CN108752423A - A kind of Cryptosporidum parvum detection TSP7 polypeptide sequences and its application - Google Patents

A kind of Cryptosporidum parvum detection TSP7 polypeptide sequences and its application Download PDF

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CN108752423A
CN108752423A CN201810519141.2A CN201810519141A CN108752423A CN 108752423 A CN108752423 A CN 108752423A CN 201810519141 A CN201810519141 A CN 201810519141A CN 108752423 A CN108752423 A CN 108752423A
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monoclonal antibody
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CN108752423B (en
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尹继刚
王东强
张天宇
焦新
邵青延
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Jilin University
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Abstract

The present invention provides a kind of Cryptosporidum parvum detection TSP7 polypeptide sequences and its applications, it is to prepare monoclonal antibody as antigen, immune Balb/c mouse using after Cryptosporidum parvum peptide T SP7 and KLH couplings, collect sample（Egg capsule）, excystation liquid excystation, at zygoblast, permeabilization liquid permeabilization is separately added into monoclonal antibody, secondary antibody, PBS buffer solution, fluorescent dye, anti-fluorescence quencher, utilizes fluorescence microscope sample for incubation at room temperature.Show that there is six kinds of Cryptosporidium specific polypeptides for being coupled KLH good immunogenicity, immune mouse the hybridoma of ideal potency can be obtained through repeated multiple times experiment；The potency that can filter out screened every time reaches 1:16000 or more monoclonal antibody, is used as fluoroscopic examination.The monoclonal antibody prepared is done using TSP7 synthesis polypeptides can sensitively to mark on the surface of Cryptosporidum parvum zygoblast very much, and cross reaction does not occur.

Description

A kind of Cryptosporidum parvum detection TSP7 polypeptide sequences and its application
Technical field
The invention belongs to Biological Detection technical fields, and in particular to a kind of Cryptosporidum parvum detection TSP7 polypeptide sequences And its application.
Background technology
Cryptosporidum parvum is a main pathogen for leading to people and other mammal Cryptosporidiosis in global range. Cryptosporidum parvum is mainly absorbed by fecal oral route and is propagated by water or things that Cryptosporidium pollutes.Its main parasitic In the microvillus edge of gastrointestinal tract epithelial cell, Symptoms weight differs after infecting host.In the normal crowd of immune function In, self limiting is generally by diarrhea caused by being proliferated in enterocyte, but this is that immunocompromised person is main lethal Reason.Cryptosporidium parvum Oocysts can long-term surviving in the environment, and conventional hypochlorite disinfectant can be resisted, this becomes Control a great problem that Cryptosporidum parvum pollutes in drinking water.It there is no the specific medicament and height for Cryptosporidium so far The vaccine of effect prevention Cryptosporidum parvum, this further increases the preventing and controlling of Cryptosporidum parvum.Currently, small hidden spore The detection method of worm includes mainly pathogeny detection, immunology detection and molecular biology for detection.But these methods are big All time-consuming and laborious, and sensitivity more demanding to detecting instrument is insufficient.Therefore it is most important to find new detection method.
But the clinical testing procedure of Cryptosporidum parvum both domestic and external is examined mainly by the methods of acid-fast stain at present It surveys.Acid-fast stain is directed to the egg capsule wall of Cryptosporidium, and specificity is low, and sensibility is poor, and can also detect that do not have The dead worm of infection ability, can not judge whether detected sample has infection ability.Therefore, it finds new sensitive, special Detection method is of great significance to Cryptosporidum parvum epidemiological survey and study on prevention.It is small hidden herein by design synthesis The polypeptide of sporozoite multiple proteins is immunized mouse and prepares monoclonal antibody, by comparing the detection result of different antibodies, selection Optimal antibody is used for the clinical examination of Cryptosporidum parvum as Cryptosporidum parvum detection antibody.
Invention content
Purpose of the present invention is to solve the problems, such as that existing detection Cryptosporidum parvum method specificity is low, sensibility is poor, and A kind of Cryptosporidum parvum detection method is provided.
