CN104342375A - Application of candida albicans SMT3 gene and candida albicans attenuated strain lack of SMT3 gene - Google Patents
Application of candida albicans SMT3 gene and candida albicans attenuated strain lack of SMT3 gene Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to application of the candida albicans SMT3 gene and a candida albicans attenuated strain lack of the SMT3 gene. The the candida albicans SMT3 gene is used for preparing the strain lack of the smt3/smt3 or used for preparing or screening candida albicans treatment medicines. The invention further discloses the candida albicans attenuated strain. The SMT3 gene in the attenuated strain is not expressed. According to the application of the candida albicans SMT3 gene and the candida albicans attenuated strain, the SMT3 gene in the candida albicans is knocked out; functions of the SMT3 gene in the cell cycle and the morphogenesis and transformation process are studied; and finally the important function of the SMT3 gene in candida albicans toxicity expressions is determined through a mice system infection experiment.
Description
Technical field
The present invention relates to genetically engineered field, be specifically related to the purposes of the SMT3 gene of a kind of Candida albicans SUMO and the Candida albicans attenuated strain of disappearance SMT3 gene.
Background technology
Candida albicans (Candida albicans) is a kind of a kind of opportunistic human body cause illness fungi be separated to clinically, special in the patient of immune down regulation, as organ transplant patients, HIV patient etc., superficial part and deep system infections widely can be caused, infection site comprises oral cavity, vagina etc., cause white mouth, vaginitis etc., also epidermis can be invaded and endotheliocyte enters blood and arrives internal organs, as kidney, brain etc., cause septicemia, seriously can cause death (Odds, F.C.1994.J.Am.Acad, Dermatol.31:S2-S5.).Candida albicans, under different growth conditionss, has different growthhabits, can change under certain condition between these different shapes.Comprise yeast state (yeast form), and mycelia state (hyphae), and white conversion between bacterium (white phase) and grey bacterium (opaque phase).This form transformation ability directly affects pathogenecity (Odds, the F.C.1985.Crit Rev Microbiol.12:45-93 of Candida albicans; Brown, A.J.P.et al.1999.Trends Microbiol.7:334-338.), its system infections ability of the bacterial strain of modality capability defect declines or disappear (Lo, H.J.et al, Cell90:939-949.) greatly.Find in the WO-1 bacterial strain that in vain-ash conversion is separated first clinically.The bacterium of these two kinds of forms also has different pathogenic, and white bacterium has stronger pathogenecity in system infections, and grey bacterium has stronger pathogenecity in epidermis infects.Thus lime conversion is two kinds of forms that candidiasis produces to adapt to different microenvironments, and these two kinds of forms play an important role for the pathogenecity of candidiasis.Meanwhile, lime conversion realizes its mating for Candida albicans is also necessary.Similar with yeast saccharomyces cerevisiae, Candida albicans, in order to realize homologous recombination, needs the process through a mating, there are some researches show that Candida albicans ash bacterium form mating efficiency is approximately 10 of white bacterium form
6doubly, therefore, Candida albicans will realize mating, first will carry out lime conversion, is converted to grey bacterium form, just can realizes mating by white bacterium.
Prior art have found the key factor of regulation and control Candida albicans lime conversion by the method had complementary functions, Wor1(White-Opaque Regulator1), Wor1 plays critical regulating and controlling effect in candidiasis lime modality.In Wor1 regulated and control network research subsequently, we are by yeast two-hybrid screening and identify some interactional albumen with it, comprising two SUMO E3 ligase enzymes containing distinctive SP-RING structure.
SUMO(small ubiquitin-related modifier, small ubiquitin-related modifier proteins) be modify after a kind of reversible protein translation found before two more than ten years, after this, the enzyme of the modification of many participation SUMOization and SUMO process is found and identifies.Meanwhile, the searching for SUMOization substrate also draws a series of albumen that SUMOization can occur and modify, and these albumen participate in the links of cell function.
SUMOization modification is the process of a height mobilism, and its result is different because of concrete albumen, comprises the location changing albumen, the activity of modulin, and the stability affecting albumen in some cases.At first sight these results seem do not have what common point; But these results are all derived from the change of SUMOization modification for the interaction property of protein in fact.
The research of SUMO in yeast saccharomyces cerevisiae is very ripe, but existing research in Candida albicans is also more rarely seen.
