CN112011564A - Novel cryptococcus nuclear indication vector and construction method and application thereof - Google Patents
Novel cryptococcus nuclear indication vector and construction method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
Abstract
The invention discloses a novel cryptococcus nuclear indication vector and a construction method and application thereof, wherein the vector fuses and expresses a nuclear localization gene CWF19 and a green fluorescence gene EGFP, and the specific position and number change of a nucleus can be indicated through green fluorescence. The vector obtained by the invention has the advantages of high safety, lasting and stable expression and simple preparation, and has important indication significance for the reproduction of cryptococcus neoformans, particularly sexual reproduction.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a novel cryptococcus nuclear indicator vector, and also relates to a method and application of the indicator vector.
Background
Cryptococcus neoformans is a yeast-type opportunistic pathogenic fungus widely existing in nature, mainly infects organ transplant patients with low immunity, AIDS patients with immunity loss and the like, and causes diseases of different degrees such as fungal pneumonia and cryptococcal meningitis. The 2017 survey data of Rajasingham et al show that worldwide there are up to 278000 people infected with cryptococcus annually and that 18 million people die, with a mortality rate of up to 64.7%. At present, three clinical medicines comprise amphotericin B, 5-flucytosine and fluconazole, but the prognosis of patients receiving treatment is very poor, which also brings great troubles to clinical treatment and resident life.
Cryptococcus neoformans is usually in the form of yeast when it is nutritious, has both a and a mating types, and is now represented by budding and is less infectious. However, in the wild, the cryptococcus can resist the extreme environment together in a sexual reproduction mode under the condition of dry and lack of nutrition, and a propagule-spore with strong stress resistance is generated. The spore is small, can be inhaled into the lung by a human body, generates field planting after reaching a certain amount, and buries hidden troubles for later health. Based on the mating characteristics and transmission pathway of cryptococcus, the process and mechanism of sexual reproduction are always the focus of attention of various research institutions and researchers. The current research base shows that in the sexual reproduction process of cryptococcus, two cells are fused to form a clavicle combination, and after four cell nucleuses are generated by meiosis after cell nucleus fusion, the top of hyphae expands to correspondingly generate 4 spore chains. However, the mechanisms and associated genes that affect fusion and division in cryptococcus nuclei are not completely understood. In order to better study the change of the number of nuclei in each structure of each cell period, the DAPI staining method has great damage to the cell structure in operation and is complicated in operation. Thus, an expression vector of fusion of the gene specifically positioned in the cell nucleus and the fluorescent protein is constructed, and then the fusion is transformed into the cryptococcus, so that the dynamic change of the cell nucleus in cell structures such as cells, binuclear hyphae, basidiophore heads and the like can be directly and clearly observed.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a novel cryptococcus nuclear indicator vector; the second purpose of the invention is to provide a construction method of a novel cryptococcus nuclear indication vector; the invention also aims to provide the application of the carrier as a cryptococcus neoformans sexual reproduction indicator carrier.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the cryptococcus neoformans nuclear indication vector contains a nuclear localization gene CWF19 and an expression frame of green fluorescence gene EGFP fusion expression, and the nucleotide sequence of the nuclear localization gene CWF19 is shown as SEQ ID No. 5.
In the invention, the indicator vector is connected into the BamHI and SpeI enzyme cutting sites of the pCN19 vector by a sequence shown in SEQ ID NO. 5.
2. The construction method of the novel cryptococcus nuclear indication vector comprises the following specific steps: cloning or synthesizing a cell nucleus localization gene CWF19, and then connecting the cell nucleus localization gene CWF19 to the downstream of the EGFP gene of a fungus expression vector containing the EGFP gene to form an expression frame containing fusion expression of the cell nucleus localization gene CWF19 and the green fluorescence gene EGFP.
In the present invention, the nuclear localization gene CWF19 was cloned or synthesized and ligated into the BamHI and SpeI cleavage sites of pCN19 vector.
3. The carrier is used as an indication carrier for sexual reproduction of cryptococcus neoformans.
In the invention, the carrier is used as an indicating carrier for indicating the change of the position and the number of the cell nucleus by the cryptococcus neoformans.
