CN105368865A - Controllable genome-modified plasmodium, recombinant expression vector and construction method and application of controllable genome-modified plasmodium and recombinant expression vector - Google Patents
Controllable genome-modified plasmodium, recombinant expression vector and construction method and application of controllable genome-modified plasmodium and recombinant expression vector Download PDFInfo
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Abstract
The invention relates to a controllable genome-modified plasmodium, a recombinant expression vector and the construction method and application of the controllable genome-modified plasmodium and the recombinant expression vector. The recombinant expression vector comprises a gene targeting long homologous arm, a gene targeting short homologous arm, a tetracycline repression protein gene expression cassette, a pyrimethamine resistance gene expression cassette and a target gene expression cassette, wherein the tetracycline repression protein gene expression cassette, the pyrimethamine resistance gene expression cassette and the target gene expression cassette are located between the gene targeting long homologous arm and the gene targeting short homologous arm, and tetracycline operator gene sequences are inserted in multiple transcriptional start sites of a target gene promoter, so that the recombinant expression vector can be used for conditional research of the functions of a certain functional gene in a plasmodium genome. Furthermore, a functional gene expression sequence, corresponding to a target gene, in the plasmodium genome is knocked out by means of the gene knockout technique; meanwhile, the recombinant expression vector is transfected into a plasmodium with genes knocked out, so that the controllable genome-modified plasmodium is obtained; a new technical scheme is provided for further research of the functions of all functional genes in the plasmodium genome, and application prospects are broad.
Description
Technical field
The present invention relates to genetically engineered field, especially relate to a kind of controlled genomic modification plasmodium, recombinant expression vector and construction process, application.
Background technology
The malaria caused by Infected With Plasmodium is one of the whole world three large main infection diseases.Have several hundred million people to be infected with malaria every year, exceed million people because of malaria death, wherein majority is the children of less than 5 years old.The one of the main reasons that malaria is difficult to effectively to control and eradicate is to understand limited to its pathogenic agent-plasmodial biology complicacy.Plasmodium has the complicated life history, it grows needs to complete in the host such as female Anopheles mosquitoes and people (or other vertebratess): plasmodium carries out sexual propagation in anopheles body, in people or other vertebrate host bodies, then carry out vegetative propagation.The growth course of plasmodium in host is divided into liver phase (Exoerythrocytic Stage) and erythrocytic phase (erythrocytic stage).Liver phase malaria does not generally show clinical symptom, and the main pathologic reaction of malaria infection is caused by erythrocytic stage.The Exoerythrocytic Stage merozoite discharged by liver cell enters in blood flow intrusion red corpuscle and carries out erythrocytic stage propagation, polypide propagation and release cause erythrocyte fragmentation, the merozoite disengaged from the red corpuscle that breaks invades healthy red corpuscle, go round and begin again, cause a large amount of red corpuscle to be destroyed, patient shows serious clinical symptom.The examining order of current plasmodium gene group completes substantially, and thus a large amount of function of sequenced genes needs to be illustrated.People to participate in plasmodium life history each stage differentiated, to grow and important gene in breeding and regulation and control thereof are also understood very few.The concrete function verifying the various gene of plasmodium and gene product thereof has great importance for the effective malaria vaccine of exploitation and discovery new antimalarial agent thing target position.
Therefore, the focus that the method analyzing plasmodium gene function has become malaria research is expanded.By biochip technology and suppression subtractive hybridization technique (Suppressivesubtractivehybridization, the combine with technique bioinformatic analysis such as SSH), can screen and obtain many and growth of malaria parasites, breeding and relevant gene is acted on to host and predicts that it may function, but the concrete function of the exploitation of vaccine and new drug significantly being confirmed to plasmodium important gene.Early stage scientist by the function of enzyme level molecular studies zymoprotein, but limits the development of this method due to the low specificity of enzyme inhibitor and the limitation of range of application.Along with the development of plasmodium gene rotaring dyeing technology, experimenter can carry out target gene inactivation by transgenic technology and directly study this gene function.RNA disturbs (RNAinterference, RNAi) with gene knockout (geneknockout) technology be apply more gene inactivation method Mammals at present, but, lack the critical elements needed for RNAi in plasmodium gene group, thus the application of RNAi in plasmodium still has difficulties.Gene knockout is the classical genetic method of research gene function, but because erythrocytic stage plasmodium is monoploid, polypide will be caused dead after the gene having a critical function to erythrocytic stage plasmodium is knocked and its function cannot be studied, therefore, set up plasmodium gene conditionality knock out technology will greatly facilitate research its gene function.Primary structure system combined with gene knockout is set up gene conditionality to knock out one of most effective means of technology.Separately have research to be combined with gene knockout by Cre/loxP or Flp/FRT system to realize gene condition and knock out, but this gene condition knocks out Technical Board and is limited to and must uses specificity or tissue-specific promoter, and this conditionality is irreversible after knocking out and occurring in this given period or particular organization, therefore, for major part vegetative period be haploid plasmodium, still there is important gene and lack the difficult problem that rear polypide is dead or be badly damaged and can not carry out the screening of transgenosis worm and clone, and be more difficult to study in this way to the gene that plasmodium all plays an important role each vegetative period.The Primary structure mode used in zooblast often utilizes some special promotors, these promotors can induce startup under some physico chemical factor, as heat-shock promoters can at high temperature be induced, glucocorticosteroid and heavy metal reaction promotor etc., there is following defect in these genetic expression induction modes: abduction delivering poor specificity, express when system is in closing condition and have leakage, inductor itself is toxic causes damage etc. to cell.1992, Gossen etc. utilized the tet-off Primary structure system constructing based on the tetracycline-resistance operon of intestinal bacteria Tn10 transposon and can be used for the Tetracycline regulation studying mammal gene function.This system comprises regulon and response unit two part.The activating transcription factor (tTA) that adjustment portion is expressed is deposited in case not activating genes of interest at tsiklomitsin and is expressed; And after tsiklomitsin is removed, tTA can activate destination gene expression in response unit.Then Gossen etc. transform tet-off system, construct the Tet-on Primary structure system by Tetracycline regulation, and this system promotor under the condition not having inductor is not activated, and after adding inductor goal gene high expression.The advantages such as tet-on system has tightly, efficient, controllability is strong and inducedvelocity is fast, are widely used in gene functional research.But be widely used in Eukaryotic tet-on system and be not suitable for plasmodium, because the key element hsv HSV albumen (herpessimplexvirusvirionprotein16 in the regulon that system comprises and response unit, VP16) and tsiklomitsin response element (tetracycline-responsiveelement, TRE) do not work in plasmodium.Many protozoon class biologies close with plasmodium kind successful Application tsiklomitsin inducible expression regulate gene expression: as trypanosoma bocagei (WirtzE. etc., Science, 1995, 268:1179-1183), ameba (HamannL. etc., Mol.Biochem.Parasitol.1997, 84:83-91.), graceful protozoon (the YanS. etc. of Li Shi, WoodsHoleMol.Parasitol.Meet., 10th, 1999.), Giardia lamblia (SunCH. etc., Mol.Biochem.Parasitol.2000, 105:51-60.), toxoplasma gondii (MeissnerM. etc., Science, 2002, 298:837-840.) etc.But plasmodial genome is rich in the analysis of AT base interference to its gene promoter sequence, also makes troubles in colibacillary amplification to the plasmid vector comprising plasmodium gene; And, plasmodium main parasitic is in the red corpuscle of vertebrates and people, ectogenic DNA is incorporated in plasmodium gene group by transfection to be needed through erythrocyte membrane, Na Chongpao, worm cytolemma and nuclear membrane four tunic, transfection efficiency is extremely low, and these cause difficulty all to the research of plasmodial genetic modification.In recent years, along with the raising of plasmodium consideration convey dyeing technique, plasmodial genetic modification research starts dynamic.2005, the tet-off system that Meissner etc. build has regulated and controled green fluorescent protein (GFP) genetic expression (Proc.Natl.Acad.Sci.USA.2005 in Plasmodium falciparum, 102 (8): 2980-2985), but, transfected plasmids is not incorporated into plasmodium gene group and exists with episome (episome) form, and episome is easily lost and is caused transfection results unstable, modulated Primary structure rate very low (about 20%) and short duration, plasmodium endogenous gene keeps complete nothing to knock out.In the end of the year 2012, PacoPino etc. set up tet-off system condition and knock out PRF and NMT gene (CellHost & Microbe2012,12:824 – 834.).Tet-off is subject to the impact of organism metabolism inductor speed when promotor gene is expressed, express quick, efficient not as good as tet-on system promotor gene, and tet-off system needing to continue to inductor when closing genetic expression, bringing inconvenience to gene functional research process, plasmodium and host thereof being existed to the unfavorable factors such as potential side effect and experimental result may be disturbed.Further, develop the full worm vaccine of controlled genomic modification plasmodium, the unavailable tet-off system of tet-on system can only be selected, because body can not give Fourth Ring after giving vaccine usually cause weak plasmodium simultaneously.The AT content of plasmodium gene is very high; Also there is many gene expression characteristicses different with other eukaryote in addition, the CMV promoter that such as can work in most eukaryotic cell and SV40 promotor inoperative in plasmodium cell, the promotor of plasmodium gene also usually lacks real TATA box, plasmodium gene promotor often has multiple transcription initiation site, and these are all that the carrying out of research work of plasmodium gene group adds difficulty.