In people's dialogue Candida cell cycles such as Stephen W.Martin, the behavior of Septin is studied.Their research shows, although SUMOization modification does not occur Septin in Candida albicans, SUMO is positioned Septin specifically; Further research shows, the component Cdc11 of Septin can with the protein binding of a SUMOization, thus make it be positioned Septin.
The people such as Michelle D.Leach have found the target protein that about 30 SUMOization are modified in Candida albicans, and wherein great majority have typical SUMOization modification pattern Ψ-K-X-E, and these albumen are mainly concerned with the answering of various stimulation to external world.
Summary of the invention
The object of the invention is to the method by gene knockout, the function of research SMT3 gene in cell cycle, form generation and switching process, and a kind of Candida albicans attenuated strain lacking SMT3 gene is provided.
First the present invention discloses the purposes of Candida albicans SMT3 gene, is the Candida albicans smt3/smt3 gene-deleted strain of not expressing for the preparation of SMT3 gene, or for the preparation of, screening Candida albicans medicine.
SMT3 gene of the present invention is containing, for example the nucleotide sequence shown in SEQ ID NO:18, and its SUMO(small ubiquitin-related modifier ubiquitin of encoding in Candida albicans is correlated with little modified protein).
Preferably, the aminoacid sequence of described SUMO albumen is as shown in SEQ ID NO:19.
Preferably, smt3/smt3 gene-deleted strain of the present invention is the attenuated strain of Candida albicans.In described Candida albicans smt3/smt3 gene-deleted strain, SMT3 gene is not expressed.
Candida albicans medicine of the present invention is with SMT3 gene for therapeutic target gene, the medicine that SMT3 gene is not expressed or the medicine being antagonism object with the albumen of SMT3 genetic expression (SUMO); Or using SMT3 gene and other genes jointly as the medicine that target gene is in addition reticent.By with SMT3 gene for target spot, screening or prepare this gene is not expressed medicine, for making the perform toxic attenuation of Candida albicans, thus treatment infection by Candida albicans.Such as, this Candida albicans medicine can be disturbed by RNA or the method for gene recombination makes SMT3 gene not express; Or by preparation SMT3 protein antagonist, the SUMO of SMT3 genetic expression is not played a role.
Second aspect present invention discloses a kind of Candida albicans attenuated strain, and be Candida albicans smt3/smt3 gene-deleted strain, in described attenuated strain, SMT3 gene is not expressed.
Further, Candida albicans attenuated strain of the present invention belongs to ubiquitin and to be correlated with little modification protein gene gene-deleted strain.
Preferably, in Candida albicans smt3/smt3 gene-deleted strain of the present invention, SMT3 gene is knocked out by the method for homologous recombination.Compared with wild-type Candida albicans, there is major defect in attenuated strain of the present invention in growth, even if also can only slowly grow in rich medium, and the exception of adjoint bacterium colony and cellular form aspect.Simultaneously, in lime transition experiment, smt3/smt3 is to promoting that the incentive condition of lime conversion is insensitive, and lime efficiency of conversion significantly reduces than the wild-type of same process, and the grey bacterium bacterium colony formed is unstable, also can be transformed into white bacterium state or filamentous growth state under cryogenic.
Third aspect present invention discloses the construction process of aforementioned Candida albicans attenuated strain, and for two copies of method to SMT3 gene in Candida albicans by homologous recombination knock out, concrete steps are as follows:
1) the knocking out of SMT3 gene first copy: adopt the primer of sequence shown in SEQ ID NO:1-2 to increase from Candida albicans wild type strain genomic dna the upstream sequence fragment of SMT3, utilize XhoI+PstI enzyme to cut to be connected in carrier pCUB6, obtain plasmid pKO-SMT3Up; Then increase from Candida albicans wild type strain genome with the primer of sequence shown in SEQ ID NO:3-4 and obtain the downstream sequence I of SMT3, cut with BamHI+NotI enzyme and be connected in aforementioned pKO-SMT3Up, obtain knocking out plasmid pSMT3KO I; Described plasmid pSMT3KO I enzyme that knocks out is cut rear conversion Candida albicans, and in cell, homologous recombination obtains single knock-out bacterial strain that the first copy SMT3 knocks out;
2) the knocking out of SMT3 gene second copy: adopt the primer of sequence shown in SEQ ID NO:1-2 to increase from Candida albicans wild type strain genomic dna the upstream sequence fragment of SMT3, utilize XhoI+PstI enzyme to cut to be connected in carrier pCUB6, obtain plasmid pKO-SMT3Up; Then increase from Candida albicans wild type strain genome with the primer of sequence shown in SEQ ID NO:3,5 and obtain the downstream sequence II of SMT3, cut with BamHI+NotI enzyme and be connected in aforementioned pKO-SMT3Up, obtain knocking out plasmid pSMT3KO II; Described plasmid pSMT3KO II enzyme that knocks out is cut rear conversion Candida albicans, and in cell, homologous recombination obtains the two knock-out bacterial strain of smt3/smt3 that the second copy SMT3 knocks out.
