CN102703489A - Construction method for prokaryotic secretory expression vector of protein transduction domain-Apoptin (PTD-Apoptin) fusion protein and application of prokaryotic secretory expression vector - Google Patents

Abstract

The invention relates to a construction method and application of a secretion expression vector of PTD-Apoptin fusion protein. The PTD-Apoptin sequence was linked to the pET22b(+) secretion expression vector to construct the prokaryotic secretion expression vector pET22b(+)-PTD-Apoptin, which was induced by IPTG to secrete the expressed target protein into the periplasmic space of E. coli by infiltration method The periplasmic space protein was extracted, and the remaining cell components were ultrasonically disrupted, and the supernatant and precipitate were separated for SDS-PAGE analysis. The PTD-Apoptin fusion protein in the periplasmic space was co-incubated with gastric cancer 823 cells, and the inhibitory effect of the fusion protein on gastric cancer 823 cells was detected by MTT method. Using the method of the present invention, the recombinant PTD-Apoptin protein can be secreted into the periplasmic space of Escherichia coli, and exists in a soluble state. The secreted and expressed recombinant PTD-Apoptin protein has biological activity, and the maximum inhibitory rate to gastric cancer 823 cells is 82.95%. .

Description

Construction method and application of prokaryotic secretory expression of PTD-Apoptin fusion protein

technical field

The invention belongs to the field of microorganisms, and in particular relates to the construction and application of a PTD-Apoptin fusion protein secretion expression system.

Background technique

Today, the treatment of tumors is still a difficult problem. Therefore, finding and preparing preparations with strong selectivity and strong action on tumor cells is the current research direction of tumor therapeutic drugs. The vp3 gene (Apoptin) isolated from chicken anemia virus can induce the apoptosis of abnormal cells such as human tumor cells or transformed cells, but has no effect on normal diploid cells. The apoptosis of tumor cells induced by Apoptin does not require the expression of tumor suppressor gene p53, and is not inhibited by the apoptosis factor Bcl-2, showing the application prospect of Apoptin as an anti-tumor agent.

In order for Apoptin to effectively enter tumor cells, it is necessary to construct a drug molecule with the ability to penetrate the membrane. A large number of protein molecules can be obtained by using the prokaryotic expression system. However, the cytoplasmic expression of prokaryotic expression systems mostly exists in the form of inclusion bodies. Inclusion bodies are denatured proteins, generally inactive, and require renaturation to restore activity. However, the renaturation efficiency of inclusion bodies is low. It is less than the requirement, and only a very small number of proteins are folded into the natural state when the inclusion body is refolded, which limits its activity. Therefore, to avoid the renaturation process of inclusion bodies, realize the protein expression in an active soluble state, and secrete a large amount into the periplasmic space of E. coli, which has few types and contents of miscellaneous proteins, is an urgent problem to be solved now.

Contents of the invention

The purpose of the present invention is to construct a fusion protein PTD-Apoptin that induces tumor cell apoptosis and can pass through the cell membrane. When expressed, it is secreted into the periplasmic space of E. coli in a soluble state, retains the function of penetrating the membrane and specifically induces tumors The apoptotic function does not require renaturation, and after the fusion protein is secreted into the periplasmic space, the content of the fusion protein accounts for 80% of the total protein in the periplasmic space.

The technical scheme adopted in the present invention is: the construction method of PTD-Apoptin fusion protein is as follows:

1) Artificially synthesize the positive and negative strands of the PTD sequence with BamH Ⅰ and EcoR Ⅰ sticky ends at both ends, mix the positive and negative strands, and prepare BamH Ⅰ at the 5' end and EcoR Ⅰ sticky at the 3' end PTD double-stranded sequence at the end;

2) Insert the obtained PTD double-strand sequence into the BamH Ⅰ/ EcoR Ⅰ site of the secretion expression plasmid pET22b(+), and construct the secretion expression plasmid pET22b(+)-PTD expressing PTD;

3) Amplify the Apoptin sequence;

4) Digest the Apoptin sequence with EcoR Ⅰ and Sal Ⅰ restriction endonucleases, recover the digested products with a gel extraction kit, and insert the recovered fragments into the EcoR of the secretory expression plasmid pET22b(+)-PTD Ⅰ/ Sal Ⅰ site, construct the secretory expression plasmid pET22b(+)-PTD-Apoptin expressing the fusion protein PTD-Apoptin;

5) Transfer the secretory expression plasmid pET22b(+)-PTD-Apoptin into Escherichia coli BL21 competent cells to obtain recombinant Escherichia coli;

