CN109295094A

CN109295094A - Recombinant expression method of source of people chemotactic factor (CF) MIP-1 α or the SDF-1 α in eukaryocyte

Info

Publication number: CN109295094A
Application number: CN201811034129.9A
Authority: CN
Inventors: 周志诚; 柴梦莹
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-09-05
Filing date: 2018-09-05
Publication date: 2019-02-01

Abstract

The invention discloses a kind of recombinant expression method of source of people chemotactic factor (CF) MIP-1 α or SDF-1 α in eukaryocyte, this method is using vector plasmid pSFV2 as the carrier for the foreign gene for carrying coding MIP-1 α or SDF-1 α, after packaging plasmid pHelper transfecting eukaryotic cells, the destination protein of exogenous gene expression is modified and there is bioactivity, and the destination protein for using this method expression and purification to go out is with high purity, yield is big, physiology and pharmacological activity are strong.

Description

Recombinant expression method of source of people chemotactic factor (CF) MIP-1 α or the SDF-1 α in eukaryocyte

Technical field

The present invention relates to bioengineering fields, exist in particular to a kind of source of people chemotactic factor (CF) MIP-1 α or SDF-1 α Recombinant expression method in eukaryocyte.

Background technique

Currently, biologically active albumen is to eukaryocyte, especially in terms of biological medicine and in human life All there is important physiological significance in system.Due to, existing procaryotic cell expression system do not have after protein synthesis translation and The ability of modification, and the protein translation and modification of the protein translation of lower eukaryotes and modification system and higher eucaryote are System has very big difference, and therefore, prokaryotes, such as bacterium, the protein after modifying that is beyond expression out such as virus is for example special The biologically active eukaryotic protein such as glycosyl modified.

Summary of the invention

The purpose of the present invention is to provide a kind of recombination of source of people chemotactic factor (CF) MIP-1 α or SDF-1 α in eukaryocyte Expression, this method can effectively give expression to biologically active albumen.

The present invention solves its technical problem and adopts the following technical solutions to realize.

A kind of recombinant expression method of source of people chemotactic factor (CF) MIP-1 α or SDF-1 α in eukaryocyte comprising:

The foreign gene for encoding MIP-1 α or SDF-1 α is connected to vector plasmid, then vector plasmid and packaging plasmid are turned Incasing cells is contaminated, the incasing cells after culture transfection obtains viral vectors, vector plasmid pSFV2, packaging plasmid is PHelper is also connected with special signature after the terminator of foreign gene, and special signature is for encoding at least one specificity Label co-continuous repeats polypeptide；

Viral vectors is transfected into host cell, after cultivation, collects supernatant, host cell is eukaryocyte；With And

Centrifugation, purifying supernatant, obtain the biologically active destination protein of exogenous gene expression.

The beneficial effect of recombinant expression method of coding MIP-1 α or the SDF-1 α provided by the invention in eukaryocyte is: By using the pSFV2 vector plasmid and pHelper packaging plasmid corotation of the foreign gene for carrying coding MIP-1 α or SDF-1 α Incasing cells is contaminated, after being packed, obtains the viral vectors of foreign gene-carrying, then viral vectors transfection eucaryon host will be obtained Cell is recombinantly expressed.In this way, the destination protein for giving expression to foreign gene in eukaryotic host cell have passed through Eukaryotic protein translation and modification system and have bioactivity；Further, since being also connected with after the terminator of foreign gene Special signature can encode at least one special signature's co-continuous and repeat polypeptide, this special signature's co-continuous repeats Polypeptide and high-effect ionic exchanger resinBetween compatibility it is stronger so that prepared MIP-1 α or SDF-1 α purity is higher and yield is big, and bioactivity is strong.

Detailed description of the invention

In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

Fig. 1 is the map of the vector plasmid pSFV2 of the embodiment of the present invention 1；

Fig. 2 is the map of the packaging plasmid pHelper of the embodiment of the present invention 1；

Fig. 3 is what MIP-1 α and the SDF-1a albumen of the embodiment of the present invention 1 was developed the color with coomassie brilliant blue staining solution Gel electrophoresis figure；

Fig. 4 is rejection ability of the MIP-1 α that provides of the embodiment of the present invention 1 to CCR5 preferendum hypotype AIDS virus；

Fig. 5 is saturation curve of the MIP-1 α of the offer of the embodiment of the present invention 1 in conjunction with the CCR5 on HEK-CCR5 cell；

Fig. 6 is the method SDF-1 α obtained using the offer of the embodiment of the present invention 1 to AIDS CXCR4 preferendum hypotype disease The suppression curve of strain；

Fig. 7 is on the method SDF-1 α obtained and A3-01T lymphocyte provided using the embodiment of the present invention 1 The saturation curve that CXCR4 is combined；

Fig. 8 is the SDF-1 α Chemotaxis test illustraton of model for the recombinant expression in experimental example 4；

Fig. 9 is that the SDF-1 α recombinantly expressed in experimental example 4 migrates the influence diagram of quantity to lymphocyte chemotactic；

Figure 10 is the MIP-1 α Chemotaxis test illustraton of model for the recombinant expression in experimental example 5；

Figure 11 is that the MIP-1 α recombinantly expressed in experimental example 5 migrates the influence diagram of quantity to lymphocyte chemotactic.

Specific embodiment

It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

The recombinant expression method to the biological activity protein of the embodiment of the present invention in eukaryocyte carries out specifically below It is bright.

A kind of recombinant expression method of source of people chemotactic factor (CF) MIP-1 α or SDF-1 α in eukaryocyte comprising following step Suddenly.

Step S1: viral vectors is prepared

The foreign gene for encoding MIP-1 α or SDF-1 α is connected to vector plasmid, then vector plasmid and packaging plasmid are turned Incasing cells is contaminated, the incasing cells after culture transfection obtains viral vectors.

Wherein, preferably, being cell lineage for the host cell in the incasing cells and subsequent step of packaging.

Wherein, vector plasmid pSFV2, structure are as shown in Figure 1；Packaging plasmid is pHelper, structure such as Fig. 2 institute Show.

Specifically, digestion processing is carried out to vector plasmid and packaging plasmid with restriction enzyme, forms linear DNA, then Linear DNA is obtained into RNA through transcription, RNA transfection incasing cells after cultivation, obtains carrying coding MIP-1 α or SDF-1 α Foreign gene viral vectors.

Wherein, preferably, restriction enzyme is SapI, this restriction endonuclease can more effectively carry out digestion, so that by The insertion genetic fragment of digestion and the cohesive end of pSFV2 carrier are preferably adhered.

