CN108192904A

CN108192904A - One kind wears film fluorescin and its encoding gene and application

Info

Publication number: CN108192904A
Application number: CN201810085975.7A
Authority: CN
Inventors: 李洪波
Original assignee: Huaihua University
Current assignee: Huaihua University
Priority date: 2018-01-29
Filing date: 2018-01-29
Publication date: 2018-06-22
Anticipated expiration: 2038-01-29
Also published as: CN108192904B

Abstract

The present invention relates to one kind to wear film fluorescin and its encoding gene and application, and gene provided by the invention is as follows（1）‑（3）Any one of DNA molecular：（1）As the DNA molecular shown in sequence in sequence table 1；（2）Under strict conditions with（1）The DNA sequence dna hybridization of restriction and coding penetrate the DNA molecular of the green fluorescent fusion protein of insect cell membrane with specificity；（3）With（1）The DNA sequence dna of restriction is at least with 90%, at least with 95%, at least with 96%, at least with the 97%, DNA molecular at least with 98% or at least with the albumen of 99% homology and coding with poisoning insect active.Present invention optimizes it is a kind of can specificity penetrate insect cell cell membrane green fluorescent protein fusion protein DNA sequence dna, and provide it is a kind of can be with high efficient expression and the method for purifying the recombinant protein.The recombination fluorescin can specifically penetrate the cell membrane of insect cell, to exogenous molecules are imported insect cell and convenient for being quantitatively of great significance.

Description

One kind wears film fluorescin and its encoding gene and application

Technical field

The present invention relates to biotechnologies, and in particular to a kind of encoding gene and its albumen for wearing film green fluorescent protein Preparation method and application.

Background technology

Global scholars have established 500 polyclonal cellular systems from 100 various insects.At home, has dozens of Insect cell line is established.These cell lines overwhelming majority comes from the agricultural pests of Lepidoptera and Diptera, derived tissues packet Ovary, spermary, embryo, imaginal discs, haemocyte, middle intestines, fat-body etc. are included, wherein being established with ovary tissue and embryonic tissue thin Born of the same parents system is most.The in vitro culture of insect cell is increasingly subject to the attention of people.The in vitro culture of insect cell is increasingly becoming life Production is for agricultural and the important means of human health biological product for health care.

Exogenous molecules are exactly imported insect cell by the main application of insect cell, by the use of insect cell as host cell To produce required product or generate other purposes.And at present by the various methods of exogenous molecules or channel genes insect cell It all can not accurately and visually detect that how many exogenous molecules of bottom have been directed in insect cell.In biological study, Scientists are usually used as the label of organism using this fluorescent molecular that oneself can be shone.And fluorescin is exactly a kind of Most suitable fluorescent tag molecule.At present, fluorescin has very extensive application, can be applied to transfectional cell really It is fixed, the measure of gene expression in vivo, the dynamic monitoring that the positioning of protein molecule is exchanged with iuntercellular molecule, immunoassay, core Base pair probe analysis etc..Therefore, fluorescin may be used as mark molecule, and external source has been divided and has been connect with fluorescin Come, just can determine that how many exogenous molecules has been directed in insect cell by fluorescence power.But fluorescin itself nothing Method penetrates the cell membrane of insect cell, need to can just be entered into the cell by the help of other methods.Therefore, it is necessary to fluorescence is transformed Protein molecular allows it to penetrate the cell membrane of insect cell.

By screening, planted from more than ten and a kind of process transformation green fluorescent protein point is obtained in improved fluorescin Son, the improved green fluorescent protein molecule can efficiently penetrate the cell membrane of insect cell.Therefore, the transformation is largely prepared Protein molecular be carry out the protein molecular application basis.At present the production method of artificial protein mainly have chemical synthesis and Exogenous gene expression method.And chemical synthesis is with high costs for the longer albumen of the chain length for obtaining high-purity and time-consuming length, builds The preparation method for founding efficient heterologous gene expression system and its albumen is the unique channel for solving to obtain a large amount of albumen.The present invention Albumen can largely be prepared by not only obtaining a kind of process transformation green fluorescent protein molecule and its gene but also providing one kind Feasible method, to exogenous molecules are imported insect cell and convenient for being quantitatively of great significance.

