CN105777878B - A kind of eperythrozoon recombination epitope antigen and its immunity detection reagent - Google Patents

A kind of eperythrozoon recombination epitope antigen and its immunity detection reagent Download PDF

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CN105777878B
CN105777878B CN201610187839.XA CN201610187839A CN105777878B CN 105777878 B CN105777878 B CN 105777878B CN 201610187839 A CN201610187839 A CN 201610187839A CN 105777878 B CN105777878 B CN 105777878B
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eperythrozoon
epi
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hspa1
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陈兆国
胡盼
米荣升
黄燕
陆珂
韩先干
王香平
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Shanghai Veterinary Research Institute CAAS
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Abstract

The present invention provides a kind of eperythrozoon recombination epitope antigen and its immunity detection reagents.The present invention has carried out the prediction of B cell antigen epi-position to eperythrozoon suis HspA1 albumen, screen and intercept the B cell antigen epi-position enrichment region of HspA1 albumen, recombination HspA1 epitope enrichment region albumen is obtained by prokaryotic expression, use recombination HspA1 epitope enrichment region albumen after purification as envelope antigen, establish the indirect ELISA method of detection eperythrozoon suis, diagnostic kit is constructed, provides effective means to detect eperythrozoon suis infection and the epidemiological survey of eperythrozoon suis of humans and animals.

Description

A kind of eperythrozoon recombination epitope antigen and its immunity detection reagent
Technical field
The present invention relates to a kind of eperythrozoon recombination epitope antigen and its immunity detection reagents.
Background technique
The eperythrozoonosis (eperythrozoonosis) of pig is posted by eperythrozoon (Mycoplasma spp.) It is born in caused a kind of communicable disease in erythrocyte surface, blood plasma and marrow.It is clinically main with fever, anaemia, jaundice Feature.The disease distribution is extensive, infection host is more, and the development of health and animal husbandry to people causes huge harm.Pig Eperythrozoon (Mycoplasma suis) is wherein to endanger one kind the most serious, can cause pig hemolytic anemia, or even danger And life, it can also cause chronic anaemia, show as that piglet slow growth, sow be infertile and immunosupress.
Eperythrozoon can not carry out in vitro culture so far, in addition small volume, form of diverse, limit to its characteristic and Control Technology research is carried out in a deep going way.Currently, mainly using pig blood smear for microscopic examination, being built using full cell soluble protein as antigen Vertical serological method and molecular biology method diagnoses the eperythrozoonosis of pig, and these diagnostic methods exist There are some shortcoming and defect when clinical application.Due to eperythrozoon suis small volume, directly very with micro- sem observation Difficulty, and eperythrozoon suis has close preferendum to red blood cell, can cause serious Erythrocyte Damage and deformation, this often by with It is diagnosed, however, other factors can also cause deformation of red blood cell, for example smear technique, osmotic pressure change etc., therefore Blood microscopy technology has very big one-sidedness.Molecular biology method is not only costly, detection cycle is longer, moreover, because Some primer specificities are poor, false positive often occur.Eperythrozoon suis Serum Antibody Detection method reported at present is main There are complement fixation test (CFT) (complement fixation test, CFT), indirect hemagglutination test (indirect Hemagglutination assay, IHA) and enzyme-linked immunosorbent assay (enzyme linked immunosorbent Assay, ELISA), wherein ELISA method has the characteristics that easy to operate, sensitivity is high, quick, easy standardization, it has also become preferred Routine diagnostic method, antigen used is mostly conventional eperythrozoon thallus soluble antigen.However, due to there is no at present The Vitro Culture Techniques of eperythrozoon, it is difficult to a large amount of antigens are obtained, so that ELISA detection method is extremely limited.
Heat shock protein (heat shock proteins, Hsps) is that the generally existing one kind of living nature is highly conserved Protein, gene have during evolution it is well-conserved, in recent years many scholars apply its sequence research biology base Because of the Phylogenetic analysis of type, the Molecule Epidemiology Investigation of progress disease and cause of disease.In addition, the albuminoid is alternatively arranged as Main antigen protein, induction host generate antibody, kill the pathogenic microorganism of invasion.Hoelzle etc. (2007) uses serum Protein science (serological proteome) combines analytical technique of mass spectrum, identifies 6 eperythrozoon suis functional proteins, One of them is Dnak sample albumen heat_shock protein A 1 (heat shock proteins A1, HspA1), and HspA1 is that have to exempt from The functional protein of epidemic focus.HspA1 belongs to heat shock protein A subunit family, and molecular size 67ku is located in swine red cell On body endochylema and after birth, have Surface accessible (surface accessibility), ATP enzyme (ATPase) active and anti- Originality may participate in sticking host's red blood cell.Author then constructs eperythrozoon suis DNA library, to library part Clone carries out shotgun sequencing, obtains the HspA1 protein coding gene sequence that overall length is 1830bp, is named as swine red cell Body a1 gene.Due in eperythrozoon suis, codon UGA codes for amino acid tryptophan, and in escherichia coli (E.coli), UGA is terminator codon, if carrying out pronuclear recombination expresses HspA1 albumen, needs the inclined preferendum of codon according to E.coli, gram The obstacle of UGA codes for amino acid tryptophan, need to be transformed a1 gene nucleotide series by the degenerate of codon, then when clothes translation Subsequent research can just be carried out by carrying out full genome synthesis.Hoelzle etc. (2007) is carried out after a1 gene nucleotide series are transformed Full genome synthesis, and detection pig is established using recombination HspA1 (recombinant HspA1, the rHspA1) albumen of prokaryotic expression The ELISA diagnostic techniques of eperythrozoonosis, sensibility and specificity, which is equal to or higher than, utilizes the full cell of eperythrozoon suis The elisa technique of foundation.Due to above-mentioned steps not only very complicated, and it is costly.In addition, eperythrozoon suis a1 gene Up to 1830bp, not only expression is difficult but also easily mutates.Therefore, clonal expression carried out to a1 gene at present, used It is also considerably less in the research institution of immunodiagnosis.
