CN102352373A - Method for realizing caenorhadits elegans in vivo functional protein rescue by utilizing escherichia coli prokaryotic expression system and special expression vector - Google Patents
Method for realizing caenorhadits elegans in vivo functional protein rescue by utilizing escherichia coli prokaryotic expression system and special expression vector Download PDFInfo
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Abstract
The invention discloses a method for realizing caenorhadits elegans in vivo functional protein rescue by utilizing an escherichia coli prokaryotic expression system and a special expression vector. The vector is a prokaryotic expression vector containing one or more same or different protein transduction peptides. The method comprises the following steps of: inserting coding genes of the functional protein with obvious caenorhadits elegans in vivo phenotype into the special expression vector; transforming into the escherichia coli prokaryotic expression system and inducing expression; feeding recombinant escherichia coli expressing the functional protein with functional protein deleted caenorhadits elegans or wild type strain caenorhadits elegans; and performing protein rescue functional verification through caenorhadits elegans phenotype screening and functional protein regulation factor expression level detection. The method has a wide application prospect, is expected to become an important technical method for screening recombinant protein medicines, and is one of important technologies for performing protein function research.
Description
Technical field
The invention belongs to the method for albumen rescue in the biological technology products Application Areas, particularly relate to a kind of method and dedicated expression vector therefor thereof that utilizes the escherichia coli prokaryotic expression system to realize functional protein rescue in the beautiful nematode body.
Background technology
Beautiful nematode is accomplished the many cells eucaryon model animals that whole genome sequence is measured as first; 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 H3K4trimethylation 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 receive 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.).The interior functional protein of beautiful nematode body is not expressed mainly because its vivo gene sudden change and/or genetically deficient cause under the natural condition; Result of study based on the unusual beautiful nematode of this genoid is unreliable, and the reparation research to beautiful nematode itself does not launch as yet based on protein level.
Summary of the invention
Find in the proteic prokaryotic expression research of contriver's function in being engaged in beautiful nematode body for a long time; Raise the external answer that proteic intestinal bacteria of function in the beautiful nematode body not only can be realized its mutating strain series phenotype that efficiently expresses through directly feeding; And can change the protein function disappearance that it causes because of transgenation, and then reach the purpose of albumen rescue (protein rescue).The purpose of this invention is to provide a kind of dedicated expression vector therefor that utilizes the escherichia coli prokaryotic expression system to realize functional protein rescue 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.
Said 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 said prokaryotic expression carrier can be the prokaryotic expression carrier of any one foreign gene, like 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 method of utilizing the escherichia coli prokaryotic expression system to realize functional protein rescue in the beautiful nematode body.
The method of utilizing the escherichia coli prokaryotic expression system to realize functional protein rescue in the beautiful nematode body provided by the present invention; Be in the encoding sox insertion claim 1 or 2 or 3 said dedicated expression vector therefors with the tangible functional protein of phenotype in the beautiful nematode body; The prokaryotic expression carrier that obtains is transformed into escherichia coli prokaryotic expression system and abduction delivering, the recombination bacillus coli that obtains is fed raise functional protein to lack beautiful nematode or wild-type strain be beautiful nematode and realize functional protein rescue in the beautiful nematode body.
Can comprise following concrete steps:
1) the tangible functional protein of phenotype and clone its encoding sox in the beautiful nematode body of screening;
2) the functional protein encoding sox is inserted in the above-mentioned dedicated expression vector therefor, obtain containing the prokaryotic expression carrier that protein transduction peptide-coding region and functional protein encoding sox 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;
5) feed the beautiful nematode of raising the functional protein disappearance with the proteic recombination bacillus coli of expressive function;
6) beautiful nematode phenotypic screen and functional protein regulatory factor expression level detect.
Utilize in the method that the escherichia coli prokaryotic expression system realizes the rescue of functional protein in the beautiful nematode body above-mentioned, the host bacterium of escherichia coli prokaryotic expression system can be in the said step 3): BL21, DH5 α, HB101, JM109, OP50 or the strange coccus of radiation hardness etc.
