CN110208553A - Application of the bull with tapeworm Antigen B - Google Patents

Application of the bull with tapeworm Antigen B Download PDF

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CN110208553A
CN110208553A CN201910561192.6A CN201910561192A CN110208553A CN 110208553 A CN110208553 A CN 110208553A CN 201910561192 A CN201910561192 A CN 201910561192A CN 110208553 A CN110208553 A CN 110208553A
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antigen
bull
tapeworm
serum
coenosis
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CN110208553B (en
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杨光友
刘俞辰
古小彬
谢跃
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Sichuan Agricultural University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

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Abstract

The present invention relates to field of biotechnology, bull band tapeworm Antigen B is disclosed as a series of related applications such as coenosis diagnostic antigen, related experiment is as the result is shown, bull identifies without reacting with negative serum there is good immunogenicity and reactionogenicity with the tapeworm Antigen B lowlenthal serum that can be infected cenurus cerebralis；It is shown in indirect ELISA method compared with hypersensitivity and specificity simultaneously, and other opposite albumen have preferable thermal stability, various results prove that bull can be used as the diagnostic antigen of coenosis with tapeworm Antigen B, and are applied in detection kit.

Description

Application of the bull with tapeworm Antigen B
Technical field
The present invention relates to field of biotechnology, in particular to application of the bull with tapeworm Antigen B.
Background technique
Coenosis (Cerebral coenurosis) is middle silk ribbon phase of the bull with tapeworm (Taenia multiceps) A kind of pair of cattle and sheep caused by larva endanger serious parasitic disease.Cenurus cerebralis main parasitic is in the central nervous system of cattle and sheep System, can also parasitize and subcutaneously or intramuscularly organize, and in the case where eating bull band tapeworm worm's ovum by mistake, the mankind can also act as intermediate place It is main.This disease often results in the direct death of cattle and sheep, causes huge economy to the animal husbandry in Europe, the U.S., Africa and Asia Loss, the Accurate Diagnosis to infection animal is one of the emphasis direction studied at present.
Diagnostic method for coenosis includes anatomy, iconography and laboratory diagnosis.With science and technology Development, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) are also gradually applied to the clinical diagnosis of the disease In.In recent years, it has reported based on bull band tapeworm recombinant antigen (Tm-P2, Tm-HSP70, Tm-GP50, Tm-GST and Tm- HSP60 ELISA diagnostic method) achieves good detection effect, but the thermal stability one that the antigen for production has It is straight to be improved.
Summary of the invention
In view of this, the purpose of the present invention is to provide bull bands tapeworm Antigen B (Tm-AgB) to be used as cenurus cerebralis The diagnostic antigen of disease and the application in the diagnostic antigen for preparing coenosis, so that bull band tapeworm Antigen B has There are higher specificity and sensibility；
Another object of the present invention is that it is more in preparation detection brain to provide bull band tapeworm Antigen B (Tm-AgB) Head larva of a tapeworm or the cercaria of a schistosome disease kit in application so that with the bull with the tapeworm Antigen B ELISA method established show its compared with High specificity and sensibility can be used for ELISA detection；
Herein, bull band tapeworm Antigen B can be non-natural, e.g. synthesizing or by artificial carrier (being frequently referred to recombinant protein rTm-AgB in the industry) of expression.It is that nature is naturally deposited that term " non-natural ", which refers to target substance not, , the non-natural substances and naturally occurring material structure and/or composition having the same are not precluded in this.
Antigen B (Antigen B) is a kind of lipoprotein specific to a kind of tapeworm, and Monteriro etc. has found 3 of AgB Subunit AgB8/1, AgB8/2 and AgB8/3 molecule can spontaneous polymerization at 120~120kDa oligomer, the oligomer to heat it is steady It is fixed, there is spiral ring dichroscope spectral property, similar natural A g B.Existing research discovery shows Antigen B with respect to bull Band other albumen of tapeworm have a good thermal stability, but correlative study of the Antigen B in coenosis there is not yet Report.
