CN102241778B - PPA-linker-Thanatin fusion protein and preparation method thereof - Google Patents
PPA-linker-Thanatin fusion protein and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a PPA-linker-thanatin fusion protein of which the amino acid sequence is as shown in SEQ ID NO: 2. The invention also discloses a preparation method of the PPA-linker-thanatin fusion protein, comprising the following steps of: acquiring target genes of recombinant pinellia pedatisecta schott agglutinin and thanatin by a two-step PCR (Polymerase Chain Reaction) method, cloning the target genes onto an E.coli expression vector pET-28a-c(+), and acquiring the expressed protein by IPTG (isopropyl thiogalactoside) induction, wherein the expressed protein is the PPA-linker-thanatin fusion protein, namely the expression product of the pinellia pedatisecta schott agglutinin-thanatin. The expression product of the pinellia pedatisecta schott agglutinin-thanatin has biological activity and has inhibiting effect on the growth of tumor cells.
Description
Technical field
The present invention relates to the preparation of escherichia coli expression recombinant protein and the value in clinical application thereof.
Background technology
International top magazine Cell, Nature, Science etc. deliver progress and the meaning thereof of summary, monograph, treatise commentary inflammation and tumour Relations Among one after another in recent years, have caused various countries scholars' interest and attention.Studies show that: inflammation and relation between tumor are close, and chronic inflammatory diseases is relevant with 1/4 above Carciuogenesis.The inflammatory response media such as cytokine, free radical, prostaglandin(PG), somatomedin can inducing DNAs methylate in the inflammatory microenvironment, the gene such as cancer suppressor gene point mutation and posttranslational modification and apparent changes, and cause the change of keeping the crucial path of normal cell homeostasis and generation and the evolution that causes cancer.General antitumor drug rarely has antibiotic effect.So in the exploitation of new drug, the research and development of the antitumor drug of antisepsis and anti-inflammation have huge using value.In order better to bring into play the effect of drugs of different types of polypeptide, adopt the technology of chemosynthesis or molecular cloning Restruction polypeptide drugs very ripe.The research group of Boman couples together by the 1-7 amino acid of wherein a kind of antibacterial peptide-cecropin A (Cecropin) N end of chemosynthesis and the N end 5-12 amino acid of melittin, the sizableness of the T1249 that couples together like this (CAM) is in 40% of cecropin A, but therefore anti-microbial activity does not reduce, and strengthens on the contrary.The engineered method of the usefulness such as Li Yanhong has obtained this T1249, chemosynthesis T1249 CA (1-7) M (5-12) gene out is connected to the outer membrane protein A (OmpA of E.coli secretion expression carrier pIN by joint
2) on the gene order of signal peptide, under the regulation and control of Lpp promotor and Lac promotor/operon, carry out secreting, expressing.Recombinant expressed in order to reach predetermined polypeptide, the clone gene of attacin and N,O-Diacetylmuramidase (Mdc-hly) is cloned on the pET32a, expresses at E.coli bacterial strain BL21 (DE3), and the antibacterial ability of recombinant protein has obtained significant raising.On the whole, using the gene engineering expression recombinant polypeptide is a kind of feasible method.The method that the present invention utilizes gene clone is at the fusion rotein of expression in escherichia coli Rhizome of Pedate Pinellia lectin and dead element, and the antibacterial effect of specific modification Rhizome of Pedate Pinellia lectin is for the research of novel antibiotic antitumor drug provides the basis.
Rhizome of Pedate Pinellia (Pinellia pedatisecta Schott) is the Araeceae per nnial herb, mainly is distributed in China, Korea and Japan.In China's traditional Chinese medicine research, Rhizome of Pedate Pinellia mainly is used as medicine with stem tuber, and it has the pharmacological actions such as blood coagulation, antibechic, analgesia, antiulcer agent, anti-arrhythmia, reducing blood-fat, antitumor, pest-resistant, tranquilizing soporific.Rhizome of Pedate Pinellia lectin (Pinellia pedatisecta agglutinin, PPA) be a kind of vegetalitas Rhizome of Pedate Pinellia albumen with blood coagulation activity of from Rhizome of Pedate Pinellia, separating, have the character with seminose and the single-minded combination of polymkeric substance thereof, the important physiological functions such as blood coagulation, antitumor, desinsection are arranged.Antibacterial peptide (Protegrin) is a kind of animal proteinum with anti-microbial activity in the organism, extensively be present in the plant and animal body, it is to give birth to immunogenic molecular mechanism in the class that develops the earliest, and it has represented the host and has resisted external microorganism the first layer system of defense.Dead element (Thanatin), claim again dead peptide, it is the antibacterial peptide that is formed by 21 amino-acid residues of discovery in insect spot ventral spine benefit stinkbug (Podisus maculiventris), simple in structure, gram-positive microorganism, Gram-negative bacteria and some fungi there is restraining effect, but little on the yeast impact, mammalian cell is not shown hemolytic yet.
