WO2020253400A1 - 一种通过增强子促进fgf-2在293t细胞中表达的方法 - Google Patents

一种通过增强子促进fgf-2在293t细胞中表达的方法 Download PDF

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WO2020253400A1
WO2020253400A1 PCT/CN2020/087967 CN2020087967W WO2020253400A1 WO 2020253400 A1 WO2020253400 A1 WO 2020253400A1 CN 2020087967 W CN2020087967 W CN 2020087967W WO 2020253400 A1 WO2020253400 A1 WO 2020253400A1
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fgf2
sequence
plasmid vector
dna sequence
pnc1
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钟树根
邝纬阳
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梦芊科技知识产权有限公司
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Definitions

  • the invention relates to an enhancer sequence, a plasmid vector pNC1, a preparation method of the plasmid vector pNC1, the use of the plasmid vector pNC1 in expressing FGF-2, and a transformant.
  • FGF2 is a very valuable protein in the pharmaceutical and healthcare industries.
  • FGF2 is an effective therapeutic protein for treating neurodegenerative diseases, heart disease, angiogenesis, difficult-to-heal wounds and fractures; it also plays an important role in the mass production of stem cells.
  • E. coli Due to the low preparation cost, high replication rate, and high productivity, E. coli has been used for the purification of recombinant proteins for a long time [1-4].
  • the use of prokaryotic expression systems often encounter obstacles in the purification of mammalian proteins [5,6].
  • scientists have been using eukaryotic hosts such as yeast and insect cells to overcome these limitations [7]. Nonetheless, it seems intuitive to use cultured cells of human origin for protein expression of human origin. In fact, there is an upward trend in the use of mammalian cells to produce recombinant proteins [8,9].
  • one aspect of the present invention provides an enhancer, which can greatly increase the expression of FGF2 with biological activity in mammalian cells.
  • the present invention provides a plasmid vector pNC1 containing the enhancer.
  • the present invention provides a method for preparing the plasmid vector pNC1 of the present invention.
  • the present invention provides the application of the plasmid vector pNC1 of the present invention in expressing FGF-2.
  • the present invention provides transformants containing the plasmid vector pNC1 of the present invention.
  • the present invention provides an enhancer sequence, which is characterized in that the enhancer sequence includes the DNA sequence of the NF- ⁇ B binding site and the DNA sequence of the CREB binding site.
  • the invention also provides a plasmid vector pNC1, which is characterized in that the plasmid vector includes the enhancer sequence provided by the invention.
  • the present invention also provides a method for preparing the plasmid vector pNC1, which is characterized in that the method includes the following steps:
  • a Synthesize a DNA sequence containing the DNA sequence of the NF- ⁇ B binding site and the DNA sequence of the CREB binding site, wherein the end of the DNA sequence of the NF- ⁇ B binding site has a NheI restriction site, and CREB is bound
  • the DNA sequence of the site has a HindIII restriction enzyme cut site at the end;
  • step d Transfect cells with the ligation product obtained in step c, select positive clones, and extract plasmids.
  • the plasmid vector pNC1 provided by the present invention can significantly increase the expression level of FGF-2, and the expressed FGF-2 is basically soluble, and its biological activity is equivalent to that of commercially available FGF-2. Therefore, using the plasmid vector pNC1 provided by the present invention to express FGF-2 can significantly increase the expression of FGF-2 with biological activity, thereby greatly reducing the cost of preparing and purifying FGF-2. In addition, the plasmid vector pNC1 can be used to express a variety of valuable proteins.
  • Figure 1 is a schematic diagram of the FGF2 construct and the his-DnaE-FGF2 construct.
  • A Synthesize the DNA of the mature functional fragment of the fgf2 gene and clone it into the pcDNA 3.1 (+) vector under the control of the CMV enhancer/promoter to form a pcDNA3.1-FGF2 construct.
  • B1 Synthesize the NF- ⁇ B binding site and CREB binding site and clone them into the CMV promoter to form the pNC1 vector;
  • B2 Then clone the DNA of the mature functional fragment of the fgf2 gene into the pNC1 vector to form pNC1-FGF2 Construct.
  • FIG. 2 shows the expression of mature functional fragment FGF2 protein in HEK293T cells.
  • A The pcDNA3.1-FGF2 and pNC1-FGF2 constructs were transfected into HEK293T cells, and the whole cell lysate was analyzed by Western blot at designated time points.
  • C Analysis of supernatant (S) and pellet (P) from cell lysates by Western blot. Commercially purchased FGF2 (+ve) was used as a positive control. For the pcDNA3.1-FGF2 and pNC1-FGF2 constructs, the FGF2 expressed by HEK293T is quite soluble.
  • Figure 3 is the purification of FGF2 expressed in HEK293T cells.
  • A The whole cell lysate and the eluate from the size exclusion chromatography sample were subjected to SDS-PAGE and stained with Coomassie brilliant blue.
  • B The sample from (A) was lyophilized and reconstituted in 0.1 x PBS, and then analyzed by silver staining after SDS-PAGE. Only 1 weak band (indicated by the arrow) was observed in the purified FGF2.
  • Figure 4 is a bioassay of purified FGF2.
  • B Commercially purchased and purified FGF2 at a concentration of 2ng/mL was added to the culture medium for 3 days. Phase contrast micrographs were taken on day 0 and day 3. Prolonged neurite outgrowth (indicated by arrows) in PC12 cells was observed after 3 days of FGF2 treatment.
  • Figure 5 shows the expression of mature functional fragment FGF2 protein in HEK293T cells and the purification of FGF2 by intein-assisted cleavage.
  • the pNC1-6xhis-DnaE-FGF2 construct was transfected into HEK293T cells, the medium was collected and spun at 2,000 g for 10 minutes to remove cell debris, and filtered through a 0.45 ⁇ m filter.
  • the protein was purified by Ni-NTA affinity chromatography. Induces the cleavage of the DnaE intein in the cleavage buffer. Starting from 2 hours after induction, the full-length his-DnaE-FGF2 protein (band indicated by *) was cleaved into FGF2 (band indicated by #). The lysis is complete 6 hours after induction.
  • Figure 6 is a bioassay of purified and endoprotein-cleaved FGF2.
  • B 2ng/mL purified and intein-cut FGF2 was added to the culture medium for 3 days. Phase contrast micrographs were taken on day 0 and day 3. Prolonged neurite outgrowth (indicated by arrows) in PC12 cells was observed after 3 days of FGF2 treatment.
  • the present invention provides an enhancer sequence, which is characterized in that the enhancer sequence includes the DNA sequence of the NF- ⁇ B binding site and the DNA sequence of the CREB binding site.
  • the DNA sequence of the NF- ⁇ B binding site is shown in SEQ ID NO. 3: GGAAATCCCCGGAAATCCCC, or its complementary sequence; the CREB binding site The DNA sequence of is shown in SEQ ID NO. 4: TGCGTCAACACTGCTCAAC, or its complementary sequence.
  • the enhancer sequence is 47 bp.
  • the enhancer sequence is shown in SEQ ID NO. 5: GGAAATCCCCGGAAATCCCCGTAAAATTTGCGTCAACACTGCTCAAC, or its complementary sequence.
  • the invention also provides a plasmid vector, which includes the enhancer sequence provided by the invention.
  • the enhancer sequence is located between the NheI and HindIII restriction sites of the plasmid vector pcDNA 3.1(+).
  • the DNA sequence of the NF- ⁇ B binding site is connected to the NheI restriction site, and the DNA sequence of the CREB binding site is restricted to HindIII. Restriction site connection.
  • the present invention provides a method for preparing plasmid vector pNC1, which is characterized in that the method comprises the following steps:
  • a Synthesize a DNA sequence containing the DNA sequence of the NF- ⁇ B binding site and the DNA sequence of the CREB binding site, wherein the end of the DNA sequence of the NF- ⁇ B binding site has a NheI restriction site, and CREB is bound
  • the DNA sequence of the site has a HindIII restriction enzyme cut site at the end;
  • step d Transfect cells with the ligation product obtained in step c, select positive clones, and extract plasmids.
  • the DNA sequence of the NF- ⁇ B binding site is shown in SEQ ID NO. 3 or its complementary sequence
  • the DNA sequence of the CREB binding site is SEQ ID NO. 4 Sequence shown or its complement.
