.3-TOPO-CD33 carrier and the primers of human cystatin C genes mature peptide:
Forward primer:
GTGGGCAGGGGCCCTGGCTATGTCCAGTCCCGGCAAGCCGCCG(SEQ ID NO:3)
Downstream primer:
CATTACTAACCGGTGAATTCTTAATGATGATGATGATGATGGGCGTCCTGACAGGTGGA(SEQ ID NO:4)。
Press down expanding people's Guang under 55 DEG C of annealing conditions with the upstream and downstream primer of carrier homologous sequence (underscore part) Element C code mature peptide genetic fragment (79-438bp), glue reclaims, and uses NovRec seamless cloning test kit seamless It is connected to pcDNATMOn 3.3-TOPO-CD33 carrier after double digestion linearization process.Convert DH5a, utilize bacterium colony PCR to enter Row positive identification, carries out order-checking by the recon being accredited as the positive and identifies.By checking order, correct clone arranges to take out in plasmid, is used for The transfection of HEK293 cell.
3. human cystatin C expression in HEK293 cell (purchased from Roche Holding Ag)
3.1. transfecting first 1 day and passing on density is 0.6 × 106It is 0.3 × 10 that individual/ml or front passes on density for 2 days6Individual/ml；
3.2. transfection carried out cell density statistics, when density is 1-1.4 × 10 same day6Individual/ml, vigor > 80% time, be used for Transfection；
3.3. transfection composite preparation: each project needs standby two/centrifuge tube/culture bottle, as a example by 20ml, puts respectively Put:
PipeMiddle addition 600 μ l PBS/ culture medium, 20 μ g plasmids, mixing, stand 5min；
PipeMiddle addition 600 μ l PBS/ culture medium, 80 μ g Polyetherimide (PEI) (PEI/ plasmid=4:
1), mixing, stand 5min；
3.4. the PEI after dilution is added in the plasmid to dilution, the most reverse 6-10 time, mix homogeneously, it is configured to (during transfection composite preparation, action must be softly rapid, mix homogeneously, to prevent partial dna-PEI to be combined for transfection composite The generation of thing)；
3.5. transfection composite stands after 15-20min, and single at the uniform velocity adds in cell culture that (dropping limit, limit is rocked carefully Born of the same parents' culture)；
3.6. in 37, 8%CO2, 130rpm, shaking table is cultivated, is carried out product collection detection after 5 days.
4. the purification of recombinant human cystatin C albumen
4.1 sample pretreatment
20ml culture supernatant adds 20mM PB, 200mM NaCl and regulates pH to 7.5.
4.2 affinity purification
Pillar (Column): Chelating SFF (column volume 0.3ml)
Buffer A (Buffer A): 20mM PB, 150mM NaCl, pH7.5
Buffer B (Buffer B): 20mM PB, 150mM NaCl, 500mM Imizadole, pH7.5
Purge process: process with Buffer A level pad.Loading, collects effluent.Loading is complete, then uses Buffer A balances pillar at least 10ml.With the imidazole buffer eluting containing 50mM, 500mM after balance, and collect eluent respectively.Often Individual gradient separate collection 0.3ml, 0.9ml, 0.7ml.The albumen collected is analyzed through SDS-PAGE, SDS-PAGE analysis-reduction and non- Electrophoretogram under reducing condition, result is as in figure 2 it is shown, gained albumen size is about 14kd, in the same size with theory, and through egg Single through gel imaging instrument scanning display band in vain, purity higher (Fig. 2)；Additionally, by prepared albumen through Sec-HPLC (Shimadzu LC_20AT) (by detector (SPD_20A), detection wavelength is 280nm, and chromatographic column is MAbPac SEC-1, with 20mM in detection Phosphate buffer solution, 150mM sodium chloride, PH7.4 is as flowing phase, and flow velocity is 0.2mL/min, and purity assay reaches more than 95% (Fig. 3), show to have obtained human cystatin C albumen by the method purification, and purity is high.
Further, it is calculated recombinant human cystatin C albumen according to A280 (nm) UV Absorption method mensuration protein concentration to exist In HEK293 cell, expression and purification gained yield is 23mg/L, improves 7-8 times than commonsense method of the prior art, improves Expressing yield, and purifying process is simple, low cost, yield is high, and purity of protein is high.
Embodiment 3
By embodiment 2 checks order correct in take out plasmid, turn according to the method mentioning transfected HEK 293 in embodiment 2 Transfected cho cell (Roche), carries out expressing lab scale, recombinates Chinese hamster ovary celI juice by the purification process mentioned in embodiment 2 The purification of human cystatin C albumen.Result shows, calculates through A280 (nm) detection, and recombinant human cystatin C albumen is at Chinese hamster ovary celI Expression can arrive 15mg/L.
Embodiment 4
With PTT5 as expression vector, seamless clone by CD33 protein signal peptide and human cystatin C protein maturation peptide gene and C-6His is connected to PTT5 expression vector (being presented by Yves Durocher laboratory).Similarly, in correct for clone, matter is taken out The transfection method that grain is mentioned by embodiment 2 carries out transfected HEK 293 (Roche), and the purification process mentioned by embodiment 2 is to carefully Intracrine liquid carries out protein purification, and result shows, recombinant human cystatin C expressing quantity is about 18mg/L.The above is only The preferred embodiment of the present invention, is not limited to the present invention, it is noted that for those skilled in the art For, on the premise of without departing from the technology of the present invention principle, it is also possible to make some improvement and modification, these improve with modification also Should be regarded as protection scope of the present invention.