6) Inoculate recombinant Escherichia coli in LB medium, and culture overnight in shake flasks, then inoculate the cultured bacteria solution in 2×YT medium with 5% inoculum amount for expanded culture in shake flasks, when the absorbance value of the bacteria solution reaches At 0.6, induce with 1.2 mM IPTG for 10 hours at 20°C, and then centrifuge at 6000 rpm for 10 minutes at 4°C to collect the bacterial cells;

7) Resuspend the collected bacterial cells in 30mL Tris-HCl containing 20% sucrose, pH 8, concentration 30mM, add EDTA pH 8, concentration 0.5M until the final concentration of EDTA is 1mM, add Stir slowly at room temperature for 10 min with a magnetic stirring bar; centrifuge at 6000 rpm at 4°C for 10 min to collect cells and remove the supernatant;

8) Resuspend the collected cells in 30 mL of frozen MgSO ₄ with a concentration of 5 mM, stir the suspension slowly for 10 min; centrifuge at 6000 rpm at 4°C for 10 min to collect the supernatant, which is the PTD-Apoptin fusion protein.

The application of the PTD-Apoptin fusion protein obtained by the above method in the preparation of antitumor drugs.

The beneficial effects of the present invention are as follows: ① The present invention fuses the protein transduction domain (PTD) of human immunodeficiency virus (HIV-1) with Apoptin to help Apoptin transmembrane into tumor cells. ② In the present invention, a large number of target proteins are expressed in the periplasm, supernatant and precipitate. Since the content of foreign proteins in the periplasmic space of E. coli is small, the content of the target protein is large, which is beneficial to the downstream separation and purification. The invention optimizes the expression condition, avoids the renaturation process of the inclusion body, and enables the target protein to be secreted into the periplasmic space of Escherichia coli in a large amount in a soluble state. ③ The present invention connects the PTD-Apoptin sequence to the pET22b(+) vector containing the signal peptide sequence to construct the prokaryotic secretion expression vector pET22b(+)-PTD-Apoptin, which is induced by IPTG to secrete the expressed target protein into Escherichia coli In the cytoplasmic space, proteins in the periplasmic space were extracted by osmosis, and the remaining cell components were ultrasonically broken, and the supernatant and precipitate were separated for SDS-PAGE analysis. The PTD-Apoptin fusion protein in the periplasmic space was co-incubated with gastric cancer 823 cells, and the inhibitory effect of the fusion protein on gastric cancer 823 cells was detected by MTT method. The results showed that the recombinant PTD-Apoptin protein could be secreted into the periplasmic space of Escherichia coli and existed in a soluble state, and the maximum inhibition rate on gastric cancer 823 cells was 82.95%. The secreted and expressed recombinant PTD-Apoptin protein has biological activity.

The fusion protein constructed by the present invention that can induce tumor cell apoptosis and can pass through the cell membrane is secreted into the periplasmic space of Escherichia coli in a soluble state when expressed, retaining the function of penetrating the membrane and specifically inducing tumor cell apoptosis , without renaturation, and after the fusion protein is secreted into the periplasmic space, the content of the fusion protein accounts for 80% of the total protein in the periplasmic space, and the content of miscellaneous proteins is very small.

Description of drawings

Figure 1 is a schematic diagram of the construction process of the secretory expression plasmid pET22b(+)-PTD.

Fig. 2 is a schematic diagram of the construction process of the secretory expression plasmid pET22b(+)-PTD-Apoptin.

Figure 3 is a PTD double-strand electrophoresis diagram;

In the figure, M: DL2000 Marker; 1: PTD double strand.

Fig. 4 is pET22b (+)-PTD plasmid electrophoresis picture;

In the figure, M: DL10000 Marker; 1: pET22b(+)-PTD plasmid; 2: pET22b(+)-PTD plasmid.

Fig. 5 is the electrophoresis figure of Apoptin gene PCR product;

In the figure, M: DL2000 Marker; 1: control group; 2: amplification product.

Figure 6 is a PCR verification electrophoresis diagram of the pET22b(+)-PTD-Apoptin recombinant plasmid;

In the figure, M: DL2000 Marker; 1: PCR amplification product of recombinant plasmid; 2: control group.

Fig. 7 is the electrophoresis diagram of pET22b(+)-PTD-Apoptin recombinant plasmid digestion and identification;

In the figure, M: DL10000 Marker; 1: Recombinant plasmid digestion product; 2: Recombinant plasmid digestion product.