Cryptosporidum parvum peptide T SP7, amino acid sequence is as shown in sequence table SEQ ID No.1；
Cryptosporidium parvum Oocysts indirect immunofluorescent detection kit, it includes：Cryptosporidum parvum peptide T SP7 and KLH is even The monoclonal antibody prepared as antigen after connection；
The Cryptosporidium parvum Oocysts indirect immunofluorescent detection kit, it further includes secondary antibody, PBS buffer solution, fluorescence Dyestuff, anti-fluorescence quencher；
The fluorescent dye is 4', 6- diamidinos -2-phenylindone DAPI；
The Cryptosporidium parvum Oocysts indirect immunofluorescent detection kit further includes excystation liquid, paraformaldehyde, permeabilization Liquid；
The excystation liquid is 0.75% cholaic acid sodium salt and 0.25% pancreatin；
The permeabilization liquid is formaldehyde：Acetone volume ratio is 1：1.
Cryptosporidium parvum Oocysts indirect immunofluorescent detection kit is in terms of detecting Cryptosporidum parvum zygoblast Using.
A kind of Cryptosporidum parvum detection method, includes the following steps：
1）Sodium hypochlorite is added in sample, most of bacterium and impurity is removed, collects concentrate；
2）Excystation liquid is added in the concentrate being collected into, 0.5 ~ 1.5h is acted in 36 ~ 38 DEG C；
3）4500 ~ 5500g is centrifuged 8 ~ 12 minutes and is removed excystation liquid；
4）Paraformaldehyde is added in mixture after centrifugation, room temperature acts on 25 ~ 35min；
5）4500 ~ 5500g is centrifuged 8 ~ 12 minutes and is removed paraformaldehyde；
6）PBS buffer solution is added to have hanged and be coated onto on the coverslip of poly-D-lysine processing, stands 1 ~ 2h；
7）Extra PBS buffer solution is sucked, the zygoblast and egg capsule not being stained with are removed；
8）Permeabilization liquid is added, in -20 DEG C of 3 ~ 7min of permeabilization；
9）Monoclonal antibody, volume ratio 1 are diluted with the PBS buffer solution containing 1% fetal calf serum FBS：40 ~ 60, incubation at room temperature 0.5~1.5h；
10）FBS-PBS washes 3 times, every time 3 ~ 7min；
11）With FBS-PBS dilution Alexa fluorTM488 goat anti-mouse IgG secondary antibodies, volume ratio 1：1800~ 2200, it is incubated at room temperature 0.5 ~ 1.5h；
12）PBS is washed three times, every time 3 ~ 7min；
13）1 is pressed with PBS：20000 dilution DAPI, 2 ~ 5min of room temperature；
14）PBS is washed three times, every time 3 ~ 7min；
15）Extra PBS is sucked, anti-fluorescence quencher is added, covered utilizes fluorescence microscope sample；
The monoclonal antibody is the monoclonal antibody being coupled prepared by the polypeptide immune Balb/c mouse of KLH；Described Polypeptid acid sequence is as shown in sequence table SEQ ID No.1；
The excystation liquid is 0.75% cholaic acid sodium salt and 0.25% pancreatin；
In the permeabilization liquid, formaldehyde：Acetone volume ratio is 1：1.
The present invention provides a kind of Cryptosporidum parvum detection methods, it is to utilize Cryptosporidum parvum peptide T SP7（Its Amino acid sequence is as shown in sequence table SEQ ID No.1）It is used as antigen after being coupled with KLH, Balb/c mouse are immunized, prepares single Clonal antibody collects sample（Egg capsule）, excystation liquid excystation, at zygoblast, permeabilization liquid permeabilization is separately added into monoclonal for incubation at room temperature Antibody, secondary antibody, PBS buffer solution, fluorescent dye, anti-fluorescence quencher, utilize fluorescence microscope sample.Through repeated multiple times reality Testing six kinds of Cryptosporidium specific polypeptides for showing to be coupled KLH, there is good immunogenicity, immune mouse can be obtained The hybridoma of ideal potency；The potency that can filter out screened every time reaches 1:16000 or more monoclonal antibody, all It can be used as fluoroscopic examination.The monoclonal antibody prepared is done using TSP7 synthesis polypeptides can sensitively be marked in small hidden spore very much The surface of sporozoite, does not occur cross reaction.
Description of the drawings
Fig. 1 part of polypeptide is coupled result figure with BSA；
Six kinds of antibody titer detection figures of Fig. 2；
Six kinds of indirect immunofluorescene assay design sketch of Fig. 3.
