CN101418311A - A kind of structure and screening method of new rna interference vector - Google Patents
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Abstract
The present invention proposes a kind of structure and screening method of new rna interference vector, it mainly comprises: the transformation of carrier, contain the generation of the dna sequence dna that disturbs target sequence and the above-mentioned dna sequence dna that disturbs target sequence that contains is cloned into three parts of carrier, complex steps of the present invention, cloning efficiency is low, is fit to high-throughout operation.This method does not need to order the primer of length greater than 60nt, does not need that the PCR product is carried out complicated enzyme and cuts purification process, is connected reorganization but rely on carrier with the external source fragment, thereby produces complete lacO sequence (lacoperator), at lac
+Coli strain in rely on blue hickie screening to judge recon.Its screening method is convenient, directly perceived, quick, efficient, and the recon that obtains does not need direction of travel to identify, is easy to realize the operation of automatization and high-throughput.
Description
Technical field
What the present invention relates to is biological gene clonal expression technology, particularly a kind of fast, efficient, high flux construction animals and plants RNA disturbs (RNA interference, RNAi) method of carrier.Be particularly suitable for making up on a large scale RNA interference units, be used for the functional study of animal-plant gene, and screen convenient and swift at each gene of animals and plants.
Background technology
RNA disturbs and is meant in normal organism, caused that by double-stranded RNA the specificity of homologous mRNA is degraded, thereby inhibitory phase is answered the process of genetic expression.It is a kind of post-transcriptional level gene silencing, ubiquity in vivo.Along with investigator's going deep into to biological phenomena research, the RNA perturbation technique has become one of important means in the researchs such as current animal-plant gene function, gene therapy, drug target screening and evaluation, plant metabolism and cell signaling path because of its high specific, high-level efficiency, easy and simple to handle, good reproducibility.
Conclusion is got up, and existing rna interference vector construction process mainly contains following three kinds: 1. oligonucleotide annealing method.By the part complementation of chemosynthesis, contain two oligonucleotide strands of the target sequence that disturbs goal gene, annealing forms double-stranded DNA under isolated condition, annealing produces the outstanding sticky end in two ends, can with the protruding terminus complementation of the carrier of handling well, after connecting conversion, obtain containing the unitary recombinant plasmid of rnai expression.The oligonucleotide of chemosynthesis is generally inverted repeats, and transcribing the single stranded RNA that obtains like this can the inflection complementary pairing, forms loop-stem structure, forms siRNA (siRNA) after treatment and cause the RNA interference effect in cell.2. pcr amplification method.When making up the zooblast rna interference vector, the PCR primer of a pair of amplification control of design earlier RNA interference units expression promoter, forward primer and promotor specificity coupling, reverse primer 5 ' the terminal interference target sequence that additionally adds at goal gene, the PCR product cloning to corresponding carrier, is obtained the carrier of expressed rna interference units.When making up plant manpower fine RNA (miRNA) expression vector, design earlier three pairs of primers as required, carry out independently PCR of three-wheel, three kinds of PCR products of acquisition contain respectively: treat the target sequence of interferential goal gene, the ring sequence of miRNA and the complementary sequence for the treatment of interferential goal gene target sequence; Three kinds of PCR products that adopt the method for overlapping extension PCR to obtain then are spliced into complete artificial mi RNA loop-stem structure sequence, it are cloned on the plant binary carrier again.The another kind of mode that adopts PCR method to make up plant artificial mi RNA carrier is to design and synthesize a pair of primer earlier, primer 5 ' hold to be the extra restriction enzyme site that adds and interference target sequence or its complementary sequence of goal gene, 3 ' hold into skeleton miRNA in ring sequence annealing region, the PCR product of acquisition cuts back to close rear clone to carrier through enzyme.3. oligonucleotide annealing extension method.Two part complementary of chemosynthesis oligonucleotide strand, contain target sequence and the complementary sequence thereof that disturbs goal gene respectively in non-complementary strand zone, make two strand annealing under given conditions, extend to form complete little double chain DNA molecule then, cut through enzyme and handle rear clone to carrier.