The invention has the beneficial effects that: the novel cryptococcus nuclear indication vector provided by the invention is fused and expressed with the EGFP gene by utilizing the characteristic of Cwf19, so that the continuous observation of a target in the sexual reproduction process can be effectively met, and Cwf19 is the cryptococcus gene, so that the expression effect is stable, the cryptococcus gene is not easy to miss, the safety is high, the cost is low, and the daily research is greatly facilitated.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a plasmid map of recombinant vector pTBL 177.
FIG. 2 shows the verification of the recombinant vector (A: PCR verification for constructing the recombinant vector; B: restriction enzyme digestion verification for constructing the recombinant vector, two bands of 5575bp and 4213bp are generated after EcoRV restriction enzyme digestion; C: subcellular localization result for transforming the recombinant vector into cryptococcus neoformans).
FIG. 3 shows the results of the localization of Cryptococcus neoformans (A: 1000X Cwf19 indicates the result of the nucleus; B: the result of the co-localization of the fluorescent strain with DAPI, re-verifying its nuclear localization, Bar 10 μm).
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1 design and amplification of PCR primers for Gene of interest CWF19
The gene sequence number CNAG _01362 of CWF19 in the cryptococcus neoformans searched from the NCBI website is found, the CDS sequence of the target gene is found in a Fungi DB database, the sequence of the CDS sequence is shown as SEQ ID NO.5, a primer is designed according to the obtained sequence, an upstream primer contains a BamHI enzyme cutting site, and a downstream primer contains a SpeI enzyme cutting site.
TL1106:5’-gacgagctgtacggatccatggggagagacagcaccaacacg-3’(SEQ ID NO.1)；
TL1107:5’-ctggcggccgttactagtttaaacgccgtttcccacggtcca-3’(SEQ ID NO.2)；
The PCR reaction system with the amplified target fragment size of 3004bp is prepared according to the reaction requirements of DreamTaq enzyme (Dream Taq Green PCR Master Mix 2 x).
The reaction template is an extracted genome of a wild type strain H99 of the Cryptococcus neoformans grubbs variety, and the specific extraction steps are as follows:
1) taking 2 sterile 2mL disruption tubes, and adding 600 μ L425-600 nm sterile glass beads and 600 μ L TENTS Buffer in the tubes in advance;
2) taking a proper amount of fresh growing thallus from a sterilized toothpick, fully stirring, adding 600 mu L phenol chloroform isoamyl alcohol (25:24:1), balancing, and mechanically crushing (the speed is 6m/s, 6 rounds in total, and the time is 40 s/round);
3) centrifuging the crushed sample at 15000rpm for 20 min;
4) putting 180 mu L of supernatant into a new 1.5mL centrifuge tube, adding 1/10 volume (20 mu L of 3M NaAc) and 2 times volume (400 mu L of absolute ethyl alcohol), fully mixing uniformly, and then placing at-20 ℃ for 20min for precipitation of nucleic acid;
5) centrifuging at 15000rpm for 10min, discarding supernatant to obtain precipitate as genome, washing with 200 μ L75% ethanol, centrifuging at 12000rpm for 5min, and discarding waste liquid; reversely buckling the centrifugal tube on clean absorbent paper, absorbing the redundant liquid, and rightly placing to volatilize the ethanol; add 100. mu.L of ddH preheated to 65 ℃ in advance2Dissolving O to obtain H99 genome; after measuring the concentration by using Nanodrop, the solution was diluted to 200 ng/. mu.L and used as a PCR template.
PCR in vitro amplification: 50. mu.L of the reaction mixture contained 25. mu.L of premixed Taq enzyme, 0.5. mu.L of each primer (10. mu.M), and 1. mu. L, ddH of template2And (3) filling the O23 mu L to the total volume, wherein small details exist, and when the target gene is prepared in a large quantity, reagents except the template are uniformly mixed according to multiples and then are subpackaged, and then the template is added.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, renaturation at 72 ℃ for 3min, and final elongation at 72 ℃ for 7 min.
Electrophoresis and recovery of PCR products: and (3) carrying out 1% agarose gel electrophoresis on the PCR product, cutting and recovering the target band gel according to Marker instructions, and performing recovery operation according to the instruction of an Omega gel recovery kit.
Example 2 preparation of vector backbone by cleavage
The vector backbone is exemplified by pCN19 containing EGFP (Price, M.S., et al, 2008.The Cryptococcus neoformans Rho-GDP disassociation inhibitor peptides intracellular and viral. infection. Immun.76, 5729-5737.) and is cleaved using its single cleavage site BamHI and SpeI located structurally downstream of EGFP.
