CN110437341A - One kind having the active detection albumen of red fluorescence and its application - Google Patents

Abstract

The present invention provides a detection protein with red fluorescent activity and its application, characterized in that the detection protein includes a streptococcal protein G (SPG) fragment and a fluorescent protein, and the streptococcal protein G (SPG) fragment is C3 of SPG In the section, the fluorescent protein is an optimized red fluorescent protein, and the detection protein has dual activities of fluorescent activity and binding to antibodies of different species. The detection protein disclosed by the present invention can broadly bind human and various animal total IgG and IgG of different subclasses. Protein binding molecules are high.

Description

A detection protein with red fluorescent activity and its application

technical field

The invention relates to the field of immunoassay, in particular to a detection protein with red fluorescence activity and application thereof.

Background technique

The widespread spread of various infectious diseases poses a serious threat to my country's national security and population health. The development of sensitive and accurate pathogen analysis methods and detection technologies is of great significance for the rapid diagnosis and timely treatment of related diseases, the effective prevention and rapid treatment of bioterrorism and public health emergencies. The classic isolation and identification of microorganisms is cumbersome and time-consuming, and it is difficult to apply to the on-site rapid detection of pathogenic microorganisms; most of the detection methods based on pathogenic nucleic acids have high detection sensitivity, but require complex nucleic acid extraction processes, and are prone to false positives; immunology Because the method is based on the specific recognition of antibodies to pathogens, it has good specificity and is easy to operate. Therefore, it is widely used in various fields of clinical and basic research. Although the conventional immunoassay method can achieve rapid detection, its sensitivity is low because the method usually recognizes the aggregation of gold nanoparticles by naked eyes. For this, we urgently need to develop a rapid and highly sensitive immunoassay method.

Streptococcal protein G (SPG) is a streptococcal cell wall protein that can bind to IgG antibodies from humans and various animals. It was first reported by Kronvall in 1973. The cell wall and culture supernatant of group A, C, and G streptococci all contain this protein, and the characteristics of SPG and staphylococcal protein A (SPA) are obviously different. In 1984, Bjorck et al. collectively referred to this protein as streptococcal protein G, and isolated and purified it. At present, although SPA has the characteristic of binding to the Fc end of antibodies, it is widely used in immunological experiments. However, compared with SPA, SPG has a stronger binding force to IgG and a wider binding spectrum (Cao Zhifang, 1989).

Starting from the N segment, SPG has three homologous structural regions A1, A2, and A3, and each homologous structure is composed of 24 amino acids, and these three homologous structures are separated by homologous regions B1 and B2 composed of 51 amino acids. followed by a spacer region S, followed by a homologous structural region C1, C2, and C3 composed of 55 amino acids, which are separated by D1 and D2 regions, followed by a hydrophilic region W after the C3 region, and finally For the M area (Sjobring, 1991). Studies have shown that the three homologous amino acid sequences C1, C2 and C3 of SPG are related to the binding of antibody IgG Fc terminal, and only 2 amino acid sequences are different between C1 and C2 regions, and 6 amino acid sequences are different between C1 and C3 regions. And the binding capacity of the C3 region to antibody IgG is equivalent to 7 times that of the C1 region (Kobatake, 1990). The A and B regions of SPG have the activity of binding to antibody Fab fragments and serum albumin, which are considered to interfere with the effect of antibodies and antigens and cause non-specific reactions.

Red fluorescent protein (RFP) is a bioluminescent protein isolated from the mushroom coral. It is a protein composed of about 248 amino acids. The fluorescence intensity emitted by RFP is higher than that of the best green fluorescent protein (GFP) mutation. higher body. It has the characteristics of strong penetrating power and long duration. DsRed2 is an artificial mutant gene derived from wild-type DsRed. The expression product of the mutant gene, RFP2, has a protein structure similar to that of green fluorescent protein (GFP). It can emit red fluorescence under natural light, which is a new visibility reporter gene with great potential.

