CN110511283A - A kind of detection kit with the active detection albumen of green fluorescence - Google Patents

A kind of detection kit with the active detection albumen of green fluorescence Download PDF

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CN110511283A
CN110511283A CN201910792893.0A CN201910792893A CN110511283A CN 110511283 A CN110511283 A CN 110511283A CN 201910792893 A CN201910792893 A CN 201910792893A CN 110511283 A CN110511283 A CN 110511283A
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protein
antibody
detection
egfp
schistosoma japonicum
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CN110511283B (en
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朱传刚
沈元曦
纪荣毅
林矫矫
洪炀
岳永程
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Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
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Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
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    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

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Abstract

The present invention provides a kind of antibody of Schistosoma japonicum detection kit with the active detection albumen of green fluorescence, includes a kind of antibody of Schistosoma japonicum detection reagent item and fusion protein in the antibody of Schistosoma japonicum detection kit；The fusion protein includes streptococcus protein G (SPG) segment and fluorescin.In antibody of Schistosoma japonicum detection fluorescent test paper strip of the invention, schistosoma japonice ovum soluble antigen (SEA) and streptococcus protein G (SPG) albumen are respectively as detection line (T), the envelope antigen of nature controlling line (C).Test strips select nitrocellulose filter (CN95) to be used as chromatographic material, and glass fibre element film is assembled into test strips as sample pad, with PVC bottom plate and blotting paper etc. together.

Description

A kind of detection kit with the active detection albumen of green fluorescence
Technical field
The present invention relates to field of immunodetection, in particular to a kind of detection examination with the active detection albumen of green fluorescence Agent box.
Background technique
Schistosoma japonicum is the parasitics flat allosome fluke of infecting both domestic animals and human, and Schistosoma japonicum has the complicated history of life, Widely distributed intermediate host and a variety of propagation or reservoir host seriously endanger global human and livestock health and affect various countries' herding Industry development, is one of five big parasitic diseases of the long-term keypoint control in China.The Deterioration mechanism of Schistosoma japonicum is not fully understood, But the pathogenic factors such as existing research discovery worm's ovum deposition, polypide stimulate, the proteantigen of miracidium glandular secretion, which can cause, posts Various lesions (Garcia EG, Mitchell GF, Rivera PT, Evardome RR, the Almonte RE, Tiu at raw position WU. Evidence of anti-embryonation immunity and egg destruction in micesensitized with immature eggs of Schistosoma japonicum.Asian Pacific Journalof Allergy&Immunology,1987,5:137)。
Streptococcus protein G (SPG) is can a kind of streptococcal cell wall egg in conjunction with the IgG antibody of people and many animals It is white, it was reported earliest by Kronvall in 1973.A, contain the egg in the cell wall of C, G group streptococcus and culture supernatant White, the characteristic of SPG and staphylococcal protein A (SPA) are significantly different.In 1984, this albumen was referred to as by Bjorck et al. Streptococcus protein G, and it is separated and has been purified.Currently, although SPA is due to having characteristic in conjunction with antibody Fc end, Relatively broad is applied in immunological experiment.However, SPG, compared with SPA, the binding force of SPG and IgG are stronger, bind profile Wider (Cao Zhifang, 1989).
GFP is the protein being made of about 238 amino acid, its excitation can be made from blue light to ultraviolet light, is issued Green fluorescent.It since fluorescin can be stablized in descendant inheritting, and can specifically be expressed according to promoter, be often used as reporting Accuse gene.Slowly instead of traditional chemical dye in needing quantitative or other experiments.Enhanced green fluorescence protein (Enhanced Green Fluorescent Protein, EGFP) is GFP mutantion line, the fluorescence intensity ratio that can launch Big 6 times of GFP or more, therefore, a kind of reporter gene is more suitable for than GFP to study gene expression, regulation, cell differentiation And protein is positioned and is transported in vivo etc..
The antigen of schistosomiasis diagnosis is mainly natural worm sources antigen at present.Indirect hemagglutination, ELISA, glue has been established The detection methods such as body gold test paper strip.With the decline of schistosoma infective rate and gradient of infection, the sensitivity of these methods need It improves, the diagnostic method that hypersensitivity must be continually looked for and can be quantified.
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.
