CN102311969B - Method and its special expression vector for realizing functional protein interference in Caenorhadits elegans by using prokaryotic expression system of escherichia coli - Google Patents
Method and its special expression vector for realizing functional protein interference in Caenorhadits elegans by using prokaryotic expression system of escherichia coli Download PDFInfo
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Abstract
The invention discloses a method and its special expression vector for realizing functional protein interference in Caenorhadits elegans by using prokaryotic expression system of escherichia coli. The vector comprises prokaryotic expression systems of one or more same of different protein transduction peptide. The method comprises the following steps: inserting respective coding gene of interactional target protein to be identified in the special expression vector any one of claim 1-3 to obtain the prokaryotic expression vector, converting the prokaryotic expression vector and inducing and expressing in escherichia coli to obtain recombinant escherichia coli, using the recombinant escherichia coli to feed normal Caenorhadits elegans, and according to the detection results of the biological and biochemical indicators of Caenorhadits elegans, estimating the capacity and strength of the occurrence of interference of the interactional protein. The invention has a wide application prospect, is expected to be an important technique of screening recombinant protein drugs, and is one of the important techniques of studying functions of protein.
Description
Technical field
The invention belongs to the method that albumen is interfered in the biological technology products Application Areas, particularly relate to a kind of escherichia coli prokaryotic expression system that utilizes and realize functional protein is interfered in the beautiful nematode body method and dedicated expression vector therefor thereof.
Background technology
Protein is the important agent of body vital movement, and its disappearance or overexpression all will cause serious defective illness, studies proteinic function by direct approach and will provide important means for the treatment of its relative disease.The model animals that is usually used in studying protein function at present mainly contains beautiful nematode, mouse and eukaryotic cells etc.Wherein, beautiful nematode is clear and definite because of its cell quantity, genetic background is clear, genome is little and advantage such as easy handling has been widely used in a plurality of research fields (Greer et al.Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C.elegans.Nature.2010 Jul 15,466 (7304): 383-7) such as genetics, developmental biology, neurobiology and molecular biology.Become the focus of present research based on functional proteic effect in beautiful its body of nematode platform scale selection.Though interfering, microinjection on the tradition gene level and RNA can arrive the proteic effect of function in the beautiful nematode body of research, but because there are shortcomings such as complex operation, cost height, cycle length and destructiveness are big in it, the phenomenon that functional protein is not expressed can appear more even, make the screening of interior proteic research of critical function of beautiful nematode body and corresponding phenotype be subjected to very big restriction (Rieckher et al.Transgenesis in Caenorhabditis elegans.Methods Mol Biol.2009,561:21-39; Shyu et al.Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis.Nat Protoc.2008,3 (4): 588-96.).Generally, a lot of protein are to be so-called protein by having an effect with its interacting proteins---and protein interaction (protein-protein interaction, thus PPI) realize its specific function.Based on this, thereby introducing a kind of can the interaction with existing protein generation in the beautiful nematode body in beautiful nematode body causes the unusual function that forms of original protein regulation network to be interfered, can in beautiful nematode body, cause the unusual protein that forms the function interference of original protein regulation network thereby perhaps directly in beautiful nematode body, introduce two or more by protein interaction, might be for disclosing the interaction between the protein and determining that important proteic function is significant.This thinking is not seen in as yet in existing document.
Summary of the invention
The purpose of this invention is to provide a kind of escherichia coli prokaryotic expression system that utilizes and realize the dedicated expression vector therefor that functional protein is interfered in the beautiful nematode body.
Dedicated expression vector therefor provided by the present invention is the prokaryotic expression carrier that contains one or more identical or different protein transduction peptides.
Described protein transduction peptide can be: TAT, Antp, VP22, Poly (Arg) and/or Poly (Lys) etc. are preferably TAT.
The carrier that sets out that is used to make up described prokaryotic expression carrier can be the prokaryotic expression carrier of any one foreign gene, as pET28, pBV220, pET30, pCRT7/CT-TOPO, pET101/D-TOPO or pQE30 etc., is preferably pET28.
Second purpose of the present invention provides a kind of escherichia coli prokaryotic expression system that utilizes and realizes the method that functional protein is interfered in the beautiful nematode body.
The escherichia coli prokaryotic expression system that utilizes provided by the present invention realizes the method that functional protein is interfered in the beautiful nematode body, be with to be identified have interactional target protein separately encoding gene insert in the aforementioned dedicated expression vector therefor, the prokaryotic expression carrier that obtains is transformed into intestinal bacteria and abduction delivering, the recombination bacillus coli that obtains is fed and raised normal strain is beautiful nematode, assesses ability and the intensity that interaction protein interferes according to beautiful nematode biology and biochemical indicator detected result.