In addition, effective vaccines against malaria is the important means of mankind's malaria control.In past 50 years, various countries investigator once attempted kinds of schemes research and development malaria vaccine, but so far yet obtain can be used for clinical effective vaccine.Early stage research shows, with the sporozoite immunization experiment animal or human of radiation exposure attenuation, can induce the immune protective efficiency of scavenging.But this radioactive rays attenuated sporozoites is difficult to a large amount of preparation and produces, thus its full worm cannot as vaccine at mankind's large-scale inoculation, and the method for radioactive rays attenuation is difficult to control its quality.Eighties of last century eighties, the foundation of molecule clone technology makes people that subunit vaccine is placed hope in the research and development of malaria vaccine.Find in past 40 years and identified tens of kinds of malaria vaccine candidate antigens, the multiple different subunit vaccine scheme of these antigens and various vaccine carrier or adjuvant matching design.A large amount of tests shows, the general Vaccine effectiveness of malaria subunit vaccine is low.Because plasmodium has the complicated life history, and its antigen has polymorphism and variability, and many scholars of current malaria vaccine research field think, the subunit's malaria vaccine based on one or more antigens may be difficult to induce the immunizing power with clinical meaning.For this reason, the visual cognitive ability of Recent study is to full worm vaccine.Ultra low-volume erythrocytic stage plasmodium repeatedly inoculation body can induce very strong immune protective efficiency.But the distinct disadvantage of this vaccine to cause infection, particularly at immunologic hypofunction or impaired individuality.Along with the foundation of the progress of Protocols in Molecular Biology, plasmodium gene group sequencing and plasmodium transfection and gene Knockout; make plasmodium genetic inactivation prepare full worm vaccine by knocking out some important gene relevant to differentiation and development of plasmodium to become possibility, the report of scavenging immune protective efficiency after recently having the sporozoite infection animal of gene knockout, can be induced.But; the plasmodium be knocked the vital gene that grows is owing to can not grow to bring and be difficult to mass-produced problem by normal growth for the strain of defect worm; and it is similar with the sporozoite vaccine of radioactive rays attenuation; there is the problem that sporozoite is difficult to the aspects such as a large amount of production, purifying and storage, and just will enter the pathogenic important stage of plasmodium once the immunizing power intrusion blood rbc having a small amount of sporozoite to escape from for the liver phase thus cause vaccine to be protected unsuccessfully.
Summary of the invention
Based on this, for above-mentioned technical problem, be necessary to provide a kind of controlled genomic modification plasmodium, recombinant expression vector and construction process, application, for the research of plasmodium gene function and vaccine.
A kind of recombinant expression vector, is characterized in that, comprises long homology arm and short homology arm and tetracycline repressible protein gene expression frame, Pyrimethamine hcl tolerant gene expression frame and destination gene expression frame between described long homology arm and described short homology arm; Wherein, goal gene promotor and goal gene is contained in described destination gene expression frame; Described goal gene promotor is inserted with tetracycline operator sequence at multiple transcription initiation site place; Described goal gene is selected from the one in plasmodium gene group in functional gene encoding sequence; Described long homology arm is DNA sequence dna corresponding with described functional gene in plasmodium gene group with the sequence of short homology arm.
A construction process for recombinant expression vector, comprises the steps:
The expressed sequence of amplification tetracycline repressible albumen, and be inserted in the plasmid vector containing Pyrimethamine hcl tolerant gene expression frame, obtain the plasmid A containing tetracycline repressible protein gene expression frame and Pyrimethamine hcl tolerant gene expression frame;
Insert the long homology arm of goal gene at 3 ' end of the tetracycline repressible protein gene expression frame of described plasmid A, obtain plasmid B;
The promotor of synthesis after transcription initiation site place is inserted with the modification of tetracycline operator sequence, and the promotor after this modification is inserted into eliminate polyclone restriction enzyme site plasmid vector on, obtain plasmid C, wherein, promotor after described modification at least one site in ef1 α a promoter fragment-205 site ,-184 sites and-163 sites is inserted with described tetracycline operator sequence, and the sequence of described ef1 α a promoter fragment is as shown in SEQIDNo.9;
The downstream of the promotor after modifying described in described plasmid C is inserted goal gene and is built destination gene expression frame, described goal gene is selected from a kind of in plasmodium gene group in functional gene encoding sequence and this goal gene can be started by the promotor after described modification and expresses, and obtain plasmid D;
Described in described plasmid D, the short homology arm of goal gene is inserted in the downstream of destination gene expression frame, obtains plasmid E;
Be inserted in described plasmid B after described plasmid E enzyme is cut, and make tetracycline repressible protein gene expression frame, Pyrimethamine hcl tolerant gene expression frame and destination gene expression frame between described long homology arm and described short homology arm, obtain described recombinant expression vector;
Wherein, the sequence of described long homology arm is that 3 ' of described functional gene corresponding with described goal gene in plasmodium gene group holds non-coding area sequence, and the sequence of described short homology arm is that 5 ' of described functional gene corresponding with described goal gene in plasmodium gene group holds non-coding area sequence.
A kind ofly the construction process described in above-mentioned any embodiment is used to build the recombinant expression vector obtained.
It is interior containing tetracycline repressible protein gene expression frame, Pyrimethamine hcl tolerant gene expression frame and destination gene expression frame that use aforesaid method builds the recombinant expression vector obtained, and multiple transcription initiation site places of goal gene promotor are inserted with tetracycline operator sequence, thus this recombinant expression vector may be used for the function of certain functional gene in conditionality research plasmodium gene group.In addition, coordinate gene Knockout, functional gene expressed sequence corresponding with goal gene in plasmodium gene group is knocked out, again this recombinant expression vector transfection is entered in the plasmodium of gene knockout, controlled genomic modification plasmodium can be obtained, and be that the function studying each functional gene in plasmodium gene group further provides new technical scheme, be with a wide range of applications, as the application of above-mentioned recombinant expression vector in preparation treatment Infected With Plasmodium medicine or diagnosis Infected With Plasmodium reagent.