The method of labeling active substances such as antibodies or G proteins such as red fluorescent protein involves chemical coupling, purification after coupling, dialysis, concentration and other cumbersome steps; and due to the characteristics of biological macromolecules, the coupling binding sites are diverse. Not only may cause the inactivation of biological macromolecules, but also affect the stability.

Contents of the invention

The technical problem mainly solved by the present invention is to provide a fast and highly sensitive immunoassay product and method to realize efficient and high-density immunoassay.

In order to realize the above technical solution, the present invention provides a tool protein capable of efficiently binding IgG, remodeling the IgG binding fragment of the SPG gene, and only retaining the C3 region where protein G can specifically bind to the Fc end of antibody IgG, And connected with RFP, the prepared recombinant protein has dual activity of fluorescent activity and binding to antibodies of different species.

In order to realize the purpose of the present invention, the present invention adopts following technical scheme:

A detection protein with red fluorescent activity, comprising a streptococcal protein G (SPG) fragment and a fluorescent protein, the streptococcal protein G (SPG) fragment is the C3 segment of SPG, and the fluorescent protein is red fluorescent protein;

Preferably, the nucleotide sequence of the C3 segment of SPG is shown in SEQ ID NO.6, and the amino acid sequence is shown in SEQ ID NO.7.

The nucleotide sequence of the optimized red fluorescent protein is shown in SEQ ID NO.8; the amino acid sequence is shown in SEQ ID NO.9.

The nucleotide sequence of the fused C3-RFP is shown in SEQ ID NO.10; the amino acid sequence is shown in SEQ ID NO.11.

Preferably, a detection protein with red fluorescent activity, the nucleotide sequence of the detection protein is shown in SEQ ID NO.5.

The present invention also provides a method for constructing a detection protein with red fluorescent activity, the method being:

(1) According to the gene sequence of the C3 fragment, PCR amplifies the C3 sequence from the bacterial solution containing the C3 fragment;

(2) Amplify the RFP sequence from the bacterial liquid containing the RFP sequence by PCR;

(3) The amplified RFP sequence was double-enzymatically digested and connected to the expression vector. After identification, the C3 fragment was double-digested and connected to construct the prokaryotic expression plasmid of the recombinant protein;

(4) Transfer the co-expression vector into the expression host for culture, and add inducible epialbumin after activation to the logarithmic growth phase;

(5) The fusion protein C3-RFP was prepared after fragmentation and purification

Wherein, the primer pair used in step (1) is shown as SEQ ID NO.1, 2;

The primer pair used in step (2) is shown in SEQ ID NO.3 and 4;

In the above method, the expression and purification of the fusion protein gene can be performed by conventional methods used in the art for protein expression and purification, such as cloning the fusion protein gene into an expression vector, transferring the expression vector and/or co-expression vector into an expression host for cultivation, After activation to the logarithmic growth phase, the induced epialbumin was added, and the fusion protein was obtained after crushing and purification. Among them, the present invention does not limit the types and types of expression vectors, co-expression vectors, and expression hosts. Vectors and hosts commonly used in the field for genetic modification can be selected. Specifically, the expression vectors can be pET-28, pET-32, For pET-15 or pET-11, etc., the co-expression vector can be pCDFDuet-1, etc.; the expression host can be selected from Escherichia coli, Bacillus subtilis, Bacillus megaterium, coryneform bacteria, Saccharomyces cerevisiae, Pichia pastoris or mammalian cells.

In the present invention, cloning can be accomplished by, for example, polymerase chain reaction (PCR).

The present invention also provides a product for immune analysis, which includes the fusion protein C3-RFP of the present invention.

Wherein, the form of product can be probe (sensor), test strip, chip, test kit etc., when using, the fusion protein C3-RFP of the present invention and other existing commercial reagent (such as enzyme-labeled antibody , fluorescently labeled antibodies, chromogenic reagents, substrates, etc.), can be used in various forms of immune analysis, such as antibody detection, antibody screening, antigen detection, pathogen detection, protein detection, protein interaction screening, high-throughput targets Protein detection, protein-nucleic acid interaction analysis, drug screening, etc.