  • the DNA sequence synthesized in step a is the sequence shown in SEQ ID NO. 5 with NheI and HindIII restriction enzyme cleavage sites or its complementary sequence.
  • the original plasmid vector is pcDNA 3.1(+).
  • the plasmid vector pNC1 of the present invention can be used for the expression of a variety of proteins.
  • the present invention also provides the use of the plasmid vector in expressing FGF-2.
  • the present invention also provides a transformant, characterized in that the transformant comprises the plasmid vector pNC1 provided by the present invention.
  • the host cell of the transformant is not particularly limited, and may be HEK293T, HeLa, C2C12, etc., for example. However, in a preferred embodiment, the host cell used is HEK 293T.
  • PCR using GeneArt Strings gene synthesis service (Thermo-Fisher Scientific, Waltham, MA) to synthesize the enhancer sequence, which is the DNA sequence containing the NF- ⁇ B binding site and the DNA sequence of the CREB binding site DNA sequence;
  • pcDNA 3.1(+) was purchased from Thermo Scientific, USA, and its vector map is shown in Figure 1A. Restriction endonucleases NheI and HindIII, and T4 DNA Ligase were purchased from NEB. Plasmid extraction kit and DNA fragment recovery kit were purchased from Thermo Scientific.
  • the enhancer template is synthesized by GeneArt string service (thermos scientific), and its DNA sequence is shown in SEQ ID NO. 5: 5'GGAAATCCCCGGAAATCCCCGTAAAATTTGCGTCAACACTGCTCAAC.
  • the forward and reverse primers used are shown in Table 1.
  • Reaction conditions 95°C, 5min; 95°C, 15s; 55°C, 10s; 72°C, 10s; 35 cycles; 72°C extension for 10min, 4°C to terminate the reaction.
  • the PCR amplification product was electrophoresed in a 3% agarose gel for result detection, and the inserted band was recovered with a DNA gel recovery and purification kit. A 59bp fragment was obtained, which was consistent with the expected size.
  • the PCR product recovered above was ligated to the plasmid vector pcDNA 3.1(+).
  • the fragments recovered by PCR and the plasmid vector pcDNA 3.1(+) were digested with NheI and HindIII, then ligated with T4DNA ligase, and the ligated product was transformed into chemically competent cell DH5a strain, and then coated on ampicillin resistant solid medium On a petri dish, pick several monoclonal colonies and inoculate them into the ampicillin-resistant liquid medium, and culture them overnight on a constant temperature shaker at 37°C and 250 rpm.
  • the plasmid was extracted with a plasmid extraction kit to obtain the recombinant plasmid vector pNC1.
  • Recombinant plasmid DNA 5 ⁇ L, 10 ⁇ buffer 2 ⁇ L, NheI 0.5 ⁇ L, HindIII 0.5 ⁇ L, ddH2O 11 ⁇ L
  • the total volume is 20 ⁇ L. 37°C, 15min.
  • the positive clones verified by restriction digestion were selected for sequencing analysis, and the results were analyzed by FinchTV and compared with BLAST software to verify the correctness of the recombinant plasmid vector.
  • the analysis results showed that the enhancer nucleotide sequence was completely correct, and the recombinant plasmid vector pNC1 was successfully obtained.
  • FGF2-pcDNA3.1(+) construct FGF2-pNC1 construct, and his-DnaE-FGF2-pNC1 construct
  • the DNA sequence (amino acids 143-288, PRO_0000008933) of the fgf2 gene without the propeptide sequence (that is, the mature functional fragment of the fgf2 gene) was designed for human codon optimization.
  • the present invention uses 6x his tags and uses the DNA polymerase III (DnaE) intein of Nostoc punctiforme PCC73102 (Npu) fused with the fgf2 gene to promote human fibroblast growth factor 2 ( FGF2) purification.
  • DnaE DNA polymerase III
  • Npu Nostoc punctiforme PCC73102
  • GeneArt Strings gene synthesis service was used to synthesize enhancer sequences containing NF- ⁇ B binding sites and CREB binding sites, and cloned into pcDNA 3.1(+) with NheI and HindIII sites (Thermo- Fisher Scientific, Waltham, MA)) to form the expression plasmid vector pNC1.
  • the synthetic 6xhis-DnaE-FGF2) was cloned into pNC1 with EcoRI and NotI sites to form a his-DnaE-FGF2-pNC1 construct, as shown in Figure 1C. All sequences were confirmed by Sanger sequencing.
  • Antibodies used in Western blotting mouse FGF-2 (clone C-2, Santa Cruz Biotechnology, Dallas, TX), mouse anti- ⁇ -actin (Sigma, St. Louis, MO).
  • FGF2-pcDNA3.1(+) construct The specific process of constructing FGF2-pcDNA3.1(+) construct is as follows:
  • the forward and reverse primers used are shown in Table 1. Add the following reactants in the EP tube in sequence: 0.5 ⁇ l template, 1x buffer, 1 ⁇ M forward primer, 1 ⁇ M reverse primer, 0.5 ⁇ l polymerase, 4 ⁇ l dNTP, add ddH2O to 50 ⁇ l reaction system for reaction.
  • Reaction conditions 95°C, 5min; 95°C, 15s; 55°C, 10s; 72°C, 30min; 35 cycles; 72°C extension for 10min, 4°C to terminate the reaction.
  • the PCR amplification product was electrophoresed in a 1% agarose gel for result detection, and the inserted band was recovered with a DNA gel recovery and purification kit. A 460bp fragment was obtained, which was consistent with the expected size.
  • the PCR product recovered above was ligated to the plasmid vector pcDNA 3.1(+).
  • the fragments recovered by PCR and the plasmid vector pcDNA 3.1(+) were digested with EcoRI and NotI, and then ligated with T4DNA ligase.
  • the ligated product was transformed into chemically competent cell DH5a strain, and then coated on ampicillin resistant solid medium On a petri dish, pick several monoclonal colonies and inoculate them into the ampicillin-resistant liquid medium, and cultivate overnight on a constant temperature shaker at 37°C and 250 rpm with shaking.
  • the plasmid was extracted with a plasmid extraction kit to obtain FGF2-pcDNA3.1(+) construct.
  • restriction enzyme digestion with EcoRI and NotI was used for identification.
  • the restriction digestion identification system and reaction conditions are as follows:
  • FGF2-pcDNA3.1(+) construct 5 ⁇ L, 10 ⁇ buffer 2 ⁇ L, EcoRI 0.5 ⁇ L, NotI 0.5 ⁇ L, ddH2O 11 ⁇ L, total volume 20 ⁇ L. 37°C, 15min.
  • the positive clones verified by restriction digestion were selected for sequencing analysis, and the results were analyzed by FinchTV and compared with BLAST software to verify the correctness of the FGF2-pcDNA3.1(+) construct.
  • the analysis results showed that the nucleotide sequence of fgf2 was completely correct, and the FGF2-pcDNA3.1(+) construct was successfully obtained.
  • FGF2-pNC1 construct The specific process of constructing FGF2-pNC1 construct is as follows:
  • the forward and reverse primers used are shown in Table 1.
  • Reaction conditions 95°C, 5min; 95°C, 15s; 55°C, 10s; 72°C, 30min; 35 cycles; 72°C extension for 10min, 4°C to terminate the reaction.
  • the PCR amplification product was electrophoresed in a 1% agarose gel for result detection, and the inserted band was recovered with a DNA gel recovery and purification kit. A 460bp fragment was obtained, which was consistent with the expected size.
  • the PCR product recovered above was ligated to the plasmid vector pNC1.
  • the fragments recovered by PCR and the plasmid vector pNC1 were digested with EcoRI and NotI, and then ligated with T4DNA ligase.
  • the ligated product was transformed into chemically competent cell DH5a strain, and spread on the ampicillin resistant solid medium plate, and picked Several monoclonal colonies were inoculated into the ampicillin resistant liquid medium and cultured overnight on a constant temperature shaker at 37°C and 250 rpm with shaking.
  • the plasmid was extracted with a plasmid extraction kit to obtain the pNC1 construct.