Existing method for purifying proteins can not reach during protein purification due to causing in default of specific marker To biomedical and levels of drugs functional purity or physiological activity, therefore, in order to improve the mesh that exogenous gene expression goes out Purity of the albumen (MIP-1 α or SDF-1 α) in subsequent purification process.Therefore, it is necessary in destination protein (MIP-1 α or SDF- 1 α) C-terminal fusion have special signature's polypeptide for purifying and separate.

The C-terminal of destination protein merges special signature's co-continuous repetition polypeptide, and (hereinafter also referred to special signature is more Peptide), i.e. Twin-Strep-tag, for amino acid sequence as shown in SEQ ID NO.1, which is by two strep-tags The comparison Chinese patent formed after (i.e. Strep tag-1 and Strep-tag II in Chinese patent CN105861557) condensation CN105861557 amino acid residue is less, the smaller polypeptide of spatial volume.Compared to simultaneously with Strep tag-1 and Strep- Tag II uses Twin-Strep-tag as special signature's polypeptide as tag polypeptide, in following purification steps from The compatibility of sub-exchange resin is higher, can specifically be incorporated on the ion exchange resin with double volume, Jin Eryou Conducive to the purity and yield for further increasing purifying protein.

Therefore, correspondingly, in this step, close to hydroxyl after the terminator of the foreign gene of coding MIP-1 α or SDF-1 α Base end reconnects special signature.Special signature's (being named as tag) is for encoding above-mentioned special signature's peptide T win- The nucleotide sequence of Strep-tag, tag are as shown in SEQ ID NO.2.

It should be noted that the quantity for special signature's albumen that the C-terminal of destination protein merges can be according to destination protein Molecular weight increased, the molecular weight of destination protein is bigger, fusion special signature's albumen quantity it is more.Such as two A or four (with 2 multiple) etc., the compatibility of the obtained ion exchange resin in fusion protein and subsequent step is higher (but due to space structure, compatibility and binding ability cannot be with strep-tag albumen (twin-strep- Tag) increase without limitation.

Step S2: transfection host cell

Above-mentioned viral vectors is transfected into host cell, host cell after cultivation, the purpose egg of exogenous gene expression White (MIP-1 α or SDF-1 α) is secreted into the culture solution outside host cell, collects supernatant.Certainly, it before transfection, needs Inactivation processing is carried out to viral vectors.

Wherein, preferably, host cell is eukaryocyte.

Wherein, more preferably, eukaryocyte is mammalian cell.

Specifically, mammalian cell is baby hamster kidney cell (BHK-21), and BHK-21 cell has infinite multiplication Ability.

Existing prokaryotic expression system can be overcome as the host cell of exogenous gene expression using BHK-21 cell System do not have protein synthesis after translate and modification the shortcomings that, and foreign gene can be given expression in BHK-21 cell it is high-purity The destination protein of degree, high specific goes to meet the biomedical research and medicament research and development demand being continuously improved.

Step S3: centrifugation, purifying supernatant

Obtained supernatant is centrifuged, purification process, obtains destination protein (MIP-1 α or SDF-1 α), is had relatively strong Physiology and pharmacological activity.

Specifically, preferably, using having with the ion exchange resin of special signature's polypeptide high-affinity to purpose egg White to be purified, the purity is high of the destination protein of acquisition can be used as different kind organism, medicine, pharmacy clinic and basic research (such as drug screening, neutrality antibody screening, Molecular mapping, protein standard substance preparation).

Step S4: the quantitative detection of albumen

The method (such as BCA, double uric acid titrations etc.) of existing protein quantification detection is needed to albuminous degeneration to be detected It could quantitative detection.And using existing protein quantification detection method total protein content can not directly reactive protein it is pure Degree can not also confirm correct expression, the formation of expression status and polymer of albumen, and it is most objective to realize to purified product Quality and quantity control.

To solve disadvantages mentioned above, in the present invention, obtained destination protein is subjected to vertical native gel electrophoresis and is examined Mas bright blue dye marker mode quantifies destination protein obtained.

Specifically, the gel after electrophoresis is placed in coomassie brilliant blue staining liquid and is impregnated 14~16 hours, developed the color.

Specifically, by albumen Marker, bovine serum albumin (BSA) balf serum albumin of known concentration (being purchased from Thermofischer, the series with various concentration, for drawing standard curve) and to quantitative destination protein Vertical native gel electrophoresis is carried out on same gel.After the completion of electrophoresis, by soak in coomassie brilliant blue staining liquid It 14~16 hours, develops the color.Then, the shade intensity and known concentration of the band according to destination protein on gel The shade intensity contrast of BSA protein band, is analyzed by imager, can obtain the concentration of destination protein, realizes albumen Non denatured quantitative detection.

Feature and performance of the invention are described in further detail with reference to embodiments.

Embodiment 1

The present embodiment provides the gene recombination methods of expression and purification source of people chemotactic factor (CF) MIP-1 α and SDF-1 α a kind of:

The first step prepares the viral vectors that foreign gene-carrying encodes MIP-1 α gene, the nucleotide of MIP-1 α gene For sequence as shown in SEQ ID NO.3, MIP-1 α gene encodes MIP-1 α albumen.

It should be noted that in order to improve in following purification steps, it is ensured that the purification degrees of MIP-1 α albumen are higher, at this In embodiment, special signature's albumen (Twin-Strep-tag) is merged in the C-terminal of MIP-1 α albumen.The Twin-Strep-tag Characteristic in conjunction with capable of having high-affinity and high specific with the ion exchange resin in following purification steps, utilizes the spy Property can by destination protein carry out high-purity purifying.Therefore, it needs to connect special signature after the terminator of MIP-1 α gene (tag, for nucleotide sequence as shown in SEQ ID NO.2, which encodes Twin-Strep-tag), obtains DNA piece Section MIP-1 α-tag.The nucleotide sequence of MIP-1 α-tag segment is as shown in SEQ ID NO.4.

Firstly, building transfer vector plasmid pSFV2-MIP-1 α-tag.

Into the pipe of the vector plasmid pSFV2 containing purifying, restriction enzyme BssHII and XmaI is added, is placed in 37 DEG C Digestion 4 hours, 1% agarose gel electrophoresis of digestion products, gel extraction obtains large fragment, and (pSFV2 was after double digestion with glutinous The linear fragment of property end).