Though above method can successfully make up various rna interference vectors, obviously there is deficiency, mainly show the following aspects:
One, complex steps, cloning efficiency is low.More than three kinds of methods all need clone's purpose fragment and carrier are carried out special processing, need the ligation of purpose fragment and carrier.1. method needs two external annealing of oligonucleotide strand of chemosynthesis, and the product that obtains needs purification process, then with the carrier ligation of handling well.Because purpose fragment to be cloned usually less than 100bp, therefore is difficult to purifying, yield is low, and obtains difficulty of correct recombinant plasmid, false positive rate height.2. method need add the interference sequence at target gene in the PCR primer, need the pcr amplification reaction of long primer (usually greater than 70nt) or many wheels usually, and the PCR product after the amplification also needs enzyme to cut purifying, carries out ligation with the carrier of handling well then; Method just can be cloned into respective carrier after the annealing of two oligonucleotide strands extends the product that obtains and must cut step such as purifying through complicated recovery purifying, enzyme in 3..Method is 3. spended time not only 2., and operation is loaded down with trivial details, and carrier to connect the probability of going up non-purpose fragment or connecting certainly very high, make follow-up Screening and Identification process complicated.
Two, be not suitable for high-throughout operation.
More than the cloned sequence for the treatment of in three kinds of methods all need purification process in advance, all depend on the segmental ligation of carrier and external source, this is for making up single or the minority rna interference vector may be feasible, but for making up but unusual difficulty of rna interference vector on a large scale, and inefficiency.The oligonucleotide strand that method is used in 1. and 2. is generally 60-100nt, and synthetic such oligonucleotide strand is the experimental cost height not only, and base resultant fault rate height, and then has increased follow-up screening operation amount greatly, wastes time and energy.Method 2. and the external source fragment 3. all need through complicated double digestion, reclaim step such as purifying, because of external source fragment generally all less (in the 200bp), it is lower to reclaim purification efficiency, and these two kinds of methods are the experimental cost costliness not only, and follow-up screening difficulty, workload is big.
(referring to publication " RNA.2002 8:1454-1460 "; " Plant Cell.2006 May; 18 (5): 1121-33. "; " Nat Biotechnol.2006 Nov; 24 (11): 1420-8. "; ＂ Physiol Genomics.2007 Nov14; 31 (3): 554-62. ＂ is referring to webpage www.invitrogen.com or Invitrogen company products catalogue)
Summary of the invention
The purpose of this invention is to provide a kind of new RNA and disturb (RNA interference, RNAi) construction of carrier.This method mainly comprises: the transformation of carrier, contain the generation of the dna sequence dna that disturbs target sequence and the above-mentioned dna sequence dna that disturbs target sequence that contains is cloned into three parts of carrier.This method does not need to order the primer of length greater than 60nt, does not need that the PCR product is carried out complicated enzyme and cuts purification process, is connected reorganization but rely on carrier with the external source fragment, thereby produces complete lacO sequence (lacoperator), at lac
+Coli strain in rely on blue hickie screening to judge recon.Its screening method is convenient, directly perceived, quick, efficient, and the recon that obtains does not need direction of travel to identify.
The concrete grammar that makes up rna interference vector among the present invention is:
One) transformation of carrier
An at first selected destination carrier is carried out mutagenesis with Bfu I (or the Bfi I) site and the lacO sequence that have existed on this carrier, and the carrier after the mutagenesis no longer contains Bfu I (or Bfi I) site and lacO sequence.Have the stuffer (as: resistant gene, gfp etc.) that Bfu I (or Bfi I) site and an end have part lacO sequence then at introducing two ends, the multiple clone site place of carrier.
