CN110511282A

CN110511282A - A kind of bifunctional protein SPG-ALP and its ELISA detection kit for enzyme-labeled antibody

Info

Publication number: CN110511282A
Application number: CN201910792463.9A
Authority: CN
Inventors: 朱传刚; 纪荣毅; 荆怡; 沈元曦; 林矫矫; 洪炀; 马以桐
Original assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Current assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center; Shanghai Veterinary Research Institute CAAS
Priority date: 2019-08-27
Filing date: 2019-08-27
Publication date: 2019-11-29

Abstract

The present invention provides a kind of bifunctional protein SPG-ALP and its ELISA detection kit for enzyme-labeled antibody, measures ox, sheep, rabbit sample using ELISA kit of the invention, it was demonstrated that several species serodiagnosis is feasible.Kit preparation process of the invention is simple, easy, has many advantages, such as that high sensitivity, Stability and veracity are good.

Description

A kind of bifunctional protein SPG-ALP and its ELISA detection examination for enzyme-labeled antibody Agent box

Technical field

The present invention relates to protein detection fusion protein and protein detection methods, in particular to a kind of to be used for abzyme Target bifunctional protein SPG-ALP, further to the ELISA detection kit for containing above-mentioned bifunctional protein SPG-ALP.

Background technique

Currently, enzyme-linked immunosorbent assay (ELISA) is an important technology in immunology diagnosis, not only apply The immunology diagnosis of infectious disease, parasitic disease and non-infective disease etc. caused by multiple pathogenic microorganisms, is also applied to The measurement of molecular antigen and antibody, application range are growing, and have been increasingly becoming the master of 21 century field of biomedicine research Stream.The essential characteristic of existing ELISA kit is, probe card be fixed in a random distribution on internal surface of hole it is anti- Former or antibody plastics or glass microporous plate, tagging system contain the antigen that enzyme marks or antibody or antiantibody and substrate, Using being in test sample antibody corresponding with immobilization probe or antigen.It is different in tagging system on root Ju probe card Ag-Ab combination, ELISA method can be subdivided into indirect method, double antibody sandwich method and dual-antigen sandwich method etc..For example, confrontation Primordial covering plate (on probe card fixed be specific antigen), target antibody and antigen of probe when detection in sample occur special Property combine, if the enzyme mark object in tagging system be enzyme mark specific antigen if be dual-antigen sandwich method, if in tagging system Enzyme mark object is that enzyme mark antiantibody is then indirect method；Target if fixed on probe card is specific antibody, when detection in sample Specific reaction occurs for the antibody probe on antigen and probe card, and the marker in tagging system is that enzyme mark specific antibody is then double Antibody sandwich.Regardless of that method, after washing away unbonded enzyme mark object, the substrate solution in tagging system, colour developing inspection is added 0D value is surveyed, the target antibody or target antigen of combination can qualitatively or quantitatively be detected.

The structure of streptococcus protein G (SPG) is since N sections, and there are three homologous structure region A1, A2, A3, each homologous knots Structure is all made of 24 amino acid, while these three homologous structures are separated by homology region B1, B2 that 51 amino acid form, and are connect Be an interval region S, homologous structure area C1, C2, C3 of 55 amino acid composition are followed by, again by D1 and D2 between them Area is separated, and is hydrophilic region W after the area C3, is finally the area M (Sjobring, 1991).Some researches show that three of SPG are same Amino acid sequence C1, the C2 in source are related to the combination at the end C3 domain antibody IgG Fc, and the area C1 and C2 only has 2 amino acid sequences Column are different, and there are 6 amino acid sequence differences in the area C1 with C3, and the binding ability of the area C3 and IgG antibody is equivalent to 7 times of the area C1 (Kobatake, 1990).And the area A, B of SPG then has the activity in conjunction with Antibody Fab fragment and in conjunction with seralbumin, It is believed to play the role of the problem of interfering antibody and antigen and bringing nonspecific reaction.

