CN102643826A - Thymosin extrasin beta4 quadruplet gene and expression method thereof in Escherichia coli - Google Patents

Abstract

The present invention relates to a thymosin β4 quadruple gene and its expression method in Escherichia coli, the base sequence of the gene is shown in SEQ ID NO.1, the method comprises the following steps: The cloning vector plasmid of the gene is digested, the DNA fragment of the 4×Tβ4 gene is recovered, and the 4×Tβ4 gene with the corresponding sticky end is obtained, and then the 4×Tβ4 gene with the corresponding sticky end is cloned into In the prokaryotic expression vector, ensure that the reading frame of the 4×Tβ4 gene in the expression vector is correct by sequencing or enzyme digestion; transform the expression vector into Escherichia coli by heat shock method, and obtain the 4×Tβ4 gene containing Escherichia coli engineering bacteria expressing the vector; extracting 4×Tβ4 functional protein. The 4×Tβ4 functional protein provided by the invention has important biological activity and function in the healing and repairing of skin and corneal wounds.

Description

Thymosin β4 quadruple gene and its expression method in Escherichia coli

technical field

The invention relates to a gene in the field of genetic engineering and a method for expressing it in Escherichia coli, in particular to a thymosin β4 quadruple gene and a method for expressing it in Escherichia coli.

Background technique

Studies have shown that thymosin β4 (thymosin β4, Tβ4) is an important G-actin sequestering factor (G-actin sequestering factor) in eukaryotic cells, and has many important physiological and cellular functions (Goldstein et al. Advances in Molecular Medicine, 2005, 11:421-429), including: promotion of cell migration, angiogenesis, cell survival, stem cell differentiation, regulation of cytokines, chemokines, specific proteases, upregulation of gene expression of cytoplasmic molecules and downregulation of gene expression of nuclear transcription factors (Huff et al. 2001, International Journal of Biochemistry and Cell Biology, 33: 205-220). It plays an important role in the clinical and application of skin diseases, corneal diseases, heart diseases, central nervous system diseases and lung diseases. At present, there are two main sources of Tβ4 for scientific research and medical use: one is extracted from calf thymus, and the other is chemical synthesis. Extracting Tβ4 from calf thymus is not only costly, but also limited by material sources and extraction techniques, its purity is not high, and its content is low; at the same time, due to zoonotic diseases, Tβ4 extracted from animal bovine thymus has risks in clinical application. Tβ4 chemical synthesis, technology and cost limit its application. With the development of genetic engineering, it becomes possible to produce Tβ4 by means of genetic engineering, which will also become a new direction. The production of recombinant Tβ4 protein by means of genetic engineering can overcome the shortcomings of low extraction rate of biological raw materials or high cost of chemical synthesis.

The literature "Acta Biochemistry and Biophysics, 2002, 34 (4): 502-505" cloned and expressed Tβ4 in Escherichia coli, and its expression level reached 30% of the total protein of the bacteria, but its purification steps were relatively complicated, and it had undergone separate purification procedures. Ammonium sulfate salting out, Source RPC reverse phase chromatography column and Q Sepharose HP anion exchange column were used to purify the target protein. In the study of the literature "Journal of Southeast University (Medical Edition), 2007, 26(4): 295-298", His6-Tβ4 was highly expressed in Escherichia coli, and the expression product accounted for 40% of the total protein of the bacteria. Nickel column affinity chromatography can obtain separation and purification. The literature "Journal of Fourth Military Medical University, 2008, 29(16): 1451-1454" selects eukaryotic secretory expression vector PsecTag2B, and uses liposome transfection technology to transiently transfect eukaryotic SW480 cells, and uses Western blot to detect Tβ4 The expression of recombinant protein realizes the expression in eukaryotic cells. According to the prokaryotic expression vector’s short production cycle, simple expression protein types, and convenient operation, the 4×Tβ4 gene was constructed into the prokaryotic expression vector, and then transformed into Escherichia coli strains to express the target protein under the induction of IPTG, and the target protein was processed Purification and analysis. There are 6 histidine sequences at the 5' end of the multiple cloning site of the pET28a vector and at the 5' end of 4×Tβ4, and the N-terminus of the target protein is tagged with histidine. Therefore, the fusion protein can be specifically combined with the Ni-NTA affinity column, so that the target protein can be purified using the metal affinity chromatography medium, which is convenient to operate, and the obtained protein has good purity and high yield. The purified 4×Tβ4 protein can not only be used as a positive protein in subsequent experiments, but more importantly, it can provide reference and reference for the purification of the target protein expressed in the plant system in the future. In the prior art, there are no related reports on designing and synthesizing the gene Tβ4 according to plant-preferred codons, concatenating the gene Tβ4 to form a quadruplex, namely 4×Tβ4, and expressing the gene 4×Tβ4 in Escherichia coli.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a 4×Tβ4 gene and a method for expressing it in Escherichia coli. The 4×Tβ4 gene was double-enzymatically excised from the plant expression vector (35S::4×Tβ4) by genetic engineering, the gel was recovered to obtain a small fragment, and then 4×Tβ4 and prokaryotic after double-enzyme digestion were used by genetic engineering The large fragments of the expression vector (pET-28a) were connected to obtain a recombinant plasmid (pET-28a-4×Tβ4). The recombinant plasmid was transformed into Escherichia coli by heat shock method, and the fusion protein containing 4×Tβ4 was expressed in E. coli. This protein has important biological activity and function in skin and cornea wound healing and repair.