When the product exists in the form of test strips, the complex of the present invention can be placed on the detection line for capturing target molecules, and gold-labeled nanoparticles can be used for detection; further, when the complex is fused with different functional ligands, it can be Realize the simultaneous detection of multiple target molecules.

In the present invention, immune analysis can be indirect immunization, sandwich immunization, etc., and can be used in various forms of immune analysis, such as antibody detection, antibody screening, antigen detection, pathogen detection, protein detection, protein interaction screening, high-throughput Target protein detection, protein-nucleic acid interaction analysis, drug screening, etc.

Beneficial effect

(1) The present invention adopts the method of molecular biology to reconstruct a new bifunctional molecule that does not exist in nature, the recombinant G protein and red fluorescent protein C3-RFP, not only the connecting fragment is designed to ensure that the two activities do not interact with each other. Interference, and codon optimization and purification tags. Since the protein is obtained through prokaryotic expression, the yield is high, and affinity chromatography can be performed through a purification tag, and the purity is guaranteed. The purified protein has been tested and has binding activity to IgG of multiple species, as well as red fluorescent activity. When applied to the detection of test strips and the like, it shows the high sensitivity of the inventive protein.

(2) The present invention remodels the IgG-binding fragment of the SPG gene, retains only the C3 region where protein G can specifically bind to the Fc end of antibody IgG, and connects RFP, and the prepared recombinant protein has fluorescence activity and is compatible with different species Dual activity of antibody binding.

(3) Based on the fusion protein C3-RFP, the present invention can efficiently and densely capture target molecules to achieve rapid and highly sensitive detection.

(4) The preparation process of the fusion protein C3-RFP of the present invention is simple and easy, and can be applied to different immunoassay modes, such as indirect ELISA, sandwich ELISA, etc., and is especially suitable for immunoassays in liquid phase. A reagent in the original detection method does not change the original operation steps and does not require additional equipment and instruments.

Description of drawings

Fig. 1 Schematic diagram of the structure of SPG.

Figure 2 PCR identification (A) and double enzyme digestion identification (B) of the prokaryotic expression plasmid pET-28a(+)-C3-RFP of the recombinant protein, in which,

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

B: M: DNA marker; 1: C3-RFP sequence obtained by double digestion of plasmid pET-28a(+)-C3-RFP, the size is 852bp; 2: Plasmid pET-28a(+)-C3-RFP The RFP sequence obtained by double enzyme digestion is 687bp in size.

Figure 3 SDS-PAGE analysis of pET-28a(+)-C3-RFP expression protein, in which, Figure A. SDS-PAGE analysis of the phase expression of pET-28a(+)-C3-RFP; Figure B. SDS-PAGE Analysis of pET-28a(+)-C3-RFP protein purification

A: M: protein markers; 0h: no IPTG induction; 1h-8h: IPTG induction 1h, 2h, 4h, 6h, 8h;

B: M: protein marker; 1: sonicated precipitate; 2: sonicated supernatant; 3: after sample loading; 4: Binding Buffer; 5: Wash Buffer; 6: Strip Buffer.

Figure 4 The expression of pET-28a(+)-C3-RFP and the observation of fluorescence activity after induction, where, 0h: no IPTG induction; 1h-8h: IPTG induction 1h, 2h, 4h, 6h, 8h

Figure 5 Western-blot analysis of pET-28a(+)-C3-RFP binding activity to IgG, in which,

A: M: Protein marker; 1: Binding activity of recombinant protein to sheep; 2: Binding activity of recombinant protein to donkey; 3: Binding activity of recombinant protein to mouse; 4: Binding activity of recombinant protein to rabbit

B: M: protein marker; 1: binding activity of recombinant protein to His antibody

Figure 6 Elisa analysis of affinity constants between pET-28a(+)-C3-RFP and IgG of multiple species, among them, Figure A: Elisa analysis of affinity constants between pET-28a(+)-C3-RFP and rabbit IgG Constant; Figure B: Elisa analysis of the affinity constant of pET-28a(+)-C3-RFP and donkey IgG; Figure C: Elisa analysis of the affinity of pET-28a(+)-C3-RFP and mouse IgG Constant; Figure D: Elisa analysis of the affinity constant between pET-28a(+)-C3-RFP and sheep IgG.