Preparation has the MIP-1 α-tag segment of BssHII and XmaI restriction enzyme site.According to the sequence of MIP-1 α gene, if Meter is respectively provided with the forward primer of BssHII restriction enzyme site, and the reverse primer with tag sequence and XmaI restriction enzyme site.With MIP-1 α gene order is that template carries out PCR amplification and obtains the DNA fragmentation for being connected with tag after the terminator of MIP-1 α gene, i.e., MIP-1 α-tag segment, then with BssHII and XmaI double digestion to pcr amplification product MIP-1 α-tag segment double digestion.After digestion 1% agarose gel electrophoresis of product, then gel extraction small fragment (through double digestion have cohesive terminus,cohesive termini MIP-1 α-tag Segment).Certainly, the MIP-1 α-tag segment containing restriction enzyme site can also directly be synthesized by the method for gene chemical synthesis, then be passed through It is connected on pSFV2 after double digestion.

Large fragment is connected by 1:3 with T4DNA ligase with small fragment；By connection product conversion E. coli impression TOP10, the XL-10 of Agilents company of state such as Life Technologies company, for example, anti-using ammonia benzyl mycin Property screening (ammonia benzyl mycin be purchased from Interchim), picking positive clone molecule extracts plasmid and simultaneously carries out restriction enzyme BssHII It is identified with XmaI double digestion, transfer vector plasmid pSFV2-MIP-1 α-tag is obtained by DNA sequencing determination.

Secondly, transfer vector plasmid pSFV2-MIP-1 α-tag and packaging plasmid pHelper cotransfection incasing cells.

Into the pipe containing pSFV2-MIP-1 α-tag and pHelper, restriction enzyme SapI is added and carries out single endonuclease digestion, PSFV2-MIP-1 α-the tag and pHelper of annular forms linear DNA by SapI single endonuclease digestion, according to mMESSAGEThe operational manual of SP6Transcription Kit transcript reagent box operates, with this obtain it is linear DNA is template, is transcribed.Wherein, each component volume ratio in reaction system is as follows, linear DNA template: transcriptase: reaction Buffer (Reaction Buffer): 100mM dNTP is 3:1:5:1, obtains linear RNA.

Then, packaging virus particle obtains viral vectors.

Incasing cells, that is, BHK-21 cell (have " immortality ", that is, have unlimited multiplication capacity) is seeded in GMEM In culture medium, it is placed in 37 DEG C, 5%CO₂Under the conditions of Multiplying culture.The GMEM culture medium, every 500mL contain 2% 1M HEPES Buffer, 1% (be purchased from Life Technologies) 100X penicillin/streptomycin and 2.5% Calf serum.After culture, after being digested, being centrifuged, BHK-21 cell suspending liquid is obtained, then diluted with PBS buffer solution, obtained Every 800 μ L contains the suspension of 10*106 BHK-21 cell.RNA is added into the suspension, by Gene Pulser The operational manual of XcellElectroporation Systems instrument (electroporation of Biorad) carries out electrotransfection operation, RNA is transcribed in vitro to be transferred in BHK-21 cell, setup parameter voltage 800V, resistance 30Us, electric current 4mA.

The packaging that inoculation of suspension liquid after electrotransfection is carried out to virion into fresh GMEM culture medium, is placed in 37 DEG C, 5%CO₂Under the conditions of co-culture 30h.After co-cultivation, co-culture media is transferred to SW32Ti centrifuge tube (purchased from Beckman Coulter company), it is centrifuged 2 hours under the conditions of 28000rpm, 4 DEG C, abandons supernatant, retain precipitating, which is after packing Virion, the Tris-NaCl-EDTA solution after 4 DEG C of ice baths are added into pipe are resuspended precipitating, obtain viral suspension, then 2 hours are stood under the conditions of 4 DEG C, is resuspended again, which saves in -80 DEG C.

Second step, viral vectors transfection host cell

By host cell, that is, BHK-21 cell inoculation in GMEM culture medium, it is placed in 37 DEG C, 5%CO₂Under the conditions of be proliferated training Support until cell confluency degree be 80% or so, then remove supernatant.The GMEM culture medium, every 500mL contain 10mL 1M HEPES Buffer, 1% 100X penicillin/streptomycin and 2.5% calf serum.

The viral suspension of -80 DEG C of preservations is taken out, chymotrypsin is added, and (Chymotrypsin is purchased from Bohenringer Mammhem) and the sterile CaCl of 50mM is added in the ratio of 2:1 by volume₂Solution activates virus 30 under room temperature Minute.It is subsequently added into Aprotinin (aprotinin is purchased from Sigma company), according to the Ki inhibition concentration pair suggested on its specification Virus carries out inactivation processing.

8mL 2%GMEM culture medium is added into the host cell after the Multiplying culture for removing supernatant (containing handling through inactivation Virus afterwards), 37 DEG C are placed in, is cultivated 1 hour；Then the 2%GMEM culture medium for adding equivalent is placed in 37 DEG C, and culture 1 is small When；Then remove supernatant.Cell is cleaned twice with 0%GMEM culture medium；Then the GMEM that 30mL is free of calf serum is added Culture medium is placed in 37 DEG C, cultivates 28-30 hours.Exogenous genetic fragment (MIP-1 α-tag) is stablized with the proliferation of host cell Ground expressed fusion protein, that is, MIP-1 α albumen-Twin-Strep-tag, and be secreted into supernatant, obtain albumen supernatant.It needs It is noted that the special signature's albumen, that is, Twin-Strep-tag has no effect on the bioactivity of MIP-1 α albumen.

Third step collects albumen supernatant and purifies destination protein

After culture, albumen supernatant is collected.By albumen supernatant be transferred to 50mL 5kDa cut-off centrifuge tube and 50kDa cut-off centrifuge tube (will be filtered off, the fusion protein, that is, MIP- of the present embodiment less than 5kDa, the albumen greater than 50kDa The molecular weight of 1 α-twin-Strep-tag is equal to 10kDa can finally be concentrated protein liquid and collect so can be trapped), It is centrifuged 45 minutes under the conditions of 4500rpm, 4 DEG C, repeated centrifugation 4 times, until albumen supernatant is left 0.5mL.Wherein, 3 times In centrifugal process, every time after centrifugation, the filtrate of collecting pipe below centrifuge tube is removed in cleaning, then freezes PBS with 4 DEG C of preservations Buffer carries out equivalent supplement, that is, the PBS buffer solution being added mixes in equal volume with remaining albumen supernatant, and the PBS is buffered The 1M sodium chloride nacl of the avidin (Avidin) and 1:10 that have that volume ratio is the 1mg/ml of 1:100 is added in liquid.

It should be noted that because cell juice and a large amount of biotins generated containing cell culture medium supernatant can be non- High-effect ionic exchanger resin in specific bond subsequent stepIt is serious to reduce fusion twin-Strep- The purification process of the MIP-1 α albumen of tag.Avidin (being contained in eluent) can be used for combining and inhibit on cell The biotin secreted out of clearly helps to improve the purification degrees of MIP-1 α-twin-Strep-tag albumen.

