CN105924518B

CN105924518B - Taeniasis suis TsSerpin B6 recombinant protein and its application

Info

Publication number: CN105924518B
Application number: CN201610248978.9A
Authority: CN
Inventors: 骆学农; 刘光学; 梁盼红; 张少华; 侯俊玲; 郭爱疆; 郑亚东; 才学鹏
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2016-04-20
Filing date: 2016-04-20
Publication date: 2019-05-28
Anticipated expiration: 2036-04-20
Also published as: CN105924518A

Abstract

The present invention provides the application and taeniasis suis TsSerpin B6 recombinant protein of taeniasis suis TsSerpin B6 recombinant protein and its application more particularly to taeniasis suis TsSerpin B6 recombinant protein as cysticercosis cellulosae detection antigen and is preparing the application in cysticercosis cellulosae detection kit.Taeniasis suis TsSerpin B6 recombinant protein of the invention can specific diagnosis cysticercosis cellulosae, and with the parasitic diseases such as pig cysticercus tenuicollis, trichina without any cross reaction.

Description

Taeniasis suis TsSerpin B6 recombinant protein and its application

Technical field

The present invention relates to taeniasis suis TsSerpin B6 recombinant protein and its applications more particularly to TsSerpin B6 to recombinate Application and TsSerpin B6 recombinant protein of the albumen as cysticercus cellulosae detection antigen are in preparation cysticercosis cellulosae detection kit In application.

Background technique

Cysticercosis (Cysticercosis) be by taeniasis suis (Taenia solium) parasitized larvae people and pig skin Under, the food-borne parasitic zoonoses of one kind caused by the positions such as muscle and brain.The disease not only causes pig breeding industry huge Economic loss, and seriously threaten food safety and human health.The study found that serpin is posted in worm Raw host immune plays key player in adjusting, and the serpin of pathogen is adjusted by interference host immune Signal inhibits the activation of host immune system.Therefore, the immunological regulation function of taeniasis suis serpin is studied Can, undoubtedly the immune evasion mechanism in the host processes diagnosis and vaccine antigen new with exploration will be being infected to illustrate taeniasis suis With directive significance.

Serpin (Serpin) is the globular preteins enzyme suppression that a kind of important structure is similar, vdiverse in function Preparation superfamily, eucaryote, virus, have been found that there are serpin genes in archeobacteria at bacterium.Serpins participates in many weights The physiological activity wanted, such as signal transduction, blood clotting, fibrinolysis, inflammatory reaction and complement activation etc. are endogenous adjusting It is played an important role in property protease and exogenous protease.Moreover, Serpins also invades host's mistake in helminth It is also played an important role in journey.The Serpins of parasitic worm protects it from place by inhibiting host serine proteases activity The degradation of enzyme of proteolysis in main body, to escape the immune attack of host.Existing research shows to post from roundworm, hookworm etc. Infested isolated serpin can inhibit the Activity of Digestive Enzymes in Intestine such as trypsase, chymotrypsin.Egyptian blood is inhaled The immunogenicity that worm is combined by the polypide surface serpin factor with the trypsase of people to reduce itself, To escape the immune attack of host.Merckelbach etc. has found the serine stretch protein through prokaryotic expression in Echinococcus multilocularis Enzyme inhibitor can inhibit trypsase, chymotrypsin and elastase activity, thus it is speculated that Echinococcus multilocularis can resist host Damage of the enteron aisle digestive ferment to polypide, the immune attack of host is escaped with this.Therefore, Serpin be expected to become important vaccine and Diagnostic antigen candidate molecules.The correlative study for carrying out taeniasis suis serpin will be helpful to illustrate taeniasis suis Immune evasion mechanism is provided fundamental basis for research and development new generation vaccine, diagnostic preparation and drug.

Summary of the invention

In order to solve the problems, such as cross reaction present in existing cysticercosis cellulosae diagnostic techniques, the present invention provides pig band silk ribbons Worm TsSerpin B6 recombinant protein and its application more particularly to TsSerpin B6 recombinant protein are as taeniasis suis/cysticercus Detect the application of the application and TsSerpin B6 recombinant protein of antigen in preparation cysticercosis cellulosae detection kit.

