CN102181450B - Trichina Ts27 gene, encoding protein and application thereof - Google Patents
Trichina Ts27 gene, encoding protein and application thereof Download PDFInfo
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Abstract
The invention relates to a trichina gene Ts27 and a protein encoded by the same and also relates to a recombinant vector and a recombinant cell containing the gene Ts27 and an antibody specifically combined with the protein encoded by the gene Ts27. The invention also relates to an application of the gene Ts27, the protein encoded by the gene Ts27 and the antibody to preparation of a detection reagent for diagnosing trichinization of humans and animals, and a detection kit. After the gene Ts27 recombinant protein is immune to animals, an antibody with a high titer can be generated; and the gene Ts27 recombinant protein can be specifically identified by animal or human serums infected by trichinas.
Description
Technical field
The present invention relates to a kind of Trichinella spiralis gene, be specifically related to a kind of Trichinella spiralis Ts27 gene, its encoded protein, and said gene and proteic purposes.
Background technology
Trichina(Trichinella spiralis) (Trichinella spiralis; Be called for short Trichinella spiralis), but infected person and more than 150 kind of animal, and its caused trichonematosis is a kind of infecting both domestic animals and human parasitosis; Be global distribution, seriously threatened human health and livestock industry is caused the tremendous economic loss.Trichinella spiralis is summarized as follows the life history: when human or animal host eats raw or half a lifetime food contains the meat (pork, dog meats etc.) of Trichinella spiralis Nang Bao, in the capsule bag larva under gastric juice, intestinal juice effect, overflows and in small intestine growth be adult.Female, male worm After mating worm produces newborn larvae.Newborn larvae is invaded intestines mucosa lymphatic vessel or vein, arrives host's voluntary muscle with lymph and circulation of blood and continues to grow.The pathogenic course of Trichinella spiralis was divided into for three phases: stage of invasion, larval migration and Nang Bao form the phase, and promptly each link in this worm life history all can make the host cause a disease.
The trichonematosis complicated clinical manifestation is various, and diagnosis is difficulty, gives timely that treatment causes certain difficulty, and immunodiagnosis and prevention that therefore should disease become the task of top priority.Trichinous diagnostic method at present commonly used has two kinds: a kind of is that etiological examination is a muscle biopsy, from patient's muscle tissue, finds trichinella larvae and be diagnostic method the most accurately.But the muscle tissue position is circumscribed to be influenced because of being won, early stage and low-grade infection person's muscle biopsy positive rate is not high in morbidity.And owing to be that wound inspection, patient's this kind inspection method beyond affordability are arranged.Second kind is Serological testing, detects the IgG level.The serological method that is used to detect anti-Trichinella spiralis antibody has indirect hemagglutination test (IHAT), IFA (IFAT), latex agglutination test (LAT), immunoenzyme stain test (IEST), enzyme linked immunological seal stain technological (ELIB) and ELISA etc.Wherein ELISA is the serological method of conventional sense trichinzation because of it has advantages such as economy, detection method stdn, susceptibility is more stable now.At present antigen commonly used has muscle larvae drainage-secretion (excretory-secretory, ES) tyvelose (tyvelose, 3, the 6-dideoxyhexose) antigen of antigen, different worm phase somatic antigen or recombinant protein such as 53kDa and synthetic etc. in ELISA detects.But there are cross reaction in ES and somatic antigen and paragonimus, clonorchis sinensis, Schistosoma japonicum and cysticercus etc., so specificity is relatively poor.Though and recombinant protein can improve specificity, its susceptibility is not high.
Because the trichonematosis substance can not limit antigenic obtaining in the external cultivation of going down to posterity in a large number; The antigenic complicacy of Trichinella spiralis, variety cause the antigen that does not still have good high sensitive high specific at present as the diagnosis candidate antigens in addition.
Summary of the invention
In order to obtain better to diagnose candidate antigens; The contriver seeks and has cloned the new gene Ts27 of Trichinella spiralis antigen gene through a large amount of experiments; Obtained the recombinant protein of Ts27 gene through recombinant expressed and purifying; Further obtained specific antibody, and had good immunogenicity and specificity with experiment proof Ts27 gene coded protein.
