CN101205538B - Clone, expression and use of Schistosoma Japonicum signal transduction protein Sjwnt-4 gene - Google Patents
Clone, expression and use of Schistosoma Japonicum signal transduction protein Sjwnt-4 gene Download PDFInfo
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Abstract
The invention discloses a Wnt family gene which is amplified for the first time from Schistosoma japonicum 19-day schistosomulum by utilization of RACE technology, wherein, sequence analysis indicatesthat: a complete coding frame of the gene comprises 1311bp, 436 amino acids coded; and theoretical molecular weight of 49.6kD. Homology analysis results indicate that: amino acid sequences of the gen e have typical Wnt family protein characteristics; similarity of the amino acid sequences with amino acid sequences of Dugesia japonica and human Wnt4 is relatively 43 percent and 37 percent; the geneis presumed to be a Wnt4 gene of schistosome and named as Sjwnt4 (GenBanK Log-On No.: DQ643829). Real-time quantitative PCR analysis indicates that: the gene is expressed all in 14-day schistosomulu m, 19-day schistosomulum, 31-day imago, 44-day female worms and 44-day male worms. The invention constructs a pronucleus expression vector pGEX-4T-2-Sjwnt4 of the gene and applies an Escherichia coli system for expression; expression proteins exist in the form of inclusion bodies; Western blottings indicate that expression products can be identified by crude antigen immune serums of Schistosoma japonicum imago.
Description
Technical field:
The invention belongs to the gene engineering field, be specifically related to clone, expression and application that a kind of Japanese blood is inhaled signal transducer Sjwnt-4 gene.
Background technology:
The secretor type glycoprotein that Wnts can both find being a class from hydra, insect to vertebrates, after coming out by emiocytosis, combine with the membrane receptor of self or adjacent cells, excite the signal path in downstream, regulate target gene expression in the nuclear, the differentiation destiny of decision cell plays regulating effect widely in animal development.The spatial and temporal expression of Wnt abnormal protein or unusual activation are often relevant with the generation of tumour.There are some researches show, the Wnt signal path plays crucial regulating effect to the normal development of mammalian reproductive system, it has mainly participated in Miao Le Shi pipe and has derived from the formation of organ, growth, ovulation and the luteinization of regulation and control ovarian follicle, in addition with the foundation of normal pregnancy and pregnant process in the variation of mammary gland also relevant.Wnt4 is one of Wnt family protein member, and in mouse, Wnt4 is to the crucial meaning that is formed with of Miao Le Shi pipe, and female, the male mouse of disappearance Wnt4 does not all have Miao Le Shi pipe; The female mouse of Wnt4 sudden change does not have the derivation organ (being uterine tube, uterus, uterine cervix and portio supravaginalis) of Miao Le Shi pipe; Mesonephric duct then exists.The growth of its testis of buck of Wnt4 disappearance is unaffected, in a single day but in the female sexual gland of growing, lack the Wnt4 signal, all present certain masculine feature on its 26S Proteasome Structure and Function, though sexual gland is positioned at their correct positions, but its profile is round than normal ovary, and does not wrap quilt as testis.In addition, the sexual gland of sudden change can break up MIS (Miao Le Shi manages inhibitory substance) and testosterone.In the mankind, the male sex's the pulsating diploid of karyomit(e) 1p31～p35 has caused Wnt4 to increase, and the overexpression of Wnt4 causes the male sex of XY to have women's phenotype.Therefore, Wnt4 may be a kind of decision gene of sex phenotype.
To the grow research of signal transduction of schistosomicide is the effective way of separating and be familiar with the schistosomicide characteristic of life.But up to the present, very few to the grow research of signal transduction system of schistosomicide so that the efficient candidate vaccine molecule of exploitation and screen newtype drug aspect making slow progress.And the research of schistosomicide Wnt gene be at home and abroad there is no the research report.
Summary of the invention:
Technical problem to be solved by this invention is to design the relevant expression of schistosomicide Wnt gene.
The present invention utilizes the RACE technology to be cloned into the cDNA sequence of the proteic ORF of containing of coding Schistosoma japonicum Wnt4 first, analyzed the expression of this gene in the Schistosoma japonicum different developmental phases, use intestinal bacteria and express, and expression product has been carried out antigenic determination.
The invention provides the clone and the expression of a kind of Schistosoma Japonicum signal transduction protein Sjwnt-4-4 gene.
