CN102565398B

CN102565398B - Clonorchiasis ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic reagent kit

Info

Publication number: CN102565398B
Application number: CN201010619492.4A
Authority: CN
Inventors: 余新炳; 徐劲; 邓传欢; 胡旭初; 吴英松; 董志宁; 伍忠銮; 胡慧霞; 赵俊红; 梁炽; 李来庆; 王征
Original assignee: Foshan Shunde Zhiyan Biotechnology Co Ltd
Current assignee: Foshan Shunde Zhiyan Biotechnology Co., Ltd.
Priority date: 2010-12-31
Filing date: 2010-12-31
Publication date: 2015-06-24
Anticipated expiration: 2030-12-31
Also published as: CN102565398A

Abstract

The invention belongs to the technical field of biology, and discloses a clonorchiasis ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic reagent kit, which comprises an ELISA reaction plate, a negative control, a positive control, a substrate liquid A, a substrate liquid B, a sample diluent, a concentrated washing liquid and a stopping solution, wherein the ELISA reaction plate is coated with a clonorchiasis glutathione transferase 2; the clonorchiasis glutathione transferase 2 is a polypeptide which has an amino acid sequence shown as SEQ ID No.1 or a conserved variation polypeptide; and an enzyme label is horse radish peroxidase-labeled antihuman IgG4 (immunoglobulin G4) second antibody. The clonorchiasis ELISA diagnostic reagent kit disclosed by the invention has the characteristics of high specificity, high sensitivity, high accuracy and the like, and can be well applied to diagnosis of clonorchiasis; a major reagent of the reagent kit is in the form of a working liquid, and can be used directly; and compared with the conventional aetiology diagnosis method, the clonorchiasis ELISA diagnostic reagent kit has the advantages of operation accuracy, easiness and convenience and believable results.

Description

A kind of clonorchiasis ELISA diagnostic kit

Technical field

The invention belongs to biological technical field, specifically, the invention describes a kind of clonorchiasis ELISA diagnostic kit.

Background technology

Clonorchiasis is a kind of parasitic zoonoses, adult colonizes in hepatic duct, and adult can destroy biliary tract epithelium and submucosal blood vessel, and produces secretion excretion antigen, cause the inflammatory reaction around bile duct and bile duct, cause bile duct limitation to be expanded and bile duct epithelial cell hyperplasia.Some antigenic components can also enter hepatic tissue by bile duct epithelial cell, and cause inflammation reaction and liver dysfunction.Long-term severe infections can cause proliferation of fibrous tissue and hepatocellular atrophy sex change, even causes canceration, ascites and cirrhosis.Liver fluke blocks bile duct, bile is flowed out not smooth, easily merges bacteriological infection and occur cholelithiasis.

The control of liver rot mainly relies on comprehensive preventive health measures.At the mass screening and mass treatment of Endemic Area strengthening to crowd, and strengthen the development of vaccine, to reduce the infection sources and protection Susceptible population.At present, the emphasis of liver rot study on prevention concentrates on diagnosis and vaccine candidate antigen, drug targets, pathogenic molecular mechanism research.

Transferase carries out the transfer of some group or the enzyme of exchange between catalytic substrate, is one of catalyzer important in liver cell association reaction in biosome (genus second-phase reaction).Glutathione transferase (glutathione S-transferase, GST) (EC2.5.1.18) be extensively be present in various biosome by multiple gene code, multi-functional isodynamic enzyme, it is the enzyme family that catalysis glutathione is connected with multiple electrophilic compound, the subunit forming enzyme has 7 at least, and most of enzyme is present in tenuigenin with homodimer or the existence of heterodimer form.

One of GSTs critical function in biosome is the detoxication of internal, exogenous poisonous substance.The biosome comprising people is all exposed in the environment of a large amount of xenobiontics existence, some compounds have electrophilic reactivity after metabolism activation, cause and endogenous molecule covalent bond, protein is important biomacromolecule, through often changing its function after exogenous molecules and its combination.Glutathione transferase impels glutathione to be combined with xenobiotic mainly through enzyme catalysis, or in non-enzymatic binding mode, chemical substance, dyestuff, the carcinogenic substance with genotoxic potential various in body is discharged in body, thus reach removing toxic substances object, discovered in recent years glutathione transferase for research liver diseases generation, develop, lapse to and prevent significant.Its catalytic reaction characteristic determine its preventing lipid peroxidation injury from expanding, resist and DNA plerosis damage to reduce the vital role in tumour generation and cancer cell drug resistance formation etc.

