CN108250304A - A kind of preparation method of the cyanobacteria phytochrome fluorescent marker of fluorescent orange - Google Patents

A kind of preparation method of the cyanobacteria phytochrome fluorescent marker of fluorescent orange Download PDF

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CN108250304A
CN108250304A CN201711460150.0A CN201711460150A CN108250304A CN 108250304 A CN108250304 A CN 108250304A CN 201711460150 A CN201711460150 A CN 201711460150A CN 108250304 A CN108250304 A CN 108250304A
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fusion protein
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ala
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王小明
宋建勋
夏坤
付卫雷
佟顺刚
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Guangzhou Tianbao Songyuan Biology Science & Technology Development Co Ltd
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • 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
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

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Abstract

The invention discloses a kind of preparation method of the cyanobacteria phytochrome fluorescent marker of fluorescent orange, fusion protein includes the 1st structural proteins and Streptavidin of the GAF structural domains of cyanobacteria phytochrome protein all2699.The fusion protein sequence of the present invention is easy to stablize expression in microorganism, and the acquisition for having evaded high-purity natural phycobniliprotein and cyanobacteria phytochrome is difficult, and manufacturing cost is higher；The use of chemical modifier；The shortcomings of naturally phycobniliprotein polymeric species are unstable.The expression of the present invention is participated in without phycobniliprotein lyase, reduces the gene dosage of conversion, improves strain stability and screening efficiency.Simultaneously by optimizing fermentation medium and fermentation condition, the yield of fusion protein is substantially increased.

Description

A kind of preparation method of the cyanobacteria phytochrome fluorescent marker of fluorescent orange
Technical field
The present invention relates to expressing fusion protein field, more particularly to a kind of preparation method of phytochrome fluorescent marker.
Background technology
Fluorescence probe (or fluorescent marker) is the important foundation raw material of fluorescence immunoassay detection technique, mainly passes through chemistry The same biotin of fluorogenic substrate-Streptavidin system is combined and obtained, fluorescence signal can be further amplified, raising is exempted from by modification method The sensitivity of epidemic disease detection.
Natural phycobniliprotein is one of most common fluorogenic substrate, and natural structure is generally six aggressiveness, and subunit is by de- auxiliary Base albumen and phycobilin are formed by lyase catalyzed combination.Phycobilin then passes through heme oxidase by ferroheme (HO1) and various biliverdin reductases are catalyzed to be formed, and common phycobilin has a phycoerythrobilin (PEB), phycourobilin (PUB), Phycocyanobilin (PCB) and algae purple Choline (PVB).By combining different phycobilins, phycobniliprotein absorbs 400nm~700nm In the range of different-waveband light after, different fluorescence can be sent out.
Traditional Zao Bie Ruan Gang Tang preparation flow is to obtain high-purity natural phycobniliprotein first, then passes through Chemical modification method is combined with biotin-Streptavidin system, immune detection is used further to after being further purified.But high-purity Natural phycobniliprotein obtain difficult, manufacturing cost is high, while structural instability, polymeric species are changeable.This causes phycobniliprotein The cost of fluorescence probe remains high.
It is a kind of lower-cost protein preparation method by microbial expression fusion protein.However the expression of fusion protein The design of fusion and the limitation of zymotechnique are suffered from, the expression that can not stablize, it is difficult to obtain the conjunction of stable quality Lattice product.
Suitable albumen segment how to be selected to be merged so that it can more steadily fermenting and producing, be have it is to be solved The technical issues of.
Invention content
It is an object of the invention to overcome the deficiencies of the prior art and provide it is a kind of be easy to expression fusion protein sequence and its Expression.
The technical solution used in the present invention is：
A kind of fusion protein, the 1st structure egg of the GAF structural domains including cyanobacteria phytochrome protein all2699 White and Streptavidin.
As being further improved for above-mentioned fusion protein, affinity labeling is also added in fusion protein.