Specifically can may further comprise the steps:
1) determines that at least one pair of has interactional functional protein (comprising bait protein and prey albumen) and also clones its encoding gene respectively for target protein;
2) the target protein encoding gene is inserted respectively in the above-mentioned dedicated expression vector therefor, obtain containing the prokaryotic expression carrier that protein transduction peptide-coding region and target protein encoding gene merge mutually;
3) with step 2) prokaryotic expression carrier that makes up is transformed into the escherichia coli prokaryotic expression system respectively;
4) abduction delivering of prokaryotic expression carrier and protein expression level detect;
5) with the recombination bacillus coli of each target protein of vivoexpression separately or mix and feed that to raise normal strain be beautiful nematode;
6) beautiful nematode biology and biochemical indicator detect and assess ability and the intensity that interferes between the target protein.
Target protein can be one functional protein in the described step 1), also can be two or more interactional functional proteins that have.This albumen can be beautiful nematode oneself protein, also can be to have potential interactional albumen in other species.
Utilize in the method that the escherichia coli prokaryotic expression system realizes that functional protein in the beautiful nematode body interferes above-mentioned, the host bacterium of escherichia coli prokaryotic expression system can be in the described step 3): BL21, DH5 α, HB101, JM109, OP50 or the strange coccus of radiation hardness etc.
Above-mentioned recombinant expression vector and reorganization bacterium all can make up according to ordinary method.
Can adopt the chemical reagent induction exogenous gene to express in the escherichia coli prokaryotic expression system in the described step 4), wherein, chemical reagent abduction delivering condition can be: adopt 500mL shake flask fermentation (150mL/ bottle); Inductor IPTG abduction delivering (final concentration 1mM); The OD of abduction delivering
600=0.6-1.0; 6-8 hour abduction delivering time.
Also can adopt temperature-induced foreign gene to express in the escherichia coli prokaryotic expression system in the described step 4), wherein, the temperature-induced expression condition can be: adopt 500mL to shake bottle 30 ℃ of fermentations (150mL/ bottle); The OD of abduction delivering
600=0.4-0.6; 42 ℃-44 ℃ of abduction delivering temperature; 6-8 hour abduction delivering time.
In addition, the protein expression level in the described step 4) detects and can adopt SDS-PAGE and/or immunoblotting.
Feed the intestinal bacteria raise beautiful nematode in the described step 5) and be identify the bacterial strain of expressing target protein in the step 4) and be applied to the NGM plate in advance or the LB plate in; Its feed the mode of raising can for feed separately raise or by a certain percentage as 1: 5,1: 2,1: 1,2: 1,5: 1 mode mix to feed and raise.
Beautiful nematode biology and biochemical indicator detect in the described step 6), and Biological indicators comprise specifically that morphological structure changes and cell levels variation etc., as whether occurring that build diminishes or obesity, the cavity phenomenon whether occurring, motor behavior imbalance etc. whether occurs; Biochemical indicator comprises that specifically target protein or its regulatory factor expression level detect, as protein, and DNA and RNA etc.Its expression level detects can be immunoblotting, gel shift experiment and polymerase chain reaction etc.
The research of the conjugated protein transduction of the present invention territory family, and utilize the main food source intestinal bacteria of beautiful nematode to be intermediary, provide a kind of escherichia coli prokaryotic expression system that utilizes to realize functional protein is interfered in the beautiful nematode body method and dedicated expression vector therefor thereof.Albumen of the present invention interfere (protein interfere) be meant by feed for beautiful nematode to raise can express can with the microorganism such as the intestinal bacteria of the interactional functional protein of the intravital target protein of beautiful nematode, thereby can cause in beautiful nematode body, occurring this functional protein and be interfered generation phenotype unusual technology in effect back by interaction, also comprise simultaneously directly two or more can be expressed can interactional functional protein microorganism such as intestinal bacteria directly feed and raise beautiful nematode, can cause beautiful nematode the technology of particular phenotype to occur by the interaction that in beautiful nematode body, takes place between two kinds of albumen, promptly react based on beautiful nematode " double cross " (C.elegans two-hybridization, C2H).The present invention not only can overcome conventional defective based on the beautiful nematode research of model animals functional protein method, and the functional protein that can efficiently express outward by microorganism such as the direct transductant of intestinal bacteria, the phenomenon of having avoided functioning gene not express, and then saved cost of examination of exceptional function albumen and broad scale research etc.Simultaneously, do not see the spanning transduction membrane effect that utilizes the protein transduction peptide and rely on the research report that the escherichia coli prokaryotic expression system realizes that functional protein is interfered in the beautiful nematode body up to now.The escherichia coli prokaryotic expression system that utilizes provided by the invention interferes the proteic method of function in the beautiful nematode body from the protein angle, prospect is widely used, be expected to become the important technical of recombinant protein class drug screening, also might become one of important technology of protein function research simultaneously.