This method comprises the following steps:
(1) material
Trizol, GeneRacer
TMKit is available from Invitrogen company; Ex Taq Hot Start archaeal dna polymerase, restriction enzyme BamH I, Xho I, T4DNA ligase enzyme, fluorescent quantificationally PCR detecting kit and random primer are available from TaKaRa biotechnology (Dalian) company limited; Sheep anti-mouse igg-HRP is available from TIANGEN Biotech (Beijing) Co., Ltd.; Anti-GST monoclonal antibody is available from SIGMA company; Goat anti-rabbit igg-HRP is available from Huamei Bio-Engrg Co.; Reversed transcriptive enzyme is available from Stratagen company; The RNA enzyme inhibitors is available from Promega company; SYBR GreenI and Calibration are available from BIO-RAD company;
(2) bacterial classification, plasmid and laboratory animal
Plasmid pGEX-4T-2, bacillus coli DH 5 alpha, BL21 (DE3) are provided by this institute (Shanghai veterinary institute), and the pUCm-T carrier is available from TIANGEN Biotech (Beijing) Co., Ltd..(male, 2.5～3.0kg) fly to reach laboratory animal plant available from the Shanghai Luojing to New Zealand white rabbit.The schistosoma japonicum Chinese strain cercaria is provided by this institute (Shanghai veterinary institute) oncomelania chamber.New Zealand white rabbit infects 20 000 with the belly paster method respectively, and 5000,2000 schistosomulums cutd open after 14 days, 19 days, 31 days and 44 days and kill, and collected polypide with the hepatic vein perfusion, and liquid nitrogen cryopreservation is standby;
(3) method
1. 19 days virgin worm 200mg of Schistosoma japonicum frozen in the liquid nitrogen are got in the extraction of total RNA, carry out the extraction of total RNA by Trizol test kit specification sheets;
2. the design of primers of RACE and this laboratory journey state cutting edge of a knife or a sword of amplification etc. utilize two-dimensional electrophoresis binding peptide quality fingerprinting graphical spectrum technology to obtain one section peptide sequence of a Wnt family protein, are the corresponding EST segment (GenBank that inquiry sequence (http://www.ncbi.nlm.nih.gov/BLAST) in Schistosoma japonicum EST storehouse searches a Schistosoma japonicum with this peptide sequence
TMAccession number AAM89872), long 727bp does not contain complete ORF.With this est sequence is the synthetic RACE primer of stencil design (can widely collect Bioisystech Co., Ltd by the Shen, Shanghai synthesizes).Two nested primer: 3GSP1:5 '-TATGCTGTGGTCGAGGATTTAAACG-3 ' of 3 ' RACE, 3GSP2:5 '-GGTGTTGCAAAGTTGTCTGTAAAACA-3 '; Two nested primer: 5GSP1:5 '-GGTGTTGCAAAGTTGTCTGTAAAACA-3 ' of 5 ' RACE, 5GSP2:5 '-TTGTCGTTTAAATCCTCGACCACAG-3 '; Concrete steps are undertaken by GeneRacerTM test kit operational manual.In the pCR4-TOPO carrier that the RACE product cloning is provided to test kit, select positive colony, by the order-checking of handsome Bioisystech Co., Ltd.Utilize DNAStar software search 3 ', the lap of 5 ' RACE product sequence and former est sequence, with three sections sequence assemblies;
3. contain the pulsating amplification of ORF cDNA and contain the pulsating amplification of ORF cDNA according to the cDNA sequences Design primer (F1 and F2) that splices.F1:5 '-CCG
GGATCCATGAATCTAACTCAGCTAGAA-3 ' introducing restriction enzyme site BamHI (underlining); F2:5 '-CGG
CTCGAGTTAATTACATGTAGATATAAC-3 ' introducing restriction enzyme site (XhoI) (can widely collect Bioisystech Co., Ltd by the Shen, Shanghai synthesizes).Getting the 3 μ l cDNA that reverse transcription obtains in 3 ' RACE is that template is carried out pcr amplification, and the PCR reaction conditions is respectively 94 ℃ of pre-sex change 5min, 94 ℃ then, 30s, and 55 ℃, 30s, 72 ℃, 1min30s, totally 30 circulations, 72 ℃ are extended 10min after the loop ends.The PCR product reclaims test kit with sepharose DNA and reclaims, connect the pUCm-T carrier behind the purifying, be converted into the bacillus coli DH 5 alpha competent cell, a menu bacterium colony enzyme is cut evaluation, and positive plasmid called after pUCm-T-Sjwnt4 also send the order-checking of handsome Bioisystech Co., Ltd;
4. bioinformatic analysis cDNA that order-checking is obtained carries out similarity comparison (http://www.ncbi.nlm.nih.gov/BLAST) on NCBI; Utilize ORF finder on the NCBI to find out the reading frame of new gene; Utilize DNAstar software that parameters such as total number of atnino acid, composition, protein relative molecular weight are analyzed; It is right to utilize clustalW software that different plant species Wnt albumen is carried out multiple ratio; Utilize NetAcet software (http://www.cbs.dtu.dk/services/NetAcet/) to carry out the glycosylation site prediction.Utilize the MHCII phenotype on-line prediction instrument (http://WWW.uni-tuebingen.de/uni/kxi/) of SYFPEITHI to carry out the t cell epitope prediction.
5. (α-tubulin) is confidential reference items to select bilharzial housekeeping gene alpha-tubulin in the fluorescence real-time quantitative RT-PCR test; Extract the virgin worm of 14d and 19d, 31d polypide, 44d male worm and the total RNA of the female worm of 44d respectively, utilize random primer to synthesize cDNA first chain behind the removal genomic dna; Fluorescence quantification PCR primer: the primer of amplification Sjwnt4: S1:5 '-ACATACAAAATCGTCTGGTCTC-3 ', S2:5 '-GATGGTAAAGGCGATGTAGTC-3 ', expanding fragment length 214bp; Amplification α-tubulin primer: T1:5 '-CTGATTTTCCATTCGTTTG-3 ', T2:5 '-GTTGTCTACCATGAAGGCA-3 ', expanding fragment length 213bp; It is synthetic that primer is given birth to the worker by Shanghai, adopts the fluorescence dye method to carry out real-time quantitative PCR, and reaction system comprises: 5 * R-PCR Buffer5 μ l, 250mM Mg
2+0.3 μ l, 10mM dNTP0.75 μ l, 10 μ M primers, 1.0 μ l, 25 * SYBR Green I1.0 μ l, 10-3 * Calibration1.0 μ l, 5U/ μ l Ex Taq Hs0.25 μ l, ddh
2O14.7 μ l, template cDNA1.0 μ l, totally 25 μ l; Reaction parameter: 95 ℃ of sex change 3min, 95 ℃ of 5s, 58 ℃ of 30s, 40 circulations, wherein 58 ℃ of 30s concluding time points are the fluorescent signal check point, each reaction is all done 3 holes and is repeated; Adopt the BIO-RAD iCycler of company
TMIQ version3.0 software carries out computational analysis, is confidential reference items markization reaction result with α-tubulin, draws the goal gene content with respect to per 1,000,000 housekeeping genes;
6. the structure of recombinant expression plasmid cuts out the cDNA segment that Sjwnt4 contains ORF with restriction enzyme BamHI, Xho I from the recombinant plasmid pUCm-T-Sjwnt4 that the back of checking order is determined, with the multiple clone site district of this complete sequence directed cloning in prokaryotic expression carrier pGEX-4T-2, make up recombinant expression plasmid pGEX-4T-2-Sjwnt4, and transform expressive host bacterium BL21 (DE3);
7. the expression in intestinal bacteria will identify that good pGEX-4T-2-Sjwnt4/BL21 (DE3) is inoculated in liquid LB substratum, and 37 ℃ of concussions are cultivated, and the OD value is to add the IPTG abduction delivering that final concentration is 1mM at 0.6 o'clock.Induce back 0h at IPTG, 0.5h, 1h, 2h, 4h, 6h collect thalline respectively, use SDS-PAGE and analyze tropina, determine best induction time;
The phase tropina carried out the SDS-PAGE electrophoresis when 8. Western blotting detected high expression level, transfer on the nitrocellulose filter, resist with anti-GST monoclonal antibody or through the Schistosoma japonicum adult antigen immune rabbit anteserum work one that pGEX-4T-2/BL21 intestinal bacteria lysate adsorption treatment is crossed respectively, the sheep anti mouse of horseradish peroxidase-labeled, goat anti-rabbit igg are two anti-, and DAB develops the color as substrate.