In order to achieve the object of the present invention, the present invention adopts the following technical scheme:
One kind having the active detection albumen of green fluorescence, including streptococcus protein G (SPG) segment and fluorescin, the chain Pneumococcal proteins G(SPG) segment be the C3 section containing SPG sequence, preferably one in C3, C3-D-C3, C3-D-C3-D-C3 Kind, the fluorescin is green fluorescent protein；
Preferably, a kind of to have the active detection albumen of green fluorescence, the nucleotides sequence of the detection albumen is classified as SEQ ID Shown in one of NO.5,7,9；The amino acid sequence of the detection albumen is shown in one of SEQ ID NO.6,8,10.
Present invention simultaneously provides a kind of product for immunoassay, the product includes fusion egg of the present invention It is white.
Preferably, the present invention provides a kind of antibody of Schistosoma japonicum detection kit, the antibody of Schistosoma japonicum detection The fusion protein of G-protein and green fluorescent protein in kit including a kind of recombination and a kind of detection of antibody of Schistosoma japonicum are glimmering Light test strips.
Further, in antibody of Schistosoma japonicum of the invention detection fluorescent test paper strip, schistosoma japonice ovum is soluble Antigen (SEA) and streptococcus protein G (SPG) albumen are respectively as detection line (T), the envelope antigen of nature controlling line (C).Test strips choosing Use nitrocellulose filter (CN95) as chromatographic material, glass fibre element film is as sample pad, with PVC bottom plate and blotting paper etc. one It is same to be assembled into test strips.
Present invention simultaneously provides a kind of antibody of Schistosoma japonicum fluorescence detection method of non-diagnostic purpose, by sample to be tested into Fusion protein, loading after mixing, ultraviolet lower detection is added in row gradient dilution.
Beneficial effect
(1) the present invention is based on fusion protein can efficient, highdensity capture target molecule, reach quick, highly sensitive detection.
(2) raw material sources of antibody of Schistosoma japonicum detection kit of the invention are convenient, manufacturing cost and popularization cost It is lower.
(3) antibody of Schistosoma japonicum detection kit accuracy of the invention is high, stability is good.
Detailed description of the invention
The structural schematic diagram of Fig. 1 A:SPG；B: protein structure schematic diagram after splicing.
The SDS-PAGE of Fig. 2 recombinant expression protein is analyzed, wherein
The phase of A:C3-EGFP.M: protein marker；0h: no IPTG induction；1,2,4,6,7:IPTG induction 1h, 2h, 4h, 6h, 7h；
The soluble analysis of B:C3-EGFP.M: protein marker；1: ultrasonication sediment；2: ultrasonication supernatant；3: on Protein Detection after sample；Filtrate after B:Binding buffer elution；Filtrate after W:Wash buffer elution；S:Strip Destination protein after buffer elution；
The phase of C:C3DC3-EGFP.M: protein marker；0h: no IPTG induction；1,2,4,6,8:IPTG induction 1h, 2h, 4h, 6h, 8h；
The soluble analysis of D:C3DC3-EGFP.M: protein marker；1: ultrasonication supernatant；2: ultrasonication sediment； 3: Protein Detection before loading；4: Protein Detection after loading；Filtrate after B:Binding buffer elution；W:Wash buffer Filtrate after elution；Destination protein after S:Strip buffer elution；
The phase of E:C3DC3DC3-EGFP.M: protein marker；0h: no IPTG induction；1,2,4,6:IPTG induction 1h, 2h, 4h, 6h；
The soluble analysis of F:C3DC3DC3-EGFP.M: protein marker；1: ultrasonication supernatant；2: ultrasonication precipitating Object；3: Protein Detection before loading；4: Protein Detection after loading；Filtrate after B:Binding buffer elution；W:Wash Filtrate after buffer elution；Destination protein after S:Strip buffer elution；
Fig. 3 A: the recombinant protein of purifying scans the excitation spectrum of recombinant protein with sepectrophotofluorometer；B: with maximum excitation light The emission spectrum of wavelength acquisition recombinant protein.
The combination determination of activity of Fig. 4 recombinant protein and different plant species IgG
A: the anti-goat of donkey；B: goat anti-mouse；C: mouse anti-rabbit；D: rabbit-anti goat；
Wherein, M:Marker；1:C3-EGFP albumen；2:C3DC3-EGFP albumen；3:C3DC3DC3-EGFP albumen
Fig. 5 Elisa method analysis recombinant protein and the IgG(of several species are followed successively by donkey, mouse, rabbit, goat) affinity costant, In, A:C3-EGFP and different plant species IgG affinity costant measure；B:C3DC3-EGFP and different plant species IgG affinity costant measure； C:C3DC3DC3-EGFP and different plant species IgG affinity costant measure.