The present invention utilizes beautiful nematode platform and prokaryotic expression system to propose the effect that albumen is interfered first, for research and vector selection based on protein function provide the important techniques platform, has important originality and novelty.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1: the structure synoptic diagram of prokaryotic expression carrier pET28b-Tat-EGFP and pET28b-EGFP.
Fig. 2: SDS-PAGE and Western blot detect Tat-EGFP and the proteic expression of EGFP.
Fig. 3: fluorescence microscope is expressed the fluorescent signal of the somatic cells of EGFP.
Fig. 4: fluorescence microscope TAT-EGFP and EGFP albumen fluorescent signal changes in distribution in beautiful nematode body.
Fig. 5: the bacterium liquid PCR qualification result that prokaryotic expression carrier pET28-Tat-Hif-1 and pET28-Hif-1 make up.
Fig. 6: the bacterium liquid PCR qualification result that prokaryotic expression carrier pET28-Tat-VHL-1 and pET28-VHL-1 make up.
Fig. 7: SDS-PAGE detects Tat-Hif-1 and the proteic expression of Hif-1 changes, and arrow is depicted as target protein Tat-Hif-1 and Hif-1 position among the figure.
Fig. 8: SDS-PAGE detects Tat-VHL-1 and the proteic expression of VHL-1 changes, and arrow is depicted as target protein Tat-VHL-1 and VHL-1 position among the figure.
Embodiment
Embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Method therefor is ordinary method if no special instructions among the following embodiment.
Can adopt any known method to make up utilizes the escherichia coli prokaryotic expression system to realize the dedicated expression vector therefor that functional protein is interfered in the beautiful nematode body, this dedicated expression vector therefor is the prokaryotic expression carrier that contains one or more identical or different protein transduction peptides, described protein transduction peptide is: TAT, Antp, VP22, Poly (Arg) and/or Poly (Lys) etc., the carrier that sets out that is used to make up described prokaryotic expression carrier can be the prokaryotic expression carrier of any one foreign gene, as pET28, pBV220, pET30, pCRT7/CT-TOPO, pET101/D-TOPO or pQE30 etc.Present embodiment is that the recombinant expression vector that vector construction contains TAT that sets out is an example with pET28b, introduced a kind of method that makes up the dedicated expression vector therefor that functional protein is interfered in the beautiful nematode body, the recombinant expression vector that contains other protein transduction peptide also can make up with reference to this method, and concrete construction process is:
At first synthesize and contain the core encoder sequence fragment that restriction enzyme site is the double-stranded tat peptide section (GenBank:ADK70247.1) of NcoI (upstream) and NdeI (downstream) by the method for chemosynthesis, adopt this TAT core fragment of restriction enzyme NcoI and NdeI double digestion and empty carrier pET28b (U.S. Novagen company) then, after reclaiming the purpose fragment respectively, connect the TAT dna fragmentation and the empty carrier fragment of cutting through enzyme through the T4 dna ligase, to connect product again is transformed among the competent escherichia coli cell BL21 (DE3), the screening recon, extract the plasmid DNA of recon and carry out NcoI and the evaluation of NdeI double digestion according to ordinary method, send Ying Jun company directly to check order at last, the prokaryotic expression carrier that conclusive evidence contains the TAT core fragment successfully constructs, with this recombinant vectors called after pET28b-TAT.