  • restriction enzyme digestion with EcoRI and NotI was used for identification.
  • the restriction digestion identification system and reaction conditions are as follows:
  • FGF2-pNC1 construct 5 ⁇ L, 10 ⁇ buffer 2 ⁇ L, EcoRI 0.5 ⁇ L, NotI 0.5 ⁇ L, ddH 2 O 11 ⁇ L, total volume 20 ⁇ L. 37°C, 15min. After identification by restriction enzyme digestion, the product obtained is consistent with the expected analysis size.
  • the positive clones verified by restriction digestion were selected for sequencing analysis, and the results were analyzed by FinchTV and compared with BLAST software to verify the correctness of the FGF2-pNC1 construct.
  • the analysis results showed that the nucleotide sequence of fgf2 was completely correct, and the FGF2-pNC1 construct was successfully obtained.
  • his-DnaE-FGF2-pNC1 construct is as follows:
  • Reaction conditions 95°C, 5min; 95°C, 15s; 55°C, 10s; 72°C, 30min; 35 cycles; 72°C extension for 10min, 4°C to terminate the reaction.
  • the PCR amplification product was electrophoresed in a 1% agarose gel for result detection, and the GFP band was recovered with a DNA gel recovery and purification kit. A fragment of 1097bp was obtained, which was consistent with the expected size.
  • the PCR product recovered above was ligated to the plasmid vector his-DnaE-FGF2.
  • the fragments recovered by PCR and the plasmid vector his-DnaE-FGF2 were digested with EcoRI and NotI, and then ligated with T4DNA ligase.
  • the ligated product was transformed into chemically competent cell DH5a strain, and then coated on ampicillin resistant solid medium On a petri dish, pick several monoclonal colonies and inoculate them into the ampicillin-resistant liquid medium, and cultivate overnight on a constant temperature shaker at 37°C and 250 rpm with shaking.
  • the plasmid was extracted with a plasmid extraction kit to obtain his-DnaE-FGF2 construct.
  • restriction enzyme digestion with EcoRI and NotI was used for identification.
  • the restriction digestion identification system and reaction conditions are as follows:
  • His-DnaE-FGF2 construct 5 ⁇ L, 10 ⁇ buffer 2 ⁇ L, EcoRI 0.5 ⁇ L, NotI 0.5 ⁇ L, ddH2O 11 ⁇ L, total volume 20 ⁇ L. 37°C, 15min.
  • the positive clones verified by restriction digestion were selected for sequencing analysis, and the results were analyzed by FinchTV and compared with BLAST software to verify the correctness of the his-DnaE-FGF2 construct.
  • the analysis results showed that the nucleotide sequence of his-DnaE-fgf2 was completely correct, and the his-DnaE-FGF2 construct was successfully obtained.
  • the DNA containing the NheI and HindIII restriction enzyme cut site enhancers synthesized by PCR is SEQ ID NO.11 (that is, the DNA containing the NheI and HindIII restriction enzyme cut site SEQ ID NO.5):
  • the DNA sequence of FGF2 containing EcoRI and NotI restriction enzyme sites synthesized by PCR is SEQ ID NO.12 (that is, SEQ ID NO.1 containing EcoRI and NotI restriction enzyme sites):
  • SEQ ID NO.13 The DNA sequence of his-DnaE-FGF2 with EcoRI and NotI restriction enzyme sites synthesized by PCR is SEQ ID NO.13 (that is, SEQ ID NO. 2 containing EcoRI and NotI restriction enzyme sites):
  • the HEK 293T and C2C12 cells were maintained in DMEM containing 10 vol% FBS and 1 vol% penicillin-streptomycin solution (Thermo-Fisher Scientific, Waltham, MA) at a temperature of 37° C. and containing 5% CO2.
  • the PC12 cells were maintained in DMEM containing 10 vol% HS, 5% FBS and 1 vol% penicillin streptomycin solution, the temperature in the environment was 37° C. and 5% CO2.
  • the pcDNA3.1-FGF2, pNC1-FGF2 and pNC1-6xhis-DnaE-FGF2 constructs were transfected into HEK 293T with Lipofectamine 2000 (Thermo-Fisher Scientific, Waltham, MA).
  • the medium was collected and rotated at 2,000 g for 10 minutes to remove cell debris, and filtered through a 0.45 ⁇ m filter.
  • the filtrate was then passed through a pre-packed heparin-agarose column (BioRad Laboratories, Hercules, CA) equilibrated with 50 mM Tris-HCl (pH 7.5). Wash the column thoroughly with 50mM Tris-HCl (pH 7.5), 0.2M NaCl.
  • FGF2 was eluted with a NaCl gradient from 0.3M to 3M (4-5 bed volumes used for the gradient).
  • the protein was passed through a pre-packed Sephadex G25 column equilibrated with 50 mM Tris-HCl (pH 7.5) and eluted with the same buffer.
  • FGF2 expressed in cells the cells were washed three times with ice-cold TBS, and then sonicated in lysis buffer (TBS provided with cOmpleteTM Protease Inhibitor Cocktail). Then FGF2 is purified in the culture medium.
  • the histidine-labeled DnaE-FGF2 was purified through a prepackaged Ni-NTA column. After washing, the column was incubated in 50 mM Tris-HCl (pH 6.2), 10 mM EDTA, 200 mM NaCl at 22°C for different durations of 0, 1, 2, 4, 6, and 10 hours, respectively, to induce DnaE The C-terminus of the intein is excised.
  • the protein was separated in 15% by volume Tris-glycine SDS-PAGE. The gel was stained with silver to obtain purified protein. On the gel, the band corresponding to FGF2 was cut, washed and incubated with 1 ⁇ g trypsin in 50 mM NH 4 HCO 3 at 4° C. overnight. The hydrolyzed samples were analyzed by the LTQ Velos Linear Ion Trap Mass Spectrometer (Thermo Fisher Scienctific, San Jose) combined with the Accela HPLE system. Obtain a complete MS scan (300-2000m/z) and use the Mascot search engine (Matric Science, Boston, MA) to identify the peptide sequence.
  • the sample was spotted or transferred to 0.2 ⁇ m NC membrane (BioRad Laboratories, Hercules, CA), and then blotted with antibody (mouse FGF-2 (clone C-2, Santa Cruz Biotechnology, Dallas, TX), Mouse anti- ⁇ -actin (Sigma, St. Louis, MO)).
  • NC membrane BioRad Laboratories, Hercules, CA
  • antibody mouse FGF-2 (clone C-2, Santa Cruz Biotechnology, Dallas, TX), Mouse anti- ⁇ -actin (Sigma, St. Louis, MO)
  • MTT assay was performed as described above.
  • C2C12 cells were seeded on a 96-well plate, and DMEM+0.5% FBS and 1ng/mL of commercial FGF2, purified FGF2 and endopeptide excised FGF2 were provided.
  • the cell activity was measured by adding MTT to a final concentration of 1 mg/mL, and incubated at 37°C for 6 hours. The medium was then replaced with DMSO, and the absorbance was measured at 540 nm in a microplate reader.
  • PC12 cells were cultured with a medium provided with 2ng/mL FGF2 for 3 days. Observe the morphology of the cells and take images through a phase contrast optical microscope.
  • the full-length FGF2 protein consists of 288 amino acids, of which amino acids 1-142 are cleaved and removed to produce functional FGF2. Therefore, the protein sequence of amino acids 143-288 is codon optimized for use in Homo sapiens.
  • the NF- ⁇ B binding site and CREB binding site enhancer sequences were cloned into the pcDNA3.1 vector containing the natural CMV immediate early promoter/enhancer sequence.
  • the synthesized fgf2 gene was then cloned into the pcDNA3.1 and pNC1 vectors ( Figure 1) to compare the expression of the two clones in mammalian cells.
  • pNC1 vector greatly increases the expression level of FGF2 in mammalian cells, as shown in Figure 2.
  • the neomycin resistance gene also provides a selection marker through G418 sulfate. Therefore, stably transfected cells can be used to purify the same exogenous human FGF2.
  • HEK293T cells were chosen for purification because of their ease of transfection.
  • the SV40 large T antigen also provides cells with the ability to replicate the transfected plasmid, which contains the SV40 origin of replication.