Alkaline phosphatase (alkaline phosphatase, ALP) is non-specific phosphomonoesterase, can be catalyzed almost The hydrolysis of all phosphate monoesters generates inorganic phosphate and corresponding alcohol, phenol, sugar etc., can be with catalytic phosphatase group Transfer reaction.ALP is present in addition to higher plant in almost all of organism, can directly participate in phosphorus metabolism, disappears in calcium, phosphorus Change, absorb, played an important role in secretion and ossific process.

G-protein is marked using enzyme obtained by combining the enzymes such as alkaline phosphatase and G-protein by chemical reaction, to replace marking Remember secondary antibody.G-protein is the protein from streptococcic cell wall, is had in conjunction with the IgG of nearly all mammal Property.If using such enzyme mark G-protein, can with an anti-binding of panimmunity kind, even if to plurality of target In the case that substance (antigen) is detected, the antibody specifically bound respectively with each substance can not also be prepared, versatility is high. But there is a problem of that detection sensitivity is low using the method for the enzyme label G-protein marked with enzymes such as alkaline phosphatases.In addition, There is also the types of thermal stability difference, inactivate in processes sometimes.Alkali phosphatase enzyme mark antibody or G-protein isoreactivity substance Method, be related to chemical coupling, purified after coupling, dialyse, concentration etc. tedious steps；And due to large biological molecule itself Feature, the binding site multiplicity of coupling, so that the product for obtaining coupling complicates, it is difficult to guarantee the unification of product, it is not only possible to The inactivation for causing enzyme, in conjunction with large biological molecule inactivation, and influence stability.And ELISA detection in ELIAS secondary antibody spy The opposite sex combines problem to expect to solve.For the primary antibody specifically bound with target substance (antigen), need to specifically bind with it Label secondary antibody, when detecting plurality of target substance (antigen), need prepare respectively with a variety of primary antibodies specific binding label Secondary antibody, poor universality.

Summary of the invention

It is real the invention mainly solves the technical problem of providing a kind of quick, highly sensitive immune detection product and method Now efficient, highdensity immune detection, and the specific antibody of a variety of domestic animals and people can be detected.

The characteristics of in order to realize that above-mentioned technical proposal, the present invention provide a kind of fusion protein, be based on the area SPG C3 and ALP, This two sections of genes are connected, one kind is reconstructed and has IgG combination activity and ALP concurrently in conjunction with active bifunctional protein SPG- ALP。

Above-mentioned bifunctional protein SPG-ALP, including streptococcus protein G (SPG) segment and ALP albumen, the streptococcus egg White G(SPG) segment be the C3 section containing SPG sequence；

Wherein, the catenation sequence between streptococcus protein G (SPG) segment and ALP albumen is as shown in SEQ ID NO.8

The nucleotide sequence of streptococcus protein G (SPG) segment is as shown in SEQ ID NO.6；

The nucleotide sequence of ALP albumen is as shown in SEQ ID NO.7；

The nucleotide sequence of the bifunctional protein SPG-ALP is as shown in SEQ ID NO.5；

Codon optimization is carried out to SPG and ALP sequence, expressing quantity improves after splicing, and solubility increases.

Present invention simultaneously provides a kind of product for immunoassay, the product includes fusion protein of the present invention SPG-ALP。

Preferably, the present invention provides a kind of kit containing above-mentioned detection albumen, and the kit includes:

ELISA Plate；

The recombinant protein of enzyme label: SPG-ALP；

Dilution: PBST；

Confining liquid: 5% skimmed milk power；

Cleaning solution: PBST；

Positive control: Schistosoma japonicum positive serum；

Negative control: Schistosoma japonicum negative serum；

Developing solution: PNPP developing solution；

Terminate liquid: 2M H₂SO₄。

In addition, the present invention also provides a kind of answering in immunoassay of the present invention based on fusion protein S PG-ALP With.