Invention content

It is an object of the present invention to obtaining a kind of green fluorescent protein molecule by transformation, this is improved green Color fluorescent protein molecule can efficiently penetrate the cell membrane of insect cell, which is as shown in sequence in sequence table 1 A kind of protein molecule.

In addition, for the defects in the prior art, it is a kind of glimmering by transformation green another object of the present invention is to provide The gene of photoprotein and the preparation method of recombinant protein and application.

The present invention provides gene, is as follows to encode a kind of gene by transformation green fluorescent protein（1）-（3）In appoint It anticipates a kind of DNA molecular：

（1）As the DNA molecular shown in sequence in sequence table 2；

（2）Under strict conditions with（1）DNA points of the albumen of DNA sequence dna hybridization and coding with poisoning insect active of restriction Son；

（3）With（1）The DNA sequence dna of restriction is at least with 95%, at least with 96%, at least with 97%, at least with 98% or extremely DNA molecular less with the albumen of 99% homology and coding with poisoning insect active.

The stringent condition can be as follows：50 DEG C, in 7% lauryl sodium sulfate（SDS）、0.5 M Na₃PO₄With 1 mM Hybridize in the mixed solution of EDTA, rinsed in 50 DEG C, 2 × SSC, 0.1% SDS；Can also be：50 DEG C, in 7% SDS, 0.5 M Na₃PO₄Hybridize in the mixed solution of 1 mM EDTA, rinsed in 50 DEG C, 1 × SSC, 0.1% SDS；Can also be：50 DEG C, 7% SDS, 0.5 M Na₃PO₄Hybridize in the mixed solution of 1 mM EDTA, floated in 50 DEG C, 0.5 × SSC, 0.1% SDS It washes；Can also be：50 DEG C, in 7% SDS, 0.5 M Na₃PO₄Hybridize in the mixed solution of 1 mM EDTA, at 50 DEG C, 0.1 × It is rinsed in SSC, 0.1% SDS；Can also be：50 DEG C, in 7% SDS, 0.5 M Na₃PO₄With it is miscellaneous in the mixed solution of 1 mM EDTA It hands over, is rinsed in 65 DEG C, 0.1 × SSC, 0.1% SDS；Or：In 6 × SSC, the solution of 0.5% SDS, 65^oIt is miscellaneous under C It hands over, then with 2 × SSC, 0.1% SDS and 1 × SSC, it is primary that 0.1% SDS respectively washes film.

Wherein, the sequence 2 in sequence table is made of 810 deoxynucleotides, this sequence includes engineered protein's gene Reading frame, clone's double enzyme site, histidine tag and only codon, 252 ammonia of the coding with sequence 1 in sequence table The protein of base acid residue sequence.

Obtained albumen is encoded by the sequence 1 in above-mentioned sequence table to belong to the scope of protection of the present invention.

The present invention provides albumen, is as follows（1）Or（2）：

（1）The protein being made of the amino acid sequence shown in sequence in sequence table 1；

（2）By substitution of the amino acid sequence shown in sequence in sequence table 1 by one or several amino acid residues and/or missing And/or add and have the protein as derived from sequence 2 of poisoning insect active.

Wherein, the sequence 2 in sequence table is made of 810 deoxynucleotides, this sequence includes engineered protein's gene Reading frame, clone's double enzyme site, histidine tag and only codon.

It the substitution of one or several amino acid residues and/or lacks and ors add and refers to not more than ten amino acid It the substitution of residue and/or lacks and ors add.

Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing said gene also belong to the protection of the present invention Range.

The recombinant vector is specially that said gene is inserted into expression vector, and the recombination for obtaining expressing above-mentioned albumen carries Body.The recombinant vector is particularly preferred as said gene being inserted into I restriction enzyme site of Xho I and Xba of expression vector pPICZ α A Between, obtain expressing the recombinant vector of above-mentioned albumen.

