CN102040658A - Schistosoma japonica polypeptide with immunogenicity and application thereof - Google Patents
Schistosoma japonica polypeptide with immunogenicity and application thereof Download PDFInfo
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Abstract
The invention discloses a schistosoma japonica polypeptide with immunogenicity, which can act with the gynecophoral canal protein of the schistosoma japonica mutually, and is provided with an amino acid sequence shown as SEQ ID NO: 1. The invention also discloses an application of the schistosoma japonica polypeptide with immunogenicity in preparing vaccines for preventing schistosomiasis or drugs for curing the schistosomiasis. The schistosoma japonica polypeptide with immunogenicity has a good immunoprophylaxis effect for the schistosomiasis, and is used as new vaccines for resisting the schistosomiasis.
Description
Technical field
The present invention relates to technical field of bioengineering, relate in particular to a kind of have immunogenic Schistosoma japonicum polypeptide and application thereof.
Background technology
Schistosomicide is a kind of parasitosis of serious harm people, animal health, and its susceptible animal comprises domestic animal and wildlifes such as people, ox, sheep, relates to more than 40 kind of Mammals.The animal infection schistosomicide is not only caused heavy losses to livestock industry production, and ill domestic animal still is the topmost contagium of China's human body schistosomicide, and prevention and cure of schistosomiasis situation is still severe.Clinical practice for many years proves that chemotherapy is not eliminated the area in schistosomicide can only temporarily reduce morbidity, and thorough blocking propagation.International experience shows: chemotherapy must combine with a kind of secular assist measure and just can produce a desired effect.Therefore, development is the assist measure that prevention and cure of schistosomiasis has strategic importance with the development blood fluke vaccine.Seek new vaccine candidate antigen and be still one of emphasis of current blood fluke vaccine research.
The bilharzial very special complexity of growing, it is a dioecism polypide unique in the Trematoda, it is the basis that female worm reaches maturity, lays eggs that female male worm is filled the span of a man's arms.Gynecophoric canal albumen is a kind of sex-specific material that transmits between the female male worm of schistosomicide that people such as Gupta find in Schistosoma mansoni.The Jin Yamei of China Agriculture Academe Shanghai Veterinary Institute, Cheng Guofeng, Fu Zhiqiang, Zhu Jianguo etc. are studied Schistosoma japonicum gynecophoric canal albumen, at first the clone obtains Schistosoma japonicum gynecophoric canal protein gene, and finds the sex-specific of its transcriptional expression and differ the opposite sex when growing.This gynecophoric canal protein gene is the specific female fluke expressing gene, and behind the schistosoma japonicum cercariae infection host the 14th day, promptly female male worm grew visible transcriptional expression in the male worm body before will fill the span of a man's arms.Utilize RNAi technology and schistosomicide vitro culture system that the proteic function of gynecophoric canal is studied, discovery gynecophoric canal albumen has influences the function that the female male worm of schistosomicide is filled the span of a man's arms, and this research also is that clear and definite first certain material can influence filling the span of a man's arms of the female male worm of schistosomicide both at home and abroad.
Display technique of bacteriophage originates from the research of Smith GP in 1985 etc., it is appropriate location and its fusion that exogenous peptide or protein coding gene is inserted into bacteriophage coat protein structure gene, makes foreign protein re-assemblying and be shown to the biotechnology of phage surface with phage.
Up to the present, the acceptor molecule that does not also have appearance to combine with Schistosoma japonicum gynecophoric canal albumen is reported as the research of vaccine application.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of immunogenic Schistosoma japonicum polypeptide that has, and this polypeptide can be used as antischistosomal vaccine candidate antigen.
In addition, also need to provide a kind of application of immunogenic Schistosoma japonicum polypeptide in the medicine that schistosomicide is treated in the vaccine or the preparation of preparation prevention schistosomicide that have.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of immunogenic Schistosoma japonicum polypeptide that has, described polypeptide can with Schistosoma japonicum gynecophoric canal protein-interacting, this polypeptide has the aminoacid sequence shown in the SEQ ID NO:1.
