CN103436557A - Visual tracking method for rapid expression of NS1 protein by bombyx mori bioreactor - Google Patents

Abstract

The invention relates to a visual tracking method for rapid expression of an NS1 protein by a bombyx mori bioreactor. The method comprises the steps: a donor plasmid carrying an egfp expression cassette and an nsl gene expression cassette is constructed, an scherichia coli DH10B competent cell is converted by the donor plasmid, the egfp expression cassette and the nsl gene expression cassette which are between Tn7R-Tn7L of sequences of the donor plasmid can be specifically transposed onto a Bm-Bacmid vector, an integrated recombinant eukaryotic expression vector for expressing an egfp gene and an nsl gene is constructed, a bombyx mori is infected by recombinant buddedvirus BV particles produced after insect cells are transfected with the integrated recombinant eukaryotic expression vector, and real-time monitoring of a situation of virus multiplication in a bombyx mori body is carried out through visual green fluorescent signals. The method can perform large-scale and real-time monitoring of expression dynamic situation of an exogenous gene in the bombyx mori body, and lays the foundation for revealing characteristics of the NS1 protein and other functional proteins.

Description

A kind of silkworm biological reactor of visual tracking is expressed the method for NS1 albumen fast

Technical field

The present invention relates to genetic engineering technique, disclose structure flow process and the application thereof of the visual tracking expression system of silkworm biological reactor.Particularly, refer in particular to the preparation method of a kind of budded virus particle BV that recombinates and utilize two minutes densovirus non-structural protein NS 1s of quick in silkworm biological reactor, the high efficient expression silkworm of this virus, for follow-up function and the structural research of NS1 albumen lays the foundation, the expression that the method equally also is applicable to other foreign gene and has the recombinant protein medicine of critical function.

Background technology

Shape virus-insect cell expressioning system (BEVS) is one of current genetically engineered four large expression systems, and the rhabdovirus expression vector utilized is mostly to select Autographa californica multicapsid nucleopolyhedrosisvirus nucleopolyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhydrosis virus (BmNPV).In numerous expression systems, expression output in view of foreign gene in prokaryotic expression system is relatively high, therefore first-selected prokaryotic expression system often when expression alien gene, but in view of expression product is to exist with the inclusion body form, product does not have biological activity and lacks posttranslational modification, has limited the further application of this system; The unicellular yeast expression system is a kind of eukaryotic expression system, but it is merely able to expression product is carried out that some are simply glycosylation modified, and expression product has certain biological activity; Mammalian cell is the eukaryotic expression system in a kind of ideal, have the product after translation is processed and translated, the albumen produced outclass other eukaryotic expression system aspect active, closer to natural protein, but because the exogenous protein expression amount in this system is on the low side, and the needed cost of culturing cell is very high, also be difficult at present meet large-scale practical application.And in BEVS, expressed product not only has correct folding, also there is the characteristics such as translation post-treatment, glycosylation and phosphorylation modification, expressed product is corresponding natural products close to it all in biological activity, antigenicity and immunogenicity, these characteristics make BEVS become a kind of widely used expression system (Kato et al., Appl Microbiol Biotechnol, 2010,85:459-470; Prabhakar et al., Mol Biotechnol, 2010,46:80 – 89).

The characteristic and the security that have numerous uniquenesses due to BEVS are good, now be widely used in numerous research fields such as medicament research and development, production of vaccine and Recombinant insect virus, it is a kind of ideal eukaryotic expression system, but the expressed in insect cells foreign protein that this expression system need to cultivated, required somewhat expensive, be unsuitable for expressing on a large scale foreign protein, utilize silkworm can overcome this unfavourable condition as bio-reactor.Silkworm is a kind of Important Economic insect of China, mainly take mulberry leaf as food, easily extensive raising, and needed culture device is simple, with low cost, with short production cycle, is very suitable for the extensive recombinant protein with critical function of expressing, in addition, the silkworm whole genome sequence is measured completes (Xia et al., Science, 2009, 326:433 – 436.), also contribute to us to silkworm, to carry out genetic modification, can cultivate the new variety that more are conducive to exogenous protein expression thus, up to the present, at least existing 30 laboratories, the whole world are utilizing silkworm to carry out the research aspect protein drug as bio-reactor, human interferon-alpha (IFN-α) (the Maeda et al. that utilized first the high efficient expression of silkworm biological reactor from 1985, Nature, 1985, 315:592 – 594) since, more than 30 kind of recombinant protein comprises human growth factor, the people 2, the 6-sialytransferase, the functional protein medicines such as people's granulocyte-macrophage colony-stimulating factor and people's antialbumin immunoglobulin G 1 have all successfully obtained expression (Kadono-Okuda et al. in silkworm biological reactor, Biochem Biophys Res Commun, 1995, 213:389 – 396, Ogata et al., BMC Biotechnol, 2009b, 9:54, Chen et al., J Biotechnol, 2006,123:236 – 247, Park et al., J Biotechnol, 2009,139:108 – 114), the huge commercial promise based on silkworm biological reactor, a lot of countries all strengthen dynamics of investment silkworm biological reactor are researched and developed in the world.