One aspect of the present invention relates to isolating polynucleotide molecule, and it contains and is selected from following nucleotide sequence:
Encoding sequence shown in sequence shown in the a.SEQ ID NO:1 or the SEQ ID NO:1 in the sequence;
B. with the protein of the nucleotide sequence coded identical sequence of a, but because of the degeneracy of genetic code and the nucleotide sequence different sequences of a;
C. under high stringent condition can with the nucleotide sequence of the nucleotide sequence hybridization of above-mentioned a or b;
D, nucleotide sequence shown in above-mentioned a or the b carried out replacement, disappearance, the interpolation modified nucleotide sequences of one or more bases;
E, the nucleotide sequence that has at least 90% identity with nucleotide sequence shown in above-mentioned a or the b; With
F. with the sequence of sequence complementary shown in above-mentioned a～e.
In one embodiment of the invention, be sequence or its encoding sequence shown in the SEQ ID NO:1, promptly in the SEQ ID NO:1 sequence the 1st to the sequence shown in the 693rd bit base.
Another aspect of the present invention relates to isolating albumen, and its aminoacid sequence is encoded by polynucleotide molecule of the present invention.
In one embodiment of the invention, its aminoacid sequence is shown in SEQ ID NO:2.
Another aspect of the present invention relates to recombinant vectors that contains polynucleotide molecule of the present invention and the reconstitution cell that contains said recombinant vectors.In one embodiment of the invention, said recombinant vectors is for inserting the carrier that PET-28a (+) is obtained with the Ts27 gene fragment.In one embodiment of the invention, said reconstitution cell is for to be transformed into the resulting cell of e. coli bl21 strain with recombinant vectors of the present invention.
Another aspect of the present invention relates to antibody, and it can combine albumen of the present invention specifically, and said antibody can be monoclonal antibody or polyclonal antibody.
Said mono-clonal or polyclonal antibody can be through the conventional immunization method preparations in this area.For example can from the serum of immunized animal, obtain and separate polyclonal antibody, and use the ordinary method test its to antigenic specificity.Perhaps, also can use any technology preparation and the separation monoclonal antibody of producing antibody molecule by the continuous cell line of cultivating.
In one embodiment of the invention,, can obtain high titer antibody serum, obtain polyclonal antibody to the Ts27 gene coded protein with Ts27 gene recombinant protein immune mouse.
Another aspect of the present invention relates to pharmaceutical composition, and it contains albumen of the present invention, antibody perhaps of the present invention, and pharmaceutically acceptable carrier.
Another aspect of the present invention relates to oligonucleotide molecules, and it is nucleotide sequence designed primer or probe according to polynucleotide molecule of the present invention, the said primer polynucleotide molecule that can increase, and said probe can be hybridized with polynucleotide molecule specifically.
In one embodiment of the invention, said oligonucleotide molecules is a primer, and said primer sequence is a sequence shown in SEQ ID NO:3 and the SEQ ID NO:4.
Another aspect of the present invention relates to a kind of test kit, and it comprises polynucleotide molecule of the present invention, albumen of the present invention, antibody of the present invention or oligonucleotide molecules of the present invention.
Another aspect of the present invention relates to polynucleotide molecule of the present invention, albumen, antibody or oligonucleotide molecules and is used for preparing the purposes in the detection reagent of diagnosing animal or human's trichinzation.
Can use above-mentioned polynucleotide molecule as diagnostic reagent, detect by Trichinella spiralis specificity polynucleotide in the animal body fluid of trichinzation or the tissue sample.
Above-mentioned albumen can be used as diagnostic reagent, with detect recently by trichinzation or existing by Trichinella spiralis specific antibody in the animal blood of trichinzation or the serum sample; Perhaps can be used as antigen and produce Trichinella spiralis specific antibody (for example mono-clonal or polyclonal antibody); Can use said antibody as diagnostic reagent, for example with the Trichinella spiralis specific protein in the screening of standard techniques such as Western trace detection method zooblast, tissue or the humoral sample.
Oligonucleotide molecules of the present invention can be used for the differential diagnosis of disease, for example can use suitable designed primer to detect the existence of Trichinella spiralis specificity polynucleotide molecule in the samples such as animal tissues or body fluid.The generation of specific amplification products can be supported the diagnosis of trichinzation, and the product that lacks amplification then possibly not indicated not infection.
Nucleotide sequence of the present invention can obtain from Trichinella spiralis cDNA gene library with immunoscreening.The invention still further relates to the varient of this sequence, the for example disappearance of one or more bases, interpolation or replacement, but do not change coded proteinic function.Varient can be the varient that naturally occurring equivalent varient or non-natural exist.
Albumen of the present invention can be transformed in the suitable host cell through making up the protokaryon or the carrier for expression of eukaryon of said gene, and purifying protein prepares from culture.Albumen of the present invention comprises albumen shown in the SEQ ID NO:2, and its verivate, fragment or the analogue with equivalent function.