The present invention provides taeniasis suis TsSerpin B6 recombinant protein, the taeniasis suis TsSerpin B6 recombinant protein Amino acid sequence be sequence table SEQ ID No.2.

The present invention also provides taeniasis suis TsSerpin B6 recombinant proteins as taeniasis suis/cysticercus detection antigen Using；The amino acid sequence of the taeniasis suis TsSerpin B6 recombinant protein is sequence table SEQ ID No.2.

The present invention also provides taeniasis suis TsSerpin B6 recombinant proteins in preparation cysticercosis cellulosae detection kit Using；The amino acid sequence of the recombinant protein is sequence table SEQ ID No.2.

The present invention also provides a kind of cysticercosis cellulosae ELISA detection kit, envelope antigen is pig band in the kit Tapeworm TsSerpin B6 recombinant protein, the amino acid sequence of the recombinant protein are sequence table SEQ ID No.2.

Preferably, further including the rabbit-anti pig IgG of HRP label in the kit.

The present invention also provides a kind of taeniasis suis ELISA detection methods, and the method is not with the disease of patent statute For the purpose of diagnosing and treating, with the 1 μ g of TsSerpin B6 recombinant protein of purifying in 37 DEG C of coating enzyme reaction plate 1h, confining liquid For 1%BSA, sample to be tested and pig negative serum are separately added into after sufficiently being washed with PBST, 37 DEG C of incubation 1h, PBST washings are three times Afterwards, the rabbit-anti pig IgG of 1:5000 times of diluted HRP label is added, DAB colour developing is sufficiently added after washing, is terminated with the concentrated sulfuric acid It measures absorbance after reaction, is positive serum, conversely, being negative serum when P (positive)/N (feminine gender) > 2.

The present invention utilizes taeniasis suis genome and transcript profile design data TsSerpin B6 special primer, with pig capsule tail Larva of a tapeworm or the cercaria of a schistosome total serum IgE is template, and RT-PCR expands TsSerpin B6 gene, and is identified by bioinformatic analysis.Construct pET- 30a-TsSerpin B6 recombinant expression carrier carries out inducing expression in e. coli bl21 (DE3).The destination protein of purifying Carry out SDS-PAGE and Western-blotting analysis.As a result are as follows: the ORF of TsSerpin B6 is made of 1131 nucleotide, Encode 376 amino acid residues；It encodes the reaction center ring (RCL) and characteristic that amino acid has Serpins more conservative Structural domain, and there are 9 potential linear bone-marrow-derived lymphocyte epitopes.The expression product of TsSerpin B6 is mainly to forgive Body form exists, and can react with cysticercus cellulosae positive serum, and specific band is generated at 53kDa.With purifying Diagnostic antigen of the 1 μ g of TsSerpin B6 recombinant protein as indirect ELISA, can specific detection cysticercus cellulosae positive serum, and With pig cysticercus tenuicollis, Trichinella sui, toxoplasma without any cross reaction.Conclusion: the segment cloned is TsSerpin B6, expression product can be used as the specific antigen of cysticercosis cellulosae ELISA diagnosis.

Detailed description of the invention

Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:

Fig. 1 is the PCR amplification result of the SerpinB6 gene of cysticercus cellulosae；Wherein, M:DNA molecular mass standard； 1.SerpinB6；

Fig. 2 is the Tertiary structure predictions of TsSerpin B6；

Fig. 3 is the alignment of TsSerpin B6 partial sequence and other helminth serpins；

Fig. 4 is the phylogenetic analysis of different plant species Serpins；

Fig. 5 is TsSerpin B6 fusion protein purification result；Wherein, M: protein standard；1:50mM miaow Azoles；2:200mM imidazoles；3:300mM imidazoles；4:400mM imidazoles；5:500mM imidazoles；

Fig. 6 is TsSerpin B6 fusion protein Western-blotting result, wherein M: protein mark It is quasi-；1: pig negative serum；2: pig positive serum.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified city It sells.

Embodiment 1

1 materials and methods

The strain of 1.1 worms and serum

Cysticercus cellulosae, cysticercus cellulosae are positive and negative serum is by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's verminosis Seminar saves.