At silkworm expression in vivo foreign protein, must preparation there is infective recombinant virus, need to be through too much bye spot screening and prepare recombinant virus by traditional method, its technological operation is loaded down with trivial details, only identify with the purification of Recombinant virus particle and just need to expend the time of a wheat harvesting period, also easily obtain the false positive recombinant virus.From (the Luckow et al. such as Luckow in 1993, J Virol, 1993, 67:4566-4579) built a kind of can in Bacillus coli cells, modify the transformation silkworm shuttle expression carrier (Bm-Bacmid) after, can be transformed and be transformed in the intestinal bacteria DH10B cell that contains Bm-Bacmid donor plasmid (pFastBacI), under the effect of locus specificity transposase, DNA fragmentation on donor plasmid between Tn7R-Tn7L just can the specificity swivel base to Bm-Bacmid, and then will identify the BmN cell that correct restructuring Bm – Bacmid transfection is cultivated, collect the culture supernatant of transfection after 5 days and just can obtain restructuring budded virus particle, thus, the structure flow process of recombinant virus has obtained very large simplification.Transfection supernatant infected silkworm after collecting or continuation are infected to the BmN cell of cultivating, metainfective silkworm or BmN total protein of cell are carried out to immunoblotting (Western blot) analysis, just can whether express and be detected target protein, and then shorten target gene and expressed the needed time in BEVS.

Yet, the genome of Bm-Bacmid is larger, that the form of hanging down copy is copied in intestinal bacteria DH10B cell, secondly, during liposome-mediated restructuring Bm-Bacmid transfection BmN cell, efficiency is lower again, simultaneously, research show to also have some albumen with special physico-chemical character in BEVS often difficult express or expression amount on the low side, therefore, whether having produced the recombinant virus particle in the cell to the restructuring Bm-Bacmid transfection that builds, to carry out Rapid identification be the key factor that foreign gene is expressed smoothly.In the past in the eukaryotic expression process, can't detect the target protein of expression by Western blot, can make us suspect design and the flow process thereof of whole experiment, especially after can querying restructuring Bm-Bacmid transfection BmN cell, produced in cells and supernatant and there is infective restructuring budded virus particle? can in BEVS, express fast, be also a bottleneck that restricts at present target protein function and structural research because this not only is related to target gene simultaneously.And only by the variation on cellular form, unavoidably transfection success or not meeting is produced to erroneous judgement, therefore be necessary to create one and can effectively follow the tracks of the visual signals that restructuring budded virus particle produces and spreads, so just can avoid repeatedly by RT-PCR and Western blot, goal gene being transcribed and whether being expressed and verified, accomplish the time saving and energy saving reagent of saving again.

Summary of the invention

Technical problem to be solved by this invention is: set up a kind of foreign gene of following the tracks of and whether enter the identification of means of being expressed in Silkworm, Bombyx mori.Main technological route is by donor plasmid pFastBacI is transformed, build the restructuring donor plasmid that exogenous dna fragment inserts that can be used under egfp expression cassette and the control of polyhedron promotor, the mode of cutting connection by enzyme is connected to polyhedron promotor downstream by the ns1 gene, this recombinant plasmid built is under the effect of transposase, swivel base is in Bm-Bacmid specifically, Bm-Bacmid transfection BmN cell by liposome Cellfectin mediation restructuring, utilize the situation that in the BmN cell after the green fluorescence signal can be judged rapidly transfection, the recombinant virus particle produces, by the transfection supernatant after collecting to 4 age silkworm carry out subcutaneous injection, the silkworm biological reactor that creates visual tracking is expressed NS1 albumen on a large scale.