In embodiments of the invention; The contriver utilizes Protocols in Molecular Biology; As: cDNA library immunoscreening, molecular cloning, polymerase chain reaction (PCR), determined dna sequence, Southern hybridization, Western blot hybridization method, the clone has identified new gene Ts 27 genes of Trichinella spiralis antigen; Recombinant protein and the height that utilizes genetic expression and immunological technique to obtain this gene immune serum of tiring; And this proteic immunogenicity, susceptibility and specificity analyzed.
The technical indicator that specifically reaches is:
1. adopt cDNA library immunoscreening to obtain the cDNA sequence of this gene in the present invention.Long 1096bp, encoded protein matter contains 231 amino acid.
2. 231 of Trichinella spiralis Ts27 gene amino acid recombinant proteins obtain to express and purifying in the e. coli bl21 strain.
3. Trichinella spiralis Ts27 gene recombinant protein immune animal is obtained the anti-Ts27 immune serum of high titre.
4. Trichinella spiralis Ts27 gene recombinant protein is discerned by the patients serum, sick porcine blood serum, sick rabbit, the sick mouse serum that infect Trichinella spiralis.And react with Ts27 gene recombinant protein immune serum.
The beneficial effect of the invention
The present invention has found Trichinella spiralis Ts27 gene cDNA; Obtained the Ts27 gene recombinant protein of purifying and carried out immunology research, experiment proof Ts27 gene coded protein has better immunogenicity, susceptibility and specificity, for trichinous diagnosis provides new candidate antigens.
Description of drawings
The expression of Fig. 1 Ts27 gene recombinant protein in intestinal bacteria and the SDS-PAGE electrophorogram of purifying, wherein
The arrow indication is the recombinant protein band.
Fig. 2 identifies the Western trace figure of the immunological characteristic of Ts27 gene recombinant protein, wherein
Fig. 3 identifies Ts27 gene recombinant protein susceptibility and specific Western trace figure, and one anti-is different people serum, wherein
Among the figure A, swimming lane 1-19 is the trichinzation patients serum
Among the figure B, swimming lane 1-15 is that (wherein 1-3 is the ascaridosis human serum to different parasitosis human serums; 4-5 uncinariasis human serum; 6-7 is the trichuriasis human serum; 8-10 clonorchis sinensis patients serum; 11-13 is the schistosomicide human serum; 14-15 is the teniasis human serum)
Among the figure C, swimming lane 1-10 is a healthy subjects serum
Embodiment
In the present invention, typically, " stringency " degree of used condition was classified when " hybridization conditions " hybridized according to measurement.It is foundation that the stringency degree can combine the melting temperature(Tm) (Tm) of mixture or probe with for example nucleic acid.For example, " maximum stringency " typically occurs in about Tm-5 ℃ (being lower than 5 ℃ of probe Tm); " high stringency " occurs in following about 5-10 ℃ of Tm; " medium stringency " occurs in following about 10-20 ℃ of probe Tm; " low stringency " occurs in following about 20-25 ℃ of Tm.Alternatively, perhaps further, hybridization conditions can with salt or the ionic strength conditions of hybridization and/or one or repeatedly stringency washing be foundation.For example, the extremely low stringency of 6 * SSC=; 3 * SSC=is low to moderate medium stringency; The medium stringency of 1 * SSC=; 0.5 stringencies such as * SSC=height.Say from function, can adopt maximum stringency condition to confirm and the tight same or near tight same nucleotide sequence of hybridization probe; And adopt high stringency condition to confirm to have an appointment 80% or the nucleotide sequence of multisequencing identity more with this probe.
For the application that requires highly selective, typically expectation adopts relatively restricted condition to form crossbred, for example, selects low relatively salt and/or high-temperature condition.Sambrook etc. (Sambrook, J. etc. (1989) molecular cloning, laboratory manual, Cold Spring Harbor Press, Plainview N.Y.) provides the hybridization conditions that comprises medium stringency and high stringency.
For ease of explanation, the suitable moderate stringent condition that is used to detect polynucleotide of the present invention and other multi-nucleotide hybrid comprises: with 5 * SSC, 0.5%SDS, the prewashing of 1.0mM EDTA (pH8.0) solution; Under 50-65 ℃, in 5 * SSC, hybridize and spend the night; Subsequently with contain 2 of 0.1%SDS *, 0.5 * with 0.2 * SSC 65 ℃ of following each washed twice 20 minutes.It will be appreciated by those skilled in the art that and easily to operate the hybridization stringency, as changing the saltiness and/or the hybridization temperature of hybridization solution.For example, in another embodiment, the tight hybridization conditions of suitable height comprises above-mentioned condition, and difference is that hybridization temperature is elevated to for example 60-65 ℃ or 65-70 ℃.