Fourth aspect present invention discloses the application of aforementioned Candida albicans attenuated strain in the growth of research Candida albicans and toxicity function, or the application in preparation or screening Candida albicans medicine.
The present invention finally also discloses a kind of method preparing Candida albicans medicine, for the SMT3 gene in Candida albicans for target, the expression of reticent SMT3 gene.
Further, be the target of the target that disturbs using the SMT3 gene in Candida albicans as RNA or gene recombination, the expression of reticent SMT3 gene.
The present invention utilizes URA-BLAST strategy to carry out gene knockout to the gene SMT3 of SUMO albumen.There is serious growth defect in the smt3/smt3 after knocking out, comprises the speed of growth slow, paramophia and unhomogeneity; Meanwhile, in modality, because of cell fission obstacle, smt3/smt3 occurs that under non-filamentous growth conditions pseudohypha grows, and under the incentive condition promoting lime conversion, then occur obvious response lag, compare wild-type, its lime efficiency of conversion significantly reduces.Therefore can reach a conclusion: SUMO gene act as important role in Candida albicans cell cycle, modality and toxicity.
Beneficial effect: the present invention have studied the impact of Candida albicans gene SMT3 on mycelial growth and bacterial strain toxicity, and successfully constructs a kind of Candida albicans attenuated strain, and SMT3 gene is not expressed in attenuated strain of the present invention.Candida albicans hyphal of the present invention growth and toxicity correlation factor gene SMT3 and Candida albicans attenuated strain all can be used for preparation or screen Candida albicans medicine.
Accompanying drawing explanation
Fig. 1: the qualification of gene deletion strains
The experimental result that Fig. 2: SUMO gene-deleted strain is slow in growth
The bacterium colony of Fig. 3: SUMO gene-deleted strain and the experimental result of cellular form
Fig. 4: checking SUMO take part in the experimental result of the posttranslational modification of Wor1
The experimental result of Fig. 5: SUMO effect in Candida albicans toxicity
Embodiment
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, instead of in order to limit the scope of the invention.
Candida albicans (Candida albicans) is a kind of human body opportunistic pathomycete, can cause and infect widely, but its real virulence factor and pathogenesis are not also studied clear completely.There are some researches show, there are with it pathogenic closely-related two kinds of modality modes in Candida albicans: yeast-hyphal development and lime modality.The present invention utilizes URA-BLAST strategy to carry out gene knockout to SUMO gene SMT3 unique in Candida albicans.Major defect is there is in the smt3/smt3 after knocking out in growth, even if also can only slowly grow in rich medium, and the exception of adjoint bacterium colony and cellular form aspect; Simultaneously, in lime transition experiment, smt3/smt3 is to promoting that the incentive condition of lime conversion is insensitive, and lime efficiency of conversion significantly reduces than the wild-type of same process, and the grey bacterium bacterium colony formed is unstable, also can be transformed into white bacterium state or filamentous growth state under cryogenic.Mouse toxicological experiment result shows, the virulence of smt3/smt3 in system infections significantly declines.
Candida albicans SMT3 gene of the present invention can represent with CaSMT3, also represents with SMT3 when obscuring with Saccharomyces Cerevisiae in S MT3 gene, and Candida albicans SMT3 gene-deleted strain smt3/smt3 represents.
Basic experiment method:
Method 1: Candida albicans gene group DNA extracting
Candida albicans overnight incubation ddH
2o washes 1 time, be suspended in (1M sorbyl alcohol in 500 μ l solution A, 100mM EDTA pH8.0), add the Zymolase of 5 μ l 20mg/ml, place after 1 hour for 37 DEG C, high speed centrifugation removes supernatant, cell 500 μ l of TE buffer(20mM TrisHCl pH7.5, 1mM EDTA) wash once, be suspended in 350 μ l of TE buffer, and add 90 μ l of solution B (250mM EDTA pH8.0, 400mM TrisHCl pH8.0, 2% SDS), place 30 minutes for 65 DEG C, add 80 μ l of 5M KAc, place 1 hour on ice, high speed centrifugation 5 minutes, draw supernatant and add the dehydrated alcohol of 1ml, place 20 minutes for-20 DEG C, high speed centrifugation 5 minutes, precipitation is washed once with 70% ethanol, centrifugal post-drying.