Two) contain the formation (detailed process is seen Fig. 1) of the dna sequence dna that disturbs target sequence
According to the difference of rna interference vector kind, take diverse ways to finish this step:
1) at siRNA carrier, shRNA carrier, at first design a pair of primer that comprises target sequence, it is 9-20nt homologous sequence each other that this primer 3 ' end contains length, wherein a primer 5 ' is held the partial sequence (just in time reconstituting complete lacO sequence with the part lacO sequence on the carrier) that additionally adds lacO, utilization has the active hot resistant DNA polymerase of terminal enzyme (DNA) and (is specially Taq archaeal dna polymerase, Ex archaeal dna polymerase, LA archaeal dna polymerase etc., down together) two primer annealings are extended, the product that obtains is desired sequence (seeing Figure 1A for details);
2) at artificial microRNA (artificial microRNAs, amiRNAs) carrier, at first with the microRNA (microRNA that sets out, miRNA) 3 ' the distolateral wing sequence is a template, design a universal primer (if 3 ' the distolateral wing sequence is shorter, then not needing universal primer), 5 ' end of this primer additionally adds the partial sequence (just in time reconstituting complete lacO sequence with the part lacO sequence on the carrier) of lacO, according to the miRNA ring sequence length difference of setting out, be divided into following two kinds of situations again then:
A) when encircling sequence short (usually below the 30nt), design a pair of primer that comprises target sequence, its 3 ' end has part ring sequence (two portions ring sequence is reconfigurable into complete ring sequence) respectively, and make this primer 3 ' end sequence part (being generally 9-20nt) homology each other, (the distolateral wing sequence of miRNA3 ' is shorter if set out wherein to contain the 15-20nt homologous sequence with that primer 5 ' end of miRNA3 ' end annealed that sets out with the universal primer that designs above, then need not design this homology part), utilization has the active hot resistant DNA polymerase of terminal enzyme (DNA) makes this to primer and the universal primer three extension of annealing mutually, and the product that obtains is desired sequence (seeing Figure 1B for details);
B) when encircling sequence long (usually more than the 30nt), design a pair of primer that comprises target sequence, its 3 ' terminal have respectively can with the ring sequence annealed partial sequence of the miRNA that sets out, the universal primer of wherein holding with 5 ' of that primer of miRNA3 ' end annealed that sets out and designing above contains the 15-20nt homologous sequence, and (the distolateral wing sequence of miRNA3 ' is shorter if set out, then need not design this homology part), with the dna sequence dna that contains the respective rings sequence is template, with three primers (a pair of special primer and a universal primer) of above-mentioned design with have the active hot resistant DNA polymerase of terminal enzyme (DNA) and carry out PCR, the product that obtains is desired sequence (seeing Fig. 1 C for details);
Three) carrier and external source are segmental is connected conversion
At first with step 1) in the carrier of reincarnate through Bfu I (or Bfi I) complete degestion, obtain the linearized vector dna molecule that 3 ' distal process goes out a base, the linear carrier of gained is through reclaiming purifying, the step 2 of crossing with purifying then) the PCR product that contains target sequence that obtains in is connected, transformed into escherichia coli bacterial strain DH10 β, coating contains the LB solid medium of 100 μ g/ml penbritins (or other microbiotic) and 30 μ g/ml X-gal, cultivated 12-16 hour for 37 ℃, present blue bacterium colony and be recon.
Four) RNAi that contains target sequence expresses the unit and is cloned on the plant binary expression vector
For being directly used in step 1) the RNAi carrier transformed (as: be used in the animal and plant cells moment detect RNAi expression vector) then can omit this step.For can not or being inconvenient to be used for step 1) the direct plant binary expression vector of transforming, can select a smaller cloning vector set by step earlier) transform (as pBluescript SK, pMAGIC etc.), the methods such as connection, Gateway recombinant clone and conjugal transfer of cutting by enzyme are then expressed the unit with RNAi and are cloned on the plant binary expression vector.
Ultimate principle of the present invention is: 1. all contain the variable base of some amount in the recognition sequence of IIs type restriction endonuclease Bfu I (or Bfi I), and double-stranded DNA can be in 3 ' terminal outstanding base after this enzyme cutting; 2. the carrier that has complete lacO sequence imports lac
+Coli strain after, the lacO site on the carrier makes Lac operon in the thalline express in conjunction with the LacI aporepressor in the thalline to open that owing to competitive the beta-galactosidase enzymes of expression makes bacterium colony be blue because of decomposing semi-lactosi analogue X-gal.According to above two principles, we are transformed into certain specific support in the carrier that carries a stuffer (as: resistant gene, gfp etc.), the stuffer two ends have a Bfu I (or Bfi I) restriction enzyme site respectively, simultaneously, near the part base sequence that has lacO a Bfu I (or the Bfi I) site therein, the residue base sequence of lacO is at the segmental end of external source, have only the external source fragment to be connected on the carrier according to correct direction, could constitute complete lacO sequence, and then filter out recon.
BfuI of the present invention or BfiI restriction endonuclease comprise that also the DNA after all enzymes are cut has the IIs type restriction endonuclease that 5 ' distal process goes out characteristics.