The present invention is realized through the following technical solutions:

A 4×Tβ4 gene, the base sequence of which is shown in SEQ ID NO.1.

A kind of recombinant vector, comprises target gene and carrier, is characterized in that, the base sequence of described target gene is as shown in SEQID NO.1, and described carrier is selected from a kind of in plasmid, cosmid or lambda phage.

A protein that is (a) or (b):

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO.2,

(b) A protein derived from (a) in which the amino acid sequence in (a) has been substituted, deleted or added with one or several amino acids and has cell proliferation promoting activity.

A method for expressing 4×Tβ4 functional protein in Escherichia coli, the method comprising the steps of:

Step 1. Obtaining 4×Tβ4 gene:

The 4×Tβ4 gene was double-digested from the plant expression vector (35S::4×Tβ4) by the method of genetic engineering, and the small fragment was recovered by gel to obtain the 4×Tβ4 gene fragment (615bp).

Step 2. Construction of prokaryotic expression vector pET28a-4×Tβ4:

Use the same double-digestion prokaryotic expression vector pET-28a glue to recover the large fragment, and then use DNA ligase to insert the 4×Tβ4 gene into the pET-28a to obtain the recombinant vector pET28a-4×Tβ4, and insert the The recombinant vector was transformed into Escherichia coli DH5α competent, after resuscitation, plated on LB solid medium containing 100mg/L kanamycin, and cultured upside down at 37°C overnight.

Step 3. Screening and sequencing identification of DH5α positive clones:

Positive clones were identified by the boiling lysis method, and a single colony was picked and inoculated in LB liquid medium containing 100 mg/L kanamycin. After culturing overnight at 37°C, 2 μL of the bacterial liquid was boiled for 5 minutes and used as a template for PCR detection. The colony amplified in the PCR detection with the same size as the 4×Tβ4 gene was picked and preserved, and 200 μL of the bacterial liquid was taken out for sequencing analysis.

Step 4. Transform Escherichia coli BL21:

Colonies with correct PCR detection and sequencing were cultured overnight, and after the recombinant vector was extracted, the recombinant vector was transformed into Escherichia coli BL21 competent, and the BL21 positive spots were detected by PCR according to the method in the third step above.

Step 5. Induced expression of target protein:

Pick a single clone from the streaked plate of Escherichia coli BL21 detected correctly by PCR, put it into 50 mL of LB liquid medium containing 100 mg/L kanamycin, and culture it at 37°C until the OD ₆₀₀ is 0.6;

Take out 1mL of the bacterial liquid, as a control before induction, and store it in a -20°C refrigerator for later use;

Add 100mM IPTG to the medium to a final concentration of 1mM, and continue to culture for 4h;

Take out 1mL of bacterial liquid, and centrifuge with the bacterial liquid before induction (4°C, 5,000rpm, 10min), discard the supernatant, and resuspend the pellet with 50μL of 2×SDS Buffer;

Samples were boiled at 95°C for 5 min and analyzed by SDS-PAGE.

Step six, extraction of 4×Tβ4 protein.

Preferably, in said step 4, said transformation comprises the following steps:

Step (1), take the competent cells prepared by the CaCl ₂ method just now, or take out the competent cells prepared by the CaCl ₂ method from the -70°C refrigerator and thaw on ice for 10 minutes;

Step (2), add 100 ng of purified expression vector DNA per 200 μL of bacterial solution under sterile conditions, add 5-10 μL of the expression vector into the competent cells, mix gently with a sampler, and place on ice 30 minutes on;

Step (3), heat shock at 42°C for 90s, then place on ice for 1-2min;

In step (4), add 800 μL of LB liquid medium containing 100 mg/L kanamycin preheated at 37°C, and culture at 37°C and 180 rpm for 90 minutes;

Step (5), centrifuge at 5000rpm for 5min, suck off 900μL of supernatant, and mix the remaining 100μL of bacterial solution;

In step (6), apply the bacterial solution evenly on the LB solid plate containing 100mg/L kanamycin with a coating stick, invert at 37°C for 16h-24h until colonies grow, and use a sterile toothpick from the Pick resistant colonies on LB solid plates for identification.

Further preferably, the components of the LB liquid medium and the mass percentages of each component are: 0.5% of yeast extract, 1% of tryptone, 1% of sodium chloride, and the balance is distilled water. The LB liquid The pH of the medium was 7.0. The components of the LB solid medium and the mass percentages of each component are: 0.5% of yeast extract, 1% of tryptone, 1% of sodium chloride, 1.5% of agar powder, and 0.01% of kanamycin , the balance is distilled water, and the pH value of the LB solid medium is 7.0.