Method 2: the conversion of Candida albicans
Prepare PEG/LiAc solution (pH 7.5) 10m1; Plasmid 5 μ g is added successively, 10 μ l10mg/ml milt DNA, mixing in 1.5mI Eppendof pipe; Add 0.1ml competent cell and 0.6m1 PEG/LiAc, votex mixing; 30 DEG C of 200rpm cultivate 30min; Add 70 μ l DMSO, mix gently; 42 DEG C of thermal shocking 15min, period shakes up frequently gently; Ice bath 2min: high speed centrifugation 15s, sops up supernatant, with 0.2m1 TE re-suspended cell; Coat on suitable nutrition screening SD flat board.
Method 3: mouse system infects
With body weight at 16-18g ICR male mice for experimental subjects, by the various bacterial strain of tail vein injection 100 μ l Candida albicans, and cultivate observation 25 days.Each bacterial strain divides height two concentration injections, and injection concentration is respectively 5 × l0
7cells/ml and 5 × l0
6cells/ml.
Method 4: co-immunoprecipitation and Western are hybridized
Candida albicans expression strain is cultured to saturated, by A in 30 DEG C in YPD substratum
6000.05 transfers in 25 DEG C of cultivation 6h in YPD substratum, to A
600cultivate 3.5h, 50ml culture collected by centrifugation for 0.6-1.0 or in YPD+10%Serum substratum in 37 DEG C, the aseptic washing of 10ml once.Following steps are all carried out at 4 DEG C.Cell is resuspended in 0.5ml yeast cell lysate (200mM Tris-HCl pH8.0,400mM (NH
4)
2sO
4, 10mM MgCl
2, 1mM EDTA, 10% Glycerol, 7mM β-mercaptoethanol, 2mM benzamidine, 1mM PMSF, 2 μ g/ml pepstatin A, 2 μ g/ml leupeptin, 4 μ g/ml antipain), add 0.4g pickling glass pearl (425-600 μm, Sigma).Vibrator FP120 shakes 3 times, each 30s, interval ice bath 5min.The centrifugal 5min of lysate 12,000rpm, supernatant is centrifugal 5min again, collects supernatant and measures protein concentration ,-70 DEG C of preservations.
Get 500 μ l cell extracts, mix with isopyknic IP Buffer, add the protein G agarose beads of 10 μ l bed volumes, mixing low-speed centrifugal after 1 hour is rotated at 4 DEG C, draw the FLAG monoclonal antibody that supernatant adds 5 μ g, rotate mixing 1 hour at 4 DEG C, then add the protein G agarose beads of 30 μ l bed volumes, continue mixing 2 hours.Wash co-immunoprecipitation thing five times with IP Buffer, be then resuspended in 2 × protein electrophorese sample-loading buffer, carry out SDS-PAGE electrophoresis.Transfer on nitrocellulose filter by wet method by protein band, with the TBS-T solution closing membrane 2-3 hour containing 5% skim-milk, primary antibodie 4 DEG C of combinations are spent the night.Film is washed 1 time, 5 minutes by TBS-T room temperature.Resist in room temperature in conjunction with 1 hour with corresponding two, then wash film four times with TBS-T, each 5 minutes, use ECL reagent colour development.
The two structure knocking out Candida albicans strain of embodiment 1 smt3/smt3
Knocked out by two copies of method to the SMT3 gene of expressing SUMO in Candida albicans of homologous recombination.Knock out strain and carried out genotype identification by PCR, concrete grammar is as follows:
1. the knocking out of Article 1 karyomit(e) SMT3 gene
With primer SMT3-UP-F(SEQ ID NO:1) and primer SMT3-UP-R(SEQ ID NO:2) the upstream sequence fragment of the SMT3 that increases from wild type strain SC5314 genomic dna is about 300bp, utilize XhoI+PstI enzyme to cut to be connected in carrier pCUB6, obtain plasmid pKO-SMT3Up.Again with primer SMT3-DOWN-F(SEQ ID NO:3) and primer SMT3-DOWN-R(SEQ ID NO:4) amplification obtains SMT3 from wild type strain SC5314 genome downstream sequence is about 300bp, cutting with BamHI+NotI enzyme is connected in pKO-SMT3Up, obtains knocking out plasmid pSMT3KO I.Gained plasmid is transformed into wild-type bacteria (JYC5, the MTLa/a type bacterium from SC5314) after cutting with XhoI and SstI enzyme after amplification.