In another aspect of this invention, provide a kind of above-mentioned nucleotide sequence of encoding with immunogenic Schistosoma japonicum polypeptide.Preferably, described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:2 as.
In another aspect of this invention, also provide a kind of phage that comprises above-mentioned nucleotide sequence.
In another aspect of this invention, also provide a kind of host cell that comprises above-mentioned phage.
In another aspect of this invention, also provide a kind of schistosomicide vaccine, comprised the above-mentioned immunogenic Schistosoma japonicum polypeptide that has.Preferably, described schistosomicide vaccine also comprises the phage as expression vector.
In another aspect of this invention, also provide the above-mentioned antibody that immunogenic Schistosoma japonicum polypeptide produces that has.
In another aspect of this invention, also provide the above-mentioned application of immunogenic Schistosoma japonicum polypeptide in the medicine that schistosomicide is treated in the vaccine or the preparation of preparation prevention schistosomicide that have.
In another aspect of this invention, also provide the above-mentioned application of immunogenic Schistosoma japonicum polypeptide in the product of preparation diagnosis schistosomicide that have.
The present invention has immunogenic Schistosoma japonicum polypeptide and can be used as diagnostic antigen the schistosomicide ill domestic animal is diagnosed.
In another aspect of this invention, also provide the application of above-mentioned phage in the vaccine of preparation prevention or treatment schistosomicide.Phage can effectively strengthen the immunogenicity of vaccine not only as expression vector but also as immunological adjuvant.
The present invention has immunogenic Schistosoma japonicum polypeptide; in twice mouse immune protectiveness experiment; make worm reduction rate and the liver egg reduction rate of immune group mouse reach 40.53%/40.2% and 35.31%/76.45% respectively, show that the present invention has immunogenic Schistosoma japonicum polypeptide schistosomicide is had good immunoprophylaxis effect.
Description of drawings
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Fig. 1 is the embodiment of the invention 1 Schistosoma japonicum adult soluble antigen specific antibody detected result figure;
Fig. 2 is the OD of the embodiment of the invention 5
260nmLinear relationship chart with phage titre;
Fig. 3 is the embodiment of the invention 6 phage clone sample SDS-PAGE electrophorograms;
Fig. 4 is the embodiment of the invention 6 phage clone sample Western blot detected result figure;
Fig. 5 is specific antibody detected result figure in the embodiment of the invention 7 experimentation on animalies.
Embodiment
Following examples only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The present invention is on the basis in Schistosoma japonicum adult phage display cDNA library, select Schistosoma japonicum gynecophoric canal albumen as probe, utilize display technique of bacteriophage screening Schistosoma japonicum adult phage display cDNA library, in the hope of screening the acceptor molecule with Schistosoma japonicum gynecophoric canal protein-interacting, for exploitation schistosomicide novel vaccine and medicine provide the candidate antigens molecule.
The preparation of embodiment 1 anti schistosoma adult soluble antigen antibody
1. the preparation of Schistosoma japonicum adult soluble antigen
1) New Zealand white rabbit infects 2000 schistosoma japonicum cercariaes with the belly paster method, cuts open behind the 42d and kills, and collects polypide with the portal vein perfusion method, and is frozen standby in liquid nitrogen.
2) from liquid nitrogen, take out two pipe Schistosoma japonicum polypides, with PBS (1 * PBS, PH 7.0,0.5Mm PMSF, 0.1mM EDTA) cracking polypide.
3) utilize glass homogenizer that polypide is ground, place ultrasonic degradation 15min in the ice bath ,-70 ℃ of multigelations three times.
4) ice-bath ultrasonic cracking 15min again.
5) the centrifugal 1h of 12000g, collecting supernatant is adult soluble antigen (SWA).
6) detect OD260 and the OD280 value of SWA with spectrophotometer.
7) calculate SWA protein concentration, protein concentration=1.55 * OD260-0.68 * OD280.
