CN103145804A

CN103145804A - Porcine parvovirus VP2 affinity peptide and coding nucleotide thereof

Info

Publication number: CN103145804A
Application number: CN2013100281481A
Authority: CN
Inventors: 任晓峰; 朱卫娟; 斯琴高娃; 任玉东; 李广兴
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2013-01-25
Filing date: 2013-01-25
Publication date: 2013-06-12

Abstract

The invention relates to porcine parvovirus VP2 affinity peptide and coding nucleotide of the porcine parvovirus VP2 affinity peptide. An amino acid sequence of porcine parvovirus VP2 affinity peptide A is shown in a sequence table SEQ ID No.1, and a nucleotide sequence of the porcine parvovirus VP2 affinity peptide A is shown in a sequence table SEQ ID No.2. An amino acid sequence of porcine parvovirus VP2 affinity peptide B is shown in a sequence table SEQ ID No.3, and a nucleotide sequence of the porcine parvovirus VP2 affinity peptide B is shown in SEQ ID No.4. The invention further relates to nucleotide sequences of a pair of primers VP2-3 and VP2-4. Due to the fact that the molecular weight of polypeptide in the porcine parvovirus VP2 affinity peptide is small, the porcine parvovirus VP2 affinity peptide can penetrate into tissue easily, the porcine parvovirus VP2 affinity peptide is stronger than proteins in infiltration capacity, the half-life period of the porcine parvovirus VP2 affinity peptide is short, accumulation in the tissue is little, the structure is stable and production cost is low.

Description

Pig parvoviral VP2 affinity peptide and coding nucleotide thereof

Technical field

The present invention relates to a kind of pig parvoviral VP2 affinity peptide and coding nucleotide thereof, belong to biological technical field.

Background technology

Phage display technique (phage display random peptide library) is a molecular biology new technology utilizing the phage expression foreign gene that grows up the eighties in 20th century.The selected phage vector of display technique of bacteriophage is filobactivirus, comprises f1, fd and M13 phage, and wherein the M13 phage is the most commonly used.The phage of this class strand ring-type DNA virus particle, the 11 kinds of protein of encoding, pIII, pVI, pVII, pVIII, pIX are its capsid structure albumen, wherein pVIII albumen is main capsid protein, consist of the tubular structure of phage, be helical symmetry and arrange, pIII and pVII albumen are positioned at the tail end of phage ghost, are the structures of phage absorption Host Strains.Most mainly concentrates on PPV diagnosis and immunology based on the research of VP2; Less about the data aspect the Study on mechanism of VP2 in the PPV course of infection.

Porcine parvovirus is very serious in China's infection at present, and positive rate can reach more than 90%, has caused huge financial loss to pig industry.PPV VP2 is the main component that consists of capsid protein, and the polypeptide of its coding can the oneself be assembled into the viroid particle, has very strong immunogenicity, is the major protein of control parvovirus.Based on some drawbacks of traditional vaccine, the synthetic polypeptide vaccine of artificial design will become possibility.Display technique of bacteriophage has unique advantage in the new generation vaccine development, and the one: the epitope that phage is showed can keep certain space conformation, can effective stimulus produce immunne response in several days; The 2nd: phage itself both can be used as again good immunological adjuvant as carrier, can strengthen immune effect.Simultaneously, the epitope of phage display peptide library technology to elutriation PPV, the acceptor of research pig parvoviral surface molecular and combination thereof, and the molecular mechanism of Virus entry host cell, the medicine of the more effective anti-parvovirus of development is significant.

1985, Smith confirmed that for the first time filobactivirus fd genome can pass through genetic engineering means, and the polypeptide of goal gene coding is illustrated in phage surface with the form of fusion rotein.Its ultimate principle is: selecting suitable phage is carrier, utilize genetic engineering technique with exogenous gene cloning to its carrier, the fusion rotein of simultaneously foreign gene being encoded at the phage particle surface display out, the polypeptide that is demonstrated keeps relatively independent space structure and biological activity, be conducive to identification and the combination of acceptor, thereby form the storehouse of containing multiple small peptide.The genotype of foreign protein or polypeptide and phenotypic corresponding relation.