The plasmodial construction process of controlled genomic modification, comprises the steps:
Described recombinant expression vector is built according to the construction process of the recombinant expression vector described in above-mentioned any embodiment;
Described recombinant expression vector transfection to be entered in plasmodium gene group to replace described functional gene expressed sequence, obtain the described controlled genomic modification plasmodium that gene condition knocks out.
A kind ofly built the controlled genomic modification plasmodium obtained by the plasmodial construction process of controlled genomic modification.
Above-mentioned recombinant expression vector or controlled genomic modification plasmodium are preparing the application in Vaccins of plasmodium.
Above-mentioned recombinant expression vector or controlled genomic modification plasmodium are in preparation diagnosis Infected With Plasmodium reagent, treatment Infected With Plasmodium medicine or the application in research purpose gene function.
The controlled genomic modification plasmodium obtained is built by above-mentioned construction process, after being screened by Pyrimethamine hcl, goal gene can be expressed in containing the environment of tsiklomitsin, when lacking tsiklomitsin in environment, goal gene is not expressed, thus form hereditary defect plasmodium, the function of this goal gene in plasmodium body can be studied thus, and finally provide technical support for preparing Vaccins of plasmodium.
Accompanying drawing explanation
Fig. 1 is the structural representation of the recombinant expression vector of an embodiment;
Fig. 2 is the structural representation of plasmid pL0016;
Fig. 3 is ef1a α promoter sequence+GFP encoding sequence;
Fig. 4 is the structure schematic flow sheet of plasmid pL0016-TetRmut;
Fig. 5 is the structure schematic flow sheet of the promotor-GFP-3 ' DHF after plasmid pT-modifies;
Fig. 6 is the gene targeting plamid vector construction schematic flow sheet for detecting promoter transcription initiation site;
Fig. 7 is Chao Shi PCR result schematic diagram;
Fig. 8 a and Fig. 8 b is the transgenosis plasmodium GFP detection of expression result figure of the tet-on system effect that the plasmodium for detecting structure is suitable for, wherein, Fig. 8 a is for giving GFP expression after ATc, and Fig. 8 b is not for giving GFP expression after ATc, and point brighter in figure represents GFP expression product;
Fig. 9 be with the transgenosis plasmodium obtained after the different plasmid transfection of flow cytomery to ATc (ATc+) with not to the condition of ATc (ATc-) under the comparing result figure of GFP expression;
Figure 10 is the structure schematic flow sheet of cKO expression vector;
Figure 11 is the controlled plasmodial genotype call results figure of genomic modification;
Figure 12 a and Figure 12 b is controlled genomic modification plasmodial GFP detection of expression result figure, and wherein, Figure 12 a is for giving GFP expression after ATc, and Figure 12 b is not for giving GFP expression after ATc, and point brighter in figure represents GFP expression product;
Figure 13 is controlled genomic modification plasmodial Sugar intake ability detected result figure;
Figure 14 is the controlled plasmodial immunology detection result figure of genomic modification.
Embodiment
Below in conjunction with drawings and the specific embodiments, the controlled genomic modification plasmodium of the present invention, recombinant expression vector and construction process, application are described in further detail.
Term of the present invention " gene expression frame " refers to the gene structure be at least made up of promotor, encoding sequence and non-coding sequence.Term of the present invention " functional gene " refers to that the albumen of translation can exercise the gene of certain function.Term of the present invention " goal gene " refers to the expressed sequence for functional study.Unless otherwise defined, all technology used in the present invention and scientific terminology are identical with belonging to the implication that those skilled in the art of the present invention understand usually.The object of the term used in specification sheets of the present invention just in order to describe specific embodiment, is not used in restriction the present invention.
As shown in Figure 1, the recombinant expression vector of an embodiment comprises the long homology arm of goal gene, short homology arm and tetracycline repressible albumen (TetRmut) gene expression frame between long homology arm, short homology arm, Pyrimethamine hcl tolerant gene expression frame (tgdhfr/ts expression cassette) and destination gene expression frame.
Present embodiment additionally provides a kind of construction process of above-mentioned recombinant expression vector, and each several part gene structure of this recombinant expression vector is described below.This construction process comprises the steps:
Step one: the expressed sequence of amplification tetracycline repressible albumen, and be inserted in the plasmid vector containing Pyrimethamine hcl tolerant gene expression frame, obtain the plasmid A containing tetracycline repressible protein gene expression frame and Pyrimethamine hcl tolerant gene expression frame.
Specifically in the present embodiment, plasmid A can use following concrete grammar step structure to obtain:
With T-REx
tMthe genome of-293 cell strains (293 monoclonal cell strain of tsiklomitsin induced cell cycle element B1 (CyclinB1) controlled expression) is as template, primer is TRmut1 and TRmut2, pcr amplification obtains the TetRmut fragment that two ends are all connected with BamHI restriction enzyme site, wherein, the sequence of TRmut1 is as shown in SEQIDNo.7, the sequence of TRmut2 is as shown in SEQIDNo.8, and the sequence of TetRmut fragment is as shown in SEQIDNo.2;
Be inserted into after the TetRmut fragment being connected with BamHI restriction enzyme site obtained is cut by restriction enzyme BamHI is mono-and cut and on dephosphorylized plasmid pL0016 with BamHI is mono-equally, to replace the egfp expression gene on plasmid pL0016, screening TetRmut forward inserts clone and obtains plasmid A.
As shown in Figure 2, plasmid pL0016 carries the sequential structure such as Green Fluorescent Protein Gene Expression frame and SSUrRNA of Pyrimethamine hcl tolerant gene expression frame, 3 base mutations, and with SacII restriction enzyme site on SSUrRNA.The promoter sequence of Green Fluorescent Protein Gene Expression frame is ef1 α a promoter sequence in plasmodium, as shown in SEQ ID No .1; 3 ' end non-coding area sequence is 3 ' end non-coding area sequence (3 ' pbdhfr/ts) of Pyrimethamine hcl resistant gene, and its sequence, as shown in SEQ ID No .3, is below designated as 3 ' DHF.Sequence (5 ' the pbdhfr/ts of promotor in Pyrimethamine hcl tolerant gene expression frame, below be designated as 5 ' DHF) as shown in SEQ ID No .4, encoding sequence (tgdhfr/ts in Pyrimethamine hcl tolerant gene expression frame, below be designated as DHF) as shown in SEQ ID No .5, in Pyrimethamine hcl tolerant gene expression frame, 3 ' end non-coding area sequence is as shown in SEQ ID No .3.Plasmid pL0016 is also outer with HindIII and SapI restriction enzyme site at Pyrimethamine hcl tolerant gene expression frame.Above-mentioned primer TRmut1 and TRmut2 has 1 and 2 base same sense mutations respectively, to facilitate selectional restriction restriction endonuclease.
Step 2: the long homology arm inserting goal gene at 3 ' end of the tetracycline repressible protein gene expression frame of plasmid A, obtains plasmid B.
Specifically in the present embodiment, plasmid B can use following concrete grammar step structure to obtain:
With plasmodium gene group DNA for template, 3 ' of the functional gene corresponding with goal gene that increase holds non-coding area sequence, and connect upper NotI restriction enzyme site at 3 ' end of this 3 ' end non-coding area sequence, again this 3 ' end non-coding area sequence is inserted in plasmid A the SSUrRNA fragment of replacing on plasmid A, obtain plasmid B, wherein, functional gene is HT1 gene, DHODH gene, Lp1A1 gene, OMD gene, SUB1 gene, Rab11A gene or the TrxR gene in plasmodium gene group.