Problem to be solved by this invention is achieved through the following technical solutions:

A kind of silkworm biological reactor of visual tracking is expressed the method for NS1 albumen fast, by structure, carries egfp expression cassette and ns1 expression casette

donor plasmid, this donor plasmid transforms intestinal bacteria DH10B competent cell, but the egfp expression cassette on the donor plasmid sequence between Tn7R-Tn7L and ns1 expression casette specificity swivel base are to the Bm-Bacmid carrier, the integrated recombinant eukaryon expression vector of construction expression egfp and ns1 gene, by the restructuring budded virus particle BV subcutaneous injection silkworm produced after its transfection insect cell, situation by visual green fluorescence real time monitoring signals Silkworm, Bombyx mori inner virus propagation, the silkworm biological reactor that utilizes that creates a kind of novelty is expressed the method for NS1 albumen on a large scale, for function and the structural research that further discloses NS1 albumen provides basis.

Described egfp expression casette and ns1 expression casette are arranged in series.

Described

donor plasmid builds through the following steps: (1) take silkworm baculovirus BmNPV genome is template, increase and obtain the DNA fragmentation of a 560bp by IE1-F/IE1-R, amplified production is carried out to SnaB I and BamH I double digestion, recovery product after enzyme is cut is connected with the plasmid pFastHTB through same double digestion, produces recombinant plasmid pFastHTB-P _ie1; (2) with EGFP-F and EGFP-R, from the pMD18T-egfp plasmid, amplification obtains the egfp full length gene of 720bp, and this amplified production is carried out to BamH I/Kpn I double digestion, the pFastHTB-P of the DNA fragmentation after enzyme is cut back to close and the same double digestion of warp _ie1connected, obtained recombinant plasmid pFastHTB-P _ie1-egfp; (3) by IE1-EGFP-SV40-F and IE1-EGFP-SV40-R, from pFastHTB-P _ie1-egfp amplification obtains the egfp expression cassette of 1544bp: P _ie1-egfp-sv40, to P _ie1-egfp-sv40 carries out EcoR I/SnaB I double digestion, and the recovery product after enzyme is cut is connected with the pFastBacI through same double digestion, obtains

(4) take Pph-F and Pph-R as primer, the P of specific amplification 232bp from the pFastHTB plasmid _polh-6 * His-TEV sequence, carry out Spe I and Xba I carries out double digestion to amplified production, by enzyme cut back to close product with through same double digestion

connected, obtained (5) increase from the pMD18T-ns1 plasmid and obtain the ns1 gene of 951bp by ns1-F and ns1-R, the ns1 gene that amplification is obtained carries out Nhe I (with Xba I isocaudarner each other) and Xho I double digestion, to the DNA fragmentation after reclaiming with through Xba I and Xho I enzyme, cut

connected, obtained there is equally a tailing signal sv40 sequence in ns1 gene downstream due to this plasmid, therefore this plasmid called after

The invention also discloses the preparation method of the restructuring budded virus particle BV of integrative gene expression egfp and ns1 gene, utilize improved donor plasmid method by swivel base by egfp expression casette and ns1 expression cassette simultaneously swivel base to molecular vehicle Bm-Bacmid, build the integrated restructuring rod granule of integrative gene expression egfp and ns1, but by the BmN cell after the green fluorescence signal Rapid identification transfection of expressing and the situation that produces recombinant virus particle BV in the Silkworm, Bombyx mori after subcutaneous injection.

The present invention utilizes the visual of green fluorescent protein, create a kind of silkworm biological reactor of following the tracks of exogenous gene expression, for this reason, the utmost point early gene ie1 promotor that the present invention has built the BmNPV source control lower EGFP expression cassette and polyhedron promotor control lower can be for the two-cistron expression vector (see figure 1) of foreign gene insertion, expression cassette under by swivel base, these two promotors being controlled is inserted in Bm-Bacmid, and then utilize the green fluorescence visual signals to judge fast the transfection situation of Bm-Bacmid in the silkworm cells BmN, and the propagation situation after recombinant virus particle infected silkworm, express on a large scale two minutes densovirus (BmBDV) Nonstructural Proteins (NS1) of silkworm by the silkworm reactor built, and then the Expression of phosphorylated pattern of announcement NS1 albumen and the mutual relationship between functional mechanism, also for utilizing silkworm biological reactor efficient, express on a large scale other foreign gene and establish solid scientific basic.