Third object of the present invention is to provide a kind of method for preparing albumen, includes the following steps：

S1：Gene described in claim 2 and expression vector pPICZ α with Xho I and I double digestions of Xba and are purified back respectively It receives, is then connected using ligase at 16 °C, obtain recombinant vector pPICZ α A-RGFP；

S2：The recombinant vector pPICZ α A-RGFP I single endonuclease digestions of Sac are linearized, and are transformed into lithium chloride conversion method complete In red yeast host bacterium, screen to obtain positive colony using Zeocin；

S3：The positive colony is forwarded to the YPD tablets containing 1000 ~ 1500 μ g/mL Zeocin, screening obtains height The transformant of Zeocin resistances, by the resistance transformant BMGY medium cultures to OD₆₀₀It is 10 ~ 15, is collected by centrifugation thin Born of the same parents are precipitated, and cell is resuspended with BMMY culture mediums, add in methanol and its mass fraction is made to be 1.0% ~ 1.5%, induce collect within 72 hours The supernatant of fermentation；

S4：Expand culture and continue Fiber differentiation 72 hours using at 28 °C, adding within every 24 hours methanol keeps its mass fraction 1.0% ~ 1.5%；

S5：The zymotic fluid that S4 cultures obtain is adjusted into pH 7.5 ~ 8.5 to be greater than or equal to turning for 15000 g using NaOH The supernatant of acquisition is loaded to the nickel parent through pH after 7.5 ~ 8.5 50 mM PBS buffer solution balance by speed centrifugation 10 ~ 20 minutes Chromatographic column is closed, is buffered with 7.5 ~ 8.5 PBS containing 20 mM imidazoles and 100 ~ 400 mM NaCl of 5 ~ 10 times of chromatography column volumes Liquid rinses the nickel affinity chromatographic column；

S6：It is chromatographed with the nickel affinity is eluted containing the PBS buffer solution of 100 ~ 400 mM imidazoles and 100 ~ 400 mM NaCl Column dialyses obtained eluent using the bag filter that molecular weight is 10 kDa in 20 mM PBS buffer solution, and then ultrafiltration is dense Contracting, the product that the ultrafiltration concentration is obtained is quick-frozen under -80 °C, is then freeze-dried.

The albumen that the method for preparing albumen according to any of the above-described kind is prepared also belongs to protection scope of the present invention.

The yeast of green fluorescent protein molecule is transformed by the step S3 stabilizations screened and energy high-level secretory expression Transformant also belongs to the scope of protection of the invention.

Above-mentioned albumen, said gene or above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium are in pest-resistant field In application be also the scope of protection of the invention.

Technical solution provided by the invention has the following advantages：First, the expression secretion according to the technical program The transformation green fluorescent protein with insect cell specificity membrane penetration effect that expression and purifying obtain, can effectively prevent host Degradation of the bacterium to expression product, mitigate host cell metabolism load and expression product to the toxic effect of host；Second is that it utilizes The gene secretion expression of secretion signal α-factor signal peptide guiding destination protein on yeast vector pPICZ α A-RGFP, purpose egg It can largely be secreted into culture solution in vain；Third, by screening is stablized and can high-level secretory expression transformation green fluorescence egg The yeast transformant of white molecule；Fourth, the transformation green fluorescent protein molecule of purifying specific can penetrate the cell of insect cell Film can simultaneously observe directly under fluorescence microscope.

The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description It obtains significantly or is recognized by the practice of the present invention.

Description of the drawings

Fig. 1 is the green fluorescent protein of the different molecular transformation for the chemical synthesis observed under fluorescence microscope and natural sequence Whether row green fluorescent protein wears film and the picture of green fluorescence is presented.

Fig. 2 is the expression vector establishment schematic diagram in the embodiment of the present invention；

Fig. 3 is the SDS-PAGE that green fluorescent protein molecule yeast transformant is transformed in difference Zeocin resistances in the embodiment of the present invention Testing result figure；

Fig. 4 is the SDS-PAGE detection knots of the different time points target protein expression under methanol induction in the embodiment of the present invention Fruit is schemed；

Fig. 5 is the SDS-PAGE of transformation green fluorescent protein that the imidazoles of various concentration in the embodiment of the present invention is purified by flash Testing result figure；

Fig. 6 is the SDS-PAGE testing result figures of transformation green fluorescent protein molecule that purifying obtains in the embodiment of the present invention；

Fig. 7 wears film result for the transformation green fluorescent protein molecule after optimizing in the embodiment of the present invention.