Embodiment 2, utilize the escherichia coli prokaryotic expression system to realize that green fluorescent protein is a spanning transduction membrane in the beautiful nematode body in normal strain
1) structure of prokaryotic expression carrier pET28b-TAT-EGFP and pET28b-EGFP and abduction delivering
Be template at first and utilize primer (italicized item is respectively BamHI and XhoI restriction enzyme site: pU_5 '-CGC with plasmid pEGFP-N1 (available from Clontech company)
TGGTGAGCAAGGGCGAG-3 ' (sequence 1 in the sequence table); PD_5 '-CCG
TTGTACAGCTCGTCCAT-3 ' (sequence 2 in the sequence table) carries out pcr amplification and obtains enhanced green fluorescence protein (EGFP, GenBank number: ADQ73885.1) cDNA encoding sequence (717bp), adopt restriction enzyme BamHI/XhoI double digestion goal gene EGFP and plasmid vector pET28b and pET28b-TAT then; Enzyme is cut and is adopted the T4 dna ligase to spend the night in 16 ℃ of connections after product reclaims; Next day, converted product all is transformed into e. coli bl21 (DE3) and adopts digestion with restriction enzyme and directly order-checking evaluation.Enzyme is cut and the qualification result that checks order shows the prokaryotic expression carrier pET28b-TAT-EGFP of the green fluorescent protein that has obtained to carry protein transduction peptide TAT core peptide section and only carry the prokaryotic expression carrier pET28b-EGFP of green fluorescent protein, and the construction process synoptic diagram as shown in Figure 1.
2) subsequently, conversion is had the host bacterium BL21 (DE3) of plasmid vector pET28b-TAT-EGFP and pET28b-EGFP to be applied to contain the LB solid medium of kantlex (final concentration 100 μ g/ml), 37 ℃ of thermostat containers are hatched 10-12 hour clone to be grown.Next day, the picking positive colony is inoculated into 5ml and contains in the LB liquid nutrient medium of kantlex (final concentration 100 μ g/ml), and 37 ℃, 220rpm shake bacterium and spend the night thalline is recovered fully; Bacterium liquid is inoculated in the LB liquid nutrient medium that contains kantlex (final concentration 100 μ g/ml) of the new configuration of 150ml according to 1: 100 ratio then, 37 ℃, 220rpm shook the about 4-5 of bacterium hour, treated OD
600Add inductor isopropyl-(Isopropyl β-D-1-Thiogalactopyranoside during for 0.6-1.0, IPTG), final concentration 1mM), placed 30 ℃, 220rpm shaking table abduction delivering rapidly 6 hours, and in the 2nd hour, collected somatic cells in the 4th hour and the 6th hour, the part somatic cells is treated fluorescence microscope, directly prepares the supernatant protein sample after all the other somatic cells ultrasonications and treats that SDS-PAGE and Western blot detect.SDS-PAGE with Western blot detection method is: A, with after the above-mentioned ultrasonication directly the supernatant protein sample of preparation adopt 15%SDS-PAGE to separate, adopt Xylene Brilliant Cyanine G R-250 dyeing and decolouring then, adopt gel imaging instrument collection image at last; B, subsequently is transferred on the pvdf membrane 5% skimmed milk room temperature sealing 30 minutes with supernatant protein sample described to specifications method after 15%SDS-PAGE separates of above-mentioned preparation; Next adopts EGFP (TBST dilution in 1: 3000) and GAPDH (TBST dilution in 1: 500) monoclonal antibody room temperature to hatch altogether 1.5 hours, and the TBST room temperature is washed film three times (10 minutes/time); (TBST dilution in 1: the 5000) incubated at room that adopts the goat anti-mouse IgG two of horseradish peroxidase-labeled anti-then 30 minutes, the TBST room temperature is washed film three times (10 minutes/time); Adopting the ECL chemiluminescence detection kit to develop at last and scanning, is the empty carrier contrast with pET28b and pET28b-TAT.The result is (mark Protein G APDH in being respectively shown in the arrow among the figure as shown in Figure 2, TAT-EGFP and EGFP protein band position), show to detect and find that the highly-soluble of having realized green fluorescent protein expresses by external evoked expression and SDS-PAGE and immunoblotting.
3) fluorescence microscope of abduction delivering thalline
Adopt 10 μ l transfering loops to be uniformly applied on the clean slide glass somatic cells of above-mentioned abduction delivering, treat to adopt fluorescence microscope somatic cells fluorescent signal behind its natural air drying, gather the fluoroscopic image and the differential interference image of abduction delivering different time points somatic cells respectively, observing TAT-EGFP and the expression of EGFP in Bacillus coli cells by the image folding at last, is the empty carrier contrast with pET28b and pET28b-TAT.Abduction delivering thalline fluorescence microscope result is (expression is carried out the detection of fluorescent signal: 0h (A), 2h (B), 4h (C), 6h (D) at the somatic cells of abduction delivering different time among the different induction time somatic cells of the fluorescence microscope fluorescent signal figure) as shown in Figure 3, along with the increase of abduction delivering time, the green fluorescent protein of expressing in the somatic cells is increasing.