  • the CMV promoter is one of the strongest promoters and has constitutive activity in HEK293T cells.
  • HEK293T cells are adherent cells, they are universally applicable to suspension culture, so they are beneficial to expand the production of FGF2.
  • the pcDNA3.1-FGF2, pNC1-FGF2 and pNC1-6xhis-DnaE-FGF2 constructs were transiently transfected into HEK293T cells and the expression was monitored. Experiments show that the pNC1 vector greatly increases the expression level of FGF2 ( Figure 2A, 2B). The expression of FGF2 reached a peak 48 hours after transfection and remained stable for a long time.
  • HEK293T cells were transfected with pNC1-FGF2. 48 hours after transfection, the cells were harvested and lysed to purify FGF2 expressed in the cells. FGF2 was purified by heparin-agarose affinity chromatography and then subjected to size exclusion chromatography. The eluted FGF2 was electrophoresed in 15% SDS-PAGE ( Figure 3A). The samples were lyophilized and reconstituted in 0.1 x PBS. In order to check the purity of FGF2, silver staining was performed after electrophoresis and showed very high purity, only 1 weak band was found above the purified FGF2 ( Figure 3B). The yield is also satisfactory.
  • FGF2 expressed by HEK293T has biological activity
  • FGF2 has been shown to stimulate cell growth in many cell types. After confirming the primary structure of the purified FGF2, we treated the cultured C2C12 with 1ng/mL purified and commercially available FGF2, and PBS was used as a control. The viability of the cultured cells was then measured by the MTT assay (Figure 4A). Both FGF2 samples can stimulate the proliferation of C2C12 cells. Purified FGF2 showed similar or even higher biological activity than commercial FGF2, but the difference was not significant. FGF2 also exhibits neurotrophic activity in neuronal cell lines. Therefore, the neurotrophic activity of purified FGF2 was tested in PC12 cells. After 3 days of FGF2 treatment, prolonged neurite outgrowth was observed, which was not present in the PBS control group ( Figure 4B). The data confirms that the purified FGF2 has biological activity and has similar activity to commercial FGF2.
  • the recombinant protein cascade containing 6x histidine-tagged DnaE-FGF2 was cloned into the pNC1 vector ( Figure 1C), and then transfected into HEK293T cells.
  • the medium was collected and rotated at 2,000 g for 10 minutes to remove cell debris, and filtered through a 0.45 ⁇ m filter.
  • the protein was purified by Ni-NTA affinity chromatography. FGF2 excision was performed on Ni-NTA column with lysis buffer (50mM Tris-HCl pH 6.2, 10mM EDTA, 200mM NaCl), and performed at different time points at 22°C.
  • FGF2 is a very valuable protein with a wide range of potency, including angiogenesis, neurogenesis and wound healing.
  • One of the reasons hindering the research on the use of FGF2 in medicine is the high cost of purification and biological activity of FGF2.
  • HEK 293T cells we successfully expressed and purified human FGF2 ( Figure 3). The silver-stained gel proved very satisfactory in purity.
  • purified FGF2 exhibited the same mitogenic or neurotrophic activity as commercially available FGF2. This simple protocol allows for the production of human exogenous FGF2 on a laboratory scale in a mammalian system, and can be easily scaled up for large-scale production.
  • endotoxin triggers the body's immune response and may lead to septic shock.
  • kits and protocols for removing endotoxins endotoxin contamination is usually unavoidable.
  • using mammalian cells can easily overcome the above-mentioned problems.
  • DnaE is a fast-cutting intein in HEK293T cells.
  • intein the primary structure (Table 3) and biological activity ( Figure 6) of excised FGF2 are the same as its natural counterpart.
  • the medical applications of purified FGF2 will be extensive.
  • the plasmid vector pNC1 can be used to express a variety of valuable proteins.

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Abstract