The accompanying drawing explanation

Fig. 1. recombinant plasmid

the structure schematic flow sheet.

Fig. 2. recombinant plasmid evaluation.

1: by IE1-EGFP-SV40-F/IE1-EGFP-SV40-R primer pair amplification egfp expression casette, extension increasing sequence length is 1544bp in theory, and amplification is consistent with theoretical expectation; 2: by Pph-F/IE1-EGFP-SV40-R primer pair amplification ns1 expression casette, extension increasing sequence is 1426bp in theory, and amplification is consistent with theoretical expectation; 3: by the ns1-F/ns1-R ns1 that increases, theoretical sequence length is 967bp, amplification with theoretical estimate consistent.

In above-mentioned PCR reaction, DNA profiling used is this recombinant plasmid

Fig. 3. the restructuring rod granule evaluation.

1: by the M13-F/M13-R primer pair not Bm-Bacmid of swivel base that increases, extension increasing sequence is the 280bp left and right in theory, and amplification is consistent with theoretical expectation; 2: by the restructuring Bm-Bacmid after M13-F/M13-R primer pair amplification swivel base, theoretical extension increasing sequence is the 4850bp left and right, and amplification is consistent with theoretical expectation; 3: the ns1 that increases from restructuring Bm-Bacmid by ns1-F/ns1-R, theoretical sequence length is 967bp, amplification is consistent with theoretical expectation; 4: by the restructuring Bm-Bacmid after M13-F/ns1-R primer pair amplification swivel base, theoretical sequence length is the 4600bp left and right, and amplification is consistent with theoretical expectation; 5: by the restructuring Bm-Bacmid after ns1-F/M13-R primer pair amplification swivel base, the theoretical sequence of amplification is 1570bp, and amplification is consistent with theoretical expectation.

In above-mentioned PCR reaction, 1 swimming lane DNA profiling used is Bm-Bacmid, and in 2,3,4 swimming lanes, DNA profiling used is this restructuring rod granule

Fig. 4. restructuring

fluorescence microscopy after transfection BmN cell is observed.A: by the recombinant shuttle plasmid built

transfectional cell, the cell after different time points is to transfection carries out the fluorescence microscopy observation; B: will virus particle solution infects cultured cells, at different time points, metainfective cell is carried out to the fluorescence microscopy observation.

Fig. 5. the fluorescence microscopy observation after recombinant virus particle supernatant subcutaneous injection silkworm and the immunoblotting assay of NS1 albumen.A: the silkworm body surface fluorescence microscopy of subcutaneous injection recombinant virus particle is observed; B: the silkworm blood total protein that has infected recombinant virus is carried out to Western blot analysis.

Embodiment

Experiment material SnaBI, BamH I, Kpn I, EcoR I, Spe I, Xba I, Nhe I, Xho I, T ₄dNA ligase, Taq enzyme and pMD18-T carrier be purchased from precious biotechnology (Dalian) company limited, and the synthetic and sequencing of primer is completed by Shanghai Sheng Gong bio-engineering corporation, shuttle vectors Bm-Bacmid, plasmid pFastHTB, pFastBac I and

(Cat.no.10362-100) purchased from Invitrogen company, plasmid extraction test kit and cut glue and reclaim test kit purchased from Omega company, pMD18T-egfp and pMD18T-ns1 plasmid, coli strain DH5 α and DH10B bacterial strain are preserved for this laboratory, kantlex, gentamicin and tsiklomitsin are purchased from Sigma company, and other reagent is domestic analytical pure.