The present invention by prokaryotic expression obtain recombination bull band tapeworm Antigen B (rTm-AgB, and Tm-AgB have Identical amino acid sequence, as shown in SEQ ID NO:1), carry out immunoblotting to it, ELISA and diagnostic method It establishes.Immunoblot results show that recombinant antigen can be infected the identified generation specific band of lowlenthal serum of cenurus cerebralis, And control group negative serum is reactionless, illustrates that recombinant antigen has stronger immunoreactivity and good immunogenicity.
Result of indirect ELISA is shown, is up to 95.8% (23/24) using the ELISA method sensibility that rTm-AgB is established, Specificity is that each 3 parts of serum generates friendship in 87.5% (21/24), with moniezia, Fasciola hepatica and haemonchus contortus 2 parts of serum generate cross reaction in fork reaction, with blister band tapeworm, generate cross reaction with 8 parts of serum of Echinococcus granulosus.With The lower sensibility of its rTm-TPx being compared is only 75.0% (18/24), and specificity is 91.7% (22/24)；rTm- The sensibility of HSP70 is 87.5% (21/24), and specificity is 83.3% (20/24)；And the sensibility of rTm-GST is 83.3% (20/24), specificity is 91.7% (22/24)；In conclusion rTm-AgB while sensibility and specificity with higher, and Other opposite albumen have preferable thermal stability, have preferable diagnosis effect, and the diagnosis for being suitable as coenosis is anti- It is former and prepare relevant detection kit as diagnostic antigen.
Diagnostic antigen based on above content, the present invention provides bull with tapeworm Antigen B as coenosis Application, and preparing the application in coenosis diagnostic antigen.Meanwhile the present invention also provides bull band tapeworms Application of the Antigen B in the kit of preparation diagnosis coenosis；Wherein, the kit is preferably ELISA reagent Box, more specifically, the ELISA kit is the kit based on ELISA indirect method.
From the above technical scheme, the present invention provides bull band tapeworm Antigen B diagnoses as coenosis A series of related applications such as antigen, related experiment is the results show that bull can be infected the mountain of cenurus cerebralis with tapeworm Antigen B Sheep blood serum is identified without reacting with negative serum, has good immunogenicity and reactionogenicity；Simultaneously in indirect ELISA side It shows in method compared with hypersensitivity and specificity, and other opposite albumen have preferable thermal stability, various results prove more Headband tapeworm Antigen B can be used as the diagnostic antigen of coenosis, and be applied in detection kit.
Detailed description of the invention
Fig. 1 show the SDS-PAGE and immunoblotting assay of Tm-AgB；Wherein, M: Protein standards；A: after purification Recombinant protein rTm-AgB；B: recombinant protein rTm-AgB after purification reacts with the lowlenthal serum of infection coenosis； C: recombinant protein is reacted with negative lowlenthal serum；
Fig. 2 show the sensibility, specificity and cross reaction situation of indirect ELISA method；Gray level line generation in figure The critical value of table indirect ELISA method；Statistical analysis is carried out using SPSS version 20.0, between different serum groups Difference uses Mann Whitney U test method statistic；P value indicates there is statistical significance less than 0.05.
Specific embodiment
Application the invention discloses bull with tapeworm Antigen B, those skilled in the art can use for reference present disclosure, It is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications carry out those skilled in the art Say it is it will be apparent that they are considered as being included in the present invention.Application of the present invention is retouched by embodiment It states, related personnel can obviously not depart from the content of present invention, be modified in spirit and scope to application described herein or suitably Changes and combinations carry out implementation and application the technology of the present invention.
The present invention is by extracting bull band tapeworm adult, protoscolex and hexacanth embryo total serum IgE, and reverse transcription is cDNA, from Tm-AgB gene is expanded in cDNA.After T is cloned, amplified production is imported in expression vector in a manner of digestion connection, with big Enterobacteria carries out the rTm-AgB that prokaryotic expression obtains recombination.