Beneficial effect

(1) the IgG binding fragment of SPG gene is reconstructed the present invention, and only retaining Protein G can be with the Fc terminal specific of IgG antibody Property the area C3 that combines, and connect ALP, it is active difunctional that there is the recombinant protein of preparation IgG activity and ALP to be combined to combine.

(2) fusion protein S PG-ALP of the invention can efficient, highdensity capture target molecule, reach quick, highly sensitive Detection.

(3) fusion protein S PG-ALP preparation process of the invention is simple, easy, is applicable to different immune detection moulds Formula, such as can be used for indirect ELISA, sandwich ELISA, immuno-PCR, one of original detection method reagent is only replaced, Do not change original operating procedure, does not need additional equipment and instrument.

Detailed description of the invention

The structural schematic diagram of Fig. 1 SPG.

Alkaline phosphatase (ALP) reaction mechanism of Fig. 2 Escherichia coli

The double digestion of Fig. 3 recombinant plasmid is identified, wherein M: nucleic acids marker；1: complete sequence segment；2: double digestion cuts ALP sequence Column, running cementing fruit is that ALP segment and complete plasmid cut the segment left after ALP segment.

Fig. 4 carries out PCR identification, wherein M: nucleic acids marker to the prokaryotic expression plasmid of recombinant protein；C3: add in system The primer sequence entered is primer sequence designed by C3 segment both ends；ALP: the primer sequence being added in system is ALP segment two The designed primer sequence in end；Complete: the primer sequence being added in system is primer sequence designed by SPG-ALP segment both ends.

Fig. 5 SDS-PAGE analyzes pET-28a(+)-SPG-ALP expression, wherein M: protein marker；0h: nothing IPTG induction；1h-6h:IPTG induces 1h, 2h, 4h, 6h.

Fig. 6 recombinant protein reacts vitality test result ALP: recombinant protein SPG-ALP with pNPP

Fig. 7 Western blotting detects SPG-ALP albumen and the combination activity of different plant species IgG.

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 It is commercially available from routine biochemistry reagent shop.

The building of 1 recombinant protein G of embodiment

1.1 biomaterial

Bacillus coli BL21 is purchased from Nanjing Vazyme Biotechnology Co., Ltd.；Plasmid pET-28a (+) is that laboratory is protected It deposits, the ALP and SPG of codon optimization are that laboratory saves ([1], [2]).

[1] Xu Rui, Zhao Dengyun, Hong Yang, Lu Ke, Li Hao, woods are rectified, Feng Jintao, Xu Yumei, Zhu Chuangang streptococcus protein G Structural domain reconstruct, expression and identification [J] China zoonosis journal, 2015,23 (05): 46-52.

[2]E. coli alkaline phosphatase gene, complete cds。 GenBank: M13345.1

The building and synthesis of 1.2 recombinant protein SPG-ALP

Drawn respectively in upstream and downstream according to the C segment and ALP gene order of spG using Primer5 software Design primers Restriction enzyme site is added in object, primer sequence is as shown in table 1.

1 amplimer of table

Have the escherichia coli DH5a bacterial strain for saving and containing C3 segment as template using laboratory, above-mentioned C3 upstream and downstream primer into Row PCR amplification obtains C3 gene order.

ALP sequence after optimization is as shown in SEQ ID NO.7, wherein carrying out signal peptide analysis, function distinguishing to ALP sequence Analysis etc., obtains the basic condition of the gene.Selection represents the end of gene order 3 ' the addition TAA terminator codon of enzyme function.Detection It is uniformly substituted for the password that coding is had a preference for the Escherichia coli of monoamino-acid by the Escherichia coli rare codon of gene order Son finally obtains new gene order and is inserted into PET-28-a plasmid using the area ALP upstream and downstream primer PCR amplification, converts DH5a It is identified.(ALP sequence is the synthesis of Jin Weizhi company)

After above-mentioned plasmid identification is correct, the C3 segment both ends of PCR amplification are cut out into cohesive end with restriction endonuclease, are contained There is the PET28-a plasmid of ALP sequence to cut out with the notch that can be connected with the cohesive end of C3 segment, reaction system is as follows:

37℃ 4h

In order to make the target fragment in system keep preferable state, two reaction systems are carried out changing system

(1) plus TE supplies 100 microlitres, then plus 400 microlitres of Solition SN(Promega kits Binding Buffer Instead of) and 100 microlitres of Solution B, it mixes.