Rhizome of Pedate Pinellia lectin and dead element are all with successful at expression in escherichia coli: the Rhizome of Pedate Pinellia lectin of expression has good blood coagulation activity, and grows at vitro inhibition human liver cancer cell QGY7703-3; The dead element of expressing has good antibacterial effect, respectively gram positive bacterial strain and gram negative strain is had the effect that suppresses growth.These are all for the invention provides technical foundation.
In the fusion rotein that utilizes escherichia coli expression Rhizome of Pedate Pinellia lectin and dead element, also overcome some problems: three step PCR processes are in the structure of fusion gene, the disappearance of base, sudden change all can cause the expression of fusion rotein, will be cloned into and carry out sequencing analysis on the T carrier so must carry out the optimization of annealing temperature of each step PCR and PCR result; Utilize in the IPTG abduction delivering process at 37 ℃, the expressing fusion protein activity is lower, attempts utilizing low temperature induction to express, and after induction time and IPTG concentration suitably increased, the activity of fusion rotein was significantly improved.Simultaneously, in order to give full play to separately different biological function of Rhizome of Pedate Pinellia lectin and dead element, we merge two polypeptide with flexible peptide (Linker).
In a word, utilize the method for gene clone, with different sources two in albumen carry out amalgamation and expression, for the research of novel antibiotic antitumor drug provides certain basis.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of PPA-linker-Thanatin fusion rotein and preparation method thereof, and this Rhizome of Pedate Pinellia lectin-dead plain expression product has biologic activity, and inhibited to the growth of tumour cell.
In order to solve the problems of the technologies described above, the invention provides a kind of PPA-linker-thanatin fusion rotein, it is the aminoacid sequence shown in the SEQ ID NO:2.
The present invention also provides the preparation method of above-mentioned PPA-linker-thanatin fusion rotein simultaneously: the goal gene that obtains restructuring Rhizome of Pedate Pinellia lectin and dead element by the method for two-step pcr, and be cloned into coli expression carrier pET-28a-c (+) with goal gene, induce the acquisition expressing protein by IPTG, this expressing protein is PPA-linker-thanatin fusion rotein (also being Rhizome of Pedate Pinellia lectin-dead plain expression product).
Improvement as the preparation method of PPA-linker-thanatin fusion rotein of the present invention: obtaining by the RT-PCR method of Rhizome of Pedate Pinellia lectin goal gene is synthetic, by designing two pairs of primers dead plain 63bp gene of 3 ' end amplification before the ORF terminator codon of Rhizome of Pedate Pinellia agglutinin gene total length; Second pair of design of primers introduced restriction enzyme site Nco I and Xho I, by the encoding gene connection restructuring of two-step pcr method with Rhizome of Pedate Pinellia lectin and the element of being connected, obtains PCR goal gene product, is connected in the T carrier and preserves.
Further improvement as the preparation method of PPA-linker-thanatin fusion rotein of the present invention: with the pMD18-T vector plasmid through Nco I, Xho I double digestion and pET-28a-c (+) plasmid behind double digestion, be connected to become recombinant expression vector plasmid pET-28a-c (+).
Comprehensively described, advantage of the present invention is as follows: vegetable protein Rhizome of Pedate Pinellia lectin and the dead element of animal protein are expressed in the pET prokaryotic expression system first, and obtain Rhizome of Pedate Pinellia lectin-dead plain fusion protein, this fusion rotein has the effect of Rhizome of Pedate Pinellia lectin antitumor cell and the effect of dead plain broad-spectrum antimicrobial concurrently, for the anti-cancer agent exploitation lays the foundation, has very great New drug market development prospect.