Cenurus cerebralis packing is obtained from the goat of Sichuan Province's natural infection.Aseptically, using disposable injection Device extracts packing cyst fluid, is centrifuged 5min with 3000rpm, is washed respectively 3 times with sterile saline and PBS afterwards, obtains protoscolex. Bull is derived from the 2 monthly age dogs of the protoscolex 30 days of 4 artificial challenges 20000 with tapeworm adult.Hexacanth embryo is by trypsin treatment It is discharged after worm's ovum in the fresh gravid proglottid of adult.
Bull provided by the present invention is described further with the application of tapeworm Antigen B below.
Embodiment 1: gene magnification
1, bull synthesizes with the extraction of tapeworm total serum IgE with cDNA
Bull band tapeworm adult, protoscolex and the hexacanth embryo for taking out Liquid nitrogen storage, are ground with mortar, referring next to day The animal tissue RNA extracts kit specification of root extracts total serum IgE.Referring to Suo Laibao mammalian proteins extracts kit explanation Book extracts total protein from adult.
2, amplification of the bull with tapeworm Tm-AgB gene
The primer reference transcript profile data Unigene17133 of Tm-AgB, it is set with 5.0 software of Primer Premier Primer, all primers are synthesized by the raw work in Shanghai:
The upstream Tm-AgB: 5 '-CGGGATCCATGAAAGCCTACATTGTT-3 ' BamHI
The downstream Tm-AgB: 5 '-CCAAGCTTCTAGTTCTCCTCATCCAT-3 ' HindIII
Amplification program is shown in Table 1:
Table 1
3, the clone of target gene and sequencing
Above-mentioned PCR product is subjected to glue recycling by Tiangeng plastic recovery kit specification, target gene and pMD19-T are carried Body connection, and be transferred to DH5 α Escherichia coli and be sequenced.
4, the amplification of Tm-AgB gene and basic physical and chemical attribute
CDNA using bull with tapeworm protoscolex goes out the band of 260bp or so as template amplification, obtains through sequencing Tm-AgB (Unigene17133) sequence homology reaches 100% in sequence and transcript profile.
Open reading frame in Tm-AgB sequence includes that (last 3 bases are to terminate to 261 codings, 86 amino acid polypeptides Codon does not encode amino acid).Signal peptide is predicted present in the 1 to 20th amino acid of the sequence, and seems have Trans-membrane region.The subcellular localization of the Tm-AgB of prediction shows that the albumen belongs to secretory protein.
5, the building and identification of recombinant plasmid
After cloning and sequencing identification is correct, extract the plasmid of pET-32a bacterial strain and target gene respectively, and with BamHI and The fast enzyme cutting of HindIII carries out double digestion, recycles digestion products.Product segment and pET-32a (+) expression vector are attached, Conversion and double digestion identification.
6, expression and identification of the recombinant plasmid in Escherichia coli
6.1, the expression and purification of recombinant protein
Correct recombinant plasmid pET32a-Tm-AgB will be sequenced and be transferred to BL21 (DE3) expression bacterium.
Expression bacterium is inoculated in two bottles of fresh LB (the 100 μ g/mL containing AMP) culture solutions containing 100mL, 37 DEG C of shaking tables It cultivates 6h (160r/min), until inducer IPTG (1mmol/L) is added after when bacterium solution OD590 is 0.8,37 DEG C of induction 6h (160r/ Min), another bottle is not added IPTG and does control culture.
It is taken to bacterium solution 1.5mL later in two new EP pipes respectively, 4 DEG C of centrifugation 1min (12,000r/min) are collected Thallus is separately added into 10 μ L 5 × SDS loading Buffer and 40 μ L PBS solutions, mixes well.
Boiling water boiling 10min, so that thallus sufficiently ruptures, 4 DEG C of centrifugation 10min (12,000r/min) take supernatant to carry out SDS- PAGE。
With coomassie brilliant blue staining 1h, expression is observed after decoloration.
6.2, the soluble analysis of albumen is expressed
Expression bacterium containing recombinant plasmid Pet32a-Tm-AgB is inoculated in the fluid nutrient medium of 500mL benzyl containing ammonia, 37 DEG C culture (160rpm/min) is 0.8 or so to OD590, is added best IPTG concentration, induces 6h.
Bacterium solution is centrifuged 10min (8000rpm), abandons supernatant, precipitating is outstanding with lysate (20mM Tris-HCl, pH=8.0) It is floating, ultrasonication thallus.
Broken cellular lysate liquid is centrifuged 10min (12,000rpm), precipitation and separation and supernatant under the conditions of 4 DEG C；It is heavy It forms sediment and suitable 8M urea dissolution is added.
Supernatant precipitating respectively takes 40 μ L, respectively plus 10 μ L 5 × sds gel sample-loading buffers, boils 10min, is centrifuged 10min (12,000rpm) carries out SDS-PAGE electrophoresis, analyzes whether it is solubility expression.