(2) adsorption column is put into EP pipe, liquid is transferred on adsorption column, uncapped and be stored at room temperature 2min.It closes the lid, 10000rpm room temperature is centrifuged 1min, abandons waste liquid.

(3) 600 microlitres of Wash Solution, 10000rpm room temperatures are added and are centrifuged 1min, abandon waste liquid, this step repeats one It is secondary.

(4) 10000rpm room temperature sky is from 2min, the alcohol that volatilizees of uncapping.(about 5 ~ 10min)

(5) adsorption column is transferred to a new EP to manage, adds water (30 microlitres) in column film center, uncaps, be stored at room temperature 2 ~ 5min.It (mentions High wash temperature helps to improve the elution efficiency of DNA)

(6) 10000rpm high speed centrifugation 1min, centrifugation liquid in pipe is the DNA fragmentation recycled, can be used immediately or -20 ° It is spare.

Connection

(1) DNA fragmentation of plasmid vector DNA PET-28a(+) and insertion are prepared by mixing into the DNA solution of 5 ~ 10ul.Use TE Buffer(10mM Tris-HCL, 1mM EDTA, pH8.0), dissolving DNA.The mole ratio of carrier DNA and the DNA of insertion are general For 0.03pmol:0.03 ~ 0.3pmol.

(2) Solution 1 of isometric (5 ~ 10ul) is added in Xiang Shangshu DNA solution, mixes.

(3) 16 ° of reaction 30min.

(4) reaction can be directly used for bacterium conversion, utilize LB culture medium (K+) screening and identification positive plasmid.

The identification of 1.3 recombinant plasmids

The SPG-ALP sequence of reconstruct is carried out to carry out PCR reaction, reaction system is as follows:

Component	Institute's expense (μ l)
		TaKaRaTaq™ Hot Start Version	25
Gene order	2
		Upstream primer (10 μM) SEQ ID NO.1	2
Downstream primer (10 μM) SEQ ID NO.4	2
		ddH2O	19
Total	50

After each component is mixed, of short duration centrifugation, which is placed in PCR instrument, is reacted, and response parameter is as follows:

Temperature	Reaction time
		94℃	3min
94℃	30s
		56℃	30s 30 cycles
72℃	1min
		72℃	10min
4℃	infinite

Electroresis appraisal (Fig. 4) and double digestion identification (Fig. 3) are carried out to PCR product, sequencing qualification result shows the sequence with design Unanimously.The full clip size of target gene is about 1518bp.C3 clip size is about 165bp, and ALP clip size is about 1353bp.

(C3 segment corresponds to upstream and downstream primer: SEQ ID NO.1,2；ALP corresponds to upstream and downstream primer: SEQ ID NO.3,4； Full sheet section corresponds to upstream and downstream primer: SEQ ID NO.1,4)

The expression and purifying of 2 recombinant protein of embodiment

The expression of 2.1 recombinant plasmids

(1) picking is inoculated in the LB liquid medium of 5ml and 150ml containing Kan+ respectively in right amount containing the BL21 of target fragment, It is placed in 37 DEG C of concussion and cultivate cases, 250rpm shake culture.

(2) when growing to logarithmic phase (when OD600 is about 0.6), the IPTG that final concentration of 1mmol/L is added carries out induction table It reaches.1h, 2h, 4h, 6h take 0.5ml bacterium solution respectively before inducing expression, after inducing expression, best using SDS-PAGE electrophoretic analysis Induction time.