GSTs is the potential drug targets of anti-plasmodium falciparum, the GSTs of worm participates in the removing toxic substances to the lipid peroxide produced by polypide metabolism and host immune system and Cytotoxic carbonyl, is also considered to immunotherapy and a chemotherapeutic potential target.Wherein, being mainly positioned blood fluke GST-26 and GST-28 of adult essence, is one of candidate vaccine molecule of 6 most potentiality that WHO proposes.Catalytic activity is had at the restructuring GST of expression in escherichia coli plasmodium falciparum.The 26KDa GST of clonorchis sinensis and the encoding gene of 28kDa GST also have catalytic activity at prokaryotic expression, and the 26kDaGST of restructuring can be used for detecting IgG and IgE antibody.Thus, prokaryotic expression activated restructuring GST is feasible. and be easy to obtain a large amount of recombinant proteins for functional study.

The present inventor, through extensive and deep research, has been separated coding for glutathion transferase 2DNA from clonorchis sinensis, and expression and purification glutathione transferase 2, and further disclose the application based on these polynucleotide and polypeptide.

Summary of the invention

The present invention, on the basis of the clonorchis sinensis glutathione transferase 2 and preparation and application thereof that provide separation, provides a kind of clonorchiasis ELISA diagnostic kit.

The invention discloses a kind of clonorchis sinensis glutathione transferase 2 of separation, it is polypeptide or its conservative variation's polypeptide of the amino acid sequence with SEQ ID NO.1.

Method well-known to those having ordinary skill in the art can be used for building containing clonorchis sinensis glutathione transferase 2 DNA sequences encoding and the suitable expression vector of transcribing/translating control signal.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Described DNA sequence dna can be effectively connected in the suitable promoter in expression vector, synthesizes to instruct mRNA.The representative example of these promoters has: colibacillary lac or trp promoter; Enter bacteriophage PL promoter; Eukaryotic promoter comprise CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promoter, retroviruse LTRS and some other known can the promoter expressed in protokaryon or eukaryotic or its virus of controlling gene.Expression vector also comprises ribosome bind site and the transcription terminator of translation initiation.In addition, expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as dihyrofolate reductase, neomycin resistance and green fluorescent protein (GFP) that eukaryotic is cultivated, or for colibacillary tetracycline or amicillin resistance.Comprise the carrier of above-mentioned suitable DNA sequence dna and suitably promoter or control sequence, may be used for transforming suitable host, with can marking protein.

Host cell can be prokaryotic, as bacterial cell e. coli bl21/DE3; Or the eukaryotic such as low, as yeast cells; Or higher eucaryotic cells, as mammalian cell.Representative example has: Escherichia coli, streptomyces; The bacterial cell of salmonella typhimurium; Fungal cell is as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast etc. of CHO, COs.293 cell or Bowes melanoma cells.

Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotes as Escherichia coli time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaCl ₂method process, step used is well-known in this area.Another kind method uses MgCl ₂.If needed, transform and also can be undertaken by the method for electroporation.When host is eucaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical methods is as microinjection, electroporation, liposome packaging etc.

The preparation method of the clonorchis sinensis glutathione transferase 2 of aforementioned separation, in general there are following steps: (1) uses the polynucleotide (or variant) of coding clonorchis sinensis glutathione transferase 2 of the present invention, or transform or suitable host cell of transduceing with the recombinant expression carrier containing these polynucleotide; (2) host cell cultivated in suitable nutrient culture media; (3) separation, protein purification from nutrient culture media or cell.

The transformant obtained can be cultivated by conventional method, expresses the polypeptide of coded by said gene of the present invention.According to host cell used, nutrient culture media used in cultivation can be selected from various conventional medium.Cultivate under the condition being suitable for host cell growth.When after host cell growth to suitable cell density, the promoter selected with the induction of suitable method (as temperature transition or chemical induction), cultivates a period of time again by cell.Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in cell or on cell membrane.If needed, can utilize its physics, the albumen of being recombinated by various separation method abstraction and purification with other characteristic of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) and other various liquid chromatography (LC) technology and these methods.