As being further improved for above-mentioned fusion protein, the sequence of fusion protein is： MGSSHHHHHHSQDPEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWT VAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGTDDDDKAMADG LELEDIITATTAEVRALLGTDRVMIYKFHPDGSGQVIAESIYENRLPSLLGLNFPADDIPPQARELLVKSKVRSIVD VATGMIGQSPVHDLETGELISEDICYRPVDSCHVEYLTAMGVKSSVVAPIFCQDELWGLLVSHHSENRTVSEDELEA MQMIVDQLAVAIAQSH。
Express the nucleotide sequence of above-mentioned fusion protein.Further, nucleotide sequence carries out codon according to host strain Optimization.
Plasmid inserted with above-mentioned nucleotide sequence.
As being further improved for above-mentioned plasmid, plasmid is pET Duet vector plasmids, and nucleotide sequence passes through digestion position Point BamH I, EcoR I are inserted into the multiple cloning sites 1 of pET Duet vector plasmids.
A kind of preparation method of fusion protein, includes the following steps：
1) according to the sequence of above-mentioned fusion protein, design obtains antigen-4 fusion protein gene sequence；
2) antigen-4 fusion protein gene sequence is inserted into plasmid, obtains fusion protein plasmid；
3) by fusion protein plasmid and the related gene of expression phycoerythrobilin synthesis：Heme oxidase gene ho1, courage are green The plasmid corotation of plain reductase gene pebS enters Escherichia coli, obtains genetic engineering bacterium；
4) it is inoculated with genetic engineering bacterium in the medium using three-level inoculation method, 37 DEG C of fermented and cultureds are to growing early period；
5) 20 DEG C are cooled to, cultivates 0.8~1.2h；
6) 25 DEG C are warming up to, adds in 14~16h of lactose Fiber differentiation that whole mass concentration is 1.3~1.6%；
7) thalline extraction is collected, purifying obtains fusion protein.
As being further improved for above-mentioned preparation method, the quality group of fermentation medium becomes：Peptone 1%, dusty yeast 0.5%, sodium chloride 1%, glycerine 0.4%, 0.004%~0.04% proteinic powder of porcine or ferroheme, surplus is water, pH value tune To 7.2.
As being further improved for above-mentioned preparation method, the lactose Fiber differentiation that whole mass concentration is 1.5% is added in.
The beneficial effects of the invention are as follows：
The fusion protein sequence of the present invention is easy to stablize expression in microorganism, has evaded high-purity natural phycobniliprotein Acquisition with cyanobacteria phytochrome is difficult, and manufacturing cost is higher；The use of chemical modifier；Natural phycobniliprotein polymeric species The shortcomings of unstable.
The expression of the present invention is participated in without phycobniliprotein lyase, reduces the gene dosage of conversion, and it is steady to improve strain Qualitative and screening efficiency.Simultaneously by optimizing fermentation medium and fermentation condition, the yield of fusion protein is substantially increased.
Description of the drawings
Fig. 1 is the spectral signature figure of phytochrome fluorescent marker；
Fig. 2 is the spectral signature figure of phytochrome fluorescent marker (control)；
Fig. 3 is the ferment effect comparison of different fermentations culture medium.
Specific embodiment
With reference to embodiment, the technical solution further illustrated the present invention.
Embodiment 1
The selection of cyanobacteria phytochrome GAF domain sequences
Cyanobacteria phytochrome gene order all2699 (algae PCC7120), there are the higher GAF knots of 3 homologys Structure domain, the GAF domain genes sequence that this programme is chosen are the 1st domain gene sequence, hereinafter referred to as gaf gene orders. Protein amino acid sequence after it is expressed is as follows：
LELEDIITATTAEVRALLGTDRVMIYKFHPDGSGQVIAESIYENRLPSLLGLNFPADDIPPQARELLVKSKVRSIVD VATGMIGQSPVHDLETGELISEDICYRPVDSCHVEYLTAMGVKSSVVAPIFCQDELWGLLVSHHSENRTVSEDELEA MQMIVDQLAVAIAQSH(SEQ ID NO：1).