Target gene fragment is successfully connected on pET-32a carrier, and conversion enters inducing expression in BL21 Escherichia coli.? Under the conditions of 37 DEG C, expression quantity is maximum when inducing 6h with 1mM IPTG.The recombinant protein size of expression is that 26kDa or so (includes The label protein of 18kDa or so), meet expected size.Soluble analysis is the results show that be expressed as soluble protein.After purification Recombinant protein be single band (Fig. 1).
Embodiment 2: immunoblotting
With the immunoreactivity of immune-blotting method rTm-AgB and infected animal serum, the specific steps are as follows:
(1) after protein electrophorese, take the corresponding gel position where protein, be put into transferring film buffer carry out it is flat Weighing apparatus, totally 3 times, each 4min.
(2) nitrocellulose filter (NC film) and 24 layers of filter paper are placed in transfering buffering liquid and impregnate 5min.
(3) cathode electrode plate, 24 layers of filter paper, gel, NC film, 24 layers of filter paper Bio-Rad half dry type is placed in order to turn It prints in slot, covers anode electrode plate.
(4) electrotransfer device is connected on electroporation, and transfering buffering liquid 35mA transfer 30min is added.
(5) after shifting, NC film is taken out, is immersed in the TBST of 5% skimmed milk power, 4 DEG C of closings are overnight.
(6) after closing, NC film is cut off, the negative and positive is separated, and the diluted primary antibody of 1:1000 is added, and room temperature is incubated After educating 2h, primary antibody is outwelled, is quickly washed with TBST film 3 times, 5min/ times.
(7) after by goat-anti-rabbit the or rabbit-anti-goat IgG of HRP label by 1:1000 dilution, add Enter NC film, after being incubated at room temperature 2h, outwells secondary antibody, quickly washed with TBST film 3 times, 5min/ times.
(8) NC film is placed in plate, is rinsed with fresh substrate developing solution until developing the color.
(9) after developing the color, NC film color development stopping is rinsed with distilled water, and photograph to record to result.
Recombinant antigen can be infected the identified generation specific band of lowlenthal serum of cenurus cerebralis as the result is shown, and compare Group negative serum is reactionless, illustrates that recombinant antigen has stronger immunoreactivity and good immunogenicity (Fig. 1).
Embodiment 3: the foundation of indirect ELISA method
1, indirect ELISA operating procedure
(1) it is pressed with antigen coat liquid and is diluted than column, every 100 μ L of hole is added in 96 ELISA Plates and is coated with；
(2) coating buffer is outwelled, liquid in hole is patted dry, is washed with PBST, each 5min is repeated four times；
(3) confining liquid closing is added.
(4) with after PBS in proportion dilute serum after washing, every 100 μ L of hole is added enzyme mark hole and is incubated for, and outwells liquid.
(5) goat or the sheep anti-rabbit secondary antibody for the HRP label that 100 μ L have diluted is added in every hole after washing, and is incubated for.
(6) solubility one pack system substrate TMB is added into hole under the conditions of being protected from light and carries out chromogenic reaction；
(7) 100 μ L 2M H2SO4 are added in hole and terminate reaction, measure its OD value when ultraviolet absorptivity is 450nm.
A kind of kit can be formed according to the indirect ELISA reagent of the present embodiment.
2, condition optimizing
(1) best antigen and serum diluted concentration are determined with Checkerboard titration method, 6 antigen concentration gradients are set, serum from 1:20 to 1:640 makees doubling dilution, using the maximum condition of P/N as most preferably.
(2) 1%BSA, 5%BSA, 1% skim milk are used in the determination of best confining liquid respectively, and 5% skim milk is closed, Using the maximum condition of P/N as most preferably.
(3) most preferably incubating for positive and negative serum is screened in the determination of positive and negative seroreaction time according to determining condition The time is educated, is set as 37 DEG C, tri- groups of 0.5h, 1h, 1.5h, using the maximum concentration of P/N as most preferably.
(4) determination of the optium concentration of secondary antibody effect sets 1:2000,1:3000,1:4000,1:5000 tetra- dilutions respectively Concentration is groped, using the maximum condition of P/N as most preferably.