Another object of the present invention has provided the application of Sjwnt-4 encoding gene in preparation control schistosomicide disease medicament.
The clone of SjWnt-4 encoding gene of the present invention and bioinformatic analysis
This research and utilization RACE technology is to EST segment (GenBank
TMThe number of landing is AAM89872) carry out 3 ' end and 5 ' the end extension, take turns pcr amplification through 2 respectively, PCR product order-checking back splicing is obtained the dna segment of 2044bp, further utilize pcr clone to obtain to contain the encoding gene of entire reading frame, its open reading frame is 1311bp, and 436 amino acid of encoding utilize the Blast software of NCBI that this sequence is carried out the homology search, other known of result and schistosomicide does not have remarkable homology, is the new gene of schistosomicide.The similarity comparative result of aminoacid sequence shows with the Wnt family protein to have high homology, wherein the highest with the proteic homology of Wnt4, and aminoacid sequence analyzed find that also it has very typical Wnt family protein feature: the conservative site that is being dispersed in more than 100 Wnt family protein in the whole protein sequence; But be rich in the cysteine residues that commissure forms disulfide linkage, about 23～24 conservative halfcystines, wherein 50% is positioned at proteic carboxyl terminal; Have three or four glycosylation sites, it is right to have selected respectively Wnt4 albumen from 7 species of Japanese triangle turbellarian worm (number of landing BAD93239.1), people (the GeneBank number of landing NP_110388.2), mouse (the GeneBank number of landing NP_033549.1), Eugene kangaroo (the GeneBank number of landing AAY18780.1), chicken (the GeneBank number of landing JC2451), nematode (the GeneBank number of landing NP_493668.1), fruit bat (the GeneBank number of landing NP_476810.1) to carry out the multiple ratio of aminoacid sequence again.The result shows that this coded by said gene aminoacid sequence is up to 43% (E=1e-100) with the Wnt4 similarity that belongs to the Japanese triangle turbellarian worm of Platyhelminthes together, with the similarity of people Wnt4 be 37% (E=9e-72), all the other are all between 36%～38%.In view of the above, the albumen of inferring this genes encoding is Schistosoma japonicum Wnt4 albumen, is Schistosoma japonicum wnt4 (Sjwnt4) (the GeneBank number of landing DQ643829) with this unnamed gene.
To the aminoacid sequence of this genes encoding carry out predicting the outcome of t cell epitope show contain in this sequence a plurality of can with the higher antigen peptide section (table 1) of tiring that combines of specific HLA molecule tool.
Table 1Sjwnt4 and HLA have the potential antigen peptide of higher joint efficiency
Table1The?peptide?binding?to?HLA?with?high?efficiency?in?Sjwnt4
Other, sex expression analysis of the phase of SjWnt4 gene of the present invention
In order to understand the expression of SjWnt4 gene Schistosoma japonicum different developmental phases polypide, extract 14 days virgin worms, 19 days virgin worms, 31 days adults, 44 days male worms and 44 days total RNA of stage polypide such as female worm, select housekeeping gene α-tubulin as confidential reference items, utilize fluorescence quantitative PCR method to detect the expression of this gene the several different developmental phases polypides of Schistosoma japonicum.Experimental result shows, SjWnt4 all has expression (Fig. 1) in the virgin worm of Schistosoma japonicum, adult and female male worm, and wherein expression amount is the highest in 19 days virgin worms, and female worm expression amount was obviously than male worm height in 44 days.
The structure of recombinant prokaryotic expression vector pGEX-4T-2-Sjwnt4 is identified
Identify that through PCR, BamHI and Xho I double digestion (Fig. 2) and order-checking confirm pGEX-4T-2-Sjwnt4 construction of recombinant expression plasmid success (Fig. 3).
Sjwnt4 gene of the present invention is at expression in escherichia coli
Recombinant expression plasmid pGEX-4T-2-Sjwnt4 obtains to express in e. coli bl21 (DE3), and the SDS-PAGE electrophoresis result shows that 1mM IPTG induces 4h promptly to reach maximum expression amount; The recombinant protein molecular weight is about 76kD, conforms to (Sjwnt4 albumen infers that molecular weight is about 49.6kD, and the about 26kD of vector expression Protein G ST is so the recombinant protein molecular weight is about 75.6kD) with expected results (Fig. 4).Recombinant protein exists with the inclusion body form, dissolves in the PBS that contains 8M urea.
Above-mentioned expression product antigenicity analysis
Carry out the SDS-PAGE electrophoresis with recombination expression product, through electrotransfer to the NC film, make an anti-Western blot that carries out with anti-GST monoclonal antibody with through the Schistosoma japonicum adult antigen immune serum of pGEX-4T-2/BL21 e. coli protein absorption respectively, the result all has one significantly to discern band (Fig. 5 arrow place) at the 75kD place, show that recombination expression product is gst fusion protein and has good antigenicity.