提供了一种增强子序列，该增强子序列包括NF-κB结合位点的DNA序列和CREB结合位点的DNA序列。还提供了一种含有所述增强子序列的质粒载体pNC1，该质粒载体pNC1的制备方法、该质粒载体pNC1在表达FGF-2中的用途，以及含有该质粒载体pNC1的转化体。所述质粒载体pNC1能够显著提高FGF-2的表达水平，而且表达的FGF-2基本上是可溶的，其生物活性与商购的FGF-2的生物活性相当。

Description

[根据细则37.2由ISA制定的发明名称]　一种通过增强子促进FGF-2在293T细胞中表达的方法 技术领域
本发明涉及一种增强子序列、一种质粒载体pNC1、该质粒载体pNC1的制备方法、该质粒载体pNC1在表达FGF-2中的用途，以及一种转化体。
背景技术
FGF2是制药和保健行业中非常有价值的蛋白质。FGF2是治疗神经退行性疾病，心脏病，血管生成，难以愈合的伤口和骨折的有效治疗性蛋白质；它还在干细胞的大规模生产中发挥重要作用。
由于制备成本低，复制率高，生产率高，大肠杆菌长期用于纯化重组蛋白[1-4]。然而，由于外源蛋白质引起的毒性，缺乏正确的蛋白质折叠和翻译后修饰，使用原核表达系统在纯化哺乳动物蛋白质时经常遇到障碍[5,6]。多年来，科学家一直在使用真核宿主如酵母和昆虫细胞来克服这些局限[7]。尽管如此，使用人类来源的培养细胞进行人类来源的蛋白质表达似乎是直观的。实际上，出现了使用哺乳动物细胞产生重组蛋白的上升趋势[8,9]。
目前，FGF2制备和纯化最广泛使用的宿主系统之一是细菌。然而，简单的原核生物缺乏必要的翻译后修饰，包括剪接，糖基化和二硫键，用于纯化的蛋白质的活性和溶解度。纯化蛋白质的天然折叠对其功能和溶解性至关重要。当在细菌系统中表达真核蛋白时经常发现蛋白质聚集或包涵体。包括在较低温度下诱导蛋白质或使蛋白质聚集体变性和复性的方法并不总是产生良好的产率。因此，制备和纯化具有生物活性的FGF2极其昂贵(4000美元/毫克)，这阻碍了其广泛的应用。
发明内容
为了克服现有技术的缺陷，本发明一方面提供了一种增强子，该增强子能够在哺乳动物细胞中大大增加具有生物活性的FGF2的表达。
另一方面，本发明提供了含有所述增强子的质粒载体pNC1。
另一方面，本发明提供了本发明质粒载体pNC1的制备方法。
另一方面，本发明提供了本发明质粒载体pNC1在表达FGF-2中的应用。
另一方面，本发明提供了含有本发明质粒载体pNC1的转化体。
因此，本发明提供了一种增强子序列，其特征在于，该增强子序列包括NF-κB结合位点的DNA序列和CREB结合位点的DNA序列。
本发明还提供了一种质粒载体pNC1，其特征在于，该质粒载体包括本发明所提供的增强子序列。
本发明还提供了一种质粒载体pNC1的制备方法，其特征在于，该方法包括以下步骤：
a.合成含有NF-κB结合位点的DNA序列和CREB结合位点的DNA序列的DNA序列，其中，NF-κB结合位点的DNA序列的末端带有NheI限制性酶切位点，CREB结合位点的DNA序列的末端带有HindIII限制性酶切位点；
b.用NheI和HindIII消化原始质粒载体，回收大片段；
c.采用连接酶连接步骤a获得的DNA序列与步骤b获得的大片段；
d.将步骤c获得的连接产物转染细胞，筛选阳性克隆，提取质粒。
本发明提供的质粒载体pNC1能够显著提高FGF-2的表达水平，而且表达的FGF-2基本上是可溶的，其生物活性与商购的FGF-2的生物活性相当。因此，采用本发明提供的质粒载体pNC1表达FGF-2， 能显著提高具有生物活性FGF-2的表达，从而能大大降低制备和纯化FGF-2的成本。此外，质粒载体pNC1可用于表达多种有价值的蛋白质。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1是FGF2构建体和his-DnaE-FGF2构建体的示意图。(A)合成fgf2基因的成熟功能片段的DNA，并克隆到在CMV增强子/启动子的控制下的pcDNA 3.1(+)载体中，形成pcDNA3.1-FGF2构建体。(B1)合成NF-κB结合位点和CREB结合位点并克隆在CMV启动子后，形成pNC1载体；(B2)然后将fgf2基因的成熟功能片段的DNA克隆到pNC1载体中，形成pNC1-FGF2构建体。(C)通过融合PCR将6xhis标签和Npu DnaE内含肽基因与fgf2融合，并克隆到pNC1载体中，形成pNC1-6xhis-DnaE-FGF2构建体。
图2是成熟功能片段FGF2蛋白在HEK293T细胞中的表达。(A)将pcDNA3.1-FGF2和pNC1-FGF2构建体转染到HEK293T细胞中，并在指定的时间点通过蛋白质印迹分析全细胞裂解物。(B)为(A)相对β-肌动蛋白标准化的光密度分析。pNC1-FGF2构建体在24小时，36小时和48小时显示出比pcDNA3.1-FGF2构建体更高的表达水平(**p<0.01，***p<0.001；在每个时间点n＝5)。(C)通过蛋白质印迹分析来自细胞裂解物的上清液(S)和沉淀(P)。商购FGF2(+ve)作为阳性对照。对于pcDNA3.1-FGF2和pNC1-FGF2构建体，HEK293T表达的FGF2都是相当可溶的。
图3是在HEK293T细胞中表达的FGF2的纯化。(A)将全细胞裂解物和来自尺寸排阻色谱样品的洗脱物进行SDS-PAGE并用考马斯亮蓝染色。(B)将来自(A)的样品冻干并在0.1 x PBS中重构，然后在SDS-PAGE后通过银染色分析。在纯化的FGF2中仅观察到1 个弱带(由箭头指示)。
图4是纯化的FGF2的生物测定。(A)MTT测定以测量FGF2相对于PBS对照对于C2C12细胞的细胞活力的影响。在指定的时间点可以观察到在培养基中1ng/mL的商购和纯化的FGF2都能够诱导细胞生长(n＝3)。(B)将浓度为2ng/mL的商购和纯化的FGF2加入培养基中3天。在第0天和第3天拍摄相差显微照片。在FGF2处理3天后观察到PC12细胞中的延长的神经突生长(用箭头表示)。
图5是成熟功能片段FGF2蛋白在HEK293T细胞中的表达以及通过内含肽辅助切割纯化FGF2。将pNC1-6xhis-DnaE-FGF2构建体转染到HEK293T细胞中，收集培养基并以2,000g旋转10分钟以除去细胞碎片，并通过0.45μm过滤器过滤。通过Ni-NTA亲和层析纯化蛋白质。在切割缓冲液中诱导DnaE内含肽的切割。从诱导后2小时开始，将全长his-DnaE-FGF2蛋白(由*表示的条带)切割成FGF2(用#表示的条带)。诱导后6小时后裂解完成。
图6是纯化的和内切蛋白切割的FGF2的生物测定。(A)MTT测定以测量FGF2相对于PBS对照对于C2C12细胞的细胞活力的影响。在指定的时间点观察到在培养基中1ng/mL的纯化和内切蛋白切割的FGF2均能够诱导细胞生长(n＝3)。(B)将2ng/mL的纯化和内含肽切割的FGF2加入培养基中3天。在第0天和第3天拍摄相差显微照片。在FGF2处理3天后观察到PC12细胞中的延长的神经突生长(用箭头表示)。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
本发明提供了一种增强子序列，其特征在于，该增强子序列包括NF-κB结合位点的DNA序列和CREB结合位点的DNA序列。
为了获得更好的增强效果，在一种优选的实施方式中，所述NF- κB结合位点的DNA序列为SEQ ID NO.3所示：GGAAATCCCCGGAAATCCCC，或其互补序列；所述CREB结合位点的DNA序列为SEQ ID NO.4所示：TGCGTCAACACTGCTCAAC，或其互补序列。
为了获得更好的增强效果，在另一种优选的实施方式中，所述增强子序列为47bp。
为了获得更好的增强效果，在一种优选的实施方式中，所述增强子序列为SEQ ID NO.5所示：GGAAATCCCCGGAAATCCCCGTAAAATTTGCGTCAACACTGCTCAAC，或其互补序列。
本发明还提供了一种质粒载体，该质粒载体包括本发明所提供的增强子序列。
在一种优选的实施方式中，所述增强子序列位于质粒载体pcDNA 3.1(+)的NheI和HindIII限制性酶切位点之间。
为了获得更好的增强效果，在另一种优选的实施方式中，所述NF-κB结合位点的DNA序列与NheI限制性酶切位点连接，CREB结合位点的DNA序列与HindIII限制性酶切位点连接。
本发明提供一种质粒载体pNC1的制备方法，其特征在于，该方法包括以下步骤：
a.合成含有NF-κB结合位点的DNA序列和CREB结合位点的DNA序列的DNA序列，其中，NF-κB结合位点的DNA序列的末端带有NheI限制性酶切位点，CREB结合位点的DNA序列的末端带有HindIII限制性酶切位点；
b.用NheI和HindIII消化原始质粒载体，回收大片段；
c.采用连接酶连接步骤a获得的DNA序列与步骤b获得的大片段；
d.将步骤c获得的连接产物转染细胞，筛选阳性克隆，提取质粒。
在一种优选的实施方式中，其中，所述NF-κB结合位点的DNA 序列为SEQ ID NO.3所示或其互补序列，所述CREB结合位点的DNA序列为SEQ ID NO.4所示或其互补序列。
在一种优选的实施方式中，其中，所述步骤a合成的DNA序列为带有NheI和HindIII限制性酶切位点的SEQ ID NO.5所示序列或其互补序列。
