CN110183527A

CN110183527A - A kind of neospora NcMIC26 antigen and its application

Info

Publication number: CN110183527A
Application number: CN201910422519.1A
Authority: CN
Inventors: 刘晶; 王先梅; 刘群; 许建海
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2019-08-30
Anticipated expiration: 2039-05-21
Also published as: CN110183527B

Abstract

The invention discloses a kind of neospora NcMIC26 antigen and its applications.The present invention discloses a kind of neospora NcMIC26 antigen first, and the amino acid sequence of the antigen is as shown in SEQ ID NO.1.The present invention further discloses the recombinant proteins of above-mentioned antigen to apply in building neospora indirect ELISA diagnostic reagent kit.The present invention has obtained a kind of new diagnostic antigen suitable for neosporosis by screening, indirect ELISA reagent kit is established using its recombinant protein as envelope antigen, it is easy to operate, rapidly and efficiently, specificity is good, sensibility is high, it is reproducible, can be used for detecting cows neospora infections situation, can detect common NcSRS2 Protein Detection less than positive sample, it can get more accurate detection data, be of great significance for the prevention and treatment of neosporosis.

Description

A kind of neospora NcMIC26 antigen and its application

Technical field

The present invention relates to a kind of field of biotechnology, especially with regard to a kind of neospora NcMIC26 antigen and its application.

Background technique

Neosporosis (neosporosis) is a variety of by that can infect caused by neospora (Neospora caninum) The protozoosis of animal, the disease that neospora causes have global distribution trend.The disease is the most serious to the harm of ox and dog, is One of cause ox miscarriage, produce the main reason for weak tire, stillborn foetus, the mummification of fetus and the nervous system disorders of dog disease, give herding Industry brings serious economic loss.The infection intensity for the ox neosporosis that country variant and area are reported exists certain poor Different, positive rate of antibody is 13.5%~82%.Currently, to neospora, there are no any effective drugs and vaccine.

Neosporosis predominantly detect method have etiological diagnosis, histopathological diagnosis, diagnosis of molecular biology with And serodiagnosis etc..Since neospora is special sexual cell endoparasitism protozoon, aetology and pathological diagnosis are more difficult, and Serological diagnostic method can apply to living animal detection, and the zoogenetic infection stage can be provided during epidemiological survey Information, and its infect neospora after antibody level height, it may be possible to provide about animal with neosporosis, ruminate it is dynamic The advantages that logistics produces and propagates the information of neospora risk through placenta, it has also become the important hand of epidemiological investigation Section.The serological technique for being applied to detection neospora antibody earliest is indirect immunofluorescence assay (IFAT), and new at present " goldstandard " of sporozoite Serology test.But reagent (such as matter of specificity fluorescent labelled antibody that the experiment needs Amount) and instrument (such as fluorescence microscope) and operating procedure it is complex, it is critical to result to the more demanding of experiment operator Value is determined with certain difficulty, therefore is not suitable for clinically field high-volume screening experiment.With extensive epidemiology The development of research, ELISA detection method due to it is simple, quickly, efficiently, as a result determine not influenced etc. by subjective factor it is excellent It puts and is widely used, be increasingly becoming the main method of Serologic detection.

Early stage indirect ELISA is with the diagnosis of tachyzoite, the neospora antigen of cracking and immunostimulating complex (ISCOM) Antigen is detected, and shows good Diagnostic parameters during neospora diagnosis, but due to neospora and belong to top The toxoplasma affiliation of multiple subphylum protozoon is close, is easy to appear cross reaction and causes specificity not high, and prepared by the antigen Higher cost.With recombinant protein expression and the development of purification technique, native antigen conduct is replaced using high-purity recombinant protein The diagnostic techniques of detection antigen compensates for disadvantages mentioned above.Generally acknowledged at present is also that most widely used neospora diagnostic antigen is NcSRS2 shows good specificity and sensibility in Serologic detection.But due to the different times of cattle infected neospora And there are certain fluctuations in infected cattle body for antibody level.It is found in some researchs, neospora positive babies The reason of cow serum is negative, and causes this phenomenon may be due to caused by the fluctuation of cow internal antibody level, or Person is the false negative due to the not high formation of the sensibility of detection method.Reported detection antigen is in the neosporosis to cows Practical diagnosis during there are still limitation, the result that right pop disease is investigated causes certain error.