Fig. 6: antibody of Schistosoma japonicum detects fluorescent test paper strip schematic diagram, wherein 1. sample pads, 2. nature controlling lines, 3. Quality Controls Line, 4.NC film, 5. water absorption pads, 6.PVC plate.
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 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, SPG(is perhaps auspicious, Zhao Dengyun, Hong Yang, Lu Ke, Li Hao, and woods is 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.) EGFP corresponds on NCBI Sequence number: U55762
The building and synthesis (as shown in Figure 1B) of 1.2 recombinant protein gene orders
It is found from GenBank, the genetic fragment in the area C of the coding EGFP and SPG announced is found out the area C1, C2, C3 and the area D, obtained To the genetic fragment in the area C3.Signal peptide analysis is carried out to EGFP sequence, is detected whether rare containing Escherichia coli in gene order Codon, i.e. frequency of use < 10% are substituted for and encode the codon that the Escherichia coli of unified amino acid are had a preference for, and last 3 ' End addition TAA terminator codon.Restriction enzyme site, structure is added in Primer5 software Design primers in upstream and downstream primer respectively Build following three kinds of plasmid (see Table 1)s:
PET-28a(+) wherein, the nucleotides sequence of C3-EGFP fusion protein is classified as shown in SEQ ID NO.1-C3-EGFP: being compiled The amino acid sequence of code is shown in SEQ ID NO.2:
PET-28a(+ the nucleotides sequence of)-C3DC3-EGFP, C3DC3-EGFP fusion protein is classified as shown in SEQ ID NO.3: The amino acid sequence of coding is shown in SEQ ID NO.4:
PET-28a(+ the nucleotides sequence of)-C3DC3DC3-EGFP, C3DC3DC3-EGFP fusion protein is classified as SEQ ID NO.5 Shown: the amino acid sequence of coding is shown in SEQ ID NO.6:
1 recombinant C 3-EGFP sequence information of table
Abbreviation Full name Sequence length Encode amino acid Molecular weight Isoelectric point
1C3-EGFP PET-28(a)-C3-EGFP 1029 bp 343 aa 37.7KDa 6.14
2C3-EGFP PET-28(a)-C3DC3-EGFP 1239 bp 413 aa 45.4 KDa 5.9
3C3-EGFP PET-28(a)-C3DC3DC3-EGFP 1455 bp 485 aa 53.3 KDa 5.76
The expression and purifying of 2 recombinant protein of embodiment
The expression and purifying of albumen are carried out using conventional Protein expression and purification means, SDS-PAGE is analysis shows that (Fig. 2), weight Group plasmid pET-28a(+)-C3-EGFP successful expression in e. coli bl21 (DE3), and 1- after 1mmol/L IPTG induction 7h expression quantity increases with the growth of time, and expression quantity after 6h is induced to reach highest and tend towards stability.Three kinds of albumen are in ultrasound Expressed by having in supernatant precipitating, the protein content in precipitating is higher than the protein content in supernatant, indicates that the albumen has one Fixed water solubility, inclusion body also exist simultaneously.Albumen hanging column, Binding and Wash buffer step can be confirmed before and after loading Suddenly a large amount of foreign proteins are all eluted, finally obtains albumen after purification after Strip Buffer removing.
3 recombinant protein activity identification of embodiment
The observation of 3.1 fluorescence spectrums
The recombinant protein of purifying prepares various concentration, and the excitation spectrum (Fig. 3 A) of recombinant protein is scanned with sepectrophotofluorometer, Obtain the maximum excitation wavelength of recombinant protein；The emission spectrum (Fig. 3 B) of recombinant protein is obtained with maximum excitation optical wavelength.Observation The height of three kinds of recombinant protein fluorescence intensities.Standard is above from the visible three kinds of recombinant proteins rSPG-EGFP fluorescence intensity of the following figure Product EGFP, wherein C3-EGFP fluorescence intensity is most strong, and C3DC3-EGFP takes second place, and C3DC3DC3-EGFP is most weak, this may be with recombination 3rd area of PROTEIN C is related with EGFP clip size, and C3 segment is only the one third of EGFP segment in C3-EGFP, leads to two panels segment table The protein reached interfered with each other in structure it is less, with the increase of C3 quantity, the functional areas C3 of the protein of expression and EGFP Functional areas are folded from each other causes EGFP fluorescence intensity to weaken.