1.2 reagent

Small amount plasmid extraction kit, TRIzol Reagent, DNA plastic recovery kit, pMD-T-19 (simple) Vector, restriction enzyme BamH I, Hind III, T4DNA ligase, it is purchased from Takara company；Revert Aid First Strand cDNA Synthesis Kit kit, Prestained Protein Ladder are purchased from Thermo public affairs Department；Competent escherichia coli cell, pET-30a, isopropyl-β-D-thiogalactoside (IPTG), X-Gal, 5 × albumen loading Buffer is purchased from Beijing Quanshijin Biotechnology Co., Ltd；Rabbit-anti pig IgG (HRP) is purchased from Sigma company；Ni Sepharose 6Fast Flow protein purification kit is purchased from GE company.

The extraction of 1.3 total serum IgEs and cDNA synthesis

The total serum IgE of cysticercus cellulosae gene is extracted, with TRIzol method with Revert Aid First Strand cDNA Synthesis Kit kit synthesizes the first chain of cDNA.

The synthesis of 1.4 design of primers

The taeniasis suis genome sequencing annotation information and transcriptome analysis completed according to this seminar, and combine The open reading frame sequence of the taeniasis suis SerpinB6 of GeneDB warehouse publication designs a pair using software Primer 5.0 Bam H I restriction enzyme site, sequence are as follows: 5 '-CGC is added in specific primer, upstream primerGGATCCATGCTAGATGAATATT ACTTTAATCACC-3'；Hind III digestion site, sequence are as follows: 5 '-is added in downstream primer CCCAAGCTTGGCAGGCCAGGGGATTGAC-3'.Primer send to Jiangsu Jin Weizhi Bioisystech Co., Ltd and synthesizes.

1.5 gene clonings and conversion

Using the cDNA of reverse transcription as template, PCR amplification is carried out with TsSerpin B6 gene-specific primer.PCR reactant System are as follows: 0.5 μ L, 5 × PS Buffer of cDNA, 10 μ L, dNTP Mix, 4 μ L, each 1 μ L of 1 μ L, Easy Taq enzyme of upstream and downstream primer, Moisturizing is to 50 μ L.PCR amplification program is as follows: 95 DEG C of 5min；94 DEG C of 40s, 67 DEG C of 40s, 72 DEG C of 1min, 30 circulations；72℃ 10min.PCR product is cloned into pMD-T-19simple carrier after purification, converts bacillus coli DH 5 alpha competent cell.Selection is single Clone strain carries out PCR and digestion identification, and positive colony send Jiangsu Jin Weizhi Bioisystech Co., Ltd to be sequenced.The amplified production Nucleotide sequence are as follows:

ATGCTAGATGAATATTACTTTAATCACCTGGTACTGTTCCCTCGAGGCTCGAAAAAAAACTGCTTCGTC TCACCACTGAGCATTTACTGCGGCTTGTCTTTGGCCCTCTCTGGGAGCTCCGGCAGATCACTTCATGAGATCGCTCA GGTCTTGCAGGTTCCTAAGCAGCAATTCAAGGATTACGACAGCATTTTAGCACATTTGGGTAGTCATCTGGATGCTG TTTCTGATGGAGATACCGGCAAAACCTTGGTTCTGGCAAACGGCCTTTTTTTGGAATCCACCATCGAGGTTAAGCAG CTCTTTAAAAAAGCCCTAGAAAAGCATTTCCATACAGATTTACATATGGTCGATTTCGCGTCAAACGCTGAGAAGGC CAGAAAGCAAATCAACGCCTGGGTCTCTGATCACACCTGCAAGAAGATCTCCGACCTCATGACTCGTGAATCCATCG ACAGTCAAACCCGCCTCGTTTTGGCTAATGCAATTTACTTTAAAGGTACTTGGAAGAATGTGTTTCACCGCGACGCA ACAACGGATTGGCCTTTTCAAACCCTTCATGACGGAGAGGTTCAGGTTCCAATGATGAATGACAATGGGGTCTATGA GATGTCTCGTTTCGATGAACTTAGCGCAACGGCACTAAAGATTCCCTTTAAATTCCATGAGATGCTTATCGTGTTAC CCGACGCAAAAGACGGTTTGATGGAGTTGTTGGAAAAGATATTCGACCCCAACCACATTTTGCGCTTCAAGGAGCTC TTCGACAAGAGCAAATACGACCCGAGCAGAGTGGAACTGCATCTACCCAAATTCAAACTTGGGGGTGTTGAAAGCCT GAATATGGAGAAACCGCTGTCTGCTATGGGTCTGGAGACTGTTTTTAACCATGATCACGCTGATTTCTCCCGCATTA CCAAGAAGGAAAAGTTGCACATCTCGAGTGTGGTTCATCAGGCTGTGATTGAGGTTAATGAGGAAGGAGCGGAGGCA GCAGCAGCAACTGGAATGGCCATTGCGCCCATGTGTCTGTGCCCAGATTTTACCGTAGATCATCCCTTTCTGTTCTT TATTGTCACCGAAACTGGTGTGCCCGCTTTCATAGGATATGTGGTCAATCCCCTGGCCTGA