Knocking out of 2.SMT3 gene second copy
The structure and first that second copy knocks out plasmid copy similar, be in order to avoid second take turns conversion time recombinate and replace the locus that the first round correctly knocks out, the SMT3 segments downstream for knocking out restructuring has increased 400bp, and concrete grammar is as follows.
Knock out the structure of plasmid pSMT3KO II: with primer SMT3-UP-F(SEQ ID NO:1) and SMT3-UP-R(SEQ ID NO:2) the upstream sequence fragment of the SMT3 that increases from wild type strain SC5314 genomic dna is about 300bp, utilize XhoI+PstI enzyme to cut to be connected in carrier pCUB6, obtain plasmid pKO-SMT3Up.Again with primer SMT3-DOWN-F(SEQ ID NO:3) and primer SMT3-DOWN400-R(SEQ ID NO:5) amplification obtains SMT3 from genome downstream sequence is about 400bp, cutting with BamHI+NotI enzyme is connected in pKO-SMT3Up, obtains knocking out plasmid pSMT3KO II.Gained knocks out plasmid and after enzyme is cut, is transformed into first of step 1 gained copies the bacterial strain knocked out, and obtains that smt3/smt3 that the second copy SMT3 knocks out is two knocks out Candida albicans strain after homologous recombination in cell.
3. the qualification of pair copy deletion mycopremna
Identified knocking out son by the method for PCR: respectively with SMT3 locus upstream primer (SEQ ID NO:6 and SEQ ID NOA:11), open reading frame inner primer (SEQ ID NOA:8 and SEQ ID NOA:10, SEQ ID NOA:9 and SEQ ID NOA:11) and knock out Plasmid Primer (SEQ ID NOA:8 and SEQ ID NOA:7) and carry out PCR qualification, concrete qualification result is shown in accompanying drawing 1.
Table 1 SMT3 knocks out and identifies the primer
The function of embodiment 2 SUMO in Candida albicans Growth of Cells
There is major defect in SUMO gene-deleted strain smt3/smt3, even if in abundant YPD substratum, it is significantly slow that its growth also occurs in growth.The present embodiment illustrates the impact of SUMO for the Candida albicans speed of growth respectively in solid and liquid nutrient medium, and concrete grammar is as follows:
1. experimental subjects: the smt3/smt3 of embodiment 1 preparation is two knocks out Candida albicans strain as experiment sample, with untreated wild-type Candida albicans strain (WT and SC5314) sample in contrast.
2. experimental technique
Obtain time smt3/smt3 knocks out strain and find the growth of its transformant slowly, therefore after gene-deleted strain has been identified, in order to the function of SUMO in Candida albicans cell growth process is described definitely, its growth curve is measured.
Getting wild-type SC5314 (Wildtype) and SUMO gene-deleted strain (smt3/smt3) the bacterium liquid of incubated overnight, is OD according to final concentration
600=0.1 was transferred in the fresh YPD liquid nutrient medium of 3mL, is placed in 30 DEG C, cultivates under 240rpm, every 45 minutes its OD of sampling monitoring
600value, experimental result is shown in Fig. 2 A.
Meanwhile, solid YPD flat board has done colony growth experiment.Get wild-type and the gene-deleted strain bacterium liquid of incubated overnight, be diluted to 10 by concentration gradient respectively
7, 10
6, 10
5, 10
4, 10
3, 10
2cell/mL, then respectively gets 1mL point on YPD flat board, is placed in 30 DEG C of incubators, and cultivate to take out afterwards for about 2 days and observe colony growth situation, result as shown in Figure 2 B.
3. experiment conclusion
A and B of Fig. 2 to be respectively in liquid and solid medium SUMO for the impact of the Candida albicans speed of growth.As shown in Figure 2, there is major defect in SUMO gene-deleted strain smt3/smt3 in growth; Even if in abundant YPD substratum, it is significantly slow that its growth also occurs.