For this reason, the present invention adopts the pET prokaryotic expression system to have the Rhizome of Pedate Pinellia lectin of high clinical value-dead fibroin at expression in escherichia coli, does antiserum(antisera) and detect in new zealand white rabbit, for new type antineoplastic medicine is laid firm basis.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is that total RNA reverse transcription is the electrophoretogram of cDNA in the Rhizome of Pedate Pinellia stem tuber;
M：15kb DNA marker；1：cDNA；
Fig. 2 is coding Rhizome of Pedate Pinellia ppa gene cloning;
M:15kb DNA marker; 1,2:Oligo group reverse transcription cDNA group PCR result;
4,5.6: random groups reverse transcription cDNA organizes PCR result;
Fig. 3 is that the ppa gene enzyme is cut identification and analysis;
M:5kb DNA marker; 1:pEasy T1-ppa (Oligo group); 2:pEasy T1-ppa (Random group)
Fig. 4 ppa gene sequencing result;
Fig. 5 ppa gene order BLAST result;
Fig. 6 is that ppal1 PCR product and ppal1-pMD18-T Hind III, EcoR I double digestion are identified;
Fig. 7 is that Hind III, the EcoR I double digestion of ppal2-pMD18-T plasmid and ppal2-pMD18-T identified;
Fig. 8 is ppal3 PCR product and ppal3-pMD18-T Hind III, and EcoR I double digestion is identified;
Fig. 9 is the evaluation of pET28a-pt;
M:1kb Marker; 1: take pET28a-pt as template PCR product; 2: negative control; The SalI of 3:pET28a-pt, the XhoI double digestion is identified;
Figure 10 is ppa-linker-thanatin (pt) gene;
The gene for coding thanatin of runic (and band double underline), italic (and with single underscore) be the linker gene, all the other are the ppa gene of encoding;
Figure 11 is that the expression-form of pET28a-ppa-linker-thanatin (pt) recombinant vectors is identified;
M: low molecular weight protein (LMWP) marker; 1:pET28a-pt/BL21 induces group; 2: induce group pET28a-pt/BL21 supernatant; 3: induce group pET28a-pt/BL21 precipitation; 4:pET28a-pt/BL21 does not induce group;
Figure 12 is the purifying of Rhizome of Pedate Pinellia lectin-dead plain expression product;
M: low molecular weight protein (LMWP) marker; 1: the albumen behind the purifying;
Figure 13 is that the blood coagulation activity of Rhizome of Pedate Pinellia lectin-dead plain expression product detects;
1: negative control; Coagulation analysis after 2:PPA-linker-Thanatin processes;
Figure 14 is that the mtt assay antitumous effect of Rhizome of Pedate Pinellia lectin-dead plain expression product detects.
Embodiment
The acquisition of the cDNA gene of embodiment 1, Rhizome of Pedate Pinellia lectin
Extract total RNA from Rhizome of Pedate Pinellia (Pinellia pedatisecta Schott) stem tuber, with reference to TransScript First-Strand cDNA Synthesis SuperMix working instructions, the total RNA reverse transcription of Rhizome of Pedate Pinellia becomes strand cDNA.
The synthetic system of First-Strand cDNA is as follows:
50 ℃ of PCR instrument temperature controls, 30min; 85 ℃, 5min inactivation RT Enzyme.CDNA behind electrophoresis (Fig. 1) shows that cDNA is 1kb smear once, meets expected results.Take cDNA as template, according to the two couples of primer PPAL1 of Pinellia pedatisecta agglutinin (PPA) mRNA complete sequence (GeneBank:HM593586.1) design that reported on the NCBI, PPAL2 is respectively:
PPAL1 wherein, PPAL2 introduces respectively restriction enzyme site Nco I, Xho I.With reference to primer Tm value, grads PCR is optimized annealing temperature.The PCR system is as follows:
The PCR product advanced 1% agarose gel electrophoresis analysis (Fig. 2), and oligo group and random group amplify bright band simultaneously at 750bp.The PCR product returns test kit through Axygen glue and reclaims, and connects the T carrier, and linked system is as follows:
PCR Product 2μl
pEASY T1 1μl
Hatch 15min for 25 ℃, to connect product is transformed in the 50 μ l T1 competent cells, in the LB solid medium, cultivate after 12 hours after transforming, picking white clone, be inoculated in and contain in the 100 μ g/ml LB liquid nutrient mediums, shake bacterium 12h, the extracting plasmid in the 37 degree constant incubators, cut identification and analysis (Fig. 3) through enzyme, oligo group and random group are recombinated successfully.
After bacterium liquid is delivered to the Sangon sequential analysis, obtain coding Rhizome of Pedate Pinellia agglutinin gene (Fig. 4).Be committed to NCBI and carry out sequence alignment (Fig. 5), the result shows that clone ppa gene has 99% homology with the gene of having reported on the NCBI.
The clone of embodiment 2, ppa-linker-thanatin recombination
Ppa coding Rhizome of Pedate Pinellia agglutinin gene goes on foot pcr amplification recombination ppa-linker-thanatin by P1, P2, P3, P4 primer through three.Overlapping gene is connected to ppa ORF 5 ' end by P1, P2, P3 respectively.Each step PCR product to the T carrier, after plasmid PCR, enzyme are cut evaluation, is entrusted the Sangon order-checking through rubber tapping purifying rear clone.