The bioinformatic analysis of 1.6 TsSerpin B6 genes

The Theoretical molecular quality of protein, amino acid composition, signal peptide, second level and three are predicted and analyzed using online software Level structure, B cell antigen epi-position, structural domain etc..

The sequence alignment and phylogenetic analysis of 1.7 TsSerpinB6 albumen

With BlastP software by other species amino acid sequences in the amino acid sequence and GenBank of TsSerpinB6 into Row compares, and is evolved using sequence construct of the Test Maximum Likelihood Tree method in 6.0 software of MEGA to selection It sets and analyzes.

The building of 1.8 recombinant expression carriers and expression

It will identify that correct pMD19-T-TsSerpinB6 and prokaryotic expression carrier pET-30a plasmid use BamH I+ respectively Xho I carries out double digestion.Digestion products carry out purification and recovery after agarose gel electrophoresis, to target fragment.Connected using T4DNA It connects enzyme and connect target fragment with pET-30a, and convert BL21 (DE3) competent cell, extract plasmid and carry out PCR and digestion mirror Fixed, positive colony carries out sequence verification.

The correct positive colony bacterium solution 1:100 of sequencing is taken to be inoculated in 100mL LB (blocking that resistance 100mg/mL) Liquid Culture In base, 37 DEG C of 220r/min shaken cultivations to bacterium solution OD_600nmWhen=0.6-1, the IPTG of 100 μ L 500mmol/L of addition, 37 DEG C 220r/min shaken cultivation 6h.The bacterium solution for collecting inducing expression carries out SDS-PAGE analysis.It is arranged simultaneously and induces preceding and pET-30a The inducing expression product of transformed bacteria solution is as control.

The purifying of 1.9 recombinant proteins and Western-blot analysis

The bacterium solution of 50mL inducing expression is taken, 8000r/min is centrifuged 15min, abandons supernatant, adds isometric PBS that thallus is resuspended After precipitating, multigelation 3 times.Thallus suspension 20min is handled using Ultrasonic Cell Disruptor, until solution clarification is bright.4 DEG C, 12000r/min is centrifuged 10min, collects supernatant precipitating respectively, carries out SDS-PAGE analysis.Utilize nickel NTA Ago-Gel FF Destination protein is purified under different imidazole gradients.Albumen after purification carries out Western- with cysticercus cellulosae positive serum Blotting detection.The amino acid sequence of the recombinant protein after purification are as follows:

MLDEYYFNHLVLFPRGSKKNCFVSPLSIYCGLSLALSGSSGRSLHEIAQVLQVPKQQFKDYDSILAHL GSHLDAVSDGDTGKTLVLANGLFLESTIEVKQLFKKALEKHFHTDLHMVDFASNAEKARKQINAWVSDHTCKKISD LMTRESIDSQTRLVLANAIYFKGTWKNVFHRDATTDWPFQTLHDGEVQVPMMNDNGVYEMSRFDELSATALKIPFK FHEMLIVLPDAKDGLMELLEKIFDPNHILRFKELFDKSKYDPSRVELHLPKFKLGGVESLNMEKPLSAMGLETVFN HDHADFSRITKKEKLHISSVVHQAVIEVNEEGAEAAAATGMAIAPMCLCPDFTVDHPFLFFIVTETGVPAFIGYVV NPLA*

2 results

The amplification of 2.1 TsSerpin B6 genes and bioinformatic analysis

Using the cDNA of cysticercus cellulosae as template, by PCR amplification, the segment that size is 1131bp or so is obtained, with expection As a result consistent (Fig. 1).