Figure 8 is the SDS-PAGE detection of PTD-Apoptin expression;

In the figure, 1: pET22b(+)-PTD-Apoptin did not induce the supernatant of the control group; 2: pET22b(+)-PTD-Apoptin did not induce the precipitation of the control group; 3: protein molecular weight standard; 4: pET22b(+)-PTD -Apoptin: periplasm with an induction time of 10 h; 5: supernatant with an induction time of pET22b(+)-PTD-Apoptin for 10 h; 6: precipitate with an induction time of pET22b(+)-PTD-Apoptin for 10 h.

Figure 9 shows the inhibitory effect of different concentrations of PTD-Apoptin fusion protein on gastric cancer 823 cells at 48 hours.

Detailed ways

Example 1 Construction and secretory expression method of PTD-Apoptin fusion protein

(1) Materials and methods

1. Materials: T ₄ DNA ligase, restriction endonuclease BamH Ⅰ, EcoR Ⅰ and Sal Ⅰ, plasmid mini-extraction kit, and gel recovery kit are all products of TaKaRa; IPTG (isopropyl-β- D-thiogalactopyranoside), MTT (thiazolium blue), DMSO (dimethylsulfoxide) were purchased from SIGMA.

2. Method

2.1 PTD sequence preparation

According to the method of primer design, the PTD sequence was artificially synthesized, and the restriction site BamH Ⅰ partial recognition site was introduced at the 5' end of the PTD sequence positive strand, and the EcoR Ⅰ partial recognition site was introduced at the 3'end; the PTD sequence negative strand 5' The partial recognition site of the restriction site EcoR Ⅰ was introduced at the end, and the partial recognition site of BamH Ⅰ was introduced at the 3' end. After annealing to form strands, the introduced restriction enzyme recognition sites create cohesive ends. The artificially synthesized positive and negative strands containing BamH Ⅰ and EcoR Ⅰ cohesive ends were mixed, kept at 95°C for 5 minutes, and then naturally cooled to room temperature to anneal the positive and negative strands to prepare a PTD double-stranded sequence.

2.2 Construction of pET22b(+)-PTD  

The prokaryotic secretory expression vector pET22b(+) was digested step by step with BamH Ⅰ and EcoR Ⅰ restriction endonucleases respectively, and the digested products were recovered with a gel recovery kit, and the recovered digested fragments were combined with artificially synthesized PTD The sequences were ligated overnight at 16°C to construct plasmid pET22b(+)-PTD.

2.3 PCR amplification of Apoptin gene

Two primers were designed according to the nucleotide sequence of Apoptin gene. Restriction sites EcoR Ⅰ, Sal Ⅰ and protective bases were respectively introduced into the 5' ends of the upstream and downstream primers.

The upstream primer is 5'-CGGAATTCGATGAACGCTCTCC-3'

The downstream primer is 5'-GCGTCGACTTACAGTCPTTDACGCCTT-3'

Using the plasmid pET30a-Apoptin as a template, the complete sequence of Apoptin was obtained by PCR amplification. PCR amplification conditions were pre-denaturation at 94°C for 5 minutes, 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 3 min, and extension at 72°C for 10 minutes.

2.4 Construction of PTD-Apoptin prokaryotic secretion expression vector

The prokaryotic secretory expression vector pET22b(+)-PTD was digested step by step with EcoR Ⅰ and Sal Ⅰ restriction endonucleases respectively, and the digested products were recovered with a gel recovery kit, and the recovered digested fragments were combined with the Apoptin gene After ligation at 16°C overnight, the recombinant plasmid pET22b(+)-PTD-Apoptin was constructed. The ligation product was transformed into E.coli DH5α strain, positive clones were picked and cultured in shake flasks, the strains were preserved, and recombinant plasmids were extracted for PCR verification and BamH Ⅰ and Sal Ⅰ enzyme digestion verification. The positive plasmids verified by PCR and enzyme digestion were sent to Shanghai Sangon Biotech Co., Ltd. for nucleic acid sequence determination.

2.5 Induction and identification of prokaryotic secretory expression of PTD-Apoptin fusion gene

Transform the correctly sequenced prokaryotic secretion expression plasmid pET22b(+)-PTD-Apoptin into E.coli BL21 (DE3) strain, inoculate the positive secretion expression strain in LB liquid medium containing Amp, and culture overnight at 37°C; The resulting bacterial solution was inoculated in 2×YT liquid medium containing Amp at an inoculum of 5%, cultured at 37°C until the _OD600 was 0.6, added with IPTG to a final concentration of 1.2mM, and induced at 20°C for 10h. Under the same conditions, uninduced pET22b(+)-PTD-Apoptin was used as a control. Centrifuge at 6000rpm for 10min to collect bacterial cells.