The PCR system is as follows:
P1, P4 product (ppal1, primer P1, P4, annealing temperature 55 degree), P2, P4 product (ppal2, primer P2, P4, annealing temperature 65 degree), P3, P4 product (ppal3, primer P3, P4, annealing temperature 56 degree) be connected respectively to pEasy T1 carrier, linked system is cut evaluation (Fig. 6 through PCR evaluation and enzyme respectively with embodiment one, 7,8) order-checking after, analytical results is correct.Introduce restriction enzyme site SalI, XhoIPCR by primer P5, P6 and obtain restructuring ppa-linker-thanatin gene.
Same through sequencing analysis, obtain expecting recombination (Figure 10, SEQ ID NO:1).
The structure of embodiment 3, ppa-linker-thanatin prokaryotic expression carrier
Ppa-linker-thanatin (pt) gene and pET28a gene pass through SalI, reclaim the purpose fragment behind the XhoI double digestion, under the effect of T4DNA ligase enzyme, construction of expression vector pET28a-pt, carrier is transformed in the T1 competent cell, extracting plasmid behind 37 ℃ of cultivation 12h, PCR identifies and enzyme is cut the success of evaluation (Fig. 9) demonstration carrier construction.
The corresponding aminoacid sequence of this expection recombination is shown in SEQ ID NO:2 (being the 2nd of sequence table).
The pET28a-pt expression vector is transformed in the intestinal bacteria among the BL21, after 37 degree are cultivated 12h, and the thalline list spot that picking contains recombinant plasmid 37 ℃ of incubated overnight in 3ml LB (the containing Amp50 μ g/ml) liquid nutrient medium.By 1: 50 dilution proportion bacterium that spends the night, generally the 1ml bacterium is joined in the 300ml culturing bottle that contains the 50mlLB substratum, 37 ℃ of concussions are cultured to OD600 ≌ 0.4-1.0.Add the IPTG inductor to final concentration 0.4mM, 9h is cultivated in 16 ℃ of concussions, and induced product is expressed.Get the 1.5ml expression product in the Eppendorf pipe, abandon supernatant through the centrifugal 3min of 12000g, add 150 μ l PBS damping fluid mixings, induce group through ultrasonic disruption, 12000g is divided into supernatant after 3min is centrifugal and precipitates very much, gets respectively inducing of 30 μ l and organizes total protein, induces group supernatant, precipitation, do not induce group, with 2 * albumen Loading Buffer mixing, after the sex change in the SDS-PAGE albumin glue electrophoresis (Figure 11) show that fusion rotein expresses successfully in e. coli bl21.
The purifying of embodiment 5, Rhizome of Pedate Pinellia lectin-dead plain expression product:
Press the method enlarged culturing abduction delivering that embodiment 4 gained are described, the bacterium liquid that 2ml contains the pET28a-pt that recombinates is inoculated in the nutrient solution of 1000ml, cross induction expression protein through IPTG, collect thalline by centrifuging, be resuspended in 40ml Binding Buffer (20mM PBS pH 7.8,500mM NaCl1% (v/v) Triton X-100 and 1% (w/v) proteinase inhibitor), through ultrasonic disruption, 3000g, 30min, 4 degree are centrifugal, and the Binding Buffer that uses of precipitation washs three times at similarity condition.Precipitation is dissolved with 40ml Bingding Buffer (containing final concentration 8M urea) afterwards, loading Ni-NTA post after the filter of 0.45uM filters, balance Binding Buffer, His-tag albumen Washing Buffer (20mM PBS pH 6.0, the 500mM NaCl) wash-out that contains 150mM imidazoles and 8M urea.Albumen is through SDS-PAGE electrophoresis (Figure 12) afterwards, and the albumen behind the purifying presents single band.