Enumerate according to the embodiment of the present invention, an appropriate method preparing clonorchis sinensis glutathione transferase 2 is:

1) encoding gene of clonorchis sinensis glutathione transferase 2 is cloned into prokaryotic expression carrier pET-30a (+);

2) by screening positive clone after the expression vector transformation of E. coli BL21/DE3 competent cell of restructuring;

3) abduction delivering of e. coli bl21/DE3;

4) collect the nutrient solution supernatant after the abduction delivering of e. coli bl21/DE3, obtain the albumen of purifying through affinity chromatography.

Above-mentioned clonorchis sinensis glutathione transferase 2 can be used for preparation clonorchiasis diagnostic kit.

The diagnosis of clonorchiasis is except the Pathogen Biology inspection of routine, and main method uses immunological diagnostic reagent box.Mainly contain two classes: enzyme linked immunological kit (ELISA) and tachysynthesis colloidal gold kit (ICT).Namely the diagnosis of clonorchiasis judges Current Infection by the circulating antigen detected in serum or urine, also carrys out rapid screening by the specific antibody detected in serum or saliva.Because clonorchis sinensis mainly colonizes in outside tissue, the antigenic component overwhelming majority produced enters enteron aisle along with bile, and only have infectiosity high, polypide is serious to bile duct obstruction, when causing epithelial duct serious damage, its Secretory product just can enter in liver and peripheral blood.Enter circulating antigen major part in peripheral blood neutralize by antibody, only have a small amount of free circulating antigen to be detected.Therefore, although the method detecting circulating antigen can reflect Current Infection, susceptibility is lower.At present, the clinical mainly antibody assay kit for auxiliary diagnosis.The method detecting antibody is divided into two kinds, and one is combine with the antibody that the antigen be coated on reaction plate or reaction film is caught as detection reagent with mark two is anti-, and a kind of is as detection reagent with the antigen marked.Two is anti-as detecting reagent, generally can only detect a kind of antibody, and non-specific binding is higher; Labelled antigen is as detection reagent, and can detect Multiple Antibodies, specificity is higher.

The invention discloses a kind of clonorchiasis ELISA diagnostic kit, comprise: ELISA reaction plate, negative control, positive control, enzyme marker, sample diluting liquid, concentrated cleaning solution, substrate A liquid, substrate B liquid and stop buffer, wherein, ELISA reaction plate is coated with above-mentioned clonorchis sinensis glutathione transferase 2, described enzyme marker is that horseradish peroxidase-labeled anti-human igg 4 two resists.

Described clonorchis sinensis glutathione transferase 2 is the recombinant expressed clonorchis sinensis glutathione transferase 2 of genetic engineering.

Preferably, the formula of sample diluting liquid is: 0.1%BSA (0.1g/100ml)+0.05% Tween-20 (v/v)+PBS (PH7.2).Further, antiseptic is also contained in sample diluting liquid as thimerosal 0.01% (0.01g/100ml).The formula of concentrated cleaning solution is: the Tris-HCl damping fluid of pH7.8.Negative control can be: the non-Endemic Area of clonorchiasis, and without eating sashimi (raw fish) history, empirical tests does not suffer from the normal human serum of clonorchiasis; Positive control can be: clonorchiasis Endemic Area, eats sashimi (raw fish) history, and empirical tests suffers from the patients serum of clonorchiasis.

The using method of clonorchiasis diagnostic kit of the present invention, comprises the following steps:

1). dosing: concentrated cleaning solution dilution is mixed with cleansing solution;

2). application of sample: add in the respective aperture of ELISA reaction plate respectively testing sample or positive and negative contrast and dilute with sample diluting liquid;

3). hatch: hatch 30 minutes with rearmounted 37 DEG C of shrouding film shrouding;

4). add enzyme marker with hole every after cleansing solution washing;

5). hatch: hatch 30 minutes with rearmounted 37 DEG C of shrouding film shrouding;

6). with cleansing solution washing, pat dry after rear every hole adds substrate A, the mixing of B liquid, the colour developing of 37 DEG C of lucifuges;

7). measure: every hole measures each hole OD value after adding stop buffer mixing.