Detailed ways

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

The construction of embodiment 1 recombinant protein G

1.1 Biomaterials

Escherichia coli BL21 was purchased from Nanjing Novizan Biotechnology Co., Ltd.; the plasmid pET-28a(+) was stored in the laboratory, and the bacterial liquid containing the C3 fragment[1] and the bacterial liquid containing the RFP sequence[2] were used for the experiment Room preservation.

[1] Xu Rui, Zhao Dengyun, Hong Yang, Lu Ke, Li Hao, Lin Jiaojiao, Feng Jintao, Xu Yumei, Zhu Chuangang. Domain remodeling, expression and identification of streptococcal protein G [J]. Chinese Journal of Animal Infectious Diseases ,2015,23(05):46-52.

[2] Kang Zeran. Cloning, prokaryotic expression characteristics and biological information analysis of monomeric red fluorescent protein gene DsRed2[D]. Yanbian University, 2016.

1.2 Construction and synthesis of recombinant protein G gene sequence

According to the gene sequence of the C3 fragment, the primers were designed as shown in Table 1, and the C3 sequence was extracted from the bacterial solution containing the C3 fragment. At the same time, the RFP sequence was amplified from the bacterial solution containing the RFP sequence by PCR. Firstly, the amplified RFP sequence was double-enzymatically digested and then connected to pET-28a(+). After identification, the C3 fragment was double-enzymatically digested. After connecting, construct the prokaryotic expression plasmid pET-28a(+)-C3-RFP of the recombinant protein. (C3 fragment PCR corresponds to upstream and downstream primers: SEQ ID NO.1, 2; RFP PCR corresponds to upstream and downstream primers: SEQ ID NO.3, 4; full fragment PCR corresponds to upstream and downstream primers: SEQ ID NO.1, 4; C3 fragment Double digestion: BamH1, EcoR1; RFP fragment double digestion: EcoR1, Xhol1; full fragment double digestion: BamH1, Xhol1)

Table 1 Primer sequences

Note: The underlined part is the enzyme cutting site

1.3 Identification of recombinant plasmids

Perform PCR reaction on the synthesized gene sequence, the reaction system is as follows:

After mixing the components, briefly centrifuge and place in a PCR instrument for reaction. The reaction parameters are as follows:

The PCR product was identified by electrophoresis; at the same time, the product was identified by BamH1 and Xho1 double enzyme digestion (Figure 2). The electrophoresis results showed that the size of the entire fragment of the target gene was about 852bp; the size of the C3 fragment was about 165bp, and the size of the RFP fragment was about 687bp , same as predicted. At the same time, the PCR product was sequenced and identified, and the sequence was shown in SEQ ID NO.5 in the sequence table, and the sequence identification result showed that it was consistent with the designed sequence. (C3 fragment corresponds to upstream and downstream primers: SEQ ID NO.1, 2; RFP corresponds to upstream and downstream primers: SEQ ID NO.3, 4; full fragment corresponds to upstream and downstream primers: SEQ ID NO.1, 4)

Expression and purification of embodiment 2 recombinant protein G

2.1 Expression of recombinant plasmids

phase

(1) Transfer the pET-28a(+)-C3-RFP recombinant plasmids with correct identification results into BL21(DE3), inoculate them in 5ml LB liquid medium containing Kan+, and place them in a shaking incubator at 37°C. Shake culture at 250rpm.

(2) (2) When growing to the logarithmic phase (OD600 is about 0.6), add IPTG with a final concentration of 1 mmol/L to induce expression. Take 0.5ml bacterial liquid before induction and 1h, 2h, 4h, 6h, 8h after induction, and analyze the best induction time by SDS-PAGE electrophoresis. (Figure 3A)

Massive expression:

(1) Transform the pET-28a(+)-C3-RFP recombinant plasmids with correct identification results into BL21(DE3), inoculate them in 150ml LB liquid medium containing Kan+, and place them in a shaking incubator at 37°C. Shake culture at 250rpm.