Specific embodiment

Below in conjunction with the attached drawing in the embodiment of the present invention, the technical solution in the embodiment of the present invention is carried out clear, complete Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, those of ordinary skill in the art are obtained every other without making creative work Embodiment shall fall within the protection scope of the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

% in following embodiments is mass percentage unless otherwise specified.Quantitative test in following embodiment, Three repeated experiments are respectively provided with, data are the average value or mean+SD of three repeated experiments.

The present invention selects Pichi strain and conformability expression plasmid pPICZ α A to be purchased from U.S. Invritrogen public affairs Department.

Used medium formula is as follows：

1）Yeast growth medium（BMGY）：

It is completely dissolved 10 g yeast extracts, 20 g peptones, constant volume to 800 mL.121 DEG C of steam high pressure sterilization 15-20 Min is cooled to room temperature, adds in 100 mL, 1 M potassium phosphate solutions, 100 mI YNB, 2 500 × biotins of mL, 20 mL 50% Sterile glycerol；

2）Yeast inducing culture（BMMY）

It is completely dissolved 10 g yeast extracts, 20 g peptones, constant volume to 800 mL.121 DEG C of steam high pressure sterilization 15-20 Min is cooled to room temperature, adds in 100 mL, 1 M potassium phosphate solutions, 100 mL YNB, 2 mL500 × biotin, 10 mL methanol；

3）YPD culture mediums

It is completely dissolved 10 g yeast extracts, 20 g peptones, constant volume to 900 mL, 121 DEG C of steam high pressure sterilization 15-20 Min is cooled to 70 DEG C or so and adds 100 mL20% sterile dextrose solution.Adding in the agar of 1.5-1.8% wherein can make Obtain YPD solid mediums；

YPG culture mediums

It is completely dissolved 10 g yeast extracts, 20 g peptones, 20 g glycerine, constant volume to 1000 mL, 121 DEG C of steam high pressures Sterilize 15-20 min.

Embodiment one

A kind of artificial reconstructed egfp is present embodiments provided, this artificial reconstructed albumen is through changing from 8 kinds It is screened in the green fluorescent protein made.These albumen are to utilize the polypeptide being chemically synthesized.It is a kind of artificial reconstructed Green fluorescent protein particular sequence is as shown in the sequence 1 in sequence table.Artificial reconstructed green fluorescent protein with it is not engineered or The green fluorescence of other molecular modifications is compared, and the green fluorescent protein of work transformation only provided by the invention can efficiently penetrate elder brother The cell membrane of worm cell.

The present embodiment is respectively obtained into 8 artificial reconstructed green fluorescent proteins and natural using the method that chemiluminescent polypeptide synthesizes The green fluorescent protein of sequence, and more than albumen compares the film ability of wearing of insect cell, specific analytical method and knot Fruit is as follows.

Experimental method one：Insect cell SF9 is cultivated in insect cell medium to 90% life in 10 cm culture dishes Long density, pancreatin digest and adjust cell concentration to 5 × 10⁵Cell/mL is added in into 96 porocyte culture plates per hole After overnight incubation, the artificial reconstructed green of different final concentrations is separately added into each culture hole for the insect SF9 cell suspensions of 100 μ l Color fluorescin and native sequences green fluorescent protein（10 ng/mL) it is incubated afterwards after five minutes, using PBS by the cell in each hole Then whether washing 3 times carries fluorescence in fluorescence microscopy Microscopic observation cell.

Experimental result one：Fig. 1 is that observed the green fluorescent protein of different molecular transformation and natural under fluorescence microscope Whether sequence green fluorescent protein has green fluorescence, as can be seen from the figure except the present invention obtain it is a kind of artificial reconstructed green Color fluorescence protein can observe outside fluorescence (G) with penetration cell film, others transformation green fluorescent protein and native sequences Green fluorescent protein cannot penetrate the cell membrane of insect cell, therefore do not observe fluorescence.And under visible light, Ke Yiqing It is clear to see in all pictures with the presence of a large amount of cell；Since all cells are all by washing, cell cannot be entered Interior fluorescin can be all eluted, this figure further confirms that can whether can see that fluorescence determines be because penetration cell Film enters intracellular and generation.Should the experimental results showed that, screening obtained a kind of artificial reconstructed egfp, It can be with penetration cell film.