Display technique of bacteriophage combines genetic expression with affine selection, its process is that target protein is adsorbed on solid phase carrier, as enzyme plate or particulate matter.Original peptide storehouse phage and target molecule short period of time hatch, and wash away unconjugated phage, then will elute with the phage of target protein specific binding with specific elutriant.Phage after the amplification wash-out, then carry out the next round elutriation, after " absorption-wash-out-amplification " through 3～4 circulations, finally obtain highly enriched sequence of being combined with target protein, then by the further evaluation and screening of enzyme linked immunosorbent assay, obtain real purpose clone, by measuring DNA sequence dna, its aminoacid sequence of deriving.Synthetic polypeptide is further studied its biological activity at last.

Summary of the invention

The object of the invention is to the epitope by phage display peptide library technology elutriation PPV, a kind of pig parvoviral VP2 affinity peptide A and B and coding nucleotide thereof are provided.

Purpose of the present invention is achieved through the following technical solutions:

1, a kind of pig parvoviral VP2 affinity peptide A, aminoacid sequence is as shown in sequence table SEQ ID No.1.

2, the Nucleotide of a kind of pig parvoviral VP2 affinity peptide A as above, nucleotide sequence is as shown in sequence table SEQ ID No.2.

3, another pig parvoviral VP2 affinity peptide B, aminoacid sequence is as shown in sequence table SEQ ID No.3.

4, the Nucleotide of a kind of pig parvoviral VP2 affinity peptide B as above, nucleotide sequence is as shown in sequence table SEQ ID No.4.

5, a kind of VP2 sequence amplimer VP2-3 and VP2-4, nucleotides sequence is classified as:

VP2-3：5’-GGGGATCCATGAGTGAAAATGTGGAA-3’；

VP2-4：5’-CCCCGTCGACTTAGTATAATTTTCTTGG-3’。

This research has the following beneficial effect that a bit reaches: utilize the storehouse technology elutriation of phage random dodecapeptide to go out the polypeptide of being combined with the VP2 protein-specific with respect to prior art.This small peptide of synthetic provides certain theoretical basis and test basis to the control porcine parvovirus.We intend utilizing the polypeptide of the analysis of virusology experimental technique and CHARACTERISTICS IDENTIFICATION in the effect of parvovirus infections in the cycle on this basis, for PPV infects and the research of pathogenesis provides Important Theoretic Foundation, also provide important theoretical foundation for searching PPV VP2 epitope and Virus entry cell mechanism.Polypeptide of the present invention easily penetrates into tissue due to molecular weight, and is stronger than large protein penetrating power; Transformation period is short, accumulates seldom (reducing the risk that its metabolite leads to complications) in tissue; Stability Analysis of Structures and production cost are lower.

Description of drawings

Fig. 1 is the pcr amplification result of VP2 gene;

Wherein M is Marker, the amplified fragments of the 1st, VP2 gene, the 2nd, negative control;

Fig. 2 is the PCR qualification result of pET32a-VP2 recombinant expression vector, the amplified fragments of the 1st, VP2 gene;

Fig. 3 is the double digestion qualification result of pET32a-VP2 recombinant expression vector, the 1st, BamH I and Sal I double digestion result;

Fig. 4 is the SDS-PAGE electrophorogram of VP2 fusion rotein;

M wherein: lower protein standard molecular weight; 1: empty carrier bacterium liquid; 2: do not induce bacterium liquid; 3～7: the recombinant bacterium of inducing 1～5h; 8: bacterial sediment after ultrasonication; 9: the thalline supernatant;

Fig. 5 is that phage is in conjunction with the purification result of clone's template DNA;

M:DL8000DNA Marker wherein; 1～10: respectively corresponding 1～10 phage is in conjunction with the purifying of clone's template DNA;

Fig. 6 is phage PCR purified product figure;

M:DL8000DNA Marker wherein; 1～10: respectively corresponding 1～10 phage pcr amplification product;

Fig. 7 is that phage is in conjunction with clone's ELISA detected result (OD490);