Specific implementation mode
The screening and synthesis of 1 Cryptosporidum parvum specific polypeptide of embodiment
1, the screening of Cryptosporidum parvum specific polypeptide
1）In the professional website of Cryptosporidium（http://cryptodb.org/cryptodb/）Plurality of target gene is searched, is made For alternative antigen；
2）The amino acid sequence of the target gene found is subjected to the pre- of three-dimensional structure by swissmodel.expasy.org It surveys, finds the relatively stronger random coil region of immunogenicity, recycle the prediction of iedb.org prediction B cell epitopes, most Other albumen of the polypeptide of design and Cryptosporidium are compared afterwards, take immunogenicity strong, the good peptide fragment of specificity is closed At；
3）In order to enhance the coupling efficiency of polypeptide and KLH and BSA, design polypeptide when, selectively avoid containing half Guang ammonia The polypeptide of acid, and C-terminal does not have manually to add a cysteine behind the polypeptide of cysteine again.
2, the synthesis of Cryptosporidum parvum specific polypeptide
Designed six kinds of Cryptosporidium specific polypeptides are synthesized by Qiang Yao biotech firms；Six kinds of Cryptosporidium specificity are more Peptide is respectively TSP7, TRAP-C2, TSP3, TSP6, TSP10 and GP900, amino acid sequence such as No.1 ~ 6 sequence table SEQ ID It is shown；Due to the haptens that the polypeptide fragment of design synthesis is small molecule, only have reactionogenicity without immunogenicity, so It needs to be coupled with a macromolecular substances in immune animal, stimulates animal body to generate antibody with this.Finally, polypeptide and blood are blue Albumen（KLH）Coupling is as animal antigen is immunized, with bovine serum albumin(BSA)（BSA）Coupling is used anti-as ELISA detection coatings It is former.The step of polypeptide is completed, is coupled with BSA by Qiang Yao biotech firms with KLH couplings is as follows：
1）1mgBSA is dissolved in the deionized water of 200 μ L, the m- maleimidobenzoyl-N- hydroxysuccinimidyl acyls of 0.2mg Imines（MBS）It is added in BSA solution after being dissolved in the DMF of 40 μ L, room temperature rotation mixing 2h；
2）The PBS dialysed overnights of large volume；
3）1mg polypeptides are dissolved in the PBS of 200 μ L, the MBS-BSA mixtures of dialysed overnight are then added to polypeptide solution In, it is incubated at room temperature 4h；
4）The PBS dialysed overnights of large volume, -20 DEG C of preservations after 50 μ L packing.
3, the coupling effect of polypeptide and BSA is detected
Following steps are taken to be detected：
1）Sample preparation：5 × SDS-PAGE Loading Buffer of the peptide-BSA and 10 μ L of 40 μ L are taken to mix, boiling water Bathe 5min；
2）SDS-PAGE is denaturalized the preparation of glue：Lower layer's separation gel that 10% is prepared according to formula prepares 5% again after coagulating completely well Upper layer concentrates glue；
3）Electrophoresis：Add in 15 μ L samples to well, the concentration glue on upper layer uses 75 V constant pressure electrophoresis, after into separation gel Voltage is adjusted to 145V and continues electrophoresis, until being moved to gel bottom；
4）Dyeing：Gel is put into the coomassie brilliant blue staining liquid of Fresh, horizontal shaker shakes stained over night；
5）Decoloration：Stained gel is put into destainer, decoloration is shaken on horizontal shaker to gel without background colour.
Electrophoretograms of the Fig. 1 for polypeptide and the SDS-PAGE of BSA couplings after coomassie brilliant blue staining；Wherein 1 He of swimming lane Swimming lane 2 is the electrophoresis result after BSA and BSA and MBS couplings respectively, and swimming lane 3 ~ 7 is that polypeptide is coupled with MBS-BSA respectively Electrophoresis result later；It can be seen from the figure that polypeptide is more somewhat larger than the molecular weight of BSA after being coupled with BSA, show more Peptide is coupled successfully with BSA.
The preparation of 2 animal immune of embodiment and monoclonal antibody
One, experimental animal immune program
Coupling six kinds of Cryptosporidium specific polypeptides of KLH are mixed with isometric Freund's adjuvant, Balb/c mouse are immunized；For the first time It is immune mix with Freund's complete adjuvant, amount is immunized as 100 μ g, latter 4 times immune to mix with Freund's incomplete adjuvant, immune to measure as 50 μ g；Twice Immune interval time is 15 days；Immune 20 μ g polypeptides of Balb/c mouse tail vein injections are carried out in first three day of cell fusion Booster immunization.