The above results shows Sjwnt4 gene the 14th day virgin worm, 19 days virgin worms, 31 days polypides, 44 days female worms and in the male worm body expression was arranged all in 44 days behind the schistosoma japonicum infection host, but the expression amount of the virgin worm of 19d is higher than other each stage significantly; Meanwhile, the expression amount of this gene in the female worm of 44d is higher than the 44d male worm.Behind the known schistosoma japonicum infection host of the research in past, in the pairing of filling the span of a man's arms of 15-16 days female male worms, the female male worm back yolk gland of filling the span of a man's arms germinates yolk cell differentiation, synthetic yolk droplet; Express chorionin in the 22nd day female polypide; After the 24th day, keep the ovulation state for a long time.Above-mentioned Sjwnt4 expression of gene status analysis result shows that this expression of gene changes with the growth variation of japonicam sex phenotype closely related, has disclosed the Wnt4 gene in the developmental vital role of schistosomicide reproductive system from a side.The biological significance that this result and this gene are expressed in mouse and wallaby has certain similarity.In mouse, Wnt4 expresses in kidney duct mesenchyme and undifferentiated sexual gland at first, and it is all expressed in the undifferentiated sexual gland of both sexes, but through after the special differentiation of sex, Wnt4 expresses and descends in male gonad, and keeps in female sexual gland always.In marsupial such as Ta Maer sandbag mouse, Wnt4mRNA has expression in undifferentiated both sexes sexual gland; Female wallaby Wnt4mRNA expression level in the ovary atomization significantly rises, and arrives the peak about mouse birth back after 9～13 days, begins to descend when all female sex cells enter postmeiotic then; The Wnt4mRNA expression level of male wallaby descends after vas deferens forms immediately.Therefore, if can pass through human intervention, as use the RNAi perturbation technique to suppress the expression of schistosomicide Wnt4 or be the activity that the medicine target suppresses it, thereby the control japonicam sex is grown, laid eggs with Wnt4 albumen, to alleviating the pathological lesion of schistosomicide, reach blocking propagation with significant.
The present invention clones first and obtains schistosomicide Wnt4 gene, for the Wnt signal path in schistosomicide grows reproductive development particularly effect and develop efficiently that the vaccine and the novel antischistosomal drug of screening of anti-schistosomiasis have bigger using value.
The Nucleotide of SjWnt-4 and speculating acid sequence.
1?AATATGATGCTACTCCTTATGATGATTCAACATCAGCCAGTAGATTAAACAAAAATAATTACAACACTGAAGGATACCGTCGATCACCAA
91?ATTCTGATTTACGTTTTGTTGAAGCATCAAAATCACAAGATTTGGATTATCCTCATACAAGTAAAAAATATATAAATTCAGACTTAAATA
181?CAAATAATTATAATCATGAACGA
204?
AATCTAACTCAGCTAGAACAAACGATTCAAGACATGAATAATAATGATAGTAACAATATAATGACATCAGAAACATTTGTTGGTGCC
N D S N N I M T S E T F V G A M N L T Q L E Q T I Q D M N N
294?GATGGAAAATTACAAATGAGTATATGCGATCATCCAAATGGATTTTTACGGAGACAAAAGAAATTATGCCGTCAGTATTTACATTTGATG
D G K L Q M S I C D H P N G F L R R Q K K L C R Q Y L H L M
384 GAGAGTGTAATCCGTGGTTATTTTATGGGTTTAAAAGAATGTGAATATCAATTTTCTGCACATCGATGGAATTGTCAGGGTCATAACTTA
E S V I R G Y F M G L K E C E Y Q F S A H R W N C Q G H N L
474 ACTATTCAAGCACCAACTAGTCGAAAACAGAAACGTTTAAGATATAGAGAATCTGAGTTGAAAAATGATATGGATAATTCACGAAGAAAA
T I Q A P T S R K Q K R L R Y R E S E L K N D M D N S R R K
564 TCTGTACGTATACAAACATACTTAGACAAGCTTTTATCTAAAGGAACAAGAGAATCAGCTTATGTTTTGGCTGTTACATCCGCCGGTGTA
S V R I Q T Y L D K L L S K G T R E S A Y V L A V T S A G V
654 TCACATGCAGTCACTAAAGCATGCAGTAGTGGTCTACATGATAACTGTGGATGTGACAGAACAATATACGATCATCCTAGAGAACCAAAT
S H A V T K A C S S G L H D N C G C D R T I Y D H P R E P N
744 TTTGAATGGTCAGGATGTTCAGATAATATACATTTTGGAGCAGCATTTTCAAGACAATTTCTTGATGTACGAGAACGTAACAGACTGAAA
F E W S G C S D N I H F G A A F S R Q F L D V R E R N R L K
834 CGTAATCCAAAATTAGGACTGACAAATTTACATAATAATCATGTGGGAAGACATATGGTAATCAATAAAATGGAAGTCCAGTGCAAATGT
R N P K L G L T N L H N N H V G R H M V I N K M E V Q C K C
924 CATGGTGTAAGTGGTTCATGTGAAATGCGTACATGTTGGCGATCGTTACCGAAATTTCGGCATTTAGGTGCACAATTACAAGAAAGATTT
H G V S G S C E M R T C W R S L P K F R H L G A Q L Q E R F
1014?CATGAAGCAATACAAGTCACTTACATACAAAATCGTCTGGTCTCGATGAAAGCACTAGAACAATTAAGTAAAGAATCAAATGGAAACGCA
H E A I Q V T Y I Q N R L V S M K A L E Q L S K E S N G N A
1104?CTATTACTTTCTCATTCTGCATTATTATCATCAGTAGTATCAGGAGTATCATCATCCGATGAATTACCGGCATCACCGCGTATTAATAGA
L L L S H S A L L S S V V S G V S S S D E L P A S P R I N R
1194?AATGCGTTTGATACTTTAACTCGTTCTACATCATTGACTACATCGCCTTTACCATCACCAACTGAAAATGATTTGATTTATATTAGTGAA
N A F D T L T R S T S L T T S P L P S P T E N D L I Y I S E
1284?TCACCAACATTTTGTCATCATGATCCAAGATATGGTAGTATTGGTACATATGGTCGACAGTGTGATGAAAATTCTCAAGGTTTAAACAGT
S P T F C H H D P R Y G S I G T Y G R Q C D E N S Q G L N S
1374?TGTAATTATTTATGCTGTGGTCGAGGATTTAAACGACAAACGTTTGTTCAACAAGAGAGATGTGATTGTAAATTTCAGTGGTGTTGCAAA
C N Y L C C G R G F K R Q T F V Q Q E R C D C K F Q W C C K
1464?GTTGTCTGTAAAACATGTCGTAAAACAGTAGTTATATCTACATGTAAT
TATATATATATATAAGGTATCTTTTTATCGTTCGCAATC
V V C K T C R K T V V I S T C N *
1554?ACTTTGTGTGTTTATTTTCATTTTCGTACAATGAACTCCCCTACCTCTGTAGATCTCTTCATAACTATTCCAAAAGTTCTTCCTTATTGC
1644?ATTTGTATTACAAAAGATCTGAATTAAAGTAACAACTCCTGATAATCCTAATCAAAAGCAAAACAAGTTAACTGAGTTTGCAGGGGATGA
1734?AGAAGATTTGAAAACATAAAATTTACTAAGTTGATGACAATTTACTCCAAAGGAAATAAGGAAACGATAAAGAATAACCGAAAGACAA
1824TATATTATTGGTGAATTCACTGTGCTTAAAATAAAAATATCAAGAGAGAACAATCGAATTTGCACACGTTTTTCCTTTCTTCCTAAATTC
1914?TATCTTTCTATAACACTGTTTCTTGTATATGGTTATTTTCTCAATATACCGATTACATTGTGTATATTCTGTTTACAGTCAATTACTTAT
2004?TAAATCAGTCAGTAACCAAAAAAAAAAAAAAAAAAAAAAA
Description of drawings:
Fig. 1: real-time quantitative RT-PCR detects the SjWnt4 gene, and the same period is not and the expression in the sex polypide Schistosoma japonicum;
14d:14 days virgin worms; 19d:19 days virgin worms; 31d:31 days polypides; 44d (M): 44 days male worms; 44d (F): 44 days female worms.