(3) the bacterium solution 12000rpm centrifugation 20min after induction is abandoned into supernatant, precipitating is resuspended with 1 × PBS of 20ml, is frozen repeatedly After melting three times, ice-bath ultrasonic be crushed the super 2s of 20min(every 9s) afterwards 12000rpm be centrifuged 15min, collect precipitate and supernatant.

(4) the 5ml 8mol urea of the precipitating after centrifugation is resuspended, repeats above-mentioned centrifugation step, collect supernatant.

(5) supernatant after supernatant, precipitating after ultrasound being resuspended, is separately added into isometric protein electrophoresis buffer, uses SDS- The solubility of PAGE electrophoretic analysis expression product.

SDS-PAGE is analysis shows that (Fig. 5), recombinant plasmid pET-28a(+)-SPG-ALP success in e. coli bl21 (DE3) Expression, and 1-6h expression quantity increases with the growth of time after 1mmol/L IPTG induction, and expression quantity after 2h is induced to reach one Determine height and later period incrementss are unobvious.

The purifying of 2.2 recombinant proteins

(sequence number: 70666-3) is purified to recombinant protein using Ni-NTA Hisbind Resin, is illustrated according to kit Book is operated, and steps are as follows for ease of Use:

(1) it takes 5ml resin to be added in new void column and stands balance, when liquid level drops to resin surface, successively follow the steps below；

(2) 2CV ddH is used₂O*2, charge buffer * 3, binding buffer*2;

(3) 50ul is stayed before loading；

(4) 3CV Binding Buffer*3 (just clear under 50ul plus needing to stay)；

(5) 3CV Wash Buffer*2, it is same to stay 50ul;

(6) 2CV Elution Buffer*2, it is same to stay 50ul;

(7) a small amount of Strip Buffer (3ml), loading, is stayed entirely repeatedly, same to take 50ul.

(8) solution of above-mentioned collection is subjected to SDS-PAGE electrophoresis, analyzes protein purification situation.

The dialysis of 2.3 recombinant proteins

(1) protein content that basis is finally collected into, the bag filter of clip suitable length, water-bath, 80 DEG C or more, 5min.

(2) prepare 2L PBS dialyzate, bag filter is carefully transferred in dialyzate, with clamp both ends, leak detection.

(3) purified albumen is added in the clip for removing one end, drives bubble out of as far as possible, then use clamp.

(4) be put into lesser trochanter in dialyzate, be placed on magnetic stirrer, slowly run, it is ensured that liquid in flowing, It may cause protein crystal if revolving speed is too big.

(5) after for 24 hours, albumen is carefully transferred in a new centrifuge tube, -20 DEG C, is saved backup.

SDS-PAGE analysis shows that, recombinant plasmid pET-28a(+)-G-ALP expression albumen ultrasonic supernatant precipitating in Exist, the protein content in precipitating is higher than the protein content in supernatant, shows that the albumen has certain water solubility.Recombinate egg Bai Chaosheng supernatant after purification, after Strip Buffer elution, is purified by Ni-NTAHisbindResin.

The enzyme activity of 3 recombinant protein SPG-ALP of embodiment

3.1 PNPP developing solution no-PNPP are prepared

Component	Institute's expense (g)
		Glycine	7.51
MgCl₂	0.203
		ZnCl₂	0.136

Deionized water is settled to 1L, adjusts PH to 7.9.It is aobvious to be made into PNPP for addition 1mg PNPP solid powder in every 1ml developing solution Color liquid, it is ready-to-use.

The alkaline phosphatase activity of enzyme reaction of 3.2 ELISA method detection recombinant proteins.

Reaction content is added by following scheme in elisa plate, and 37 DEG C are protected from light, and takes out the OD value at measurement 405nm.

ELISA method reacts recombinant protein SPG-ALP with PNPP, measures the alkaline phosphatase activity of recombinant protein, As a result as shown in Figure 6.Should be the results show that ALP catalysis PNPP develop the color, and exclusion developing solution self decomposes possibility and recombinant protein Self color influences final result possibility.Display reaction has significant difference, the enzyme mark alkalinity phosphorus with commercialization with control group The OD value indifference of the chromogenic reaction of sour enzyme.