3.2 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, 130mA, 75min.
(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) PBST 1:2000 is used to dilute the different plant species IgG(that NC film uses HRP to mark) as antibody, 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 three kinds of recombinant proteins all have and donkey, and mouse, rabbit, the binding ability (Fig. 4) of sheep IgG
3.3 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) it by the mountain sheep anti mouse of HRP label, rabbit-anti goat, mouse anti-rabbit, the anti-goat of donkey these four secondary antibodies, uses respectively PBST carries out doubling dilution, the 100 every holes μ l, 37 DEG C of incubation 2h by 1:500,1:1000,1:2000,1:4000.
(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.
Three kinds of recombinant proteins after dialysis are measured into concentration respectively, different concentration is used to be coated with 96 orifice plates respectively as anti- Original the secondary antibody of doubling dilution is added in plate later, with antigen binding.Using OD450 value as ordinate, with antigen concentration Logarithm carries out curve fitting as abscissa, and affine curve is as shown in Figure 5.
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 three kinds of recombinant proteins, the results are shown in Table 2.
The affinity costant of 2 three kinds of recombinant proteins of table
C3-EGFP C3DC3-EGFP C3DC3DC3-EGFP
Goat 1.0×108 1.8×108 1.9×108
Donkey 3.8×107 1.2×108 1.3×108
Mouse 2.6×107 6.0×107 1.2×108
Rabbit 3.2×107 9.2×107 1.3×108
The affine active number correlation with the area C3 respectively of three kinds of albumen and different plant species as the result is shown.Three kinds of albumen Also not all with the affine activity of the IgG of different plant species, with the affine active highest of goat, next is donkey, mouse and rabbit respectively.
The Preliminary development of 4 recombinant protein fluorescent test paper strip of embodiment
Prepare antibody of Schistosoma japonicum detection fluorescent test paper strip.Schistosoma japonice ovum soluble antigen (SEA) and streptococcus egg White G (SPG) albumen is respectively as detection line (T), the envelope antigen of nature controlling line (C).Test strips select nitrocellulose filter (CN95) it is used as chromatographic material, glass fibre element film is assembled into test paper as sample pad, with PVC bottom plate and blotting paper etc. together Item.The principle and assembly model figure of fluorescent test paper strip are as shown in Figure 6.
Fluorescent test paper strip detects cow's serum evaluation
In fluorescence immune chromatography test paper bar detection, the IgG in test serum forms the IgG of label in conjunction with rSPG-EGFP. With SEA specific binding IgG in detection line with SEA in conjunction with, rather than specific IgG in C line in conjunction with SPG, ultraviolet light photograph It penetrates down, forms the distinguishable green florescent signal of naked eyes.
The ox Schistosoma japonicum feminine gender blood of 127 parts of excrement inspections positive ox Schistosoma japonicum positive serum and 82 parts of excrement inspection feminine genders Sorting does not use two methods of indirect ELISA and fluorescence immune chromatography test paper bar to detect.Compare fluorescence immune chromatography test paper bar and Connect the coincidence rate of ELISA method testing result.The results are shown in Table 3, it is assumed that fluorescence immune chromatography test paper bar result and indirectly ELISA testing result has significant difference, X2=170.079 > X2 0.01=6.63, P < 0.01 illustrates that null hypothesis is invalid, two Method correlation is significant.Kappa=0.9173 illustrates that the consistency of two methods is good.Using excrement inspection result as goldstandard, fluorescence examination The detection sensitivity that paper slip detects this batch of cow's serum is 98.42%；Detection specificity is 97.56%(table 3).
Table 3, ELISA and fluorescent test paper strip coincidence rate detect
Fluorescence immune chromatography test paper bar and indirect elisa method detect simultaneously 127 parts of positive ox Schistosoma japonicum serum of excrement inspection and 82 parts of ox Schistosoma japonicum negative serums, fluorescence immune chromatography test paper bar and ELISA testing result it is almost the same (Kappa= 0.9173).It is 98.42% and 97.56% that the sensitivity and specificity of fluorescence immune chromatography test paper bar detection reach respectively.Display tool There is higher practical value.