Therefore, there is an urgent need to screen the better dominant antigen of new diagnosis effect, more sensitive, special serology is established Diagnostic method can apply to animal population detection, provide foundation for the diagnosis of neosporosis.

The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without answering When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.

Summary of the invention

One of the objects of the present invention is to provide a kind of new neospora dominant antigens, and the antigen is to detection neospora With high sensibility and good specificity.

Another object of the present invention is to provide include indirect ELISA of the recombinant protein of above-mentioned antigen as envelope antigen Kit establishes simple, fast and efficient serological diagnostic method.

To achieve the above object, the present invention provides a kind of neospora NcMIC26 antigen, the amino acid sequences of the antigen Column are as shown in SEQ ID NO.1.

In a preferred embodiment, the present invention prepares the screening of neospora NcMIC26 antigen and identification includes such as Lower step:

Neospora is cultivated, polypide is proliferated and collects, is incubated for after precipitating is resuspended, tachyzoite excretory-secretory antigen is collected With tachyzoite whole worm antigen；

The tachyzoite excretory-secretory antigen being collected into and tachyzoite whole worm antigen are subjected to immunoblotting, screening obtain not with The neospora specific protein band of toxoplasma positive serum reaction；

Neospora specific protein band is collected by SDS-PAGE electrophoresis and is analyzed by mass spectrometry, to obtain this hair Bright neospora NcMIC26 antigen.

Invention further provides a kind of genes for encoding above-mentioned neospora NcMIC26 antigen.

In a preferred embodiment, the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

The present invention also provides a kind of neospora NcMIC26 antigen above-mentioned or coding neospora NcMIC26 antigens Gene preparation treatment, diagnosis or prevention of neosporosis product in application.

The present invention further also provides a kind of neospora indirect ELISA diagnostic reagent kit, wraps in the diagnostic kit Include amino acid sequence recombinant protein rNcMIC26 as shown in SEQ ID NO.1.

In a preferred embodiment, the preparation method of the recombinant protein rNcMIC26 includes the following steps:

1) design primer F and R:

F:5 '-AGCAAATGGGTCGCGGATCCGTTCTGGATTTCATAGACTTGG-3'；

R:5 '-TCGAGTGCGGCCGCAAGCTTCGAAGTCCATTCGCCCCACGTT-3'；

2) PCR amplification is carried out using primers F and R using the cDNA of neospora as template, obtains target fragment；

3) target fragment and carrier pET-28a are attached, connection product is converted to Escherichia coli Trans1-T1 and is felt By state cell, recombined pronucleus expression plasmid pET-28a-MIC26 is obtained；

4) the recombined pronucleus expression plasmid pET-28a-MIC26 built is converted to Escherichia coli Transetta and is experienced State cell induces the expression of recombinant protein, collects and purifies to obtain recombinant protein rNcMIC26.

Neospora indirect ELISA diagnostic reagent kit of the present invention further includes detection antibody, the coating buffer, closing of HRP label Liquid, antibody diluent, cleaning solution, substrate solution, terminate liquid, neospora positive serum, negative serum.

In a preferred embodiment, the detection antibody of the HRP label is the goat-anti Niu Kangti of HRP label；It is described Coating buffer is the carbonate buffer solution of the pH=9.6 of 0.05M；The confining liquid is 5% horse serum-PBST；The antibody dilution Liquid is 2% horse serum-PBST；The cleaning solution is 0.5%PBST；The substrate solution is tmb substrate liquid A, substrate solution B；Institute Stating terminate liquid is the 2M concentrated sulfuric acid.

