CN110511281B

CN110511281B - Schistosoma japonicum antibody detection kit for detection protein with red fluorescence activity

Info

Publication number: CN110511281B
Application number: CN201910765647.6A
Authority: CN
Inventors: 朱传刚; 柴瑞; 沈元曦; 纪荣毅; 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
Priority date: 2019-08-19
Filing date: 2019-08-19
Publication date: 2022-05-17
Anticipated expiration: 2039-08-19
Also published as: CN110511281A

Abstract

The invention provides a schistosoma japonicum antibody detection kit with a red fluorescence activity detection protein, which comprises a schistosoma japonicum antibody detection reagent strip and a fusion protein C3-RFP; furthermore, in the schistosoma japonicum antibody detection fluorescent test strip, the schistosoma japonicum egg soluble antigen (SEA) and the Streptococcal Protein G (SPG) are respectively used as the coating antigens of the detection line (T) and the quality control line (C). The test paper strip is formed by combining a nitrocellulose membrane (CN 95) as a chromatographic material, a glass cellulose membrane as a sample pad, a PVC base plate, absorbent paper and the like.

Description

Schistosoma japonicum antibody detection kit for detection protein with red fluorescence activity

Technical Field

The invention relates to the field of immunodetection, in particular to a schistosoma japonicum antibody detection kit for detecting proteins with red fluorescence activity.

Background

Schistosomiasis is a parasitic zoonosis with serious harm caused by schistosomiasis, and still remains one of public health problems worldwide to date. Schistosome infection can cause morbidity and mortality in humans and livestock, affect the health of humans and livestock and thus cause significant economic losses. Schistosomiasis is mainly caused by organ damage and malnutrition and complications caused by infection, and researches show that the adjustment life of the disability caused by schistosomiasis is almost equal to that of AIDS, and exceeds that of malaria and tuberculosis, so that the damage of schistosomiasis can be seen. Livestock is the main host of schistosomiasis. Research shows that the infection rates of livestock and human are closely related, and the infection rate of human is higher in the area with higher schistosomiasis japonica infection rate of cattle, so if the epidemic of schistosomiasis japonica of livestock can not be effectively controlled, schistosomiasis japonica can not be finally controlled and eliminated in China.

The conventional methods for diagnosing the schistosomiasis japonica comprise a pathogenic diagnosis method and an immunological diagnosis method, wherein the pathogenic diagnosis method comprises a feces hatching method and a feces detection method, the missed detection rate is high, and the infection is often detected to a certain degree and possibly only in the later infection stage, so that the early diagnosis and treatment are not facilitated. The immunological diagnosis method comprises test strips, ELISA, IHA and the like, and the judgment subjectivity of the IHA method is larger because an ELISA detection needs a special instrument. The test strip is a simple, convenient and quick diagnosis method with intuitive result, and has high practical value in large-scale diagnosis.

The conventional test paper for detecting the schistosome comprises a colloidal gold immunochromatographic test paper and a percolation test paper. Since the development of the test strip, the test strip becomes the first choice for quickly diagnosing schistosomiasis based on the characteristics of convenience in detection, rapidness in detection, strong specificity and the like. However, the immunochromatographic test strip can only be used for qualitative experiments, the detection sensitivity needs to be improved, and the fluorescence immunochromatographic test strip can be used for qualitative or semi-quantitative detection and has higher sensitivity.

Disclosure of Invention

The invention mainly solves the technical problem of providing a quick and high-sensitivity immunoassay product and a method, and realizing high-efficiency and high-density immunoassay.

In order to realize the technical scheme, the invention provides a schistosoma japonicum antibody detection kit with a red fluorescence activity detection protein.

In order to realize the purpose, the invention firstly utilizes the genetic engineering technology to clone and efficiently express the recombinant fusion protein C3-RFP of G protein and red fluorescent protein, and the nucleotide sequence of the fusion protein C3-RFP is shown as SEQ ID NO. 1; the amino acid sequence is shown as SEQ ID NO.2, and the immunogenicity and specificity of the polypeptide are detected by an immunological technique. Experiments show that the fusion protein C3-RFP obtained by the method has good high sensitivity and specificity.