The selection of transgenosis plasmid combinations
This programme is combined using Duet series double-mass model, sa::The multiple cloning sites of gaf fusions access pET Duet 1；Phycoerythrobilin synzyme plasmid uses pACYC Duet-ho1-pebS.
Streptavidin sa sequences and gaf sequences, 6his-taq affinity labelings sequence carry out Combined design, and for large intestine Bacillus expression system carries out gene order optimization.Final sa::Fused protein amino acid sequence expressed by gaf gene orders It is as follows：
MGSSHHHHHHSQDPEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWT VAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGTDDDDKAMADG LELEDIITATTAEVRALLGTDRVMIYKFHPDGSGQVIAESIYENRLPSLLGLNFPADDIPPQARELLVKSKVRSIVD VATGMIGQSPVHDLETGELISEDICYRPVDSCHVEYLTAMGVKSSVVAPIFCQDELWGLLVSHHSENRTVSEDELEA MQMIVDQLAVAIAQSH(SEQ ID NO：2).
Gene order after fusion passes through restriction enzyme site BamH I (GGATCC), EcoR I (GAATTC) access pET Duet The multiple cloning sites 1 (MCS1) of vector plasmid, obtain plasmid pET Duet-sa::gaf.
Strain prepares screening
1) from picking coli strain BL21 (DE3) single bacterium colony on the tablet of fresh coating, 5mL LB cultures are inoculated in Base, 37 DEG C of shaken cultivations are stayed overnight；
2) 100 μ L saturated cultures are taken, it is sterile to be forwarded to 5mL LB culture mediums, 37 DEG C of shaken cultivations to OD600=0.3~ When 0.4, bacterium solution is transferred in 5mL sterile centrifugation tubes, places 10min on ice；Centrifugation 1min (10000g, 4 DEG C) is discarded supernatant, Collect precipitation thalline；
3) 0.1mol/LCaCl being pre-chilled with 1mL2Thalline is resuspended in sterile solution, and centrifugation 30s (10000g, 4 DEG C) removes supernatant, The 0.1mol/L CaCl that bacterial sediment is pre-chilled with 100 μ L2It is resuspended, is positioned on ice, adds in a certain amount of plasmid built, Ice bath places 30min after mixing, and 300 μ L LB culture mediums, 37 DEG C of low-speed oscillation trainings are added in after 42 DEG C of heat shock 90s, ice bath 5min 45min is supported, on the sterile LB culture medium flat plates being coated on containing corresponding antibiotic, it is visible to being formed to be inverted in 37 DEG C of incubators Monoclonal bacterial plaque.
The experiment proved that the clump count of this programme culture is probably 1~102, and the Zao Bie Ruan Gang Tang under the conditions of Three plasmid combinations conversion scheme culture clump count probably 1~10.The genetic engineering bacterium screening efficiency higher of this programme.
A small amount of expression
The monoclonal strain that picking is come out, is inoculated in by sterile working in 4 shaking flasks, each shaking flask 300mL LB trainings Support base, 37 DEG C of shaken cultivations to OD600It is 0.3 to 0.4；Then shaking flask ice-water bath cooling 0.5h；Addition isopropylthio-β- To final concentration of 1mmol/L, 20 DEG C vibrate D- galactosides (isoprophylthio- β-D-galactoside, abbreviation IPTG) Expression about 12 hours；8000g thalline were collected by centrifugation cells.