Fig. 2: recombinant plasmid pGEX-4T-2-Sjwnt4 double digestion is identified and PCR identifies;
M1:DNA Marker DL15 000; The 1:pGEX-4T-2-Sjwnt4 recombinant plasmid is through BamHI, Xho I double digestion; The 2:pGEX-4T-2 empty carrier is through BamHI, Xho I double digestion; M2:DNA marker DL2 000; 3:pGEX-4T-2-Sjwnt4 recombinant plasmid PCR product.
Fig. 3: pGEX-4T-2-Sjwnt4 recombinant plasmid synoptic diagram;
Fig5The?recombinant?plasmid?pGEX-4T-2-Sjwnt4。
Fig. 4: SDS-PAGE analyzes the not expressing protein of phase simultaneously of pGEX-4T-2-Sjwnt4/BL21 (DE3);
M: protein standard molecular weight; 1,2,3,4,5,6: recombinant plasmid pGEX-4T-2-Sjwnt4 induces back 0h, 0.5h, 1h, 2h, 4h, the expression product of 6h at IPTG; 7:pGEX-4T-2-Sjwnt4 does not add the product that IPTG cultivates 6h
Fig. 5: the Western blot of pGEX-4T-2-Sjwnt4 recombinant protein analyzes;
M: dye the standard protein molecular weight in advance; 1:pGEX-4T-2-Sjwnt4 is induced product not; 2:pGEX-4T-2-Sjwnt4 abduction delivering product; A.: with mouse-anti GST monoclonal antibody is an anti-Western blot who carries out; B: make an anti-Western blot who carries out with anti schistosoma adult antigen rabbit anteserum.
Embodiment:
The clone of embodiment 1 Schistosoma Japonicum signal transduction protein Sjwnt-4-4 gene
1. experiment material
1.1 material
Trizol, GeneRacer
TMKit is available from Invitrogen company; Ex Taq Hot Start archaeal dna polymerase, T4DNA ligase enzyme are available from TaKaRa biotechnology (Dalian) company limited.
1.2 bacterial classification, plasmid and laboratory animal
Bacillus coli DH 5 alpha is provided by this institute, and the pUCm-T carrier is available from TIANGEN Biotech (Beijing) Co., Ltd..(male, 2.5～3.0kg) fly to reach laboratory animal plant available from the Shanghai Luojing to New Zealand white rabbit.The schistosoma japonicum Chinese strain cercaria is provided by this institute oncomelania chamber.New Zealand white rabbit infects 20000,5000,2000 schistosomulums with the belly paster method respectively, cuts open after 14 days, 19 days, 31 days and 44 days and kills, and collects polypide with the hepatic vein perfusion, and liquid nitrogen cryopreservation is standby;
2. method
2.1 19 days virgin worm 200mg of Schistosoma japonicum frozen in the liquid nitrogen are got in the extraction of total RNA, carry out the extraction of total RNA by Trizol test kit specification sheets;
2.2RACE design of primers and amplification this laboratory journey state cutting edge of a knife or a sword etc. utilize two-dimensional electrophoresis binding peptide quality fingerprinting graphical spectrum technology to obtain one section peptide sequence of a Wnt family protein, be the corresponding EST segment (GenBank that inquiry sequence (http://www.ncbi.nlm.nih.gov/BLAST) in Schistosoma japonicum EST storehouse searches a Schistosoma japonicum with this peptide sequence
TMAccession number AAM89872), long 727bp does not contain complete ORF.With this est sequence is the synthetic RACE primer of stencil design (can widely collect Bioisystech Co., Ltd by the Shen, Shanghai synthesizes).Two nested primer: 3GSP1:5 '-TATGCTGTGGTCGAGGATTTAAACG-3 ' of 3 ' RACE, 3GSP2:5 '-GGTGTTGCAAAGTTGTCTGTAAAACA-3 '; Two nested primer: 5GSP1:5 '-GGTGTTGCAAAGTTGTCTGTAAAACA-3 ' of 5 ' RACE, 5GSP2:5 '-TTGTCGTTTAAATCCTCGACCACAG-3 '; Concrete steps are pressed GeneRacer
TMThe test kit operational manual carries out.In the pCR4-TOPO carrier that the RACE product cloning is provided to test kit, select positive colony, by the order-checking of handsome Bioisystech Co., Ltd.Utilize DNAStar software search 3 ', the lap of 5 ' RACE product sequence and former est sequence, with three sections sequence assemblies;
Contain the pulsating amplification of ORF cDNA 2.3 contain the pulsating amplification of ORF cDNA according to the cDNA sequences Design primer (F1 and F2) that splices.F1:5 '-CCG
GGATCCATGAATCTAACTCAGCTAGAA-3 ' introducing restriction enzyme site BamHI (underlining); F2:5 '-CGG
CTCGAGTTAATTACATGTAGATATAAC-3 ' introducing restriction enzyme site (Xho I) (can widely collect Bioisystech Co., Ltd by the Shen, Shanghai synthesizes).Getting the 3 μ l cDNA that reverse transcription obtains in 3 ' RACE is that template is carried out pcr amplification, and the PCR reaction conditions is respectively 94 ℃ of pre-sex change 5min, 94 ℃ then, 30s, and 55 ℃, 30s, 72 ℃, 1min30s, totally 30 circulations, 72 ℃ are extended 10min after the loop ends.The PCR product reclaims test kit with sepharose DNA and reclaims, connect the pUCm-T carrier behind the purifying, be converted into the bacillus coli DH 5 alpha competent cell, a menu bacterium colony enzyme is cut evaluation, and positive plasmid called after pUCm-T-Sjwnt4 also send the order-checking of handsome Bioisystech Co., Ltd;
2.4 the cDNA that bioinformatic analysis obtains order-checking carries out similarity comparison (http://www.ncbi.nlm.nih.gov/BLAST) on NCBI; Utilize ORF finder on the NCBI to find out the reading frame of new gene; Utilize DNAstar software that parameters such as total number of atnino acid, composition, protein relative molecular weight are analyzed; It is right to utilize Genetool software that different plant species Wnt albumen is carried out multiple ratio; Utilize NetAcet software (http://www.cbs.dtu.dk/services/NetAcet/) to carry out the glycosylation site prediction.Utilize the MHCII phenotype on-line prediction instrument (http://www.uni-tuebingen.de/uni/kxi/) of SYFPEITHI to carry out the t cell epitope prediction.