After centrifugation, remaining 0.5mL albumen supernatant Gravity flow Column ion exchange resin column (being purchased from IBA company) carried out column purification, crosses column method and carries out by its specification；In filtration egg After white supernatant, with 2ml 1X cleaning buffer solution (Wash Buffer, is purchased from IBA company) cleaning ion exchange resin column, it is repeated 5 times；After cleaning, using 5~10ml 1X eluent (Elution Buffer with Biotin is purchased from IBA company) by destination protein from ion exchange resin It is filtered out on column, obtains albumen filter liquor.

It should be noted that this, which crosses column purification operation, is utilized ion exchange resin column Gravity flowColumn in conjunction with the high-affinity of special signature's albumen and high specific, The other impurities albumen being effectively filtered to remove in albumen supernatant, it is ensured that the MIP-1 α egg that obtained albumen filter liquor contains White purity is very high, and purity is close to 100%.Compare IBA company second generation Gravity flow Strep-Tactin SuperColumn product, the application is using newest third generation Gravity flow Superflow column: it is suitble to continuous two strep-tag strep-tags and ion exchange resin its main feature is that having The binding site of pillar height affine combination can make the twin-strep-tag strep-tag in above-mentioned expressed recombinant protein It increases exponentially with the affinity of ion exchange resin column, and the volume of the ion exchange column is bigger, theoretically can at least mention Rise 4-10 times of affine combination ability.

Obtained albumen filter liquor is transferred to the 1kDa cut-off centrifuge tube (purchased from Vivaspin company) of 50mL, in 4500rpm, it is centrifuged 10 minutes under the conditions of 4 DEG C, repeated centrifugation 4 times until albumen filter liquor is left 0.2~0.4mL.Wherein, preceding In 3 centrifugal processes, every time after centrifugation, the filtrate of collecting pipe below centrifuge tube is removed in cleaning, is then freezed with 4 DEG C of preservations PBS buffer solution carries out equivalent supplement, that is, the PBS buffer solution being added mixes in equal volume with albumen filter liquor remaining in centrifuge tube. After centrifugation, protein concentrate filter liquor is obtained.It can be saved in -80 DEG C of encapsulation.The MIP-1 α that the protein concentrate filter liquor contains Recombinant protein purity is high, has bioactivity.

In addition, the present embodiment also carries out expression and purification by purpose albumen of SDF-1 α, feature of the invention is further illustrated And performance.The principle and step of expression and purification SDF-1 α with expression and purification MIP-1 α, the difference is that, preparing viral vectors In step, with SDF-1 α gene replacement MIP-1 α gene, special signature is also connected in the C-terminal of SDF-1 α gene Tag genetic fragment, the tag genetic fragment encode Twin-Strep-tag.The nucleotide sequence such as SEQ of the gene of SDF-1 α albumen ID NO.5.Obtained SDF-1 α purity is very high according to the above method, and has very strong bioactivity and pharmacological activity.

In addition, in other examples, also using expression of the biological activity protein of the present invention in eukaryocyte Other kinds of AIDS virus memebrane protein such as R5 type or X4 type or other kinds of needs can be expressed after translation Just there is the albumen of bioactivity after modification, it is only necessary to clone the corresponding foreign gene for encoding the type albumen according to a conventional method On to pSFV2 viral vectors of the invention, then carries out, can give expression to high-purity by cycle and taking corresponding operation provided in this embodiment Degree, biologically active albumen.

Experimental example 1:

To the non denatured quantitative detection of MIP-1 α and SDF-1

Resulting MIP-1 α and SDF-1 is purified to embodiment 1 in the detection example and carries out non denatured quantitative detection, together When, using MIP-1 α and SDF-1 expressed by the recombinant expression method that is provided by Chinese patent CN105861557 as control, Use Strep-Tag1 and Strep-Tag2 for special signature's polypeptide simultaneously.Compare the above patent, the method for the present embodiment In combined coefficient, generated time, level of purification has greater advantage on Product Activity.

Firstly, proteins gel electrophoresis is carried out using 18 porin running gels (Criteron 18well), loading albumen: Compare that (Accuruler, PM-001X are purchased to the protein concentrate filter liquor containing destination protein of quantitative detection, protein labeling The BSA albumen of Biopioneer, molecular weight for 3.5~240kDa) and for making standard curve reference (is purchased from Thermofischer company, BSA original liquid concentration 2mg/mL, when loading, are diluted to the critical field of 0.5-10 μ g/ μ L), electrophoresis ginseng Number: 150V, 1 hour.

Electrophoresis terminates, and gel is first cleaned once with ddH20, and gel is then placed in coomassie brilliant blue staining liquid (protein Stain buffer) in develop the color, in 4 DEG C of soaked overnights, every other day after purpose band appearance after, Reusability ddH20 rocks It cleans (30min/ time, 5-6 times or so), up to BSA albumen does not have the other impurities to be referring to band and destination protein band Only.Then the analysis software provided using ChemiDoc XRS+System imager is deep referring to band color to each BSA albumen The corresponding relationship of shallow intensity and its BSA protein concentration is analyzed, and (totally 6 BSA albumen are referring to band, corresponding BSA concentration Value is 4 μ g/ μ L, 2 μ g/ μ L, 1 μ g/ μ L, 0.5 μ g/ μ L, 0.25 μ g/ μ L, 0.125 μ g/ μ L respectively), with BSA concentration for horizontal seat Mark is that standard curve is made in ordinate referring to band shade intensity, in conjunction with the standard curve, by obtaining destination protein item The shade intensitometer of band calculates the concentration of destination protein, realizes the purpose of the non denatured quantitative detection of albumen.Protein gel Electrophoresis result is as shown in Figure 3.

From the figure 3, it may be seen that the concentration of MIP-1 α albumen of the MIP-1 α molecular weight of albumen in 8kDa, protein concentrate filter liquor is 0.25 μ g/ μ L or so.The inconsistent reason in the position of the destination protein band of #1-#5, which is that otherness is glycosyl modified, leads to purpose The size of albumen is variant.Compared to Chinese patent CN105861557 provide recombinant expression method (see the in Fig. 3 the 2nd and the 3rd Band), the content and purity of destination protein expressed by this method in the embodiment of the present invention 1 are bigger (see the 4th He in Fig. 3 5th band).It is shown by the stainable bands of protein gel, the purifying process that the present invention uses, significantly reduction production cost, The yield of at least 2-4 times of recombinant protein MIP-1 α and SDF-1 α is improved, and is maintained close to 100% high-purity.