Antigen is special to activate in conjunction with antigenic determinant receptor-specific corresponding with corresponding antibodies or lymphocytic cell surface Different immune response.Therefore, antigenic determinant is the material base of immune response and immune response (reactionogenicity).In recent years, The Antigen Epitope Prediction for carrying out antigen using Epitope prediction tool is begun trying both at home and abroad, constructs multi-epitope antigen (multi- Epitope antigen), i.e., multiple epitopes relevant to target antigen and the antigen of helper epitope are carried simultaneously, as Diagnostic antigen, at present in the helminths such as Schistosoma japonicum, toxoplasma, the bacteriums such as mycobacterium tuberculosis, haemophilus parasuis, adenopathy Start to apply in the virus such as poison, has achieved good effect.But so far there is not yet application report on eperythrozoon.
Currently, in terms of eperythrozoon, the B cell antigen epi-position report biology of only one coded by said gene albumen Informatics software has carried out the B cell antigen epi-position of one coded by said gene albumen of prediction, and without carrying out subsequent research, This is because the Unknown Function of overwhelming majority coding albumen, as hypothesis albumen are only few in the genome of eperythrozoon suis Number albumen has predicted its function according to its sequence homology, such as: eperythrozoon suis glyceraldehyde 3-phosphate dehydro-genase sample egg White 1 (M.suis glyceraldehyde-3-phosphate dehydrogenase-like protein 1, MSG1), DnaK Sample heat_shock protein A 1 (Heat Shock Protein A1, HspA1), GroEL, α-enolase (α-Enolase), pyruvic acid Dehydrogenase (Pyruvate Dehydrogenase) and RNA helicase etc., and most shortage experimental verifications.Research at present is most More albumen is HspA1, MSG1 and α-Enolase.Due in eperythrozoon suis, codon UGA codes for amino acid tryptophan, and In escherichia coli (E.coli), UGA is terminator codon.Therefore, prokaryotic expression or when eukaryotic expression, foreign countries are all made of full base Because of synthesis, domestic g1 gene (encoding gene of MSG1) is synthesized using full genome, and a1 gene and α-Enolase encoding gene are to keep away An open reading frame has been selected after opening TGA.Due to above-mentioned status, Epitope prediction the relevant technologies are not applied to attached so far On red cell body.
Foreign countries, Hoelzle etc. (2007) carries out full genome synthesis after a1 gene nucleotide series are transformed, and utilizes protokaryon Recombination HspA1 (recombinant HspA1, rHspA1) albumen of expression establishes the ELISA of detection swine eperythrozoonosis Diagnostic techniques (the entire albumen of HspA1).The country, although utilizing prokaryotic expression part weight in imperial political affairs etc. (2012) Group HspA1 albumen, but the coded sequence of its HspA1 albumen is used for the purpose of avoiding terminating in Escherichia coli, and select One open reading frame (0RF), do not consider the encoded albumen of this section of sequence whether be B cell epitope.Mesh Before, domestic scholar not yet is attached red thin with recombination HspA1 (recombinant HspA1, rHspA1) albumen foundation detection pig The ELISA diagnostic techniques of cell space disease.So in the prior art, as long as the enzyme linked immunosorbent detection based on HspA1, is using it Entire albumen is difficult to expect that the Partial Fragment for using HspA1 does coating antigen, and there are serious technology prejudice.
Summary of the invention
For the defects in the prior art, the object of the present invention is to provide a kind of eperythrozoon recombination epitope antigen and its Immunity detection reagent.The present invention has carried out the prediction of B cell antigen epi-position to eperythrozoon suis HspA1 albumen, and screening is simultaneously The B cell antigen epi-position enrichment region (abbreviation HspA1 epitope area) for intercepting HspA1 albumen obtains recombination HspA1 by prokaryotic expression Epitope area albumen uses recombination HspA1 epitope area's albumen after purification as envelope antigen, establishes detection eperythrozoon suis Indirect ELISA method constructs diagnostic kit, and to detect, the eperythrozoon suis of humans and animals infects and pig is attached red thin The epidemiological survey of cell space provides effective means.
The purpose of the present invention is what is be achieved through the following technical solutions:
In a first aspect, the present invention provides a kind of eperythrozoon recombination epitope antigen rHspA1, sequence such as SEQ ID No.1 It is shown.
Second aspect, the present invention provide a kind of preparation method of eperythrozoon recombination epitope antigen, include the following steps:
B cell antigen epi-position enrichment region is screened from eperythrozoon suis HspA1 protein amino acid sequence, is denoted as HspA1 Epitope area；
Its volume is screened from eperythrozoon suis HspA1 protein coding gene according to the amino acid sequence in HspA1 epitope area Code sequence, is denoted as a1-epi；
A1-epi sequence is expanded, and recovery product is connect with pMD18-T carrier, obtains recombinant plasmid pMD18-T-a1- epi；
I He of restriction enzyme BamH is used respectively to the recombinant plasmid pMD18-T-a1-epi plasmid and I plasmid of pCold EcoR I carries out double digestion identification, glue recycling, and the glue recovery product of a1-epi and the glue recovery product of digestion pCold I are connected It connects, obtains I-a1-epi of recombinant plasmid pCold；
E. coli strain bl21 is converted with the I-a1-epi of recombinant plasmid pCold, obtains pColdI-a1-epi- E.coliBL21 bacterial strain；
By pColdI-a1-epi-E.coli BL21 strain inoculated in LB liquid medium, training is induced under the conditions of IPTG It supports, collect thallus and cracks, collect supernatant to get eperythrozoon recombination epitope antigen rHspA1.