Preferably, in step six, the extraction includes the following steps:

12000rpm, centrifuge at 4°C for 5min to collect the cells induced to express the 4×Tβ4 protein, discard the supernatant, and keep the cells on ice;

Dissolve the bacteria collected by centrifugation with PBS (140mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ , 1.8mM KH ₂ PO ₄ ) at a ratio of 5:1, and ultrasonically disrupt the bacteria, 6×10s, once every 10s (200w-300w), the sample has been kept on ice;

Resuspend the bacterial pellet (20-200mL cell culture) with buffer B, the amount of buffer B is 5mL/g of the wet heavy bacterial pellet, stir at room temperature for 15-60min, or shake gently, be careful to avoid foaming , the solution is translucent, that is, the cracking is good;

Centrifuge at 10,000 g for 30 min at 4°C, discard the precipitate, and collect the supernatant for column purification;

Suspend 50% Ni-NTA solution, pack the column to avoid air bubbles, the amount of Ni-NTA is 5-10mg protein/mL resin;

After the resin settles naturally, wash the column with 5 times the column volume of ddH ₂ O, and then add 5-10 times the column volume of 1×Ni-NTA buffer B to equilibrate the column;

Put the sample on the column, wash the column with 5-10 times the column volume of 1×Ni-NTA buffer B, the flow rate is 1mL/min, collect the effluent, and perform SDS-PAGE detection;

Wash the column with 5-10 times the column volume of 1×Ni-NTA buffer C (mainly to wash away unbound proteins), collect the effluent, and perform SDS-PAGE detection;

Wash the column with 5 times the column volume of 1×Ni-NTA buffer E to elute the 4×Tβ4 protein, collect the eluate with 2mL EP tubes respectively, and store the eluate at -20°C or directly carry out SDS-PAGE detection;

The protein concentration was determined according to the Bradford method. After mixing 2 μL protein sample with 98 μL Bradford reagent, the absorbance value of OD ₅₉₅ was measured in a microplate reader (Bio-TEK, USA).

Further preferably, the composition of the 1×Ni-NTA Buffer B is: 8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, the rest is distilled water, and the pH of the 1×Ni-NTA Buffer B is 8.0.

Further preferably, the composition of the 1×Ni-NTA Buffer C is: 8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, the rest is distilled water, and the pH of the 1×Ni-NTA Buffer C is 6.3.

Further preferably, the composition of the 1×Ni-NTA Buffer E is: 8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, the rest is distilled water, and the pH of the 1×Ni-NTA Buffer E is 4.5.

The present invention provides a new way to prepare 4×Tβ4 protein. In the present invention, the Tβ4 gene is concatenated into 4×Tβ4 gene by means of genetic engineering, and the His-tag contained in the expressed 4×Tβ4 fusion protein can combine with Ni -NTA-specific binding characteristics, purification of 4×Tβ4 fusion protein with high purity and yield, the fusion protein has important biological activity and function in the healing and repair of skin and corneal wounds. The invention also provides a research method for detecting and analyzing whether 4×Tβ4 gene exists in Escherichia coli genome DNA and whether protein samples contain 4×Tβ4 protein. The method provided by the invention has the characteristics of short production cycle, simple types of expressed proteins and convenient operation.

Description of drawings

Fig. 1 is the polymerase chain reaction (PCR) detection result figure of DH5α transformant;

Fig. 2 is the polymerase chain reaction (PCR) detection result figure of BL21 transformant;

Fig. 3 is the induced expression analysis chart of pET-28a-4×Tβ4 strain;

Fig. 4 is the expression analysis diagram of the 4×Tβ4 fusion protein induced at different times in the prokaryotic system;

Figure 5 is an analysis diagram of the expression site of the 4×Tβ4 fusion protein;

Fig. 6 is the immunoblotting (Western blot) analysis figure of prokaryotic system expression 4×Tβ4 fusion protein;

Figure 7 is the SDS-PAGE detection of the purified 4×Tβ4 target protein;

Fig. 8 is the lymphocyte proliferation and cell viability assay (MTT) of the purified 4×Tβ4 fusion protein.

Detailed ways

The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as molecular cloning such as Sambrook: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions. suggested conditions.