Clonorchiasis diagnostic kit of the present invention is using glutathione transferase 2 as diagnostic antigen, employing enzyme linked immunosorbent assay (indirect method) detects the IgG4 antibody in clonorchis sinensis patients serum, and the incubation time of one antiserum and two antienzyme labels respectively needs 30 minutes.

Accompanying drawing explanation

Product-the time curve of different volumes restructuring GST2 is added in the catalytic activity detection of figure-1 recombinant protein GST2

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that example is not for limiting the scope of the invention.

Embodiment 1: the clone of glutathione transferase 2

By open reading frame (ORF) complete in the ORF FINDER program looks Unigene of NCBI, whether be full-length gene with BLASTx programmed decision, full-length gene is wherein known or new gene, the maximum ORF being numbered c003el0a is 639bp, we by it referred to as GST2.

1) the genomic extraction of clonorchis sinensis:

From cat liver, collect 10 clonorchis sinensis adults, PBS centrifugally removes supernatant after cleaning.Add the lysis buffer (STE, containing 2%SDS, 0.2mg/ml, Proteinase K) of 10 times of volumes after homogenate, mixing, 37 DEG C of water-baths are spent the night.Next day, solution is put to room temperature, add equal-volume balance phenol, on, under put upside down about 10min gently, the centrifugal 15min of room temperature 10000rpm, get upper strata aqueous phase, repeat extracting 2 times, get upper strata aqueous phase, add the 3mol/L sodium acetate (pH5.2) of 0.1 times of volume and the absolute ethyl alcohol of 2 times of volumes, mixing, the centrifugal 10min of 12000rpm, abandon supernatant, 70% ethanol washes twice, the centrifugal 5min of 12000rpm, abandon supernatant, after ethanol volatilization to the greatest extent, add 30 μ l TE (10mmol/LTris-HCI, 1mmol/L EDTA, pH8.0) dissolve, get 5ul 1% agarose gel analysis, remaining-20 DEG C, sample saves backup.

2) preparation of competent cell:

From culture plate, the mono-bacterium colony of picking BL21/DE3 enters in 5ml LB nutrient solution, and 37 DEG C 250 revs/min (rpm) joltings are spent the night.Getting 150ul next day adds in 3ml LB nutrient solution, and 37 DEG C of 250rpm joltings are to A ₆₀₀=0.6.Take out bacterium liquid, ice bath 30min, 4 DEG C, the centrifugal 5min of 4000rpm, abandons supernatant, adds the lime chloride 750 μ l of 0.1mol/L, ice bath 30min, 4 DEG C, the centrifugal 5min of 4000rpm, abandons supernatant, add the lime chloride 400 μ l of 0.1mol/L, packing 100 μ l/ manages, 4 DEG C preserve in 24h with or often pipe add-70 DEG C of preservations after 30% sterile glycerol mixing, use in 3 months.

3) amplification of GST2 gene and clone:

The amplification of 1.1 genes, the structure of prokaryotic expression plasmid and qualification

Respectively with Library plasmid cDNA and genomic DNA for template,

Upstream primer P1 is 5 ' GCGAATTCCACATGAAACACAGACACTGTG3 ', introduces two protectiveness bases and EcoR I restriction enzyme site;

Downstream primer P2 is 5 ' CGGTCGACGTTAATCGTCGCCACAGTC3 ', introduces two protectiveness bases and Sal I restriction enzyme site.

With TaKaRa Ex Taq ^tMthe reaction conditions of this gene of enzymatic amplification is as follows: 95 DEG C of denaturation 3min, 94 DEG C of sex change 1min, 61 DEG C of annealing 50sec, and 72 DEG C extend 1min, totally 30 circulations, and last 72 DEG C extend 8min.The agarose gel electrophoresis analysis verification of PCR primer 12g/L, obtaining molecular weight is the fragment of 639bp.PCR primer reclaims, and carries out EcoR I and Sal I double digestion, connects 18h with the prokaryotic expression carrier pET-30a (+) carrying out same double digestion by thing mass ratio 3-4:1 T4DNA ligase in 16 DEG C.Get 100 μ l BL21/DE3 competent cells, add above-mentioned connection product, gently ice bath 1h after mixing, 42 DEG C of water-bath 90sec, ice bath 5min, add LB nutrient solution 400 μ l, put into 37 DEG C of water-bath 2min after mixing, 37 DEG C of 150rpm shake 1h.All bacterium liquid paving to through preheating containing in the LB flat board of kanamycins, be inverted at 37 DEG C of incubators and cultivate 12-16h.Bacterium liquid containing positive plasmid is frozen for subsequent use with 30% sterile glycerol-70 DEG C.