Two, prepared by monoclonal antibody
1, the preparation of feeder cells
The previous day of fused cell makes word and supports confluent monolayer cells；A common BALB/c mouse is taken, draws neck to put to death, is put into 75% alcohol Impregnate 5min.It is transferred to superclean bench, abdominal cut skin is pulled open to both sides, and exposure peritonaeum takes the 5mL syringes after sterilizing, 6 ~ 8mLHAT culture solutions are extracted, mouse peritoneal is injected, is extracted out after kneading abdomen more than 10 times repeatedly, add HAT to 12mL, it is every after mixing Hole 1 is added dropwise into 2 piece of 96 well culture plate, places 37 °C, 5%C02Culture is spare in cell incubator.
2, cell fusion
1) sterile to take spleen
Immune mouse tweezers are extractd into eyeball, mouse blood sampling is gently squeezed, the mouse after blood sampling is put into 75% alcoholic solution Middle immersion 5min.It is transferred to superclean bench, gauze on pad cuts off skin with the scissors of high-temperature sterilization, breaks peritonaeum, stripping successively From fat, spleen is taken to be put into the culture dish of the washing lotion containing 10mL, it is dazzling on spleen with syringe needle, then used from the side of spleen 1mL syringes inject washing lotion, inject 2 ~ 3 times repeatedly, are then lightly ground spleen with 5mL syringe needle stamens, until spleen only stays Until outer membrane.Suspension is filtered with copper mesh several times, by contaminant filter repeatedly.It sucks in 10mL spleen lapping liquids to centrifuge tube, 1500r/m centrifuges 6min；.
2) SP2/0 cells are handled
The culture bottle supernatant of the cell containing SP2/0 is outwelled, bottle wall is blown and beaten repeatedly with washing lotion, SP2/0 cells are drawn to 50mL fusions With in glass centrifugal bottle, 1500r/m centrifuges 6min；
3) it is mixed after being blown afloat splenocyte and SP2/0 cells with washing lotion, 1500r/m centrifuges 6min；
4) with hand gently percussion bottom of bottle, so that cell is become single layer, be put into 37 °C of water-baths, 1mL50%PEG is slowly added drop-wise to from It is dripped in 1min in heart bottle, keeps bottle content constantly to rotate during being added dropwise；
5) 90s is stood；
6) slowly add washing lotion 4mL in 3min；Four steps are divided to complete, it is as follows：
Washing lotion 1ml is added in 60s
Washing lotion 1ml is added in 60s
Washing lotion 1ml is added in 30s
Washing lotion 1ml is added in 30s
7) a large amount of washing lotions are added, 1500r/m centrifuges 6min, washes away PEG；
8) supernatant is abandoned, 35mL HAT are added in precipitation；
9) it is added in 96 well culture plates that feeder cells are added in the previous day, is dripped per hole 3, place 37 °C of 5%C02 cells trainings Support culture in case；
10) after merging 24 hours, I1AT training bases are added-dripped per hole, mat woven of fine bamboo strips 4d, 6d, 8d and 10d change liquid after fusion, fusion 10 ~ 12d can detect potency afterwards.
3, the screening of hybridoma
In 100 times of microscopic observations, when on tissue culture plate cell growth to a visual field 1/3 when can by cell liquid supernatant into Row indirect ELISA detects.By the high cell expansion culture of potency, freeze.Through repeated multiple times experiment, it is coupled six kinds of hidden spores of KLH Mouse, which is immunized, in sub- worm specific polypeptide can filter out the high hybridoma of valid value, illustrate six kinds of Cryptosporidiums for being coupled KLH There is specific polypeptide good immunogenicity, immune mouse the hybridoma of ideal potency can be obtained.