The bioinformatic analysis of 2.2 taeniasis suis TsSerpin B6 albumen

TsSerpin B6cDNA sequence contains an open reading frame by 1131bp, encodes by 376 amino acid residues The polypeptide of composition, Theoretical molecular quality are 42.3kDa.Hydrophilicity analysis shows that the albumen is hydrophobic proteins. PredictProtein prediction shows that in the Protein secondary structure, α spiral accounts for 35.37%, β-pleated sheet 25.27%, rest part For random coil.Helical structure and coiled structure are dispersed in entire protein molecular, constitute TsSerpin B6 secondary structure Basic framework.Comprehensive analysis show the epitope of TsSerpin B6 albumen may be predominantly located at amino acid sequence the 12nd~ 20,36~46,54~64,74~83,123~130,138~156,175~186,188~197,253~267. MotifScan analysis shows, there may be 1 potential cAMP and cGMP deopendent protein kinase phosphorylations by TsSerpin B6 Site, 10 potential casein kinase phosphorylation sites, 5 potential N- myristolation sides, 6 potential protein kinases C phosphorylation site and 1 potential tyrosine protein kinase phosphorylation site.SMART analysis shows that, TsSerpin B6 amino 7-343 are TsSerpin B6 structural domain, the conserved sequence FXVDHPFLFFI with Serpin albumen in acid sequence. TsSerpin B6 Tertiary structure predictions show that the albumen has 13 α spirals, and 15 β-pleated sheets, remaining is random coil (Fig. 2). Wherein 327Glu, 328Gly, 329Ala, 330Glu；347Asp, 348Phe, 349Thr, 350Val, 351Asp, 352His, 353Pro is that serpin contains more conservative reaction center ring.

The analysis of 2.3 phylogenetic evolutions

It is found by homologous sequence similarity system design, the Serpins of TsSerpin B6 and other species possesses common work Property site, that is, Serpins reaction center ring (RCL), the conservative knot of the characteristic with serpin superfamily member Structure: NAVYFKG and DVNEEG (Fig. 3).

In Fig. 3, underlined in red indicates that serpins characteristic conserved region sequence, black box indicate serpins more Conservative reaction center ring.Sh (Schistosoma haematobium), Emu (Echinococcus Multilocularis), SjB6 (S.japonicum), Smpi56and Sm2 (S.mansoni), Cs (Clonor Chissinensis), Pw (Paragonimus westermani), Hc (Haemon chuscontortus), Tv (Trichostrong ylusvitrinus), Bm1and Bm2 (Brugia malayi).

Systematic evolution tree is constructed using Test Maximum Likelihood Tree method in 6.0 software of MEGA.As a result Show that the Serpin of TsSerpin B6 and Echinococcus multilocularis is located at same branch, affiliation is nearest, and with other species Affiliation farther out.As a result such as Fig. 4.Sh(Schistosoma haematobium),Emu(Echinococcus multilocularis),SjB6(S.japonicum),Smpi56and Sm2(S.mansoni),Cs(Clonorchis sinensis),Pw(Paragonimus westermani),Hc(Haemonchus contortus),Tv (Trichostrongylus vitrinus),Bm1and Bm2(Brugia malayi)。

The expression and analysis of 2.4 recombinant bacterial strains

Recombinant plasmid pET-30a-TsSerpinB6 obtains the segment of about 1131bp through PCR identification and digestion identification, says It is bright successfully to construct the prokaryotic expression carrier for carrying TsSerpin B6 gene.PET-30a-TsSerpin B6 is recombinated with IPTG Bacterium inducing expression, and tested and analyzed through SDS-PAGE, occur albumen purpose band near expected 53kDa, through ultrasonication Afterwards thallus SDS-PAGE analysis the result shows that, destination protein mainly exists with inclusion bodies.