3. result
Utilize the RACE technology to EST segment (GenBank
TMThe number of landing is AAM89872) carry out 3 ' end and 5 ' the end extension, take turns pcr amplification through 2 respectively, PCR product order-checking back splicing is obtained the dna segment of 2044bp, further utilize pcr clone to obtain to contain the encoding gene of entire reading frame, its open reading frame is 1311bp, and 436 amino acid of encoding utilize the Blast software of NCBI that this sequence is carried out the homology search, other known of result and schistosomicide does not have remarkable homology, is the new gene of schistosomicide.The similarity comparative result of aminoacid sequence shows with the Wnt family protein to have high homology, wherein the highest with the proteic homology of Wnt4, and aminoacid sequence analyzed find that also it has very typical Wnt family protein feature: the conservative site that is being dispersed in more than 100 Wnt family protein in the whole protein sequence; But be rich in the cysteine residues that commissure forms disulfide linkage, about 23～24 conservative halfcystines, wherein 50% is positioned at proteic carboxyl terminal; Have three or four glycosylation sites, it is right to have selected respectively Wnt4 albumen from 7 species of Japanese triangle turbellarian worm (number of landing BAD93239.1), people (the GeneBank number of landing NP_110388.2), mouse (the GeneBank number of landing NP_033549.1), Eugene kangaroo (the GeneBank number of landing AAY18780.1), chicken (the GeneBank number of landing JC2451), nematode (the GeneBank number of landing NP_493668.1), fruit bat (the GeneBank number of landing NP_476810.1) to carry out the multiple ratio of aminoacid sequence again.The result shows that this coded by said gene aminoacid sequence is up to 43% (E=le-100) with the Wnt4 similarity that belongs to the Japanese triangle turbellarian worm of Platyhelminthes together, with the similarity of people Wnt4 be 37% (E=9e-72), all the other are all between 36%～38%.In view of the above, the albumen of inferring this genes encoding is Schistosoma japonicum Wnt4 albumen, is Schistosoma japonicum wnt4 (Sjwnt4) (the GeneBank number of landing DQ643829) with this unnamed gene.
The expression of Schistosoma japonicum Sjwnt4 gene in intestinal bacteria
1. experiment material
1.1 materials limitations restriction endonuclease BamH I, Xho I, T4DNA ligase enzyme are available from TaKaRa biotechnology (Dalian) company limited.
1.2 bacterial classification, plasmid plasmid pGEX-4T-2, BL21 (DE3) are provided by this institute.
2. method
2.1 the structure of recombinant expression plasmid cuts out the cDNA segment that Sjwnt4 contains ORF with restriction enzyme BamHI, Xho I from the recombinant plasmid pUCm-T-Sjwnt4 that the back of checking order is determined, with the multiple clone site district of this complete sequence directed cloning in prokaryotic expression carrier pGEX-4T-2, make up recombinant expression plasmid pGEX-4T-2-Sjwnt4, and transform expressive host bacterium BL21 (DE3);
2.2 the expression in intestinal bacteria will identify that good pGEX-4T-2-Sjwnt4/BL21 (DE3) is inoculated in liquid LB substratum, 37 ℃ of concussions are cultivated, and the OD value is to add the IPTG abduction delivering that final concentration is 1mM at 0.6 o'clock.Induce back 0h at IPTG, 0.5h, 1h, 2h, 4h, 6h collect thalline respectively, use SDS-PAGE and analyze tropina, determine best induction time.
3. result
Recombinant expression plasmid pGEX-4T-2-Sjwnt4 obtains to express in e. coli bl21 (DE3), and the SDS-PAGE electrophoresis result shows that 1mM IPTG induces 4h promptly to reach maximum expression amount; The recombinant protein molecular weight is about 76kD, conforms to (Sjwnt4 albumen infers that molecular weight is about 49.6kD, and the about 26kD of vector expression Protein G ST is so the recombinant protein molecular weight is about 75.6kD) with expected results (Fig. 4).Recombinant protein exists with the inclusion body form, dissolves in the PBS that contains 8M urea.
Claims (2)
1. Schistosoma Japonicum signal transduction protein Sjwnt-4-4 gene is characterized in that the Nucleotide of described Sjwnt-4 gene and speculating acid sequence are as follows:
204?