Fermentation
1) level-one kind liquid is prepared：Qualified 20 μ L of monoclonal strain kind liquid are taken, are accessed in 25mL LB culture mediums, 37 DEG C low Fast shaken overnight culture is to saturated concentration；
2) two level kind liquid is prepared：5mL level-one kind liquid is transferred in 1.5L LB culture mediums, 37 DEG C of middling speed shaken cultivations 3~ 4h, OD600Control is below 0.5；
3) it is inoculated with：1.5L two level kind liquid is forwarded in 200L fermentation tanks, culture medium 150L, fermentative medium formula：Egg White peptone 1%, dusty yeast 0.5%, sodium chloride 1%, glycerine 0.4%, 0.04% proteinic powder of porcine, pH value are adjusted to NaOH solution 7.2；
4) it cultivates：37 DEG C, the culture of 300rpm middling speeds to growing early period, when be about 1h；
5) cool down：The setting of tank temperature is down to 20 DEG C, and rotating speed is down to 150rpm, and when low speed culture is about 1h；
6) it induces：Add in lactose final concentration about 1.5%, 25 DEG C, 150rpm low speed cultures, when induced expression is about 16h；
7) tank under：Wet thallus is collected in centrifugation.
Ferment effect is evaluated：Take the wet thallus of 1g weight, with 5mL phosphate buffers (20mM KPP, 0.5M NaCl, PH7.2 after) being resuspended, sampling carries out fluorescence emission spectrum detection.Ferment effect is assessed according to the fluorescence volume of unit mass wet thallus.
Extraction
1) wet thallus is resuspended according to 20% ratio with the phosphate buffer (20mM KPP, 0.5M NaCl) of pH7.2；
2) ultrasonication is extracted, power 250W, time 1s, is spaced 3s, total duration 5min；(or freeze-thaw method extraction, repeatedly Freeze thawing 3 times freezes at least 2h every time)；
3) 10000g, 10min, repeated centrifugation twice, take supernatant；
4) 0.45 μm of aperture water system membrane filtration, takes supernatant.
Purifying
Nickel ion chelating gel affinity chromatography.After loading, 50mM imidazole solutions elution impurity, fraction collection sample.pH7.2 Phosphate buffer (20mM KPP, 0.5M NaCl) dialysis remove imidazoles.
Sample after purification, carries out fluorescence emission spectrum detection and absorption spectrum scans, scanning range 300nm~800nm, Detection data can be used for the fluorescence property of assessment product.Its characteristic absorption and fluorescence spectrum are as shown in Figure 1.In figure, solid line is glimmering Optical emission spectroscopy, excitation wavelength 520nm, scanning range 350nm~800nm；Dotted line is visible absorption spectra, scanning range 350nm~800nm.The GAF structural domain fluorescence properties of marker selection are splendid, are suitable for immune labeled.
Comparative example 1：
With embodiment 1, the protein amino acid sequence after being expressed the difference lies in GAF structural domains is as follows：
QQYQHTLLLKQITEEIRQSLQWEKILKTTVTEVQRILQVDRALIFQINSDGSGKVVQEAVMPGWSVTLDQDIYDPCL KDGYLNMYRDGRITAIADVYQGGLKPCYVEFLQQFQVKANLVVPIRVRRNLWGLLIVHQCDRPRQWTELELDLLKHL ADQMGIALTQSQLLAQETRQA(SEQ ID NO：3), the fused protein amino acid sequence expressed by final gene order is such as Under：
MGSSHHHHHHSQDPEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWT VAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGTDDDDKAMADG QQYQHTLLLKQITEEIRQSLQWEKILKTTVTEVQRILQVDRALIFQINSDGSGKVVQEAVMPGWSVTLDQDIYDPCL KDGYLNMYRDGRITAIADVYQGGLKPCYVEFLQQFQVKANLVVPIRVRRNLWGLLIVHQCDRPRQWTELELDLLKHL ADQMGIALTQSQLLAQETRQA(SEQ ID NO：4)
It purifies obtained fusion protein characteristic absorption and fluorescence spectrum is as shown in Figure 2.In Fig. 2, solid line is fluorescent emission Spectrum, excitation wavelength are respectively 460nm and 520nm, scanning range 300nm~800nm；Dotted line is visible absorption spectra, scanning Range 300nm~800nm.