Experimental example 2

To the bioactivity research of MIP-1 α albumen:

The bioactivity for the recombinant expression method MIP-1 α albumen obtained that above-described embodiment 1 provides is studied, People's CD4+T lymphocyte primary is inhibited to be felt by AIDS virus (zoo virus strain and Strain primary) in MIP-1 α albumen The anti-virus ability of dye.Simultaneously with MIP-1 α expressed by the recombinant expression method that is provided by Chinese patent CN105861557 As control (using Strep-Tag1 and Strep-Tag2 for the recombinant expression method of special signature's polypeptide simultaneously).

One, experimentation:

Source of people cell primary is to be provided after the PBMC of healthy human body peripheral blood separation using Mitenyli Biotec CD4 Beads enrichment is purified into the T lymphocyte with the T4 antigen determinant positive (CD3+CD4+100% is positive).

The I type AIDS virus strain (zoo virus strain JR-CSF and wild-type strain ADA01) of two kinds of CCR5 hypotypes For the infection of source of people CD4+ cell primary, expand and break up in the following ways: 1% penicillin is added in RPMI culture medium With the recombination human source interleukin I L-2 (being purchased from Mitenyli Biotec) of streptomysin (deriving from GIBCO)+20ng/ml.Culture 3 After it, in the state of culture plate cell dense growth, and cell density is maintained to cultivate between 1500000~2000000/ milliliters Between base, cell enters logarithmic growth phase.

The cell into logarithmic growth phase is taken, 50 μ l culture medium (+1% penicillin of RPMI culture medium and chains are added in every hole The recombination human source interleukin I L-2 of mycin+20ng/ml), the lymphocyte primary containing 200000 CD4+T purifying, 50ul contain It is dense with different MIP-1 α that infection experiment titrates 10ng P24 AIDS virus nucleoprotein/hole AIDS virus and 100ul Spend (itself being the natural chemotactic factor (CF) of CCR5) or the culture medium without test medicine.After mixing, it is then placed in cell Incubator is incubated for 48 hours.

After cultivation, centrifugation elutes twice and is added what 100 μ l Promega Renila luciferase kits provided 1 times of lysate mixes, and is tested in 24 hours.Using 96 orifice plates (PerkimElmer is opaque, white), 50 μ l are added The cell lysate containing lysate enough Renila Luciferase colour developing is added on the lance head of spectrophotometer Reagent (chromogenic substrate of 1 times of volume mixes 1000 times of diluted substrate buffer solutions), mixes, accesses spectrophotometer The spray gun input port of (Molecule Devices Spectramax Plus 384), instruction allow photometer to spray into automatically to every hole The colour reagent of 50 μ l.Then spectrophotometer is allowed to be read.Then the RLU fluorescence reading shown according to Screen Program, into Row is quantitative.

Two, experimental result

I type AIDS virus strain for both CCR5 preferendums of zoo virus strain and wild-type strain, it is different Place is that the sequence in certain the several section virus membrane antigen (gp120) is inconsistent, uses CCR5 as invasion T lymphocyte Core mechanism is constant.The physiological mechanism that the two invades source of people lymphocyte primary is identical, the sense to CD4+ T lymphocyte primary Dye ability is also similar.

As shown in figure 4, for the infection of two kinds of AIDS viruses strain, the inhibition virus capable of MIP-1 α is close to (K_dIt is located at About 10nM is horizontal), meanwhile, on inhibiting performance, since CCR5 is less in the expression of cell primary, the ability of cell infection is more Add weak, each tested molecule is bigger to the influence power of cell infection.MIP-1 α is better than pair the rejection ability of zoo virus strain The restraint of wild-type strain, thus illustrates, the MIP-1 α recombinant protein synthesized through the invention is inhibiting AIDS CCR5 Infecing for preferendum subtype virus strain shows powerful rejection ability.

Saturation curve of the MIP-1 α albumen in conjunction with the CCR5 on HEK-CCR5 cell is as shown in Figure 5.Due to stablizing cell The CCR5HEK cell line of Membrane surface expression can be coupled in vitro the recombination MIP-1 α of AF647 fluorophor label with specific recognition Albumen allows to the CCR5 of the cell membrane surface of stable bond HEK-CCR5 cell, and determines its maximum molecular recognition amount Bmax.The specific recognition capability of MIP-1 α is very strong, and twin-strep-tag recombinant protein especially used in the present invention is matched MIP-1 α obtained by IBA third generation strep-tactin purification system is closed, expression quantity and affinity are better than Chinese patent The purifying expression system that CN105861557 is provided, this difference can be from the Bmax maximum knot of identical Kd value and 50% difference Resultant could see.Thus illustrate, recombinant protein MIP-1 α synthesized by the present invention can effectively inhibit CCR5 preferendum hypotype AIDS The infection of virus has good bioactivity.

Experimental example 3

To bioactivity research of the SDF-1 α albumen in terms of inhibiting HIV infection:

The bioactivity for the recombinant expression method SDF-1 α albumen obtained that above-described embodiment 1 provides is studied, I.e. SDF-1 α albumen inhibits people's CD4+T lymphocyte primary by AIDS virus CXCR4 preferendum (zoo virus strain and disease primary Strain) infection anti-virus ability.Simultaneously expressed by the recombinant expression method that is provided by Chinese patent CN105861557 SDF-1 α (uses Strep-Tag1 and Strep-Tag2 for the recombinant expression side of special signature's polypeptide simultaneously as control Method).

One, experimentation:

The AIDS virus strain of I type (zoo virus strain NL4.3 and the wild-type strain gp120# of two kinds of CXCR4 hypotypes 99) be used for the infection of source of people CD4+ cell primary, expand and break up in the following ways: 1% disk Buddhist nun is added in RPMI culture medium The recombination human source interleukin I L-2 (being purchased from Mitenyli Biotec) in XiLin and streptomysin (deriving from GIBCO)+20ng/ml.Training After supporting 3 days, in the state of culture plate cell dense growth, and cell density is maintained to be situated between 1500000 and 2000000/ milliliters Culture medium, cell enter logarithmic growth phase.

The cell into logarithmic growth phase is taken, 50ul culture medium (+1% penicillin of RPMI culture medium and chain is added in every hole The recombination human source interleukin I L-2 of mycin+20ng/ml), the lymphocyte primary containing 200000 CD4+T purifying, 50ul contain It is dense with different SDF-1 α that infection experiment titrates 10ng P24 AIDS virus nucleoprotein/hole AIDS virus and 100ul Spend (itself being the natural chemotactic factor (CF) of CXCR4 receptor) or the culture medium without test medicine.After mixing, it is then placed in Cell incubator is incubated for 48 hours.