The present invention compared with prior art has remarkable advantages:
One, among the present invention, in improved carrier, inserted the stuffer (as: resistant gene, gfp etc.) that is easy to screen, stuffer is lost after Bfu I (or Bfi I) enzyme is cut, and makes in the recombinant screen process that like this cutting the background that not exclusively causes because of enzyme very easily distinguishes.
Two, the present invention carrier with contain the PCR product end that disturbs target sequence and respectively comprise a part of lacO sequence, have only and work as both according to correct direction successful connection, could produce complete lacO sequence on the recombinant plasmid, recon just might present blueness, and the method for the lacO sequence reconstruct of this forward screening makes carrier very easily distinguish from the bacterium colony of generations such as connecting, non-aim sequence is connected with carrier and closure is incorrect and the correct recon that we want.
Three, the present invention is in the process that makes up siRNA and shRNA interference carrier, utilize the extension of annealing mutually of two primers, the PCR product that obtains does not need complicated recovery purifying (available in case of necessity two volumes ethanol or monoploid amass isopropanol precipitating) just can directly be connected with the carrier of handling well, simplified the experiment operation greatly, improve conventional efficient, reduced experimental cost.
Four, the present invention has utilized a universal primer to put up a bridge in the process that makes up artificial microRNA (amiRNA) carrier dexterously, make the primer length of all designs not be subjected to the restriction of flanking sequence length, and all be controlled in the 60nt, so not only save experimental cost, and greatly reduced the base error rate of primer in synthetic.
Five, whole process operation of the present invention is simple and convenient, and experimental period is short, the recombination efficiency height, and cost is low, and screening is convenient, directly perceived, quick, is easy to realize the operation of automatization and high-throughput.
Description of drawings
Fig. 1: contain RNAi and express unitary vector construction process
Wherein 1. 2. represent to comprise a pair of primer that disturbs target sequence; 3. represent universal primer; LacO ' expression is positioned at the part laoO sequence on the carrier, and base sequence is 5 '-agcgctcacaatt-3 '; " expression is positioned at the part laoO sequence on the universal primer to lacO; Gene X represents stuffer; BfuI represents restriction enzyme site, and its recognition sequence and cleavage site are: 5 '-GTATCCNNNNNN|3 '
3′-CATAGGNNNNN|
-5′
The destination carrier structure iron of transforming among Fig. 2: the embodiment 1
Wherein 5 ' flanking seq represents 5 ' distolateral wing sequence of selected miRNA skeleton, and BfuI represents restriction enzyme site; The partial sequence of the complete laoO of lacO ' expression; Promoter represents promoter sequence; Terminator represents the terminator sequence; Gene X represents stuffer.
Embodiment
The present invention is further described with embodiment below:
Embodiment 1:
The miR319a that utilizes the present invention to make up with Arabidopis thaliana is a skeleton, with plant manpower fine RNA (amiRNA) expression vector of chalcone synthase (CHS) gene as the interference target gene.
1) transformation of cloning vector
For the needs of subsequent experimental, selected cloning vector is for can be used for engaging auxiliary genetic integration clone (mating-assisted genetically integrated cloning, the MAGIC) donor plasmid of method.The analysis showed that, carry two BfuI sites on this plasmid dna sequence, a lacO sequence becomes the BamHI site with two BfuI site mutagenesis, and the lacO sequence deletion is suddenlyd change.Multiple clone site place at the carrier of reincarnate inserts following fragment: " promotor (P35S)-5 ' flanking sequence-BfuI site-gfp express unit-BfuI site-lacO partial sequence-terminator (Tnos)-" obtains transforming carrier pDONOR-gfp.(seeing Fig. 2 for details)
2) contain the formation of the artificial microRNA-miR319aCHS that disturbs target sequence
According to concrete grammar step 2 of the present invention) in principle, analyze the miR319a sequence in Arabidopis thaliana source, save universal primer, design and synthesize a pair of Auele Specific Primer pF ﹠amp; PR is a template with the carrier that contains the miR319a sequence, carries out PCR with the Taq archaeal dna polymerase, and the product that obtains is miR319aCHS.The primer sequence of design is as follows:
pF：5’ag
3’
pR：5’cacaatt
3’
In the above primer, among the pF following stroke
Part is represented two bases that 5 ' the distolateral wing sequence is cut away by BfuI; Among the pR following stroke
Part is represented 3 ' the distolateral wing sequence; Following stroke
The ring sequence annealing portion of part expression and miR319a; Gray shade is partly represented the interference target sequence of selected goal gene; Italicized item represents to form with the part lacO sequence of carrying on the carrier residue sequence of complete lacO.