Furthermore, the invention develops a schistosoma japonicum antibody detection kit by utilizing the fusion protein C3-RFP prepared by the method;

the schistosoma japonicum antibody detection kit comprises a schistosoma japonicum antibody detection reagent strip and a fusion protein C3-RFP;

furthermore, in the schistosoma japonicum antibody detection fluorescent test strip, the schistosoma japonicum egg soluble antigen (SEA) and the Streptococcal Protein G (SPG) are respectively used as the coating antigens of the detection line (T) and the quality control line (C). The test paper strip is formed by combining a nitrocellulose membrane (CN 95) as a chromatographic material, a glass cellulose membrane as a sample pad, a PVC base plate, absorbent paper and the like.

The final working concentration of the fusion protein C3-RFP was 200 ng/ml.

The invention also provides a schistosoma japonicum antibody fluorescence detection method, which comprises the steps of carrying out gradient dilution on a sample to be detected, adding the fusion protein C3-RFP, mixing, loading the sample, and detecting under ultraviolet.

The schistosoma japonicum antibody fluorescence detection method has the following advantages:

(1) the detection is quick: the result is obtained in 10 minutes;

(2) specificity: positive to blood fluke serum sample only and negative to blood fluke serum sample infected by other pathogens;

(3) sensitivity: the sensitivity and specificity of the fluorescence immunochromatographic test strip detection respectively reach 100 percent and 96.34 percent, and the fluorescence immunochromatographic test strip has higher practical value.

(4) The storage and transportation are convenient, and the product can be stored for 18 months at room temperature;

(5) is convenient for clinical and household use and has industrialization.

The fluorescent immunochromatography technology is focused on not only having higher sensitivity, but also being capable of semi-quantitative or quantitative analysis. The novel quantum dot fluorescent microsphere of the marking material has the following unique characteristics: the characteristics of wide excitation wavelength, strong fluorescence brightness, high stability and the like are increasingly paid more attention to various research fields. In the conventional fluorescence chromatography, fluorescent microspheres are mainly used as marker molecules, but the molecular weight of the fluorescent microspheres is large and nonuniform, coupling with IgG is required, and the change of the coupled microspheres is large, so that the flow rate and the like during chromatography are difficult to keep consistent, and the coefficient of variation during quantitative detection is increased. The recombinant C3-RFP is used for replacing fluorescent microspheres, the preparation is simple, 10mg of recombinant protein can be extracted from 500ml of bacterial liquid, the cost is low, the bacterial liquid has great advantages in large-scale mass production, and the fluorescent microsphere has the characteristics of high and stable fluorescence intensity, light background dyeing, high sensitivity and the like, and has a good application prospect. On one hand, the recombinant fluorescent protein avoids the process of coupling a fluorescent marker and IgG, and can be directly combined with the IgG through an IgG combination fragment of the recombinant protein; on the other hand, the size of the marker molecules is uniform, the flow rate is consistent during chromatography, the high background caused by residue on the NC membrane is not easy to occur when the NC membrane with the proper pore diameter is selected, chromatography can be completed in a short time for detection, quantitative semi-quantitative detection is facilitated in practical application, and the accuracy and the repeatability are improved.

Streptococcal protein G has great application prospect due to the characteristic of wide specific combination with IgG of different species. The reconstructed protein C3-RFP not only has the binding property with IgG, but also has the fluorescence property of the RFP, and the experiment mainly verifies the application of the fluorescence immunochromatographic test strip established on the basis of the protein, and determines the wide potential of the recombinant protein C3-RFP in the test strip detection and the application prospect of the established test strip.