The present invention provides the application methods of above-mentioned neospora indirect ELISA diagnostic reagent kit, specifically include following step It is rapid:

A) recombinant protein rNcMIC26 antigen coat: is diluted to 10 μ with the carbonate buffer solution of the pH=9.6 of 0.05M G/mL, after coating condition is 37 DEG C of 1h, 4 DEG C of 14~16h of placement；

B) it washs: liquid in hole is discarded, the 0.5%PBST that 300 μ L are added is washed 4 times, every minor tick 5min；

C) close: confining liquid uses 5% horse serum-PBST, every hole 100 μ L, 37 DEG C of closing 1h；

D) wash: operating procedure is same b)；

E) primary antibody is incubated for: 2% horse serum-PBST solution 1:200 dilutes ox serum to be checked, every 100 μ L of hole, every plate setting mark Quasi- ox neospora positive serum, negative serum are as positive negative control；37 DEG C of incubation 1h；

F) wash: operating procedure is same b)；

G) secondary antibody is incubated for: being diluted with the goat-anti Niu Kangti 1:25000 of 2% horse serum-PBST solution dilution HRP label, often Hole 100 μ L, 37 DEG C of incubation 0.5h；

H) wash: operating procedure is same b)；

I) develop the color: tmb substrate liquid A, substrate solution B are mixed according to the ratio of 1:1, every 100 μ L of hole, color development at room temperature 10min；

J) terminate: every hole 2M concentrated sulfuric acid terminates reaction, every 50 μ L of hole；Every hole double wave is read in microplate reader after reaction Long OD_450/630nmValue；

H) result judgement: as the OD of serum sample ELISA to be checked_450/630nmValue >=0.165 when, can 99.9% level On be determined as positive findings.

Compared with prior art, it has the following beneficial effects: according to the present invention

The present invention screens to have obtained a kind of new neospora diagnostic antigen, builds using its recombinant protein as envelope antigen Found indirect ELISA reagent kit, easy to operate, rapidly and efficiently, specificity is good, sensibility is high, and it is reproducible, it can be used for detecting ox Group's neospora infections situation, can detect common NcSRS2 Protein Detection less than positive sample, can get more accurate Detection data is of great significance for the prevention and treatment of neosporosis.

Detailed description of the invention

Fig. 1 is the SDS-PAGE figure and Western Blot of neospora secretory protein according to an embodiment of the present invention Qualification figure；Wherein, 1: tachyzoite excretory-secretory antigen esa；2: tachyzoite whole worm antigen pellet.

Fig. 2 is the amplification figure of MIC26 gene according to an embodiment of the present invention；Wherein, M:DL-2000plus DNA Marker；1:MIC26 gene amplification product.

Fig. 3 is the protokaryon inducing expression and purifying figure of rNcMIC26-His albumen according to an embodiment of the present invention；Its In, M: albumen Marker；1: not inducing bacteria liquid sample；2: bacteria liquid sample after induction；3: supernatant protein sample；4: inclusion body protein Sample；5: rNcMIC26-His albumen after purification.

Fig. 4 is rNcMIC26-His reactionogenicity and specificity identification figure according to an embodiment of the present invention；Wherein, M: Pre-dyed albumen Marker；1: source of mouse Nc negative serum；2: source of mouse positive serum；3: source of mouse Tg positive serum；4: source of mouse His is mono- It is anti-.

Specific embodiment

With reference to the accompanying drawing, specific embodiments of the present invention will be described in detail, it is to be understood that guarantor of the invention Shield range is not limited by the specific implementation.

The experimental method involved in the present invention arrived is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Neospora Nc1 (Conrad P A, Barr B C, Sverlow K W, et al.In vitro isolation and characterization of a Neospora sp.from aborted bovine foetuses[J] .Parasitology, 1993,106.) public can obtain the biomaterial from applicant, which only attaches most importance to duplicate hair Used in bright related experiment, it not can be used as other purposes and use.

Bacterial strain: E.coli Transetta (DE3), Trans1-T1 competent cell are purchased from Beijing Quan Shijin biotechnology Co., Ltd；M5DH5a Competent Cell polymerize U.S. Biotechnology Co., Ltd purchased from Beijing.

Carrier: pET28a prokaryotic expression plasmid, kalamycin resistance start expression fusion His label by T7 promoter Destination protein.