Embodiment two

The artificial synthesized C- ends for present embodiments providing a kind of optimization carry a kind of artificial reconstructed green of 6 × His labels Fluorescence protein gene, particular sequence is as shown in the sequence 2 in sequence table.DNA sequence dna after optimization is compared through NCBI, without apparent Similitude.

DNA of the C- ends of optimization with 6 × His labels is respectively connected to Pichia pastoris secreted expression carrier pPICZ α On A, recombinant vector is obtained, the lithium chloride conversion method then provided using Invitrogen companies operation manual is respectively carried recombination Body is transformed into Pichia pastoris host strain X-33, respectively with the YPD tablets containing 100 μ g/mL Zeocin antibiotic after conversion It is screened, transformant is verified using PCR, the Pichia pastoris transformant scribing line after PCR is verified is seeded to respectively to be contained There are the YPD tablets of the Zeocin antibiotic of various concentration, screening respectively is obtained containing a kind of green fluorescent protein base by transformation The resistance Pichia pastoris transformant of cause then respectively with a small amount of induced expressions of BMMY, then carries out SDS-PAGE analyses.Analysis As a result showing can be expressed and be secreted with a kind of DNA of the green fluorescent protein by transformation Pichia pastoris transformants built Target protein.

Embodiment three

The present embodiment provides a kind of methods for preparing albumen, specifically comprise the following steps：

S1：Construction of expression vector and conversion：DNA of the artificial synthesized C- ends of embodiment one with 6 × His labels is connected to Pichia pastoris secreted expression carrier pPICZ α A, obtain recombinant vector pPICZ α A-RGFP, vector construction is as shown in Fig. 2, Fig. 2 Schematic diagram is built for the carrier for expression of eukaryon pPICZ α A-RGFP in the embodiment of the present invention.Main vector construction step is preferably such as Under：

（1）With a kind of plasmid of the green fluorescence protein gene by transformation of Xho I and I double digestions of Xba containing synthesis, obtain Purpose segment, reaction system are as follows（Restriction endonuclease and buffer solution used are purchased from Dalian TAKARA companies）：

The 15 μ L of plasmid of AaIT genes containing synthesis

10 × M buffer solutions, 5 μ L

XhoⅠ 5 U

XbaⅠ 5 U

Sterile water is to 50 μ L

（2）With I double digestion pPICZ α A of Xho I and Xba, carrier segment is obtained, reaction system is as follows（Restriction endonuclease used and buffering Liquid is purchased from Dalian TAKARA companies）：

15 μ L of plasmid pPICZ α A

10 × M buffer solutions, 5 μ L

XhoⅠ 5U

XbaⅠ 5U

Sterile water is to 50 μ L

（3）By step（1）With（2）Obtained purpose segment and carrier segment DNA gel withdraws kit recycling, the kit Purchased from Dalian TAKARA companies, concrete operations are carried out by kit specification.

（4）By step（3）Recycle obtained purpose segment and carrier T4DNA ligases（Purchased from Dalian TAKARA companies） Reaction is attached, target gene is properly inserted in the excretion vector reading frame containing secretion signal α-factor, reactant System is as follows：

1 μ L of carrier pPICZ α A segments

Target fragment falls 3 μ L

10 × buffer solution, 1 μ L

0.5 μ L of T4 ligases

Sterile water is to 10 μ L

S2：The conversion of recombinant plasmid：By the recombinant vector pPICZ α A-RGFP SacI single endonuclease digestion linearizes, according to The lithium chloride conversion method that Invitrogen companies operation manual provides, recombinant vector is transformed into Pichia pastoris host strain, this That embodiment is selected is X-33.It is screened, is utilized with the YPD tablets containing 100 μ g/mL Zeocin antibiotic after conversion PCR verifies transformant；