The function of embodiment 3SUMO in the generation of Candida albicans form and maintenance process
1. experimental technique
In view of the exception of SUMO gene-deleted strain smt3/smt3 in the speed of growth, its phenotype in the generation of Candida albicans form is further examined or check.
The smt3/smt3 bacterium liquid of incubated overnight is transferred on abundant substratum YPD flat board, cultivate about 7 days in 30 DEG C of incubators after, observes its colonial morphology; Compare with wild-type Candida albicans strain (SC5314).
2. experimental result
Experimental result is as shown in Figure 3: the two knock-out bacterial strain of smt3/smt3 occurs bacterium colony unhomogeneity under air jet flow condition, be classified as white bacterium bacterium colony (Wh), ash bacterium bacterium colony (Op) and corrugated bacterium colony (as shown in b, d, e in Fig. 3), Fig. 3 illustrates this three kinds of colonial morphologies.
Sample respectively these three kinds of colonial morphologies and observe cellular form, see g, i, j, k in Fig. 3 respectively, the cellular form that these three kinds of bacterium colonies are corresponding differs from corresponding wild-type bacteria.Also find that the growthhabit of similar pseudohypha appears in smt3/smt3 under air jet flow condition simultaneously.Relate to before this for the result of study of Septin, infer that this is the tissue disorder due to Septin, the misgrowth form that cell fission is obstructed and is caused.
In lime modality, also there is obvious defect in smt3/smt3.Concrete manifestation is delayed to the stimuli responsive of common promotion lime conversion: such as, at CO
2under culture condition, compare wild-type bacteria, the lime transfer capability of smt3/smt3 significantly reduces.Meanwhile, the grey bacterium of smt3/smt3 is unstable, can return white bacterium form with very high rate conversion.
Comprehensive conclusions is known, and the disappearance of SUMO have impact on lime conversion in two: one is that the efficiency causing lime to be changed reduces; Two is cause grey bacterium state labile.
The effect of embodiment 4SUMO in regulatory factor Wor1 posttranslational modification
One, the preliminary identification of SUMO posttranslational modification effect
1. experiment material
For the ease of detecting endogenous Wor1 albumen, testing the sequence by incorporating one section of coding HA label at 3 ' end of this protein open reading frame, thus obtaining Wor1-HA fusion rotein.
Incorporate the structure of the wild type strain of WOR1-HA: design packet is containing WOR1orf end 60bp and two primers (shown in table 2SEQ ID NO:12-13) comprising WOR1 downstream 60bp, with pJI HA-CaURA3-HA(from Jinmi Kim laboratory, K.-H.Lee et al.Biochemical and Biophysical Research Communications 337,2005) be masterplate, PCR method amplification HA-URA3-HA fragment.PCR primer is transformed into parent bacterium (the MTLa/a type bacterium from SC5314) after concentrated, drawing to the flat board containing 5 '-FOA, impelling URA3 gene ring to go out, so that subsequent transformation through being accredited as positive transformant.
Incorporate the structure of the smt3/smt3 absence type bacterial strain of WOR1-HA: design packet is containing WOR1orf end 60bp and two primers (shown in table 1SEQ ID NO:12-13) comprising WOR1 downstream 60bp, with pJI HA-CaURA3-HA for masterplate, pcr amplification HA-URA3-HA fragment.PCR primer is transformed into the smt3/smt3 absence type bacterial strain that embodiment 1 builds after concentrated, drawing to the flat board containing 5 '-FOA, impelling URA3 gene ring to go out, so that subsequent transformation through being accredited as positive transformant.
WOR1-HA strain construction the primer integrated by table 2
2. experimental technique:
In order to obtain the clone that Wor1 expresses, the bacterial strain that WOR1 in above-mentioned wild-type and smt3/smt3 incorporates HA label is passed through CO
2stimulate and obtain grey bacterium bacterium colony, enlarged culturing is carried out to grey bacterium bacterium colony, collect thalline and extract total protein and detect and immunoprecipitation analysis for Western blot.
Concrete grammar is: the wild-type and smt3/smt3 that incorporate WOR1-HA are lined the SD flat board that pH is 6.8, be placed in 20%CO
2, cultivate, until grow grey bacterium bacterium colony under normal temperature.To be transferred to by the grey bacterium bacterium colony of gained in liquid and enlarged culturing, to gained culture extracting total protein, carry out Western blot detection to the total protein of grey bacterium culture, experimental result is shown in Fig. 4 B.