Qualification: sequencing result shows, the gene order being cloned into the clonorchis sinensis glutathione transferase 2 of pET-30a (+) is SEQ ID NO.2.

AATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCG

GTTCTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGA

CGACAAGGCCATGGCTGATATCGGATCCGAATTCCACATGAAACACAGACACTGTGCGCTATTCTATTTCAACGTCCGT

GGCAGAGCTGAGGCGATTAGGATGGTACTGCACGCTAACGATGTTTCGTTTGAGGATGTGCGTTTCGATAAGGACCAAT

GGTCTGAACGCAAACACGAGTTTCCGGGTGGTAAATTACCGCTTCTCCAAGTAAGAGAAGAGGGATCACAAGAGAAGAA

GACTTATACAGAGAGCATGGCGATCGCCCGAGTGCTCGCAAAACACTACAGTATGATGGGCGATTCTGAAGAGGAATAT

TACAAGATTGAACGAATGATTGGGCAGTGTGCCGATTTGGATAAGGAGTTTGTCAATGTCTTTTTCGCACGAGAAGACC

AAAAGAAAGAAGTACTTGAGAAAGCAATGGGTGGAGAGGTGCCGAGACTACTGGAACTTATTTGCAAATCACTCTCTGA

ATCTGGTGGCAAGTTCGTCGCTGGTAACAAAGTAACTCTTGGAGACATATGCCTTATGGCATCCATGGAAAATGTACGG

AGAGCGGACCCTCAGCTTTTGAAAACGAAATATTCGACATTATTGGCCCTCGAGGCGGAGGTGTTCAAGGTCCTGCCGA

AACTTGCGGACTACGTCAAAACACGACCAGAAACCGTCCTGTGAAGCAGACTGTGGCGACGATTAACGTCGACAAGCTT

GCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGT

1.2CsGST2 gene is at the abduction delivering of e. coli bl21/DE3

With the BL21/DE3 containing empty plasmid pET-30a (+) for negative control.BL21/DE3 containing pET-30a (+)-CsGST2 of recombinant plasmid is inoculated in the nutrient culture media containing kanamycins, 37 DEG C, 250rpm, and shaken cultivation is transferred by 10ml/L after spending the night, and at 37 DEG C, 250rpm, shaken cultivation is to A ₆₀₀=0.6, take out 1ml as pre-induction sample, all the other bacterium liquid add IPTG (final concentration is 1mmol/L) Fiber differentiation 4-5h, take out 1ml as postinduction sample.All samples 4 DEG C, 8000rpm, 15min collected after centrifugation thalline, a little precipitation adds 5 μ l DTT, 95 μ l 1 × SDS, 30 μ l resets and add 2 μ l DTT, 8 μ l 4 × SDS, resuspended mixing, the centrifugal 1min of boiling water bath 5min, precipitation 13000rpm, supernatant is crawl slightly, get the capable SDS-PAGE of upper liquid 10 μ l (separation gel 150g/L, concentrated glue 50g/L), with coomassie brilliant blue staining, after observation has expression, albumen has expression in supernatant, without expressing in precipitation, carries out great expression and purifying.

1.3CsGST2 gene is at a large amount of abduction deliverings of e. coli bl21/DE3

37 DEG C, 250rpm, the bacterium liquid that shaken cultivation is spent the night is forwarded in 1000ml LB nutrient solution in the ratio of 20ml/L, and 37 DEG C, 250rpm, shaken cultivation is to A ₆₀₀=0.6, add IPTG (final concentration is 1mmol/L) Fiber differentiation 4-5h, with 4 DEG C, after 8000rpm, 15min are centrifugal, abandon most supernatant nutrient solution, collect thalline, add 1 × binding buffer liquid (5mM imidazoles+0.5M NaCl+20mM Tris-HCl, PH7.9) the resuspended mixing of 12-15ml complete, collect, be stored in-20 DEG C, next day ultrasonic degradation bacterium (power 150W, continue 1sec, stop 2sec, 15min altogether), 4 DEG C, 13000rpm, 15min, collect supernatant, 0.45 μm of filtering with microporous membrane.