(2) When growing to the logarithmic phase (OD600 is about 0.6), add IPTG with a final concentration of 1 mmol/L to induce expression. After 8 hours of induction, centrifuge at 12000rpm for 20min and discard the supernatant, resuspend the pellet with 20ml 1×PBS, freeze and thaw three times, sonicate in an ice bath for 20min (over 2s every 9s), then centrifuge at 12000rpm for 15min, collect the precipitate and supernatant .

(4) Resuspend the centrifuged pellet with 5ml 8mol urea, repeat the above centrifugation steps, and collect the supernatant.

(5) Add an equal volume of protein electrophoresis buffer to the supernatant after sonication and the supernatant after resuspension of the precipitate, and analyze the solubility of the expressed product by SDS-PAGE electrophoresis.

2.2 Purification of recombinant protein

Use the Ni-NTA Hisbind Resin kit (serial number: 70666-3) to purify the recombinant protein, and operate according to the product manual. The simple operation steps are as follows:

(1) Add 5ml of resin to a new empty column and let it stand for equilibrium. When the liquid level drops to the resin surface, add 15ml of ddH ₂ O, 25ml of 1×Charge Buffer, and 15ml of 1×Binding Buffer for washing.

(2) When the liquid level drops to the surface of the resin, add the supernatant solution after ultrasonic crushing, repeat the sample loading 3 times, and collect the solution after passing through the column.

(3) Add 50ml 1×Binding Buffer and 30ml 1×Wash Buffer successively for washing, and collect the solutions after passing through the column respectively.

(4) Add 20ml 1×Stripe Buffer to wash away Ni ions, and collect the solution after passing through the column.

(5) Add an appropriate amount of 1×Stripe Buffer to preserve the resin.

(6) Perform SDS-PAGE electrophoresis on the above collected solution to analyze the protein purification.

SDS-PAGE analysis showed (Figure 3) that the recombinant plasmid pET-28a(+)-C3-RFP was successfully expressed in Escherichia coli BL21(DE3), and the expression level increased with time 1-6h after induction with 1mmol/L IPTG The expression level reached the highest and tended to be stable after 8 hours of induction. At the same time, SDS-PAGE analysis showed that the protein expressed by the recombinant plasmid pET-28a(+)-C3-RFP was expressed in both the ultrasonic supernatant and the precipitate, and the protein content in the precipitate was higher than that in the supernatant, indicating that the The protein has a certain water solubility, and the inclusion body also exists at the same time.

Example 3 Activity identification of C3-RFP recombinant protein

3.1 Observation of fluorescence activity

Centrifuge the bacterial solution collected in the above phase at 10,000rpm for 5min, discard the supernatant, add 200ul ddH ₂ O to resuspend the bacterial cells, after resuspending, pipette 10ul onto a clean glass slide, cover with a cover glass, and place in the fluorescent electron Under the microscope, use RFP excitation light to excite, and observe whether the cells have red fluorescence. Fluorescent electron microscope observation (Figure 4) showed that the fluorescence of the recombinant plasmid pET-28a(+)-C3-RFP in Escherichia coli BL21(DE3) increased with time at 1-8 hours after the expression was successfully induced, and the fluorescence increased after 8 hours of induction reached a maximum and stabilized.

3.2 Western blotting to detect the binding activity of recombinant protein and IgG

(1) Perform SDS-PAGE electrophoresis on the purified protein, then transfer the protein to NC membrane, 130mA, 75min.

(2) Soak the NC membrane in 5% skimmed milk powder diluted with PBST, and seal at room temperature for 2 hours.

(3) Wash the blocked NC membrane three times with PBST, 5 min each time.

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

(5) Wash the incubated NC membrane three times with PBST, 10 min each time.

(6) The NC membrane was developed with DAB two-component chromogenic solution kit, and after color development, it was washed with running water to terminate the reaction.