The Western-blot of 2.5 destination proteins is analyzed

8M urea dissolves inclusion body precipitating, is purified with Ni-NTA Purification System.SDS-PAGE points Analysis shows that destination protein obtains preferable purification effect (result such as Fig. 5).It is anti-with the TsSerpin B6 recombinant protein of purifying Original carries out Western-blot analysis.The results show that TsSerpin B6 albumen can be anti-with cysticercus cellulosae positive serum specificity It answers, illustrates that TsSerpin B6 albumen has good immunoreactivity (Fig. 6).

3 discuss

Conformational polymerphism is presented in serpin, and functional diversity, most of they belong to non-classical class Kazal inhibitor family.Most of serpin is single-stranded glycoprotein, and nuclear structure contains 8-9 α-spiral shell Rotation and 3 β-pleated sheets, the structure of outstanding feature be more conservative reaction center ring (reactive center loop, RCL).RCL structure is the target spot that serine protease identification serine protease inhibits, when RCL is identified by serine protease And after being cut open, the conformation of serpin changes, and also occurs so as to cause the conformation of serine protease Change, the activated centre of enzyme is caused to be destroyed, plays inhibiting effect.Homologous sequence compares display, and TsSerpin B6 has serine The characteristic conserved structure of protease inhibitors superfamily member: NAVYFKG and DVNEEG.System carries out analysis and also indicates that and obtained The sequence obtained is closer with echinococcus Serpin affiliation, and the sequence for illustrating that the present invention obtains is taeniasis suis Serpin B6 gene.

The present invention predicts TsSerpin B6 albumen using bioinformatic analysis, shows the albumen n end without letter Number peptide sequence, may be nonsecreting type albumen.Existing research show Serpin gene family member both can intracellular expression, can also It is secreted into extracellular.But lacks signal peptide sequence only according to TsSerpin B6 and decide that it may be less for non-secretory albumen Accurately.Because existing research shows tapeworm, although many protein sequences lack signal peptide sequence, can detect in its cyst fluid To the protein component, even if illustrating that these albumen still can be secreted into extracellularly without signal peptide.The present invention is in prokaryotic expression system The TsSerpin B6 albumen of high efficient expression in system can be with cysticercus cellulosae positive serum although mainly existing with inclusion bodies Specific reaction occurs, illustrates that recombinating TsSerpin B6 has good immunoreactivity, and the albumen may be secretory Albumen can be secreted into polypide external stimulus host and generate the specific antibody for being directed to the component.The above result shows that TsSerpin B6 With the potentiality as pork measles serodiagnosis candidate molecules.In addition, by TsSerpin B6 protein B lymphocyte The prediction of epitope shows that the albumen has 9 potential Linear B Cell Epitopes, illustrates that TsSerpin B6 is also possible to become The candidate molecules of new generation vaccine research and development.

Confirmation has been studied, all there is serpin in worm, protozoan and other helminths, Serpins is played a very important role in helminth-host's interaction process, by adjusting endogenous protease and outside The effect of endogenous binding protein enzyme makes helminth successfully escape the defense mechanism of host.Helminth is during infecting host, silk Serine protease inhibitor can be by inhibiting the activity of host serine proteases, so that the protease of itself be made to drop from host Solution escapes host immune attack.Therefore, further research taeniasis suis serpin enters to invade parasitism in polypide Function and effect in the process, will be illustrate taeniasis suis invasion and immune evasion mechanism and taeniasis suis new generation vaccine and The research and development of bio-pharmaceutical are provided fundamental basis.

Embodiment 2 is detected based on the cysticercosis cellulosae ELISA of recombinant antigen TsSerpinB6

With the 1 μ g of TsSerpinB6 of purifying in 37 DEG C of coating enzyme reaction plate 1h, it is coated with the carbonic acid that buffer is pH9.6 Salt buffer, envelope antigen concentration are 10 μ g/ml, confining liquid 1%BSA, and it is dilute to be separately added into multiple proportions after sufficiently being washed with PBST The pig negative serum released and cysticercus cellulosae, pig cysticercus tenuicollis, Trichinella sui and pig toxoplasma positive serum, 37 DEG C of incubation 1h, After PBST washing three times, the rabbit-anti pig IgG of 1:5000 times of diluted HRP label is added, DAB colour developing is sufficiently added after washing, Absorbance is measured after terminating reaction with the concentrated sulfuric acid.Testing result is as shown in table 1.