2. the preparation of anti schistosoma antigen-antibody
1) it is standby to get-20 ℃ of preservations of blood and separation of serum before the immunity;
2) with PBS dilution Schistosoma japonicum soluble antigen, every New Zealand white rabbit hindlimb muscle injection imago of blood fluke soluble antigen 500ug is complete or Freund with Fu Shi;
3) every two all immunity once, carry out three immunity altogether, each immunity back one all ear veins are got blood, detect antibody with the agarose diffusion experiment and produce level;
4) three exempted from for two weeks after, cut open and kill to collect and separation of serum ,-20 ℃ of preservations are standby;
5) carry out the ELISA experiment with the Schistosoma japonicum adult soluble antigen as detecting antigen, detect antibody and produce level, the result as shown in Figure 1, in Fig. 1, A: preimmune serum antibody horizontal; B: infect back serum antibody level.As shown in Figure 1, after the immunity of Schistosoma japonicum adult soluble antigen, the antibody horizontal of anti schistosoma adult soluble antigen is apparently higher than the preimmune serum antibody horizontal in the New Zealand white rabbit serum, and this anti schistosoma adult soluble antigen antibody is used for following immunogenicity test experience.
The titre of phage (tiring) is exactly the quantity of contained phage alive in one milliliter of nutrient solution.Owing to containing on the agar plate of special host bacteria, phage produces macroscopic plaque, therefore can carry out the counting of phage.But because of its actual efficiency of plaque count method is difficult near 100% (generally on the low side, because have minority phage alive may not cause infection), so for the concentration of expressing viral suspension exactly (titre or tire), generally without the absolute quantity of virus particle but represent with plaque-forming unit (Plague-forming units writes a Chinese character in simplified form into pfu).The concrete steps that phage titre is measured are as follows:
1) host bacterium BLT5403 is inoculated in the 50ml M9LB nutrient solution, 37 ℃, 200rpm/min shaking culture make its OD
600nmValue reaches about 0.5;
2) get original phage library 10 μ L and carry out gradient dilution in 1: 100 ratio: 10 with the LB liquid medium
2, 10
4, 10
6, 10
7, 10
8, 10
9, 10
10, 10
11
3) each the gradient dilution liquid 100 μ L that gets phage library add 250 μ L BLT5403 (OD
600nmBe about 0.5), hatch 20min for 37 ℃ behind the mixing;
4) add 3.75mL again and be cooled to 55 ℃ top-agar, be tiled in behind the mixing and contain Amp
+The LB solid medium on, be inverted to cultivate 2～3h in 37 ℃ of incubators;
5) the plaque number on the counting flat board, phage titre (pfu/mL)=plaque number * extension rate * 10.
Embodiment 3 Schistosoma japonicum adult (Sj44d) phage display cDNA library screening
(Schistosoma japonicum phage display cDNA library is preserved by this laboratory, and its storage capacity is 4.98 * 10 based on Schistosoma japonicum adult phage display cDNA library
6Pfu, recombination fraction is 93.8%), select Schistosoma japonicum gynecophoric canal albumen as probe, screening Schistosoma japonicum adult phage display cDNA library, in the hope of screening and the relevant acceptor molecule of Schistosoma japonicum gynecophoric canal albumen, the concrete steps of screening with affine elutriation method are as follows:
1) the Schistosoma japonicum gynecophoric canal albumen with purifying is 1 μ g/mL with the TBS weaker concn;
3) Schistosoma japonicum gynecophoric canal diluted protein solution is added elisa plate with 100 μ L/ holes, 4 ℃ of bags are spent the night;
4) next day, the coating buffer that inclines pats dry, and cleans 5 times with TBST, is put in shaking table vibration 10min at every turn;
5) add 37 ℃ of sealings of 150 μ L 1h with the every hole of 3%BSA;
6) the deblocking liquid that inclines pats dry, and cleans 5 times with TBST, is put in shaking table vibration 10min at every turn;
7) original library (or every screening back elutriant amplified production of taking turns) being diluted to phage concentration with TBST is 10
10Pfu/mL, every hole adds 100 μ L and hatches 1h for 37 ℃;
6) the unconjugated phage that inclines pats dry, and cleans 5 times with TBST, is put in shaking table vibration 10min at every turn.