S3：The screening of high-level secretory expression yeast transformant and the expression of albumen：Pichia pastoris transformant after PCR is verified The YPD tablets of the streak inoculation extremely Zeocin antibiotic containing various concentration, screening obtain the transformant of high Zeocin resistances, By the resistance transformant a small amount of induced expressions of BMMY, screening obtains the transformant of high-level secretory expression target protein, selects The yeast transformant single bacterium colony of high-level secretory expression is taken, is seeded to the 250 mL triangles equipped with 50 mL YPD fluid nutrient mediums In bottle, cultivated under 28 DEG C, 300 rpm condition of culture to thalline OD₆₀₀=6.0, above-mentioned bacterium solution is taken, is seeded to containing 500 mL , Mei Ping are inoculated with 5 mL in 2000 mL shaking flasks of BMGY culture mediums, and in 28 DEG C, 250 rpm are cultivated to OD₆₀₀=12；At room temperature 2500 G centrifuges 5 min, collects thalline, and thalline is resuspended with the BMMY of 1/7 former volume of culture, 100% methanol is added into culture medium to end Concentration（Mass fraction）It is 1.0% ~ 1.5%, induces 72 hours, continues Fiber differentiation 72 hours at 28 DEG C, add first within every 24 hours Alcohol makes its mass fraction be maintained at 1.0% ~ 1.5%, collects the supernatant of fermentation.

It should be noted that by the transformant of the high Zeocin resistances a small amount of induced expressions of BMMY, analyzed through SDS-PAGE, Screening obtains 4 plants of stabilizations and height from ten plant height resistances (resistance level is greater than or equal to 1500 μ g/mL Zeocin) transformant A kind of yeast transformant of green fluorescent protein by transformation of level secretion expression, the SDS-PAGE results of beggar screening are such as Shown in Fig. 3, wherein swimming lane 1-4 is the albumen of high level expression transformant expression screened, and swimming lane 5 is the table of starting strain Up to albumen, illustrate that target protein realizes high-level secretory expression.

Different time target protein expression under methanol induction is detected with SDS-PAGE to analyze, electrophoresis result such as Fig. 4 institutes Show.Through induction 1 ~ 4 day, there is apparent destination protein to express, and by continuing to add methanol, induce in 30 kDa or so Destination protein content higher, the total amount result that specific purposes albumen accounts for whole bacterial protein are as shown in table 1 below.

The destination protein that the different induction times of table 1 obtain accounts for the degree result of Supernatant protein

Induction time	1 day	2 days	3 days	4 days
					Degree（%）	12	28	41	37

The present embodiment provides a kind of methods for preparing albumen, specifically also need to include the following steps：

S4：High-level transformant, by obtained supernatant in 4 DEG C of centrifugations, was collected the supernatant after centrifugation, is utilized through induction 3 days Tris alkali adjusts pH to 7.5 ~ 8.5, is centrifuged 15 minutes with the rotating speed of 15000 g, the supernatant of acquisition is added to through pH 7.5 ~ 8.5 PBS buffer solution balance after nickel affinity chromatographic column in, with 3 times chromatography column volume pH 7.5 ~ 8.5 contain 50 mM 7.5 The buffer solution of ~ 8.5 PBS and 20 mM imidazoles rinses nickel affinity chromatographic column.

S5：Nickel affinity is eluted with the buffer solution containing 50 mM 7.5 ~ 8.5 PBS and 100,200,300 and 400 mM imidazoles Chromatographic column simultaneously collects eluent, by eluent using the bag filter of 10 kDa of molecular weight in 20 mM, 7.5 ~ 8.5 PBS buffer solution It is concentrated by ultrafiltration after middle dialysis.

S6：The green of transformation is passed through in freeze-drying to get the recombination of high-purity after concentrated product is quick-frozen in -80 DEG C of refrigerators Fluorescin freeze-dried powder.

It should be noted that the purifying protein that step S5 is eluted carries out SDS-PAGE analyses, as a result such as Fig. 5 institutes Show, Fig. 5 is the green fluorescent protein by transformation that the imidazoles of various concentration in the embodiment of the present invention is purified by flash SDS-PAGE testing result figures.From the results, it was seen that the rinsing liquid first with 20 mM imidazoles can remove a large amount of foreign proteins It removes, when buffer solution elution chromatography column for recycling 100,200,300 and 400 mM imidazoles can afford the mesh of high-purity Mark albumen.It is utilized after eluent is dialysed using the bag filter of 10 kDa of molecular weight in 50 mM, 7.5 ~ 8.5 PBS buffer solution The 15 mL super filter tubes of 10 kDa of molecular cut off are by 20 times of sample concentration.Take the purifying protein of 20 micrograms, SDS-PAGE detections The results are shown in Figure 6, only has protein band in target position, illustrates to have obtained the recombination egg of the green fluorescence by transformation of High Purity In vain.