Embodiment 1. recombinant plasmids

structure

For the promotor of utmost point early gene ie1 in amplification baculovirus BmNPV genome, by two Auele Specific Primer IE1-F-:5'-of Primer Premier5.0 software design tACGTAgTAAGTTATTAATAAAATGCACTGACACG-3'(SnaBI) (SEQ ID NO.1) and IE1-R:5'- gGATCCaTCGTGTCGCCGCCA-3'(BamH I) (SEQ ID NO.2), the DNA fragmentation of 560bp increases from the BmNPV genome by PCR, DNA fragmentation after amplification is carried out respectively to SnaBI and BamH I single endonuclease digestion, target DNA after enzyme is cut is cut glue purification, and the DNA clone after purifying is produced to recombinant plasmid pFastHTB-P to the pFastHTB carrier _ie1; For amplification egfp gene order total length, by two Auele Specific Primer EGFP-F:5'-of Primer Premier5.0 software design gGATCCaTGGTGAGCAAGGGC-3'(BamHI) (SEQ ID NO.3) and EGFP-R:5'- gGTACCtTACTTGTACAGCTCGTCCATG-3'(KpnI) (SEQ ID NO.4), the egfp full length gene sequence that increases from pMD18T-egfp by PCR and obtain 720bp, the target DNA fragment that amplification is obtained carries out BamH I/Kpn I double digestion, product after enzyme is cut is cut glue and is reclaimed, and the product after reclaiming and carrier pFastHTB-P _ie1connected, result produces recombinant plasmid pFastHTB-P _ie1-egfp.

For amplification comprises the egfp expression casette that gathers tail signal SV40 sequence, by two Auele Specific Primer IE1-EGFP-SV40-F:5'-GC of Primer Premier5.0 software design gAATTCgTAGGTTATTGATAAAATGAAC-3'(EcoR I) (SEQ ID NO.5) and IE1-EGFP-SV40-R:5'-GC tACGTAgATCCAGACATGATAAGATACATTG-3'(SnaBI) (SEQ ID NO.6), by PCR from recombinant plasmid pFastHTB-P _ie1the whole expression cassette of amplification egfp: P in-egfp _ie1-egfp-sv40, by the P of amplification _ie1-egfp-sv40 is connected to plasmid pFastBacI, to the recombinant plasmid called after produced

in addition, by two Auele Specific Primer Pph-F:5'-GC of Primer Premier5.0 software design aCTAGTaTCATGGAGATAATTAAAATGATAA-3'(Spe I) (SEQ ID NO.7) and Pph-R:5'-TA tCTAGAgCCCTGAAAATACAGGTTTTC-3'(Xba I) (SEQ ID NO.8), take the pFastHTB plasmid as template, by the PCR P that increases specifically _polh-6 * His-TEV sequence (sequence label and the TEV enzyme that include polyhedron promoter sequence, 6 Histidines in this sequence are cut sequence).P after enzyme is cut back to close _polh-6 * His-TEVDNA fragment and carrier

connected, to the recombinant plasmid called after produced

this recombinant plasmid built is checked order, and result shows in full accord with theoretic DNA sequence dna, polyhedron promotor P _polhdownstream has the polyclone restriction enzyme site that can insert for exogenous dna fragment, and polyadenylic acid tailing signal sv40 sequence.

In above-mentioned PCR reaction: required baculovirus BmNPV genome and egfp gene template all are stored in this laboratory; PFastBacI, pFastHTB carrier are purchased from Invitrogen company.

Embodiment 2. recombinant plasmids

structure

For disclosing the relation between silkworm densovirus NS1 protein phosphorylation modification in two minutes and its function, utilize silkworm biological reactor to express on a large scale NS1 albumen.By two Auele Specific Primer ns1-F:5'-AT of Primer Premier5.0 software design gCTAGCaTGGAATCGAAGTCAAATTT-3'(Nhe I) (SEQ ID NO.9) and ns1-R:5'-TA cTCGAGcTACCCATAATATTTATTATATACG-3'(xho I) (SEQ ID NO.10), take the BmBDV viral genome as template, by PCR amplification ns1 gene from the pMD18T-ns1 plasmid specifically.Target DNA fragment to amplification carries out Nhe I/xho I double digestion, the enzyme after reclaiming is cut to the plasmid recombinant plasmid of product and above-mentioned structure

connected, produced recombinant plasmid, carry out the PCR evaluation by different primer pairs, and result as shown in Figure 2, shows successfully to have produced the restructuring donor plasmid, can be used for next step swivel base.