7) every hole adds 200 μ L 3M urea, is put in room temperature vibration 10min on the shaking table, discards reaction solution.
8) every hole adds 200 μ L 4M urea, incubated at room 20min.(confirm that through preliminary experiment other 4 kinds of elutriant 1%SDS, 10mM EDTA, 5M NaCl and 2M Guanidinium hydrochloride wash-out are after the original position amplification all can amplify a large amount of phages, elution efficiency significantly is lower than 4M urea)
9) collect elutriant, at once elutriant is carried out titer determination and amplification;
10) picking phage clone PCR identifies the pulsating size distribution situation of clone's external source, to detect the enrichment condition of phage;
11) measure the amplification phage titre and also carry out the next round screening as stated above.Carry out 3 altogether and take turns screening.
Wherein, the step of the amplification of elutriant and titer determination is as follows:
1) host bacterium BLT5403 is inoculated in the 50ml M9LB nutrient solution, 37 ℃, 200rpm/min shaking culture make its OD
600nmValue reaches about 0.5;
2) get elutriant 50ul and join (OD among the BLT5403
600nmBe about 0.5), continue shaking culture;
3) treat that bacterium liquid is clarified fully after, collect amplification phagocytosis body fluid, 4 ℃, the centrifugal 10min of 8000g low-temperature and high-speed;
4) get amplification phagocytosis body fluid 10 μ L and carry out gradient dilution with the LB liquid medium in 1: 100 ratio, carry out titer determination according to embodiment 2 methods and be used for the next round screening, it is standby that all the other add-20 ℃ of preservations of 5%～20% glycerine placement.
Through the affine elutriation of three-wheel, specific phage obtains higher degree enrichment (90 times), the picking positive phage clones checks order and bioinformatic analysis from screening on the back amplification flat board at random, the final polypeptide be made up of Schistosoma japonicum SJCHGC06360 protein part encoding sequence (aminoacid sequence shown in the SEQ ID NO:1) of obtaining.
The amplification and the purifying of embodiment 4 phage samples
1) host bacterium BLT5403 is inoculated in 50mL M9LB nutrient solution, 37 ℃, 200rpm/min shaking culture make its OD
600nmValue is about 0.5;
2) add each clone phagocytosis body fluid 50 μ L respectively, after the continuation shaking culture treats that bacterium liquid is clarified fully, collect amplification phagocytosis body fluid;
3) 4 ℃, the centrifugal 10min of 8000g low-temperature and high-speed are transferred to supernatant in another centrifuge tube and add the PEG/NaCl of 1/6 volume, and 4 ℃ of precipitations are spent the night behind the mixing;
4) 4 ℃, the centrifugal 20min of 12000g low-temperature and high-speed, supernatant discarded;
5) with the resuspended precipitation of 1mL phage leach liquor, the centrifugal 5min of 12000g low-temperature and high-speed;
6) supernatant is transferred in the 1.5mL centrifuge tube, adds the PEG/NaCl of 1/6 volume again, 4 ℃ of precipitation 1h behind the mixing;
7) 4 ℃, the centrifugal 10min of 12000g low-temperature and high-speed are dissolved in precipitation among 100 μ L, 1 * PBS, and 4 ℃ of preservations are standby.
The titer determination of embodiment 5 phage clones
1) the phage clone 10 μ L that get purifying carry out gradient dilution with the LB liquid nutrient medium in 1: 100 ratio, carry out phage titre according to the method for embodiment 2 and measure.
2) get each gradient dilution liquid of this phage simultaneously, the OD behind each phage gradient dilution of UV spectrophotometer measuring
260nm, OD
280nmAnd OD
320nmValue.