Example 1 Construction of prokaryotic expression vector pET-28a-4×Tβ4:

The preparation of step (1), pET-28a plasmid

After a single colony was cultured to a suitable concentration, the plasmid was extracted using a plasmid extraction (small amount) kit (Shanghai Huashun Bioengineering Co., Ltd.). The specific process is as follows:

Pick a single colony and inoculate it into 10 mL of LB liquid medium containing 100 mg/L kanamycin and culture overnight;

Take the bacterial solution cultivated overnight to the logarithmic phase in a centrifuge tube, centrifuge at 10,000 g for 90 sec, discard the supernatant, add 250 μL P1 solution, resuspend the bacterial cells until uniform, and place at room temperature for 4 min;

Add 250 μL of P2 solution, and immediately invert gently for 5-10 times to mix well, within 4 minutes until the bacteria are dissolved and the liquid is clear;

Add 350μL of P3 solution, and immediately invert gently for 5-10 times to mix well, a large amount of white feathery precipitate appears, centrifuge at 12,000g for 5min;

Take the supernatant and add it to the column, do not absorb the precipitate (you can pass through the same adsorption column in multiple tubes);

Centrifuge at 10,000rpm for 15sec, discard the liquid in the collection tube, and add 500μL B1 solution;

Centrifuge at 10,000rpm for 15sec, discard the liquid in the collection tube, and add 500μL W1 solution;

Centrifuge at 10,000rpm for 15sec, discard the liquid in the collection tube, add 500μL W1 solution, and let stand for 1min;

Centrifuge at 10,000rpm for 15sec, discard the liquid in the collection tube, and centrifuge at 10,000rpm for 1min;

Transfer the adsorption column to a new 1.5mL Eppendorf tube, and add 50 μL T1 solution to the center of the adsorption column;

55℃ water bath adsorption column for 3min, centrifuge at 12,000g for 1min;

Discard the adsorption column, and store at -20°C for later use after gel electrophoresis detection.

Step (2), pET-28a plasmid is double-digested with BamHI and SacI

Add in a centrifuge tube: 5 μg of pET-28a plasmid, 5 μL of 10×NEB Buffer BamHI, 20 U of BamHI, 0.5 μL of 1 mg/ml BSA, supplemented with ddH ₂ O to a final volume of 50 μL; Plasmid, column recovery;

Add in a centrifuge tube: 3 μg of pET-28a plasmid digested with BamHI, 5 μL of 10×NEB Buffer1, 20 U of SacI, 0.5 μL of 1 mg/ml BSA, supplemented with ddH ₂ O to a final volume of 50 μL; digestion at 37°C for 2 hours, Take a 3 μL sample and electrophoresis to check whether the enzyme digestion is complete; after the enzyme digestion is completed, warm bath at 65°C for 15 minutes to completely inactivate the enzyme.

Step (3), the recovery of enzyme digestion product

The digested products were subjected to agarose (1%) gel electrophoresis, and then purified and recovered using Gel Extraction Mini Kit (Shanghai Huashun Bioengineering Co., Ltd.). The specific steps are as follows:

Cut the segment from the agarose gel with a clean scalpel blade, put it into a clean Eppendorf tube, weigh the gel weight, and add 300 μL of S1 solution per 100 mg of gel;

Water bath at 55°C for 10 minutes until the glue is completely dissolved, and mix by inverting every 2 minutes to promote the dissolution of the glue;

Transfer the melted liquid into the adsorption column, centrifuge at 12,000rpm for 30s, pour off the liquid in the collection tube, and then put the adsorption column into the same collection tube;

Add 500μL of W1 solution to the adsorption column, let it stand for 1min, centrifuge at 12,000rpm for 15s, pour off the liquid in the collection tube, and then put the adsorption column into the same collection tube;

Repeat the previous step;

Centrifuge at 12,000rpm for 1min;

Put the adsorption column into a clean Eppendorf tube, add 30 μL T1 solution to the center of the adsorption membrane, bathe in 55°C water for 1 min, and centrifuge at 12,000 rpm for 1 min;

A 3 μL sample was taken to detect the recovery effect by 1% agarose gel electrophoresis, and the carrier fragment recovered after enzyme digestion was stored at -20°C until use.

Step (4), acquisition of target gene 4×Tβ4

The vector 35S::4×Tβ4 was digested with Bam HI and Sac I respectively, the reaction conditions were the same as step (3), and the digested 4×Tβ4 gene fragment (613bp) was recovered from the gel.

Step (5), connection of recovery fragment and carrier

The recovered target gene fragment and vector fragment were ligated overnight at 16°C. Reaction system: 4×Tβ4 6 μL, pET-28a 2 μL, 10×ligase buffer (ligase buffer) 1 μL, T4DNA ligase (T4DNA ligase) 1 μL; transform the ligation product into E. On the LB plate, incubate upside down in a constant temperature incubator at 37°C for 14-20 hours, and pick well-growing monoclonal positive colonies for colony PCR and sequencing verification.