4, ascites preparation and antibody purification
1）At first 7 days of inoculation hybridoma, need to the isometric normal saline dilution of balb/c mouse peritoneal injections Freund's incomplete adjuvant, every mouse peritoneal injection 1 × 106 Hybridoma.It injects hybridoma and collects ascites after 5-7 days；
2）The ascites being collected into is purified with " octanoic acid-ammonium sulfate precipitation " method, and steps are as follows：
A：With the acetate buffer solution of 4 times of volumes（60mM, pH=4.0）Ascites is diluted, then with 0.1M NaOH tune pH to 4.5；
B：It is slowly added to octanoic acid（25μl/ml of diluted water）, and it is made fully to dissolve, room temperature shakes 30min, 10000g centrifuges 30min, draws supernatant；
10 × PBS buffer solution of 1/10 volume is added in C, with 1M NaOH tune pH to 7.4；
Supernatant is cooled to 4 DEG C by D, and ammonium sulfate is added（0.277g/ml, 45% saturation degree）30min is stirred, 5000g centrifuges 15 points Clock collects precipitation IgG；
E abandons supernatant, with 1/10 PBS suspensions IgG of former ascites volume；
The PBS dialysed overnights of 50 times of volumes of F；
Sample after dialysis is heated to 55 DEG C by G, heats 20min, and 5000g centrifuges 20min, collects supernatant in -80 DEG C of preservations；
HSDS-PAGE detects antibody purity.
5, antibody titer is detected by indirect ELISA method by different extension rates, steps are as follows：
1）Antigen coat：The peptide-BSA that coupling obtains is diluted to 5 μ g/mL with Coating Buffer, per 50 μ of hole L, 37 DEG C of placement 1h, 4 DEG C overnight；
2）Board-washing：Washing Buffer are prepared, using board-washing machine washing plate 3 times, per 4 min of minor tick；
3）Closing：The confining liquid that 100 μ L contain 3% BSA, 37 DEG C of 1 h of closing are added per hole；
4）Board-washing：Same step（2）；
5）Primary antibody：1 is pressed using Tween Buffer：200,1:500,1:1000,1:2000,1:4000,1:8000,1:16000, 1：32000 extension rates dilute primary antibody, per hole 50 μ L, 37 DEG C of 1 h of incubation；
6）Board-washing：Same step（2）；
7）Secondary antibody：1 is pressed using Tween Buffer:20000 extension rates dilute secondary antibody, per hole 50 μ L, 37 DEG C of 1 h of incubation；
8）Board-washing：Same step（2）；
9）Colour developing：The developing solution of 1 mg/mL is prepared, per 50 μ L of hole, 37 DEG C are protected from light 20 min of colour developing, and microplate reader detects OD405 and reads Number.
Six kinds of antibody titer detection figures are as shown in Fig. 2, antibody titer reaches 1:16000 or more, it is used as Fluoroscopic examination.
3 indirect immunofluorescene assay clinical sample of embodiment
Six kinds of monoclonal antibodies that above-described embodiment is prepared use, specific steps respectively as the primary antibody in fluoroscopic examination It is as follows：
1, a certain amount of sodium hypochlorite is added in the clinical sample being collected into and carries out sterilization processing, the side floated followed by sucrose Method removes most of bacterium and impurity；
2, excystation liquid is added in the concentrate being collected into（Including 0.75% cholaic acid sodium salt, 0.25% pancreatin）, 1h is acted in 37 DEG C, The purpose is to be incubated the egg capsule of Cryptosporidum parvum, 4 zygoblasts inside egg capsule are released, it is prepared to detect with this Monoclonal antibody whether can act on zygoblast；
3,5000g is centrifuged 10 minutes and is removed excystation liquid；
4,4% paraformaldehyde is added in the mixture after centrifugation（2g formaldehyde is dissolved in 50ml PBS solutions）, room temperature effect 30min fixes zygoblast by paraformaldehyde, it is made to hold complete form in subsequent experiment；
5,5000g is centrifuged 10 minutes and is removed paraformaldehyde；
6, PBS buffer solution is added to have hanged and be coated onto on the coverslip of poly-D-lysine processing, stands 1h, zygoblast is made to adhere to lid glass The surface of piece；
7, extra PBS is sucked, the zygoblast and egg capsule not being stained with are removed；
8, permeabilization liquid is added（Formaldehyde：Acetone=1：1）In -20 DEG C of permeabilization 5min；
9, with six kinds of prepared monoclonal antibodies of the PBS dilutions containing 1%FBS（1：50）, it is incubated at room temperature 1h；
10, FBS-PBS is washed 3 times, each 5min；
11, with FBS-PBS 1：2000 dilution Alexa fluorTM488 goat anti-mouse IgG secondary antibodies, incubation at room temperature 1h；
12, PBS is washed three times, each 5min；
13, with PBS by 20000 times of dilution 4', 6- diamidinos -2-phenylindone（DAPI）, room temperature 3min；
14, PBS is washed 3 times, each 5min；
15, extra PBS is sucked, anti-fluorescence quencher is added, covered utilizes fluorescence microscope sample.