3) with the OD of each extent of dilution phagocytosis body fluid of adopted phage clone
260nm, OD
280nmOr OD
320nmValue is for X-coordinate, is ordinate zou with the titre of phage, analyzes actual measurement titre of phage and the linear relationship between the ultraviolet absorption value, sets up the linear relationship chart of rough determination phage titre.Fig. 2 is OD
260nmLinear relationship chart with phage titre.
4) calculate the titre of phage clone according to the linear relationship chart of setting up.
The immunogenicity of embodiment 6 phage clones detects
1) SDS-PAGE electrophoresis, the result as shown in Figure 3, in Fig. 3, M: marker protein; 1: empty phage vector; The phage clone of 2:SJCHGC06360 partial amino-acid series (polypeptide of aminoacid sequence shown in the SEQ ID NO:1), the fusion rotein that this phage clone is expressed such as the protein band of arrow indication.
2) electrophoretic blotting: cut and onesize nitrocellulose filter of blob of viscose and six metafiltration paper, and nitrocellulose filter is put into damping fluid invade bubble, filter paper is put into deionized water and is invaded bubble; Open box, downwards anodal, three metafiltration paper, nitrocellulose filter, gel, three metafiltration paper are placed in stack successively in order; Box is placed in the mould, adds biological ice, in groove, fill it up with transfering buffering liquid, 180mA, transferase 12～3h;
3) after electrophoretic transfer finishes, gel is transferred in the Kao Masi light blue dye liquor dyes, to check whether protein shifts fully;
4) will shift good nitrocellulose filter puts into 4 ℃ of sealings of 3% bovine serum albumin and spends the night;
5) discard confining liquid, it is inferior to give a baby a bath on the third day after its birth with TBST, is put in shaking table vibration 5min at every turn;
6) add the anti schistosoma adult soluble antigen antibody that dilutes at 1: 200 and resist incubated at room 1h as one;
7) TBST gives a baby a bath on the third day after its birth time, is put in shaking table vibration 5min at every turn;
8) goat anti-rabbit antibody of the HRP mark of adding dilution in 1: 2000, incubated at room 1h;
9) TBST gives a baby a bath on the third day after its birth time, is put in shaking table vibration 5min at every turn;
10) filter membrane is put into precipitation threshold tmb substrate colour developing liquid and develop the color, jog under the room temperature, after the color depth of protein band reached requirement, slightly rinsing of water was immediately transferred to filter membrane in the TBS solution then, takes pictures, writes down experimental result.
Phage clone Western blot detected result as shown in Figure 4, in Fig. 4, M: marker protein; 1: empty phage vector; The phage clone of 2:SJCHGC06360 partial amino-acid series (polypeptide of aminoacid sequence shown in the SEQ ID NO:1).As shown in Figure 4, the phage clone of aminoacid sequence has immunogenicity shown in the SEQ ID NO:1.
The immune protective experiment of embodiment 7 phage clones
1. animal immune experiment
1) buys BalB/C mouse, random packet, 10 every group.
2) the SJCHGC06360 partial amino-acid series phage clone that obtains with Schistosoma japonicum gynecophoric canal protein screening Schistosoma japonicum adult phage display library carries out the immunoprotection test, establishes the blank group simultaneously.
3) by table respectively with each group phage clone PBS dilution, with 206 adjuvants according to 450 μ L: 550 μ L ratio mixings, and to make every group sample total amount be 1.1mL.
4) every subcutaneous 3 injections in BalB/C mouse 0.1mL back, the phagocytosis scale of construction of every injected in mice is 10
13Pfu.
5) every two all immunity once, carry out three immunity altogether, exempt from all backs tail vein blood respectively, separation of serum-20 ℃ preservation with three before the immunity.
6) three exempted from for two weeks after, with 30 cercaria skin of abdomen of every mouse paster challenge infection.
7) pluck eyeball behind the 42d and get blood, hepatic vein is poured into towards worm, and counting polypide number is collected liver and ight soil.
8) serum of difference separated and collected blood ,-20 ℃ of preservations are standby.
2. counting worm reduction rate
Statistics is respectively organized the polypide number, counting worm reduction rate (worm reduction rate=control group polypide number-experimental group polypide number/control group polypide number).