Preferably, the eperythrozoon suis refers specifically to German 54/96 separation strains；The present invention is Sino-German with GenBank The a1 gene nucleotide series of 54/96 separation strains of state are designed specific primer.
Preferably, the amplification primers are as shown in SEQ ID No.3, SEQ ID No.4.Primer specificity is stronger, does not have There is miscellaneous band, grads PCR Shi Junneng amplifies purpose band, and band brightness is almost the same.
Preferably, the screening of the B cell antigen epi-position enrichment region is based on eperythrozoon suis HspA1 Argine Monohydrochloride It is carried out on the basis of sequence bioinformatic analysis.
Preferably, the B cell antigen epi-position enrichment region is specifically hydrophily, flexibility, antigenic index, surface accessibility Etc. parameter values are higher and region containing functional domain.
Preferably, the preparation method further includes the steps that purifying gained eperythrozoon recombination epitope antigen.
The third aspect, the present invention provides a kind of eperythrozoon immunity detection reagent, specifically with the eperytozoa Weight group epitope antigen is envelope antigen.
Preferably, the antigen coat concentration of the detection kit is 0.5~4 μ g/mL；It is particularly preferred that the antigen Peridium concentration is 0.5 μ g/mL.
Preferably, the serum dilution of the detection kit is 1:(50~400)；It is particularly preferred that the serum is dilute Degree of releasing is 1:400.
Preferably, the off-period of the detection kit is 1~3h；It is particularly preferred that the off-period is 2h.
Preferably, the serum action time to be checked of the detection kit is 0.5~2h；It is particularly preferred that described to be checked Serum action time is 1.5h.
Preferably, the enzyme labelled antibody working concentration of the detection kit is 1:(2000~5000)；It is particularly preferred that institute Stating enzyme labelled antibody working concentration is 1:5000.
Preferably, the enzyme labelled antibody action time of the detection kit is 0.5~1.5h；It is particularly preferred that the enzyme Labeling antibody action time is 1h.
Preferably, the substrate developing time of the detection kit is 10~20min；The substrate developing time is 15min。
Compared with prior art, the present invention have it is following the utility model has the advantages that
1, the envelope antigen of this research is the recombination HspA1 epitope area albumen of prokaryotic expression, and under low temperature induction, it is with can The form of dissolubility exists, and has His label in N-terminal, this is conducive to large-scale purification recombination HspA1 epitope area albumen, thus Keep antigenic source no longer constrained, therefore these envelope antigens are not only producible in vitro and are standardized；In addition, Use recombinant antigen to replace native antigen as envelope antigen, is applied to ELISA detection and preparation ELISA kit not only may be used To exclude pig derived component and the stability and accuracy of antigen between different batches can be significantly improved.
2, it is also difficult to exclude coli somatic egg completely even across purification and recovery due to the recombinant protein of prokaryotic expression The interference of Bai Chengfen, and during the growth process swinery may multiple ehec infection, may be containing big in serum sample The antibody of enterobacteria ingredient, it is likely that cause false positive results and reduce the specificity of indirect ELISA detection method, to avoid this One influences, this test is before blood serum sample is reacted with envelope antigen, first using E. coli lysate and blood serum sample at 37 DEG C Act on 1h.
3, the positive for the indirect ELISA method and Species specific PCR and MSA quantitative fluorescent PCR that this research institute establishes meets Rate is respectively 64.81%, 62.96%, and negative match-rate is respectively 66.67%, 100%.This research institute is prompted to establish indirect ELISA and Species specific PCR and MSA quantitative fluorescent PCR yin and yang attribute coincidence rate with higher.In addition, the indirect ELISA method With preferable specificity, sensitivity and repeatability, one kind is provided quickly and effectively for epidemiological survey and medical diagnosis on disease Detection method.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention, Objects and advantages will become more apparent upon:
The transmembrane region bioinformatic analysis of Fig. 1: HspA1 albumen；
The signal peptide bioinformatic analysis of Fig. 2: HspA1 albumen；
The hydrophobic biological bioinformatics analysis of Fig. 3: HspA1 albumen；
The functional domain bioinformatic analysis of Fig. 4: HspA1 albumen；
The prediction of Fig. 5: HspA1 Protein secondary structure；
The prediction of Fig. 6: HspA1 protein B cell antigen epitope；
The PCR amplification of Fig. 7: a1-epi gene；Wherein, M:DL2000DNA molecular mass standard；1-7:a1-epi gene piece Section amplified production；N: negative control；
Fig. 8: the PCR identification of the plasmid transformed bacteria of gene prokaryotic containing a1-epi；Wherein, M:DL2 000DNA molecular mass Standard；I-a1-epi transformed bacteria PCR product of 1-9:pCold；P: positive control；N: negative control；
The SDS-PAGE of Fig. 9: rHspA1 albumen is detected；Wherein, before the induction of I empty expression vector of 1:pCold；2:pCold I is empty After expression vector induction；Before the induction of I-a1-epi empty expression vector of 3:pCold；The induction of I-a1-epi empty expression vector of 4:pCold Afterwards；The elution of 5:50mM imidazoles；The elution of 6:100mM imidazoles；The elution of 7:100mM imidazoles；The elution of 8:150mM imidazoles；9:200mM imidazoles Elution；
The Western blot of Figure 10: rHspA1 albumen is analyzed；Wherein, M: pre-dyed protein standard molecular weight；1,2: RHspA1 recombinant protein；I empty expression vector of 3:pCold.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention Protection scope.