2.6 Protein extraction of periplasmic fraction 

Resuspend the collected bacterial cells in 30mL Tris-HCl containing 20% sucrose, pH 8, concentration 30mM, add EDTA pH 8, concentration 0.5M until the final concentration of EDTA is 1mM, add magnetic stirring Stir slowly at room temperature for 10 min; centrifuge at 6000 rpm at 4°C for 10 min to collect the cells, and remove the supernatant. The collected cells were resuspended in 30 mL of frozen MgSO ₄ with a concentration of 5 mM, and the suspension was stirred slowly for 10 min; the supernatant was collected by centrifugation at 6000 rpm at 4°C for 10 min, which was the PTD-Apoptin fusion protein.

2.7 MTT method to detect the apoptosis rate of gastric cancer cells

Gastric cancer 823 cells in the logarithmic growth phase were inoculated in a 96-well cell culture plate, and about 5×10 ³ cells were inoculated in 0.1 mL per well, and cultured in a 37°C, 5% CO ₂ incubator for 6-12 hours. After the cells adhered to the wall and the plating rate was about 70%~80%, samples containing PTD-Apoptin protein concentrations of 9.375 μg/mL and 11.25 μg/mL were added respectively, and three parallel groups were set up for each concentration, and blank control groups were added, etc. volume of culture solution and cultured in a 37°C, 5% CO ₂ incubator for 48 hours. Add 10 μL of 5 mg/mL MTT to each well, incubate at 37°C and 5% CO ₂ incubator for 3 hours, aspirate the supernatant after incubation, then add 100 μL DMSO (dimethyl sulfoxide) to each well for 5-10 minutes to dissolve the purple color Crystallization, the absorbance at 490nm was measured with a microplate reader. Calculate the cell inhibition rate, and plot the different concentrations of protein and the cell inhibition rate.

Cell inhibition rate=1－[(average OD of experimental group-average OD of background group)/(average OD of blank control group-average OD of background group)]×100%.

(2) Results

1. PTD sequence preparation

Mix the artificially synthesized positive and negative strands containing the sticky ends of BamH Ⅰ and EcoR Ⅰ, 95°C for 5 minutes, and then naturally cool down to room temperature to anneal the positive and negative strands. The results are shown in Figure 3. It can be seen from Figure 3 that in There is a target band at about 30 bp below 100 bp, indicating that the PTD double-strand synthesis is successful.

2. Construction of pET22b(+)-PTD  

The prokaryotic secretory expression vector pET22b(+) was digested step by step with BamH Ⅰ and EcoR Ⅰ restriction endonucleases respectively, and the digested products were recovered with a gel recovery kit, and the recovered digested fragments were combined with artificially synthesized PTD The sequences were ligated overnight at 16°C, and the plasmid pET22b(+)-PTD was constructed. The results are shown in Figure 4. It can be seen from Figure 4 that a band appeared around 4000bp, indicating that pET22b(+)-PTD was successfully constructed.

3. Acquisition of the Apoptin gene

The plasmid pET30a-Aptptin was used as a template for PCR amplification, and the results are shown in Figure 5. From Figure 5, it can be seen that the target band Apoptin small fragment appeared between 250-500bp, while the control group had no target band, indicating that the PCR amplification was successful Apoptin gene was obtained.

4. Verification of PTD-Apoptin prokaryotic secretion expression vector  

The recovered PCR product and the prokaryotic secretory expression vector pET22b(+) were digested with BamH Ⅰ and Sal Ⅰ restriction endonucleases respectively, and the recovered fragments were ligated to construct the recombinant plasmid pET22b(+)-PTD-Apoptin , the ligation product was transformed into Escherichia coli; positive clones were picked and expanded for culture, and recombinant plasmids were extracted for PCR and enzyme digestion verification. The results are shown in Figure 6 and Figure 7. It can be seen from Figure 6 and Figure 7 that the target band appeared between 250-500bp, indicating that the prokaryotic secretion expression vector pET22b(+)-PTD-Apoptin was successfully constructed.