为了获得更好的增强效果，在一种优选的实施方式中，其中，原始质粒载体为pcDNA 3.1(+)。
本发明的质粒载体pNC1可以用于多种蛋白质的表达，优选地，本发明还提供所述的质粒载体在表达FGF-2中的用途。
本发明还提供一种转化体，其特征在于，该转化体包括本发明提供的质粒载体pNC1。
所述转化体的宿主细胞没有特别限制，例如可以为HEK293T、HeLa、C2C12等。但在一种优选的实施方式中，所采用的宿主细胞是HEK 293T。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
实施例
材料和方法
化学品、质粒和抗体，除非另有说明，否则所有化学品均购自Sigma-Aldrich(St.Louis，MO)。
质粒载体pNC1的构建
如图1中的B1所示：
a.采用PCR(用GeneArt Strings基因合成服务(Thermo-Fisher Scientific，Waltham，MA))合成增强子序列，该增强子序列为含有NF-κB结合位点的DNA序列和CREB结合位点的DNA序列的 DNA序列；
b.用NheI和HindIII消化质粒载体pcDNA 3.1(+)；
c.用NheI和HindIII消化步骤a合成的DNA序列；
d.采用连接酶连接步骤b获得的消化后的质粒载体pcDNA 3.1(+)与步骤c获得的消化后的DNA序列。
通过Sanger测序确认序列。
具体过程如下：
材料：pcDNA 3.1(+)购自美国Thermo Scientific公司，其载体图谱如图1A所示。限制性内切酶NheI和HindIII，以及T4 DNA Ligase购自NEB公司。质粒提取试剂盒和DNA片段回收试剂盒购自Thermo Scientific公司。
增强子PCR扩增
增强子的模板通过GeneArt string service(thermos scientific)合成，其DNA序列如SEQ ID NO.5所示：5’GGAAATCCCCGGAAATCCCCGTAAAATTTGCGTCAACACTGCTCAAC。采用的正向和反向引物如表1所示。在EP管中依次加入以下反应物：0.5μl的模板，1x缓冲液，1μM正向引物，1μM反向引物，0.5μl聚合酶，4μl dNTP，加ddH 2O至50μl反应体系中进行反应。
反应条件：95℃、5min；95℃、15s；55℃、10s；72℃、10s；35个循环；72℃延伸10min，4℃终止反应。
将PCR扩增产物在3％琼脂糖凝胶中电泳进行结果检测，用DNA胶回收纯化试剂盒回收插入条带。获得59bp的片段，与预期大小一致。
将上述PCR回收产物连接至质粒载体pcDNA 3.1(+)上。
将上述PCR回收片段和质粒载体pcDNA 3.1(+)用NheI和HindIII酶切处理，然后用T4DNA连接酶进行连接，连接产物转化至化学感受态细胞DH5a菌株中，涂布到氨苄抗性固体培养基平皿，挑取若干单克隆菌落，接种到氨苄抗性液体培养基中，恒温摇床 37℃、250rpm振荡培养过夜。采用质粒提取试剂盒提取质粒，得到重组质粒载体pNC1。
重组质粒载体pNC1的鉴定
用NheI和HindIII酶切进行鉴定，酶切鉴定体系及反应条件如下表：
重组质粒DNA 5μL，10×缓冲液2μL，NheI 0.5μL，HindIII 0.5μL，ddH2O 11μL
总体积20μL。37℃，15min。
经酶切鉴定，获得产物与预期分析大小一致。
选取酶切验证正确的阳性克隆进行测序分析，结果经FinchTV解析及BLAST软件比对，以验证重组质粒载体的正确性。分析结果表明，增强子核苷酸序列完全正确，成功获得了重组质粒载体pNC1。
FGF2-pcDNA3.1(+)构建体、FGF2-pNC1构建体和his-DnaE-FGF2-pNC1构建体
没有前肽序列的fgf2基因(即fgf2基因的成熟功能片段)的DNA序列(氨基酸143-288，PRO_0000008933)被设计用于人类的密码子优化。
用GeneArt Strings基因合成服务(Thermo-Fisher Scientific，Waltham，MA)合成设计的fgf2基因，将合成的fgf2基因克隆到具有EcoRI和NotI位点的pcDNA3.1(+)和pNC1中，形成FGF2-pcDNA3.1(+)构建体和FGF2-pNC1构建体，如图1A,1B2所示。通过Sanger测序确认所有序列。
为了优化纯化，本发明使用6x his标签以及使用融合了fgf2基因的点形念珠藻(Nostoc punctiforme)PCC73102(Npu)的DNA聚合酶III(DnaE)内含肽来促进人成纤维细胞生长因子2(FGF2)的纯化。
用GeneArt Strings基因合成服务(Thermo-Fisher Scientific， Waltham，MA)合成设计的fgf2和6x his-Npu DnaE内含肽基因。通过重叠PCR将6x his-Npu DnaE内含肽与fgf2融合。
为了进一步提高fgf2的表达，使用GeneArt Strings基因合成服务合成含有NF-κB结合位点和CREB结合位点的增强子序列，并克隆到具有NheI和HindIII位点的pcDNA 3.1(+)中(Thermo-Fisher Scientific，Waltham，MA))以形成表达质粒载体pNC1。将合成6xhis-DnaE-FGF2)克隆到具有EcoRI和NotI位点的pNC1中，形成his-DnaE-FGF2-pNC1构建体，如图1C所示。通过Sanger测序确认所有序列。用于蛋白质印迹的抗体：小鼠FGF-2(克隆C-2，Santa Cruz Biotechnology，Dallas，TX)，小鼠抗β-肌动蛋白(Sigma，St.Louis，MO)。
FGF2-pcDNA3.1(+)构建体的构建具体过程如下：
fgf2基因PCR扩增
fgf2的模板通过GeneArt string service(thermos scientific)合成，其DNA序列如SEQ ID NO.1所示CCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA。采用的正向和反向引物如表1所示。在EP管中依次加入以下反应物：0.5μl的模板，1x缓冲液，1μM正向引物，1μM反向引物，0.5μl聚合酶，4μl dNTP，加ddH2O至50μl反应体系中进行反应。
反应条件：95℃、5min；95℃、15s；55℃、10s；72℃、30min；35个循环；72℃延伸10min，4℃终止反应。
将PCR扩增产物在1％琼脂糖凝胶中电泳进行结果检测，用DNA胶回收纯化试剂盒回收插入条带。获得460bp的片段，与预期大小一致。
将上述PCR回收产物连接至质粒载体pcDNA 3.1(+)上。
将上述PCR回收片段和质粒载体pcDNA 3.1(+)用EcoRI和NotI酶切处理，然后用T4DNA连接酶进行连接，连接产物转化至化学感受态细胞DH5a菌株中，涂布到氨苄抗性固体培养基平皿，挑取若干单克隆菌落，接种到氨苄抗性液体培养基中，恒温摇床37℃、250rpm振荡培养过夜。采用质粒提取试剂盒提取质粒，得到FGF2-pcDNA3.1(+)构建体。
FGF2-pcDNA3.1(+)构建体的鉴定
用EcoRI和NotI酶切进行鉴定，酶切鉴定体系及反应条件如下表：
FGF2-pcDNA3.1(+)构建体5μL，10×缓冲液2μL，EcoRI 0.5μL，NotI 0.5μL，ddH2O 11μL总体积20μL。37℃，15min。
经酶切鉴定，获得产物与预期分析大小一致。
选取酶切验证正确的阳性克隆进行测序分析，结果经FinchTV解析及BLAST软件比对，以验证FGF2-pcDNA3.1(+)构建体的正确性。分析结果表明，fgf2核苷酸序列完全正确，成功获得了FGF2-pcDNA3.1(+)构建体。
FGF2-pNC1构建体的构建具体过程如下：
fgf2基因PCR扩增
fgf2的模板通过GeneArt string service(thermos scientific)合成，其DNA序列如SEQ ID NO.1所示：CCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCA CCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA。采用的正向和反向引物如表1所示。在EP管中依次加入以下反应物：0.5μl的模板，1x缓冲液，1μM正向引物，1μM反向引物，0.5μl聚合酶，4μl dNTP，加ddH 2O至50μl反应体系中进行反应。
反应条件：95℃、5min；95℃、15s；55℃、10s；72℃、30min；35个循环；72℃延伸10min，4℃终止反应。将PCR扩增产物在1％琼脂糖凝胶中电泳进行结果检测，用DNA胶回收纯化试剂盒回收插入条带。获得460bp的片段，与预期大小一致。
将上述PCR回收产物连接至质粒载体pNC1上。
将上述PCR回收片段和质粒载体pNC1用EcoRI和NotI酶切处理，然后用T4DNA连接酶进行连接，连接产物转化至化学感受态细胞DH5a菌株中，涂布到氨苄抗性固体培养基平皿，挑取若干单克隆菌落，接种到氨苄抗性液体培养基中，恒温摇床37℃、250rpm振荡培养过夜。采用质粒提取试剂盒提取质粒，得到pNC1构建体。
FGF2-pNC1构建体的鉴定