Fig. 8 is mtt assay vitro detection polypeptide inhibition, and A is the MTT result that polypeptide suppresses PPV, and B is that polypeptide is to the inhibiting rate of PPV cells infected;

Fig. 9 is indirect immunofluorescence vitro detection polypeptide inhibition, ST cell contrast (A), and 100TCID50PPV contrasts (B), No. 1 peptide+PPV (C), No. 2 peptide+PPV (D), 1+2 peptide+PPV (E, F);

Embodiment

The present invention designs pair of primers, and amplification obtains PPV VP2 gene fragment, and construction recombination plasmid VP2-pET32a induces through IPTG, carries out high efficient expression in E.coli Rosetta.Take VP2 albumen as target molecule, utilize phage dodecapeptide storehouse immediately, the VP2 recombinant protein has been carried out five taken turns biopanning, finishing screen is selected the dodecapeptide of 6 kinds of avidity.The highest and the synthetic dodecapeptide of repeated 2 best sequences, detect the synthetic peptide extracorporeal antivirus effect by mtt assay and immunofluorescent test active good with avidity.This result of study is laid a good foundation for seeking the PPV acceptor, for providing certain theoretical basis and test basis to the control of PPV and the development of polypeptide vaccine from now on.

Embodiment one

The structure of recombinant expression plasmid pET32a-VP2

(1) amplification of VP2 sequence

(a) according to the codon of bacterium preference, design primer VP2-3 and VP2-4

VP2-3:5 '-GG GGATCCATGAGTGAAAATGTGGAA-3 ' (draw horizontal line place represent that the restriction enzyme site of introducing is BamHI)

VP2-4:5 '-CCCC GTCGACTTAGTATAATTTTCTTGG-3 ' (draw horizontal line place represent that the restriction enzyme site of introducing is SalI)

(b) carry out conventional chain polymerization enzyme reaction amplification VP2 gene

Take VP2-3 and VP2-4 as primer, take VP2-T-6 as template, carry out PCR with the ExTaq polysaccharase, the PCR reaction conditions of VP2: containing described PCR reaction system in 50 μ l systems is 50 μ L, comprise 5 μ L10 * EasyTaq Buffer, 4 μ L dNTP, 1 μ L VP2-3,1 μ L VP2-4,0.5 μ L VP2-T-6,0.5 μ L ExTaq polysaccharase, add sterilized water and mend to 50 μ L.With mixed solution at the response procedures that described PCR detects be: 95 ℃ of denaturation 5min; 94 ℃ of sex change 1min; 56 ℃ of annealing 1min; 72 ℃ are extended 2min; 30 circulations are carried out in reaction; Last 72 ℃ are extended 15min again.Obtain VP2 sequence PCR product and analyze with 1% agarose gel electrophoresis, have single target stripe near 1740bp, result such as Fig. 1 show that it is the VP2 gene that experiment has obtained specific DNA fragment in line.

(2) structure of recombinant expression plasmid pET32a-VP2

After glue purification is reclaimed, the VP2PCR product namely carries out BamH I and SalI double digestion, after glue purification reclaims, the external connection of plasmid pET32a with cutting back to close through same enzyme, builds recombinant expression plasmid pET32a-VP2.Be transformed in intestinal bacteria competence JM109 37 ℃ of overnight incubation, Amp+ screening positive clone.

PCR and the enzyme of embodiment two restructuring expression plasmid pET32a-VP2 are cut evaluation

Select positive colony, extracting plasmid pET32a-VP2 identifies as primer carries out PCR take VP2-3 and VP2-4, and BamH I and Sal I double digestion, and product detects with 1% agarose gel electrophoresis, and the position is consistent with prediction.Result such as Fig. 2 show that the VP2 gene successfully connects in the pET32a carrier.

The SDS-PAGE of embodiment three abduction delivering products analyzes

To identify correct recombinant plasmid pET32a-VP2 Transformed E .coli Rosetta (DE3) competent cell, adding IPTG to make final concentration is 0.6mmol/L, 37 ℃ of shaking culture.The Processing Test sample carries out the SDS-PAGE electrophoretic analysis.Result such as Fig. 4.