After cultivation, centrifugation elutes twice and the offer of 100ul Promega Renila luciferase kit is added 1X lysate mixes, and is tested in 24 hours.Using 96 orifice plates (PerkimElmer is opaque, white), it is added 50ul's Enough Renila Luciferase colour developing examinations are added on the lance head of spectrophotometer in cell lysate containing lysate Agent (chromogenic substrate of 1 times of volume mixes 1000 times of diluted substrate buffer solutions), mixes, accesses spectrophotometer (Molecule Devices Spectramax Plus 384) spray gun input port, instruction allow photometer automatically to every hole spray into 50ul colour developing Reagent.Then spectrophotometer is allowed to be read.Then the RLU fluorescence reading shown according to Screen Program, is quantified.

Two, experimental result

I type AIDS virus strain for both CXCR4 preferendums of zoo virus strain and wild-type strain, it is different Place is certain several section of virus membrane antigen (gp120) (as influenced gp120 memebrane protein variable region of the CCR5 in conjunction with gp120 Several amino acid sequences in V3 structure) sequence it is inconsistent.However, the physiology machine of the two invasion source of people lymphocyte primary Make it is identical, it is also similar to the infection ability of CD4+T lymph cell primary.

As shown in fig. 6, for the infection of two kinds of AIDS viruses strain, the inhibition virus capable of SDF-1 α is close to (K_dIt is located at About 5nM is horizontal), meanwhile, on inhibiting performance, since CXCR4 is big in the expression quantity of cell primary, the ability of cell infection can be more By force, each tested molecule is bigger to the influence power of cell infection.Two kinds of SDF-1 α that the present invention synthesizes are to two kinds of CXCR4 Strain Rejection ability be better than the restraint to wild-type strain, thus illustrate, the SDF-1 α recombinant protein synthesized through the invention Infecing for AIDS CXCR4 preferendum subtype virus strain is being inhibited to show powerful rejection ability.

Saturation curve of the SDF-1 α albumen in conjunction with the CXCR4 on HEK-CXCR4 cell is as shown in Figure 7.It is thin using stablizing The A3-01T lymphocytic series of after birth surface expression can be coupled in vitro the recombination of AF647 fluorophor label with specific recognition SDF-1 α albumen allows to the CXCR4 of cell membrane surface itself great expression of stable bond HEK-CXCR4 cell, and really Determine its maximum molecular recognition amount Bmax.The specific recognition capability of SDF-1 α is very strong, twin- especially used in the present invention Strep-tag recombinant protein cooperates IBA third generation strep-tactin XT purification system SDF-1 α obtained, expression quantity The purifying expression system of Chinese patent CN105861557 offer is better than with affinity, this difference can be from identical Kd value It could see the productivity and yield of two methods and the difference of quality with close to the Bmax Bmax greater than 150% difference.By This illustrates that recombinant protein SDF-1 α synthesized by the present invention can effectively inhibit the infection of CXCR4 preferendum hypotype AIDS virus And to the effective specific recognition of CXCR4.

Experimental example 4

The influence that recombinant expression SDF-1 α acts on A3.01T lymphocyte chemotactic

One, experimental principle

Chemotaxis is the movable vital signs of lymphocyte, on pathology, usually infected lymphocyte Migration, a functional embodiment of cancer cell locality diffusion and pathological index.Its chemotactic active mechanism comes from cell Adhesion molecule integrin include integrins, desintegrins and they between interact and chemotactic factor (CF) and become Change factor acceptor be combined with each other, the complexing actions such as signals-modulating.SDF-1 is as the unique agonist of CXCR4 receptor, for a large amount The T lymphocyte for expressing CXCR4 generates irreversible transferance.Damage and infected tissue and host cell can all raise CXCR4 and SDF-1 alpha expression, so that this lymphocyte is more aobvious strong because of the chemotaxis that chemotactic factor (CF) induces.Therefore SDF-1 α Recombinant expression technique for will (lymphocyte transmigration and chemotactic be living as the medical active albumen for influencing CCR5 physiological function Change function) there is apparent directiveness effect.

Two, experimental method

Cell chemotaxis experimental model is as shown in figure 8, experiment uses the Transwell of the offer of Corning company Bicarbonate filter chamber (48wells) is being separately added into 50ul and 500ul in the upper and lower respectively first Mixed liquor (streptomysin (Gibco)+10% of the penicillin+100ug/ml of RPMI1640 culture medium+100ug/ml is purchased from 100% calf serum of Biowest), it is put into 37 DEG C of incubators and cultivates 4 hours, 50ul then is added on upper layer and contains 50000 The T lymphocyte of a A3.01 expression CXCR4 from the application method and comes from lower layer's addition 50ul difference gradient concentration The recombination SDF-1 α and negative control molecule AMD3100 of Chinese patent CN105861557 method expression.

37 DEG C of incubators are put into, 5h to be migrated is waited.After 5 hours, lower confluent monolayer cells are all collected, the stream of 100ul is concentrated to In the buffer of formula Cytometric Analysis, the channel of FSC and SSC then through flow cytometer Canto-II carry out 180 seconds thin Born of the same parents' reading Analysis (every 3 Kong Weiyi concentration), statistically analyze each concentration tested molecule inducing cell from upper layer down The mobile cell quantity of layer, as a result as shown in Figure 9.

Three, experimental result:

It is marked in Fig. 9, " SDF-1alpha (previous method) " refers to be mentioned by Chinese patent CN105861557 The recombination SDF-1 α of the method expression of confession；Similarly, " SDF-1alpha (current method) " refers to by the embodiment of the present application 1 The recombination SDF-1 α of the method expression of offer.

As seen from Figure 9, the CXCR4 chemotactic process of SDF-1a induction causes a large amount of A3.01T lymphocyte to migrate from upper layer To lower layer, when SDF-1a concentration is 10^-10M~10^-8When the section M is sequentially increased, the migration quantity of A3.01T lymphocyte gradually increases It is more；When SDF-1a concentration is greater than 10^-8When M, the migration quantity of A3.01T lymphocyte is gradually decreased.Illustrate SDF-1 α to A3.01T Lymphocyte has the concentration chemotactic effect of normal distribution.The Strep-tactin XT+twin-strep- provided through the invention Tag a new generation purifies the SDF-1 α recombinant protein of expression system expression in the same concentration from 10^-9M~10^-5M is lured under stimulation The cell number for leading chemotactic is higher and poor than the cell quantity of Strep-tactin+strep-tag system in CN105861557 Different more significant, the efficiency under low concentration induction is more preferable.As negative control, from the commercialization AMD3100 of SIGMA purchase (for the micromolecular inhibitor of known CXCR4) does not generate special chemotactic effect to CXCR4.