3) carrier and external source are segmental is connected conversion
The carrier of reincarnate in the step 1) is cut through the BfuI enzyme, enzyme cut product after reclaiming purifying with step 2) in the PCR product miR319aCHS linked enzyme of purifying be connected, transformed into escherichia coli DH10 β then, coating LA+X-gal flat board, cultivate 12 hours observationss in 37 ℃, select blue single bacterium colony extracting plasmid checking, the checking result shows that blue colonies is that the probability of recon is 100%.Correct recon is given the order-checking of Invitrogen company.The correct recombinant plasmid that checks order is used for next step experiment.
4) be cloned on the plant binary RNAi expression vector RNAi unit that contains target sequence
Adopt the method for MAGIC, the RNAi unit miR319aCHS on the recombinant plasmid that order-checking in the step 3) is correct transfers to the acceptor carrier through transforming---on the plant binary carrier p1301R.Experimental result shows that the positive colony rate that acquisition contains the plant binary expression vector of miR319aCHS sequence reaches 100%.
Embodiment 2:
Utilizing the present invention to make up with mammiferous miR30 is skeleton, is the artificial microRNA of animal (amiRNA) carrier that disturbs target sequence with agtagattaccactggagtct.
1) transformation of cloning vector
Analyze carrier pBluescript SK (+) sequence, BfuI site on this sequence of mutagenesis and lacO sequence.Multiple clone site place at the carrier of reincarnate inserts following fragment: " promotor (CMV)-5 ' flanking sequence-BfuI site-gfp express unit-BfuI site-lacO partial sequence-terminator (SV40PolyA)-".
2) contain the formation of the artificial microRNA-mir30-X that disturbs target sequence
According to concrete grammar step 2 of the present invention) in principle, analyze the miR30 sequence, design and synthesize a universal primer pUR and a pair of Auele Specific Primer pF ﹠amp; PR mixes 3 primers, anneals down at 25 ℃ with the Taq archaeal dna polymerase, and 72 ℃ of extensions, the product that obtains is mir30-X.The primer sequence of design is as follows:
pUR：cacaatt
pF：
tagtgaagccacagatgta
pR：cgaggcagtaggca
tacatctgtggcttcac
In the above primer, italicized item represents to form with the part lacO sequence of carrying on the carrier residue sequence of complete lacO; Following stroke
Part is represented 3 ' the distolateral wing sequence of miR30; Gray shade partly represents to disturb target sequence (among the pF) and complementary sequence (among the pR) thereof.
3) carrier and external source are segmental is connected conversion
The carrier of reincarnate in the step 1) is cut through Bfu I enzyme, enzyme cut product after reclaiming purifying with step 2) in be connected with ligase enzyme through the PCR of alcohol precipitation product miR30-X, transformed into escherichia coli DH10 β then, coating LA+X-gal flat board, cultivate 12 hours observationss in 37 ℃, select blue single bacterium colony extracting plasmid checking, the checking result shows that blue colonies is that the probability of recon is 100%.Correct recon is given the order-checking of Invitrogen company, and the correct recombinant plasmid that checks order promptly can be used for next step mammalian cell moment detection of expression.