As in one embodiment, when goal gene is HT1 gene, primer 3 ' A1 and 3 ' A2 can be used from 3 ' end non-coding area sequence (3 ' UTR) of plasmodium gene group DNA cloning HT1 gene, wherein the sequence of 3 ' A1 is as shown in SEQ ID No .17, and the sequence of 3 ' A2 is as shown in SEQ ID No .18.With NotI restriction enzyme site (GCGGCCGC) on 3 ' A2 primer.
Step 3: synthesize the promotor after transcription initiation site place is inserted with the modification of tetracycline operator sequence, and the promotor after this modification is inserted into eliminate polyclone restriction enzyme site plasmid vector on, obtain plasmid C.As shown in Figure 2, at least one site in ef1 α a promoter fragment-205 site ,-184 sites and-163 sites of the promotor after modification is inserted with tetracycline operator sequence, and the sequence of ef1 α a promoter fragment is as shown in SEQIDNo.1.Preferably, as in one embodiment, can two tetracycline operator sequences be inserted in-184 site of ef1 α a promoter fragment and be engaged in-163 site and insert a tetracycline operator sequence.
Specifically in the present embodiment, plasmid C can use following concrete grammar step structure to obtain:
With plasmodium gene group DNA for template, primer is pbEF1a1 and pbEF1a578, amplification obtains 5 ' end and is connected with HindIII restriction enzyme site and 3 ' end is connected with the ef1 α a promoter fragment of BamHI, PmeI and SapI restriction enzyme site in turn, and ef1 α a promoter fragment is inserted on pMD18Tsimple plasmid, obtain plasmid C1, wherein, the sequence of pbEF1a1 is as shown in SEQIDNo.9, the sequence of pbEF1a578 is as shown in SEQIDNo.10, and the sequence of ef1 α a promoter fragment is as shown in SEQIDNo.1;
With 3 ' of Pyrimethamine hcl resistant gene end non-coding area sequence for template, primer is 3 ' DHF1 and 3 ' DHF2, amplification obtain 5 ' end be connected with BamHI restriction enzyme site and 3 ' end be connected with in turn XbaI and PmeI restriction enzyme site 3 ' end non-coding area sequence, and by this sequence by being inserted on the plasmid C1 of same double digestion after BamHI and PmeI double digestion, obtain plasmid C2, wherein, the sequence of 3 ' DHF1 is as shown in SEQIDNo.11, the sequence of 3 ' DHF2 is as shown in SEQIDNo.12, 3 ' end non-coding area sequence of Pyrimethamine hcl resistant gene is as shown in SEQIDNo.3,
Use the method for full genome synthesis, the promotor after synthetic modification, then be inserted into replace described ef1 α a promoter fragment in plasmid C2, thus obtain described plasmid C.Such as, in the present embodiment, after SpeI and SwaI double digestion can be used, the promotor after this modification is inserted into replace ef1 α a promoter fragment in the plasmid C2 of same double digestion, thus obtains plasmid C.In the present embodiment, the promotor after modification is preferably inserted with 2 TetO in-184 site and is inserted with the promotor of 1 TetO in-163 site.
Step 4: the downstream of the promotor after modifying in plasmid C is inserted goal gene and built destination gene expression frame, goal gene is selected from a kind of in plasmodium gene group in functional gene encoding sequence and this goal gene can be started by the promotor after modifying and expresses, and obtains plasmid D.
Containing goal gene promotor, goal gene and goal gene 3 ' end non-coding area sequence in destination gene expression frame.Wherein, goal gene promotor is the promotor after above-mentioned modification.Goal gene is selected from the one in plasmodium gene group in functional gene encoding sequence.Goal gene 3 ' holds non-coding area sequence with 3 ' end non-coding area sequence of Pyrimethamine hcl gene, as shown in SEQ ID No .3.
Step 5: the short homology arm of goal gene is inserted in the downstream of destination gene expression frame in plasmid D, obtains plasmid E.
Specifically in the present embodiment, plasmid E can use following concrete grammar step structure to obtain:
With 5 ' of functional gene corresponding with goal gene in plasmodium gene group end non-coding area sequence for template, amplification 5 ' end non-coding area sequence, then be inserted in described plasmid D, obtain described plasmid E.Such as, when functional gene is HT1 gene, upper NotI restriction enzyme site and PmeI restriction enzyme site can be connected successively at 5 ' end of 5 ' the end non-coding area sequence obtained, 3 ' end connects upper XbaI enzyme cutting site, be inserted in the plasmid D of same double digestion after re-using PmeI and XbaI double digestion, obtain plasmid E.And for example, in other embodiments, when goal gene is HT1 gene, primer 5 ' A1 and 5 ' A2 can be used from 5 ' end non-coding area sequence (5 ' UTR) of plasmodium gene group DNA cloning HT1 gene, wherein the sequence of 5 ' A1 is as shown in SEQ ID No .19, and the sequence of 5 ' A2 is as shown in SEQ ID No .20.With NotI restriction enzyme site (GCGGCCGC) on 5 ' A1 primer.
Step 6: be inserted in plasmid B after plasmid E enzyme is cut, and make tetracycline repressible protein gene expression frame, Pyrimethamine hcl tolerant gene expression frame and destination gene expression frame between long and short homology arm, obtain recombinant expression vector.
Specifically in the present embodiment, step 6 is inserted in the plasmid B of same double digestion after using SapI and HindIII double digestion to plasmid E, obtains recombinant expression vector.
Further, incorporated by reference to Fig. 1 and Fig. 2, in the present embodiment, Pyrimethamine hcl tolerant gene expression frame is between tetracycline repressible protein gene expression frame and destination gene expression frame, short homology arm is located at goal gene 3 ' and holds the downstream of non-coding area sequence and holding the end of non-coding area sequence to be provided with NotI restriction enzyme site away from goal gene 3 ', long homology arm to be located in tetracycline repressible protein gene expression frame 3 ' end non-coding area sequence downstream and in away from tetracycline repressible protein gene expression frame the end of 3 ' end non-coding area sequence be provided with NotI restriction enzyme site, thus cut this recombinant expression vector by restriction enzyme NotI enzyme, make long, tetracycline repressible protein gene expression frame between short homology arm is communicated with, Pyrimethamine hcl tolerant gene expression frame and the linear sequence of destination gene expression shaped as frame, this linear order by gene targeting be inserted into functional gene knock out after the correct position of plasmodium gene group.
Containing tetracycline repressible protein gene expression frame, Pyrimethamine hcl tolerant gene expression frame and destination gene expression frame in this recombinant expression vector, and the transcription initiation site place of goal gene promotor is inserted with tetracycline operator sequence, thus this recombinant expression vector may be used for the function of certain functional gene in conditionality research plasmodium gene group.In addition, coordinate gene Knockout, functional gene expressed sequence corresponding with goal gene in plasmodium gene group is knocked out, again this recombinant expression vector transfection is entered in the plasmodium of gene knockout, controlled genomic modification plasmodium can be obtained, and be that the function studying each functional gene in plasmodium gene group further provides new technical scheme, be with a wide range of applications, as recombinant expression vector is preparing the application in Vaccins of plasmodium, the application etc. of recombinant expression vector in preparation treatment Infected With Plasmodium medicine or diagnosis Infected With Plasmodium reagent.
In addition, present embodiment additionally provides the plasmodial construction process of a kind of controlled genomic modification, and it comprises the steps:
Recombinant expression vector is built according to the construction process of above-mentioned recombinant expression vector;
Recombinant expression vector enzyme is cut laggard line linearity process, and transfection is entered in plasmodium gene group, make long and short homology arm and between sequential structure be inserted into the functional gene position that plasmodium is knocked, obtain controlled genomic modification plasmodium.