In the present invention; Said replacement, disappearance, interpolation modified nucleotide sequences of the nucleotide sequence shown in the SEQ ID NO:1 being carried out one or more bases; Be meant respectively or simultaneously at the 5 ' end and/or the 3 ' end of said nucleotide sequence, and/or sequence inside for example is no more than 2-45, perhaps is no more than 2-30; Perhaps be no more than 3-20; Perhaps be no more than 4-15, perhaps be no more than 5-10, the replacement, disappearance, the interpolation that perhaps are no more than 6-8 the base of representing with continuous integral number are one by one respectively modified.
Describe through a kind of polynucleotide; The nucleotide sequence that it had for example with the reference nucleotide sequence of SEQID NO:1 at least " identity " of tool 95% be meant: in per 100 Nucleotide of the reference nucleotide sequence of SEQ IDNO:1; The nucleotide sequence of these polynucleotide is except containing the difference that reaches 5 Nucleotide, and the nucleotide sequence of these polynucleotide is identical with reference sequences.In other words, in order to obtain the identical polynucleotide of nucleotide sequence and reference nucleotide sequence at least 95%, nearly 5% Nucleotide can be by deletion or by another nucleotide substitution in the reference sequences; Maybe can some Nucleotide be inserted in reference sequences, the Nucleotide that wherein inserts can reach reference sequences total nucleotide 5%; Or in some Nucleotide, have deletion, insert and the combination of replacement, wherein said Nucleotide reach reference sequences total nucleotide 5%.These sudden changes of reference sequences can occur in 5 ' or 3 ' terminal position of reference nucleotide sequence; Or any place between these terminal positions; They or be dispersed in separately in the Nucleotide of reference sequences, or be present in the reference sequences with the group of one or more vicinities.
In the present invention; The algorithm that is used for confirming sequence identity and sequence similarity percentage ratio is for example BLAST and BLAST 2.0 algorithms, and they are described in (1990) J.Mol.Biol.215:403-410 such as (1977) Nucl.Acid.Res.25:3389-3402 such as Altschul and Altschul respectively.For example adopt described in the document or default parameters, BLAST and BLAST 2.0 can be used for definite nucleotide sequence homology percentage ratio of the present invention.Carrying out software that BLAST analyzes can be obtained by the public through state-run biotechnology information center.
In the present invention; The nucleotide sequence that nucleotide sequence shown in said and the SEQ ID NO:1 has at least 90% sequence identity comprises and the same basically polynucleotide sequence of the disclosed sequence of SEQ ID NO:1; For example when adopting methods described herein (for example adopting the BLAST of canonical parameter to analyze), compare with polynucleotide sequence of the present invention and to contain at least 90% sequence identity, preferred at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or those sequences of higher sequence identity.
In the present invention, said oligonucleotide molecules better is to be about 15nt at least, be more preferably and be about 25nt, and 50nt especially at least, and can be under the height stringent condition and one or more above-mentioned polynucleotide molecules hybridization.Said height stringent condition is in the 6XSSC/0.5% trisodium phosphate, to wash film, and washes film temperature and be about 37 ℃ for being about 14 base persons, is about 17 base persons and is about 48 ℃, is about 20 base persons and is about 55 ℃, is about 23 base persons and is about 60 ℃.Other hybridization conditions than the long oligonucleotide molecule of the present invention can be confirmed according to standard technique by those skilled in the art.In embodiments of the invention, oligonucleotide molecules of the present invention is complementary to the above-mentioned polynucleotide molecule of a present invention part one of at least.
Term used herein " polynucleotide molecule ", " oligonucleotide molecules ", " encoding sequence " etc. are meant strand or double-stranded DNA and RNA molecule; Can comprise one or more protokaryon sequences; The cDNA sequence; The genomic dna sequence that comprises exon and intron, the DNA of chemosynthesis and RNA sequence, and justice and corresponding antisense strand are arranged.