CDNA reverse transcription reagent box: it is purchased from Beijing Quanshijin Biotechnology Co., Ltd

2 × ExTaq Mix PCR reagent, Goldview nucleic acid dye, DL2000plus DNA marker, reverse transcription examination Agent box (EasyScript One-step gDNA Removal and cDNA synthesis Supermix): complete purchased from Beijing Shi Jin Bioisystech Co., Ltd.

His fusion protein purification column (Ni2+), pvdf membrane: it is purchased from Germany Merck Millipore Novagen company.

Low background ECL shines colour reagent box: being century biotechnology Science and Technology Ltd. purchased from Beijing health.

Single slice connects kit: being purchased from Nanjing Nuo Weizan Bioisystech Co., Ltd.

HRP marks goat-anti ox IgG antibody: being purchased from Southern Biotechnology Associates, Inc. (U.S.) Product.

Specific embodiment is described in detail below, to more fully understand the present invention；It is to be understood that of the invention Protection scope be not limited by the specific implementation.

Embodiment 1: the screening of neospora diagnostic antigen

1) collection of neospora secretory protein

By neospora Nc1 mass propgation in Vero cell, about 72 hours are bred to state to be released to neospora When, it separates, purify from Vero cell and collect polypide.Counting reaches 5 × 10⁷A/mL or more, with 200 μ L serum-free DMEM Precipitating, 37 DEG C of incubation 3-5h are resuspended；With centrifugation after the completion of being incubated for, neospora secretory protein is respectively obtained, i.e., extracellular speed Grow sub- excretory-secretory antigen (esa) and tachyzoite whole worm antigen (pellet)；To the secretory protein being collected into, SDS-PAGE is carried out Electrophoresis carries out silver staining according to silver staining kit specification, as a result such as Fig. 1.

2) the Western Blot identification of neospora secretory protein

The secretory protein being collected into using Western blot method to the above method carries out antigen-reactive originality and special Property identification, primary antibody be source of mouse neospora positive serum, rabbit source toxoplasma positive serum, secondary antibody be HRP label sheep anti mouse The goat anti-rabbit igg of IgG, HRP label；As a result as shown in Figure 1, having filtered out the neospora not reacted with toxoplasma positive serum The protein band (50~70kDa) of specificity.

3) Mass Spectrometric Identification of neospora specific proteins

According to Western blot as a result, specific band (50~70kDa) corresponding in SDS-PAGE protein adhesive is cut, It send to BeiJing HuaDa protein Research Center Co., Ltd's mass spectral analysis, corresponding polypeptide information is obtained, in conjunction with bioinformatics The immunogenicity of albumen is analyzed, the immunogenicity of each antigen gene of comprehensive analysis determines that screening neospora dominant antigen is NcMIC26, the amino acid sequence as shown in SEQ ID NO.1.

Embodiment 2: the protokaryon inducing expression and purifying of recombinant protein r NcMIC26

1) clone of NcMIC26 gene

According to the neospora dominant antigen NcMIC26 known in embodiment 1, ToxoDB online database (http: // Www.toxodb.org the coding gene sequence that neospora NcMIC26 antigen is found in) removes its signal peptide, selection and bow The shape lower part of worm homologous gene similitude (271bp-1014bp), design primer F and R, using the cDNA of neospora as mould Plate expands the genetic fragment of neospora MIC26, and the nucleotide sequence of the genetic fragment is such as tied as shown in SEQ ID NO.2 Fruit is as shown in figure 3, sequence size is 744bp；

Wherein, the cDNA of neospora extracts neospora RNA and reverse transcription by Trizol- chloroform method, specifically includes Following steps:

The neospora tachyzoite of purifying is collected up to 3~5 × 10⁷It is a, use 1mLRNA separation agent is resuspended equal It is even, it is stored at room temperature 10min；200 μ L chloroforms are added, vortex oscillation 20s is stored at room temperature 5min, 4 DEG C of centrifugation 12000rpm centrifugations 15min；Sample is transferred in new EP pipe after centrifugation, the isopropanol of isometric -80 DEG C of pre-coolings of addition, it is quiet in -20 DEG C of refrigerators 15min is set, rear 4 DEG C of centrifugation 12000rpm are centrifuged 15min；Supernatant is abandoned, 75% ethyl alcohol 1mL of -20 DEG C of pre-coolings is added, it is heavy slowly to have hanged It forms sediment, 4 DEG C of centrifugation 12000rpm are centrifuged 15min；This step is repeated, supernatant is discarded.Completely to ethyl alcohol volatilization, add the DEPC of 15 μ L or so Water dissolution precipitating, as extracts and obtains RNA.