Drawings

FIG. 1 is a schematic view of a fluorescent test strip for detecting schistosoma japonicum katsurada antibody, wherein 1 is a sample pad, 2 is a detection line, 3 is a quality control line, 4 is an NC film, 5 is a water absorption pad, and 6 is a PVC plate

FIG. 2 shows PCR identification (A) and double-restriction enzyme identification (B) of a recombinant protein prokaryotic expression plasmid pET-28a (+) -C3-RFP,

a: m: a DNA marker; 1: the plasmid pET-28a (+) -C3-RFP is subjected to PCR to obtain a C3 sequence with the size of 165 bp; 2: the RFP sequence obtained by PCR of plasmid pET-28a (+) -C3-RFP is 687bp in size; 3: PCR of plasmid pET-28a (+) -C3-RFP to obtain C3-RFP sequence with size of 852bp

B: m: a DNA marker; 1: the plasmid pET-28a (+) -C3-RFP is subjected to double enzyme digestion to obtain a C3-RFP sequence with the size of 852 bp; 2: the plasmid pET-28a (+) -C3-RFP is subjected to double enzyme digestion to obtain an RFP sequence, and the size is 687 bp.

FIG. 3 is an SDS-PAGE analysis of pET-28a (+) -C3-RFP expression protein, wherein the SDS-PAGE analysis of pET-28a (+) -C3-RFP phase expression is shown in FIG. A; SDS-PAGE analysis of pET-28a (+) -C3-RFP protein purification

A: m: a protein marker; 0 h: no IPTG induction; 1h-8 h: IPTG induction is carried out for 1h, 2h, 4h, 6h and 8 h;

b: m: a protein marker; 1: ultrasonically crushing the precipitate; 2: ultrasonically crushing the supernatant; 3: after sample loading; 4: binding Buffer; 5: wash Buffer; 6: strip Buffer.

FIG. 4 fluorescent activity observation after expression induction of pET-28a (+) -C3-RFP expression, wherein 0 h: no IPTG induction; 1h-8 h: IPTG induction for 1h, 2h, 4h, 6h and 8h

FIG. 5Western-blot analysis of binding activity of pET-28a (+) -C3-RFP to IgG,

a: m: a protein marker; 1: binding activity of the recombinant protein to sheep; 2: binding activity of the recombinant protein to donkey; 3: binding activity of the recombinant protein to mouse; 4: binding Activity of recombinant proteins to Rabbit

B: m: a protein marker; 1: binding Activity of recombinant protein with His antibody

FIG. 6 analysis of the affinity constants of pET-28a (+) -C3-RFP with multi-species IgG by the Elisa method, in which, Panel A: the Elisa method analyzes the affinity constant of pET-28a (+) -C3-RFP and rabbit IgG; and B: the Elisa method analyzes the affinity constant of pET-28a (+) -C3-RFP and donkey IgG; and (C) figure: the Elisa method analyzes the affinity constant of pET-28a (+) -C3-RFP and mouse IgG; FIG. D: the Elisa method analyzes the affinity constant of pET-28a (+) -C3-RFP with sheep IgG.

Test strip sensitivity detection of fig. 7, a: detecting the sensitivity of the fluorescence immunochromatographic test strip; the positive serum of Schistosoma japonicum is diluted by 1:100, 1:200, 1:1000, 1:10000, 1:20000, and the negative serum is diluted by 1: 100. B: detecting the ELISA sensitivity; schistosoma japonicum positive serum is diluted according to the ratio of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200 and 1: 6400.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

EXAMPLE 1 construction of recombinant protein G

Escherichia coli BL21 was purchased from Sokenozokerite Biotech, Inc. of Nanjing; the plasmid pET-28a (+) was stored in the laboratory, and the bacterial suspension [1] containing the C3 fragment and the bacterial suspension [2] containing the RFP sequence were stored in the laboratory.

[1] Series, Zhao Dengyun, hong Yang, Luke, Lihao, Lin rectified, Von gold waves, Xuyumei, Zhu-Seng-streptococcal protein G domain reconstruction, expression and identification [ J ]. Chinese animal infectious diseases journal 2015,23(05):46-52.