Produce and the method for operation disclosed polynucleotide molecule of this paper and oligonucleotide molecules is well known by persons skilled in the art, and can according to the recombinant technology of having described (referring to Maniatis etc., 1989,
Molecular cloning, laboratory manual, press of cold spring harbor laboratory, cold spring port, New York; Ausubel etc., 1989,
The molecular biology current techniques, Greene Publishing Associates & Wiley Interscience, NY; Sambrook etc., 1989,
Molecular cloning, the laboratory Handbook, the 2nd edition, press of cold spring harbor laboratory, cold spring port, New York; Innis etc. (volume), 1995,
The PCR strategy, Academic Press, Inc., San Diego; And Erlich (volume), 1992, round pcr, Oxford University Press, New York) accomplish.
In the present invention, said Ts27 gene recombinant protein, Ts27 gene coded protein and Ts27 albumen are same implication, all refer to the albumen that translation obtains according to the encoding sequence in the Ts27 gene cDNA sequence.
To combine embodiment that embodiment of the present invention are described in detail below, but it will be understood to those of skill in the art that the following example only is used to explain the present invention, and should not be regarded as limiting scope of the present invention.Unreceipted actual conditions person among the embodiment carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be through the conventional products of commercial acquisition.
The clone and the sequential analysis of embodiment 1 Trichinella spiralis Ts27 gene
1. the preparation of screening serum
Collect Trichinella spiralis muscle larvae (international standard worm strain ISS533); Experiment pig (WZSP), 20kg/ head, totally 4; 20000 of peroral infection Trichinella spiralis muscle larvaes; Each 2 of nz's large ear rabbit and BALB/C mices, per os infects 4000 of Trichinella spiralis muscle larvaes and 500 respectively, obtains artificial challenge's porcine blood serum, infected rabbits serum and infected mice serum after 4 weeks.Measure serum titer through ELISA, infected pigs's serum reaches more than 1: 10000, and infected rabbits serum and infected mice serum reach more than 1: 1000.
2. immunoscreening expression library
Trichinella spiralis adult λ ZAPIIcDNA expression library is available from Liu Mingyuan (reference " structure and the screening of Chinese Trichinella spiralis strain isolated adult and newborn larvae gene library ", Chinese animal doctor's journal, 1998,18 (2): 147-150.).
1 μ l Trichinella spiralis adult λ ZAP II cDNA expression library, diluent adds among the 600 μ l host bacterium XL1-Blue, and 37 ℃, adsorb and added top-agar in 20 minutes, be inverted in the plaque that is cultured to the needle point size in 42 ℃ of incubators and grow.Be layered on nitrocellulose filter on the flat board, be placed on 37 ℃ of incubator overnight cultures.Second day at room temperature with the film marked; Take off from flat board; The trichinzation porcine blood serum (1: 10000) of immersion embodiment 1 preparation 2 hours; Wash film 3 times with TBST (TBS contains 0.5%Tween20), reseal membrane immersed the anti-pig IgGs of two anti-working fluid-1: 10000 horseradish peroxidase-rabbits (Sigma company) 2 hours.Film is immersed among the colour developing liquid DAB (Pierce company), after the colour developing fully, add H
2The O termination reaction.Trichinzation porcine blood serum with embodiment 1 preparation in 1: 10000 further screens the positive plaque of acquisition, sieves again through three-wheel, obtains 74 positive plaques.Carry out interior shearing of body of positive plaque subsequently.The positive plaque liquid (can according to circumstances change add-on) that 500 μ l host bacterium XL1-Blue and 500 μ l screening is obtained is added among the NZY Broth of 5ml, and 4～5hr are cultivated in 37 ℃ of joltings, add 3 chloroforms again; 37 ℃, 15min, 3000rpm are cultivated in jolting; Behind 4 ℃ of centrifugal 15min; With 1.5ml centrifuge tube packing supernatant, every pipe adds 5 μ l chloroforms, and 4 ℃ of preservations are subsequent use.Cleer and peaceful 5 μ l helper phages on the phage after 200 μ l host bacterium XL1-Blue, the above-mentioned amplification of 150 μ l are mixed 37 ℃ of water-bath 15min; The LB liquid nutrient medium that adds 3ml, 37 ℃, 3hr is cultivated in jolting; 65 ℃ of water-bath 20min; 4000rpm gets the supernatant packing behind 4 ℃ of centrifugal 15min, is the supernatant after shearing in the phage body.The supernatant of getting after shearing in the 4 μ l bodies is added among the 200 μ l host bacterium SOLR, and 37 ℃, water-bath 15min; Coat on the flat board, carry out the white screening of indigo plant, the positive clone of white colony.After PCR identifies, choose the Ts27 clone and carry out determined dna sequence.