4 SPG-ALP recombinant protein activity identification of embodiment

4.1 Western blotting detect recombinant protein and the combination activity of IgG

(1) albumen after purification is subjected to SDS-PAGE electrophoresis, later by protein delivery to NC film, 140mA, 90min.

(2) NC film is soaked in diluted 5% skimmed milk power of PBST, room temperature closes 2h.

(3) the NC film after closing is washed three times with PBST, each 5min.

(4) goat anti-mouse IgG for marking NC film with HRP, rabbit-anti goat IgG, mouse anti-rabbit IgG, the anti-goat of donkey IgG uses PBST 1:2000 to dilute) it is not consistent as antibody with figure, it is incubated at room temperature 1h.

(5) the NC film after incubation is washed three times with PBST, each 10min.

(6) NC film is developed the color with DAB two-component colour developing liquid kit, is rinsed after colour developing with flowing water, terminates reaction.

The results show that recombinant protein and mouse, sheep, donkey, rabbit antibody all have in conjunction with active (Fig. 7).

4.2 ELISA methods measure recombinant protein and different plant species IgG affinity costant

(1) with BCA method measurement recombinant protein concentration, and use commercialization standard protein G(SPG) as compare.

(2) albumen is subjected to doubling dilution since 10 μ g/ml with coating buffer, carries out 8 dilutions altogether, it is every with 100 μ l Hole is coated on 96 orifice plates, and 3 repetitions are arranged in each concentration, and 4 DEG C of coatings are overnight.

(3) 96 orifice plates are washed three times with PBST, the 200 every holes μ l, each 5min.

(4) solution for using diluted 5% skimmed milk power of PBST, the 150 every holes μ l, 37 DEG C of closing 2h are added.

(5) 96 orifice plates are washed three times with PBST, the 200 every holes μ l, each 5min.

(6) by the mountain sheep anti mouse of HRP label, rabbit-anti goat, mouse anti-rabbit, the anti-goat of donkey these four secondary antibodies, PBST is used respectively Doubling dilution, the 100 every holes μ l, 37 DEG C of incubation 2h are carried out by 1:1000,1:2000,1:4000,1:8000.

(7) 96 orifice plates are washed three times with PBST, the 200 every holes μ l, each 5min.

(8) TMB is added to develop the color, the 100 every holes μ l react at room temperature 15min.

(9) 2mol/L H is added₂SO₄Reaction is terminated, OD450 value is read in the 30 every holes μ l.

Recombinant protein after dialysis is measured into concentration respectively, different concentration is used to be coated with 96 orifice plates respectively as antigen, it The secondary antibody of doubling dilution is added in plate afterwards, with antigen binding.Using OD450 value as ordinate, with the logarithm of antigen concentration Value is used as abscissa, carries out curve fitting.

It according to the curve of fitting, substitutes into corresponding formula, calculates separately affinity costant Ka, take it to put down obtained Ka value Mean obtains the affinity costant value of recombinant protein, wherein the affinity costant of recombinant protein SPG-ALP and rabbit, donkey, mouse, goat Respectively rabbit: 5.41*10^4, donkey: 4.35*10^4, mouse: 3.76*10^4, goat: 7.48*10^4.

Recombinant protein SPG-ALP and rabbit, donkey, mouse, goat IgG all have in conjunction with activity as the result is shown, have several species IgG Binding ability.Albumen has the function of the area C on SPG after recombination, and the albumen of recombination does not influence its former protein function.