Under chemotaxis, the combination of CXCR4 and chemotactic factor (CF) SDF-1 α are the interaction processes of a dosage effect； A large amount of SDF-1 α pours in the chemotaxis that will affect CXCR4, generates internal actin (actin) and filament albumen (filament) polymerization, by the stronger chemotactic of activity creep mechanism (pseudopodia) make cell formed local polarisation State migrates into lower layer (or moving in parallel) from upper layer.And its tested molecule does not then generate any chemotaxis to this. But excessive SDF-1 α accumulation will form SDF-1 α double focusing object (dimer), be unfavorable for the chemotactic effect of SDF-1 α.It causes instead The steric effect of double focusing object influences the combination of the binding site and CXCR4 of SDF-1 α.And SDF-1 α itself can be from T lymph Cell itself oneself is released, and the cyclical effect of autocrine is formed, and directly affecting the chemotactic effect of SDF-1 α itself, (actuating quantity is much Less than the recombinant protein dosage of test).

Experimental example 5

The influence that recombinant expression MIP-1 α acts on A3.01-CCR5T lymphocyte chemotactic

One, experimental principle

Similarly, MIP-1 α important as CCR5 receptor chemotactic factor (CF) and native agonist, for expressing a large amount of CCR5 T lymphocyte generate irreversible transferance.Damage and it is infected tissue and host cell can all raise CCR5 and MIP-1 alpha expression, so that the lymphocyte (especially T lymphocyte) of this CCR5 expression is made because of the chemotactic that chemotactic factor (CF) induces With more aobvious strong.CCR5 receptor can be combined with CCL4 (MIP-1 β) and CCL5 (RANTES), and wherein CCL4 and CCL5 can also be tied The chemotaxis of conjunction CCR5 induction of lymphocyte, but weak (the Kd difference of the affinity ratio CCL3 of CCL4 and CCL5 combination CCR5 For 20-50nM), and the Kd of CCL3 is about 3.3-8nM, and the natural differences of Kd are different with its according to specific different cell line Therefore expression system and acceptor quantity refer to the recombinant expression technique of MIP-1 α for will be as influencing CCR5 physiological function Medical active albumen (lymphocyte transmigration and chemotactic mobilizing function) has apparent directiveness effect.

Wherein, MIP-1 α (also referred to as CCL3) is source of people Macrophage Inflammatory Protein1α, is CCR5 receptor native agonist With lymphocyte and leukocyte chemokines.

By the inducing action of chemotactic factor (CF), cell can generate the variation of polar cell film, and tend to chemotactic factor (CF) to being formed The place polarization migration of high concentration.This chemotactic process is by chemotactic factor (CF) concentration, the expression quantity of chemokine receptors and thin The influence of the normality of born of the same parents.

Two, experimentation:

Cell chemotaxis experimental model is as shown in Figure 10, and experiment uses the Transwell of the offer of Corning company Bicarbonate filter chamber (48wells) is being separately added into 50ul and 500ul in the upper and lower respectively first Mixed liquor (streptomysin (Gibco)+10% of the penicillin+100ug/ml of RPMI1640 culture medium+100ug/ml is purchased from 100% calf serum of Biowest), it is put into 37 DEG C of incubators and cultivates 4 hours, 50ul then is added on upper layer respectively and contains The T lymphocyte and 50ul that 50000 A3.01-CCR5 express CCR5 are from the application method and from Chinese patent (abbreviation MVC, No. CAS is 376348- to the recombination MIP-1 α and negative control molecule Maraviroc of CN105861557 method expression 65-1 is the small-molecule drug of commercialization CCR5 antagonist, while being also the antagonist of CCR5).

37 DEG C of incubators are put into, 5h to be migrated is waited.After 5 hours, lower confluent monolayer cells are all collected, in the same way into Row detection, as a result as shown in figure 11.

Three, experimental result:

It is marked in Figure 11, " MIP-1alpha (previous method) " refers to be mentioned by Chinese patent CN105861557 The recombination MIP-1a of the method expression of confession；Similarly, " MIP-1alpha (current method) " refers to by the embodiment of the present application 1 The recombination MIP-1 α of the method expression of offer.

As seen from Figure 11, the CCR5 chemotactic process of MIP-1 α induction causes a large amount of A3.01-R5 lymphocyte to move from upper layer Lower layer is moved to, when MIP-1 α concentration is 10^-10M~10^-7When the section M is sequentially increased, the migration quantity of A3.01-R5 lymphocyte by It is cumulative more；When MIP-1 α concentration is greater than 10^-7When M, the migration quantity of A3.01-R5 lymphocyte is gradually decreased.Illustrate MIP-1 α couple A3.01-R5 lymphocyte has the concentration chemotactic effect of normal distribution.The strep-tactin provided by the embodiment of the present application 1 XT+twin-strep-tag a new generation purifies the MIP-1 α recombinant protein of expression system expression in the same concentration from 10^-9M~ 10^-5Cell of the cell number of M inducing chemotactic under stimulating compared with Strep-tactin+strep-tag system in CN105861557 Number wants high, and efficiency is more preferable.As negative control, the commercialization Maraviroc bought from SIGMA is not generated CCR5 special Different chemotactic effect.

Under chemotaxis, the combination of CCR5 and chemotactic factor (CF) MIP-1 α are the interaction processes of a dosage effect； A large amount of MIP-1 α pours in the chemotaxis that will affect CCR5, generates internal actin (actin) and filament albumen (filament) polymerization, by the stronger chemotactic of activity creep mechanism (pseudopodia) make cell formed local polarisation State migrates into lower layer (or moving in parallel) from upper layer.And its tested molecule does not then generate any chemotaxis to this. But excessive MIP-1 α accumulation will form MIP-1 α double focusing object (dimer), the chemotactic effect for being helpless to MIP-1 α causes instead The steric effect of double focusing object influences the combination of the binding site and CCR5 of MIP-1 α.And MIP-1 α itself can be from T lymph Cell itself oneself is released, and the cyclical effect of autocrine is formed, and directly affecting the chemotactic effect of MIP-1 α itself, (actuating quantity is much Less than the recombinant protein dosage of test).

Thus illustrate, SDF-1 α and MIP-1 α albumen expressed by the system described by this patent demonstrates current experiment Expressed product is unique；It is also the previous generation system in comparison CN105861557, it was demonstrated that this patent and test method are Optimal at present can pass through with the chemotactic factor (CF) of inducer T lymphocyte (expression CXCR4 or CCR5) and for CXCR4 and CCR5 The synthesis technology and method of the agonist of biosynthesis.