Mass preparation of pCN19 vector
1) Activating an escherichia coli carrier, culturing for one day, and then taking a single colony to be cultured in 10mL of LB liquid culture medium overnight;
2) 2 parts of thalli are collected by a 5mL centrifuge tube, centrifuged for 2min at 10000rpm, and waste liquid is discarded;
3) adding 250 mu L of Solution I for resuspension, and transferring all the Solution into a new 1.5mL centrifuge tube;
4) adding 250 mu L of Solution II for cracking, and gently mixing uniformly for 2min until the sample is transparent and viscous;
5) adding 350 mu L Solution III to renature the plasmid DNA, immediately mixing the Solution and the plasmid DNA softly to generate flocculent precipitate;
6) centrifuging at 15000rpm for 10min, collecting 750 μ L supernatant in a collecting column, standing, centrifuging at 10000rpm for 1min, and discarding waste liquid;
7) adding 500 μ L HBC Buffer, centrifuging at 10000rpm for 1min, and discarding the waste liquid;
8) adding 700 μ L Washing Buffer, cleaning for 2 times, each time at 12000rpm for 1min, and discarding the waste liquid;
9) centrifuging at 14000rpm for 2min in an empty tube, transferring into a new 1.5mL centrifuge tube, uncovering the centrifuge tube until the organic reagent is completely volatilized, adding 50 μ L ddH preheated to 65 ℃2Dissolving O, and measuring the concentration.
The plasmid extraction kit used in the experiment was sold by Omega.
Enzyme digestion and recovery of the vector skeleton: mu.g of the vector was taken and 2. mu.L of each of BamHI and SpeI, 10. mu.L of buffer, and ddH was added2Supplementing O to 100 μ L, flicking, mixing, placing in 37 deg.C water bath, 3-5 hr, taking out 1 μ L sample, and detecting whether the reaction is complete; after the enzyme digestion is completed, all samples are subjected to 1% agarose gel electrophoresis, and the gel recovery step is carried out according to the instruction of an Omega gel recovery kit.
Example 3 in vitro cloning of recombinant vectors
Ligation transformation of the fragment of interest to the vector backbone
1) Constructing a connection system according to the use instruction of a homologous recombination kit (Vazyme), and connecting for 30min at 37 ℃;
2) during the connection period, ice is taken out by an ice box in advance, competent cells at the temperature of-80 ℃ are placed on the ice for unfreezing, the connection fragments are sucked into the competent cells after the connection is finished, the competent cells are placed on the ice for 30min for full reaction, and the whole process is as gentle as possible;
3) after the reaction is finished, thermally shocking for 60s at 42 ℃, standing on ice for 3min, adding 500 mu L of LB culture medium, and resuscitating for 30min at 37 ℃ by a horizontal shaking table at 150 rpm;
4) centrifuging at 3000rpm for 2min, sucking away 400 μ L of redundant liquid, slightly blowing to blow cells, and coating on LB plate containing kanamycin;
5) after the surface of the plate was dried, the plate was incubated overnight in an incubator at 37 ℃.
Validation of recombinant vectors
1) Selecting transformation spots, performing streak amplification culture, and placing in a 37 deg.C incubator for 6-8 h;
2) checking whether the target gene is integrated on the vector by using a primer with the size of 1Kb in the target gene CWF19, wherein the primers are TL 771: 5'-ccaccccttccggcaactcttc-3' (SEQ ID NO. 3); TL 772: 5'-gcccagcttatccgcattatcat-3' (SEQ ID NO. 4); the template is transformed Escherichia coli bacterial plaque, and a system of 25. mu.L of each reaction is constructed and verified according to the reaction conditions in example 1;
3) selecting 4 positive PCR-verified vectors for culture and extracting plasmids, wherein the specific steps are shown in example 2;
4) according to the plasmid map, EcoRV is selected for enzyme digestion verification (5575bp and 4213bp), and the vector with correct enzyme digestion verification, such as A and B in the attached figure 2, is selected for sequencing in Shanghai.
5) The vector with the correct sequencing was preserved and named pTBL177, the structure of which is shown in FIG. 1, and was frozen in a refrigerator at-80 ℃ in 20% glycerol as the medium for use.