1.4 affinitive layer purification albumen

Ni-NTA resin, the wash-out capable SDS-PAGE of GST2 albumen is out verified as destination protein by gradient concentration imidazoles 20mM, 30mM, 40mM, 60mM, 200mM and 400mM imidazoles, get purity high, measure large being collected together, (dislysate is 1 × PBS to load bag filter, PH7.4), 4 DEG C of dialysis 24h (changing liquid 2-3 time).Solution after dialysis 0.22 μm of filtering with microporous membrane, packing ,-80 DEG C save backup.

Qualification: protein sequencing result shows, the sequence that purifying obtains albumen (recombinant protein GST2) is SEQ ID NO.1.

MKHRHCALFYFNVRGRAEAIRMVLHANDVSFEDVRFDKDQWSERKHEFPGGKLPLLQVREEGSQEKKTYTESMAI

ARVLAKHYSMMGDSEEEYYKIERMIGQCADLDKEFVNVFFAREDQKKEVLEKAMGGEVPRLLELICKSLSESGGK

FVAGNKVTLGDICLMASMENVRRADPQLLKTKYSTLLALEAEVFKVLPKLADYVKTRPETVL。

1.5 glutathione transferase 2 determinations of activity

The research of recombinant protein catalytic activity

1). recombinant protein GST2's is quantitative:

Use Bradford method, be that standard is (see Bradford MM.A rapid and sensitivemethod for the quantitation of protein utilizing the principle of protein-dye binding.AnalytBiochem with bovine serum albumin(BSA), 1976,72:248-254).

2). the preparation of reagent:

CDNB absolute ethyl alcohol is mixed with 20mmol/L solution.GSH water is mixed with 20mmol/L solution.

3). reaction system is:

0.1mol/L sodium phosphate buffer (pH 6.5), GSH, CDNB, recombinant protein, reaction volume is 3ml, temperature of reaction is 25 DEG C (when adding the reagent of absolute ethyl alcohol preparation, should notice that the volume of absolute ethyl alcohol is no more than 1/4th of reaction cumulative volume) (see Habig WH, Pabst MJ, Jacoby WB.Glutathione S-transferase:the firstenzymatic mercapturic acid formation.J Biol Chem, 1974,249:7130-7139.).Blank 0.1mol/L sodium phosphate buffer, all incubation reaction do three pipes, average.When surveying maximum reaction velocity, make negative control with the reaction system only not adding recombinant protein, make positive control with the thick enzyme of clonorchis sinensis adult.With being first added with damping fluid, the reaction system (final concentration of GSH and CDNB is 1mmol/L) of GSH and CDNB hatches 5min in 25 DEG C of water-baths, adds different volumes recombinant protein and starts reaction, measure the A of 1-5min ₃₄₀.After selected suitable recombinant protein amount, negative control is made by the reaction system only not adding CDNB, the reaction system being added with GSH (final concentration 1mmol/L), recombinant protein and damping fluid is hatched 5min in 25 DEG C of water-baths, add variable concentrations CDNB and start reaction, survey the A at 25 DEG C of reaction 5min ₃₄₀, according to the mM extinction coefficient Δ e=9.6Lmmol of the product S-sweet peptide of (2,4-dinitro benzene) paddy skin (P) ^-1cm ^-1calculate the active unit of enzyme (see Habig WH, Pabst MJ, Jacoby WB.GlutathioneS-transferase:the first enzymatic mercapturic acid formation.J Biol Chem, 1974,249:7130-7139.).Michaelis constant Km according to Michaelis-Menten equation statistic software SPSS 13.0 using the inverse of concentration of substrate as independent variable, the inverse of speed carries out linear regression as dependent variable and calculates and obtain.