AATCTAACTCAGCTAGAACAAACGATTCAAGACATGAATAATAATGATAGTAACAATATAATGACATCAGAAACATTTGTTGGTGCC
N D S N N I M T S E T F V G A M N L T Q L E Q T I Q D M N N
294?GATGGAAAATTACAAATGAGTATATGCGATCATCCAAATGGATTTTTACGGAGACAAAAGAAATTATGCCGTCAGTATTTACATTTGATG
D G K L Q M S I C D H P N G F L R R Q K K L C R Q Y L H L M
384?GAGAGTGTAATCCGTGGTTATTTTATGGGTTTAAAAGAATGTGAATATCAATTTTCTGCACATCGATGGAATTGTCAGGGTCATAACTTA
E S V I R G Y F M G L K E C E Y Q F S A H R W N C Q G H N L
474?ACTATTCAAGCACCAACTAGTCGAAAACAGAAACGTTTAAGATATAGAGAATCTGAGTTGAAAAATGATATGGATAATTCACGAAGAAAA
T I Q A P T S R K Q K R L R Y R E S E L K N D M D N S R R K
564?TCTGTACGTATACAAACATACTTAGACAAGCTTTTATCTAAAGGAACAAGAGAATCAGCTTATGTTTTGGCTGTTACATCCGCCGGTGTA
S V R I Q T Y L D K L L S K G T R E S A Y V L A V T S A G V
654?TCACATGCAGTCACTAAAGCATGCAGTAGTGGTCTACATGATAACTGTGGATGTGACAGAACAATATACGATCATCCTAGAGAACCAAAT
S H A V T K A C S S G L H D N C G C D R T I Y D H P R E P N
744?TTTGAATGGTCAGGATGTTCAGATAATATACATTTTGGAGCAGCATTTTCAAGACAATTTCTTGATGTACGAGAACGTAACAGACTGAAA
F E W S G C S D N I H F G A A F S R Q F L D V R E R N R L K
834?CGTAATCCAAAATTAGGACTGACAAATTTACATAATAATCATGTGGGAAGACATATGGTAATCAATAAAATGGAAGTCCAGTGCAAATGT
R N P K L G L T N L H N N H V G R H M V I N K M E V Q C K C
924?CATGGTGTAAGTGGTTCATGTGAAATGCGTACATGTTGGCGATCGTTACCGAAATTTCGGCATTTAGGTGCACAATTACAAGAAAGATTT
H G V S G S C E M R T C W R S L P K F R H L G A Q L Q E R F
1014?CATGAAGCAATACAAGTCACTTACATACAAAATCGTCTGGTCTCGATGAAAGCACTAGAACAATTAAGTAAAGAATCAAATGGAAACGCA
H E A I Q V T Y I Q N R L V S M K A L E Q L S K E S N G N A
1104?CTATTACTTTCTCATTCTGCATTATTATCATCAGTAGTATCAGGAGTATCATCATCCGATGAATTACCGGCATCACCGCGTATTAATAGA
L L L S H S A L L S S V V S G V S S S D E L P A S P R I N R
1194?AATGCGTTTGATACTTTAACTCGTTCTACATCATTGACTACATCGCCTTTACCATCACCAACTGAAAATGATTTGATTTATATTAGTGAA
N A F D T L T R S T S L T T S P L P S P T E N D L I Y I S E
1284?TCACCAACATTTTGTCATCATGATCCAAGATATGGTAGTATTGGTACATATGGTCGACAGTGTGATGAAAATTCTCAAGGTTTAAACAGT
S P T F C H H D P R Y G S I G T Y G R Q C D E N S Q G L N S
1374?TGTAATTATTTATGCTGTGGTCGAGGATTTAAACGACAAACGTTTGTTCAACAAGAGAGATGTGATTGTAAATTTCAGTGGTGTTGCAAA
C N Y L C C G R G F K R Q T F V Q Q E R C D C K F Q W C C K
1464?GTTGTCTGTAAAACATGTCGTAAAACAGTAGTTATATCTACATGTAAT
V V C K T C R K T V V I S T C N *。
2. the clone of Schistosoma Japonicum signal transduction protein Sjwnt-4-4 gene and the method for expression according to claim 1 is characterized in that this method comprises the following steps:
(1) material
Trizol, GeneRacer
TMKit; Ex Taq Hot Start archaeal dna polymerase, restriction enzyme BamHI, Xho I, T4 dna ligase, fluorescent quantificationally PCR detecting kit and random primer; The anti-GST monoclonal antibody of sheep anti-mouse igg-HRP; Goat anti-rabbit igg-HRP; Reversed transcriptive enzyme; The RNA enzyme inhibitors; SYBR Green I and Calibration;
(2) bacterial classification, plasmid and laboratory animal
Plasmid pGEX-4T-2, bacillus coli DH 5 alpha, BL21 DE3 are provided by the Shanghai veterinary institute; The pUCm-T carrier; New Zealand white rabbit is male, 2.5～3.0kg; The schistosoma japonicum Chinese strain cercaria is provided by the Shanghai veterinary institute; New Zealand white rabbit infects 20000,5000,2000 schistosomulums with the belly paster method respectively, cuts open after 14 days, 19 days, 31 days and 44 days and kills, and collects polypide with the hepatic vein perfusion, and liquid nitrogen cryopreservation is standby;
(3) method
1. 19 days virgin worm 200mg of Schistosoma japonicum frozen in the liquid nitrogen are got in the extraction of total RNA, carry out the extraction of total RNA by Trizol test kit specification sheets;
2. the design of primers of RACE and amplification
Utilizing two-dimensional electrophoresis binding peptide quality fingerprinting graphical spectrum technology to obtain one section peptide sequence of a Wnt family protein, is inquiry sequence searches a Schistosoma japonicum in Schistosoma japonicum EST storehouse corresponding EST segment GenBank with this peptide sequence
TMAccession number AAM89872, long 727bp does not contain complete ORF, is the synthetic RACE primer of stencil design with this est sequence, two nested primer: 3GSP1 of 3 ' RACE:
5′-TATGCTGTGGTCGAGGATTTAAACG-3′，3GSP2：
5 '-GGTGTTGCAAAGTTGTCTGTAAAACA-3 '; Two nested primer: 5GSP1 of 5 ' RACE:
5′-GGTGTTGCAAAGTTGTCTGTAAAACA-3′，5GSP2：
5′-TTGTCGTTTAAATCCTCGACCACAG-3′；
Concrete steps are pressed GeneRacer
TMThe test kit operational manual carries out, and in the pCR4-TOPO carrier that the RACE product cloning is provided to test kit, selects the positive colony order-checking, utilize DNAStar software search 3 ', the lap of 5 ' RACE product sequence and former est sequence, with three sections sequence assemblies;
3. contain the pulsating amplification of ORF cDNA
CDNA sequences Design primers F 1 and F2 according to splicing contain the pulsating amplification of ORF cDNA, F1:5 '-CCG
GGATCCATGAATCTAACTCAGCTAGAA-3 ' introducing restriction enzyme site BamH I underlines; F2:5 '-CGG