It carries out wearing film analysis, specific analytical method and knot by the green fluorescent protein being transformed by what comparative example purified Fruit is as follows.

Experimental method：Insect cell SF9 is cultivated in insect cell medium to 90% growth in 10 cm culture dishes Density, pancreatin digest and adjust cell concentration to 5 × 10⁵Cell/mL adds in 1 mL into 12 porocyte culture plates per hole Insect SF9 cell suspensions, culture 4 hours after, to each culture hole be separately added into 20 ng/mL of final concentration through transformation it is green Color fluorescin albumen, incubation at room temperature sop up culture solution, and PBS washing cells time remove remnants' after twenty minutes, with pipettor After green fluorescence recombinant protein by transformation, fluorescence microscopy Microscopic observation wears film situation.

Experimental result：Experimental result as shown in fig. 7, insect cell it is observed that apparent fluorescence is presented in cell.Illustrate ferment The egfp of Recombinant Artificial transformation that matrix reaches, it can equally penetrate the cell membrane of insect cell.

In the description of this specification, reference term " one embodiment ", " example ", " is specifically shown " some embodiments " The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description Point is contained at least one embodiment of the present invention or example.In the present specification, schematic expression of the above terms are not It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office It is combined in an appropriate manner in one or more embodiments or example.In addition, without conflicting with each other, the skill of this field Art personnel can tie the different embodiments or examples described in this specification and the feature of different embodiments or examples It closes and combines.

Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is impossible to limitation of the present invention is interpreted as, those of ordinary skill in the art within the scope of the invention can be to above-mentioned Embodiment is changed, changes, replacing and modification, and it does not separate the essence of the corresponding technical solution various embodiments of the present invention skill The range of art scheme should all cover in the claim of the present invention and the range of specification.

Sequence table

<110>Huaihua College

<120>One kind wears film fluorescin and its encoding gene and application

<130> 2

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 252

<212> PRT

<213> Aequorea victoria

<400> 1

Gly Arg Arg Arg Arg Arg Arg Arg Arg Arg Pro Pro Gln Met Val Ser

1 5 10 15

Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu

20 25 30

Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu

35 40 45

Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr

50 55 60

Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr

65 70 75 80

Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp

85 90 95

Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile

100 105 110

Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe

115 120 125

Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe

130 135 140

Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn

145 150 155 160

Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys

165 170 175

Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu

180 185 190

Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu

195 200 205

Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp

210 215 220

Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala

225 230 235 240

Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

245 250

<210> 2

<211> 810

<212> DNA

<213> Aequorea victoria

<400> 2

ccgctcgaga aaagaggtag aagaaggaga agaagaagga gaagaccacc acaactcgag 60

aaaagaatgg tgtctaaggg cgaagagttg ttcaccggtg ttgttccaat cttggttgag 120

ttggacggtg atgtcaacgg tcacaagttc tctgtttctg gtgaaggtga gggtgacgct 180

acttacggaa agttgacctt gaagttcatc tgcaccaccg gtaagttgcc agttccatgg 240

ccaactttgg ttaccacttt gacctacggt gtccagtgct tctcaagata cccagaccat 300

atgaagcagc acgacttctt caagtctgct atgccagagg gttacgtcca agagagaacc 360

attttcttca aggacgacgg taactacaag accagagccg aagttaagtt cgagggtgac 420

accttggtta acagaatcga gttgaagggt atcgacttca aagaggacgg aaacatcttg 480

ggacacaagt tggagtacaa ctacaactcc cacaacgtct acatcatggc cgacaagcag 540

aagaacggta tcaaggttaa cttcaagatc aggcacaaca tcgaggacgg ttccgttcaa 600

ttggctgacc actaccaaca gaacactcca attggtgacg gtccagtttt gttgccagac 660

aaccactact tgtccactca atccgctttg tctaaggacc caaacgagaa gagggaccac 720

atggttttgt tggagttcgt tactgctgcc ggtatcactc ttggtatgga cgagttgtac 780

aagcatcatc atcatcatca ttaatctaga 810

Claims

It is as follows 1. one kind wears film fluorescin and its encoding gene and application（1）-（3）Any one of DNA molecular：