It is compared by the experimental result of a variety of monoclonal antibodies, chooses specificity, sensibility is highest as final Diagnosis antibody；The results are shown in Figure 3, and the monoclonal antibody prepared using TSP7 synthesis polypeptides can be marked sensitively very much The surface of Cryptosporidum parvum zygoblast, and cross reaction does not occur with the thalline in sample.Therefore selection sensibility and spy Anisotropic high anti-TSP7 polypeptide antibodies are as Cryptosporidum parvum detection antibody.
Sequence table
<110>Jilin University
<120>A kind of Cryptosporidum parvum detection TSP7 polypeptide sequences and its application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213>Cryptosporidum parvum TSP7 (Cryptosporidium parvum)
<400> 1
Gln Tyr Ser Glu Trp Thr Met Trp Gly Glu Cys
1 5 10
<210> 2
<211> 15
<212> PRT
<213>Cryptosporidum parvum TRAP-C2 (Cryptosporidium parvum)
<400> 2
Glu Pro Lys Gly Ala Pro Leu Leu Tyr Val Asp Gly Asp Gly Cys
1 5 10 15
<210> 3
<211> 14
<212> PRT
<213>Cryptosporidum parvum TSP3 (Cryptosporidium parvum)
<400> 3
Gln Ala Asn Pro Ser Ser Ile Leu Asn Leu Ala Asn Gln Cys
1 5 10
<210> 4
<211> 15
<212> PRT
<213>Cryptosporidum parvum TSP6 (Cryptosporidium parvum)
<400> 4
Glu Leu Asp Ser Ser Arg Thr Pro Val Asn Glu Thr Ile Asn Cys
1 5 10 15
<210> 5
<211> 14
<212> PRT
<213>Cryptosporidum parvum TSP10 (Cryptosporidium parvum)
<400> 5
Ser Gly Ser Ala Thr Gln Gly Gly Pro Ser Thr Thr Glu Cys
1 5 10
<210> 6
<211> 12
<212> PRT
<213>Cryptosporidum parvum GP900 (Cryptosporidium parvum)
<400> 6
Val Pro Gly Thr Ala Ala Pro Lys Lys Gly Gly Cys
1 5 10

Claims (8)

1. a kind of Cryptosporidum parvum detection TSP7 polypeptide sequences and its application, amino acid sequence such as sequence table SEQ ID Shown in No.1.
2. Cryptosporidium parvum Oocysts indirect immunofluorescent detection kit, it includes：Small hidden spore described in claim 1 The monoclonal antibody prepared as antigen after worm peptide T SP7 and KLH couplings.
3. Cryptosporidium parvum Oocysts indirect immunofluorescent detection kit according to claim 2, it is characterised in that：It Further include secondary antibody, PBS buffer solution, fluorescent dye, anti-fluorescence quencher.
4. Cryptosporidium parvum Oocysts indirect immunofluorescent detection kit according to claim 3, it is characterised in that：Institute The fluorescent dye stated is 4', 6- diamidinos -2-phenylindone DAPI.
5. according to the Cryptosporidium parvum Oocysts indirect immunofluorescent detection kit described in claim 2,3 or 4, feature exists In：Further include excystation liquid, paraformaldehyde, permeabilization liquid.
6. a kind of Cryptosporidium parvum Oocysts indirect immunofluorescent detection kit according to claim 5, feature exist In：The excystation liquid is 0.75% cholaic acid sodium salt and 0.25% pancreatin.
7. a kind of Cryptosporidium parvum Oocysts indirect immunofluorescent detection kit according to claim 6, feature exist In：The permeabilization liquid is formaldehyde：Acetone volume ratio is 1：1.
8. the Cryptosporidium parvum Oocysts indirect immunofluorescent detection kit described in claim 2 is in detection Cryptosporidum parvum Application in terms of zygoblast.
CN201810519141.2A 2018-05-25 2018-05-25 TSP7 polypeptide sequence for detecting cryptosporidium parvum and application thereof Expired - Fee Related CN108752423B (en)

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