用EcoRI和NotI酶切进行鉴定，酶切鉴定体系及反应条件如下表：
FGF2-pNC1构建体5μL，10×缓冲液2μL，EcoRI 0.5μL，NotI 0.5μL，ddH 2O 11μL总体积20μL。37℃，15min。经酶切鉴定，获得产物与预期分析大小一致。
选取酶切验证正确的阳性克隆进行测序分析，结果经FinchTV解析及BLAST软件比对，以验证FGF2-pNC1构建体的正确性。分析结果表明，fgf2核苷酸序列完全正确，成功获得了FGF2-pNC1构建体。
his-DnaE-FGF2-pNC1构建体的构建具体过程如下：
his-DnaE-FGF2的融合PCR扩增
his-DnaE-FGF2的模板通过GeneArt string service(thermos scientific)合成，其DNA序列如SEQ ID NO.2所示：CATCATCACCATCACCACGCCGAGTACTACGAGACCGAGATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACAACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGGGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGATCAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGACCTGATGAGGGTGGACAACCTGCCCAACGCCGAGTACTACGAGACCGAGATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACAACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGGGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGATCAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGACCTGATGAGGGTGGACAACCTGCCCAACCCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGGT ACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA。以His-DnaE正向引物和FGF2反向引物，采用的正向和反向引物如表2所示。在EP管中依次加入以下反应物：0.5μl的模板，1x缓冲液，1μM正向引物，1μM反向引物，0.5μl聚合酶，4μl dNTP，加ddH2O至50μl反应体系中进行反应。
反应条件：95℃、5min；95℃、15s；55℃、10s；72℃、30min；35个循环；72℃延伸10min，4℃终止反应。将PCR扩增产物在1％琼脂糖凝胶中电泳进行结果检测，用DNA胶回收纯化试剂盒回收GFP条带。获得1097bp的片段，与预期大小一致。
将上述PCR回收产物连接至质粒载体his-DnaE-FGF2上。
将上述PCR回收片段和质粒载体his-DnaE-FGF2用EcoRI和NotI酶切处理，然后用T4DNA连接酶进行连接，连接产物转化至化学感受态细胞DH5a菌株中，涂布到氨苄抗性固体培养基平皿，挑取若干单克隆菌落，接种到氨苄抗性液体培养基中，恒温摇床37℃、250rpm振荡培养过夜。采用质粒提取试剂盒提取质粒，得到his-DnaE-FGF2构建体。
his-DnaE-FGF2构建体的鉴定
用EcoRI和NotI酶切进行鉴定，酶切鉴定体系及反应条件如下表：
his-DnaE-FGF2构建体5μL，10×缓冲液2μL，EcoRI 0.5μL，NotI 0.5μL，ddH2O 11μL总体积20μL。37℃，15min。
经酶切鉴定，获得产物与预期分析大小一致。
选取酶切验证正确的阳性克隆进行测序分析，结果经FinchTV解析及BLAST软件比对，以验证his-DnaE-FGF2构建体的正确性。 分析结果表明，his-DnaE-fgf2核苷酸序列完全正确，成功获得了his-DnaE-FGF2构建体。
表1
Figure PCTCN2020087967-appb-000001
PCR合成的含有NheI和HindIII限制性酶切位点增强子的DNA为SEQ ID NO.11(即为含有NheI和HindIII限制性酶切位点SEQ ID NO.5)：
Figure PCTCN2020087967-appb-000002
PCR合成的含有EcoRI和NotI限制性酶切位点的FGF2的DNA序列为SEQ ID NO.12(即为含有EcoRI和NotI限制性酶切位点SEQ ID NO.1)：
Figure PCTCN2020087967-appb-000003
Figure PCTCN2020087967-appb-000004
PCR合成的带有EcoRI和NotI限制性酶切位点的his-DnaE-FGF2的DNA序列为SEQ ID NO.13(即为含有EcoRI和NotI限制性酶切位点SEQ ID NO.2)：
Figure PCTCN2020087967-appb-000005
Figure PCTCN2020087967-appb-000006
细胞培养和转染
将HEK 293T和C2C12细胞维持在含有10体积％FBS和1体积％青霉素链霉素溶液(Thermo-Fisher Scientific，Waltham，MA)的DMEM中，环境中温度为37℃，含有5％CO2。将PC12细胞维持在含有10体积％HS，5体积％FBS和1体积％青霉素链霉素溶液的DMEM中，环境中温度为37℃，含有5％CO2。根据制造商的说明，用Lipofectamine 2000(Thermo-Fisher Scientific，Waltham，MA)将pcDNA3.1-FGF2，pNC1-FGF2和pNC1-6xhis-DnaE-FGF2构建体转染到HEK 293T中。
FGF2的纯化
收集培养基并以2,000g旋转10分钟以除去细胞碎片，并通过0.45μm过滤器过滤。然后将滤液通过用50mM Tris-HCl(pH7.5)平衡的预装的肝素-琼脂糖柱(BioRad Laboratories，Hercules，CA)。用50mM Tris-HCl(pH 7.5)，0.2M NaCl彻底洗涤柱子。用0.3M至3M(4-5床体积用于梯度)的NaCl梯度洗脱FGF2。洗脱后，使蛋白质通过用50mM Tris-HCl(pH7.5)平衡的预装的Sephadex G25柱，并用相同的缓冲液洗脱。
对于细胞内表达的FGF2，将细胞用冰冷的TBS洗涤三次，然 后在裂解缓冲液(提供有cOmpleteTM蛋白酶抑制剂混合物(cOmpleteTM Protease Inhibitor Cocktail)的TBS)中超声处理。然后在培养基中纯化FGF2。
为了获得内含肽切除的FGF2，通过预先包装的Ni-NTA柱纯化组氨酸标记的DnaE-FGF2。洗涤后，将柱在50mM Tris-HCl(pH6.2)，10mM EDTA，200mM NaCl中于22℃温育不同的持续时间，分别为0、1、2、4、6、10小时，以诱导DnaE内含肽的C末端切除。
蛋白质分析
在15体积％Tris-甘氨酸SDS-PAGE中分离蛋白质。凝胶用银染以获得纯化的蛋白质。在凝胶上，切下对应于FGF2的条带，洗涤并与1μg胰蛋白酶在50mM NH 4 HCO 3中于4℃温育过夜。通过与Accela HPLE系统结合的LTQ Velos线性离子阱质谱仪(LTQ Velos Linear Ion Trap Mass Spectrometer)(Thermo Fisher Scienctific，San Jose)分析水解的样品。获得完整的MS扫描(300-2000m/z)并用Mascot搜索引擎(Matric Science，Boston，MA)鉴定肽序列。对于细胞裂解物，将样品点样或转移到0.2μmNC膜(BioRad Laboratories，Hercules，CA)上，然后用抗体印迹(小鼠FGF-2(克隆C-2，Santa Cruz Biotechnology，Dallas，TX)，小鼠抗β-肌动蛋白(Sigma，St.Louis，MO))。
FGF2的生物学测定
为了监测FGF2对细胞增殖的影响，如前所述进行MTT测定。简而言之，将C2C12细胞接种在96孔板上，并提供DMEM+0.5体积％FBS和1ng/mL的商购FGF2，纯化的FGF2和内切肽切除的FGF2。通过加入MTT至终浓度1mg/mL测定细胞的活性，并在37℃下孵育6小时。然后用DMSO替换培养基，并在微量板读数器中在540nm处测量吸光度。
为了检测FGF2的神经营养作用，将PC12细胞与提供有2ng/mL的FGF2的培养基一起培养3天。观察细胞的形态，并通过相差光学显微镜拍摄图像。
结果
构建表达人相同的外源FGF2的质粒
全长FGF2蛋白由288个氨基酸组成，其中氨基酸1-142被切割并除去以产生功能性FGF2。因此，氨基酸143-288的蛋白质序列经过密码子优化，用于智人(Homo sapiens)。为了探索蛋白质最大化表达的可行性，将NF-κB结合位点和CREB结合位点增强子序列克隆到含有天然CMV立即早期启动子/增强子序列的pcDNA3.1载体中。然后将合成的fgf2基因克隆到pcDNA3.1和pNC1载体中(图1)，用于比较两个克隆在哺乳动物细胞中的表达。实验表明pNC1载体大大提高了FGF2在哺乳动物细胞中的表达水平，如图2所示。新霉素抗性基因还通过G418硫酸盐提供选择标记。因此可以选择稳定转染的细胞用于纯化人相同的外源FGF2。