By comparison diagram 1 and Fig. 2 it is found that the GAF structural domain fluorescence properties of comparative example are poor, the properties of product phase with Fig. 1 Poor 10 times or more, it is impossible to be used in immune detection.
Influence of the different fermentations culture medium to ferment effect
Embodiment 2：
The monoclonal strain that the fermentation of Example 1 uses, ferments as follows.
1) level-one kind liquid is prepared：Qualified 20 μ L of monoclonal strain kind liquid are taken, are accessed in 25mL LB culture mediums, 37 DEG C low Fast shaken overnight culture is to saturated concentration；
2) two level kind liquid is prepared：5mL level-one kind liquid is transferred in 1.5L LB culture mediums, 37 DEG C of middling speed shaken cultivations 3~ 4h, OD600Control is below 0.5；
3) it is inoculated with：1.5L two level kind liquid is forwarded in 200L fermentation tanks, culture medium 150L, fermentative medium formula：Egg White peptone 1%, dusty yeast 0.5%, sodium chloride 1%, glycerine 0.4%, 0.004% ferroheme, pH value are adjusted to 7.2 with NaOH solution；
4) it cultivates：37 DEG C, the culture of 300rpm middling speeds to growing early period, when be about 1h；
5) cool down：The setting of tank temperature is down to 20 DEG C, and rotating speed is down to 150rpm, and when low speed culture is about 1h；
6) it induces：Add in lactose final concentration about 1.5%, 25 DEG C, 150rpm low speed cultures, when induced expression is about 16h；
7) tank under：Wet thallus is collected in centrifugation.
Embodiment 3：
The monoclonal strain that the fermentation of Example 1 uses, ferments as follows.
1) level-one kind liquid is prepared：Qualified 20 μ L of monoclonal strain kind liquid are taken, are accessed in 25mL LB culture mediums, 37 DEG C low Fast shaken overnight culture is to saturated concentration；
2) two level kind liquid is prepared：5mL level-one kind liquid is transferred in 1.5L LB culture mediums, 37 DEG C of middling speed shaken cultivations 3~ 4h, OD600Control is below 0.5；
3) it is inoculated with：1.5L two level kind liquid is forwarded in 200L fermentation tanks, culture medium 150L, fermentative medium formula：Egg White peptone 1%, dusty yeast 0.5%, sodium chloride 1%, glycerine 0.4%, pH value is adjusted to 7.2 with NaOH solution；
4) it cultivates：37 DEG C, the culture of 300rpm middling speeds to growing early period, when be about 1h；
5) cool down：The setting of tank temperature is down to 20 DEG C, and rotating speed is down to 150rpm, and when low speed culture is about 1h；
6) it induces：Add in lactose final concentration about 1.5%, 25 DEG C, 150rpm low speed cultures, when induced expression is about 16h；
7) tank under：Wet thallus is collected in centrifugation.
Embodiment 1, embodiment 2 and embodiment 3 have been respectively adopted different fermentative medium formulas, and formula components are consistent Part all employs same batch raw material.Its ferment effect compares as shown in figure 3, embodiment 1 and embodiment 2 are after optimizing Culture medium, embodiment 3 are traditional culture medium.The fluorescence property of Examples 1 and 2 tunning improves at least compared with embodiment 3 50%, meet purity Coriolis mass requirement, illustrate that its culture medium composition is more advantageous to the expression of fusion protein.
Expression stability：
Respectively by the zymotechnique of embodiment 1 and the zymotechnique fermenting and producing fusion protein 3 batches of embodiment 2, find to melt The expression quantity of hop protein is highly stable, and the deviation of average expression amount is within 5%.