Embodiment 3. restructuring rod granules

evaluation

Get the recombinant plasmid that 5ng builds

add in intestinal bacteria DH10B competent cell, mix gently and be placed on 30min on ice, 42 ℃ of thermal shock 45sec place 5min on ice, add the SOC substratum of 900 μ l, and 37 ℃ of thermal agitations (225rpm) 4h gets 100 μ l suspensions and coats containing three anti-K ⁺t ⁺g ⁺(50 μ g/ml kantlex, 10 μ g/ml tsiklomitsins, 7 μ g/ml gentamicins) and on the LB flat board of 40 μ g/ml IPTG and 20mg/ml X-gal, cultivate 36～48h for 37 ℃, choosing the single bacterium colony of typical larger white is cultivated, Bacmid by different primer pairs to extracting carries out taking turns the PCR evaluation more, and result as shown in Figure 3.

In above-mentioned reaction: kantlex, tsiklomitsin and gentamicin are purchased from Sigma company, and IPTG and X-gal are purchased from Shanghai Chao Rui biotech firm.

Embodiment 4. restructuring

fluorescence microscopy after transfection BmN cell is observed

Identify through PCR that correct recombinant clone is seeded in and contain three kinds of microbiotic K above-mentioned ⁺g ⁺t ⁺in the LB liquid nutrient medium of (50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins), cultivate 48 hours under 37 ℃, the condition of 225～320rpm after; The bacterium liquid that 1.5ml is cultivated is centrifugal, gets solution I (the 15mM Tris-HCl (pH8.0) of 0.3ml; 10mM EDTA, 100 μ g/ml RNase A); Thalline after centrifugal is carried out resuspended, add subsequently solution II (the 0.2M NaOH of the fresh configuration of 0.3ml; 1%SDS), put upside down gently up and down three times, the solution in pipe is mixed, at room temperature place 5min, solution becomes after clarification at leisure; Adding lentamente the solution III (3M KAc (pH5.5)) of 0.3ml, put upside down and mix gently up and down, obvious white precipitate deposits yields is now arranged, is the mixture of genome of E.coli DNA and protein; Be inserted into standing 10min in ice; The centrifugal 10min of 12,000rpm, move to the supernatant solution after centrifugal in another EP pipe, add the Virahol of equivalent toward this EP pipe, put upside down and mix gently up and down, after ice bath 5～10min, by the centrifugal 15min of the rotating speed of 12,000rpm, make DNA be deposited in EP pipe bottom; Supernatant solution in pipe is abandoned, add the DNA washing of precipitate twice, 12 of the ethanol of 0.5ml70% to the pipe bottom, the centrifugal 1min of 000rpm; Abandon supernatant, residual ethanol is inhaled and abandoned, place in aseptic super clean bench and carry out drying at room temperature 10min, last, add the aseptic tri-distilled water of 30 μ l that the DNA precipitation is dissolved.

At liposome (Cat.no.10362-100) under mediation, restructuring Bm-Bacmid transfection BmN cell by above-mentioned extracting, cell by fluorescent microscope after to transfection carries out Fluirescence observation, result is as shown in Figure 4 A: after transfection 24h, can be observed scattered fluorescent signal in the BmN cell, can be observed subsequently increasing fluorescent signal; After cell transfecting 96h, can be observed in nearly 50% BmN cell and all be distributed with the green fluorescence signal; For further confirming whether the virus particle produced has infectivity, cells and supernatant after the collection transfection, itself and BmN cell are hatched, after 1h, abandon supernatant, add perfect medium and continue to cultivate, by fluorescence microscopy, carry out Continuous Observation, result is as shown in Figure 4 B: after infecting 24h, in 30% cell, fluorescence distribution is arranged; After infecting 48h, in 80% cell, can observe green fluorescence; After infecting 72h, almost in each cell, can observe the green fluorescence signal, budded virus particle in transfection supernatant cells infected again is described, the egfp expression box of introducing on virus expression carrier can not affect the infection ability of virus particle; Simultaneously, the situation after these visual green fluorescence signals contribute to us to judge rapidly restructuring Bm-Bacmid transfection bombyx mori cell, and the infection conditions after virus particle solution subcutaneous injection silkworm in four ages.