Above-mentioned recombinant expression vector all can make up according to ordinary method with the reorganization bacterium.
Can adopt the chemical reagent induction exogenous gene in the escherichia coli prokaryotic expression system, to express in the said 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 in the escherichia coli prokaryotic expression system, to express in the said 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 said step 4) detects and can adopt SDS-PAGE and/or immunoblotting.
Feed the intestinal bacteria raise beautiful nematode in the said step 5) and be identify the proteic bacterial strain of expressive function in the step 4) and be applied to the NGM plate in advance or the LB plate in.
Beautiful nematode phenotypic screen is meant and filters out the beautiful nematode with functional protein phenotype in the said step 6); Comprise that specifically morphological structure changes with the cell levels variation etc., as whether occur that build diminishes or obesity, whether the formation of vacuoles phenomenon, whether motor behavior imbalance etc. appears; The functional protein regulatory factor comprises protein, DNA and RNA etc., and its expression level screening 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, a kind of method and dedicated expression vector therefor thereof that utilizes the escherichia coli prokaryotic expression system to realize functional protein rescue in the beautiful nematode body is provided.Albumen according to the invention rescue (protein rescue) is meant to feed through the beautiful nematode strain system that gives the sudden change of specific function protein coding gene raises microorganism such as the intestinal bacteria that can express this functional protein, thereby corrects the technology of this functional protein disappearance phenotype in the beautiful nematode strain system.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 through microorganism such as the direct transductant of intestinal bacteria; The phenomenon of having avoided functioning gene not express, and then practiced thrift 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 functional protein rescue in the beautiful nematode body up to now.The escherichia coli prokaryotic expression system that utilizes provided by the invention saves the proteic method of function in the beautiful nematode body from the protein angle; Prospect is widely used; Not only can be used for proteic rescue research of function and application in the beautiful nematode body; But also can be used for the for example proteic rescue research of the interior function of animal bodies such as fruit bat, zebra fish, mouse, rat of other materials; Therefore 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 of albumen rescue 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 explained 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: prokaryotic expression carrier pET28-Tat-SOD-1,2,3,4,5 and pET28-SOD-1,2,3,4, the 5 bacterium liquid PCR qualification results that make up.
Fig. 6: SDS-PAGE detects Tat-SOD-1, and 2,3,4,5 and SOD-1,2,3,4,5 proteic expression change.
Embodiment
Embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
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 saved 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; Said 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 said prokaryotic expression carrier can be the prokaryotic expression carrier of any one foreign gene, like 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 of functional protein rescue 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 the method by chemical synthesis synthetic to contain restriction enzyme site be the NcoI(upper reaches) and the NdeI(downstream) the core encoder sequence fragment of double-stranded tat peptide section (GenBank:ADK70247.1); 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 TATDNA fragment and the empty carrier fragment of cutting through enzyme through the T4DNA ligase; To connect product again and be transformed into competent escherichia coli cell BL21(DE3) in; The screening recon; Extract the DNA of recon and carry out NcoI and the evaluation of NdeI double digestion according to conventional method; Send Ying Jun company directly to check order at last; The prokaryotic expression carrier that conclusive evidence contains the TAT core fragment makes up successfully, with this recombinant vector called after pET28b-TAT.
1) structure of prokaryotic expression carrier pET28b-TAT-EGFP and pET28b-EGFP and abduction delivering
First, the plasmid pEGFP-N1 (purchased from Clontech) as a template and using primer pairs (italics respectively BamHI and XhoI restriction sites): pU_5'-CGC?
ATGGTGAGCAAGGGCGAG-3 '(the sequence of Sequence 1); pD_5 '-CCG?