4). the catalytic activity of recombinant protein GST2

In 1-5min, add the recombinant protein GST2 of 1,5,10 μ l, generate product amount different, as shown in figure-1, react 5min when adding 5 μ l or 10 μ l GST2, the amount of product prolongation no longer in time and increasing, the active unit of GST2 is 22.76 ± 0.096 μm of olmg ^-1min ^-1.5 μ l GST2 react 5min A at 25 DEG C of catalysis variable concentrations CDNB ₃₄₀rising value in Table-1, the average Km that the same process data obtain GST2 is 111 μm of ol.

The A of 5 μ l GST2 catalysis variable concentrations CDNB ₃₄₀

Table-1

Embodiment 2: the physicochemical property of glutathione transferase 2

The glutathione transferase 2 of the affinitive layer purification that embodiment 1 is obtained, after enterokinase enzyme excision carrier sequence, through SDS-PAGE electrophoresis, and standard molecule discharge curve, record its molecular weight and be about 30kDa; Utilize Amerham isoelectric focusing electrophoresis instrument, through the isoelectric focusing electrophoresis of pH3-10 and pH7-10 gradient fixing glue, recording its isoelectric point is 6.97.The Stability Analysis of Structures of protein.

Embodiment 3: prepare diagnostic kit

The detection of clonorchis sinensis antibody before this adopts crude antigen or the antigen coated reaction plate of secretion excretion more, susceptibility, specificity are all not satisfactory, and the genetic engineering recombinant antigen GST2 that the present invention adopts embodiment 1 to obtain wraps by reaction plate, detect the IgG4 antibody in clonorchis sinensis patient body with indirect elisa method.

The assembling of kit:

1. the preparation (96 holes/plate) of reaction plate:

The clonorchis sinensis glutathione transferase-2 (GST2) of purifying embodiment 1 obtained is buffered liquid (pH9.6) with carbonate bag and is made into 5 μ g/ml, 96 hole reaction plates, every hole adds 100 μ l, wrap by rear room temperature yawing 30 minutes, 4 DEG C are spent the night, morning next day room temperature yawing after 30 minutes again, outwell coating buffer, pat dry, 0.5%BSA (w/v)+3% trehalose (w/v)+PBS (PH7.2) closes, 200 μ l/ holes, 4 DEG C close spend the night after outwell confining liquid.The liquid of falling deblocking, pats dry, and drains aluminium-foil paper and packages spare.

2. sample diluting liquid (11ml/ bottle):

0.1%BSA (w/v)+0.05% Tween-20 (v/v)+PBS (PH7.2), adds thimerosal 0.01% (w/v), 0.22 μm of membrane filtration.

3. blood sample positive and negative control treatment (0.10ml/ bottle):

Positive control:

Feces of Patients confirms there is worm's ovum through microscopy (excrement three is examined), and get venous patient whole blood room temperature static 2 hours, 4 DEG C are spent the night, 4000rpm, centrifugal 10 minutes, is separated supernatant-20 DEG C and saves backup, through the serum of ELISA experimental verification OD value 1.9-2.0;

Negative control:

Ight soil finds no worm's ovum through microscopy (excrement three is examined), static 2 hours of venous whole room temperature, and 4 DEG C are spent the night, 4000rpm, centrifugal 10 minutes, is separated supernatant-20 DEG C and saves backup, and through the serum of ELISA experimental verification OD value about 0.1.

Positive and negative contrast adds thimerosal 0.01% (w/v), and once, every bottle of 0.10ml packing is for subsequent use for 0.22 μm of membrane filtration.

4. enzyme marker thing (11ml/ bottle):

Anti-1: 1500 dilution of horseradish peroxidase-labeled anti-human igg 4 two, the packing of 11ml/ bottle.

5. concentrated cleaning solution (30ml/ bottle, 25 ×):

Tris-HCl damping fluid (PH7.8), with 0.22 μm of membrane filtration, used time adding distil water or deionized water do 25 times of dilutions.

6. stop buffer (6ml/ bottle):

2M H ₂SO ₄。

7. substrate solution A and substrate solution B all has the complete sale of commodity.

8. shrouding film:

3.

9. instructions:

1 part.

Embodiment 4: the use of kit

4.1 kits use step:

1). dosing: concentrated cleaning solution distilled water or deionized water are done 1: 25 dilution.

2). application of sample: add testing sample respectively or positive and negative contrast 5 μ l in respective aperture, then every hole adds 95 μ l sample diluting liquids.