[2] Clone, prokaryotic expression characteristics and biological information analysis of the monomer red fluorescent protein gene DsRed2 [ D ]. Yanbian university, 2016.

Obtaining a fusion protein C3-RFP of the expression recombinant G protein and the red fluorescent protein by utilizing a PCR means, wherein the nucleotide sequence of the fusion protein C3-RFP is shown as SEQ ID NO. 1; the amino acid sequence is shown as SEQ ID NO. 2.

Carrying out electrophoretic identification on the PCR product; meanwhile, the product is subjected to double enzyme digestion identification of BamH1 and Xho1 (FIG. 2), and the electrophoresis result shows that: the size of the whole target gene fragment is about 852 bp; the C3 fragment size was about 165bp, the RFP fragment size was about 687bp, as predicted. And simultaneously, sequencing and identifying the PCR product, wherein the sequencing and identifying result shows that the sequence is consistent with the designed sequence.

EXAMPLE 2 expression and purification of recombinant protein G

2.1 expression of the recombinant plasmid

Time phase

(1) The pET-28a (+) -C3-RFP recombinant plasmids with correct identification results are respectively transferred into BL21 (DE 3), inoculated into 5ml LB liquid culture medium containing Kan +, placed in a shaking incubator at 37 ℃ and subjected to shaking culture at 250 rpm.

(2) When the growth reached the logarithmic phase (OD 600 was about 0.6), IPTG was added to a final concentration of 1mmol/L for induction of expression. 0.5ml of bacterial liquid is respectively taken before induction expression and 1h, 2h, 4h, 6h and 8h after induction expression, and SDS-PAGE electrophoresis is applied to analyze the optimal induction time. (FIG. 3A)

And (3) large-scale expression:

(1) the pET-28a (+) -C3-RFP recombinant plasmids with correct identification results are respectively transferred into BL21 (DE 3), inoculated into 150ml LB liquid culture medium containing Kan +, placed in a shaking incubator at 37 ℃ and subjected to shaking culture at 250 rpm.

(2) When the growth reached the logarithmic phase (OD 600 was about 0.6), IPTG was added to a final concentration of 1mmol/L for induction of expression. Centrifuging the bacteria solution after 8h induction at 12000rpm for 20min, discarding the supernatant, resuspending the precipitate with 20ml of 1 XPBS, repeatedly freezing and thawing for three times, ultrasonically crushing for 20min (2 s over 9 s), centrifuging at 12000rpm for 15min, and collecting the precipitate and the supernatant.

(4) The centrifuged pellet was resuspended in 5ml of 8mol urea, the centrifugation step was repeated, and the supernatant was collected.

(5) Adding the supernatant after ultrasonic treatment and the supernatant after the sediment is resuspended into equal volume of protein electrophoresis buffer solution respectively, and analyzing the solubility of the expression product by SDS-PAGE electrophoresis.

2.2 purification of the recombinant protein

Recombinant protein was purified using Ni-NTA Hisbind Resin kit (SEQ ID NO: 70666-3), according to the instructions for the product, and the simple procedure was as follows:

(1) adding 5ml resin into a new empty column, standing for balancing, and sequentially adding 15ml ddH when the liquid level is reduced to the resin surface₂O，25ml 1×Charge Buffer, 15ml 1 × Binding Buffer solution washing.

(2) When the liquid level dropped to the resin surface, the supernatant solution after ultrasonication was added and the sample application was repeated 3 times, and the solution after column chromatography was collected.

(3) 50ml of 1 XBinding Buffer and 30ml of 1 XBash Buffer are added in sequence for washing, and the solution after passing through the column is respectively collected.

(4) The Ni ions were washed off by adding 20ml of 1 XSlipe Buffer and the solution after column chromatography was collected.

(5) The resin was saved by adding the appropriate amount of 1 XScript Buffer.