The detection of the bacteriostatic activity of embodiment 6, Rhizome of Pedate Pinellia lectin-dead plain expression product and the analysis of blood coagulation activity:
Carry out in the analysis liquid medium within of purifying rear fusion protein antibacterial ability among the embodiment 5, the bacterium of growth is resuspended in the nutrient solution that contains microbiotic (Streptomycin sulphate concentration 10ug/ml) with the semilog phase, and the bacterium final concentration is 10
6Individual/ml.Sample dissolution behind the purifying adds 100ul bacterial suspension mixing in the phosphate buffered saline buffer (pH 6.0) of 10mM, 37 degree are hatched 5h on 96 orifice plates, measures 650nm place absorbancy with microplate reader.The measurement of minimum inhibition concentration (Minimal inhibitory concentrations, MIC) is undertaken by the method for nutrient solution Microdilution.The microorganism of each test is diluted to 10 with nutrient solution
5CFU/ml gets 100ul and isopyknic antibacterial peptide solution mixing, the bacto peptone solution dilution of continuous usefulness 1% on 96 orifice plates then, and 37 degree are hatched 10h, measure the absorbancy at 650nm place.Tested person gram-positive microorganism (genus bacillus, streptococcus aureus), Gram-negative bacteria (e. coli k12-594) and fungi (yeast) arranged, positive control is natural Thanatin.Fusion rotein behind the purifying shows that for the Analysis of Antimicrobial Activity (table 1) of three kinds of bacterial strains of tested person the albumen of expression has obvious anti-microbial activity to the bacterium of tested person.
Table 1, Rhizome of Pedate Pinellia lectin-dead plain expression product Analysis of Antimicrobial Activity
The antibacterial ability analysis of PPA-linker-Thanatin
The analysis of the blood coagulation activity of purifying rear fusion protein adopts the method for isoconcentration dilution to carry out among the embodiment 5, and it is 40 μ gml that the albumen behind the purifying is diluted to concentration
-1, make 2 times of gradient dilutions at 96 orifice plates with 0.85% physiological saline.Add 10 μ l samples at slide glass, equal-volume mixes 2% rabbit erythrocyte, and room temperature is placed about 5-10min, makes blank with physiological saline.Establish 3 parallel laboratory tests, results averaged at every turn.The lower fusion rotein blood clotting effect (Figure 13) that detects of high power lens (40 *), the result shows that fusion rotein has obvious blood coagulation effect.
Embodiment 7, the active detection of the antineoplastic mtt assay of Rhizome of Pedate Pinellia lectin-dead plain expression product:
The liver cancer hum7 cell of cultivating is inoculated in sub 96 well culture plates, and concentration is 2 * 10
5/ ml. inoculates in every hole 180 μ l, continues to add the fusion rotein (sample dissolves with PBS) behind the purifying among the embodiment 5 behind the cultivation 24h.The application of sample final concentration is divided into 0.1mg/ml, 0.05mg/ml, 0.25mg/ml (volume is 20 μ l).The every hole of control group adds 20 μ l PBS, adds same extracting solution (final concentration 0.05mg/ml) 20 μ l.Negative control adds 20 μ l DMSO.Every group of 5 holes.Every hole adds MTT solution 20 μ l after continue cultivating 24h, hatches after 4h. carefully sucks nutrient solution in 96 orifice plates for 37 ℃.Every hole adds 200 μ l DMSO.At the constant temperature oscillator 5min that vibrates, measure each hole OD value.Measuring wavelength is that 792nm. calculates each extract to the growth inhibition ratio of tumour cell.Growth inhibition ratio (%)=(1-experimental group OD value-control group OD value/control group OD value) * 100%.Show that from experimental result (Figure 14) albumen behind the purifying has obvious antitumous effect, its inhibition that suppresses the growth of liver cancer hum7 cell increases along with the increase of experimental concentration.
On enumerate only for several specific embodiments of the present invention, the invention is not restricted to above embodiment.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, being is protection scope of the present invention.
Claims (3)
1. a PPA-linker-thanatin fusion rotein is characterized in that: be the aminoacid sequence shown in the SEQ ID NO:2.
2. method for preparing PPA-linker-thanatin fusion rotein as claimed in claim 1, it is characterized in that: the goal gene that obtains restructuring Rhizome of Pedate Pinellia lectin and dead element by the method for two-step pcr, and be cloned into coli expression carrier pET-28a-c (+) with goal gene, induce the acquisition expressing protein by IPTG, this expressing protein is PPA-linker-thanatin fusion rotein.
3. the method for preparing PPA-linker-thanatin fusion rotein according to claim 2, it is characterized in that: obtaining by the RT-PCR method of described Rhizome of Pedate Pinellia lectin goal gene is synthetic, by designing two pairs of primers dead plain 63bp gene of 3 ' end amplification before the ORF terminator codon of Rhizome of Pedate Pinellia agglutinin gene total length; Second pair of design of primers introduced restriction enzyme site Nco I and Xho I, by the encoding gene connection restructuring of two-step pcr method with Rhizome of Pedate Pinellia lectin and the element of being connected, obtains PCR goal gene product, is connected in the T carrier and preserves.
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