Biopanning and the amplification of embodiment four phage random dodecapeptide storehouses to VP2 albumen affinity ligand

According to the phage panning technique of this laboratory maturation, with phage random dodecapeptide storehouse, target molecule VP2 albumen is carried out 5 take turns biopanning.

(1) first round biopanning

(a) the VP2 recombinant protein passes through 0.22 μ m disposable filter filtration sterilization, and measures its concentration, with coating buffer (0.1mol/L NaHCO ₃PH8.6) VP2 albumen is diluted to 100 μ g/mL.Below operate all aseptic carrying out, use sterilization rifle head.

(b) elisa plate radiation sterilization 30min under ultraviolet lamp, every hole adds 150 μ L VP2 diluted protein solutions.4 ℃ of coated spending the night.

(c) get rid of coating buffer, back-off is patted several times on clean paper handkerchief, to remove residual liquid.Every hole adds the 200 μ L liquid of blockading, and places 4h for 4 ℃.

(d) TBST (TBS+0.1%[v/v] Tween-20) damping fluid is washed plate 6 times, rotates elisa plate at every turn the bottom in hole and edge are all washed, and discards damping fluid, and clean paper handkerchief arsis gets rid of several times to remove residual liquid.The washing process operation wants fast, and is dry to avoid plate.

(e) 1 μ L phage dodecapeptide original library adds the TBS damping fluid of 99 μ L, and mixing adds in the enzyme plate hole, and 40min is shaken at the room temperature interval slowly, namely shakes slowly 10min, stops 10min, carries out successively 40min.

(f) abandon liquid, be inverted in to pat on clean paper handkerchief and remove several times raffinate.0.1%TBST washing 10 times is got rid of bat at every turn and is all changed clean paper handkerchief and prevent crossed contamination.

(g) add elutriant (0.2mol/L Glycine-HCl pH2.2) 100 μ L, room temperature is shaken 30min slowly, then elutriant is moved in aseptic micro-EP pipe, add neutralizer (1mol/L Tris-HCl pH9.1) 15 μ L with in and elutriant, be phage first round elutriation eluate, 4 ℃ of preservations.

(h) measure the titre of first round elutriation eluate.

(2) amplification of first round elutriation eluate

(a) get the phage eluate of the 10 μ L first round, do 10 ^-1-10 ^-5Dilution, the titre of mensuration first round wash-out.

(b) E.coli ER2738 inoculates in Low-LB liquid nutrient medium (containing 1 ‰ tsiklomitsins) in 1: 100 ratio, and 37 ℃ of 200rpm shaking tables are cultivated 5h.

(c) get first round eluate 50 μ L and join in the 20mLLow-LB culture with the new E.coli ER273820 μ L that activates, 37 ℃ of shaking tables are cultivated 4.5h.

(d) with in culture packing 10mL centrifuge tube, 4 ℃, the centrifugal 10min of 10000rpm.The top 80% of getting supernatant changes in a fresh tube, adds 1/6 volume PEG8000/NaCl mixing.4 ℃ of phage sedimentations are spent the night.

(e) 4 ℃, the centrifugal 15min of 10000rpm.Abandoning supernatant, of short duration centrifugal, suck residual liquid.

(f) get the resuspended throw out of 1mL TBS, suspension moves in micro-EP pipe, and 4 ℃, the centrifugal 5min of 10000rpm remove the residual precipitate thing.Then supernatant is changed in another aseptic micro-EP pipe, add the PEG8000/NaCl of 1/6 volume, 4 ℃ of sedimentation 60min.

(g) 4 ℃, the centrifugal 10min of 10000rpm, supernatant discarded, of short duration centrifugal, suck residual liquid with micropipet.With the 200 resuspended throw outs of μ LTBS, 4 ℃, the centrifugal 1min of 10000rpm remove residual not tolerant precipitation.Supernatant changes in the micro-EP pipe of sterilization, is amplified production.

(h) get 10 μ L amplified materials and do 10 ^-8-10 ^-12Dilution, the dull and stereotyped titre of measuring first round elutriation amplified material of LB/IPTG/Xgal.