1. material and method
1.1 serum and bacterial strain
Eperythrozoon suis (Mycoplasma suis) positive serum picks up from field growing and fattening pigs, detects through Species specific PCR For eperythrozoon suis infection, indirect ELISA detects antibody positive；Involved serum is the toxoplasma that conventional technical means obtains (Toxoplasma gondii) infects Swine serum, swine fever virus (Classical swine fever virus, CSFV) infection Swine serum, porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome Virus, PRRSV) infection Swine serum, Cryptosporidium spp Swine serum.Pig field serum pick up from Changning district, Shanghai, Songjiang District and The adult growing and fattening pigs of Jiading District slaughterhouse；CN126 plants of DNA of eperythrozoon suis are studied from Chinese Academy of Agricultural Sciences Shanghai animal doctor Institute's (deposit number RA-SH-CN-ZX-13-126, Wang Xiangpei etc., Chinese veterinary science, 2014).
1.2 main agents
Prime STAR GXL DNA Polymerase, DL2 000DNA molecular mass standard, 6 × Loading Buffer, pMD18-T Vector, T4DNA Ligase, restriction enzyme BamH I and EcoR I are purchased from precious bioengineering (Dalian) Co., Ltd；Poba gene group DNA extraction kit (resin type) is purchased from Shanghai SBS Genetech gene technology Co., Ltd； AxyPrep DNA plastic recovery kit is purchased from Axygen company；Trans1-T1Phage Resistant Competent cell It is purchased from Beijing Quanshijin Biotechnology Co., Ltd；It is limited that 2 × Taq PCR MasterMix is purchased from Tiangeng biochemical technology (Beijing) Company；Protein Marker,BCA Protein Assay Kit is purchased from Thermo company；6×Protein Loading Buffer is purchased from the steady Biotechnology Co., Ltd in upper Haikang；Ni-NTA His Bind Resin chromatographic column, pvdf membrane Purchased from German Merck company；Tryptone, yeast extract, skimmed milk power are purchased from Britain OXOID company；Kanamycin sulfate Purchased from Sangon Biotech (Shanghai) Co., Ltd.；TMB is purchased from TIANGEN Biotech (Beijing) Co., Ltd.；Persulfuric acid Ammonium, acrylamide, TEMED are purchased from Sigma company；His-Tag (27E8) Mouse mAb (HRP Conjugate) is purchased from Cell Signaling Technology；Rabbit-anti pig IgG-HRP is purchased from Abcam company；ELISA ELISA Plate is U.S. CoringCostar Products；Other reagents are that domestic analysis is pure.
The bioinformatic analysis of 1.3 eperythrozoon suis HspA1 amino acid sequences
It (is stepped on from the amino acid full length sequence of the German 54/96 separation strains HspA1 albumen of GenBank downloading eperythrozoon suis Record AM265536), using TMpredServer (http://www.ch.embnet.org/software/TMPRED_ form.html) software to HspA1 albumen carry out transmembrane region prediction；Using Signal P 4.1Server (http:// www.cbs.dtu.dk/ ser vices/SignalP/) software predicts the signal peptide of albumen；It is soft using Protscale Part (http://web.expasy.org/cgi-bin/protscale/protscale.pl hydrophobicity prediction) is carried out to albumen； Functional structure domain analysis is carried out to albumen using SMART (http://smart.embl-heidelberg.de/) software.
With the secondary structure and its characteristic of DNAStar software prediction albumen: using Gamier-Robson and Chou- The secondary structure of Fasman method prediction albumen；The hydrophilic region of Kyte-Doolittle method prediction albumen；Karplus- The flexibility region in Schulz method prediction peptide backbone area；The antigenic index of Jameson-Wolf method prediction albumen；Emini method is pre- Survey the accessibility that specific region is located at protein molecular surface.
As a result: TMpredServer software analysis the result shows that, HspA1 has 6 transbilayer helixes (Fig. 1)；Signal P 4.1 Server softwares analysis the result shows that, HspA1 no signal peptide (Fig. 2)；Protscale software analysis the result shows that, HspA1 Maximum hydrophobicity is+2.278, and minimum hydrophobicity is -2.933 (Fig. 3)；SMART software analysis shows, HspA1 has typical HSP70 spline structure (Fig. 4).
In terms of secondary structure, Gamier-Robson and Chou-Fasma method prediction result is shown, alpha-helix, beta sheet Section coincidence factor is relatively high, and β-corner area otherness is larger；Kyte-Doolittle method prediction result shows, hydrophilic section It is more, it is in uneven distribution, compares comparatively dense in c-terminus distribution；Karplus-Schulz method prediction result shows, flexibility area Section is more, is unevenly distributed, and compares concentration in c-terminus distribution；Emini method prediction result shows, surface accessibility section compared with It is more, it is in uneven distribution, aminoterminal and c-terminus are distributed equal comparatively dense；Jameson-Wolf method prediction result shows, Gao Kangyuan Index section is more, and distribution is relatively uniform, especially compares concentration (Fig. 5) in c-terminus distribution.
The prediction of 1.4HspA1B cell antigen epitope
According in http://tools.immuneepitope.org/tools/bcell/iedb-input network address Antibody Epitope Prediction software carries out the prediction of mean antigen epitope index to HspA1, and uses Kolaskar-Tongaonkar method calculates the mean antigen epitope index numerical value of HspA1.
As a result, Kolaskar-Tongaonkar method prediction result is shown, HspA1 albumen mean antigen epitope index is (1.017 Fig. 6).