4) normal strain is that feeding of beautiful nematode raised and fluorescence microscope
E. coli bl21 (DE3) somatic cells of abduction delivering TAT-EGFP and EGFP fusion rotein is applied in the LB plate, treating that its room temperature is dried slightly is placed on 20 ℃ of biochemical incubators and cultivates and treated that a large amount of clones grew in 72 hours, to be 20 of beautiful nematodes cultivate and adopt fluorescence microscope TAT-EGFP and EGFP fluorescent signal to distribute in beautiful nematode body in 20 ℃ of biochemical incubators the normal strain of picking, and gathered fluoroscopic image and differential interference image on the 48th hour in feeding to raise, passing through image folding observation TAT-EGFP and EGFP fusion rotein at last in the intravital distribution situation of beautiful nematode, is the empty carrier contrast with pET28b and pET28b-TAT.Fluorescence microscope TAT-EGFP and EGFP albumen fluorescent signal be changes in distribution (arrow is depicted as TAT-EGFP and EGFP fusion rotein at the intravital distributing position of beautiful nematode among the figure) as shown in Figure 4 in beautiful nematode body, raise beautiful nematode and express the Bacillus coli cells of target protein after 48 hours by directly feeding, the TAT-EGFP fluorescent signal obviously is distributed in beautiful nematode intestines parietal cell, the EGFP fluorescent signal then is distributed in beautiful nematode enteric cavity, the empty carrier control group is not seen any fluorescent signal, illustrates that TAT protein transduction peptide can efficiently mediate foreign protein spanning transduction membrane in beautiful nematode body.Simultaneously, by relatively empty carrier control group and the beautiful nematode differential interference image of experimental group do not see that it tangible cellular form occurs and changes, illustrate that thus TAT protein transduction peptide has comparatively safe spanning transduction membrane effect, can mediate the foreign protein spanning transduction membrane and enter beautiful nematode intestines parietal cell, study based on beautiful nematode platform and prokaryotic expression system for follow-up that the proteic interference of function provides technical support in the beautiful nematode body.
Embodiment 3, utilize the escherichia coli prokaryotic expression system to realize that normal strain is the interference of interaction protein Hif-1 and VHL-1 in the beautiful nematode body
1. method
1) part protein-interacting relationship analysis in the beautiful nematode body
The organism interaction protein can deeply excavate acquisition or make up acquisition voluntarily according to existing biological data storehouse based on the information biology means.The present invention is directed to the interior part functional protein of beautiful nematode body analyzes its interaction relationship, find to exist in the online polypide albumen that interacts and verify by experiment that Hif-1/VHL-1 and gpd-2/gpd-3 are arranged by analyzing, so follow-up Hif-1 of choosing of the present invention and VHL-1 carry out the research that albumen is interfered as having interactional target protein.
2) structure of prokaryotic expression carrier pET28-Tat-Hif-1/Hif-1 and pET28-Tat-VHL-1/VHL-1 and abduction delivering
Carrier construction method structure with reference to embodiment 2 carries the cDNA encoding sequence of beautiful nematode Hif-1 and VHL-1 and the prokaryotic expression carrier of Tat transduction peptide sequence, is contrast with the prokaryotic expression carrier that does not contain the peptide of transduceing.Be that beautiful nematode is extracted total RNA and reverse transcription at first at normal strain, with the reverse transcription product be then template and in conjunction with following primer to (italicized item is respectively restriction enzyme site):
PU_BamHI-Hif-1:5 '-CGC
TGGAAGACAATCGGAAAAGAAACA-3 ' (sequence 3 in the sequence table)
PD_XhoI-Hif-1:5 '-CCG
GAGAGCATTGGAAATGGGGAAT-3 ' (sequence 4 in the sequence table)
PU_BamHI-VHL-1:5 '-CGC
TGTCAGACGGTTCGATGGATGA-3 ' (sequence 5 in the sequence table)
PD_XhoI-VHL-1:5 '-CCG