CTTGTACAGCTCGTCCAT-3 '(the sequence of Sequence 2) obtained by PCR amplification of enhanced green fluorescent protein (EGFP, GenBank No.: ADQ73885.1) cDNA coding sequence (717bp), and using the BamHI restriction endonuclease / XhoI digested plasmid gene EGFP and pET28b and pET28b-TAT; digestion products recovered using T4DNA ligase overnight at 16 ℃ connection; next day, all the transformation products were transformed into E. coli BL21 (DE3) and use restrictions endonuclease digestion and direct sequencing.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 appearance collection image at last; B, subsequently is transferred on the pvdf membrane 5% skimmed milk room temperature sealing 30 minutes with supernatant protein sample said 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/inferior); (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/inferior); 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 through external evoked expression and SDS-PAGE and immunoblotting.
3) abduction delivering thalline fluorescence microscope
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 through 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) to 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) the wild-type 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 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 fluorescence signal in beautiful nematode body, to distribute in 20 ℃ of biochemical incubators the strain of picking wild type; And gathered fluoroscopic image and differential interference image on the 48th hour in feeding to raise; Observing TAT-EGFP and the distribution situation of EGFP fusion in beautiful nematode body by the image folding at last, is the empty carrier contrast with pET28b and pET28b-TAT.Fluorescence microscope TAT-EGFP and EGFP albumen fluorescence signal be changes in distribution (arrow is depicted as TAT-EGFP and the distributing position of EGFP fusion in beautiful nematode body among the figure) as shown in Figure 4 in beautiful nematode body; Raise Bacillus coli cells that beautiful nematode expresses target protein after 48 hours by directly feeding; The TAT-EGFP fluorescence signal obviously is distributed in beautiful nematode intestines parietal cell; The EGFP fluorescence signal then is distributed in beautiful nematode enteric cavity; The empty carrier control group is not seen any fluorescence signal, illustrates that TAT protein transduction peptide can efficiently mediate foreign protein spanning transduction membrane in beautiful nematode body.Simultaneously; Through 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 get into beautiful nematode intestines parietal cell, study based on beautiful nematode platform and prokaryotic expression system for follow-up that the proteic rescue of function provides technical support in the beautiful nematode body.
1. method
1) prokaryotic expression carrier pET28-Tat-SOD-1,2,3,4,5 and pET28-SOD-1,2,3,4,5 structure and abduction delivering
Carrier construction method structure with reference to embodiment 2 carries beautiful nematode SOD-1 respectively, and the prokaryotic expression carrier of the cDNA encoding sequence of 2,3,4,5 different lengthss and 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, be template then with the reverse transcription product and combine following primer to (italicized item is respectively restriction enzyme site) to the wild-type strain:
pU_BamHI-SOD-1: 5'-CGC?
ATGTTTATGAATCTTCTCACTCAGGT-3 '(the sequence of Sequence 3)
pD_XhoI-SOD-1: 5'-CCG?
CTGGGGAGCAGCGAGAG-3 '(the sequence of Sequence 4)
pU_EcoRI-SOD-2: 5'-CCG?
ATGCTTCAAAACACCGTTCGCTG-3 '(the sequence of Sequence 5)
pD_XhoI-SOD-2: 5'-CCG?
TTGCTGTGCCTTTGCAAAACGC-3 '(the sequence of Sequence 6)
pU_EcoRI-SOD-3: 5'-CCG?
ATGCTGCAATCTACTGCTCGCAC-3 '(the sequence of Sequence 7)
pD_XhoI-SOD-3: 5'-CCG?
TTGTCGAGCATTGGCAAATCTCT-3 '(the sequence of Sequence 8)
pU_BamH? I-SOD-4: 5'-CGC?
ATGAAAACTCGTGTTGTTTTAATTCTGGC-3 '(the sequence of Sequence 9)
pD_XhoI-SOD-4: 5'-CCG?
GACGGTACCGATAGTTCCACATG-3 '(the sequence of Sequence 10)
pU_BamHI-SOD-5: 5'-CGC?