The results show that the recombinant protein has the ability to bind to IgG from donkey, goat, mouse, rabbit, etc. (Figure 5)

3.3 ELISA method to determine the affinity constant of recombinant protein and IgG of different species

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

(2) Use the coating solution to dilute the protein from 10 μg/ml to a total of 8 dilutions, coat each well on a 96-well plate with 100 μl, set 3 replicates for each concentration, and coat at 4°C overnight.

(3) Wash the 96-well plate three times with PBST, 200 μl per well, 5 min each time.

(4) Add a solution of 5% skimmed milk powder diluted with PBST, 150 μl per well, and block at 37°C for 2 hours.

(5) Wash the 96-well plate three times with PBST, 200 μl per well, 5 min each time.

(6) HRP-labeled goat anti-mouse, rabbit anti-goat, mouse anti-rabbit, and donkey anti-goat four secondary antibodies were used in PBST at 1:500, 1:1000, 1:2000, 1:4000, 1: 8000 for doubling dilution, 100 μl per well, and incubated at 37°C for 2h.

(7) Wash the 96-well plate three times with PBST, 200 μl per well, 5 min each time.

(8) Add TMB for color development, 100 μl per well, and react at room temperature for 15 minutes.

(9) Add 2mol/L H ₂ SO ₄ to terminate the reaction, 30μl per well, and read the OD450 value.

The measured data are shown in Table 2, and the affinity curve is shown in Figure 6.

Table 2 Determination of recombinant protein and IgG affinity constants of different species by ELISA

K C3-RFP rabbit 1.9*10⁵ donkey 4.1*10⁵ mouse 1.7*10⁵ sheep 5.4*10⁵

The results showed that the SPG gene and red fluorescent protein (RFP) gene fragments were reconstructed in this experiment. The C3 region in SPG that can specifically bind to the Fc end of antibody IgG was retained, and then the RFP gene fragment was connected to construct the prokaryotic expression plasmid of the rSPG-RFP recombinant protein. The constructed prokaryotic expression plasmid successfully expressed the recombinant protein rSPG-RFP. The recombinant protein has both the ability of SPG to bind to IgG of different species and the function of RFP to emit fluorescence, and is a new type of dual-functional recombinant protein.

The fluorescent activity of recombinant proteins has gradually appeared in bacteria. It is generally believed that the correct folding of proteins is the basis for fluorescence. In the early stage of bacterial expression of recombinant protein, recombinant protein has appeared in SDS-PAGE phase analysis, but the corresponding fluorescent signal is not strong, which may be due to the need for a process of protein folding. By analyzing the fluorescence characteristics of the recombinant protein, the recombinant protein began to show strong fluorescence when it was expressed for 4 hours, and the fluorescence intensity increased several times at 8 hours. It was detected that the fluorescence intensity under 532nm excitation light and 582nm reflection light was not significantly different from that of RFP, suggesting that The recombinant protein rSPG-RFP was correctly folded, and the recombinant expression of the two segments of the gene did not affect its function.

It should be understood that although the content of the present invention has been described in detail above with general descriptions and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art of. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

<110> Shanghai Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences (Shanghai Branch of China Center for Animal Health and Epidemiology)

<120> A detection protein with red fluorescent activity and its application

<160> 11

<170> PatentIn version 3.5

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<213> C region upstream primer

<400>CGCGGATCCACCTACAAAAC

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<212>DNA

<213> C region downstream primer

<400>CCGGAATTCGCTACCG

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<211> 25

<212>DNA

<213> RFP region upstream primer

<400>CCCGGAATTCGCCTCCTCCGAGAAC

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<212>DNA

<213> RFP region downstream primer

<400>GGCCTCGAGCAGGAACAG

<210>5

<211> 891

<212>DNA

<213> Nucleotide sequence of recombinant protein C3-RFP

<400>CGCGGATCCACCTACAAACTGGTGATCAACGGCAAAACTTTAAAAGGTGAGACCACCACAAAAGCAGTGGATGCCGAGACCGCACAGAAGGCCTTCAAGCAGTATGCCAACGACAATGGCGTGGATGGCGTGTGGACCTATGACGATGCCACCAAAACCTTCCGCGTGACAGAGGGCGGTGGTGGTAGTGGTGGTGGCGGTAGCGAATTCGCCTCCTCCGAGAACGTCATCACCGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCACCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCCACAACACCGTGAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCGCCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGCGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCTGCTTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTGATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGACCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTGGACGCCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCACCGAGGGCCGCCACCACCTGTTCCTGTAGCTCGAG