Step (6), screening and sequencing identification of positive clones

PCR amplification system 25 μL, including primers Tβ4 F (5′-acggtaccatgtctagaatg-3′) and Tβ4 R (5′-gagctcttaactagtcatag-3′) each 1 μL (10 μmol/L), 0.3 μL Taq DNA polymerase (5 units/μL) , 2.5 μL of 10×PCR buffer, 1.5 μL of MgCl ₂ (25mmol/L), 2 μL of template (bacteria to be tested), 1.5 μL of dNTPs (2.5mmol/L), 15.2 μL of deionized sterile water, on Cover with 20 μL sterilized paraffin oil;

PCR conditions: 94°C for 10 min; 94°C for 45 s, 60°C for 45 s, 72°C for 45 min, 35 cycles; 72°C for 10 min; electrophoresis to detect the PCR amplification product, and the length of the amplified fragment was 590 bp;

PCR product detection: use 1.0% (w/v) agarose gel with EB (ethidium bromide) to electrophoresis in 1×TAE electrophoresis buffer, then detect and take pictures under the purple light of the gel imaging system, the results are shown in Figure 1 Shown, wherein: Swimming lane M is DL2000 DNA molecular weight standard; Swimming lane + is the positive control of Escherichia coli DH5α containing plant expression vector plasmid 35S::4×Tβ4 as template; Swimming lane 10 is the negative control of empty vector Escherichia coli DH5α; Other swimming lanes are Independent DH5α-positive plaques 1-9. The data on the left are DNA molecular weight size standards.

Step (7), transformation of Escherichia coli (BL21)

Take the positive clones verified by PCR and sequencing, and extract the recombinant plasmid pET-28a-4×Tβ4 according to step (1). After gel running and verification, take 2-5 μL of the recombinant plasmid DNA and heat-shock it into BL21 competent cells. A single clone was picked, and recombinants were screened and identified by PCR. The PCR system and conditions are the same as step (6). The PCR detection results of BL21 transformants are shown in Figure 2, wherein: Swimming lane M is DL 2000 DNA molecular weight standard; Swimming lane + is the positive control of Escherichia coli BL21 containing plant expression vector plasmid 35S::4×Tβ4 as template; Swimming lane 8 It is the negative control of empty vector E. coli DH5α; other swimming lanes are independent BL21 positive spots 1-7. The data on the left are DNA molecular weight size standards.

Example 2 4×Tβ4 is expressed in Escherichia coli BL21

Step (1) Induced expression of target protein

Pick a single clone from a fresh streaked plate and put it into 50mL liquid medium containing 100mg/L kanamycin, and culture it at 37°C until the _OD600 is 0.6;

Take out 1mL of the bacterial liquid, as a control before induction, and store it in a -20°C refrigerator for later use;

Add 100mM IPTG to the medium to a final concentration of 1mM, and continue to culture for 4h;

Take out 1mL of bacterial liquid, and centrifuge with the bacterial liquid before induction (4°C, 5,000rpm, 10min), discard the supernatant, and resuspend the pellet with 50μL of 2×SDS Buffer;

Samples were boiled at 95°C for 5 min for SDS-PAGE analysis.

Step (2) SDS-PAGE detection expression product

SDS-PAGE preparation refers to "Molecular Cloning" (Sambrook et al., 1989);

After the sample is processed, take out 20 μL of the sample, connect the electrophoresis device to the power supply (the positive electrode is connected to the lower tank), the voltage used on the gel is 8v/cm, when the dye enters the separating gel, increase the voltage to 15v/cm;

After electrophoresis, take out one piece of gel, put it in staining solution (methanol: water: glacial acetic acid = 4.5:4.5:1, add Coomassie brilliant blue R-250 according to 0.25g/100mL) and stain overnight, and use the other piece for Western blot analyze;

Use decolorization solution (methanol: water: glacial acetic acid = 4.5:4.5:1) to decolorize until the protein band is clear and there is no background. Protein molecular weight standard; Lane 1 is the pET-28a-4×Tβ4 strain without TPTG induction; Lane 6 is the empty vector without the target gene induced by IPTG; Lanes 2-5 are the pET-28a- 4 x Tβ4 strain. The data on the left are protein molecular weight size markers.

Step (3), the influence of induction time on target protein expression

Select a strain from the positive clones expressing the target protein, analyze the expression of the target protein at different times, and determine the best induction time. The expression of the strain was induced by the above-mentioned induction method, and 1 mL of bacterial liquid was taken out at 0h, 2h, 4h and 6h after induction respectively. Centrifuge at 5,000 rpm for 10 minutes at 4°C to collect the cells and discard the supernatant. Add 50 μL of 2×SDS Buffer to the precipitate, and cook at 95°C for 5 minutes to denature the protein. The change of the expression product over time was detected by SDS-PAGE. The expression analysis results of the 4×Tβ4 fusion protein induced at different times in the prokaryotic system are shown in Figure 4, in which: Lane M is the protein molecular weight standard; Lane 1 is the untreated IPTG-induced strains; Lane 6 is the empty vector induced by IPTG without the target gene; 2-4 is the protein expression after 2h, 4h, and 6h induction by IPTG; the data on the left is the protein molecular weight standard.