In conclusion expression of the biological activity protein provided by the invention in eukaryocyte, obtained purpose Albumen have passed through baby hamster kidney cell protein translation and modification system translation modification, have stronger normal molecular amount, Bioactivity, physiological activity and pharmacological activity；And the destination protein being actually isolated and purified with and theoretic destination protein Similitude is with high purity close to 100%；The destination protein obtained using the expression can be by metabolic marker method mark fluorescent Label or radioactive isotope label make different kind organism, medicine, pharmacy clinic and basic research, such as drug screening, neutrality Antibody screening, Molecular mapping, protein standard substance preparation；Using the expression expression albumen also have it is with short production cycle, The features such as at low cost.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

SEQUENCE LISTING

<110>Chai Mengying

<120>recombinant expression method of a kind of source of people chemotactic factor (CF) MIP-1 α or SDF-1 α in eukaryocyte

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 30

<212> PRT

<213>artificial sequence

<400> 1

Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys

20 25 30

<210> 2

<211> 87

<212> DNA

<213>artificial sequence

<400> 2

tggagccacc cccagttcga gaagggcggc ggcagcggcg gcggcagcgg cggcagcagc 60

gcctggagcc acccccagtt cgagaag 87

<210> 3

<211> 279

<212> DNA

<213>artificial sequence

<400> 3

atgcaggtct ccactgctgc ccttgctgtc ctcctctgca ccatggctct ctgcaaccag 60

ttctctgcat cacttgctgc tgacacgccg accgcctgct gcttcagcta cacctcccgg 120

cagattccac agaatttcat agctgactac tttgagacga gcagccagtg ctccaagccc 180

ggtgtcatct tcctaaccaa gcgaagccgg caggtctgtg ctgaccccag tgaggagtgg 240

gtccagaaat atgtcagcga cctggagctg agtgcctga 279

<210> 4

<211> 366

<212> DNA

<213>artificial sequence

<400> 4

atgcaggtct ccactgctgc ccttgctgtc ctcctctgca ccatggctct ctgcaaccag 60

ttctctgcat cacttgctgc tgacacgccg accgcctgct gcttcagcta cacctcccgg 120

cagattccac agaatttcat agctgactac tttgagacga gcagccagtg ctccaagccc 180

ggtgtcatct tcctaaccaa gcgaagccgg caggtctgtg ctgaccccag tgaggagtgg 240

gtccagaaat atgtcagcga cctggagctg agtgcctgat ggagccaccc ccagttcgag 300

aagggcggcg gcagcggcgg cggcagcggc ggcagcagcg cctggagcca cccccagttc 360

gagaag 366

<210> 5

<211> 282

<212> DNA

<213>artificial sequence

<400> 5

atgaacgcca aggtcgtggt cgtgctggtc ctcgtgctga ccgcgctctg cctcagcgac 60

gggaagcccg tcagcctgag ctacagatgc ccatgccgat tcttcgaaag ccatgttgcc 120

agagccaacg tcaagcatct caaaattctc aacactccaa actgtgccct tcagattgta 180

gcccggctga agaacaacaa cagacaagtg tgcattgacc cgaagctaaa gtggattcag 240

gagtacctgg agaaagcttt aaacaagagg ttcaagatgt ga 282

<210> 6

<211> 369

<212> DNA

<213>artificial sequence

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atgaacgcca aggtcgtggt cgtgctggtc ctcgtgctga ccgcgctctg cctcagcgac 60

gggaagcccg tcagcctgag ctacagatgc ccatgccgat tcttcgaaag ccatgttgcc 120

agagccaacg tcaagcatct caaaattctc aacactccaa actgtgccct tcagattgta 180

gcccggctga agaacaacaa cagacaagtg tgcattgacc cgaagctaaa gtggattcag 240

gagtacctgg agaaagcttt aaacaagagg ttcaagatgt gatggagcca cccccagttc 300

gagaagggcg gcggcagcgg cggcggcagc ggcggcagca gcgcctggag ccacccccag 360

ttcgagaag 369

Claims

1. a kind of recombinant expression method of source of people chemotactic factor (CF) MIP-1 α or SDF-1 α in eukaryocyte, which is characterized in that its Include:

The foreign gene for encoding MIP-1 α or SDF-1 α is connected to vector plasmid, then the vector plasmid and packaging plasmid are turned Incasing cells is contaminated, the incasing cells after culture transfection obtains viral vectors, and the vector plasmid is pSFV2, the packaging Plasmid is pHelper, special signature is also connected with after the terminator of the foreign gene, the special signature is for encoding At least one special signature's co-continuous repeats polypeptide；

By the viral vectors transfection host cell, after cultivation, supernatant is collected, the host cell is eukaryocyte；With And

It is centrifuged, purifies the supernatant, obtain the biologically active destination protein of the exogenous gene expression.

2. recombinant expression side of source of people chemotactic factor (CF) MIP-1 α or the SDF-1 α according to claim 1 in eukaryocyte Method, which is characterized in that special signature's co-continuous repeats the amino acid sequence of polypeptide as shown in SEQ ID NO.1.

3. recombinant expression side of source of people chemotactic factor (CF) MIP-1 α or the SDF-1 α according to claim 1 in eukaryocyte Method, which is characterized in that the nucleotide sequence of the special signature is as shown in SEQ ID NO.2.

4. recombinant expression side of source of people chemotactic factor (CF) MIP-1 α or the SDF-1 α according to claim 1 in eukaryocyte Method, which is characterized in that the special signature is two.

5. recombinant expression side of source of people chemotactic factor (CF) MIP-1 α or the SDF-1 α according to claim 1 in eukaryocyte Method, which is characterized in that the eukaryocyte is mammalian cell.

6. recombinant expression side of source of people chemotactic factor (CF) MIP-1 α or the SDF-1 α according to claim 5 in eukaryocyte Method, which is characterized in that the mammalian cell is baby hamster kidney cell.

7. recombinant expression side of source of people chemotactic factor (CF) MIP-1 α or the SDF-1 α according to claim 1 in eukaryocyte Method, which is characterized in that the step of preparing the viral vectors further include: with restriction enzyme to the vector plasmid and described Packaging plasmid carries out digestion processing, linear DNA is formed, then the linear DNA is obtained RNA through transcription, by the RNA transfection institute State incasing cells.

8. recombinant expression side of source of people chemotactic factor (CF) MIP-1 α or the SDF-1 α according to claim 8 in eukaryocyte Method, which is characterized in that the restriction enzyme is SapI.

9. recombinant expression side of source of people chemotactic factor (CF) MIP-1 α or the SDF-1 α according to claim 1 in eukaryocyte Method, which is characterized in that further include: the obtained destination protein is subjected to vertical electrophoresis on gel, described in after electrophoresis Gel, which is placed in coomassie brilliant blue staining liquid, to be impregnated 14~16 hours, is developed the color.