Using cDNA reverse transcription reagent box, reverse transcription is carried out according to the reaction system of table 1 and obtains the cDNA of neospora.

1 neospora RNA reverse transcription of table at cDNA reaction system

Primer are as follows:

F:5 '-AGCAAATGGGTCGCGGATCCGTTCTGGATTTCATAGACTTGG-3 ' (as shown in SEQ ID NO.3) Restriction enzyme site containing BamHI (underscore mark),

R:5 '-TCGAGTGCGGCCGCAAGCTTCGAAGTCCATTCGCCCCACGTT-3 ' (as shown in SEQ ID NO.4) Restriction enzyme site containing HindIII (underscore mark)；

Reaction system are as follows: 1 μ L, 2 × ExTaq Mix PCR polymerase of 1 μ L of template cDNA, primers F 1 μ L, primer R, 25 μ L, DDH₂22 μ L of O, totally 50 μ L system；

PCR reaction process are as follows: 95 DEG C of initial denaturations 5min, 95 DEG C of denaturation 30s, 57 DEG C of annealing 1min, 72 DEG C of extension 1min, if Set 35 circulations, 72 DEG C of last extension 10min；PCR product is obtained after nucleic acid electrophoresis using plastic recovery kit recycling PCR Target fragment, be placed in -20 DEG C of preservations.

2) building of recombined pronucleus expression plasmid pET-28a-MIC26 and protein expression

Using single slice connection kit by after purification neospora MIC26 genetic fragment and pET-28a double digestion bone Frame carries out single slice connection (reaction system are as follows: 3 μ L, MIC26 genetic fragment of pET-28a double digestion carrier framework 4 μ L, 5 × CE 21 μ L of μ L, Exnase of Buffer, totally 10 μ L)；Connection product is converted into Escherichia coli Trans1-T1 competent cell, Appropriate bacterium solution is taken to be inoculated into the LB solid medium of that resistance of card, inversion is incubated overnight；It selects single bacterium colony and carries out PCR mirror It is fixed, positive bacterium solution sequencing is selected, recombined pronucleus expression plasmid pET-28a-MIC26 is obtained.

The recombined pronucleus expression plasmid pET-28a-MIC26 built is converted to Escherichia coli Transetta competence In cell, PCR is accredited as positive monoclonal bacterium and expands culture, 0.8mM IPTG in the LB liquid medium of that resistance of card Induce the expression of recombinant protein.

Collect thallus, ice-bath ultrasonic cracking, 4 DEG C of centrifuge separation supernatant inclusion body proteins, SDS-PAGE electrophoretic analysis weight The expression-form of histone, as a result as shown in Figure 3, it is known that recombinant protein rNcMIC26 is mainly expressed in inclusion body, to forgiving Recombinant protein is purified in nickel affinity chromatography method after carrying out renaturation after body washing；SDS-PAGE analyzes purity of protein, knot As shown in Figure 3, obtained recombinant protein rNcMIC26-His size after purification is 35kDa to fruit, -80 after the albumen packing of purifying It DEG C saves backup.

3) rNcMIC26-His reactionogenicity and specificity identification

Reactionogenicity identification is carried out to recombinant protein rNcMIC26-His using Western Blot, concrete operations: respectively With source of mouse neospora positive serum (1:400), source of mouse neospora negative serum (1:400), source of mouse toxoplasma positive serum (1:400), source of mouse His monoclonal antibody (1:500 is served only for the recombinant protein with His label) are incubated for as primary antibody；Secondary antibody makes Western blot test is carried out with corresponding HRP label mountain sheep anti-mouse igg (1:5000) antibody；As a result as shown in figure 4, illustrating weight Histone rNcMIC26-His has sound response originality, determine the recombinant protein rNcMIC26-His of the neospora not with bow There are cross reaction, the preliminary assessment albumen to have the diagnostic value of neosporosis for shape worm.