3). hatch: hatch 30 minutes with rearmounted 37 DEG C of shrouding film shrouding.

4). washing: rinse 4 times with cleansing solution, pat dry.

5). enzyme-added: every hole adds enzyme marker 100 μ l.

6). hatch: hatch 30 minutes with rearmounted 37 DEG C of shrouding film shrouding.

7). washing: rinse 6 times with cleansing solution, pat dry.

8). colour developing: every hole adds substrate A, each 50 μ l of B liquid, and mixing of vibrating gently, 37 DEG C of lucifuges develop the color 10 minutes.

9). measure: every hole adds 50 μ l stop buffers, pats mixing.Setting microplate reader ripple is longer than 450nm place (suggestion uses double UV check), measures each hole OD value.

Serum dilution is 1: 20, two dilutabilitys resisted of enzyme labeling are 1: 1500, the incubation time of one antiserum and two antienzyme labels respectively needs 30 minutes, comparatively, the similar reaction time shortens half, improve efficiency, reaction conditions easily realizes (37 DEG C, leave standstill), this kit is operated more convenient.

4.2 kit sensitivity Detection tests:

Get 176 parts of microscopy positive serums, adopt mentioned reagent box to detect, final ELISA result is positive 161 parts, and positive rate is 91.50%.

4.3 kit specific detection tests:

Get 258 parts of negative serums, adopt mentioned reagent box to detect, ELISA result measures 233 parts of feminine genders, and negative recall rate is 90.31%.

Use kit described in the invention, method shown in by specification, carry out cross reaction experiment, result is as table-2:

Cross reaction experimental result (Results of cross-reaction)

Table-2

Conclusion: no matter kit of the present invention is that susceptibility or specificity are all better than other similar at present kits.Experimental result shows, this kit has high specificity, highly sensitive, accuracy high, the diagnosis of clonorchiasis can be applied to preferably, the main agents of this kit all adopts working fluid form, can directly use, more traditional etiological diagnosis method has very large advantage: operate accurate, easy, credible result.

Claims

1. a clonorchiasis diagnostic kit, comprise: ELISA reaction plate, negative control, positive control, enzyme marker, substrate A liquid, substrate B liquid, sample diluting liquid, concentrated cleaning solution and stop buffer, it is characterized in that, described ELISA reaction plate is coated with clonorchis sinensis glutathione transferase 2, described clonorchis sinensis glutathione transferase 2 is polypeptide of the amino acid sequence with SEQ ID NO.1, and described enzyme marker is that horseradish peroxidase-labeled anti-human igg 4 two resists.

2. clonorchiasis diagnostic kit as claimed in claim 1, is characterized in that, described sample diluting liquid by percent weight in volume be 0.1%BSA, percent by volume is that the PBS mixed preparing of 0.05% Tween-20 and pH7.2 forms.

3. clonorchiasis diagnostic kit as claimed in claim 2, is characterized in that, also containing antiseptic in described sample diluting liquid.

4. clonorchiasis diagnostic kit as claimed in claim 1, it is characterized in that, described concentrated cleaning solution is the Tris-HCl damping fluid of pH7.8.

5. clonorchiasis diagnostic kit as described in as arbitrary in claim 1-4, is characterized in that, also comprise shrouding film and instructions in described clonorchiasis diagnostic kit.

6. as described in as arbitrary in claim 1-5, the using method of clonorchiasis diagnostic kit, comprises the following steps:

1). by concentrated cleaning solution dilution preparation cleansing solution;

2). add in the respective aperture of ELISA reaction plate respectively testing sample or positive and negative contrast and dilute with sample diluting liquid;

3). hatch with rearmounted 37 DEG C of shrouding film shrouding;

4). add enzyme marker with every hole after cleansing solution washing ELISA reaction plate;

5). hatch with rearmounted 37 DEG C of shrouding film shrouding;

6). add substrate A, the mixing of B liquid, 37 DEG C of lucifuge colour developings with every hole after cleansing solution washing ELISA reaction plate;

7). every hole measures each hole OD value after adding stop buffer mixing.

7. the using method of clonorchiasis diagnostic kit as claimed in claim 6, it is characterized in that, the incubation time in step 3 and 5 only needs 30 minutes.