The controlled genomic modification plasmodium obtained is built by above-mentioned construction process, after being screened by Pyrimethamine hcl, goal gene can be expressed in containing the environment of tsiklomitsin, when lacking tsiklomitsin in environment, goal gene is not expressed, thus form regulatable hereditary defect plasmodium, the function of this goal gene in plasmodium body can be studied thus, and finally provide technical support for preparing Vaccins of plasmodium.This controlled genomic modification plasmodium also can be applied in preparation diagnosis Infected With Plasmodium reagent, treatment Infected With Plasmodium or Other diseases (as tumour, HIV etc.) medicine.
As when doing certain functional gene research in plasmodium body, above-mentioned controlled genomic modification plasmodium can be used, when inductor exists, this genetically modified controlled genomic modification plasmodium can be beneficial to the plasmodial screening of transgenosis, clones and provide the full worm of transgenosis of abundance for subsequent gene functional study by normal growth; And when not providing inductor, plasmodium will show the phenotype of goal gene disappearance, thus this goal gene can be lacked plasmodial metamorphosis, biological nature changes and host studies aspects such as its immunoreactive changes, analysis and synthesis evaluation, thus determines goal gene function.
As by this controlled genomic modification plasmodium prepare Vaccins of plasmodium application time, not only can at machine Immune inducing in vivo scavenging protection, can also produce in a large number and stablize, and in the main phase (erythrocytic stage) that plasmodium is caused a disease, body is played a good protection, its ultimate principle is: when providing tsiklomitsin inductor, this genomic modification plasmodium can normal growth (as can be infected to animal model or carrying out vitro culture) to provide a large amount of full worm vaccines; But not giving in the body of inductor, this genomic modification plasmodium cannot normal growth and propagation, but short-lived worm alive can induce host to produce effective immune protective efficiency.
In order to more clearly understand technology contents of the present invention, be described with reference to the accompanying drawings especially exemplified by following examples.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Various conventional chemical reagent used in embodiment, is commercially available prod.
Be that the present embodiment primer sequence used gathers with following table 1.
Table 1
The present embodiment constructs a kind of controlled genomic modification plasmodium, first build cKO expression vector as shown in Figure 10, with green fluorescent protein (GFP) as reporter gene protein, then by this cKO expression vector transfection P. berghei, P. berghei is by tail vein injection infecting mouse, infecting mouse obtains genetically modified controlled genomic modification plasmodium through Pyrimethamine hcl screening, with dehydration tetracycline (ATc) as system induction agent, gene expression regulation is carried out to this genomic modification plasmodium, mainly comprises the steps:
1, transcription initiation site is determined, optimal startup:
1) plasmid construction
As shown in Figure 2, pL0016 plasmid carries Pyrimethamine hcl resistant gene (tgdhfr/ts expression cassette).As shown in Figure 4, TetR gene order by PCR from T-REx
tM-293 cell strain genome amplifications obtain, and be the convenience of follow-up restriction enzyme selection simultaneously, same sense mutation 3 bases of TetR gene in PCR primer, obtain the RetR gene order of suddenling change, its two ends are connected with BamHI restriction enzyme site, are designated as TetRmut, and primer is TRmut1 and TRmut2.Be inserted into same BamH I after TetRmut fragment singly being cut by restriction enzyme BamH I singly to cut and the GFPmut3 fragment of dephosphorylized plasmid pL0016 replacing pL0016, screening TetRmut forward inserts clone and obtains plasmid pL0016-TetRmut.
As shown in Figure 5, GFP gene coded sequence is inserted into the downstream of the plasmodium ef1 α a promotor having merged 2 or 3 TetO sequences, comprises the steps:
The first step, with plasmodium gene group DNA for template, primer is pbEF1a1 and pbEF1a578, amplification obtains ef1 α a promoter fragment, 5 ' end is introduced Hind III restriction enzyme site, 3 ' end has introduced BamH I, Pme I and Sap I restriction enzyme site successively, this fragment is inserted into pMD18Tsimple plasmid and obtains plasmid pT-pbef1 α a;
Second step, 3 ' the UTR fragment (3 ' pbdhfr/ts) of pcr amplification dhfr/ts gene, primer is 3'DHF1 and 3'DHF2, amplified fragments 5 ' end is introduced BamH I and EcoR V restriction enzyme site, 3 ' end is introduced XbaI and Pme I restriction enzyme site, and this fragment is inserted into plasmid pT-pbef1 α a and obtains plasmid pT-pbef1 α a-3 ' DHF after being cut by BamH I and Pme I couple;
3rd step, pcr amplification GFP encoding sequence, primer is GFP1 and GFP717, is inserted into plasmid pT-pbef1 α a-3 ' DHF and obtains plasmid pT-pbef1 α a-GFP-3 ' DHF after amplified fragments BamH I and EcoR V double digestion;
4th step, incorporated by reference to Fig. 2, select ef1 α a promotor in plasmodium gene group, use the method synthesis of full genome synthesis respectively in-205 of ef1 α a promotor in Jin Ruisi bio tech ltd, the site such as-184 and-163 insert the promotor after the modification of TetO sequence, build the promotor after obtaining four modifications: ef1 α a205-2TetO (namely-205 site of ef1 α a promotor are inserted with two TetO), ef1 α a184-2TetO (namely-184 site of ef1 α a promotor are inserted with two TetO), ef1 α a163-2TetO (namely-163 site of ef1 α a promotor are inserted with two TetO) and ef1 α a163-TetO+184-2TetO (i.e.-163 site of ef1 α a promotor be inserted with a TetO and-184 site are inserted with two TetO).Promotor after four of synthesis modify be added to promotor (ef1 α a-presume+2tetO) that prediction site (-45 site) inserts 2 TetO totally five modify after promoter fragment be inserted in plasmid pT-pbef1 α a-GFP-3 ' DHF respectively by SpeI and SwaI double digestion to replace ef1 α a promoter fragment, obtain the promotor-GFP-3 ' DHF after plasmid pT-modification, comprise following five plasmids: pT-pbef1 α a-presume+2tetO-GFP-3'DHF, pT-pbef1 α a205+2tetO-GFP-3'DHF, pT-pbef1 α a184+2tetO-GFP-3'DHF, pT-pbef1 α a163+2tetO-GFP-3'DHF and pT-pbef1 α a163+tetO-184-2tetO+GFP-3'DHF.
As shown in Figure 6, five plasmids obtained above are used respectively Sap I and Hind III double digestion, be inserted in the two plasmid pL0016-TetRmut cut of same enzyme, obtain gene targeting plasmid vector: p21, pATcon205, pATcon184, pATcon163 and pATcon184-163.Meanwhile, plasmid pT-pbef1a α-GFP-3 ' DHF is obtained control plasmid p20 by being inserted into plasmid pL0016-TetRmut after Sap I/Hind III double digestion.
2) plasmodium is cultivated and transfection: the gene targeting plasmid vector of all above-mentioned structures and control plasmid are respectively by transfection P. berghei after Sac II linearization for enzyme restriction and be incorporated into genomic SSUrRNA gene locus.