The screening of 1.5HspA1 epitope enrichment region segment
HspA1 protein function structural domain, secondary structure and B cell antigen epi-position prediction result are analyzed, chosen The parameter values such as hydrophily, flexibility, antigenic index and the surface accessibility of HspA1 albumen are higher and containing functional domain Region is advantage B cell antigen epi-position enrichment region (abbreviation HspA1 epitope area).According to the swine red cell logged in GenBank The sequence (accession number AM265536) of 54/96 separation strains HspA1 protein coding gene a1 of body Germany screens the volume of epitope area segment Code sequence (abbreviation a1 epitope area, a1-epi).
As a result, by referring to HspA1 protein function structural domain, secondary structure, hydrophilic index, flexibility parameter, antigen Number, surface accessibility etc. are analyzed, and filter out the 388th -609 sections of c-terminus as HspA1 epitope area, sequence is shown in SEQ ID No.1.Corresponding epitope area genetic fragment a1-epi sequence is shown in SEQ ID No.2.
HspA1 epitope region amino acid sequence (SEQ ID No.1):
RNSTIPIDKKQLFSTAVDNQPSVDIHVVQGERPMANQNKSLGTFTLQGIKQAPKGMPKIEVSFSIDANG ILTVKAEDKDTGKQNNITINQASGLSEEEINKIIREAEENLEQDKKVKEEIEIKNEAESWISMLENQMKDDSSKIPE ASIEETKKLIEEFKKLLEEKKYDELKAKMNQLKEMSQKMMQEVYQQQQAAGGQAASEEKGPEGEDIKEVELNEESN
HspA1 epitope area coded sequence a1-epi (SEQ ID No.2):
AGAAATAGTACTATTCCAATTGATAAGAAGCAATTGTTCTCAACTGCTGTAGACAATCAACCTAGTGTT GATATTCACGTAGTACAAGGTGAAAGACCTATGGCTAACCAAAACAAATCCCTAGGTACTTTCACCCTTCAAGGAAT TAAGCAAGCTCCTAAGGGAATGCCAAAAATAGAAGTATCCTTCTCTATTGACGCTAACGGTATTCTTACCGTTAAAG CAGAAGATAAGGATACTGGAAAGCAAAACAATATAACTATTAATCAAGCTTCTGGATTATCAGAAGAAGAAATTAAT AAGATAATCCGAGAAGCTGAAGAAAATCTTGAACAAGATAAGAAGGTTAAGGAAGAAATAGAAATTAAGAATGAAGC TGAATCTTGGATTTCTATGCTAGAAAACCAAATGAAGGATGATTCTTCAAAGATTCCTGAAGCAAGTATAGAAGAAA CTAAGAAGTTAATTGAAGAATTTAAGAAACTTCTTGAAGAAAAGAAGTATGATGAACTTAAGGCTAAGATGAATCAA CTAAAAGAAATGAGTCAAAAAATGATGCAAGAAGTTTATCAACAACAACAAGCTGCTGGAGGACAAGCTGCATCAGA AGAAAAAGGTCCTGAAGGAGAAGACATCAAAGAAGTAGAACTTAATGAAGAAAGTAATTAG
The building of 1.6HspA1 epitope area prokaryotic expression carrier
1.6.1a1-epi the amplification of gene
According to a1-epi sequence, with 5.0 software design 1 of Primer Premier to specific primer:
Upstream primer (SEQ ID No.3): 5 '-CGGGATCCATGAGAAATAGTACTATTCCAATTG-3 '；
Downstream primer (SEQ ID No.4): 5 '-CGGAATTCCTAATTACTTTCTTCATTAAGTTCTAC-3 '；
Primer is synthesized by Shanghai Hua Da Bioisystech Co., Ltd.
Bacterial strain uses therefor is practical be District of Shanghai separation strains (126 separation strains of Changning), because of the a1 base of 126 separation strains of Changning Because of the a1 gene order similitude highest of 54/96 separation strains German in sequence and GenBank, similitude 99.89%.
Using with CN126 plants of DNA of eperythrozoon suis in same cluster of German 54/96 separation strains a1 gene order as template It is expanded, PCR system are as follows: Prime STAR GXL DNA Polymerase (1.25U/ μ L) 0.2 μ L, 5 × Prime STAR GXL Buffer(Mg2+Plus) 5 μ L, each 4 μ L of 0.2 μ L, 2.5mmol/L dNTP of upstream and downstream primer (100 μm of ol/L), template 4 μ L of DNA adds sterilizing ddH2O to 25 μ L.PCR amplification condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45s, 58 DEG C of annealing 45s, 72 DEG C of extension 1min, 37 circulations；72 DEG C of extension 10min.
1.6.2a1-epi the building of cloning vector
The glue recovery product of a1-epi is connect with pMD18-T carrier respectively, positive connection product is accredited as through PCR and turns Change bacterium solution is sent to Shanghai Hua Da Bioisystech Co., Ltd and is sequenced, and identifies that correct recombinant plasmid is respectively designated as pMD18-T-a1-epi。
1.6.3a1-epi the building of prokaryotic expression carrier:
PMD18-T-a1-epi plasmid and I plasmid of pCold are carried out with restriction enzyme BamH I and EcoR I respectively double Digestion identification, digestion products carry out electrophoresis with 1.0% Ago-Gel and glue recycles.By the glue recovery product of a1-epi and digestion The glue recovery product of pCold I is attached.It send positive connection product transformed bacteria solution is accredited as through PCR to the life of Shanghai Hua Da The sequencing of object Technology Co., Ltd., identifies that correct recombinant plasmid is respectively designated as I-a1-epi of pCold.