3. interpretation
From Fig. 4 B, above Wor1 self band ~ position detection of 12KDa is defined as HMW(Higher Molecular Weight to a band) band, infer that it is that the SUMOization of Wor1 modifies band from stripe size; And in smt3/smt3, this HMW band do not detected.These results show, and Wor1 there occurs SUMOization and modifies in the process of its regulation and control lime conversion.Infer that Wor1 may have occurred SUMOization and modifies and regulate and control its function whereby.
Two, the further checking of SUMO posttranslational modification effect
1. experiment material
The construction process of the plasmid of his-SMT3 amalgamation and expression is: from SC5314 genome, amplify SMT3 gene with primer SEQ ID NO:14 and primer SEQ ID NO:15, pBA1 carrier (BES116+ADH1promoter is connected to after cutting process with BglII+ClaI enzyme, from the flat laboratory of Liu Hao) in, obtain his-SMT3-pBA1, under the control of Candida albicans ADH1 promotor, realize the amalgamation and expression of his-SMT3.
The preparation of Smt3 antibody: amplify SMT3 gene open reading frame fragment with the primer of sequence shown in SEQ ID NO:16 and SEQ ID NO:17 from SC5314 genomic dna, be inserted in pET-28a carrier (Novagene) after using NcoI and XhoI double digestion, obtain his-SMT3 prokaryotic expression plasmid.Above-mentioned his-SMT3 expression plasmid is transformed in prokaryotic expression bacterial strain BL21, it is induced to express with IPTG, the fusion rotein his-Smt3 expressed, through nickel post (Ni-NTA Sefinose Resin) enriching and purifying, obtains the protokaryon albumen that final concentration is about 5mg/mL, purity more than 95%.Obtain the antiserum(antisera) in rabbit source successively as antigen immune rabbit, obtain the antibody (α-Smt3) of antifungal SUMO albumen through proA-Agarose purifying.
In table 3 SMT3 prokaryotic expression plasmid and Candida albicans, his-SMT3 fusion expression plasmid builds primer used
2. experimental technique
In order to verify that the HMW band detected is that SUMOization modifies band, using the wild type strain incorporating WOR1-HA of preparation in the plasmid steps for importing one of the his-SMT3 amalgamation and expression of structure as experimental group (His-Smt3) further; Meanwhile, to have imported the wild type strain incorporating WOR1-HA of pBA1 carrier as empty vector control group (untagged).Stimulated by CO2 and obtain grey bacterium culture, enlarged culturing, extracting total protein, carries out enriching and purifying by nickel post (Ni-NTA).Whether the albumen be purified to has Wor1 albumen with HA antibody test.Carry out immunoprecipitation in contrast with the Smt3 antibody of preparation, experimental result is shown in Fig. 4 A simultaneously.
3. interpretation
As shown in Figure 4 A, in the bacterial strain of his-SMT3-pBA1 plasmid having imported amalgamation and expression, the sample that cellular lysate liquid obtains through Ni-NTA enriching and purifying detects through Western blot, obtain the band of a Wor1, the position of this band is consistent with the HMW pillar location of Wor1 described before, meanwhile, swimming lane corresponding to the control strain of his-SMT3 label is not being had Wor1 band then not detected.Therefore, the albumen obtained through Ni-NTA enriching and purifying is and in this bacterial classification, is specially His-Smt3 with SUMO() the Wor1 albumen modified.
Meanwhile, in contrast, we carry out immunoprecipitation analysis with purified anti-Smt3 antiserum(antisera) to above-mentioned lysate, and result is the Wor1 having SUMO to modify in the lysate of two bacterial strains.This proves that the state of fusion expression plasmid on Wor1 albumen that we import does not affect, and in the bacterial strain not importing label, the Wor1 of SUMOization just can not by under the enrichment of nickel post.
More than comprehensive, we pass through the method for nickel post enriching and purifying and immunoprecipitation, demonstrate Wor1 in Candida albicans, there is SUMOization modification.
The toxicity of the Candida albicans attenuated strain of embodiment 5CaSMT3 genetically deficient
In order to set forth SUMO further for the impact of modality and at the pathogenic middle role of Candida albicans, the present embodiment has carried out mouse system infection experiment to gene-deleted strain smt3/smt3.