Table 1 is to recombinate the ELISA testing result that TsSerpinB6 (1 μ g) is antigen

As it can be seen from table 1 the average OD that TsSerpinB6 antigen is reacted with 100 times of diluted cysticercus cellulosae positive serums Value up to 0.93, and with the diluted pig negative serum of same multiple, pig cysticercus tenuicollis positive serum, Trichinella sui positive serum, The OD value of pig toxoplasma positive serum reaction is 0.4 hereinafter, i.e. P (positive)/N (feminine gender)=2.65 > 2.Illustrate with TsSerpinB6 be antigen ELISA method, have well detection specificity, overcome existing Serology test easily with There are cross reactions the shortcomings that such as pig cysticercus tenuicollis, is ideal cysticercosis cellulosae specific detection antigen.

Identification method of the embodiment 3 to taeniasis suis positive serum

Kit and detection method in Application Example 2 detect to test serum and analysis detection is as a result, work as P When (positive)/N (feminine gender) > 2, testing result can be judged to the positive, conversely, being then feminine gender.

Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention, Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features. All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention Within protection scope.

<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences

<120>taeniasis suis TsSerpin B6 recombinant protein and its application

<170> PatentIn version 3.5

<210> 1

<211> 1131

<212> DNA

<213>artificial sequence

<400> 1

atgctagatg aatattactt taatcacctg gtactgttcc ctcgaggctc gaaaaaaaac 60

tgcttcgtct caccactgag catttactgc ggcttgtctt tggccctctc tgggagctcc 120

ggcagatcac ttcatgagat cgctcaggtc ttgcaggttc ctaagcagca attcaaggat 180

tacgacagca ttttagcaca tttgggtagt catctggatg ctgtttctga tggagatacc 240

ggcaaaacct tggttctggc aaacggcctt tttttggaat ccaccatcga ggttaagcag 300

ctctttaaaa aagccctaga aaagcatttc catacagatt tacatatggt cgatttcgcg 360

tcaaacgctg agaaggccag aaagcaaatc aacgcctggg tctctgatca cacctgcaag 420

aagatctccg acctcatgac tcgtgaatcc atcgacagtc aaacccgcct cgttttggct 480

aatgcaattt actttaaagg tacttggaag aatgtgtttc accgcgacgc aacaacggat 540

tggccttttc aaacccttca tgacggagag gttcaggttc caatgatgaa tgacaatggg 600

gtctatgaga tgtctcgttt cgatgaactt agcgcaacgg cactaaagat tccctttaaa 660

ttccatgaga tgcttatcgt gttacccgac gcaaaagacg gtttgatgga gttgttggaa 720

aagatattcg accccaacca cattttgcgc ttcaaggagc tcttcgacaa gagcaaatac 780

gacccgagca gagtggaact gcatctaccc aaattcaaac ttgggggtgt tgaaagcctg 840

aatatggaga aaccgctgtc tgctatgggt ctggagactg tttttaacca tgatcacgct 900

gatttctccc gcattaccaa gaaggaaaag ttgcacatct cgagtgtggt tcatcaggct 960

gtgattgagg ttaatgagga aggagcggag gcagcagcag caactggaat ggccattgcg 1020

cccatgtgtc tgtgcccaga ttttaccgta gatcatccct ttctgttctt tattgtcacc 1080

gaaactggtg tgcccgcttt cataggatat gtggtcaatc ccctggcctg a 1131

<210> 2

<211> 376

<212> PRT

<213>artificial sequence

<400> 2

Met Leu Asp Glu Tyr Tyr Phe Asn His Leu Val Leu Phe Pro Arg Gly

1 5 10 15

Ser Lys Lys Asn Cys Phe Val Ser Pro Leu Ser Ile Tyr Cys Gly Leu

20 25 30

Ser Leu Ala Leu Ser Gly Ser Ser Gly Arg Ser Leu His Glu Ile Ala

35 40 45

Gln Val Leu Gln Val Pro Lys Gln Gln Phe Lys Asp Tyr Asp Ser Ile

50 55 60

Leu Ala His Leu Gly Ser His Leu Asp Ala Val Ser Asp Gly Asp Thr

65 70 75 80