Step (4), determination of target protein expression site

Select a strain from the positive clone expressing the target protein, analyze the solubility of the target protein, and prepare for the method adopted in the next purification analysis;

After adding IPTG to the strain for induction for 4 hours, centrifuge (4°C, 4,000g, 20min) to discard the supernatant, resuspend the pellet with 5mL lysis buffer (lysis buffer), and put it on ice immediately;

Add lysozyme (final concentration: 1 mg/mL) to the resuspension, and place it on ice overnight;

Ultrasonic to disrupt cells, ultrasonic 6×10s, once every 10s (200w-300w), the sample should always be kept on ice;

Centrifuge at 10,000g for 30min at 4°C, collect the supernatant, and keep it on ice for later use;

The pellet was resuspended with 5mL lysis buffer and kept on ice for later use;

Take 5 μL each of the above supernatant and precipitate, add an equal volume of 2×SDS Buffer, cook at 95°C for 5 minutes, and analyze the expression site of the target protein by SDS-PAGE. The analysis results of the expression site of the 4×Tβ4 fusion protein are shown in Figure 5, where : Swimming lane M is the protein molecular weight standard; Swimming lane 1 is the precipitate after 4 hours of induction and broken by ultrasound; Swimming lane 2 is the supernatant after 4 hours of induction and broken by ultrasound; Swimming lane 3 is the empty vector without the target gene after 4 hours of induction ; The data on the left is the protein molecular weight size standard.

Step (5), Western blot analysis of expression product

The positive clones expressing the target protein were induced to express and separated by SDS-PAGE electrophoresis;

After SDS-PAGE electrophoresis, remove the gel, stain one of the gels with Coomassie brilliant blue, and transfer the other gel to the nitrocellulose membrane;

Transfer of protein to PVDF membrane: equilibrate the gel and PVDF membrane with transfer buffer (39mM glycine, 48mM Tris Base, 0.037% SDS, 20% methanol) for 30 minutes, transfer with semi-dry electroporator at room temperature for 1h, and gel two times. 3 layers of Whatman filter paper on each side;

PVDF membrane protein detection: soak the PVDF membrane in blocking solution, shake slowly at 37°C, and block for 60 minutes (blocking solution: take 5g skimmed milk powder and dissolve it in 100mL of 1×PBS); then soak the filter membrane in washing buffer, Wash twice at 37°C for 15 minutes each time; add primary antibody (anti-his-tag antibody), incubate at 37°C for 30 minutes, wash three times; add secondary antibody (avidin-alkaline phosphatase complex) , incubate at 37°C for 30 minutes, wash twice, add substrate for color development and observe protein bands, the results of western blot analysis of 4×Tβ4 fusion protein are shown in Figure 6, where Lane M is the protein molecular weight standard, and Lane 1 is unidentified protein. The pET-28a-4×Tβ4 strain induced by TPTG, lane 6 is the empty vector without the target gene induced by IPTG, and the lanes 2-5 are the pET-28a-4×Tβ4 strain induced by IPTG, the left Data are protein molecular weight size standards.

Step (6), purification of 4×Tβ4 fusion protein

Induce the expression of the target protein, the method is the same as above, and the induction time is 4h;

Centrifuge at 12,000 rpm at 4°C for 5 minutes to collect the cells, discard the supernatant, and keep the cells on ice;

Dissolve the bacteria collected by centrifugation with PBS (140mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO4, 1.8mM KH2PO4) at a ratio of 5:1, ultrasonically disrupt the bacteria, 6×10sec, once every 10sec (200w-300w), the sample need to be kept on ice at all times;

Use buffer B (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, the rest is distilled water, pH 8.0) to resuspend the bacterial pellet (20-200mL cell culture), the amount of buffer B is 5mL/g Ww. Stir at room temperature for 15-60 min, or shake gently, being careful to avoid foaming. The solution is translucent, that is, the cracking is good;

Centrifuge at 10,000g for 30min at 4°C, discard the precipitate, and collect the supernatant for column purification;

Suspend the 50% Ni-NTA solution and pack it into a column to avoid air bubbles. The amount of Ni-NTA is 5-10mg protein/mL resin;

Wait for the resin to settle naturally, wash the chromatography column with 5 times the column volume of ddH ₂ O, and then add 5-10 times the column volume of 1×Ni-NTA buffer B to equilibrate the chromatography column.

Put the sample on the column, wash the column with 1×Ni-NTA buffer B of 5-10 times the column volume, flow rate of 1mL/min, collect the effluent, and perform SDS-PAGE detection;

Wash the column with 1×Ni-NTA buffer C (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, the rest is distilled water, pH 6.3) with 5 to 10 times the column volume (mainly to wash off unbound protein ), collect the effluent for SDS-PAGE detection;

Wash the column with 1×Ni-NTA buffer E (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, the rest is distilled water, pH 4.5) with 5 times the column volume, and elute the target protein with The eluate was collected in a 2mL EP tube and directly detected by SDS-PAGE; the SDS-PAGE detection results of the purified 4×Tβ4 target protein are shown in Figure 7, in which: lane M is the protein molecular weight standard; lane 1 is the elution condition of pH8 The eluted effluent; lane 2 is the effluent eluted under the condition of pH 6.3; 3-9: the 4×Tβ4 fusion protein obtained by elution and purification under the condition of pH 4.5; the data on the left is the protein molecular weight standard.