When the mouse Tubifex level of infection P. berghei reaches 1 ~ 3%, get blood by mouse heart and collect containing plasmodial anticoagulated whole blood, the plasmodium overwhelming majority in blood is in ring bodies or trophont phase.The blood collected removes white corpuscle and thrombocyte by CF11, then the red corpuscle of enrichment Infected With Plasmodium on 74%Percoll layering liquid is added to, the centrifugal 20min of 5000 turns/min room temperature, cell layering, brown cellular layer (comprising the red corpuscle of the Infected With Plasmodium of more than 99%) in the middle of careful collection, wash twice with the RPMI1640 perfect medium containing 20% inactivated fetal bovine serum, in substratum, also add 25mMHEPES, 2mM glutamine, 2g/L glucose, 10mg/L xanthoglobulin and 50 μ g/mL neomycinsulphates.The red corpuscle of the Infected With Plasmodium after enrichment is with every milliliter 1 × 10
7~ 2 × 10
7the concentration of individual cell is resuspended in RPMI1640 perfect medium, and moves into culturing bottle, enters low-oxygen gas mixture (containing 5%CO to " blowing " in culturing bottle
2, 10%O
2and 85%N
2) 3 ~ 4min, screw bottle cap and culturing bottle is placed on just fully to have hanged the speed shake of RBC on shaking table, cultivation 16 ~ 18 hours in 37 DEG C of thermostat containers.Take a morsel solution of red blood cells afterwards that cultivate, centrifugal, removes supernatant, add 10 μ l serum, mixing, smear, and dyeing, whether basis of microscopic observation plasmodium reaches the schizont phase.
After cultivation, ring bodies and the trophont phase polypide grow the schizont phase, if be in the mature schizont phase more than 70% worm, namely can be used for transfection.Get 0.5 × 10
7~ 1 × 10
7infected With Plasmodium red corpuscle after individual cultivation, in the centrifugal 5min of 200 turns/min, abandons supernatant, is resuspended in the human T cells that 100 μ L add 10 μ g plasmid to be transfected in advance
in solution, re-suspension liquid moves into electric revolving cup, and electric revolving cup is placed in Amaxa consideration convey dye instrument (manufacturer: Lonza company, model
iI) in, choose U33 program and carry out electricity turn, electricity adds 50 μ L perfect mediums immediately with Cell protection after turning in transfection cup, and then this 150 μ L electricity is turned mixed solution by tail vein injection to the mouse in 8 ~ 10 week age.Electricity turned after 24 ~ 48 hours, and Recipient mice gives 0.07mg/mL Pyrimethamine hcl by tap water and continues 7 ~ 10 days.When mouse Tubifex level reaches 3 ~ 4%, collect Mouse whole blood by Culling heart blood and carry out conservation and detection.
3) 5 ' RACE and nest-type PRC
Extract the transgenosis plasmodium total serum IgE of the above-mentioned plasmid pL0016 transfection containing GFPmut expression cassette with Trizol, total serum IgE as with
test kit (Invitrogen company article No.: L1502-01) carries out the parent material of 5 ' RACE.According to
the operation steps of test kit, total serum IgE 5 ' end is by CIP (calfintestinalphosphatase, CIP) dephosphorylation, and the mRNA of CIP on band " cap sequence " does not affect, then, the 5 ' cap of mRNA is removed with TAP (tobaccoacidpyrophosphatase, TAP), and mRNA and the GeneRacerRNAOligo gone after cap is connected, the mRNA that GeneRacerRNAOligo connects exists
carry out reverse transcription reaction and nest-type PRC under the effect of III reversed transcriptive enzyme, nest-type PRC primer is GeneRacer5 ' Primer and the GeneRacer5 ' NestedPrimer that provides of test kit and GFP special primer GFP541 and the GFP333 that synthesizes voluntarily for this reason.First Nucleotide that mRNA and GeneRacerRNAOligo is connected is exactly transcription initiation site.Utilize
test kit carries out 5 ' RACE can determine transcription initiation site accurately, because
the principle of test kit determine 5 ' RACE result for the template that follow-up nest-type PRC provides be all the full-length cDNA obtained by full length mRNA molecule reverse transcription.
Nest-type PRC the first step PCR primer is: GeneRacer
tM5 ' Primer and GFP541; Reaction conditions is: 94 DEG C, 5min; 94 DEG C, 30s, 52 DEG C, 30s, 72 DEG C, 2min, 35 circulations; 72 DEG C, 5min.Nest-type PRC second step PCR primer is: GeneRacer
tM5 ' NestedPrimer and GFP333; Reaction conditions is: 94 DEG C, 5min; 94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C, 1.5min, 30 circulations; 72 DEG C, 5min.PCR primer is separated by 1.0% agarose gel electrophoresis, SYBRGreen dyeing and ultraviolet transilluminator observe with take pictures, result is as shown in Figure 7.The first step and second step PCR primer are respectively the smear of about 730bp and 520bp.NestedPCR second step product is undertaken cloning by TOPOTA Cloning Kit and got some clones and checks order.Sequence DNAMAN sequence analysis software is analyzed.46 clones are sent to check order altogether after the nested PCR product clone that 3 times independently 5 ' RACE tests.Sequencing result comparison is topmost transcription initiation site with analyzing-184 sites determining ef1 α a promotor,-205 and-163 grades be time main transcription initiation site, the controlled effect of plasmodium destination gene expression of plasmid pT-pbef1 α a163+tetO-184-2tetO+GFP-3'DHF transfection is better.
4) plasmodium cloning
The Balb/c mouse of every aseptic operation excision spleen rejects scavenger cell by tail vein injection 100 μ L clodronate liposome (clodronateliposomes), after 72 hours, mouse after process is by the Infected With Plasmodium red corpuscle of tail vein injection limiting dilution, every injected in mice 0.7 ~ 1 Infected With Plasmodium red corpuscle, each transgenosis plasmodium carries out cloning needs 20 ~ 25 mouse, about 20% will become positive infection, within 7 ~ 8 days, can Tubifex be detected after infection.
5) transgenosis plasmodium gene type detects
The blood collecting Infected With Plasmodium removes leucocyte-removing by CF11 post, then extract test kit with blood DNA and extract transgenosis plasmodium gene group DNA, analyze transfected plasmids by PCR and Southernblotting and be incorporated on the correct site of plasmodium gene group.
6) transgenosis plasmodium GFP detection of expression
The transgenosis plasmodium worm strain of recovery liquid nitrogen conservation, transgenosis plasmodium gives 1 × 10 by every mouse
6the dosage of individual parasitized erythrocyte infects two groups of mouse, and one group of mouse gives ATc with tap water, and another group mouse does not give ATc.Infect mouse after 6 days and pass through CO
2after anesthesia, Culling heart blood gets whole blood, blood by 74%percoll enrichment Infected With Plasmodium red corpuscle, to wash after 2 times with PBS and resuspended be 1 × 10
6the concentration of/mL, be added in 96 orifice plates or on slide glass in confocal fluorescent basis of microscopic observation and shooting GFP expression, result as figures 8 a and 8 b show, show the success of said gene target practice plamid vector construction, and Successful transfection enters plasmodium gene group.With the average fluorescent strength of the GFP of the p20ATc-of flow cytomery for 100, the GFP relative mean fluorescent intensity level of other sample is as shown in Figure 9 (mean value of 3 different experiments and standard deviation): the ATc+/ATc-ratio of visible pATcon184-163 is maximum and pATcon184-163ATc-value is minimum, is also that the control effects of the tetracycline-controlled expression system of pATcon184-163 gene targeting plasmid vector is best.
2, recombinant expression vector is built, and transfection is entered in plasmodium gene group, build the plasmodium that gene conditionality knocks out: adopt the tetracycline-controlled expression system optimized, promotor after modification adopts and is inserted with two TetO sequences and-163 for site is inserted with the ef1 α a promotor of a TetO sequence in-184 site, goal gene adopts HT1 gene, conditionality knocks out the expressed sequence of plasmodial HT1 gene, and the plasmodium gene group of gene knockout is entered in recombinant expression vector transfection.HT1 gene expression product is the sugar transporters on plasmodium cytolemma, is considered to the vital gene of Blood-stage Plasmodium.