5 recombinant protein SPG-ALP of embodiment detects antibody of Schistosoma japonicum in serum

(1) schistosoma japonice ovum soluble antigen is prepared, is diluted to end with carbonate buffer solution dilution worm's ovum soluble antigen Concentration 10ug/ml.Every hole 100ul is coated in ELISA Plate, and overnight, PBST is washed 3- times 4 DEG C of coatings, each 5min；

(2) 5% skimmed milk power, every hole 200ul are prepared with PBST, 37 DEG C of 2h, PBST are washed 3-5 times, each 5min；

(3) ox Schistosoma japonicum yin and yang attribute serum 1:100 dilutes, every hole 80ul, 37 DEG C of 2h, PBST washing 3-5 times, every time 5min；

(4) by recombinant protein SPG-ALP with 0.1mg/ml, every hole 60ul, 37 DEG C of incubation 2h, PBST wash 3-5 times, every time 5min。

(5) the every hole 100ul of PNPP developing solution, 2M H₂SO₄Every hole 50ul terminates reaction, measures A_562nm, arrange result.

With above-mentioned experimental procedure measurement ox, sheep, each 10 parts of serum Schistosoma japonicum positive serums of rabbit and ox, sheep, rabbit standard Negative serum, serum 1:100 dilution, measures A562nm numerical value, detects the detection feasibility of the several species serum of this method.As a result Such as table 1, rabbit, ox, sheep Schistosoma japonicum positive serum detection readings are above 0.21, and standard female serum testing result is lower than 0.1, positive serum P/N value is all larger than equal to 2.1, prompts, it is feasible to do several species serodiagnosis in this way.

The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention. Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention Within protection scope.

Table 1, the detection of several species serum

<110>China Agriculture Academe Shanghai Veterinary Institute (China Animal Health and Epidemiology Center, branch center, Shanghai)

<120>a kind of bifunctional protein SPG-ALP and its ELISA detection kit for enzyme-labeled antibody

<160> 8

<170> PatentIn version 3.5

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<211> 19

<212> DNA

<213>area C upstream primer

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<211>21

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<400> CCG GAA TTC TGA GCC CCC ACC

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<213>area ALP upstream primer

<400> CCGGAA TTCCCT GTT CTG

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<400> GGC CTC GAGTTA TTT CAG CCC

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<210>6

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<210>7

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<212> DNA

<213>nucleotide sequence of recombinant protein A LP segment after optimizing

<400> GAA TTC CCT GTT CTG GAA AAC CGG GCT GCG CAA GGC GAT ATT ACG GCC CCG GGC GGC GCC CGT CGT CTG ACG GGT GAT CAA ACC GCG GCG CTG CGT GAT AGT CTG AGC GAT AAG CCG GCG AAG AAC ATC ATT CTG CTC ATT GGC GAT GGC ATG GGC GAT AGC GAG ATC ACC GCC GCG CGT AAT TAT GCC GAA GGC GCC GGC GGC TTT TTC AAA GGT ATC GAC GCG CTG CCA CTG ACC GGC CAA TAC ACC CAC TAC GCG CTG AAC AAG AAG ACC GGC AAG CCG GAC TAT GTG ACG GAT AGT GCC GCC AGT GCC ACC GCG TGG AGT ACC GGC GTT AAA ACC TAC AAT GGC GCG CTG GGC GTG GAC ATC CAC GAG AAA GAC CAC CCG ACC ATT CTC GAA GCG AAA GCG GCG GGT CTG GCG ACG GGT AAT GTT AGC ACC GCG GAA CTG CAA GAT GCC ACC CCA GCC GCG CTG GTT GCG CAT GTT ACG AGC CGC AAA TGT TAT GGC CCG AGT GCG ACG AGT GAG AAA TGC CCG GGT AAC GCG CTG GAA AAG GGC GGT AAA GGC AGT ATT ACC GAA CAG CTG CTG AAT GCC CGT GCC GAC GTT ACG CTC GGT GGT GGT GCG AAA ACG TTC GCC GAA ACG GCG ACC GCG GGT GAG TGG CAA GGT AAA ACG CTC CGC GAA CAA GCG CAA GCC CGT GGT TAC CAA CTG GTT AGT GAT GCG GCC AGT CTG AAC AGC GTT ACG GAG GCC AAT CAG CAA AAA CCG CTG CTG GGT CTG TTC GCG GAC GGT AAT CCG GTT CGC TGG CTG GGC CCG AAA GCC ACG TAC CAC GGT AAC ATC GAC AAA CCA GCC GTG ACG TGC ACC CCG AAT CCG CAA CGC AAT GAC AGT GTT CCG ACC CTC GCG CAG ACG GAT AAA GCG ATC GAG CTG CTG AGC AAG AAC GAG AAA GGC TTC TTT CTG CAA GTT GAA GGT GCG AGC ATC GAC AAA CAA GAT CAT GCG GCC AAT CCG TGT GGC CAG ATT GGT GAG ACG GTG GAT CTG GAT GAA GCC GTT CAG CGC GCG CTG GAA TTT GCG AAG AAA GAG GGC AAC ACG CTG GTG ATT GTG ACC GCC GAT CAT GCG CAC GCC AGT CAA ATT GTT GCC CCG GAT ACC AAA GCG CCG GGT CTG ACC CAA GCG CTG AAT ACC AAA GAC GGT GCC GTG GTT AGC TAT GGC AAC AGC GAA GAG GAT AGC CAA GAA CAT ACG GGC AGT CAG CTG CGT ATC GCC GCC TAT GGT CCG CAT GCG GCG AAT GTT GTT GGT CTG ACC GAT CAG ACC GAT CTG TTT TAT ACC AAA GCC GCT CTG GGG CTG AAA TAA CTC GAG