(6) The collected solutions were subjected to SDS-PAGE to analyze protein purification.

SDS-PAGE analysis shows (figure 3), the recombinant plasmid pET-28a (+) -C3-RFP is successfully expressed in Escherichia coli BL21 (DE 3), the expression level increases with the increase of time after 1mmol/L IPTG induction for 1-6h, and the expression level reaches the highest and tends to be stable after 8h induction. Meanwhile, SDS-PAGE analysis shows that the protein expressed by the recombinant plasmid pET-28a (+) -C3-RFP is expressed in both ultrasonic supernatant and sediment, and the protein content in the sediment is higher than that in the supernatant, which shows that the protein has certain water solubility and the inclusion bodies also exist at the same time.

Example 3C 3-identification of the Activity of the RFP recombinant protein

3.1 Observation of fluorescence Activity

Centrifuging the bacteria solution collected from the time phase at 10000rpm for 5min, discarding the supernatant, and adding 200ul ddH₂And O, resuspending the thallus, sucking 10ul of the thallus on a clean glass slide after resuspension, covering the glass slide, exciting the thallus by RFP exciting light under a fluorescence electron microscope, and observing the thallus for red fluorescence. Fluorescence electron microscopy observation shows (figure 4) that fluorescence increases with time 1-8h after the recombinant plasmid pET-28a (+) -C3-RFP is successfully induced and expressed in Escherichia coli BL21 (DE 3), and the fluorescence reaches the highest and tends to be stable after 8h of induction.

3.2 Western blotting detection of the binding Activity of recombinant proteins with IgG

(1) The purified protein was subjected to SDS-PAGE, after which the protein was transferred to NC membrane at 130mA for 75 min.

(2) The NC membrane is soaked in 5% skimmed milk powder diluted by PBST and sealed for 2h at room temperature.

(3) The blocked NC membranes were washed three times with PBST for 5min each.

(4) Goat anti-mouse IgG, rabbit anti-goat IgG, mouse anti-rabbit IgG, donkey anti-goat IgG (diluted with PBST 1: 2000) labeled with HRP on the NC membrane were used as antibodies, and incubated at room temperature for 1 h.

(5) Incubated NC membranes were washed three times with PBST for 10min each.

(6) And developing the NC membrane by using a DAB two-component developing solution kit, washing with running water after developing, and terminating the reaction.

The results showed that the recombinant protein had the binding ability to IgG of goat, donkey, mouse, rabbit, etc. (FIG. 5)

3.3 determination of the affinity constant of recombinant proteins to IgG of different species by ELISA

(1) The concentration of the recombinant protein was determined by the BCA method, and commercial Standard Protein G (SPG) was used as a control.

(2) Proteins were diluted in coating solution in multiples of 10. mu.g/ml for a total of 8 dilutions, coated in 100. mu.l per well in 96-well plates, 3 replicates per concentration setting, and coated overnight at 4 ℃.

(3) The 96-well plate was washed three times with PBST, 200. mu.l per well, 5min each time.

(4) A solution of 5% skimmed milk powder diluted with PBST was added, 150. mu.l per well, and blocked at 37 ℃ for 2 h.

(5) The 96-well plate was washed three times with PBST, 200. mu.l per well, 5min each time.

(6) Four secondary antibodies, namely rabbit anti-goat, donkey anti-goat, mouse anti-rabbit and goat anti-mouse, marked by HRP are respectively added with PBST according to the weight ratio of 1:500, 1:1000, 1:2000, 1:4000 and 1: 8000 dilutions were made at 100. mu.l per well and incubated for 2h at 37 ℃.

(7) The 96-well plate was washed three times with PBST, 200. mu.l per well, 5min each time.

(8) TMB was added thereto for color development, and 100. mu.l of each well was reacted at room temperature for 15 min.

(9) 2mol/L H was added₂SO₄The reaction was stopped and 30. mu.l/well was read for OD 450.