ATGGATATTCTCTCTGATATTGCCAATGC-3 '(the sequence of sequence? column 11)
pD_XhoI-SOD-5: 5'-CCG?
TGCAGGAGCGGCAAGAGCAATG-3 '(the sequence of Sequence 12)
Carry out pcr amplification subsequently to obtain SOD-1, the cDNA encoding sequence of 2,3,4,5 different lengthss; Then goal gene and carrier enzyme are cut the back and made up prokaryotic expression carrier pET28-Tat-SOD-1,2,3,4,5 and pET28-SOD-1; 2,3,4,5, at last above-mentioned carrier enzyme is cut and directly order-checking conclusive evidence.Bacterium liquid PCR qualification result as shown in Figure 5, further sequencing analysis shows and has successfully made up prokaryotic expression carrier pET28-Tat-SOD-1,2,3,4,5 and pET28-SOD-1,2,3,4,5.
Subsequently; The prokaryotic expression carrier pET28-Tat-SOD-1 that above-mentioned structure is correct; 2,3,4; 5 and pET28-SOD-1; 2,3,4; 5 are transformed into host bacterium BL21 (DE3) 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 appearance collection image at last; B, the supernatant protein sample of above-mentioned preparation said 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/inferior); (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/inferior); 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 as shown in Figure 6, the outer highly-soluble of display body is expressed and has been prepared Tat-SOD-1,2,3,4,5 and SOD-1,2,3,4,5 albumen, particularly Tat-SOD-1,2,3,4,5.
2) SOD-1, feeding of 2,3,4, the 5 beautiful nematodes of mutating strain series raised and the statistical study of adult rate
With abduction delivering TAT-SOD-1,2,3; 4,5 and SOD-1,2; The e. coli bl21 of 3,4,5 fusion roteins (DE3) somatic cells 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; Picking SOD-1,2,3; 4,5 transgenation strains are that 20 of beautiful nematodes (being respectively the not beautiful nematode of expressive function Protein S OD-1, SOD-2, SOD-3, SOD-4, SOD-5) were cultivated 48 hours in 20 ℃ of biochemical incubators.Then beautiful nematode is transferred to and feeds on the NGM substratum that contains different concns (0.1-0.5mM) Paraquat to observe the variation of adult rate and phenotype, like beautiful elegans development, body length and anti-Paraquat toxicity etc.The result finds; Feed and raise the external expression TAT-SOD-1 that efficiently expresses of beautiful nematode; 2; 3; 4; 5 thalline has tangible resistant function to the beautiful nematode damage that Paraquat caused of 0.1-0.3mM; Its adult rate is raised the external expression SOD-1 that efficiently expresses of beautiful nematode and feed, 2 than recovering normal before the albumen rescue intervention; 3; The thalline of 4,5 (not containing protein transduction peptide TAT) has more weak resistant function relatively to the Paraquat of 0.1-0.5mM, and control group (feed and raise BL21 (DE3) the intestinal bacteria group that contains empty carrier pET28b) is not seen tangible anti-Paraquat damaging action simultaneously; Thereby illustrate and realized SOD-1; 2,3,4; 5 transgenation strains are the proteic rescue of function in the beautiful nematode body, and the effect that utilizes the expression system that contains transduction peptide TAT can bring into play albumen rescue technology better is described simultaneously.
Claims (10)
1. a dedicated expression vector therefor that utilizes the escherichia coli prokaryotic expression system to realize functional protein rescue in the beautiful nematode body is the prokaryotic expression carrier that contains one or more identical or different protein transduction peptides.
2. dedicated expression vector therefor according to claim 1 is characterized in that: said protein transduction peptide is: TAT, Antp, VP22, Poly (Arg) and/or Poly (Lys) are preferably TAT.
3. dedicated expression vector therefor according to claim 1; It is characterized in that: the carrier that sets out that is used to make up said prokaryotic expression carrier can be the prokaryotic expression carrier of any one foreign gene; Like pET28, pBV220, pET30, pCRT7/CT-TOPO, pET101/D-TOPO or pQE30, be preferably pET28.