5. Expression of PTD-Apoptin fusion gene in E.coli BL21 (DE3) strain

The prokaryotic secretory expression plasmid pET22b(+)-PTD-Apoptin with correct sequencing results was transferred into Escherichia coli BL21(DE3), and the expression was induced with a final concentration of 1.2mM IPTG, induced for 10h at 20°C, and pET22b was not induced under the same conditions. (+)-PTD-Apoptin was used as a control. Centrifuge, collect the bacteria, obtain the periplasmic fraction, sonicate the remaining fraction, and perform SDS-PAGE detection on the expression product. The results are shown in Figure 8. It can be seen from Figure 8 that there is a band at 20KDa in the periplasm, supernatant and precipitate, which is in line with the expected molecular weight. The control group has no band at the expected molecular weight, indicating that the fusion protein is successfully expressed and secreted into the periplasmic space of E. coli.

6. Inhibitory rate of gastric cancer 823 cells detected by MTT method

After adding PTD-Apoptin fusion protein and incubating for 24 hours, observed by fluorescence microscope, compared with the control group, the number of cells was significantly reduced, indicating that the addition of fusion protein has an inhibitory effect on cell growth. After continuing to incubate for 48 hours, it was observed that the cells shrank. After adding MTT and incubating for 3 hours, DMSO dissolved the purple crystals. The OD ₄₉₀ was measured with a microplate reader. After analyzing the data, it was concluded that as the concentration of the fusion protein increased, the cell survival rate increased. lower. After incubation for 48h, the IC ₅₀ is about 279μg/mL. Compared with the control group, different concentrations of fusion proteins had significant inhibitory effects on gastric cancer cells ( P <0.01). After incubation of gastric cancer cells with the fusion protein for 48 hours, the maximum inhibition rate was 82.95%. The results are shown in Table 1 and Figure 9.

Table 1 The inhibitory effect of different concentrations of PTD-Apoptin fusion protein on gastric cancer 823 cells at 48 hours

±s，

/%

±s,

/%

the control group The concentration is 9.375μg/mL The concentration is 11.25μg/mL Inhibition rate 0 67.71±0.03* 82.95±0.02*

* P <0.01 compared with the control group.

Claims (2)

1.PTD-Apoptin the construction process of fusion rotein is characterized in that method is following:

1) the synthetic two ends have BamHI with EcoRThe positive and negative chain of the PTD sequence of I sticky end mixes normal chain and minus strand, is prepared into 5 ' end and has BamHI, 3 ' end have EcoRThe double-stranded sequence of the PTD of I sticky end;

2) the double-stranded sequence of the PTD that obtains is inserted into secreting, expressing plasmid pET22b's (+) BamHI/ EcoRSecreting, expressing plasmid pET22b (+)-PTD that expresses PTD is constructed in the I site;

3) amplification Apoptin sequence;

4) the Apoptin sequence is used EcoRI with SalI digestion with restriction enzyme, enzyme are cut product and are reclaimed the test kit recovery with gel, and the endonuclease bamhi after reclaiming is inserted into secreting, expressing plasmid pET22b (+)-PTD's EcoRI/ SalSecreting, expressing plasmid pET22b (+)-PTD-Apoptin of expressed fusion protein PTD-Apoptin is constructed in the I site;

5) secreting, expressing plasmid pET22b (+)-PTD-Apoptin is changed in the e. coli bl21 competent cell, obtain recombination bacillus coli;

6) recombination bacillus coli is inoculated in the LB substratum; Shake-flask culture spends the night; Bacterium liquid after will cultivating again is inoculated in 5% inoculum size and shakes a bottle enlarged culturing in 2 * YT substratum, when bacterium liquid absorbance reaches 0.6, under 20 ℃ of conditions, induces 10h with 1.2 mM IPTG; 4 ℃ of centrifugal 10min of 6000rpm collect somatic cells afterwards;

7) somatic cells of collecting is resuspended in contains that 20% sucrose, pH are 8, concentration is among the 30mL Tris-HCl of 30mM, adding pH is 8, and concentration is that EDTA to the EDTA final concentration of 0.5M is 1mM, adds magnetic stirring bar, slowly stirs 10min under the room temperature; 4 ℃ of centrifugal 10 min collecting cells of 6000rpm are removed supernatant;

8) cell of collecting being resuspended in the freezing concentration of 30mL is the MgSO of 5 mM ₄In, slowly stir suspension 10min; 4 ℃ of centrifugal 10 min of 6000rpm collect supernatant, are the PTD-Apoptin fusion rotein.

2. the application of PTD-Apoptin fusion rotein in preparation treatment antitumor drug that obtains according to the described method of claim 1.

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