Step (7), the MTT colorimetric method of lymphocyte proliferation detects the activity of 4×Tβ4 fusion protein

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method was used to detect the biological activity of 4×Tβ4 protein expressed by Escherichia coli (Mosmann, T. (1983) J.Immunol.Methods 65, 55-63), the specific operation steps are as follows:

After the 4×Tβ4 fusion protein purified in step (6) was sterilized by a filter membrane (Millipore, 0.45 μm), the concentration was adjusted to 0.5 μg/μL with 1×PBS;

Immerse the Balb/c mouse in ether, take it out and fix it on a sterile plate with a tack, cut the abdominal skin with sterile scissors and tweezers, open the abdominal cavity, and take out the spleen;

The spleen is placed on a 200-mesh copper grid, fully ground, and washed with RPMI1640 (complete) medium while grinding;

Centrifuge at 1,000rpm for 10min, discard the supernatant;

Suspend the cell pellet with 2mL red blood cell lysate, pipette for about 5min to fully lyse, add 8mL GKN buffer solution (NaCl 8g, KCl 0.4g, Na ₂ HPO ₄ ·2H ₂ O 1.77g, NaH ₂ PO ₄ ·H ₂ O 0.69 g, glucose 2g, phenol red 0.01g, dissolved in 1000ml double distilled water) to terminate the reaction;

Centrifuge at 1,000rpm for 10min, discard the supernatant;

Add 10 mL of RPMI1640 medium to the precipitation, and wash twice;

Suspend in about 10 mL of RPMI1640 medium, count on a hemocytometer under a microscope, and adjust the cell density to 10 ⁵ cells/mL;

According to the density of 10 ⁵ cells/mL, culture on 96-well plate;

Reaction system: In the experiment, a blank control group (1×PBS), a positive control group (Tβ4, purchased from Shanghai Jier Biochemical Co., Ltd.), and purified 4×Tβ4 fusion protein were set as sample groups. Add 100 μL of standard positive protein Tβ4 (1.0 μg) and 100 μL of purified 4×Tβ4 fusion protein after filter sterilization to the 96-well culture plate containing lymphocytes, add 1×PBS 100 μL to the control wells, and make 3 batches, each Each batch has 5 replicates;

After mixing gently, place in a 5% CO ₂ , 37°C incubator for 24 hours;

Add 10 μL of MTT (5 mg/mL mother solution) solution to each well under sterile conditions, mix gently, and continue to incubate for 4 h;

Observe the 96-well plate cells regularly under an inverted microscope (Olympus CKX31, Japan). When a clearly visible purple precipitate occurs, add 150 μL of DMSO (dimethyl sulfoxide) to each well, and then gently shake the 96-well plate. And place the 96-well plate at room temperature (dark) for 15 minutes;

The absorbance value of OD ₅₇₀ was read on a microplate reader (Bio-TEK, USA), and the cell growth curve was drawn with the sample dose as the abscissa and the absorbance value as the ordinate.

According to the determined OD ₅₇₀ value, use the formula to calculate the rate of promoting cell proliferation;

Promoting cell proliferation rate% = (OD _test - OD _control ) / OD _test

The MTT test results of the purified 4×Tβ4 fusion protein are shown in Figure 8, where 4×Tβ4 (BL21) is the lymphocyte proliferation rate of the purified 4×Tβ4 fusion protein, and Tβ4 (GL Biochem) was purchased from Shanghai Gil Biochem The pro-lymphocyte proliferation rate of Tβ4. The experimental results showed that the lymphocyte proliferation rate of the 4×Tβ4 fusion protein expressed and purified by Escherichia coli was 15.09%, and the lymphocyte proliferation rate of Tβ4 purchased from Shanghai Gil Biochemical was 8.49%. The results showed that, compared with the chemically synthesized Tβ4, the 4×Tβ4 fusion protein expressed and purified by Escherichia coli had an obvious biological effect of promoting the proliferation of mouse spleen lymphocytes.

Claims (10)

1. 4 * T Beta-4 gene, its base sequence is shown in SEQ ID NO.1.

2. protein, its aminoacid sequence is shown in SEQ ID NO.2.

3. a recombinant vectors comprises goal gene and carrier, it is characterized in that, the base sequence of said goal gene is shown in SEQ ID NO.1, and said carrier is selected from a kind of in plasmid, clay or the lambda particles phage.

4. method at expression in escherichia coli 4 * T β 4 functional proteins is characterized in that this method comprises following step:

Step 1; The cloning vector plasmids that will contain the said 4 * T Beta-4 gene of claim 1 carries out enzyme and cuts; Reclaim the dna fragmentation of said 4 * T Beta-4 gene; Acquisition has said 4 * T Beta-4 gene of corresponding sticky end, then the said 4 * T Beta-4 gene that has corresponding sticky end is cloned in the prokaryotic expression carrier, cuts evaluation through order-checking or enzyme and guarantees that the said 4 * T Beta-4 gene reading frame in the said expression vector is correct;

Step 2 with in the heat shock method transformed into escherichia coli, obtains to comprise the colibacillus engineering of said 4 * T Beta-4 gene expression vector with said expression vector;

Step 3 is extracted 4 * T β, 4 functional proteins.