Fluorescence microscopy observation after embodiment 5. recombinant virus particle supernatant subcutaneous injection silkworms and the immunoblotting assay of NS1 albumen

Collect the cells and supernatant solution after above-mentioned transfection, by microsyringe to every 4 age silkworm (306 strains, the susceptible kind) cells and supernatant after subcutaneous injection 5 μ l transfections, 100 susceptible strain silkworms have altogether been injected, feed the mulberry leaf of raising fresh harvesting to them every day, under the condition of 27 ℃ of room temperatures, it is raised, after raising the 4th day or the 5th day, by body formula fluorescent microscope, the silkworm infected after virus is carried out to the observation of green fluorescence signal, found that as shown in Figure 5A: the whole body surface of silkworm can both be observed the green fluorescence signal, illustrate that the virus particle that is integrated with goal gene has entered in Silkworm, Bombyx mori, and the institute that spreads all over silkworm by subinfection more in a organized way, collect the blood of virus infection silkworm after 7 days, be placed in liquid nitrogen and preserved, blood total protein to 4 after collecting different silkworms sources carries out Western blot analysis, the fusion that monoclonal antibody by anti-6 * His label is in silkworm blood to expression has the NS1 fusion rotein of 6 * His label to be identified, result shows: the specific protein leukorrhea (as shown in Figure 5 B) that can hybridize to a big or small 36kDa in the silkworm blood of these 4 parts of different sourcess.

In sum, utilize a kind of traceable silkworm biological reactor expression system of visual green fluorescence signal creation, express two minutes densovirus NS1 albumen of silkworm, for the phosphorylation modification pattern of identifying NS1 albumen and the mechanism of action that discloses NS1 albumen provide basis; Simultaneously, the visual tracking silkworm biological reactor of this of establishment provides a kind of alternative expression platform for extensive other albumen with critical function of expressing, for the structure and function research of functional protein is given security.

Claims (2)

1. the silkworm biological reactor of a visual tracking is expressed the method for NS1 albumen fast, it is characterized in that, builds and to carry egfp expression cassette and ns1 expression casette

donor plasmid, transform intestinal bacteria DH10B competent cell with this donor plasmid, but the egfp expression cassette on the donor plasmid sequence between Tn7R-Tn7L and ns1 expression casette specificity swivel base are to the Bm-Bacmid carrier, the integrated recombinant eukaryon expression vector of construction expression egfp and ns1 gene, by the restructuring blastogenesis type BV virus particle infected silkworm produced after its transfection insect cell, by the situation of visual green fluorescence real time monitoring signals Silkworm, Bombyx mori inner virus propagation.

2. method according to claim 1, is characterized in that described

donor plasmid builds through the following steps: (1) take silkworm baculovirus BmNPV genome is template, increase and obtain the DNA fragmentation of a 560bp by IE1-F/IE1-R, amplified production is carried out to SnaB I and BamH I double digestion, recovery product after enzyme is cut is connected with the plasmid pFastHTB through same double digestion, produces recombinant plasmid pFastHTB-P _ie1; (2) with EGFP-F and EGFP-R, from the pMD18T-egfp plasmid, amplification obtains the egfp full length gene of 720bp, and this amplified production is carried out to BamH I/ Kpn I double digestion, the pFastHTB-P of the DNA fragmentation after enzyme is cut back to close and the same double digestion of warp _ie1connected, obtained recombinant plasmid pFastHTB-P _ie1-egfp; (3) by IE1-EGFP-SV40-F and IE1-EGFP-SV40-R, from pFastHTB-P _ie1-egfp amplification obtains the egfp expression cassette of 1544bp: P _ie1-egfp-sv40, to P _ie1-egfp-sv40 carries out EcoR I/SnaB I double digestion, and the recovery product after enzyme is cut is connected with the pFastBacI through same double digestion, obtains

(4) take Pph-F and Pph-R as primer, the P of specific amplification 232bp from the pFastHTB plasmid _polh-6 * His-TEV sequence, carry out Spe I and Xba I carries out double digestion to amplified production, by enzyme cut back to close product with through same double digestion

connected, obtained

(5) increase from the pMD18T-ns1 plasmid and obtain the ns1 gene of 951bp by ns1-F and ns1-R, the ns1 gene that amplification is obtained carries out Nhe I (with Xba I isocaudarner each other) and Xho I double digestion, to the DNA fragmentation after reclaiming with through Xba I and Xho I enzyme, cut

connected, obtained

there is equally a tailing signal sv40 sequence in ns1 gene downstream due to this plasmid, therefore this plasmid called after

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