3.Ts27cDNA sequence and amino acids coding analysis
The long 1096bp of the cDNA of Ts27 gene, 231 amino acid of encoding, its theoretical molecular is 26.2KD.Below be the cDNA sequence (SEQ ID NO:1) of Ts27 gene and the aminoacid sequence (SEQ ID NO:2) of its expressing protein.
GAAAATATGGCACATAGCAATGAAAATGtTGACtTTATGaAATATGCaAtTGAGGAgGCTCCAGCTGCGgCATTTTACATTCCAGAATATTTTAaTGACaAGGAGGAAGAGTTTTATACACAGCAGATATATTCAGCTCCCGCTCCGAAATGGACTAGCCTTAGTGCTAGGCGtTTGCTAAATTACGGAGGTATTGTTGGCAAAAAGGGACTCATTCAAGTTAATGATATACCTTTCTGGTTACGTGCACTGATGAAGCGGGTTTGTAAGTCAGTACCATTGTTTACTCCAGGTAATGAACCGAATCATGTTCTAATCAATGAATATTTACCTGGTCAAGGAATTATGCCTCATACTGATGGGCCAGCTTATTTTCCTGTGGTTGCCAACATTACTTTGGGGAGTCATACTGTGCTTGATCTCTATGAAACAGAAACGAGAAATTATATTGGATCATTTCTCCTTGAACGGAAAAGCTTATTAATAATTTCTGAGCAGCTGTATGTAAATTTCCAGCATGGTATCAAAGAAGTTAAAGAAGATGAAATCAGTGAAAAAATACTAAACTATAAATTATGCTCAGAAAAGTATAGTAGCGAAGAAAAAATTCCTCGTAAAACACGAATATCCATTACAATTCGAAACGTGCCGAAAACGATTACAGCTTTTACAGCTGTTTTCAATAACCGTGCA
TGAATACGCATGGGATGCTGGATTCCAGTGAAAATTTTACCGAGAGAGACTATCTCGGTTATTATTAAAGTTCGTTTCTCATTCGATGTTCATTGCCAAGTCAGATTGATTTTTTTAATCTAATTGTTCATTGATTCAAATTTCTTTTTTTTGATTAGTTGCTAAATGTTCATTTCATTGTCCAAATATTTCCATTGACGATTATGTGACTTTATCATTTATGTAATAATCGTTTTCTTTTACATTTCACACGTTTCAGCAATCAGTATTTTtAATATCAATTTTTTCGtTATTTTATATTTTGTtATTGTAcCATGATGAAATTTTATaAAATATGATAGCAATAGATAGGTCAACAAAAGCAGCAAAATATATtAAAAAAAAAAAAAAAAACtCgAGGGGC(SEQ ID NO：1)； (wherein the 1st to sequence shown in the 693rd bit base be encoding sequence, i.e. sequence between the black underscore)
ENMAHSNENVDFMKYAIEEAPAAAFYIPEYFNDKEEEFYTQQIYSAPAPKWTSLSARRLLNYGGIVGKKGLIQVNDIPFWLRALMKRVCKSVPLFTPGNEPNHVLINEYLPGQGIMPHTDGPAYFPVVANITLGSHTVLDLYETETRNYIGSFLLERKSLLIISEQLYVNFQHGIKEVKEDEISEKILNYKLCSEKYSSEEKIPRKTRISITIRNVPKTITAFTAVFNNRA(SEQ?IDNO：2)。
1.Ts27 gene prokaryotic and purifying
(1) amplification Ts27 gene:
The positive colony pBluescript/Ts27 bacterium liquid that obtains with embodiment 1 is that template is carried out full bacterium pcr amplification, and primer does
5 '-CGGGATCCGAAAA TATGGCACATAGCAATG-3 ' (SEQ ID NO:3); With 5 '-CGCTCGAGTGCACGGTTA TTGAAAACAGC-3 ' (SEQ ID NO:4).
Amplification condition: 95 ℃ of 5min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 2min, 35 circulations; 72 ℃ of 10min.