Linearized use of recombinant vectors
The effect of the circular DNA is lower than that of the linear DNA when the circular DNA is transformed, so the vector of the invention needs to be subjected to single enzyme linearization before transformation, and ScaI enzyme digestion, enzyme digestion system and gel recovery are selected in non-critical element areas according to a map as in example 2.
The constructed vector pTBL177 was transformed into Cryptococcus neoformans subcellular localization results, as shown in C in FIG. 2 and FIG. 3. The results show that the subcellular localization is in the nucleus.
The recombinant vector pTBL177 obtained by the invention is stable and not easy to miss targets, can accurately indicate the morphological number and position of cell nucleus, and is suitable for various yeasts and fungi, particularly for the requirement of sexual reproduction cells for indicating the change of cell nucleus. The recombinant vector is simple to prepare, has low cost and is suitable for laboratory and industrial production.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
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<213> Cryptococcus neoformans (Crytococcus neoformans)
<400> 5
gacgagctgt acggatccat ggggagagac agcaccaaca cggatcggga cacgcacaga 60
cacggctcaa ggcatgaccg ccacgacagg cctcataaac atcgaactca cagggatcgg 120
gacaagcaag atgcagacca gattgaagag gagcatagga gagagagaaa gcggctcaag 180
aaggagaaga agacagacga tgcccgtgat cttcaggtgc tggacgatga tcctagtatg 240
tgggtagaaa aatcattgga tcctaccaat gctgtcgcga acatccccac cgccgactct 300
cttcctctaa catccaatcc ttctggtcct aaagtttctc ttcctccctc tacagcagca 360
ggctcagaag gccgcgagcg cgattcttgg atgctggagc catcggtatc ttctgccatc 420
gtcccggtac cgagagacga tgtgccgcat agcgctgtca agtctgctgc cgatacggcg 480
gacgggtacg gagatgggca gcctgcggtc tcgaatgtgg acctcttttc ctccatggga 540
atagaacata agagaaagga tcctagatcg gataggccag acccatccca ggtcagtttt 600
gcacttggca ttcgcggata agctgataca tgcaatttga aagctcgtgg tcgacgaccg 660
tttcgagctc aacacccaac tcctcgaggg gaaaaatgtc gatgaatacg aagtcaaagg 720
tattgcaacc tatatataat atccttcgtc ctttactgat caccttccta gagaaaaaga 780
cgacatttgg tgggcctggg taccagtggc gaatgatgaa actcaagcgt ctctatgagc 840
aggcagaaga acagtctcga cccgtagaag aagtggctct tgaacgctac ggatcacttg 900
acgagttcaa cgaagctctc gaagaacgtc gttatctaga cgatcgtgag gcgcgacgca 960
ggtcccgggg cgtcagcaga cctcttgggc ctggttccga ttcttccagg cccgccaccc 1020
cttccggcaa ctcttccggt atgcggactc ccgacgcagg tcgccgattc atgtttgcta 1080
accgcaacgc tggcgagcaa acttttggca ctggcggtgg cagtggcaat cgacctggct 1140
cgcgaacggg tttccgtcgg cccggtgaag atttagaaca gggtgcgacg cctgccagca 1200
gcgctgggag gcttgataca cttcgccgtg acaatggtgg acttggaaca ccaaagcttg 1260
aaagtggggt gagatccggt tctagcggtg ttgttccgat gaaggtcggt actcctatcc 1320
ctagcgtttt caccccaacc actctcacac gttctttcac cggtcctccc ccccctgggc 1380
ctgagcatga atccagtgct gtggacccga cctcttcgaa accccctctt tctactgaac 1440
agctcaacaa acttcaggcc gccgttctcc gttccaaact tatggacgat ccgaatgcct 1500
ccgctttgga agacgaatac gaaattgagc gtgagcggag cgagcgtgca catgcgggtg 1560
tgggtgcagg tgcaggactg tgggagggaa ataatgaggg gatgcaaggc cagctcggca 1620
ggatggatga aaaaggcaat aggatagaag tgcaggtcct tcctacattg gacgcgagag 1680
ggaagctgta tgatgtaggc actggcaagg aagatgaatc ggtcgtcaga cccggaaacc 1740
ggaaacaaaa ggacgccaag ttcgagaccc gtgacaagga aggcaacctg ttacgttata 1800
acgccgatga cgatactcaa tccctggggg aacttgtgcg acaagaacgt tttggcgcag 1860
gttcatctga tcagaaaaac ctggatgctg agatggcgag ggctattgca acggatggca 1920
agtttgaaga tgaccttgac tatatggatg ataatgcgga taagctgggc aggaaaaaga 1980