Embodiment 3: the foundation of cow's serum neospora antibody indirect ELISA detection method

1) reaction condition

Cow's serum neospora antibody indirect ELISA reaction condition is as shown in table 2:

2 cow's serum neospora antibody indirect ELISA reaction condition of table

Method and step is as follows:

A) recombinant protein rNcMIC26 antigen coat: is diluted to 10 μ with the carbonate buffer solution of the pH=9.6 of 0.05M G/mL, every 100 μ L of hole in 96 hole enzyme mark micro-reaction plates, after coating condition is 37 DEG C of 1h, 4 DEG C overnight (14~16h)；

D) wash: operating procedure is same b)；

F) wash: operating procedure is same b)；

H) wash: operating procedure is same b)；

J) terminate: every hole 2M concentrated sulfuric acid terminates reaction, every 50 μ L of hole；Every hole double wave is read in microplate reader after reaction Long OD_450/630nmValue.

2) determination of ELISA detection method critical value

RNcMIC26-ELISA detects 30 parts of ox neospora negative antibody serum OD_450/630nmValue as shown in table 3, obtains Average valueIt is 0.065, standard deviation (SD) is 0.033, critical valueIt is 0.165, according to principle of statistics, when The OD of serum sample ELISA to be checked_450/630nmWhen value >=0.165, it can be determined as positive findings in 99.9% level.

The determination of 3 rNcMIC26-ELISA critical value of table

3) sensitivity experiment

By standard positive serum sample, doubling dilution, progress ELISA experiment determine serum most with critical value since 1:50 High dilution；The results are shown in Table 4, and the indirect ELISA method after optimization can detecte serum highest dilution and reach 1:3200, Illustrate that the sensitivity of this method detection is higher.

4 rNcMIC26-ELISA sensitivity experiment result of table

4) cross reaction is tested

Cross reaction test is carried out with toxoplasma, obtains that the results are shown in Table 5, shows that this method detection ox toxoplasma is positive The OD value of serum is respectively less than its critical value, illustrates that built rNcMIC26-ELISA is neospora antibody specificity indirect ELISA Detection method can distinguish ox arch insect infection during diagnosing to neosporosis.

5 rNcMIC26-ELISA specificity experiments result of table

5) repeated experiment

Test result is repeated in batch and between criticizing as shown in table 6, table 7, the coefficient of variation of all comparative samples is respectively less than 15%, Illustrate this method repeatability preferably.

Experimental result is repeated in 6 rNcMIC26-ELISA of table batches

Experimental result is repeated between 7 rNcMIC26-ELISA of table batches

6) comparative experiments

Detect that 98 parts of neosporas are positive and 39 parts of neospora feminine gender cow's serums with IFAT, respectively with MIC26 and The indirect ELISA that two kinds of antigen of SRS2 is established is detected, and the specificity and sensibility of ELISA are compared；The results are shown in Table 8, Compared with IFAT, rNcMIC26-ELISA sensibility is 76.53% (75/98) compared with SRS2 high, and specificity is 84.62% (33/ 39), two kinds of albumen diagnosis effects are close；During being detected to 98 parts of neospora positive serums, rNcMIC26- The serum of ELISA test positive has 75 parts, and the serum of rSRS2-ELISA test positive has 65 parts, the positive findings of the two And it is non-uniform；It is for statistical analysis to totally 137 parts of serum of detection, by after the association of two kinds of diagnostic antigens its with IFAT positive coincidence rate is 93.88% (92/98), and negative match-rate is still 84.62% (33/39).