(3) second and third amplification of taking turns biopanning and eluate is the same

(4) the pET-32a label protein is removed in the fourth round screening

The pET-32a label protein is target molecule, coated elisa plate 10 μ g/100 μ l, after phage and the effect of pET-32a label protein, the results supernatant is fourth round elutriation eluate, remove the phage of being combined with label protein by this process, obtain only in the phage of VP2 protein-specific combination

(5) the 5th take turns biopanning

Phage the 5th is taken turns elutriation.The package amount of target protein is 4 μ g/ holes, and the end product that this step obtains is the 5th eluate of taking turns, and it is done 10 ^-2～10 ^-8Dilution, mensuration the 5th is taken turns the titre of eluate on the LB/IPTG/Xgal flat board, 4 ℃ of sum flat boards less than 100 plaques that keep in Dark Place.

Table 1 phage is eluriated product titre pfu and measures

?	The first round	Second takes turns	Third round	Fourth round	The 5th takes turns
						Eluate	9×10 ³	10×10 ⁴	7.5×10 ⁵	8.5×10 ⁷	9×10 ⁶
Amplified material	8.2×10 ¹¹	9.1×10 ¹¹	7.1×10 ¹¹	5.0×10 ¹¹	6.3×10 ¹²

Carry out the characterized in conjunction with the clone after taking turns through five, elutriation is gone out positive mono-clonal send to and carry out sequencing, and its amino sequence of deriving.

Embodiment five synthesizes dodecapeptide according to phage in conjunction with clone's sequencing result

(1) with reference to method shown in " phage display peptide storehouse rapid screening peptide part service manual ", order-checking is carried out purifying with template.Result such as Fig. 5.

(2) the specific phage primer sequence that provides with reference to " phage display peptide storehouse rapid screening peptide part service manual ", by the synthetic pair of primers of the large Gene Tech. Company Limited of Beijing six directions China:

+130M13：5’-TCACCTCGAAAGCAAGCTGA-3’

-28M13：5’-CCCTCATAGTTAGCGTAACG-3’

(3) with reference to the method in " phage display peptide storehouse rapid screening peptide part service manual ": the 1-10 phage DNA carries out PCR as template after the purifying, amplified production is through 1% agarose gel electrophoresis, under ultraviolet lamp, visible size is about 10 single bands of 250bp, result such as Fig. 6.

(4) 1-10 PCR product is sent to Beijing large Gene Tech. Company Limited of six directions China and measured nucleotide sequence, the N-terminal sequence of its sequence with random dodecapeptide-gIII fusion rotein compared, the ELISA method filters out 10 phage dodecapeptide nucleotide sequences with the target molecule specific binding, and it is translated into corresponding aminoacid sequence, what wherein avidity was best is No. 1 combination clone's PCR product, and its aminoacid sequence is HTHQLHFLNHNP; 3,5,6,7, No. 8 is HNLHLYHYLRSA in conjunction with the coded aminoacid sequence of clone PCR products, and repeatability is best.Concrete grammar is with reference to " phage display peptide storehouse rapid screening peptide part service manual ".Result such as Fig. 7.

(5) HTHQLHFLNHNP and HNLHLYHYLRSA being sent to the synthetic purity of doctor biotech firm is 95% polypeptide.

Table 2 phage is in conjunction with clone's sequencing result

Embodiment six mtt assay vitro detection polypeptide inhibitions

PPV (100TCID50) and peptide 2 are selected in test ^-1Dilution (50 μ g/100 μ L) is divided into virus+No. 1 peptide group, virus+No. 2 peptide group, virus+1, No. 2 peptide mixing group, groups of cells and virus group.

Import the ST cell into 96 porocyte culture plates, discard nutrient solution after cell grows to 30-50%, add the mixture after PPV and polypeptide are hatched 1h, 37 ℃, 5%CO ₂Cultivate 1h in incubator, abandon the DMEM that liquid adds serum-free.Each organizes 5 repeating holes.96 orifice plates are put 37 ℃, 5%CO ₂Incubator continues to cultivate, and every day, observation of cell changed.When the cell of virus control group approximately occurs coming off more than 80%, discard 96 hole all liquids, every hole adds MTT (5mg/mL) 20 μ L, continues lucifuge and cultivates 4h.Supernatant discarded adds the every hole 150 μ L of lysate DMSO, vibration 10min, and dissolving fully to be crystallized, microplate reader records the light absorption value at 490nm place.

