CN110615844A

CN110615844A - Multi-species universal detection protein with green fluorescence activity and application thereof

Info

Publication number: CN110615844A
Application number: CN201910792298.7A
Authority: CN
Inventors: 朱传刚; 纪荣毅; 沈元曦; 林矫矫; 洪炀; 岳永程
Original assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Current assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center; Shanghai Veterinary Research Institute CAAS
Priority date: 2019-08-26
Filing date: 2019-08-26
Publication date: 2019-12-27

Abstract

The invention provides a multi-species universal detection protein with green fluorescence activity and application thereof, and is characterized in that the detection protein comprises a Streptococcal Protein G (SPG) fragment and a fluorescent protein, an IgG binding fragment of an SPG gene is reconstructed, only a C3 region of the protein G which can be specifically combined with an Fc end of an antibody IgG is reserved, the detection protein is divided into three groups, namely C3, C3-D-C3, C3-D-C3-D-C3, and the three groups are all connected with EGFP, and the prepared recombinant protein has dual activities of the fluorescent activity and the combination with antibodies of different species. The evolved immunoglobulin binding molecules disclosed herein can bind a broad spectrum of total IgG including human and various animals and IgG of different subclasses, and have a binding force higher than that of existing immunoglobulin binding molecules, as compared to immunoglobulin binding molecules of the prior art.

Description

Multi-species universal detection protein with green fluorescence activity and application thereof

Technical Field

The invention relates to the field of immunodetection, and particularly relates to a multi-species universal detection protein with green fluorescence activity and application thereof.

Background

The wide-range epidemic spread of various infectious diseases poses serious threats to national security and population health of China. The development of sensitive and accurate pathogen analysis methods and detection techniques is of great significance for the rapid diagnosis and timely treatment of related diseases, the effective prevention and rapid treatment of bioterrorism and emergent public health events. The classical microorganism separation and identification are troublesome and time-consuming, and are difficult to be applied to the field rapid detection of pathogenic microorganisms; detection methods based on pathogenic nucleic acids mostly have higher detection sensitivity, but require complex nucleic acid extraction processes and are easy to generate false positives; the immunological method has good specificity due to the specific recognition effect of the antibody on the pathogen, and is simple to operate, so the immunological method is widely applied to various fields of clinical and basic research. Although the conventional immunoassay method can realize rapid detection, the method has low sensitivity because the method usually recognizes nanogold aggregation color development by naked eyes. In view of the above, there is a need to develop a rapid and highly sensitive immunoassay method.

Streptococcal Protein G (SPG) is a streptococcal cell wall protein capable of binding to human and various animal antibodies IgG, SPG starts from segment N and has three homologous structure regions A1, A2 and A3, each homologous structure is composed of 24 amino acids, and the three homologous structures are separated by homologous regions B1 and B2 composed of 51 amino acids, then a spacing region S, then homologous structure regions C1, C2 and C3 composed of 55 amino acids, which are separated by D1 and D2 regions, and the C3 region is followed by hydrophilic region W and finally M region (Sjobring, 1991). It has been shown that three homologous amino acid sequences of the SPG, the C1, C2, and C3 regions, are associated with binding to the Fc-terminus of antibody IgG, and that the C1 and C2 regions differ by only 2 amino acid sequences, the C1 and C3 regions differ by 6 amino acid sequences, and that the binding capacity of the C3 region to antibody IgG is equivalent to 7-fold that of the C1 region (Kobatake, 1990). The A, B region of SPG has binding activity to the Fab fragment of antibodies and binding activity to serum albumin, and is thought to interfere with the action of antibodies and antigens and to cause nonspecific reactions.

GFP is a protein consisting of about 238 amino acids, which is excited by blue light to ultraviolet light and emits green fluorescence. Since fluorescent proteins are stably inherited in progeny and can be expressed specifically according to a promoter, they are often used as reporter genes. Slowly replace traditional chemical dyes in the need of quantification or other experiments. Enhanced Green Fluorescent Protein (EGFP) is a GFP mutant line, and the intensity of emitted fluorescence is more than 6 times greater than that of GFP, so that EGFP is more suitable to be used as a reporter gene to study gene expression, regulation, cell differentiation, Protein localization and transportation in organisms and the like.

A method for labeling active substances such as antibodies or G protein and the like by green fluorescent protein and the like relates to the complicated steps of chemical coupling, purification after coupling, dialysis, concentration and the like; due to the characteristics of biological macromolecules, coupled binding sites are various, so that the fluorescence activity of green fluorescent protein can be changed and even inactivated, and the activity of the marked biological macromolecules is easily changed or inactivated. And due to the uncertainty of the coupling site, the stability of the reagent is affected. The invention adopts a molecular biology method to reconstruct a novel bifunctional molecule which does not exist in the nature, the recombinant G protein and the green fluorescent protein rG-EGFP, and a connecting fragment is designed during construction to ensure that the two activities do not interfere with each other, and codon optimization and purification labels are carried out. The protein is obtained by prokaryotic expression, so that the yield is high, affinity chromatography can be performed through a purification label, and the purity is ensured. The purified protein has the binding activity with multi-species IgG and green fluorescence activity after detection. When the protein is applied to test paper and other tests, the high sensitivity of the protein is shown.