5. the method at expression in escherichia coli 4 * T β 4 functional proteins according to claim 4 is characterized in that, in said step 2, said conversion may further comprise the steps:

Step 1 is got and is just used CaCl ₂The competent cell of method preparation, or from-70 ℃ of refrigerators, take out and use CaCl ₂The competent cell of method preparation melts 10min on ice;

Step 2 adds the 100ng expression vector dna of purifying according to per 200 μ L bacterium liquid under the aseptic condition, and the said expression vector of 5～10 μ L is added in the said competent cell, with sample injector mixing gently, places 30min on ice;

Step 3,42 ℃ of heat shock 90s are put in 1～2min on ice again;

Step 4 adds the LB liquid nutrient medium that contain kantlex 100mg/L of 800 μ L through 37 ℃ of preheatings, and 37 ℃, 90min is cultivated in the 180rpm recovery;

Step 5, the centrifugal 5min of 5000rpm inhales and removes supernatant 900 μ L, with remaining 100 μ L bacterium liquid mixings;

Step 6 evenly is applied to said bacterium liquid on the LB solid plate of the kantlex that contains 100mg/L with spreading rod, is inverted 16h～24h to growing bacterium colony for 37 ℃, identifies with aseptic toothpick picking resistance bacterium colony from the said LB solid plate.

6. the method at expression in escherichia coli 4 * T β 4 functional proteins according to claim 5; It is characterized in that; The component of said LB liquid nutrient medium and the mass percent of each component are: yeast extract is 0.5%, and Tryptones is 1%, and sodium-chlor is 1%; Surplus is a zero(ppm) water, and the pH value of this LB liquid nutrient medium is 7.0.

7. the method at expression in escherichia coli 4 * T β 4 functional proteins according to claim 5 is characterized in that, the component of said LB solid medium and the mass percent of each component are: yeast extract is 0.5%; Tryptones is 1%; Sodium-chlor is 1%, and agar powder 1.5%, kantlex are 0.01%; Surplus is a zero(ppm) water, and the pH value of this LB solid medium is 7.0.

8. the method for in enterobacteria, expressing 4 * T β, 4 functional proteins according to claim 4 is characterized in that, in said step 3, said extraction may further comprise the steps:

Under 4 ℃ of conditions, the centrifugal 5min of 12000rpm collects through said 4 * T β, the 4 proteic thalline of abduction delivering, abandons supernatant, and said thalline remains on ice always;

Dissolve the thalline of said centrifugal collection in 5: 1 ratios with PBS, the said thalline of ultrasonic disruption, sample remains on ice always, and the component of said PBS and each concentration of component are: 140mM NaCl, 2.7mM KCl, 10mMNa ₂HPO ₄, 1.8mM KH ₂PO ₄

Resuspended bacterial sediment, stirring at room 15～60min, perhaps vibration gently is to the resuspended solution shape that is translucent;

Under 4 ℃ of conditions, the centrifugal 30min of 10000g discards deposition, collects supernatant and is used for column purification;

Be suspended in 50%Ni-NTA solution, the dress post, said Ni-NTA consumption is 5-10mg albumen/mL resin;

After the said resin natural subsidence, with the ddH of 5 times of column volumes ₂O crosses post and cleans chromatography column, adds the said chromatography column of 1 * Ni-NTA buffer B balance of 5-10 times of column volume again;

The sample upper prop is washed post with 1 * Ni-NTA buffer B of 5-10 times of column volume, and flow velocity is 1mL/min, collects effluent, carries out SDS-PAGE and detects;

1 * Ni-NTA buffer C with 5～10 times of column volumes washes post, collects effluent, carries out SDS-PAGE and detects;

1 * Ni-NTAbufferE with 5 times of column volumes washes post, and 4 * T β, 4 albumen are carried out wash-out, collects elutriant with the EP pipe of 2mL respectively, and said elutriant is stored in-20 ℃.

9. the method at expression in escherichia coli 4 * T β 4 functional proteins according to claim 8; It is characterized in that; The moity of said 1 * Ni-NTA Buffer B is: 8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl; All the other are zero(ppm) water, and the pH value of this 1 * Ni-NTA Buffer B is 8.0.

10. the method at expression in escherichia coli 4 * T β 4 functional proteins according to claim 8; It is characterized in that; The moity of said 1 * Ni-NTA Buffer C is: 8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl; All the other are zero(ppm) water, and the pH value of this 1 * Ni-NTA Buffer C is 6.3; The moity of said 1 * Ni-NTA Buffer E is: 8M urea, and the 0.1M sodium phosphate buffer, 0.01M Tris-Cl, all the other are zero(ppm) water, the pH value of this 1 * Ni-NTABuffer E is 4.5.

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