Claims (10)

1. a recombinant human cystatin C expression vector, including the coding base of mammalian expression vector and CD33 protein signal peptide Cause, the nucleotides sequence of the encoding gene of described CD33 protein signal peptide is classified as the nucleotide sequence shown in SEQ ID NO:1.
Recombinant human cystatin C expression vector the most according to claim 1, it is characterised in that described mammal is expressed and carries Body is pcDNATM3.3-TOPO, PTT5 or pcDNA4/His A.
Recombinant human cystatin C expression vector the most according to claim 1 and 2, it is characterised in that described CD33 protein signal The sequence of peptide is the aminoacid sequence shown in SEQ ID NO:2.
Recombinant human cystatin C expression vector the most according to claim 1 and 2, it is characterised in that in described expression vector also Including Kozac sequence, the nucleotide sequence of described Kozac sequence nucleotide sequence as shown in SEQ ID NO:5.
5. a transgenic cell, including the recombinant human cystatin C expression vector according to any one of Claims 1-4.
6. want the transgenic cell described in 5 according to right, it is characterised in that described cell be HEK293 cell, NSO cell or Chinese hamster ovary celI.
Recombinant human cystatin C expression vector the most according to any one of claim 1 to 4 presses down being used for preparing recombined human Guang Application in element C protein.
8. according to the transgenic cell described in claim 5 or 6 for preparing the application in recombinant human cystatin C albumen.
9. a preparation method for recombinant human cystatin C albumen, comprises the following steps:
1) encoding gene of CD33 protein signal peptide is inserted in mammalian expression vector, obtain recombinant human cystatin C and express Carrier, the encoding gene of described CD33 protein signal peptide is as shown in SEQ ID NO:1；
2) again by described recombinant human cystatin C expression vector and host cell infection, transgenic cell is obtained；
3) more described transgenic cell is expressed, purification, obtain recombinant human cystatin C albumen.
The preparation method of recombinant human cystatin C albumen the most according to claim 9, it is characterised in that in step 1) in, The encoding gene of described CD33 protein signal peptide obtains by cloning according to the primers of human cystatin C genes mature peptide , the gene order of forward primer is as shown in SEQ ID NO:3, and the gene order of downstream primer is as shown in SEQ ID NO:4.
CN201610647346.XA 2016-08-10 2016-08-10 Expression vector and high efficient expression thereof and the method preparing human cystatin C albumen Pending CN106119286A (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009384A1 (en) * 1987-05-22 1988-12-01 Novo-Nordisk A/S Method of producing cystatin c or modifications hereof and dna-sequence for use when carrying out the method
CN101413001A (en) * 2008-11-28 2009-04-22 四川省迈克科技有限责任公司 Recombinant human cystatin C genes, and expression and use thereof
CN102071214A (en) * 2010-06-30 2011-05-25 武汉生之源生物科技有限公司 Construction, expression and purification of human cystatin C eucaryon expression vector
CN103237560A (en) * 2010-10-15 2013-08-07 葛兰素史密丝克莱恩生物有限公司 Cytomegalovirus GB antigen
CN105254764A (en) * 2015-08-27 2016-01-20 上海康岱生物医药技术有限公司 ACVR1-Fc fusion protein, and preparation method and application thereof
CN105622752A (en) * 2016-01-28 2016-06-01 百奇生物科技（苏州）有限公司 Procalcitonin (PCT) monoclonal antibody pair, and preparation method and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009384A1 (en) * 1987-05-22 1988-12-01 Novo-Nordisk A/S Method of producing cystatin c or modifications hereof and dna-sequence for use when carrying out the method
CN101413001A (en) * 2008-11-28 2009-04-22 四川省迈克科技有限责任公司 Recombinant human cystatin C genes, and expression and use thereof
CN102071214A (en) * 2010-06-30 2011-05-25 武汉生之源生物科技有限公司 Construction, expression and purification of human cystatin C eucaryon expression vector
CN103237560A (en) * 2010-10-15 2013-08-07 葛兰素史密丝克莱恩生物有限公司 Cytomegalovirus GB antigen
CN105254764A (en) * 2015-08-27 2016-01-20 上海康岱生物医药技术有限公司 ACVR1-Fc fusion protein, and preparation method and application thereof
CN105622752A (en) * 2016-01-28 2016-06-01 百奇生物科技（苏州）有限公司 Procalcitonin (PCT) monoclonal antibody pair, and preparation method and application thereof

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