(2) the Ts27 gene fragment after the amplification purification is subcloned on prokaryotic expression carrier PET-28a (+) after BamH I and Xho I enzyme are cut.Correct recombinant plasmid transformed e. coli bl21 strain after 37 ℃ of 150 rev/mins of 1.0mM IPTG induce 3 hours, is filled to bacterium liquid branch in the 50ml centrifuge tube; 11000rpm, 4 ℃ of centrifugal 5min abandon supernatant; Collect thalline, with the fully suspension of 20mM Tris-HCl (pH7.9) of 200ml.Add 1ml proteinase inhibitor II (Protease Inhibitor Cocktail Set II).Fully ultrasonic on ice, ultrasonic output intensity 85%, each 6 seconds, 3 seconds at interval; 11000rpm, 4 ℃ of centrifugal 15min abandon supernatant, collect inclusion body and cell debris; Use 20mM Tris-HCl (pH7.9) the suspension deposition of 100ml again.Adding freshly prepared lysozyme soln to final concentration is 100 μ g/ml, places 30min on ice, during constantly stir with glass stick; Fully ultrasonic on ice, ultrasonic output intensity 85%, each 6 seconds, 3 seconds at interval; 11000rpm, 4 ℃ of centrifugal 15min abandon supernatant, obtain the Ts27 gene recombinant protein, and recombinant protein makes purity higher through electroelution appearance wash-out behind His-binding (Novagen company) affinity chromatography column purification, and purity reaches (Fig. 1) more than 80%.
2.Ts27 the preparation of gene recombinant protein immune serum
The Ts27 recombinant protein of purifying is with after adjuvant ISA 50V mixes, and through skin multi-point injection immunity BALB/c mouse (25 μ g/ only), per 2 weeks are strengthened 1 time, strengthen altogether 2 times.Initial immunity is plucked eyeball after 5 weeks and is got blood, after the blood of collection is put room temperature placement 2～3hr, and 3000rpm, 4 ℃ of centrifugal 10min; Get supernatant, 3000rpm, 4 ℃ of centrifugal 10min.Collect serum and packing ,-80 ℃ of preservations are subsequent use.ELISA measures antibody titer up to more than 1: 80000.
The immunological characteristic of embodiment 3Ts27 gene recombinant protein
1. enzyme linked immunological absorption (ELISA) detects
Ts27 gene recombinant protein with 1 μ g embodiment 2 preparation is antigen coated 96 hole enzyme plates, and one anti-ly is serum to be checked, and an anti-serum to be checked is respectively trichonematosis human serum (35 examples; The Central China University of Science and Technology provides, and working concentration is 1: 200), infect rabbit and mouse serum (each 2 example, embodiment 1 preparation of Trichinella spiralis; Working concentration is 1: 1000); Infect the porcine blood serum (working concentration is 1: 10000 for 2 examples, embodiment 1 preparation) of Trichinella spiralis.Two anti-(sheep anti mouse, goat-anti rabbit, goat-anti people two are anti-for horseradish peroxidase-labeled corresponding two anti-; Available from biotech firm of China fir Golden Bridge product in Beijing; Anti-pig two resists available from Sigma rabbit; Working concentration is 1: 5000), healthy subjects serum, the normal rabbit that does not infect Trichinella spiralis, pig and mouse serum are made as negative control.
Experimental result shows that this albumen can produce positive reaction with trichonematosis human serum, the rabbit that infects Trichinella spiralis, pig and mouse serum, and OD value and negative serum ratio are greater than 2.0.The recombinant protein that the Ts27 gene is described has special immunogenicity, is the diagnostic reagent that has potentiality.
2.Western engram analysis
The Ts27 genetic expression albumen (200ng) of the purifying of embodiment 2 preparations is behind the SDS-PAGE electrophoresis; Be transferred to pvdf membrane; Detect with ECL chemical luminescence reagent kit (Amersham company); Respectively with patients serum 1: 200 (Central China University of Science and Technology provides), infected pigs's serum 1: 10000, infected rabbits serum 1: 1000, infected mice serum 1: 1000 (above-mentioned three kinds of serum are embodiment 1 preparation), Ts27 gene recombinant protein immune mouse serum (embodiment 2 preparations) 1: 10000 as first antibody, two anti-and present embodiments 1 in identical.The result shows that about 33KDa place all presents a tangible Western blot band (Fig. 2); Show that further the Ts27 gene recombinant protein has good immunogenicity; Can be the people, pig, rabbit, mouse serum and the Ts27 gene recombinant protein immune serum that infect Trichinella spiralis discerns; And specificity is good, can be used for the diagnosis of trichinzation.