As a result, since the information obtained from GenBank shows that a1 gene is free of introne, it is attached red thin using pig The CN126 strain DNA of cell space is expanded, and a1-epi sequence amplification product size is 672bp (including ATG) (Fig. 7).Sequencing result It carries out BLAST to compare online, the similitude in the a1-epi and GenBank of amplification is 100%.To the a1-epi protokaryon table of building PCR identification is carried out up to carrier transformed bacteria solution, the results show that amplified production size and Insert Fragment are (Fig. 8) in the same size.Sequencing knot Fruit shows that a1-epi gene order is consistent with insetion sequence, Prokaryotic expression vector construction success.
Inducing expression, purifying, the measurement of content and the detection of 1.7rHspA1 albumen
1.7.1 the inducing expression of HspA1 (recombinant HspA1, rHspA1) albumen, the survey of purifying and content are recombinated It is fixed to cultivate pColdI-a1-epi-E.coli BL21 strain inoculated under the conditions of 37 DEG C, 200r/min in LB liquid medium To logarithmic growth phase, IPTG (isopropyl-beta D-thio galactopyranoside) then is added, the ultimate density of IPTG is 1mmol/ L.Bacterium is shaken with 200r/min in 16 DEG C of incubators, thallus is collected after inducing expression 10h, supernatant is collected after centrifugation in ultrasound cracking. Using His-tagged nickel column to rHspA1 albumen progress affinitive layer purification, and according toBCA Protein Assay Kit specification carries out assay to albumen after purification, and albumen after purification is saved in -20 DEG C.
1.7.2rHspA1 the SDS-PAGE detection of albumen
Albumen before purification and after purification is detected using SDS-PAGE.16 μ L and 4 μ 5 × sds gels of L are taken respectively Sample-loading buffer mixes, 100 DEG C of heating water bath 5min, takes 10 μ L loadings after moment centrifugation.80V electrophoresis 30min, then 120V is electric Swim 1h, is dyed with coomassie brilliant blue staining liquid, is then decolourized with destainer, post analysis electrophoresis result of decolourizing.
The results show that albumen after purification has apparent band through SDS-PAGE electrophoresis at 31kDa, with expected purpose Band is consistent (Fig. 9), and maximum concentration is 758.035 μ g/ μ L.
1.7.3rHspA1 the Western blot detection of albumen
Reactionogenicity detection is carried out to rHspA1 after purification with Western blot technology, antibody used is His-Tag (27E8) Mouse mAb (HRP Conjugate) (1:1000 dilution), ECL colour developing.
The results show that rHspA1 albumen can be reacted with His-Tag (27E8) Mouse mAb (HRP Conjugate), and The ultrasonic lysate of I empty expression vector of pCold cannot be reacted with His-Tag (27E8) Mouse mAb (HRP Conjugate) (Figure 10).
The foundation of 1.8 indirect ELISA detection methods
1.8.1 the selection of antigen best peridium concentration and best serum dilution
Measured using chessboard method, with 0.05mol/L sodium carbonate-bicarbonate buffer (pH9.6) by recombinant antigen successively 0.5,1.0,2.0,4.0 μ g/mL are diluted to, respectively coated elisa plate, every 100 μ L of hole, 4 DEG C of coatings are overnight.With PBST detersive enzyme Target 3 times, each 5min, then with 5% skimmed milk power, 37 DEG C of closing 2h.It is washed ELISA Plate 3 times with PBST again, with 1:50,1: 100, the diluted positive serum of 1:200,1:400 and negative serum are primary antibody, 37 DEG C of effect 1h.After PBST is washed 3 times, it is added The rabbit-anti pig IgG (1:5000 dilution) of 100 μ LHRP label, 37 DEG C of effect 1h.100 μ L TMB-H are added in every hole2O2It is protected from light colour developing 15min adds 50 μ L2mol/L sulfuric acid and terminates reaction, measures the OD value (OD450) at 450nm wavelength, and selection is positive OD450 > 1.0, feminine gender OD450 be smaller, the biggish antigen coat concentration of P/N value, serum dilution combination are dense as most suitable coating Degree and serum optimum dilution degree.
It the results are shown in Table 1, select positive serum OD value close to 1.0 and be greater than 1.0, and reacting hole when P/N value is larger is anti- Former and serum extension rate is as the two best operating condition.The measurement best peridium concentration of antigen is 0.5 μ g/ on this basis ML, and serum optimum diluting multiple is 1: 400.
Table 1: the selection of best peridium concentration and best serum dilution
1.8.2 the selection of best off-period
It with most suitable antigen concentration coated elisa plate, is closed with confining liquid, off-period is respectively 1h, 2h and 3h, so Positive serum is by the extension rate dilution determined in 1.8.1, the same 1.8.1 of other ELISA determination steps afterwards.Compare each group yin, yang The OD value and P/N value of property serum, to select suitable off-period.
It the results are shown in Table 2, show that the P/N value measured in 37 DEG C of at a temperature of closing 2h is maximum, so selecting 37 DEG C of 2h for most Good off-period.
Table 2: the selection of best off-period
1.8.3 the selection of best serum action time to be checked
ELISA Plate is coated with, after closing according to the condition of optimization, and serum to be checked is added, and 37 DEG C act on 0.5h, 1h, 1.5h respectively And 2h, carry out ELISA measurement, the same 1.8.1 of other steps.Compare the OD value and P/N value of each group positive and negative serum, to select to close Suitable serum action time.
As a result 3 are seen attached list, shows that P/N value reaches maximum when 37 DEG C of at a temperature of serum acts on 1h.So selection 1.5h For best serum action time.
Table 3: the selection of best serum action time to be checked
1.8.4 the selection of best enzyme labelled antibody working concentration
After serum to be checked is by determining time effect, enzyme labelled antibody is pressed to 1:2 000,1:3 000,000 and of 1:4 respectively 1:5000 dilution, 100 holes μ L/, the same 1.8.1 of other steps, carry out ELISA measurement, compare each group positive and negative serum OD value and P/N value, to select suitable enzyme labelled antibody working concentration.