tgaagagcga cgcattaaaa cgggcctttg ccataaatgg taggttcagt ttgactcccg 2040
tgactcgttt ctaggatatt ttactcaagg cgaaattgtg gaacagatta tgcccgaaca 2100
aagaaagcgc tcgatacatg cccattttgc taccaggacg accgtcctcc gcaaaccgcc 2160
atcgttgctc ttggtacacg cacttatatg tgctgcacgc cgtacgaaga acttgtaccg 2220
gggcactgtc tgatagtgcc tttgcagcac cacctgagta tgctggagat ggaagatgat 2280
gattgggacg aagtccgcgt gagtcacaat ctaacagaaa catgttttta gcagctcacc 2340
tcacacgctt taaccctcag aacttcatga agtgcctcat gcgcatgcac gctcaatcaa 2400
accatggcgt tatctttttc gaaaccatca cctccttcaa atcccagcgg cactcataca 2460
tagaagctat tcctgtgcct tttcacatat ttcaagacct tcctgcctat ttccgtgaat 2520
cgattctttc ctctgaagga gaatggacgc agcacaagaa gttgattgat ttctcgtcga 2580
gaccaggtgg ctttaggagg atgatggtac caaacttgcc atatttcatg gttcagtggg 2640
attataaagg cgagaagggc tacgggcacg tcatagaagg tatcaaagat agtggagcag 2700
gaggaggaga agacgaggaa gggcatgtgg gtggagcaat gtccgagagc gagttcccaa 2760
ggtaaatttg agtttgcagg cttgtcagaa ttagtacgta aacacggagg gctgactgag 2820
agaaattaga tactttgccc aagaagtcat cggcaacatc ctgggcctgg aagctcgtaa 2880
atggagaaga ccaaggaaaa tggacgtggc gttgaataaa gaaagggcac ggaagttggg 2940
aactcatttc cagccgtata attggaccgt gggaaacggc gtttaaacta gtaacggccg 3000
ccag 3004
Claims (6)
1. Novel cryptococcus nuclear indicator vector, its characterized in that: the indicating vector contains an expression frame of fusion expression of a cell nucleus localization gene CWF19 and a green fluorescence gene EGFP, and the nucleotide sequence of the cell nucleus localization gene CWF19 is shown as SEQ ID No. 5.
2. The cryptococcus neoformans nuclear indicator vector of claim 1, wherein: the indicator vector is connected into the BamHI and SpeI enzyme cutting sites of the pCN19 vector by a sequence shown in SEQ ID NO. 5.
3. The method for constructing the cryptococcus neoformans nuclear indicator vector as claimed in claim 1 or 2, is characterized by comprising the following specific steps: cloning or synthesizing a cell nucleus localization gene CWF19, and then connecting the cell nucleus localization gene CWF19 to the downstream of the EGFP gene of a fungus expression vector containing the EGFP gene to form an expression frame containing fusion expression of the cell nucleus localization gene CWF19 and the green fluorescence gene EGFP.
4. The preparation method according to claim 3, comprising the following steps: the nuclear localization gene CWF19 was cloned or synthesized and ligated into the BamHI and SpeI cleavage sites of pCN19 vector.
5. Use of a vector according to claim 1 or 2 as an indicator of sexual reproduction of cryptococcus neoformans.
6. Use according to claim 5, characterized in that: the carrier is used as an indicating carrier for indicating the change of the position and the number of the cell nucleus by the cryptococcus neoformans.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190216893A1 (en) * | 2016-06-03 | 2019-07-18 | The Scripps Research Institute | Compositions and methods of modulating immune response |
| CN111328287A (en) * | 2017-07-04 | 2020-06-23 | 库瑞瓦格股份公司 | Novel nucleic acid molecules |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190216893A1 (en) * | 2016-06-03 | 2019-07-18 | The Scripps Research Institute | Compositions and methods of modulating immune response |
| CN111328287A (en) * | 2017-07-04 | 2020-06-23 | 库瑞瓦格股份公司 | Novel nucleic acid molecules |
Non-Patent Citations (10)
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Application publication date: 20201201 |