Result such as Fig. 8.No. 1 peptide inhibiting rate is that 32.77%, No. 2 peptide inhibiting rate is about 63%, 1+2 mixed peptide inhibiting rate and is about 70%.

Embodiment seven indirect immunofluorescence vitro detection polypeptide inhibitions

(1) PPV (100TCID50) and polypeptide 2 are selected in test ^-1Dilution (50 μ g/100 μ L) is divided into virus+No. 1 peptide group, virus+No. 2 peptide group, virus+1, No. 2 peptide mixing group, groups of cells and virus group.

(2) the ST cell imports 24 porocyte culture plates (creep plate) into, treats that cell grows to 30-50%.Discard nutrient solution, add the mixture after 100TCID50PPV and polypeptide are hatched 1h, 37 ℃, 5%CO ₂Cultivate 1h in incubator, abandon the DMEM that liquid adds serum-free.5 multiple holes of each group.Continue to be cultured to the virus control hole and pathology occurs.

(3) the indirect immunofluorescence concrete steps are as follows:

(a) 24 orifice plates after taking-up is cultivated discard nutrient solution, and precooling PBS washes 3 times.

(b) add 4% paraformaldehyde of 350 μ l precoolings, room temperature is 30min fixedly.

(c) discard paraformaldehyde, add the 0.1mol/L glycine incubated at room 5min of precooling.

(d) abandon liquid, 1 * PBS washing 3 times.1%Triton-100 room temperature effect 10min.

(e) abandon liquid, 1 * PBS washing 3 times.Take out creep plate back-off to 1: the VP2 rabbit polyvalent antibody of 100 dilutions (0.1%BSA is diluent), incubated at room 1h.

(f) creep plate is put back in 24 orifice plates, and 1 * PBS washes 3 times, adds anti-(0.1%BSA is diluent) lucifuge of goat-anti rabbit FITC mark two of dilution in 1: 300 to hatch 1h.

(g) 1 * PBS washing is 3 times.Anti-fluorescence decay mountant mounting is in the fluorescence microscopy Microscopic observation.

(4) test every group at every turn choose 5 different visuals field and carry out fluorescence number statistics, 3 times parallel test fluorescence is counted the average statistics result

Groups of cells: 1; Virus group: 64; No. 1 peptide: 45; No. 2 peptides: 23; 1+2 mixed peptide: 18.

The inhibiting rate that goes out by fluorescence number rough calculation:

No. 1 peptide: 29.7%; No. 2 peptides: 64%; 1, No. 2 mixed peptides: 71.9%.

Repeatedly result is basically identical with the MTT result.Result such as Fig. 9.

If confirm the inhibition of polypeptide, need to further carry out animal vivo test.

Claims

1. a pig parvoviral VP2 affinity peptide A, is characterized in that, aminoacid sequence is as shown in sequence table SEQ ID No.1.

2. the Nucleotide of a coding a kind of pig parvoviral VP2 affinity peptide A as claimed in claim 1, is characterized in that, nucleotide sequence is as shown in sequence table SEQ ID No.2.

3. pig parvoviral VP2 affinity peptide B, it is characterized in that: aminoacid sequence is as shown in sequence table SEQ ID No.3.

4. the Nucleotide of a coding a kind of pig parvoviral VP2 affinity peptide B as claimed in claim 3, is characterized in that, nucleotide sequence is as shown in sequence table SEQ ID No.4.

A VP2 sequence amplimer VP2-3 and VP2-4, it is characterized in that, nucleotides sequence is classified as:

VP2-3：5’-GGGGATCCATGAGTGAAAATGTGGAA-3’；

VP2-4：5’-CCCCGTCGACTTAGTATAATTTTCTTGG-3’。