Although it should be appreciated that above having made with a general description of the specific embodiments to the content of present invention detailed Description, but on the basis of the present invention, can to carry out it is some modify or improve, this is to those skilled in the art Obviously.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the present invention and want Seek the range of protection.
<110>China Agriculture Academe Shanghai Veterinary Institute (China Animal Health and Epidemiology Center, branch center, Shanghai)
<120>a kind of detection kit with the active detection albumen of green fluorescence
<160> 6
<170> PatentIn version 3.5
<210>1
<211>1030
<212> DNA
<213>nucleotide sequence of recombinant protein 1C3-EGFP
<400>ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTA GCATGACTGGTGGACAGCAAATGGGTCGCGGATCCACTTACAAACTGGTTATTAATGGTAAAACCTTGAAAGGCGAA ACAACTACTAAAGCAGTAGACGCAGAAACTGCACAAAAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGG TGTTTGGACTTATGATGATGCGACTAAGACCTTTAGGGTAACTGAAGGCGGTGGGGGCTCAGGAGGTGGGGGCTCAG AATTCGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGC CACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCAC CGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCG ACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAG GACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGG CATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCA TGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTC GCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCA GTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCA CTCTCGGCATGGACGAGCTGTACAAGTAACTCGAGGCC
<210>2
<211>344
<212>protein
<213>protein sequence of recombinant protein 1C3-EGFP
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<211>1239
<212> DNA
<213>nucleotide sequence of recombinant protein c 3DC3-EGFP
<400>ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTA GCATGACTGGTGGACAGCAAATGGGTCGCGGATCCACTTACAAACTGGTTATTAATGGTAAAACCTTGAAAGGCGAA ACAACTACTAAAGCAGTAGACGCAGAAACTGCACAAAAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGG TGTTTGGACTTATGATGATGCGACTAAGACCTTTAGGGTAACTGAAAAACCAGAAGTGATCGATGCGTCTGAATTAA CACCAGCCGTGACAACTTACAAACTGGTTATTAATGGTAAAACCTTGAAAGGCGAAACAACTACTAAAGCAGTAGAC GCAGAAACTGCACAAAAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGGTGTTTGGACTTATGATGATGC GACTAAGACCTTTAGGGTAACTGAAGGCGGTGGGGGCTCAGGAGGTGGGGGCTCAGAATTCGTGAGCAAGGGCGAGG AGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC GAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTG GCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACT TCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACC CGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGG CAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACG GCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAAC ACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCC CAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGT ACAAGTAAGAGCTAGCC
<210>4
<211>413
<212> protein
<213>amino acid sequence of recombinant protein c 3DC3-EGFP
<400>MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSTYKLVINGKTLKGETTTKAVDAETAQKAFKQYA NDNGVDGVWTYDDATKTFRVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVDG VWTYDDATKTFRVTEGGGGSGGGGSEFVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT GKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKG IDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQ SALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKELA
<210>5
<211>1455
<212> DNA
<213>nucleotide sequence of recombinant protein c 3DC3DC3-EGFP
<400>ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTA GCATGACTGGTGGACAGCAAATGGGTCGCGGATCCACTTACAAACTTGTTATTAATGGTAAAACATTGAAAGGCGAA ACAACTACTAAAGCAGTAGACGCAGAAACTGCACAAAAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGG TGTTTGGACTTATGATGATGCGACTAAGACCTTTAGGGTAACTGAAGGCAAACCAGAAGTGATCGATGCGTCTGAAT TAACACCAGCCGTGACAACTTACAAACTTGTTATTAATGGTAAAACATTGAAAGGCGAAACAACTACTAAAGCAGTA GACGCAGAAACTGCACAAAAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGGTGTTTGGACTTATGATGA TGCGACTAAGACCTTTAGGGTAACTGAAGGCAAACCAGAAGTGATCGATGCGTCTGAATTAACACCAGCCGTGACAA CTTACAAACTTGTTATTAATGGTAAAACATTGAAAGGCGAAACAACTACTAAAGCAGTAGACGCAGAAACTGCACAA AAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGGTGTTTGGACTTATGATGATGCGACTAAGACCTTTAG GGTAACTGAAGGCGGTGGGGGCTCAGGAGGTGGGGGCTCAGAATTCGTGAGCAAGGGCGAGGAGCTGTTCACCGGGG TGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGAT GCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGAC CACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCA TGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAG TTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACT TCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGAC GGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAACTCGAGG CC
<210>6
<211>485
<212> protein
<213>amino acid sequence of recombinant protein c 3DC3DC3-EGFP
<400>MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSTYKLVINGKTLKGETTTKAVDAETAQKAFKQYA NDNGVDGVWTYDDATKTFRVTEGKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVD GVWTYDDATKTFRVTEGKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVDGVWTYD DATKTFRVTEGGGGSGGGGSEFVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPV PWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE DGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSK DPNEKRDHMVLLEFVTAAGITLGMDELYKLEA

Claims (4)

1. a kind of G-protein of recombination and the fusion protein of green fluorescent protein, the fusion protein are selected from: (1), C3-EGFP melts The nucleotides sequence of hop protein is classified as shown in SEQ ID NO.1: the amino acid sequence of coding is shown in SEQ ID NO.2；With or (2), the nucleotides sequence of C3DC3-EGFP fusion protein is classified as shown in SEQ ID NO.3: the amino acid sequence of coding is SEQ ID Shown in NO.4；He or the nucleotides sequence of (3), C3DC3DC3-EGFP fusion protein are classified as shown in SEQ ID NO.5: coding Amino acid sequence is shown in SEQ ID NO.6.