As a result 4 are seen attached list, shows that enzyme labelled antibody concentration P/N value at 1: 5 000 reaches maximum, thus 1: 5 000 it is dense Degree is best enzyme labelled antibody working concentration.
Table 4: the selection of best enzyme labelled antibody working concentration
1.8.5 the selection of best enzyme labelled antibody action time
After ELISA Plate is added by the extension rate determined in enzyme labelled antibody, 37 DEG C act on 0.5h, 1h, 1.5h respectively, The same 1.8.1 of his step carries out ELISA measurement, compares the OD value and P/N value of each group positive and negative serum, to select suitable enzyme mark Antibody action time.
As a result 5 are seen attached list, shows that P/N value reaches maximum when enzyme labelled antibody acts on 1h, so the enzyme labelled antibody of selection 1h is made Use the time.
Table 5: the selection of best enzyme labelled antibody action time
1.8.6 the selection of the best developing time of substrate
Enzyme labelled antibody press optimize time effect, washing, addition substrate after, 37 DEG C respectively effect 10min, 15min and 20min reads after being terminated with 50 hole μ L/ of 2mol/L sulfuric acid, compares the OD value and P/N value of each group positive and negative serum, to select to close Suitable substrate developing time.
As a result see attached list 6, develop the color 15min when, positive value reaches and reaches maximum close to 1.0, P/N value, so selecting 15min is best developing time.
Table 6: the selection of the best developing time of substrate
1.9 critical values determine
18 parts of negative Swine serums are taken, 1: 400 dilution carries out indirect ELISA measurement, these serum under the conditions of selected Data obtain OD450 average value through statistical analysisWith standard deviation S.According to Principle of Statistics, OD450 valueWhen, sentence For the positive.450 value of ODWhen, it is judged to feminine gender, the person of falling between is judged to suspicious, need to detect sample again, examine again When OD450 (sample) valueWhen be judged to the positive, OD450 (sample) value When be judged to feminine gender.
As a result, the testing result peak of 18 parts of healthy Swine serums is 0.1965, minimum is 0.117, average valueFor 0.141759, standard deviation S D are 0.031305,When sample OD450 value x >= The positive is judged to when 0.266979；Feminine gender is judged to as sample OD450 value x < 0.235674；When sample OD450 value between It is then judged between 0.235674 (containing 0.235674) and 0.266979 doubtful.When being examined to suspicious sample again, when OD450 valueFor the positive, OD450 valueFor feminine gender.
2.0 cross reactions test
Cryptosporidium spp Swine serum, arch insect infection Swine serum, swine fever virus infection Swine serum, pig blue-ear disease are taken respectively Virus infection Swine serum, 1:400 dilution, is detected, while setting up positive and negative serum control with the indirect ELISA method optimized, Every part of serum does 3 repetitions in parallel, observes whether each serum and system have cross reaction.
As a result 7 are seen attached list, with rHspA1 albumen coated elisa plate, with Cryptosporidium spp Swine serum, arch insect infection pig Serum, swine fever virus infection Swine serum, porcine reproductive and respiratory syndrome virus infect pig blood clearance response, and OD45 is respectively 0.077333,0.2245,0.04,0.230, it is below 0.235674.Positive control OD450 is 1.454667, negative control OD450 is 0.196667.
Table 7: intercrossing test
Swine fever Pig toxoplasma Pig indigo plant ear Cryptosporidium suis Positive control Negative control
OD450 0.04 0.22 0.23 0.08 1.45 0.20
2.1 repetitive test
2.1.1 repetitive test in batch
8 parts of serum are taken to carry out indirect ELISA measurement under the conditions of selected, every part of blood serum sample does 12 weights in parallel It is multiple, calculate the coefficient of variation (coefficient of variance, CV) as follows: CV=[sample OD450 value standard deviation (s)/sample OD450 value average] × 100%.
2.1.2 repetitive test between batch
It takes the rHspA1 albumen of 4 parts of different batches to be coated with, indirect ELISA is carried out to 8 parts of serum under the conditions of selected Measurement.Every part of sample is repeated 3 times, and is averaged, and observes its CV value with 2.1.1.
As a result as shown in subordinate list 8, it is coated with elisa plate with the rHspA1 albumen of same a batch preparation, 8 parts of samples are examined It surveys, repeats 12 holes, the coefficient of variation mean value that test is repeated in batch is 4.2%.With the albumen of 4 different batches to 4 parts of samples into Row detection, the coefficient of variation mean value that test is repeated between criticizing is 7.94%, is shown repeated preferable.
Table 8: repetitive test result
Serum Variation within batch coefficient (CV, n=12) (%) Interassay coefficient of variation (CV, n=4) (%)
S1 3.97 3.37
S2 2.77 3.90
S3 3.19 5.50
S4 2.04 6.86
S5 3.35 4.16
S6 4.20 6.35
S7 1.47 7.94
S8 1.51 4.24
The Preliminary Applications of 2.2ELISA method
Tested with determining various optimum conditions, 75 parts of pig anteserum samples in Shanghai City detected, respectively with Species specific PCR, the China Agriculture Academe Shanghai Veterinary Institute's animal derived food of WATANABE etc. (2012) report are raw The eperythrozoon MSA fluorescent quantitative PCR result that object security laboratory is established is compared, and calculates its coincidence rate.
As a result, detect 75 parts of field pig anteserum samples with the indirect ELISA that rHspA1 albumen is established, positive sample as the result is shown Product account for 54 parts, positive rate 72.0%.It is compared with Species specific PCR and MSA quantitative fluorescent PCR, positive recombination rate point Not Wei 64.81%, 62.96%, negative match-rate is respectively 66.67% and 100% (subordinate list 9).