TGCTGAGGTCTCTGGGGTCC-3 ' (sequence 6 in the sequence table) carries out pcr amplification to obtain the cDNA encoding sequence of Hif-1 and VHL-1, then goal gene and carrier enzyme are cut back structure prokaryotic expression carrier pET28-Tat-Hif-1/Hif-1 and pET28-Tat-VHL-1/VHL-1, at last above-mentioned carrier enzyme is cut and directly order-checking conclusive evidence, wherein pET28-Tat-Hif-1 is by 7 expressions of sequence table sequence, pET28-Hif-1 is by 8 expressions of sequence table sequence, pET28-Tat-VHL-1 is by 9 expressions of sequence table sequence, pET28-VHL-1 is by 10 expressions of sequence table sequence, bacterium liquid PCR qualification result as shown in Figure 5 and Figure 6
Subsequently, prokaryotic expression carrier pET28-Tat-Hif-1/Hif-1 that above-mentioned structure is correct and pET28-Tat-VHL-1/VHL-1 are transformed into host bacterium BL21 (DE3) respectively and are applied to the LB solid medium that contains kantlex (final concentration 100 μ g/ml), and 37 ℃ of thermostat containers are hatched 10-12 hour clone to be grown.Next day, the picking positive colony is inoculated into 5ml and contains in the LB liquid nutrient medium of kantlex (final concentration 100 μ g/ml), and 37 ℃, 220rpm shake bacterium and spend the night thalline is recovered fully; Bacterium liquid is inoculated in the LB liquid nutrient medium that contains kantlex (final concentration 100 μ g/ml) of the new configuration of 150ml according to 1: 100 ratio then, 37 ℃, 220rpm shook the about 4-5 of bacterium hour, treated OD
600Add inductor isopropyl-(Isopropyl β-D-1-Thiogalactopyranoside during for 0.6-1.0, IPTG), final concentration 1mM), placed 30 ℃, 220rpm shaking table abduction delivering rapidly 6 hours, and in the 2nd hour, directly prepare total protein after collecting somatic cells and ultrasonication in the 4th hour and the 6th hour, last white protein and protein precipitation sample treat that SDS-PAGE and Western blot detect.SDS-PAGE with Western blot detection method is: A, with after the above-mentioned ultrasonication directly the protein sample of preparation adopt 15%SDS-PAGE to separate, adopt Xylene Brilliant Cyanine G R-250 dyeing and decolouring then, adopt gel imaging instrument collection image at last; B, the supernatant protein sample of above-mentioned preparation described to specifications method after 15% SDS-PAGE separates is transferred on the pvdf membrane 5% skimmed milk room temperature sealing 30 minutes; Next adopts 6 * His (TBST dilution in 1: 3000) monoclonal antibody room temperature to hatch altogether 1.5 hours, and the TBST room temperature is washed film three times (10 minutes/time); (TBST dilution in 1: the 5000) incubated at room that adopts the goat anti-mouse IgG two of horseradish peroxidase-labeled anti-then 30 minutes, the TBST room temperature is washed film three times (10 minutes/time); Adopting the ECL chemiluminescence detection kit to develop at last and scanning, is the empty carrier contrast with pET28b and pET28b-TAT.The SDS-PAGE detected result is shown in Fig. 7 (arrow is depicted as target protein Tat-Hif-1 and Hif-1 position among the figure) and Fig. 8 (arrow is depicted as target protein Tat-VHL-1 and VHL-1 position among the figure), the outer solubility expression of display body has prepared Tat-Hif-1/Hif-1 and Tat-VHL-1/VHL-1 albumen, particularly Tat-Hif-1 and Tat-VHL-1 have established important foundation for follow-up based on the two research of carrying out albumen interference function.
3) normal strain is that feeding of beautiful nematode raised and biological and biochemical indicator detection
A) independent feeding raised the somatic cells that normal strain is beautiful nematode expression Tat-Hif-1 and Hif-1 fusion rotein
E. coli bl21 (DE3) somatic cells of abduction delivering Tat-Hif-1 and Hif-1 fusion rotein is applied in LB or the NGM plate, treating that its room temperature is dried slightly is placed on 20 ℃ of biochemical incubators and cultivates and treated that a large amount of clones grew in 72 hours, and the normal strain of picking is that 20 of beautiful nematodes were cultivated 48 hours in 20 ℃ of biochemical incubators.The beautiful nematode of picking to the sodium sulfite solution (1g/L, 2g/L and 5g/L) of different concns carries out the hypoxia stress test and adds up its mortality ratio then, and then assesses its anti-hypoxemia ability.Detect and the statistical study discovery by system, compare with pET28b with control group pET28b-Tat, feed to raise and express the colibacillary nematode of Tat-Hif-1 and show tangible anti-hypoxemia effect, show that mainly pharyngeal muscle and neuronic swelling degree reduce, mobility strengthens, express the colibacillary nematode of Hif-1 and have only faint anti-hypoxemia effect and feed to raise, show phenotype the hypoxia injury sensitivity.