2. a kind of antibody of Schistosoma japonicum detection kit, it is characterised in that wrapped in the antibody of Schistosoma japonicum detection kit A kind of G-protein of recombination and the fusion protein of green fluorescent protein and a kind of antibody of Schistosoma japonicum detection fluorescent test paper strip are included, Wherein the fusion protein is as described in claim 1.
3. a kind of antibody of Schistosoma japonicum detection kit as claimed in claim 2, wherein the antibody of Schistosoma japonicum Detect in fluorescent test paper strip, schistosoma japonice ovum soluble antigen (SEA) and streptococcus protein G (SPG) albumen respectively as Detection line (T), the envelope antigen of nature controlling line (C).
4. a kind of antibody of Schistosoma japonicum detection kit as claimed in claim 3, wherein the test strips select nitric acid fine It ties up plain film (CN95) and is used as chromatographic material, glass fibre element film is assembled into together as sample pad with PVC bottom plate and blotting paper etc. Test strips.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113504367A (en) * 2021-06-02 2021-10-15 威海市环翠区动物疫病预防控制中心(威海市环翠区动物卫生检疫中心) Hemagglutination inhibition test detection method for avian influenza and newcastle disease

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WO1998014605A1 (en) * 1996-10-04 1998-04-09 Loma Linda University Renilla luciferase and green fluorescent protein fusion genes
WO1998040477A1 (en) * 1997-03-14 1998-09-17 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
CN101124472A (en) * 2004-03-17 2008-02-13 夏威夷大学 Sensor constructs and detection methods
CN105384803A (en) * 2015-11-23 2016-03-09 中国医学科学院病原生物学研究所 Schistosoma japonicum katsurada recombinant protein SjSAPLP4 as well as encoding gene and application thereof
CN108267582A (en) * 2016-12-30 2018-07-10 中国农业科学院上海兽医研究所 Infection of Toxoplasma Gondii colloidal gold immuno-chromatography test paper strip and preparation method thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998014605A1 (en) * 1996-10-04 1998-04-09 Loma Linda University Renilla luciferase and green fluorescent protein fusion genes
WO1998040477A1 (en) * 1997-03-14 1998-09-17 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
CN101124472A (en) * 2004-03-17 2008-02-13 夏威夷大学 Sensor constructs and detection methods
CN105384803A (en) * 2015-11-23 2016-03-09 中国医学科学院病原生物学研究所 Schistosoma japonicum katsurada recombinant protein SjSAPLP4 as well as encoding gene and application thereof
CN108267582A (en) * 2016-12-30 2018-07-10 中国农业科学院上海兽医研究所 Infection of Toxoplasma Gondii colloidal gold immuno-chromatography test paper strip and preparation method thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113504367A (en) * 2021-06-02 2021-10-15 威海市环翠区动物疫病预防控制中心(威海市环翠区动物卫生检疫中心) Hemagglutination inhibition test detection method for avian influenza and newcastle disease
CN113504367B (en) * 2021-06-02 2023-08-08 威海市环翠区动物疫病预防控制中心(威海市环翠区动物卫生检疫中心) Hemagglutination inhibition test detection method for avian influenza and newcastle disease

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