SEQUENCE LISTING
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195 200 205
Val Val Gln Glu Ala Val Met Pro Gly Trp Ser Val Thr Leu Asp Gln
210 215 220
Asp Ile Tyr Asp Pro Cys Leu Lys Asp Gly Tyr Leu Asn Met Tyr Arg
225 230 235 240
Asp Gly Arg Ile Thr Ala Ile Ala Asp Val Tyr Gln Gly Gly Leu Lys
245 250 255
Pro Cys Tyr Val Glu Phe Leu Gln Gln Phe Gln Val Lys Ala Asn Leu
260 265 270
Val Val Pro Ile Arg Val Arg Arg Asn Leu Trp Gly Leu Leu Ile Val
275 280 285
His Gln Cys Asp Arg Pro Arg Gln Trp Thr Glu Leu Glu Leu Asp Leu
290 295 300
Leu Lys His Leu Ala Asp Gln Met Gly Ile Ala Leu Thr Gln Ser Gln
305 310 315 320
Leu Leu Ala Gln Glu Thr Arg Gln Ala
325

Claims (10)

1. a kind of fusion protein, the 1st structural proteins of the GAF structural domains including cyanobacteria phytochrome protein all2699 And Streptavidin.
2. fusion protein according to claim 1, it is characterised in that：Affinity labeling is also added in fusion protein.
3. fusion protein according to claim 1, it is characterised in that：The sequence of fusion protein is： MGSSHHHHHHSQDPEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWT VAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGTDDDDKAMADG LELEDIITATTAEVRALLGTDRVMIYKFHPDGSGQVIAESIYENRLPSLLGLNFPADDIPPQARELLVKSKVRSIVD VATGMIGQSPVHDLETGELISEDICYRPVDSCHVEYLTAMGVKSSVVAPIFCQDELWGLLVSHHSENRTVSEDELEA MQMIVDQLAVAIAQSH。
4. express the nucleotide sequence of fusion protein described in claims 1 or 2.
5. nucleotide sequence according to claim 3, it is characterised in that：Nucleotide sequence carries out codon according to host strain Optimization.
6. the plasmid inserted with the nucleotide sequence of claim 3 or 4.
7. plasmid according to claim 5, it is characterised in that：Plasmid is pET Duet vector plasmids, and nucleotide sequence leads to Cross the multiple cloning sites 1 that restriction enzyme site BamH I, EcoR I are inserted into pET Duet vector plasmids.
8. a kind of preparation method of fusion protein, includes the following steps：
1) sequence of fusion protein according to claim 1 or claim 2, design obtain antigen-4 fusion protein gene sequence；
2) antigen-4 fusion protein gene sequence is inserted into plasmid, obtains fusion protein plasmid；
3) by fusion protein plasmid and the related gene of expression phycoerythrobilin synthesis：Heme oxidase gene ho1, biliverdin is also The plasmid corotation of nitroreductase gene pebS enters Escherichia coli, obtains genetic engineering bacterium；
4) it is inoculated with genetic engineering bacterium in the medium using three-level inoculation method, 37 DEG C of fermented and cultureds are to growing early period；
5) 20 DEG C are cooled to, cultivates 0.8~1.2h；
6) 25 DEG C are warming up to, adds in 14~16h of lactose Fiber differentiation that whole mass concentration is 1.3~1.6%；
7) thalline extraction is collected, purifying obtains fusion protein.
9. preparation method according to claim 8, it is characterised in that：The quality group of fermentation medium becomes：Peptone 1%, dusty yeast 0.5%, sodium chloride 1%, glycerine 0.4%, 0.004%~0.04% proteinic powder of porcine or ferroheme, surplus For water, pH value is adjusted to 7.2.
10. preparation method according to claim 8 or claim 9, it is characterised in that：Add in the lactose that whole mass concentration is 1.5% Fiber differentiation.
CN201711460150.0A 2017-12-28 2017-12-28 A kind of preparation method of the cyanobacteria phytochrome fluorescent marker of fluorescent orange Pending CN108250304A (en)

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