CTCGAGTTAATTACATGTAGATATAAC-3 ' introducing restriction enzyme site Xho I; Getting the 3 μ l cDNA that reverse transcription obtains in 3 ' RACE is that template is carried out pcr amplification, the PCR reaction conditions is respectively 94 ℃ of pre-sex change 5min, 94 ℃ then, 30s, 55 ℃, 30s, 72 ℃, 1min30s, totally 30 circulations, 72 ℃ are extended 10min after the loop ends, the PCR product reclaims test kit with sepharose DNA and reclaims, connect the pUCm-T carrier behind the purifying, be converted into the bacillus coli DH 5 alpha competent cell, a menu bacterium colony enzyme is cut evaluation, positive plasmid called after pUCm-T-Sjwnt4 and order-checking;
4. bioinformatic analysis
The cDNA that order-checking is obtained carries out the similarity comparison on NCBI, utilize ORF finder on the NCBI to find out the reading frame of new gene; Utilize DNAstar software that total number of atnino acid, composition, protein relative molecular weight are analyzed; It is right to utilize clustalW software that different plant species Wnt albumen is carried out multiple ratio; Utilize NetAcet software to carry out the glycosylation site prediction, utilize the MHCII phenotype on-line prediction instrument of SYFPEITHI to carry out the t cell epitope prediction;
5. fluorescence real-time quantitative RT-PCR
Selecting bilharzial housekeeping gene alpha-tubulin in the test is confidential reference items; Extracted respectively 14 days and 19 days virgin worms, 31 days adults, 44 days male worms and 44 days total RNA of female worm, utilize random primer to synthesize cDNA first chain behind the removal genomic dna; Fluorescence quantification PCR primer: the primer of amplification Sjwnt4: S1:5 '-ACATACAAAATCGTCTGGTCTC-3 ', S2:5 '-GATGGTAAAGGCGATGTAGTC-3 ', expanding fragment length 214bp; Amplification α-tubulin primer: T1:5 '-CTGATTTTCCATTCGTTTG-3 ', T2:5 '-GTTGTCTACCATGAAGGCA-3 ', expanding fragment length 213bp; It is synthetic that primer is given birth to the worker by Shanghai, adopts the fluorescence dye method to carry out real-time quantitative PCR, and reaction system comprises: 5 * R-PCR Buffer, 5 μ l, 250mM Mg
2+0.3 μ l, 10mM dNTP 0.75 μ l, 10 μ M primers, 1.0 μ l, 25 * SYBR Green I, 1.0 μ l, 10-3 * Calibration 1.0 μ l, 5U/ μ l Ex Taq Hs 0.25 μ l, ddH
2O 14.7 μ l, template cDNA 1.0 μ l, totally 25 μ l; Reaction parameter: 95 ℃ of sex change 3min, 95 ℃ of 5s, 58 ℃ of 30s, 40 circulations, wherein 58 ℃ of 30s concluding time points are the fluorescent signal check point, each reaction is all done 3 holes and is repeated; Adopt the BIO-RAD iCycler of company
TMIQversion 3.0 softwares carry out computational analysis, are confidential reference items markization reaction result with α-tubulin, draw the goal gene content with respect to per 1,000,000 housekeeping genes;
6. the structure of recombinant expression plasmid
From the recombinant plasmid pUCm-T-Sjwnt4 that the back of checking order is determined, cut out the cDNA segment that Sjwnt4 contains ORF with restriction enzyme BamHI, Xho I, with the multiple clone site district of this complete sequence directed cloning in prokaryotic expression carrier pGEX-4T-2, make up recombinant expression plasmid pGEX-4T-2-Sjwnt4, and transform expressive host bacterium BL21DE3;
7. the expression in intestinal bacteria
To identify that good pGEX-4T-2-Sjwnt4/BL21 DE3 is inoculated in liquid LB substratum, 37 ℃ of concussions are cultivated, the OD value is to add the IPTG abduction delivering that final concentration is 1mM at 0.6 o'clock, induces back 0h, 0.5h at IPTG, 1h, 2h, 4h, 6h collect thalline respectively, use SDS-PAGE and analyze tropina, determine best induction time;
8. Western blotting detects
The phase tropina carries out the SDS-PAGE electrophoresis during with high expression level, transfer on the nitrocellulose filter, resist with anti-GST monoclonal antibody or through the Schistosoma japonicum adult antigen immune rabbit anteserum work one that pGEX-4T-2/BL21 intestinal bacteria lysate adsorption treatment is crossed respectively, the sheep anti mouse of horseradish peroxidase-labeled, goat anti-rabbit igg are two anti-, and DAB develops the color as substrate.
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| CN1286261A (en) * | 2000-08-04 | 2001-03-07 | 华东师范大学 | Mimic peptide of schistosoma japonice ovum antigen and its screening and application |
| CN1477202A (en) * | 2003-03-11 | 2004-02-25 | 中国农业科学院上海家畜寄生虫病研究 | Schistosomiasis japonica (Chinese Continental strain) MFS4 gene clone, expression amd DNA vacine |
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| CN1286261A (en) * | 2000-08-04 | 2001-03-07 | 华东师范大学 | Mimic peptide of schistosoma japonice ovum antigen and its screening and application |
| CN1477202A (en) * | 2003-03-11 | 2004-02-25 | 中国农业科学院上海家畜寄生虫病研究 | Schistosomiasis japonica (Chinese Continental strain) MFS4 gene clone, expression amd DNA vacine |
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