（1）As the DNA molecular shown in sequence in sequence table 1；

（2）Under strict conditions with（1）One kind that the DNA sequence dna hybridization of restriction and coding specific can penetrate insect cell membrane is worn The DNA molecular of film green fluorescent fusion protein；

（3）With（1）The DNA sequence dna of restriction at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least With 98% or at least with 99% homology and a kind of DNA molecular for wearing film green fluorescent protein of coding.
2. the albumen that DNA molecular according to claim 1 encodes.
3. albumen according to claim 2, it is characterised in that：It is as follows（1）Or（2）：

（1）The protein being made of the amino acid sequence shown in sequence in sequence table 2；

（2）By substitution of the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues and/or missing And/or add and penetrated with specificity the green egg white matter as derived from sequence 2 of insect cell membrane.
4. recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing gene described in claim 1；

The recombinant vector be specially will gene described in claim 1 be inserted into expression vector in, obtain expression claim 2 or The recombinant vector of 3 albumen, the recombinant vector are particularly preferred as said gene being inserted into the X of expression vector pPICZ α Aho I and Xba Between I restriction enzyme site, obtain expressing the recombinant vector of above-mentioned albumen.
5. a kind of method that albumen is prepared using gene described in claim 1, is included the following steps：

S1：Gene described in claim 1 and expression vector pPICZ α are used into X respectivelyhoI and XbaI double digestion simultaneously purifies recycling, Then it is connected using ligase at 16 DEG C, obtains recombinant vector pPICZ α A-RGFP；

S2：By the recombinant vector pPICZ α A-RGFP SacI single endonuclease digestion linearizes, and is transformed into lithium chloride conversion method complete In red yeast host bacterium, screen to obtain positive colony using Zeocin；

S3：The positive colony is forwarded to the YPD tablets containing 1500 ~ 2000 μ g/mL Zeocin, screening obtains height The transformant of Zeocin resistances, by the resistance transformant BMGY medium cultures to OD₆₀₀It is 10 ~ 15, is collected by centrifugation thin Born of the same parents are precipitated, and cell is resuspended with BMMY culture mediums, add in methanol and its mass fraction is made to be 1.0% ~ 1.5%, induce collect within 72 hours The supernatant of fermentation.
6. the method according to claim 5 for preparing albumen, it is characterised in that：After the induction 24 hours of step S3 and Before the supernatant for collecting fermentation, following steps are further included：

S31：Continue Fiber differentiation 72 hours at 28 DEG C, adding methanol within every 24 hours makes its mass fraction be maintained at 1.0% ~ 1.5%.
7. the method according to claim 5 or 6 for preparing albumen, it is characterised in that：After step s 3, it further includes as follows Step：

S4：The zymotic fluid that S4 cultures obtain is adjusted into pH 7.5 ~ 8.5 to be greater than or equal to turning for 15000 g using NaOH The supernatant of acquisition is loaded to the nickel parent through pH after 7.5 ~ 8.5 50 mM PBS buffer solution balance by speed centrifugation 10 ~ 20 minutes Chromatographic column is closed, with 7.5 ~ 8.5 50 mM containing 20 mM imidazoles and 100 ~ 400 mM NaCl of 5 ~ 10 times of chromatography column volumes PBS buffer solution rinses the nickel affinity chromatographic column；

S5：The nickel affinity chromatographic column, the elution that will be obtained are eluted with containing the buffer solution of 50 mM PBS and 100 ~ 400 mM imidazoles Liquid is dialysed using the bag filter that molecular weight is 10 kDa in 20 mM PBS buffer solution, is then concentrated by ultrafiltration.
8. the method according to claim 7 for preparing albumen, it is characterised in that：After step s 5, following step is further included Suddenly：

S6：The product that the ultrafiltration concentration is obtained is quick-frozen at -80 DEG C, is then freeze-dried.
9. the albumen being prepared according to the method that claim 5-8 any one of them prepares albumen.
10. recombinant vector, cell line described in gene described in the albumen of claim 2,3 or 9, claim 1 or claim 4 Or application of the recombinant protein in the intracellular marker field of zooblast especially insect cell.