Grope optimum reaction condition with the indirect ELISA that rTm-AgB is established the results show that best coating condition is 4 DEG C of mistakes Night, antigen optium concentration are the 1 every hole μ g/, and best serum diluted concentration is 1:640, and best confining liquid and sealing condition are 5% de- 37 DEG C of closing 1h of rouge milk powder, it is 37 DEG C of 1h that serum, which is incubated for Best Times, and it is 37 DEG C of 1.5h that secondary antibody, which is incubated for Best Times, and secondary antibody is best When diluted concentration is 1:2000, the optimum condition of substrate colour developing is that 37 DEG C of 15min measure yin and yang attribute serum in the above conditions P/N value reach highest, be 2.23.
3, the determination of critical value
At optimum conditions, the OD450 of 24 parts of goat coenosis negative serums is measured.Three repetitions are set.According to Critical value=+ 3 times of average value standard deviation calculates.The critical value for obtaining rTm-AgB is 0.309, the plate of all indirect ELISA methods The coefficient of variation is respectively less than 10% between interior and plate.
4, specificity, sensibility and cross reaction test
It is detected with the indirect ELISA of foundation to 24 parts of lowlenthal serums that coenosis is suffered from confirmation are analysed, detects it Sensibility.Sensibility calculation formula are as follows: the practical positive number in ELISA result positive number × 100/.
It is detected to confirmation is analysed without 24 parts of lowlenthal serums that parasitic disease infect with the indirect ELISA of foundation, detection Its specificity.Specific calculation formula are as follows: the practical negative number in ELISA result feminine gender number × 100/.Meanwhile with 60 parts of serum Sample infects the sheep serum of Echinococcus granulosus (12 parts) and moniezia (12 parts) respectively from confirmation is analysed, and really Recognize and infected lowlenthal serum of the blister with tapeworm (12 parts), Fasciola hepatica (12 parts) and haemonchus contortus (12 parts), is handed over Pitch reaction test.
It is up to 95.8% (23/24) using the ELISA method sensibility that rTm-AgB is established, specificity is 87.5% (21/ 24) cross reaction, is generated with 3 parts of serum each in moniezia, Fasciola hepatica and haemonchus contortus, with blister band tapeworm In 2 parts of serum generate cross reactions, it is more serious (8 parts) with Echinococcus granulosus cross reaction.It is compared with them The lower sensibility of rTm-TPx is only 75.0% (18/24), and specificity is 91.7% (22/24)；The sensibility of rTm-HSP70 For 87.5% (21/24), specificity is 83.3% (20/24)；And the sensibility of rTm-GST is 83.3% (20/24), specificity For 91.7% (22/24), cross reaction situation and above-mentioned difference are less (Fig. 2).In view of conceptual data situation, rTm-AgB is same When sensibility and specificity with higher, and other opposite albumen have preferable thermal stability, have preferable diagnosis effect Fruit is suitable as the diagnostic antigen of coenosis and prepares relevant detection kit as diagnostic antigen.
5, in criticizing, batch between repetitive test
The coated plank of same batch is taken, the lowlenthal serum for being determined as the coenosis positive is analysed in 3 parts of detection, and every part sets 3 repeating holes are set, according to the ELISA method having built up, batch interior repetition is carried out and tests, calculate the coefficient of variation, detect this method Batch in repeatability.
3 coated planks of batch are taken, at optimum conditions, detect 3 parts of coenosis positive serums, every part of setting 3 A repeating hole carries out repeating to test between criticizing, calculates the coefficient of variation, detect batch of this method according to the ELISA method having built up Between repeatability.
For interassay coefficient of variation less than 10%, variation within batch coefficient shows that the test has good repeatability less than 5%.
6, clinical detection
The goat for only being from 5, Sichuan Province small towns to 100 carries out the investigation of cenurus cerebralis infection conditions, and each sample repeats to survey Three times, wherein 18 parts of serum detect corresponding antibodies, it is determined as the positive.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.
Sequence table
<110>Sichuan Agricultural University
<120>application of the bull with tapeworm Antigen B
<130> MP1913050
<160> 1
<170> SIPOSequenceListing 1.0
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Met Lys Ala Tyr Ile Val Leu Ala Leu Ala Leu Val Ala Phe Val Ala
1 5 10 15
Val Ala Arg Ala Glu Glu Asp Ile Glu Ser Lys Ala Arg Asp Gly Val
20 25 30
Met Lys Ser Leu Ala Glu Leu Lys Asp Phe Phe Lys Asn Asp Pro Met
35 40 45
Gly Gln Lys Leu Ala Ser Ile Cys Lys Asp Leu Lys Asp Leu Phe Leu
50 55 60
Met Ala Lys Thr Lys Thr His Ser Ala Phe Asn Asp Tyr Ile Lys Arg
65 70 75 80
Leu Met Asp Glu Glu Asn
85