1) plasmid construction, as shown in Figure 10, comprises the steps:
By the 2ADNA fragment of synthesis (as shown in sequence table SEQ IDNo.6,2ADNA fragment derives from foot and mouth disease virus, its expression product can effective self cleavage (self-cleave), and its series connection albumen can both equivalent express) be inserted into pMD18T plasmid, obtain plasmid pT-2A, the introducing of 2ADNA fragment 5 ' end has BamHI and EcoRI restriction enzyme site, and 3 ' end introducing has Bgl II and Xho I restriction enzyme site.
Primer 2 AGFP1 and 2AGFP717 amplification GFP Express Sequence Tags, 5 ' end of this GFP Express Sequence Tags is introduced Bgl II restriction enzyme site, 3 ' end is introduced EcoRV and Xho I restriction enzyme site, GFP Express Sequence Tags is inserted into pT-2A by Bgl II with Xho I double digestion and obtains pT-2A-GFP, then by BamHI and EcoRV double digestion from pT-2A-GFP cut 2A-GFP fragment replace plasmid pT-modify after GFP fragment promotor-GFP-3'DHF, obtain plasmid pT-modify after promotor+2A-GFP-3'DHF.
HT1 encoding sequence is obtained from plasmodium gene group DNA cloning by primer HTC3 and HTC2, then the HT1 encoding sequence that restriction enzyme BamHI and EcoRI double digestion increase is used, and after being inserted into plasmid pT-modification on promotor+2A-GFP-3'DHF, obtaining plasmid pT-and modify rear promotor+goal gene-2A-GFP-3'DHF.
Primer 5 ' A1 and 5 ' A2 is from the 5 ' UTR of plasmodium gene group DNA cloning HT1, and after being inserted into plasmid pT-modification after this 5 ' UTR is used XbaI and PmeI double digestion, promotor+goal gene-2A-GFP-3'DHF obtains plasmid pT-and modifies rear promotor+short homology arm of goal gene-2A-GFP-3'DHF+.
Primer 3 ' A1 and 3 ' A2 is inserted into plasmid pL0016-TetRmut replacement SSUrRNA from the 3 ' UTR of plasmodium gene group DNA cloning HT1 and obtains the long homology arm of plasmid pL0016-TetRmut-.
After being modified by plasmid pT-, promotor+short homology arm of goal gene-2A-GFP-3'DHF+ is by Sap I and Hind III double digestion, the long homology arm of plasmid pL0016-TetRmut-that the fragment cut inserts same double digestion obtains recombinant expression vector cKO expression vector, is designated as pHT1cKO.Containing NotI restriction enzyme site (GCGGCCGC) on primer 5 ' A1 and 3 ' A2, thus NotI restriction enzyme site is incorporated on carrier pHT1cKO by primer 5 ' A1 and 3 ' A2 as plasmid linearization site.
2) plasmodium vitro culture and transfection:
Substantially with " 1, determine transcription initiation site; optimal startup " operation corresponding to part, different places is: pHT1cKO is by the linearizing of restriction enzyme Not I single endonuclease digestion, transfection plasmodium, Infected With Plasmodium mouse after transfection (24 ~ 48 hours in advance give 0.2mg/mlATc by tap water and continue to give after infection).Infecting in the tap water of mouse after 24 hours and add 0.07mg/ml Pyrimethamine hcl maintenance 7 ~ 9 days, when Tubifex level reaches more than 1%, collecting Mouse whole blood conservation transgenosis plasmodium by anaesthetizing rear Culling heart blood.
3) transgenosis plasmodium cloning: with the operation that " 1, determine transcription initiation site, optimal startup " part is corresponding.
4) transgenosis plasmodium gene type detects: with the operation that " 1, determine transcription initiation site, optimal startup " part is corresponding.
Southernblotting analyzes: genomic dna restriction enzyme Hind III enzyme is cut, 20 μ g enzymes cut after genomic dna be separated by 0.8% agarose gel electrophoresis, then by transfer printing, DNA is transferred on positively charged nylon membrane from agarose, with film 1kbHT1 probe 42 DEG C of hybridized overnight of DNA---HT1 probe passes through DIG on PCRDIGprobesynthesiskit (Roche) mark by HT1 gene fragment, finally, film DIGLuminescentDetectionKit (Roche) after hybridization carries out developing the color and detects, result as shown in figure 11, WT represents the strain of wild-type P. berghei worm, HT1cKO represents the P. berghei worm strain that HT1 conditionality knocks out.
5) transgenosis plasmodium morphologic detection: transgenosis plasmodium cultivates 18 hours in vitro, remarkable with the plasmodium morphological differences not to ATc to ATc, substantially grow the mature schizont phase to the plasmodium of ATc, and be substantially stuck in the trophont phase to the plasmodial development of ATc.
6) transgenosis plasmodium detects host toxicity: the transgenosis plasmodium worm strain of recovery liquid nitrogen conservation, transgenosis Infected With Plasmodium two groups of mouse, give 1 × 10 by every mouse
6the erythrocytic dosage of individual Infected With Plasmodium, one group of mouse is to ATc, another group mouse does not give ATc, after infecting, every day gets a smear of bleeding from mousetail and prepares blood smear, dry that rear methyl alcohol is fixed, basis of microscopic observation blood smear after Giemsa stain dyeing, the mean value of all erythrocytic percentage ratio is accounted for as the horizontal index of Tubifex, to the rising of Tubifex persistent levels, death after the mouse infection of ATc using the parasitized erythrocyte counting 8 visuals field (about 100, each visual field red corpuscle); Significantly low to Tubifex level after the mouse infection of ATc, survival.
7) transgenosis plasmodium GFP detection of expression: with the operation that " determining transcription initiation site; optimal startup " part is corresponding, as depicted in figs. 12 a and 12b, result proves that above-mentioned recombinant expression vector successfully constructs, and Successful transfection enters the correct position of plasmodium gene group.
8) transgenosis plasmodium Sugar intake ability detects: the transgenosis plasmodium worm strain of recovery liquid nitrogen conservation, transgenosis plasmodium gives 1 × 10 by every mouse
6the dosage of individual parasitized erythrocyte infects two groups of mouse, and one group of mouse is to ATc, and another group mouse does not give ATc, and infects and get Mouse Blood after 6 days, blood passes through percoll layering liquid enrichment Infected With Plasmodium red corpuscle, to wash after 2 times with PBS and resuspended be 1 × 10
6the concentration of/mL, 20 minutes are hatched altogether with 200 μMs of fluorescently-labeled glucose (2-NBDG), the centrifugal 18s of 10000 turns/min, abandon supernatant, re-suspended cell after PBS washes 2 times, cell moves to 96 hole enzyme plates and uses multi-functional microplate reader to detect fluorescence, and determined wavelength is 488nm (exciting)/540nm (transmitting), and result as shown in figure 13.
9) immunology detection: by this gene knockout/express regulatable genomic modification plasmodium immune mouse, immunity attacks worm to afterwards for 30 days the plasmodium of mouse infection wild-type, mouse all survives and does not occur obvious Tubifex level, and result as shown in figure 14.
What the present embodiment was used is mouse P. berghei, can understand, and in other embodiments, also may be used for other kind plasmodium as mouse Xia Shi plasmodium or people plasmodium etc.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a recombinant expression vector, it is characterized in that, comprise long homology arm and short homology arm and tetracycline repressible protein gene expression frame, Pyrimethamine hcl tolerant gene expression frame and destination gene expression frame between described long homology arm and described short homology arm; Wherein, goal gene promotor and goal gene is contained in described destination gene expression frame; Described goal gene promotor is inserted with tetracycline operator sequence at multiple transcription initiation site place; Described goal gene is selected from the one in plasmodium gene group in functional gene encoding sequence; Described long homology arm is DNA sequence dna corresponding with described functional gene in plasmodium gene group with the sequence of short homology arm.