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<213> Nucleotide sequence of the C3 fragment of SPG

<400>ACTTACAAACTTGTTATTAATGGTAAAACGCTGAAGGGTGAAACCACCACCAAGCGGTGGATGCCGAAACCGCGCAGAAGGCCTTTAAGCAGTACGCCAACGACAATGGCGT GGATGGTGTTTGGACCTACGACGACGCGACCAAAACCTTTCGTGTTACCGAA

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<213> Amino acid sequence of the C3 fragment of SPG

<400>TYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVDGVWTYDDATKTFRVTE

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<212>DNA

<213> Nucleotide sequence after RFP optimization:

<400>GAA TTC GCC TCC TCC GAG AAC GTC ACC GAG TTC ATG CGC TTC AAA GTGCGC ATG GAA GGC ACC GTG AAT GGC CAC GAG TTC GAG ATT GAA GGC GAA GGT GAA GGCCGC CCA TAC GAA GGC CAC AAC ACC GTG AAG CTG AAG GTG ACC AAA GGC GGT CCA CTGCCG TTC GCG TGG GAT ATT CTG AGC CCG CAG TTC CAG TAC GGC AGC AAG GTG TAC GTTAAG CAT CCG GCG GAC ATC CCG GAT TAC AAG AAG CTG AGC TTC CCG GAA GGC TTC AAATGG GAG CGC GTG ATG AAC TTC GAG GAC GGC GGC GTT GCC ACG GTT ACC CAA GAT AGTAGT CTG CAA GAT GGC TGC TTC ATC TAC AAG GTG AAG TTC ATC GGC GTG AAC TTC CCGAGC GAT GGC CCA GTG ATG CAG AAA AAG ACC ATG GGC TGG GAA GCG AGC ACC GAA CGTCTG TAC CCG CGC GAT GGT GTT CTG AAA GGC GAA ACG CAC AAG GCG CTG AAA CTG AAAGAC GGC GGC CAC TAC CTC GTG GAG TTC AAG AGC ATC TAC ATG GCC AAA AAG CCG GTGCAA CTG CCG GGC TAT TAC TAC GTG GAC GCC AAG CTG GAC ATC ACC AGC CAT AAC GAGGAC TAC ACG ATC GTG GAG CAG TAC GAA CGC ACC GAA GGT CGC CAC CAC CTG TTC CTG

<210>9

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<212> PROTEIN

<213> RFP amino acid sequence

<400>EFASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVKLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHHLFL

<210>10

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<212>DNA

<213> C3-RFP nucleotide sequence

<400>GGA TCC ACC TAC AAA CTG GTG ATC AAC GGC AAA ACT TTA AAA GGT GAG ACCACC ACA AAA GCA GTG GAT GCC GAG ACC GCA CAG AAG GCC TTC AAG CAG TAT GCC AACGAC AAT GGC GTG GAT GGC GTG TGG ACC TAT GAC GAT GCC ACC AAA ACC TTC CGC GTGACA GAG GGC GGT GGT GGT AGT GGT GGT GGC GGT AGC GAA TTC GCC TCC TCC GAG AACGTC ATC ACC GAG TTC ATG CGC TTC AAA GTG CGC ATG GAA GGC ACC GTG AAT GGC CACGAG TTC GAG ATT GAA GGC GAA GGT GAA GGC CGC CCA TAC GAA GGC CAC AAC ACC GTGAAG CTG AAG GTG ACC AAA GGC GGT CCA CTG CCG TTC GCG TGG GAT ATT CTG AGC CCGCAG TTC CAG TAC GGC AGC AAG GTG TAC GTT AAG CAT CCG GCG GAC ATC CCG GAT TACAAG AAG CTG AGC TTC CCG GAA GGC TTC AAA TGG GAG CGC GTG ATG AAC TTC GAG GACGGC GGC GTT GCC ACG GTT ACC CAA GAT AGT AGT CTG CAA GAT GGC TGC TTC ATC TACAAG GTG AAG TTC ATC GGC GTG AAC TTC CCG AGC GAT GGC CCA GTG ATG CAG AAA AAGACC ATG GGC TGG GAA GCG AGC ACC GAA CGT CTG TAC CCG CGC GAT GGT GTT CTG AAAGGC GAA ACG CAC AAG GCG CTG AAA CTG AAA GAC GGC GGC CAC TAC CTC GTG GAG TTCAAG AGC ATC TAC ATG GCC AAA AAG CCG GTG CAA CTG CCG GGC TAT TAC TAC GTG GACGCC AAG CTG GAC ATC ACC AGC CAT AAC GAG GAC TAC ACG ATC GTG GAG CAG TAC GAACGC ACC GAA GGT CGC CAC CAC CTG TTC CTG TAG CTC GAG

<210>11

<211> 295

<212> protein

<213>C3-RFP amino acid sequence:

<400>GSTYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVDGVWTYDDATKTFRVTEGGGGSGGGGSEFASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVKLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHHLFLLE

Claims (8)

1. A detection protein with red fluorescent activity, characterized in that the detection protein comprises a streptococcal protein G (SPG) fragment and a fluorescent protein, and the streptococcal protein G (SPG) fragment is the C3 segment of SPG, so The above-mentioned fluorescent protein is an optimized red fluorescent protein, and the detection protein has dual activities of fluorescent activity and binding to antibodies of different species. 2. A detection protein with red fluorescent activity as claimed in claim 1, wherein the nucleotide sequence of the C3 segment of SPG is shown in SEQ ID NO.6, and the amino acid sequence is shown in SEQ ID NO.7; The nucleotide sequence of the optimized red fluorescent protein is shown in SEQ ID NO.8; the amino acid sequence is shown in SEQ ID NO.9. 3. A kind of detection protein with red fluorescent activity as claimed in claim 1, the nucleotide sequence of described detection protein C3-RFP is as shown in SEQ ID NO.10; Amino acid sequence is as shown in SEQ ID NO.11 Show. 4. A construction method for preparing a detection protein with red fluorescent activity, the method is: According to the gene sequence of the C3 fragment, the C3 sequence is amplified by PCR from the bacterial solution containing the C3 fragment; Amplify the RFP sequence from the bacterial solution containing the RFP sequence by PCR; Firstly, the amplified RFP sequence was double-enzymatically digested and then connected to the expression vector. After identification, the C3 fragment was double-digested and connected to construct the prokaryotic expression plasmid of the recombinant protein; Transfer the co-expression vector into the expression host for culture, activate to the logarithmic growth phase and add inducible epibumin; Fusion protein C3-RFP was obtained after fragmentation and purification Wherein, the primer pair used in step (1) is shown as SEQ ID NO.1, 2; The primer pair used in step (2) is shown in SEQ ID NO.3,4. 5. The construction method according to claim 4, wherein the nucleotide sequence of the detection protein C3-RFP is shown in SEQ ID NO.10; the amino acid sequence is shown in SEQ ID NO.11. 6. The application of the detection protein with red fluorescent activity as claimed in claim 1 in the in vitro immunological detection of antibodies. 7. The application of the detection protein with red fluorescence activity as claimed in claim 1 in purifying antibodies. 8. the detection protein with red fluorescent activity as claimed in claim 1 prepares the application of immunoassay product, and described application is probe (sensor), test strip, chip, test kit etc., when using, claim The fusion protein C3-RFP described in 1 is mixed with other existing commercial reagents (such as enzyme-labeled antibodies, fluorescently-labeled antibodies, chromogenic reagents, substrates, etc.).