B) independent feeding raised the somatic cells that normal strain is beautiful nematode expression Tat-VHL-1 and VHL-1 fusion rotein
E. coli bl21 (DE3) somatic cells of abduction delivering Tat-VHL-1 and VHL-1 fusion rotein is applied in LB or the NGM plate, treating that its room temperature is dried slightly is placed on 20 ℃ of biochemical incubators and cultivates and treated that a large amount of clones grew in 72 hours, and the normal strain of picking is that 20 of beautiful nematodes were cultivated 48 hours in 20 ℃ of biochemical incubators.The beautiful nematode of picking to sodium sulfite solution (1g/L, 2g/L and the 5g/L) hypoxia stress of different concns is tested and is added up its mortality ratio then, and then assesses its hypoxemia ability.Detect and the statistical study discovery by system, compare with pET28b with control group pET28b-Tat, feed to raise and express the proteic colibacillary nematode of Tat-VHL-1 and show significantly to the anoxic sensitivity, mortality ratio is higher, express the proteic colibacillary nematode of VHL-1 and show as anoxic insensitive and feed to raise, mortality ratio does not have considerable change, and simultaneously, control group does not also have considerable change.
C) mixing is fed and is raised the somatic cells that normal strain is beautiful nematode expression Tat-Hif-1/Hif-1 and Tat-VHL-1/VHL-1 fusion rotein
E. coli bl21 (DE3) somatic cells of abduction delivering Tat-Hif-1/Hif-1 and Tat-VHL-1/VHL-1 fusion rotein was applied to behind the ratio mixing in LB or the NGM plate according to 1: 1, treating that its room temperature is dried slightly is placed on 20 ℃ of biochemical incubators and cultivates and treated that a large amount of clones grew in 72 hours, and the normal strain of picking is that 20 of beautiful nematodes were cultivated 48 hours in 20 ℃ of biochemical incubators.The beautiful nematode of picking to the sodium sulfite solution (1g/L, 2g/L and 5g/L) of different concns carries out the hypoxia stress damage and adds up its mortality ratio then, and then assesses its anti-anoxia ability.Detect and the statistical study discovery by system, compare with pET28b with control group pET28b-Tat, mix to feed to raise and express Tat-Hif-1 and the proteic colibacillary nematode of Tat-VHL-1 does not see obvious variation, illustrate that the Tat-Hif-1 of high expression level and the Tat-VHL-1 of high expression level are that its function is offset in the albumen interference by interaction; Mix to feed to raise and express Tat-Hif-1 and the anti-hypoxia activity appears in the proteic colibacillary nematode of VHL-1, illustrate that the Tat-Hif-1 of high expression level still can bring into play its function; Mix to feed to raise and express Tat-VHL-1 and the proteic colibacillary nematode of Hif-1 shows as the anoxic sensitivity, illustrate that the Tat-VHL-1 of high expression level still can bring into play its function; Express Hif-1 and the proteic colibacillary nematode of VHL-1 does not see obvious variation and mix to feed to raise, thereby illustrate that the Hif-1 that is low expression and VHL-1 do not form effective protein-interacting and do not bring into play the albumen interference effect.
It is the protein interference that The above results explanation promotes the Tat-Hif-1 of high expression level and Tat-VHL-1 in beautiful nematode body functional interaction to take place by the protein transduction peptide in intestinal bacteria, thereby can illustrate conversely that technology of the present invention is used in the beautiful nematode by feeding method research protein interaction very easily, is protein---the research of protein interaction (PPI) provides a kind of new technology.
Claims (11)
1. one kind is utilized the escherichia coli prokaryotic expression system to realize the method that functional protein is interfered in the beautiful nematode body, be with to be identified have interactional target protein separately encoding gene insert in the dedicated expression vector therefor, the prokaryotic expression carrier that obtains is transformed into intestinal bacteria and abduction delivering, the recombination bacillus coli that obtains is fed and raised normal strain is beautiful nematode, assesses ability and the intensity that interaction protein interferes according to beautiful nematode biology and biochemical indicator detected result; Described dedicated expression vector therefor is the prokaryotic expression carrier that contains one or more identical or different protein transduction peptides, this protein transduction peptide is TAT, Antp, VP22, Poly (Arg) and/or Poly (Lys), and the carrier that sets out of prokaryotic expression carrier is pET28, pBV220, pET30, pCRT7/CT-TOPO, pET101/D-TOPO or pQE30.