Claims (2)
1, a kind of structure and screening method of new rna interference vector, it mainly comprises: the transformation of carrier, contain the generation of the dna sequence dna that disturbs target sequence and the above-mentioned dna sequence dna that disturbs target sequence that contains is cloned into three parts of carrier, it is characterized in that concrete steps are:
One, the transformation of carrier
An at first selected destination carrier, mutagenesis is carried out in the Bfu I that existed on this carrier or Bfi I site and lacO sequence, carrier after the mutagenesis no longer contains Bfu I or Bfi I site and lacO sequence, introduces two ends at the multiple clone site place of carrier then and has the stuffer that Bfu I or Bfi I site and an end have part lacO sequence;
Two, contain the formation of the dna sequence dna that disturbs target sequence
According to the difference of rna interference vector kind, take diverse ways to finish this step:
1) at siRNA carrier, shRNA carrier, at first design a pair of primer that comprises target sequence, it is 9-20nt homologous sequence each other that this primer 3 ' end contains length, wherein primer a 5 ' end additionally adds the lacO partial sequence that can just in time reconstitute complete lacO sequence with the part lacO sequence on the carrier, utilization has the active hot resistant DNA polymerase of terminal enzyme (DNA) extends two primer annealings, and the product that obtains is desired sequence;
2) at artificial microRNA (artificial microRNAs, amiRNAs) carrier, at first with the microRNA (microRNA that sets out, miRNA) 3 ' the distolateral wing sequence is a template, 5 ' end of this primer additionally adds the lacO partial sequence that can just in time reconstitute complete lacO sequence with the part lacO sequence on the carrier, according to the miRNA ring sequence length difference of setting out, be divided into following two kinds of situations again then:
A) when encircling sequence less than 30nt, design a pair of primer that comprises target sequence, its 3 ' end has part ring sequence respectively, and make this primer 3 ' end sequence portion homologous each other, sequence length is 9-20nt, wherein contain the 15-20nt homologous sequence with the universal primer that designs above with that primer 5 ' end of miRNA 3 ' end annealed that sets out, utilization has the active hot resistant DNA polymerase of terminal enzyme (DNA) makes this to primer and the universal primer three extension of annealing mutually, and the product that obtains is desired sequence;
B) when encircling sequence greater than 30nt, design a pair of primer that comprises target sequence, its 3 ' terminal have respectively can with the ring sequence annealed partial sequence of the miRNA that sets out, wherein 5 ' end with miRNA 3 ' that primer of end annealed that sets out contains 15-20nt homologous sequence with the universal primer that designs above, with the dna sequence dna that contains the respective rings sequence is template, with a pair of special primer, a universal primer of above-mentioned design with have the active hot resistant DNA polymerase of terminal enzyme (DNA) and carry out PCR, the product that obtains is desired sequence;
Three, carrier and external source are segmental is connected conversion
At first with the carrier of reincarnate in the step 1 through Bfu I or Bfi I complete degestion, obtain the linearized vector dna molecule that 3 ' distal process goes out a base, the linear carrier of gained is through reclaiming purifying, be connected with the PCR product that contains target sequence that obtains in the step 2 that purifying is crossed then, transformed into escherichia coli bacterial strain DH10 β, coating contains the LB solid medium of 100 μ g/ml penbritins or other microbiotic and 30 μ g/mlX-gal, cultivated 12-16 hour for 37 ℃, present blue bacterium colony and be recon;
Four, the RNAi that contains target sequence expresses the unit and is cloned on the plant binary expression vector
For being directly used in the RNAi carrier that step 1 is transformed, then can omit this step, for the plant binary expression vector that can not directly transform, should select a smaller cloning vector one to transform set by step earlier, cut connection, Gateway recombinant clone and conjugal transfer method by enzyme then and RNAi is expressed the unit be cloned on the plant binary expression vector.
2, the structure and the screening method of a kind of new rna interference vector according to claim 1, it is characterized in that described BfuI or BfiI restriction endonuclease, comprise that also the DNA after all enzymes are cut has the IIs type restriction endonuclease that 5 ＇ distal process go out characteristics.
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| CN105132413A (en) * | 2015-08-17 | 2015-12-09 | 深圳大学 | Primer for preparing shRNA, preparing method, vector and building method |
| CN105132413B (en) * | 2015-08-17 | 2018-06-19 | 深圳大学 | It is used to prepare primer and preparation method, the carrier and construction method of shRNA |
| CN108342409A (en) * | 2018-01-17 | 2018-07-31 | 周口师范学院 | A kind of plant RNA i expression vectors and its construction method and application |
| CN108342409B (en) * | 2018-01-17 | 2021-05-14 | 周口师范学院 | Plant RNAi expression vector and construction method and application thereof |
| CN109652443A (en) * | 2019-02-25 | 2019-04-19 | 四川大学 | A kind of artificial microRNA interference carrier and its construction method and application |
| CN109652443B (en) * | 2019-02-25 | 2023-04-07 | 四川大学 | Artificial microRNA interference vector and construction method and application thereof |
| CN114277030A (en) * | 2021-12-28 | 2022-04-05 | 滨州医学院 | pri-miRNA (pri-microribonucleic acid) modified sequence and vector for expressing sequence |
| CN114277030B (en) * | 2021-12-28 | 2024-07-16 | 滨州医学院 | Pri-miRNA improved sequence and vector for expressing same |
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