Gly Lys Thr Leu Val Leu Ala Asn Gly Leu Phe Leu Glu Ser Thr Ile

85 90 95

Glu Val Lys Gln Leu Phe Lys Lys Ala Leu Glu Lys His Phe His Thr

100 105 110

Asp Leu His Met Val Asp Phe Ala Ser Asn Ala Glu Lys Ala Arg Lys

115 120 125

Gln Ile Asn Ala Trp Val Ser Asp His Thr Cys Lys Lys Ile Ser Asp

130 135 140

Leu Met Thr Arg Glu Ser Ile Asp Ser Gln Thr Arg Leu Val Leu Ala

145 150 155 160

Asn Ala Ile Tyr Phe Lys Gly Thr Trp Lys Asn Val Phe His Arg Asp

165 170 175

Ala Thr Thr Asp Trp Pro Phe Gln Thr Leu His Asp Gly Glu Val Gln

180 185 190

Val Pro Met Met Asn Asp Asn Gly Val Tyr Glu Met Ser Arg Phe Asp

195 200 205

Glu Leu Ser Ala Thr Ala Leu Lys Ile Pro Phe Lys Phe His Glu Met

210 215 220

Leu Ile Val Leu Pro Asp Ala Lys Asp Gly Leu Met Glu Leu Leu Glu

225 230 235 240

Lys Ile Phe Asp Pro Asn His Ile Leu Arg Phe Lys Glu Leu Phe Asp

245 250 255

Lys Ser Lys Tyr Asp Pro Ser Arg Val Glu Leu His Leu Pro Lys Phe

260 265 270

Lys Leu Gly Gly Val Glu Ser Leu Asn Met Glu Lys Pro Leu Ser Ala

275 280 285

Met Gly Leu Glu Thr Val Phe Asn His Asp His Ala Asp Phe Ser Arg

290 295 300

Ile Thr Lys Lys Glu Lys Leu His Ile Ser Ser Val Val His Gln Ala

305 310 315 320

Val Ile Glu Val Asn Glu Glu Gly Ala Glu Ala Ala Ala Ala Thr Gly

325 330 335

Met Ala Ile Ala Pro Met Cys Leu Cys Pro Asp Phe Thr Val Asp His

340 345 350

Pro Phe Leu Phe Phe Ile Val Thr Glu Thr Gly Val Pro Ala Phe Ile

355 360 365

Gly Tyr Val Val Asn Pro Leu Ala

370 375

Claims

1. taeniasis suis TsSerpin B6 recombinant protein, it is characterised in that: the amino acid sequence of the recombinant protein is sequence table SEQ ID No.2。

2. taeniasis suis TsSerpin B6 recombinant protein is preparing the application of taeniasis suis/cysticercus detection antigen；The pig band The amino acid sequence of tapeworm TsSerpin B6 recombinant protein is sequence table SEQ ID No.2.

3. application of the taeniasis suis TsSerpin B6 recombinant protein in preparation cysticercosis cellulosae detection kit；The recombination The amino acid sequence of albumen is sequence table SEQ ID No.2.

4. a kind of cysticercosis cellulosae ELISA detection kit, it is characterised in that: envelope antigen is taeniasis suis in the kit TsSerpin B6 recombinant protein, the amino acid sequence of the recombinant protein are sequence table SEQ ID No.2.

5. kit according to claim 4, it is characterised in that: further include the rabbit-anti pig of HRP label in the kit IgG。

6. a kind of taeniasis suis ELISA detection method, the method is not using the diagnosing and treating of the disease of patent statute as mesh , it is characterised in that: with the 1 μ g of TsSerpin B6 recombinant protein of purifying in 37 DEG C of coating enzyme reaction plate 1h, confining liquid is 1%BSA is separately added into sample to be tested and pig negative serum after sufficiently being washed with PBST, after 37 DEG C of incubation 1h, PBST washings three times, The rabbit-anti pig IgG of 1:5000 times of diluted HRP label is added, DAB colour developing is sufficiently added after washing, is terminated and is reacted with the concentrated sulfuric acid After measure absorbance, P (positive)/N(is negative) > 2 when, be positive serum, conversely, being negative serum；The taeniasis suis The amino acid sequence of TsSerpin B6 recombinant protein is sequence table SEQ ID No.2.