Disclosure of Invention

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

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

in order to realize the technical scheme, the invention provides a tool protein capable of efficiently combining IgG, the IgG combination fragment of an SPG gene is reconstructed, only a C3 region of protein G capable of being specifically combined with the Fc end of antibody IgG is reserved and divided into three groups, namely C3, C3-D-C3, C3-D-C3-D-C3, and all the groups are connected with EGFP, and the prepared recombinant protein has dual activities of fluorescence activity and combination with antibodies of different species.

A detection protein with green fluorescence activity, which comprises a Streptococcal Protein G (SPG) fragment and a fluorescent protein, wherein the Streptococcal Protein G (SPG) fragment is a sequence containing a C3 segment of SPG, preferably one of C3, C3-D-C3, C3-D-C3-D-C3, and the fluorescent protein is green fluorescent protein;

preferably, the optimized IgG binding fragment C3 has the nucleotide sequence shown in SEQ ID NO. 11;

the optimized D fragment nucleotide sequence of the SPG gene is shown as SEQ ID NO. 12;

the Linker nucleotide sequence between the optimized C3 and EGFP segment is shown in SEQ ID NO. 13;

the nucleotide sequence of the optimized EGFP protein is shown as SEQ ID NO. 14;

preferably, the nucleotide sequence of the detection protein with green fluorescence activity is shown in one of SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO. 9; the amino acid sequence of the detection protein is shown in one of SEQ ID NO.6, 8 and 10.

The invention also provides a construction method of the detection protein with green fluorescence activity, which comprises the following steps:

(1) according to the gene sequence of the C3 fragment, PCR amplifying C3, C3DC3 and C3DC3DC3 sequences from a bacterial liquid containing the C3 fragment;

(2) amplifying an EGFP sequence from a bacterial liquid containing the EGFP sequence by PCR;

(3) firstly, connecting the amplified EGFP sequence to an expression vector after double enzyme digestion, and respectively connecting C3, C3DC3 and C3DC3DC3 fragments after identification to construct a prokaryotic expression plasmid of the recombinant protein;

(4) transferring the expression vector and the co-expression vector into an expression host for culture, and adding induced epialbumin after the activation to a logarithmic growth phase;

(5) after crushing and purification, fusion proteins C3-EGFP, C3DC3-EGFP and C3DC3DC3-EGFP are prepared.

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

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

in the above method, the expression and purification of the fusion protein gene can be performed by methods conventionally used in the art for protein expression and purification, for example, cloning the fusion protein gene into an expression vector, transferring the expression vector and/or a co-expression vector into an expression host for culture, adding the induced surface albumin after activation to a logarithmic growth phase, and obtaining the fusion protein after crushing and purification. The invention does not limit the types and categories of the expression vector, the co-expression vector and the expression host, and can select the vector and the host which are used for genetic modification in the field, concretely, the expression vector can be pET-28, pET-32, pET-15 or pET-11, etc., and the co-expression vector can be pCDFDuet-1, etc.; the expression host may be selected from E.coli, B.subtilis, B.megaterium, Corynebacterium, Saccharomyces cerevisiae, Pichia pastoris or mammalian cells.

In the present invention, cloning can be performed by, for example, chain enzyme Polymerization (PCR).

The invention also provides a product for immunoassay, which comprises the fusion protein.

When the product is used, the fusion protein disclosed by the invention is mixed with other existing commercial reagents (such as an enzyme-labeled antibody, a fluorescence-labeled antibody, a color developing agent, a substrate and the like), and can be used for various forms of immunoassay, such as antibody detection, antibody screening, antigen detection, pathogen detection, protein interaction screening, high-throughput target protein detection, protein-nucleic acid interaction analysis, drug screening and the like.

When the product exists in the form of a test strip, the compound can be placed on a gold-labeled pad for labeling antibody molecules; furthermore, when the complex is fused with different functional ligands, the simultaneous detection of multiple target molecules can be realized.

When the product exists in the form of a kit, the kit can also comprise buffer solution, washing solution, diluent or color developing agent and the like.

In the present invention, the immunoassay can be indirect immunoassay, sandwich immunoassay, etc., and can be used for various types of immunoassays, such as antibody detection, antibody screening, antigen detection, pathogen detection, protein interaction screening, high-throughput target protein detection, protein-nucleic acid interaction analysis, drug screening, etc.

Advantageous effects

(1) According to the invention, an IgG binding fragment of the SPG gene is reconstructed, only a C3 region of protein G capable of being specifically bound with an Fc end of an antibody IgG is reserved, and EGFP is connected, so that the prepared recombinant protein has dual activities of fluorescence activity and binding with antibodies of different species.

(2) The fusion protein can efficiently and high-density capture target molecules, and achieves rapid and high-sensitivity detection.