Embodiment 4Ts27 genetic expression albumen susceptibility and specific evaluation
1. enzyme linked immunological absorption (ELISA) detects
Ts27 gene recombinant protein with 1 μ g embodiment, 2 preparations is antigen coated 96 hole enzyme plates; One anti-is serum to be checked, an anti-serum to be checked be respectively the trichonematosis human serum (35 examples, the Central China University of Science and Technology provides; Working concentration is 1: 200), other parasitic infection patients serum (each 5 example of roundworm patient, hookworm patient, whipworm patient, clonorchis sinensis patient, Schistosoma japonicum patient, taeniasis suis patients serum; The Central China University of Science and Technology provides, and working concentration is 1: 200), healthy subjects serum (10 examples; The worker of parasite teaching and research room of the Capital University of Medical Sciences, working concentration is 1: 200).Two anti-goat-anti people two anti-(available from biotech firm of China fir Golden Bridge product in Beijing, working concentration is 1: 5000) for horseradish peroxidase-labeled are made as negative control with healthy subjects serum.This albumen can produce positive reaction with the trichonematosis human serum, and OD value and negative serum ratio are greater than 2.0.Simultaneously, this albumen does not react with other parasitic infection patients serum, explains that the recombinant protein of Ts27 gene has special immunogenicity, and susceptibility is 91.4% (32/35), and specificity is 100% (0/35), is the diagnostic antigen that has potentiality.
2.Western engram analysis
The Ts27 genetic expression albumen (100ng) of the purifying of embodiment 2 preparations is behind the SDS-PAGE electrophoresis; Be transferred to pvdf membrane; Detect with ECL chemical luminescence reagent kit (Amersham company); Respectively with trichonematosis human serum, other parasitosis human serums (roundworm patient, hookworm patient, whipworm patient, clonorchis sinensis patient, Schistosoma japonicum patient, each 5 example of taeniasis suis patients serum; The Central China University of Science and Technology provides) and healthy subjects serum 10 examples (worker of parasite teaching and research room of the Capital University of Medical Sciences); Working concentration is 1: 200, two anti-goat-anti people two anti-(available from biotech firm of China fir Golden Bridge product in Beijing, working concentration is 1: 5000) for horseradish peroxidase-labeled.The result shows 32 routine trichonematosis human serums and Ts27 genetic expression albumen test; All present a tangible Western blot band at about 33KDa place; Have 3 examples not react, and hybridization signal (accompanying drawing 3) does not all appear in other patients serums and normal human serum and Ts27 genetic expression albumen.Susceptibility is consistent with ELISA with specificity, shows that further the Ts27 gene recombinant protein has good sensitivity and specificity, can be used for the diagnosis of trichinzation.
Although embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by accompanying claims and any equivalent thereof.
Claims (8)
1. isolating polynucleotide molecule, it is formed by being selected from following nucleotide sequence:
Encoding sequence shown in sequence shown in the a.SEQ ID NO:1 or the SEQ ID NO:1 in the sequence;
B. with the protein of the nucleotide sequence coded identical sequence of a, but because of the degeneracy of genetic code and the nucleotide sequence different sequences of a; With
C. with the sequence of sequence complementary shown in above-mentioned a～b.
2. isolating albumen, its aminoacid sequence is by the polynucleotide molecule coding of claim 1.
3. the albumen of claim 2, its aminoacid sequence is shown in SEQ ID NO:2.
4. recombinant vectors, it contains the described polynucleotide molecule of claim 1.
5. reconstitution cell, it contains the recombinant vectors of claim 4.
6. oligonucleotide molecules, it is the nucleotide sequence designed primer according to the polynucleotide molecule of claim 1, said primer sequence is a sequence shown in SEQ ID NO:3 and the SEQ ID NO:4.
7. test kit, it comprises polynucleotide molecule, claim 2 or 3 the albumen or the oligonucleotide molecules of claim 6 of claim 1.
8. the oligonucleotide molecules of the polynucleotide molecule of claim 1, claim 2 or 3 albumen or claim 6 is used for preparing the purposes in the detection reagent of diagnosis animal or human trichinzation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544628A (en) * | 2003-11-13 | 2004-11-10 | 首都医科大学 | Trichina TS88 gene, their protein products and uses |
| CN101481691A (en) * | 2008-04-30 | 2009-07-15 | 首都医科大学 | Trichinella spiralis 70KDa heat shock protein and use thereof |
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| CN1544628A (en) * | 2003-11-13 | 2004-11-10 | 首都医科大学 | Trichina TS88 gene, their protein products and uses |
| CN101481691A (en) * | 2008-04-30 | 2009-07-15 | 首都医科大学 | Trichinella spiralis 70KDa heat shock protein and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 诸欣平等.旋毛虫抗原基因Ts88 的克隆及鉴定.《中国人兽共患病杂志》.2004,第20卷(第1期),19-21. * |
| 魏骏飞等.抗旋毛虫副肌球蛋白单克隆抗体的制备与鉴定.《中国病原生物学杂志》.2009,第4卷(第9期),659-662. * |
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