The different antigen coat indirect ELISAs of table 8 are compared with IFAT coincidence rate

Therefore, the neospora diagnostic antigen sensibility screened in the present invention is good, and specificity is high, can be used as and generally acknowledges at present It is pure and fresh to can be applied to ox blood for one supplement antigen of diagnostic antigen SRS2 missing inspection, the sensibility of the detection improved to a greater extent The detection of sporozoite antibody, confidence level are higher.

The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention and Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.

Sequence table

<110>China Agricultural University

<120>a kind of neospora NcMIC26 antigen and its application

<130> P190459DD1F

<141> 2019-05-22

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 248

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

Val Leu Asp Phe Ile Asp Leu Val Pro Ile Ser Ser Glu Glu Val His

1 5 10 15

Leu Ser Val Val Thr Phe Ala Asp Ser Pro Gln Asp Val Phe Thr Phe

20 25 30

Lys Gln Pro Gln Ala Thr Asn Lys Gln Leu Ala Lys Glu Ala Phe Lys

35 40 45

Tyr Leu Arg Tyr Arg Arg Gly Gly Ser Thr Ala Thr Asp Lys Gly Leu

50 55 60

Ile Arg Ala Arg Arg Tyr Leu Thr Arg Pro Val Tyr Gly Thr Arg Ala

65 70 75 80

Asn Val Pro Lys Val Leu Val Leu Met Thr Asp Gly Glu Ser Asp Arg

85 90 95

His Tyr Asp Thr Ile Gln Ala Ala Asp Gln Ala Arg Ala Glu Gly Ile

100 105 110

Ser Val Phe Val Val Gly Val Gly Met Ala Asn Pro Val Glu Cys Arg

115 120 125

Gly Val Cys Gly Cys Gly Arg Tyr Gly Pro Cys Pro Gln Phe Ile Met

130 135 140

Ser Asn Trp Asn Glu Leu Val Gln Thr Val Asp Ser Ile Met Gly Glu

145 150 155 160

Val Cys Lys Lys Leu Pro Lys Asp Ala Glu Cys Ser Glu Trp Ser Glu

165 170 175

Trp Ser Gly Cys Thr Ala Ser Cys Gly Ala Gly Thr Arg Thr Lys Thr

180 185 190

Arg Lys Gln Leu Ser Pro Pro Leu Ala Gly Asp Pro Pro Cys Asp Asn

195 200 205

Cys Glu Pro Met Met Gly Lys Thr Cys Glu Asp Leu Gly Gly Leu Val

210 215 220

Arg Val Glu Asp Cys Asn Gln Gly Glu Cys Pro Gln Asp Ala Gly Cys

225 230 235 240

Gly Thr Trp Gly Glu Trp Thr Ser

245

<210> 2

<211> 744

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

gttctggatt tcatagactt ggttcccatc tcctctgaag aggttcacct ttcggtggtg 60

accttcgcgg actcaccgca ggacgtcttc actttcaagc aacctcaggc aaccaacaag 120

caactggcga aagaagcatt taaatacctg agataccgga gggggggaag tacggcgacg 180

gacaaaggcc ttatacgcgc acgccgatac ctcacccgcc ccgtgtatgg cacgcgagcg 240

aacgtaccga aggtgctggt gctcatgaca gatggcgaat cggacaggca ttacgacacc 300

atccaagctg cggaccaagc aagagctgag ggcatttcgg ttttcgttgt tggcgttgga 360

atggccaacc ctgtagagtg ccgtggtgtc tgtgggtgcg ggaggtacgg cccttgtccg 420

cagtttatca tgtccaactg gaacgaactg gtacaaactg tcgattcgat tatgggtgaa 480

gtgtgcaaga aattgcccaa agatgcagag tgcagcgagt ggagcgaatg gtcggggtgc 540

acagcctctt gtggcgccgg cacgcgcacc aaaactcgga agcaactgtc cccgccgctg 600

gctggagatc ccccatgcga caactgcgag ccgatgatgg gcaaaacgtg tgaagatctt 660

ggaggtcttg tgagggttga ggattgcaac cagggcgagt gcccccagga cgccggctgt 720

ggaacgtggg gcgaatggac ttcg 744

<210> 3

<211> 42

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

agcaaatggg tcgcggatcc gttctggatt tcatagactt gg 42

<210> 4

<211> 42

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

tcgagtgcgg ccgcaagctt cgaagtccat tcgccccacg tt 42

Claims

1. a kind of neospora NcMIC26 antigen, which is characterized in that the amino acid sequence of the antigen such as SEQ ID NO.1 institute Show.