The measured data are shown in Table 2, and the affinity curves are shown in FIG. 6.

TABLE 2 determination of the affinity constant of recombinant proteins for IgG of different species by ELISA

K	C3-RFP
		Rabbit	1.9*10⁵
Donkey meat	4.1*10⁵
		Mouse	1.7*10⁵
Sheep (sheep)	5.4*10⁵

The results show that the SPG gene was reconstituted with a fragment of the Red Fluorescent Protein (RFP) gene in this experiment. The C3 region which can be specifically combined with the Fc end of the antibody IgG in SPG is reserved, then the RFP gene segment is connected, and the prokaryotic expression plasmid constructed by the prokaryotic expression plasmid of the C3-RFP recombinant protein successfully expresses the recombinant protein C3-RFP. The recombinant protein has the ability of combining SPG with IgG of different species and the function of RFP for emitting fluorescence, and is a novel bifunctional recombinant protein.

Example 4C 3-RFP recombinant protein fluorescence test strip

Schistosoma japonicum egg soluble antigen (SEA) and Streptococcal Protein G (SPG) were streaked at a concentration of 0.5mg/ml and a speed of 4ul/cm, and fixed on a nitrocellulose membrane (CN 95) as a detection line (T) and a quality control line (C). The schistosome antibody detection test paper strip is assembled together with a glass cellulose membrane, a PVC bottom plate and absorbent paper.

Carrying out gradient dilution on the positive and negative serum of the standard bovine schistosoma japonicum, adding the recombinant protein C3-RFP, mixing, loading, and detecting under ultraviolet. When the standard positive serum is diluted to 1:10000, the detection is still positive under ultraviolet rays. During ELISA detection, the positive serum of the bovine schistosoma japonicum can be diluted to 1:400 at most, when the positive serum exceeds 1:400, the P/N detection is less than or equal to 2.1, and the detection result is negative.

Therefore, in the fluorescence immunochromatographic test strip detection, IgG in serum to be detected is combined with C3-RFP to form labeled IgG. IgG specifically bound with SEA is bound with SEA in the detection line, while nonspecific IgG is bound with SPG in the C line, and under the irradiation of ultraviolet rays, a red fluorescence signal which is distinguishable to naked eyes is formed. Compared with ELISA, the detection method has higher sensitivity, the highest ELISA dilution for detecting the positive serum of the bovine schistosoma japonicum is 1:400, and the fluorescence immunochromatographic test strip established at this time can still detect the positive serum when the serum is diluted by 1:10000, and shows higher sensitivity.

Example 5 evaluation of bovine serum by fluorescent test strip assay

127 portions of positive serum of the bovine schistosoma japonicum through the excrement detection and 82 portions of negative serum of the bovine schistosoma japonicum through the excrement detection are respectively detected by two methods of indirect ELISA and a fluorescence immunochromatographic test strip. And comparing the coincidence rate of the detection results of the fluorescence immunochromatographic test strip and the indirect ELISA method. The results are shown in Table 3, assuming that the fluorescence immunochromatographic test strip results are significantly different from the indirect ELISA detection results, X²=173.645＞X² _0.01The hypothesis is not true, and the correlation between the two methods is remarkable. Kappa =0.9660, which shows that the fluorescence immunochromatographic test strip is substantially consistent with the ELISA detection result. Taking the stool detection result as a gold standard, and detecting the bovine serum of the batch by using a fluorescent test strip with the detection sensitivity of 100 percent; the detection specificity was 96.34%.

TABLE 3 detection of ELISA and fluorescent test strip compliance

Therefore, the fluorescence immunochromatographic test strip has high practical value in sample high-flux rapid detection.