Claims (4)

1. bull as the application of the diagnostic antigen of coenosis and/or is preparing coenosis with tapeworm Antigen B Application in diagnostic antigen.
2. application of the bull with tapeworm Antigen B in the kit of preparation diagnosis coenosis.
3. applying according to claim 2, which is characterized in that the kit is ELISA kit.
4. applying according to claim 3, which is characterized in that the ELISA kit is the reagent based on ELISA indirect method Box.
CN201910561192.6A 2019-06-26 2019-06-26 Application of taenia multiceps Antigen B Expired - Fee Related CN110208553B (en)

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Publication number Priority date Publication date Assignee Title
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CN106018831A (en) * 2016-07-14 2016-10-12 四川农业大学 Marker GP50 for coenuriasis, as well as coenuriasis diagnosing kit for diagnosing coenuriasis

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103184225A (en) * 2013-03-11 2013-07-03 中国农业科学院兰州兽医研究所 Taenia multiceps antigen gene and recombinant protein and application thereof
CN106018831A (en) * 2016-07-14 2016-10-12 四川农业大学 Marker GP50 for coenuriasis, as well as coenuriasis diagnosing kit for diagnosing coenuriasis

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Title
TAHEREH MOHAMMADZADEH，ET AL: "Comparison of the usefulness of hydatid cyst fluid, native antigen B and recombinant antigen B8/1 for serological diagnosis of cystic echinococcosis", 《TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE》 *
WEN HUI LI，ET AL: "Preliminary Analysis of Taenia multiceps Metacestode Antigens by Two-Dimensional Electrophoresis", 《IRANIAN J PARASITOL》 *
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