<210>8

<211>30

<212> DNA

<213>nucleotide sequence of catenation sequence

<400>GGC GGT GGG GGC TCA GGA GGT GGG GGC TCA

Claims

1. a kind of product for immunoassay, the product includes bifunctional protein SPG-ALP, and the form of product can be spy Needle (sensor), test strips, chip, kit etc.；The bifunctional protein SPG-ALP, it is characterised in that the difunctional egg White SPG-ALP has IgG concurrently and activity and ALP is combined to combine activity, including streptococcus protein G (SPG) segment and ALP albumen, described Streptococcus protein G (SPG) segment is the sequence of the C3 section containing SPG, it is preferred that streptococcus protein G (SPG) segment and ALP Catenation sequence between albumen is as shown in SEQ ID NO.8；The nucleotide sequence of streptococcus protein G (SPG) segment is such as Shown in SEQ ID NO.6；The nucleotide sequence of ALP albumen is as shown in SEQ ID NO.7, it is furthermore preferred that the bifunctional protein Nucleotide sequence as shown in SEQ ID NO.5.

2. product as described in claim 1, the product is kit, the kit may also include buffer, cleaning solution, Dilution or color developing agent etc..

3. product as claimed in claim 2, the kit include:

ELISA Plate；

The recombination double functions Protein S PG-ALP of enzyme label；

Dilution: PBST；

Confining liquid: 5% skimmed milk power；

Cleaning solution: PBST；

Positive control: Schistosoma japonicum positive serum；

Negative control: Schistosoma japonicum negative serum；

Developing solution: PNPP developing solution；

Terminate liquid: 2M H₂SO₄。

4. application of the product described in claim 1-3 in immunoassay.

5. the method for antibody of Schistosoma japonicum, described in a kind of detection serum using the non-diagnostic purpose of recombinant protein SPG-ALP Method includes the following steps:

(1) schistosoma japonice ovum soluble antigen is prepared, is diluted to end with carbonate buffer solution dilution worm's ovum soluble antigen Concentration 10ug/ml；Every hole 100ul is coated in ELISA Plate, and overnight, PBST is washed 3- times 4 DEG C of coatings, each 5min；

(4) by recombinant protein SPG-ALP with 0.1mg/ml, every hole 60ul, 37 DEG C of incubation 2h, PBST wash 3-5 times, every time 5min；