可溶性FGF2在HEK 293T细胞中的表达
选择HEK293T细胞用于纯化，因为其易于转染。SV40大T抗原还提供细胞复制转染质粒的能力，其含有SV40复制起点。此外，CMV启动子是最强的启动子之一，并且在HEK293T细胞中具有组成型活性。虽然HEK293T细胞是贴壁细胞，但它适用于悬浮培养是通用的，因此有利于扩大FGF2的产生。将pcDNA3.1-FGF2 pNC1-FGF2和pNC1-6xhis-DnaE-FGF2构建体瞬时转染至HEK293T细胞并监测表达。实验表明：pNC1载体大大提高了FGF2的表达水平(图2A，2B)。FGF2的表达在转染后48小时达到峰值并且在较长时间点保持稳定状态。
使用细菌系统进行蛋白质制备和纯化的缺点之一是形成不溶性聚集体。为了测试在HEK293T细胞中表达的FGF2的溶解度，从 细胞裂解中收获沉淀级分。将不溶性级分在Laemmili样品缓冲液中超声处理，然后在SDS-PAGE中电泳。裂解物的蛋白质印迹显示绝大部分FGF2是可溶的(图2C)。与FGF2标准相比，在pcDNA3.1-FGF2中表达的FGF2约为20μg/mL，对于在pNC1-FGF2中表达的FGF2约为50μg/mL。
FGF2的纯化和蛋白质测序
为了使纯化的FGF2的产量最大化，用pNC1-FGF2转染HEK293T细胞。转染后48小时，收获并裂解细胞以纯化细胞内表达的FGF2。通过肝素-琼脂糖亲和层析纯化FGF2，然后进行尺寸排阻层析。洗脱的FGF2在15％SDS-PAGE中电泳(图3A)。将样品冻干并在0.1 x PBS中重构。为了检查FGF2的纯度，在电泳后进行银染色并且显示出非常高的纯度，在纯化的FGF2上方仅发现1个弱条带(图3B)。产量也令人满意。为了鉴定纯化的FGF2，切下SDS-PAGE上的条带，溶解并在胰蛋白酶消化后通过LC-MS进行分析。测序结果显示纯化的FGF2的一级结构与成熟的外源人FGF2相同(表2)。因此，我们预期纯化的FGF2表现出与人天然对应物相同的性质，活性和功能。
HEK293T表达的FGF2具有生物学活性
已经显示FGF2在许多细胞类型中刺激细胞生长。在确认纯化的FGF2的一级结构后，我们用1ng/mL的纯化和商购FGF2处理培养的C2C12，PBS用作对照。然后通过MTT测定法测量培养细胞的活力(图4A)。两种FGF2样品都能够刺激C2C12细胞的增殖。纯化的FGF2显示出与商购FGF2相似甚至更高的生物活性，但差异不显着。FGF2还在神经元细胞系中表现出神经营养活性。因此，在PC12细胞中测试了纯化的FGF2的神经营养活性。在FGF2处理3天后，观察到延长的神经突生长，其在PBS对照组中不存在(图4B)。数据证实纯化的FGF2具有生物学活性，并具有与商购FGF2相似的 活性。
在HEK293T细胞中内含肽介导的纯化
将含有6x组氨酸标记的DnaE-FGF2的重组蛋白质级联克隆到pNC1载体中(图1C)，然后转染到HEK293T细胞。收集培养基并以2,000g旋转10分钟以除去细胞碎片，并通过0.45μm过滤器过滤。通过Ni-NTA亲和层析纯化蛋白质。用裂解缓冲液(50mM Tris-HCl pH 6.2,10mM EDTA，200mM NaCl)在Ni-NTA柱上进行FGF2切除，在22℃下进行不同的时间点。将珠子和上清液在2x Laemmili样品缓冲液中煮沸以洗脱珠子结合的蛋白质，然后进行SDS-PAGE(图5)。实验结果表明，内含肽的完全切割需要至少5小时的孵育。通过LC-MS测定切除的蛋白质的鉴定，发现切除的FGF2的一级序列与成熟的人FGF2相同(表3)。还测试了切除的FGF2对C2C12细胞的生物活性，并且内切蛋白切除的FGF2的性能也与纯化的FGF2相同(图6A，6B)。
表2
液相色谱-串联质谱法分析纯化的FGF2
Figure PCTCN2020087967-appb-000007
a在胰蛋白酶部分消化纯化的FGF2后，通过Mascot搜索引擎鉴定N-末端和C-末端序列
b肽的理论质荷比
c肽的实验质荷比
表3
用液相色谱-串联质谱法分析内含肽-切除的FGF2
Figure PCTCN2020087967-appb-000008
a在胰蛋白酶部分消化纯化的FGF2后，通过Mascot搜索引擎鉴定N-末端和C-末端序列
b肽的理论质荷比
c肽的实验质荷比
FGF2是一种非常有价值的蛋白质，具有广泛的效力，包括血管生成，神经发生和伤口愈合。阻碍FGF2在医学上使用的研究的原因之一是纯化和生物活性FGF2的高成本。使用HEK 293T细胞，我们成功表达并纯化了人源FGF2(图3)。银染的凝胶证实纯度非常令人满意。在C2C12和PC12细胞培养物中，纯化的FGF2表现出与市售FGF2相同的促有丝分裂或神经营养活性。这种简单的方案允许在哺乳动物系统中实验室规模生产人外源性FGF2，并且可以容易地按比例放大以进行大规模生产。
用于FGF2纯化的最广泛使用的宿主系统之一是细菌。然而，简单的原核生物缺乏必要的翻译后修饰，包括剪接，糖基化和二硫键，用于纯化的蛋白质的活性和溶解度。纯化蛋白质的天然折叠对其功能和溶解性至关重要。当在细菌系统中表达真核蛋白时经常发现蛋白质聚集或包涵体。包括在较低温度下诱导蛋白质或使蛋白质聚集体变性和复性的方法并不总是产生良好的产率。使用细菌系统的另一个问题是内毒素。脂多糖或内毒素通常存在于大肠杆菌中。内毒素的存在会引发人体免疫反应，并可能导致感染性休克。虽然有去除内毒素的商购试剂盒和方案，但内毒素的污染通常是不可避免的。然而，使用哺乳动物细胞，可以容易地克服上述问题。
使用哺乳动物细胞表达蛋白质，我们能够纯化功能性，正确折叠和修饰的蛋白质(表2，图3、图4)。因此，我们是第一次在哺 乳动物细胞中成功纯化无标记的人成熟外源FGF2。
在本发明中，我们已经证明DnaE是HEK293T细胞中的快速切割内含肽。通过使用这种内含肽，我们已经证实切除的FGF2的一级结构(表3)和生物活性(图6)与其天然对应物相同。纯化的FGF2的医学应用将是广泛的。此外，质粒载体pNC1可用于表达多种有价值的蛋白质。
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Claims (14)

  1. 一种增强子序列，其特征在于，该增强子序列包括NF-κB结合位点的DNA序列和CREB结合位点的DNA序列。
  2. 根据权利要求1所述的增强子序列，其特征在于，所述NF-κB结合位点的DNA序列为SEQ ID NO.3所示或其互补序列，所述CREB结合位点的DNA序列为SEQ ID NO.4所示或其互补序列。
  3. 根据权利要求1所述的增强子序列，其特征在于，所述增强子序列为47bp。
  4. 根据权利要求3所述的增强子序列，其特征在于，所述增强子序列为SEQ ID NO.5所示或其互补序列。
  5. 一种质粒载体pNC1，其特征在于，该质粒载体pNC1包括权利要求1-4中任意一项所述的增强子序列。
  6. 根据权利要求5所述的质粒载体pNC1，其特征在于，所述增强子序列位于质粒载体pcDNA 3.1(+)的NheI和HindIII限制性酶切位点之间。
  7. 根据权利要求6所述的质粒载体pNC1，其特征在于，所述NF-κB结合位点的DNA序列与NheI限制性酶切位点连接，CREB结合位点的DNA序列与HindIII限制性酶切位点连接。
  8. 一种质粒载体pNC1的制备方法，其特征在于，该方法包括以下步骤：
    a.合成含有NF-κB结合位点的DNA序列和CREB结合位点的DNA序列的DNA序列，其中，NF-κB结合位点的DNA序列的末端带有NheI限制性酶切位点，CREB结合位点的DNA序列的末端带有HindIII限制性酶切位点；
    b.用NheI和HindIII消化原始质粒载体，回收大片段；
    c.采用连接酶连接步骤a获得的DNA序列与步骤b获得的大片段；
    d.将步骤c获得的连接产物转染细胞，筛选阳性克隆，提取质粒。
  9. 根据权利要求8所述的质粒载体pNC1的制备方法，其特征在于，所述NF-κB结合位点的DNA序列为SEQ ID NO.3所示或其互补序列，所述CREB结合位点的DNA序列为SEQ ID NO.4所示或其互补序列。
  10. 根据权利要求8所述的质粒载体pNC1的制备方法，其特征在于，所述步骤a合成的DNA序列为带有NheI和HindIII限制性酶切位点的SEQ ID NO.5所示序列或其互补序列。
  11. 根据权利要求8所述的质粒载体pNC1的制备方法，其特征在于，原始质粒载体为pcDNA 3.1(+)。
  12. 权利要求5-7中任意一项所述的质粒载体pNC1在表达FGF-2中的用途。
  13. 一种转化体，其特征在于，该转化体包括如权利要求5-7中任意一项所述的质粒载体pNC1。
  14. 如权利要求13所述的转化体，其特征在于，所采用的宿主细胞是HEK 293T。
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