2. a kind of gene for encoding neospora NcMIC26 antigen described in claim 1.

3. gene according to claim 2, which is characterized in that the nucleotide sequence of the gene such as SEQ ID NO.2 institute Show.

4. a kind of neospora NcMIC26 antigen as described in claim 1 is in preparation treatment, diagnosis or prevention of neosporosis Product in application.

5. a kind of gene as claimed in claim 2 or claim 3 answering in the product of preparation treatment, diagnosis or prevention of neosporosis With.

6. a kind of neospora indirect ELISA diagnostic reagent kit, which is characterized in that include amino acid sequence in the diagnostic kit Arrange the recombinant protein rNcMIC26 as shown in SEQID NO.1.

7. neospora indirect ELISA diagnostic reagent kit according to claim 6, which is characterized in that the recombinant protein The preparation method of rNcMIC26 includes the following steps:

1) design primer F and R:

F:5 '-AGCAAATGGGTCGCGGATCCGTTCTGGATTTCATAGACTTGG-3'；

R:5 '-TCGAGTGCGGCCGCAAGCTTCGAAGTCCATTCGCCCCACGTT-3'；

3) target fragment and carrier pET-28a are attached, connection product is converted to Escherichia coli Trans1-T1 competence Cell obtains recombined pronucleus expression plasmid pET-28a-MIC26；

4) the recombined pronucleus expression plasmid pET-28a-MIC26 built is converted thin to Escherichia coli Transetta competence Born of the same parents induce the expression of recombinant protein, collect and purify to obtain recombinant protein rNcMIC26.

8. neospora indirect ELISA diagnostic reagent kit according to claim 6, which is characterized in that the kit is also Detection antibody, coating buffer, confining liquid, antibody diluent, cleaning solution, substrate solution, terminate liquid, new spore including HRP label Worm positive serum, negative serum.

9. neospora indirect ELISA diagnostic reagent kit according to claim 8, which is characterized in that the HRP label Detect the goat-anti Niu Kangti that antibody is HRP label；The coating buffer is the carbonate buffer solution of the pH=9.6 of 0.05M；The envelope Closing liquid is 5% horse serum-PBST；The antibody diluent is 2% horse serum-PBST；The cleaning solution is 0.5%PBST；Institute Stating substrate solution is tmb substrate liquid A, substrate solution B；The terminate liquid is the 2M concentrated sulfuric acid.

10. a kind of application method of the neospora indirect ELISA diagnostic reagent kit as described in claim 6-9 is any, special Sign is, includes the following steps:

A) antigen coat: being diluted to 10 μ g/mL with the carbonate buffer solution of the pH=9.6 of 0.05M for recombinant protein rNcMIC26, After coating condition is 37 DEG C of 1h, 4 DEG C of 14~16h of placement；

D) wash: operating procedure is same b)；

E) primary antibody is incubated for: 2% horse serum-PBST solution 1:200 dilutes ox serum to be checked, every 100 μ L of hole, and standard ox is arranged in every plate Neospora positive serum, negative serum are as positive negative control；37 DEG C of incubation 1h；

F) wash: operating procedure is same b)；

G) secondary antibody is incubated for: being diluted with the goat-anti Niu Kangti 1:25000 of 2% horse serum-PBST solution dilution HRP label, every hole 100 μ L, 37 DEG C of incubation 0.5h；

H) wash: operating procedure is same b)；

J) terminate: every hole 2M concentrated sulfuric acid terminates reaction, every 50 μ L of hole；Every hole dual wavelength is read in microplate reader after reaction OD_450/630nmValue；

H) result judgement: as the OD of serum sample ELISA to be checked_450/630nmWhen value >=0.165, it can sentence in 99.9% level It is set to positive findings.