The comparison of 9: three kinds of testing results of table
Sample number Number positive Positive rate (%) Positive coincidence rate (%) Negative match-rate (%)
RHspA1 indirect ELISA 75 54 72.0
Regular-PCR 75 42 56.0 64.81 66.67
MSA quantitative fluorescent PCR 75 34 45.33 62.96 100
Due to eperythrozoon suis can not in vitro culture, it is more slow to the progress of eperythrozoon suis, to experiment Room diagnosis causes certain difficulty.Currently, the Serologic detection of eperythrozoon suis mainly uses ELISA, this method have it is simple, Quickly, the features such as sensitivity is high, test sample amount is more, easy standardization.
The envelope antigen of this research is the recombination HspA1 epitope area albumen of prokaryotic expression, and under low temperature induction, it is with solvable Property form exist, and N-terminal have His label, this be conducive to large-scale purification recombination HspA1 epitope area albumen, to make Antigenic source is no longer constrained.In addition, recombinant antigen is used to replace native antigen as envelope antigen, it is applied to ELISA and detects And preparation ELISA kit can significantly improve the stability of antigen between different batches.
Since the recombinant protein of prokaryotic expression is also difficult to exclude coli somatic albumen completely even across purification and recovery The interference of ingredient, and during the growth process swinery may multiple ehec infection, large intestine may be contained in serum sample The antibody of bacillus ingredient, it is likely that cause false positive results and reduce the specificity of indirect ELISA detection method, to avoid this It influences, this test is first made using E. coli lysate and blood serum sample at 37 DEG C before blood serum sample is reacted with envelope antigen Use 1h.
The positive coincidence rate of indirect ELISA method and Species specific PCR and MSA quantitative fluorescent PCR that this research institute establishes Respectively 64.81%, 62.96%, negative match-rate are respectively 66.67%, 100%.This research institute is prompted to establish indirect ELISA and Species specific PCR and MSA quantitative fluorescent PCR yin and yang attribute coincidence rate with higher.In addition, the indirect ELISA method With preferable specificity and repeatability, a kind of quickly and effectively detection side is provided for epidemiological survey and medical diagnosis on disease Method.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow Ring substantive content of the invention.

Claims (7)

1. a kind of eperythrozoon recombination epitope antigen rHspA1, which is characterized in that sequence is as shown in SEQ ID No.1.
2. a kind of preparation method of eperythrozoon recombination epitope antigen according to claim 1, which is characterized in that including Following steps:
B cell antigen epi-position enrichment region is screened from eperythrozoon suis HspA1 protein amino acid sequence, is denoted as HspA1 epitope Area；
Its code sequence is screened from eperythrozoon suis HspA1 protein coding gene according to the amino acid sequence in HspA1 epitope area Column, are denoted as a1-epi；
A1-epi sequence is expanded, and recovery product is connect with pMD18-T carrier, obtains recombinant plasmid pMD18-T-a1-epi；
Restriction enzyme BamH I and EcoR is used respectively to the recombinant plasmid pMD18-T-a1-epi plasmid and I plasmid of pCold I carries out double digestion identification, glue recycling, and the glue recovery product of a1-epi and the glue recovery product of digestion pCold I are attached, Obtain I-a1-epi of recombinant plasmid pCold；
E. coli strain bl21 is converted with the I-a1-epi of recombinant plasmid pCold, obtains pColdI-a1-epi-E.coli BL21 bacterial strain；
By pColdI-a1-epi-E.coli BL21 strain inoculated in LB liquid medium, the Fiber differentiation under the conditions of IPTG is received Collection thallus simultaneously cracks, and collects supernatant to get eperythrozoon recombination epitope antigen rHspA1；
The amino acid sequence in HspA1 epitope area is as shown in SEQ ID No.1；The nucleotide sequence of the a1-epi such as SEQ Shown in ID No.2.
3. the preparation method of eperythrozoon recombination epitope antigen according to claim 2, which is characterized in that the amplification With primer as shown in SEQ ID No.3, SEQ ID No.4.
4. the preparation method of eperythrozoon recombination epitope antigen according to claim 2, which is characterized in that the B is thin The screening of extracellular antigen epitope enrichment region is based on eperythrozoon suis HspA1 protein amino acid sequence bioinformatic analysis basis Upper progress.
5. the preparation method of eperythrozoon recombination epitope antigen according to claim 2, which is characterized in that the preparation Method further includes the steps that purifying gained eperythrozoon recombination epitope antigen.
6. a kind of eperythrozoon immunity detection reagent, which is characterized in that be specifically with eperythrozoon recombination epitope antigen RHspA1 is coating antigen；The eperythrozoon recombination epitope antigen rHspA1 is by any preparation method of claim 2~5 It obtains.
7. eperythrozoon immunity detection reagent according to claim 6, which is characterized in that the detection kit Antigen coat concentration is 0.5~4 μ g/mL.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN101701033A (en) * 2009-11-12 2010-05-05 上海交通大学 Methods for preparing antigen, antibody and sample of Eperythrozoon suis
CN104027796A (en) * 2014-06-24 2014-09-10 湖南农业大学 Pig eperythrozoonosis vaccine and preparation method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101701033A (en) * 2009-11-12 2010-05-05 上海交通大学 Methods for preparing antigen, antibody and sample of Eperythrozoon suis
CN104027796A (en) * 2014-06-24 2014-09-10 湖南农业大学 Pig eperythrozoonosis vaccine and preparation method thereof

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* Cited by examiner, † Cited by third party
Title
猪附红细胞体病间接ELISA诊断试剂盒的研制及初步应用;张守发等;《中国兽医学报》;20101231;第30卷(第12期);第1642-1645页 *

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