The result is as shown in table 1 below, from the statistic data of table 1 as can be known, in twice experimentation on animals, to compare with the blank group, the worm reduction rate of the phage clone of SJCHGC06360 partial amino-acid series (polypeptide of aminoacid sequence shown in the SEQ ID NO:1) reaches 40.53% and 40.2% respectively.
The worm reduction rate of each group in table 1 immunization experiment
3. liver egg count
1) takes by weighing the weight of every group every BalB/C mouse liver respectively;
2) every liver is added 5mL PBS, the abundant homogenate of homogenizer;
3) add 5mL 10%NaOH again, 56 ℃ of digestion 1.5h;
4) will digest the abundant mixing of liver homogenate well, every group of abundant mixing of appropriateness dilution, anatomical lens is counting liver worm's ovum number down, every three repetition;
5) calculate liver average ovum lotus number (EPG), the average ovum lotus of liver is counted the average worm's ovum number of the liver * liver cumulative volume/liver gross weight of the each microscopy volume of EPG=, and calculates every group liver egg reduction rate (liver egg reduction rate=control group liver EPG-experimental group liver EPG/ control group liver EPG).
The result is as shown in table 2 below, from the statistic data of table 2 as can be known, in twice experimentation on animals, to compare with the blank group, the liver egg reduction rate of the phage clone of SJCHGC06360 partial amino-acid series (polypeptide of aminoacid sequence shown in the SEQID NO:1) reaches 35.31% and 76.45% respectively.
The liver egg reduction rate of each group in table 2 immunization experiment
4. the serological specificity antibody horizontal detects
1) with the positive phage clones bag by elisa plate, 4 ℃ are spent the night;
2) discard coating buffer, TBST cleans 3 times, each shaking table vibration 5min;
3) with PBS preparation 3%BSA, 37 ℃ of sealings of every hole 100 μ L 1h;
4) discard confining liquid, TBST cleans 3 times, each shaking table vibration 5min;
5) serum of collecting was diluted with 1: 100 respectively with TBS, every hole 100 μ L are hatched 1h for 37 ℃;
6) discard one and resist, TBST cleans 3 times, each shaking table vibration 5min;
7) sheep anti mouse two of horseradish peroxidase-labeled is anti-, with dilution in 1: 2000, and to add final concentration be 0.5% BSA with TBST, and fully behind the mixing, every hole 100 μ L are hatched 1h for 37 ℃;
8) incline two anti-ly, TBST cleans 3 times, each shaking table vibration 5min;
9) every hole adds 37 ℃ of colour developings of 100 μ L soluble T MB substrates colour developing liquid 20min;
10) 2M H
2SO
4The 50mL/ hole stops;
11) read light absorption value with microplate reader at the 450nm place.
The result as shown in Figure 5, in Fig. 5, alphabetical A, the B of X-coordinate refer to respectively: A:SJCHGC06360 partial amino-acid series phage clone; B: empty phage vector.As shown in Figure 5, SJCHGC06360 partial amino-acid series phage clone group no matter be three exempt from a week after or infecting for 6 weeks after, the specific antibody level of generation all is higher than sky phage vector control group (B group).
Can draw from above-mentioned experimental result, SJCHGC06360 partial amino-acid series phage clone has good immunogenicity, and can develop becomes the schistosomicide novel vaccine.The Schistosoma japonicum polypeptide of this clonal expression is the proteic partial amino-acid series of Schistosoma japonicum SJCHGC06360 (aminoacid sequence shown in the SEQ ID NO:1), preferably, encode the nucleotide sequence of aminoacid sequence shown in this SEQ ID NO:1 shown in SEQ ID NO:2, and this SEQ ID NO:2 nucleotides sequence is classified one section sequence in the Schistosoma japonicum SJCHGC06360 gene as.Phage is as the carrier of expression product, himself has very strong immunogenicity, it is natural immunological adjuvant, the Schistosoma japonicum albumen of expressing at phage surface is easier near antibody, easily by immune system recognition, and the expression product of phage clone is the native conformation product that organism self translating mechanism produces, and can well bring out the Ag-Ab immune response of body.