4. one kind is utilized the escherichia coli prokaryotic expression system to realize the method that functional protein is saved in the beautiful nematode body; Be in the encoding sox insertion claim 1 or 2 or 3 said dedicated expression vector therefors with the tangible functional protein of phenotype in the beautiful nematode body; The prokaryotic expression carrier that obtains is transformed into escherichia coli prokaryotic expression system and abduction delivering, the recombination bacillus coli that obtains is fed raise functional protein to lack beautiful nematode or wild-type strain be beautiful nematode and realize functional protein rescue in the beautiful nematode body.
5. method according to claim 4 is characterized in that: specifically may further comprise the steps:
1) the tangible functional protein of phenotype and clone its encoding sox in the beautiful nematode body of screening;
2) the functional protein encoding sox is inserted in the above-mentioned dedicated expression vector therefor, obtain containing the prokaryotic expression carrier that protein transduction peptide-coding region and functional protein encoding sox 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;
5) the proteic recombination bacillus coli of expressive function is fed raise that functional protein lacks beautiful nematode or the wild-type strain is beautiful nematode;
6) beautiful nematode phenotypic screen and functional protein regulatory factor expression level detect.
6. method according to claim 5 is characterized in that: the host bacterium of escherichia coli prokaryotic expression system is in the said step 3): BL21, DH5 α, HB101, JM109, OP50 or the strange coccus of radiation hardness.
7. according to claim 5 or 6 described methods; It is characterized in that: adopt the chemical reagent induction exogenous gene in the escherichia coli prokaryotic expression system, to express in the said step 4); Wherein, chemical reagent abduction delivering condition is: inductor IPTG abduction delivering (final concentration 1mM); The OD of abduction delivering
600=0.6-1.0; 6-8 hour abduction delivering time; Or
Adopt temperature-induced foreign gene in the escherichia coli prokaryotic expression system, to express in the said step 4), wherein, the temperature-induced expression condition can be: the OD of abduction delivering
600=0.4-0.6; 42 ℃-44 ℃ of abduction delivering temperature; 6-8 hour abduction delivering time.
8. according to claim 5 or 6 or 7 described methods, it is characterized in that: the protein expression level in the said step 4) detects can adopt SDS-PAGE and/or immunoblotting.
9. according to the arbitrary described method of claim 5 to 8, it is characterized in that: feed the intestinal bacteria raise beautiful nematode in the said step 5) and be identify the proteic bacterial strain of expressive function in the step 4) and be applied to LB in advance or the NGM plate in.
10. according to the arbitrary described method of claim 5 to 9, it is characterized in that: beautiful nematode phenotypic screen comprises that morphological structure changes with cell levels and changes in the said step 6), as whether occurring that build diminishes or obesity, formation of vacuoles phenomenon etc. whether; The functional protein regulatory factor comprises protein, DNA and RNA etc., and its expression level screening can be immunoblotting, gel shift experiment and polymerase chain reaction etc.
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| CN117204398A (en) * | 2023-08-29 | 2023-12-12 | 华南农业大学 | Method for evaluating oxidation resistance and aging resistance of egg white protein polypeptide based on caenorhabditis elegans model |
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| JP2000106884A (en) * | 1998-10-07 | 2000-04-18 | Nec Corp | Recombinant tpa-1a gene for single production of tpa-1a protein |
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| CN102146417A (en) * | 2010-02-10 | 2011-08-10 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method and special expression vector thereof for promoting solubility expression of foreign protein by TAT (Trans-activator transduction) core peptide |
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| CN117204398A (en) * | 2023-08-29 | 2023-12-12 | 华南农业大学 | Method for evaluating oxidation resistance and aging resistance of egg white protein polypeptide based on caenorhabditis elegans model |
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| CN102311969A (en) | 2012-01-11 |
| CN102352373B (en) | 2013-12-04 |
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