2. method according to claim 1 is characterized in that: may further comprise the steps:
1) determines to have interactional target protein and clone its encoding gene respectively;
2) the target protein encoding gene is inserted respectively in the described dedicated expression vector therefor, obtain containing the prokaryotic expression carrier that protein transduction peptide-coding region and target protein encoding gene merge mutually;
3) with step 2) prokaryotic expression carrier that makes up is transformed into the escherichia coli prokaryotic expression system;
4) abduction delivering of prokaryotic expression carrier and protein expression level detect and identify;
5) step 3) is obtained and the recombination bacillus coli of the vivoexpression target protein identified through step 4) separately or mix and feed that to raise normal strain be beautiful nematode;
6) detect beautiful nematode biology and biochemical indicator and assess ability and the intensity that interferes between the target protein.
3. method according to claim 2 is characterized in that: described target protein is two or more interactional functional proteins that have.
4. method according to claim 3 is characterized in that: described target protein is beautiful nematode oneself protein, or has potential interactional albumen in other species.
5. method according to claim 2 is characterized in that: the host bacterium of escherichia coli prokaryotic expression system is in the described step 3): BL21, DH5 α, HB101, JM109 or OP50.
6. according to claim 2 or 3 or 4 or 5 described methods, it is characterized in that: described abduction delivering is expressed in the escherichia coli prokaryotic expression system for adopting the chemical reagent induction exogenous gene, wherein, chemical reagent abduction delivering condition is: final concentration is a 1mM inductor IPTG abduction delivering; The OD of abduction delivering
600=0.6-1.0; 6-8 hour abduction delivering time; Or
Described abduction delivering is expressed in the escherichia coli prokaryotic expression system for adopting temperature-induced foreign gene, and wherein, the temperature-induced expression condition is: the OD of abduction delivering
600=0.4-0.6; 42 ℃-44 ℃ of abduction delivering temperature; 6-8 hour abduction delivering time;
Protein expression level in the described step 4) detects and adopts SDS-PAGE and/or immunoblotting.
7. according to claim 2 or 3 or 4 or 5 described methods, it is characterized in that: feed the intestinal bacteria of raising beautiful nematode in the described step 5) and need be applied in advance in NGM plate or the LB plate; It is 1:5,1:2,1:1,2:1 or 5:1 that the ratio of raising is fed in mixing.
8. method according to claim 6 is characterized in that: feed the intestinal bacteria of raising beautiful nematode in the described step 5) and need be applied in advance in NGM plate or the LB plate; It is 1:5,1:2,1:1,2:1 or 5:1 that the ratio of raising is fed in mixing.
9. method according to claim 2 is characterized in that: biology of beautiful nematode and biochemical indicator detect in the described step 6), and Biological indicators comprise that specifically morphological structure changes and cell levels changes; Biochemical indicator comprises that specifically target protein or its regulatory factor expression level detect; The behavior of beautiful nematode variation detected by quantitative study of behaviour technique means before and after protein was interfered; Obtain ability and the intensity that interaction protein interferes according to the phenotypic difference analysis of beautiful nematode before and after albumen is interfered.
10. method according to claim 9 is characterized in that: described Biological indicators are that spawning rate changes, the adult rate changes, whether occur that build diminishes or fat, whether the cavity phenomenon occurs and the motor behavior imbalance whether occurs pharyngeal; Apparent index is judged by stereomicroscope observation.
11. method according to claim 9 is characterized in that: described biochemical indicator is protein, DNA and RNA, and its expression level detects and is immunoblotting, gel shift experiment and polymerase chain reaction.
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| 体内蛋白转导的研究进展;彭涛等;《中国药科大学学报》;20031231;第24卷(第5期);477-480 * |
| 吴国清等.多肽TAT与核定位信号介导的蛋白质入核递送.《中国生物工程杂志》.2004,(第5期),61页左栏第2段－右栏最后1段. |
| 多肽TAT与核定位信号介导的蛋白质入核递送;吴国清等;《中国生物工程杂志》;20041231(第5期);61页左栏第2段－右栏最后1段 * |
| 彭涛等.体内蛋白转导的研究进展.《中国药科大学学报》.2003,第24卷(第5期),477-480. |
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