1. experimental subjects:
Body weight is at 16 ~ 18g ICR male mice;
The smt3/smt3 of embodiment 1 preparation is two knocks out Candida albicans strain, wild-type Candida albicans strain (WT, SC5314)
2. experimental technique
Using body weight at 16 ~ 18g ICR male mice as experimental subjects, be divided into experimental group (smt3/smt3) and control group (wildtype), often organize 6 mouse, by tail vein injection 100 μ l Candida albicans bacterial strain, (experimental group injection smt3/smt3 is two knocks out Candida albicans strain, control group injection wild-type Candida albicans strain SC5314), cultivate observation 25 days.Each bacterial strain divides height two concentration injections, and injection concentration is respectively 5 × l0
7cells/ml and 5 × l0
6cells/ml.Experimental result see Fig. 5 (Fig. 5 corresponding be 5 × l0
6the result of cells/ml).
3. experimental result
As shown in Figure 5, there is significant virulence defect in smt3/smt3, and the mouse of injection smt3/smt3 is seldom dead.In the time range of observing statistics, the mouse majority of injection smt3/smt3 can be survived; And the mouse of injecting wild-type is all dead in 7 days.
Claims (10)
1. the purposes of Candida albicans SMT3 gene is the Candida albicans smt3/smt3 gene-deleted strain of not expressing for the preparation of SMT3 gene, or for the preparation of, screening Candida albicans medicine.
2. purposes as claimed in claim 1, it is characterized in that, SMT3 gene is containing, for example the nucleotide sequence shown in SEQ ID NO:18.
3. purposes as claimed in claim 1, is characterized in that, SMT3 gene ubiquitin of encoding in Candida albicans is correlated with little modified protein.
4. a Candida albicans attenuated strain, be Candida albicans smt3/smt3 gene-deleted strain, in described attenuated strain, SMT3 gene is not expressed.
5. Candida albicans attenuated strain as claimed in claim 4, is characterized in that, described Candida albicans attenuated strain is that ubiquitin is correlated with little modification protein gene gene-deleted strain.
6. Candida albicans attenuated strain as claimed in claim 5, is characterized in that, described ubiquitin little modified protein of being correlated with contains the aminoacid sequence shown in SEQ ID NO:19.
7. Candida albicans attenuated strain as claimed in claim 4, it is characterized in that, in described Candida albicans smt3/smt3 gene-deleted strain, SMT3 gene is knocked out by the method for homologous recombination.
8. the construction process of Candida albicans attenuated strain described in the arbitrary claim of claim 4-7, for two copies of method to SMT3 gene in Candida albicans by homologous recombination knock out, concrete steps are as follows:
1) the knocking out of SMT3 gene first copy: adopt the primer of sequence shown in SEQ ID NO:1-2 to increase from Candida albicans wild type strain genomic dna the upstream sequence fragment of SMT3, utilize XhoI+PstI enzyme to cut to be connected in carrier pCUB6, obtain plasmid pKO-SMT3Up; Then increase from Candida albicans wild type strain genome with the primer of sequence shown in SEQ ID NO:3-4 and obtain the downstream sequence I of SMT3, cut with BamHI+NotI enzyme and be connected in aforementioned pKO-SMT3Up, obtain knocking out plasmid pSMT3KO I; Described plasmid pSMT3KO I enzyme that knocks out is cut rear conversion Candida albicans, and in cell, homologous recombination obtains single knock-out bacterial strain that the first copy SMT3 knocks out;
2) the knocking out of SMT3 gene second copy: adopt the primer of sequence shown in SEQ ID NO:1-2 to increase from Candida albicans wild type strain genomic dna the upstream sequence fragment of SMT3, utilize XhoI+PstI enzyme to cut to be connected in carrier pCUB6, obtain plasmid pKO-SMT3Up; Then increase from Candida albicans wild type strain genome with the primer of sequence shown in SEQ ID NO:3,5 and obtain the downstream sequence II of SMT3, cut with BamHI+NotI enzyme and be connected in aforementioned pKO-SMT3Up, obtain knocking out plasmid pSMT3KO II; Described plasmid pSMT3KO II enzyme that knocks out is cut rear conversion Candida albicans, and in cell, homologous recombination obtains the two knock-out bacterial strain of smt3/smt3 that the second copy SMT3 knocks out.
9. the application of Candida albicans attenuated strain described in the arbitrary claim of claim 4-7 in the growth of research Candida albicans and toxicity function, or the application in preparation or screening Candida albicans medicine.
10. prepare a method for Candida albicans medicine, for the SMT3 gene in Candida albicans for target, the expression of reticent SMT3 gene.
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