Therefore, the SJCHGC06360 partial amino-acid series phage clone that the present invention develops by display technique of bacteriophage has outstanding immunogenicity, is very suitable for becoming antischistosomal novel vaccine.
The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Sequence table
＜110〉China Agriculture Academe Shanghai Veterinary Institute
＜120〉have immunogenic Schistosoma japonicum polypeptide and application thereof
<160>2
<170>PatentIn?version?3.3
<210>1
<211>88
<212>PRT
<213>Schistosoma?japonicum
<400>1
Asn?Ser?Glu?Ser?Asp?Val?Lys?Glu?Ile?Thr?Val?Ala?Pro?Lys?Glu?Ser
1 5 10 15
Val?Val?Thr?Gln?Pro?Lys?Ser?Glu?Asp?Asn?Ile?Asp?Thr?Phe?Glu?Gln
20 25 30
Asn?Arg?Leu?Lys?Leu?Ala?Ala?Lys?Leu?Lys?Gly?Ser?Lys?Lys?Ser?Lys
35 40 45
Glu?Ser?Lys?Ser?Pro?Ser?Ser?Ser?Pro?Met?Gln?Lys?Pro?Asn?Gly?Lys
50 55 60
Arg?Val?Lys?Glu?Ser?Arg?Arg?Trp?Asp?Asn?Ala?Ala?Thr?Gly?Glu?Glu
65 70 75 80
Ala?Ala?Ala?Leu?Asp?Phe?Ser?Gly
85
<210>2
<211>261
<212>DNA
<213>Schistosoma?japonicum
<400>2
ctgaaagtga?tgttaaggaa?attactgtcg?caccaaagga?aagcgtagtt?actcaaccta 60
aatcagaaga?taacatcgat?actttcgaac?aaaatagact?taagttagct?gccaagctta 120
aggggtctaa?gaagagtaag?gaatctaaaa?gcccttcatc?atcacctatg?caaaagccta 180
acggtaagcg?tgtcaaagaa?agccgtcgat?gggataatgc?tgcaactgga?gaggaggcag 240
cagctttaga?ttttagtggc?a 261
Claims (10)
1. one kind has immunogenic Schistosoma japonicum polypeptide, it is characterized in that, described polypeptide can with Schistosoma japonicum gynecophoric canal protein-interacting, this polypeptide has the aminoacid sequence shown in the SEQ ID NO:1.
2. coding claim 1 described nucleotide sequence with immunogenic Schistosoma japonicum polypeptide.
3. nucleotide sequence according to claim 2 is characterized in that, described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:2 as.
4. phage that comprises the described nucleotide sequence of claim 2.
5. host cell that comprises the described phage of claim 4.
6. a schistosomicide vaccine is characterized in that, comprises that claim 1 is described to have an immunogenic Schistosoma japonicum polypeptide.
7. claim 1 is described has the antibody that immunogenic Schistosoma japonicum polypeptide produces.
8. claim 1 is described has the application of immunogenic Schistosoma japonicum polypeptide in the medicine of the vaccine of preparation prevention schistosomicide or preparation treatment schistosomicide.
9. claim 1 is described has the application of immunogenic Schistosoma japonicum polypeptide in the product of preparation diagnosis schistosomicide.
10. the application of the described phage of claim 4 in the vaccine of preparation prevention or treatment schistosomicide.
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| CN102040658B CN102040658B (en) | 2012-12-19 |
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| CN113045671A (en) * | 2021-01-29 | 2021-06-29 | 长春万成生物电子工程有限公司 | Recombinant antigen for detecting schistosoma japonicum, preparation method and application thereof |
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| CN113045671A (en) * | 2021-01-29 | 2021-06-29 | 长春万成生物电子工程有限公司 | Recombinant antigen for detecting schistosoma japonicum, preparation method and application thereof |
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