It is to be understood that while the present disclosure has been described in detail hereinabove with respect to specific embodiments thereof, it is apparent that modifications and improvements may be made thereto without departing from the scope of the invention as defined by the appended claims. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

<110> Shanghai animal doctor institute of Chinese academy of agricultural sciences (Shanghai center of Chinese centers of animal health and epidemiology)

<120> schistosoma japonicum antibody detection kit with red fluorescence activity detection protein

<160> 2

<170> PatentIn version 3.5

<210>1

<211> 888

<212> DNA

<213> C3-RFP nucleotide sequence

GGA TCC ACC TAC AAA CTG GTG ATC AAC GGC AAA ACT TTA AAA GGT GAG ACC ACC ACA AAA GCA GTG GAT GCC GAG ACC GCA CAG AAG GCC TTC AAG CAG TAT GCC AAC GAC AAT GGC GTG GAT GGC GTG TGG ACC TAT GAC GAT GCC ACC AAA ACC TTC CGC GTG ACA GAG GGC GGT GGT GGT AGT GGT GGT GGC GGT AGC GAA TTC GCC TCC TCC GAG AAC GTC ATC ACC GAG TTC ATG CGC TTC AAA GTG CGC ATG GAA GGC ACC GTG AAT GGC CAC GAG TTC GAG ATT GAA GGC GAA GGT GAA GGC CGC CCA TAC GAA GGC CAC AAC ACC GTG AAG CTG AAG GTG ACC AAA GGC GGT CCA CTG CCG TTC GCG TGG GAT ATT CTG AGC CCG CAG TTC CAG TAC GGC AGC AAG GTG TAC GTT AAG CAT CCG GCG GAC ATC CCG GAT TAC AAG AAG CTG AGC TTC CCG GAA GGC TTC AAA TGG GAG CGC GTG ATG AAC TTC GAG GAC GGC GGC GTT GCC ACG GTT ACC CAA GAT AGT AGT CTG CAA GAT GGC TGC TTC ATC TAC AAG GTG AAG TTC ATC GGC GTG AAC TTC CCG AGC GAT GGC CCA GTG ATG CAG AAA AAG ACC ATG GGC TGG GAA GCG AGC ACC GAA CGT CTG TAC CCG CGC GAT GGT GTT CTG AAA GGC GAA ACG CAC AAG GCG CTG AAA CTG AAA GAC GGC GGC CAC TAC CTC GTG GAG TTC AAG AGC ATC TAC ATG GCC AAA AAG CCG GTG CAA CTG CCG GGC TAT TAC TAC GTG GAC GCC AAG CTG GAC ATC ACC AGC CAT AAC GAG GAC TAC ACG ATC GTG GAG CAG TAC GAA CGC ACC GAA GGT CGC CAC CAC CTG TTC CTG TAG CTC GAG

<210>2

<211> 295

<212> protein

<213> C3-RFP amino acid sequence:

GSTYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVDGVWTYDDATKTFRVTEGGGGSGGGGSEFASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVKLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHHLFLLE

Claims

1. a recombinant fusion protein C3-RFP of G protein and red fluorescent protein is characterized in that the nucleotide sequence of the fusion protein C3-RFP is shown as SEQ ID NO. 1; the amino acid sequence is shown as SEQ ID NO. 2.

2. A schistosoma japonicum antibody detection kit is characterized in that the schistosoma japonicum antibody detection kit comprises a schistosoma japonicum antibody detection reagent strip and a fusion protein C3-RFP, wherein the fusion protein C3-RFP is as in claim 1.

3. The schistosoma japonicum antibody detection kit according to claim 2, wherein in the schistosoma japonicum antibody detection reagent strip, the schistosoma japonicum egg soluble antigen SEA and the streptococcal protein G protein are used as the coating antigens of the detection line T and the quality control line C respectively.

4. The schistosoma japonicum antibody detection kit according to claim 3, wherein the reagent strip is formed by assembling nitrocellulose membrane CN95 as a chromatography material, a glass cellulose membrane as a sample pad, a PVC base plate and absorbent paper.

5. The schistosoma japonicum antibody detection kit according to claim 3, wherein the final working concentration of said fusion protein C3-RFP is 200 ng/ml.