2. a construction process for recombinant expression vector, is characterized in that, comprises the steps:
The expressed sequence of amplification tetracycline repressible albumen, and be inserted in the plasmid vector containing Pyrimethamine hcl tolerant gene expression frame, obtain the plasmid A containing tetracycline repressible protein gene expression frame and Pyrimethamine hcl tolerant gene expression frame;
Insert the long homology arm of goal gene at 3 ' end of the tetracycline repressible protein gene expression frame of described plasmid A, obtain plasmid B;
The promotor of synthesis after transcription initiation site place is inserted with the modification of tetracycline operator sequence, and the promotor after this modification is inserted into eliminate polyclone restriction enzyme site plasmid vector on, obtain plasmid C, wherein, promotor after described modification at least one site in plasmodium ef1 α a promoter fragment-205 site ,-184 sites and-163 sites is inserted with described tetracycline operator sequence, and the sequence of described plasmodium ef1 α a promoter fragment is as shown in SEQIDNo.1;
The downstream of the promotor after modifying described in described plasmid C is inserted goal gene and is built destination gene expression frame, described goal gene is selected from a kind of in plasmodium gene group in functional gene encoding sequence and this goal gene can be started by the promotor after described modification and expresses, and obtain plasmid D;
Described in described plasmid D, the short homology arm of goal gene is inserted in the downstream of destination gene expression frame, obtains plasmid E;
Be inserted in described plasmid B after described plasmid E enzyme is cut, and make tetracycline repressible protein gene expression frame, Pyrimethamine hcl tolerant gene expression frame and destination gene expression frame between described long homology arm and described short homology arm, obtain described recombinant expression vector;
Wherein, the sequence of described long homology arm is that 3 ' of described functional gene corresponding with described goal gene in plasmodium gene group holds non-coding area sequence, and the sequence of described short homology arm is that 5 ' of described functional gene corresponding with described goal gene in plasmodium gene group holds non-coding area sequence.
3. the construction process of recombinant expression vector as claimed in claim 2, is characterized in that, build described plasmid A and specifically comprise the steps:
With T-REx
tMthe genome of-293 cell strains is as template, primer is TRmut1 and TRmut2, pcr amplification obtains the TetRmut fragment that two ends are all connected with BamHI restriction enzyme site, wherein, the sequence of described TRmut1 is as shown in SEQIDNo.7, the sequence of described TRmut2 is as shown in SEQIDNo.8, and the sequence of described TetRmut fragment is as shown in SEQIDNo.2;
Be inserted into after the TetRmut fragment being connected with BamHI restriction enzyme site obtained is cut by restriction enzyme BamHI is mono-and cut and on dephosphorylized plasmid pL0016 with BamHI is mono-equally, to replace the egfp expression gene on plasmid pL0016, screening TetRmut forward inserts clone and obtains described plasmid A.
4. the construction process of recombinant expression vector as claimed in claim 3, is characterized in that, build described plasmid B and specifically comprise the steps:
With plasmodium gene group DNA for template, 3 ' of the described functional gene corresponding with described goal gene that increase holds non-coding area sequence, and connect upper NotI restriction enzyme site at 3 ' end of this 3 ' end non-coding area sequence, again this 3 ' end non-coding area sequence is inserted in described plasmid A the SSUrRNA fragment of replacing on described plasmid A, obtain described plasmid B, wherein, described functional gene is HT1 gene, DHODH gene, Lp1A1 gene, OMD gene, SUB1 gene, Rab11A gene or the TrxR gene in plasmodium gene group.
5. the construction process of recombinant expression vector as claimed in claim 4, is characterized in that, build described plasmid C and specifically comprise the steps:
With plasmodium gene group DNA for template, primer is pbEF1a1 and pbEF1a578, amplification obtains 5 ' end and is connected with HindIII restriction enzyme site and 3 ' end is connected with the ef1 α a promoter fragment of BamHI, PmeI and SapI restriction enzyme site in turn, and described ef1 α a promoter fragment is inserted on pMD18Tsimple plasmid, obtain plasmid C1, wherein, the sequence of described pbEF1a1 is as shown in SEQIDNo.9, the sequence of described pbEF1a578 is as shown in SEQIDNo.10, and the sequence of described ef1 α a promoter fragment is as shown in SEQIDNo.1;
With 3 ' of Pyrimethamine hcl resistant gene end non-coding area sequence for template, primer is 3 ' DHF1 and 3 ' DHF2, amplification obtain 5 ' end be connected with BamHI restriction enzyme site and 3 ' end be connected with in turn XbaI and PmeI restriction enzyme site 3 ' end non-coding area sequence, and by this sequence by being inserted on the plasmid C1 of same double digestion after BamHI and PmeI double digestion, obtain plasmid C2, wherein, the sequence of described 3 ' DHF1 is as shown in SEQIDNo.11, the sequence of described 3 ' DHF2 is as shown in SEQIDNo.12, 3 ' end non-coding area sequence of described Pyrimethamine hcl resistant gene is as shown in SEQIDNo.3,
Use the method for full genome synthesis, synthesize the promotor after described modification, then be inserted into replace described ef1 α a promoter fragment in plasmid C2, thus obtain described plasmid C.
6. the construction process of recombinant expression vector as claimed in claim 5, is characterized in that, build described plasmid E and specifically comprise the steps:
Hold non-coding area sequence for template with 5 ' of described functional gene corresponding with described goal gene in plasmodium gene group, increase described 5 ' end non-coding area sequence, then be inserted in described plasmid D, obtains described plasmid E.
7. the plasmodial construction process of controlled genomic modification, is characterized in that, comprise the steps:
Described recombinant expression vector is built according to the construction process of the recombinant expression vector according to any one of claim 1 ~ 6;
Described recombinant expression vector transfection to be entered in plasmodium gene group to replace described functional gene expressed sequence, obtain the described controlled genomic modification plasmodium that gene condition knocks out.
8. one kind is built the controlled genomic modification plasmodium obtained by the plasmodial construction process of controlled genomic modification according to claim 7.
9. recombinant expression vector as claimed in claim 1 or genomic modification plasmodium controlled are as claimed in claim 8 preparing the application in Vaccins of plasmodium.
10. recombinant expression vector as claimed in claim 1 or genomic modification plasmodium controlled are as claimed in claim 8 in preparation diagnosis Infected With Plasmodium reagent, treatment Infected With Plasmodium medicine or the application in research purpose gene function.
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| CN110168093A (en) * | 2017-09-12 | 2019-08-23 | 广州中科蓝华生物科技有限公司 | It is a kind of transfect cytozoon kit and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110168093A (en) * | 2017-09-12 | 2019-08-23 | 广州中科蓝华生物科技有限公司 | It is a kind of transfect cytozoon kit and its application |
| CN110168093B (en) * | 2017-09-12 | 2023-08-15 | 中科蓝华(广州)生物医药技术有限公司 | Kit for transfecting intracellular parasites and application thereof |
| CN109312345A (en) * | 2018-04-26 | 2019-02-05 | 广州中科蓝华生物科技有限公司 | A kind of promoter and its application |
| WO2019205057A1 (en) * | 2018-04-26 | 2019-10-31 | 广州中科蓝华生物科技有限公司 | Promoter and application thereof |
| CN109312345B (en) * | 2018-04-26 | 2021-07-20 | 广州中科蓝华生物科技有限公司 | Promoter and application thereof |
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| CN105368865B (en) | 2019-04-09 |
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