(3) The fusion protein of the invention has simple and easy preparation process, can be suitable for different immunoassay modes, such as indirect ELISA, sandwich ELISA and the like, is particularly suitable for immunoassay in liquid phase, only replaces one reagent in the original detection method, does not change the original operation steps, and does not need additional equipment and instruments.

Drawings

FIG. 1A: structural schematic diagram of SPG; b: schematic diagram of the spliced protein model structure.

FIG. 2 shows PCR identification of recombinant protein prokaryotic expression plasmid, wherein,

a: the PCR identification result of C3-EGFP (M: M: protein marker; region 1: C3; region 2: EGFP; region 3: whole fragment);

b: the PCR identification result of C3DC3-EGFP (M: M: protein marker; region 1: C3; region 2: EGFP; region 3: whole fragment);

c: the result of PCR identification of C3DC3DC3-EGFP (M: M: protein marker; region 1: C3; region 2: EGFP; region 3: whole fragment).

FIG. 3 SDS-PAGE analysis of recombinantly expressed proteins, wherein,

a: c3-phase of EGFP. M: a protein marker; 0 h: no IPTG induction; 1.2, 4, 6, 7: IPTG induction is carried out for 1h, 2h, 4h, 6h and 7 h;

b: solubility analysis of C3-EGFP. M: a protein marker; 1: ultrasonically crushing the precipitate; 2: ultrasonically crushing the supernatant; 3: detecting protein after sample loading; b: the filtrate after Binding buffer elution; w: the filtrate after Wash buffer elution; s: the target protein after elution of the Stripbuffer;

c: c3DC 3-phase of EGFP. M: a protein marker; 0 h: no IPTG induction; 1.2, 4, 6, 8: IPTG induction is carried out for 1h, 2h, 4h, 6h and 8 h;

d: solubility analysis of C3DC 3-EGFP. M: a protein marker; 1: ultrasonically crushing the supernatant; 2: ultrasonically crushing the precipitate; 3: detecting protein before sample loading; 4: detecting protein after sample loading; b: the filtrate after Binding buffer elution; w: the filtrate after Wash buffer elution; s: target protein after Strip buffer elution;

e: phase of C3DC3DC 3-EGFP. M: a protein marker; 0 h: no IPTG induction; 1.2, 4, 6: IPTG induction is carried out for 1h, 2h, 4h and 6 h;

f: solubility analysis of C3DC3DC 3-EGFP. M: a protein marker; 1: ultrasonically crushing the supernatant; 2: ultrasonically crushing the precipitate; 3: detecting protein before sample loading; 4: detecting protein after sample loading; b: the filtrate after Binding buffer elution; w: filtrate after Washbuffer elution; s: target protein after Strip buffer elution;

FIG. 4A: scanning the excitation spectrum of the rSPG-RFP by using a fluorescence spectrophotometer; b: the emission spectrum of the recombinant protein was obtained with the maximum excitation light wavelength.

FIG. 5 binding Activity assay of recombinant proteins with different species IgG

A: donkey anti-goat; b: goat anti-mouse; c: mouse anti-rabbit; d: rabbit anti-goat;

m: marker; 1: C3-EGFP protein; 2: c3DC3-EGFP protein; 3: c3DC3DC3-EGFP protein

Fig. 6 Elisa assay analyzes affinity constants of recombinant proteins with multi-species IgG, wherein a: determining the affinity constant of C3-EGFP and IgG of different species; b: determining the affinity constant of the C3DC3-EGFP and IgG of different species; c: c3DC3DC3-EGFP was determined with IgG affinity constants for different species.

FIG. 7: and D, carrying out fluorescence staining observation on the metacercaria A, carrying out observation on the metacercaria under natural light, and carrying out observation under the excitation wavelength of green fluorescence.

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 proteins

1.1 biological Material

Escherichia coli BL21 was purchased from Nanjing Novozam Biotech, Inc.; plasmid pET-28a (+) for laboratory preservation, SPG (Series, Zhao Dengyun, hong Yang, Lu Ke, Lihao, Lin correction, Von gold Tao, Xu Yumei, Zhu Xing streptococcal protein G domain reconstruction, expression and identification [ J ] Chinese animal infectious disease journal, 2015,23(05): 46-52.) EGFP corresponds to the sequence number on NCBI: u55762

1.2 construction and Synthesis of recombinant protein Gene sequences (see FIG. 1B)

From GenBank, published gene fragments of the C region coding EGFP and SPG are searched, and the C1, C2, C3 and D regions are found to obtain a gene fragment of the C3 region. And (3) carrying out signal peptide analysis on the EGFP sequence, detecting whether the gene sequence contains escherichia coli rare codons, namely, the frequency of the EGFP sequence is less than 10%, replacing the EGFP sequence with codons which are preferred by escherichia coli and used for coding uniform amino acids, and finally adding a TAA stop codon at the 3' end. Primer5 software design primers, add cleavage sites to the upstream and downstream primers respectively, the Primer sequences are as follows:

primer name	Primer sequences
		C region upstream primer	CGCGGATCCACCTACAAAC(SEQ ID NO.1)
C-region downstream primer	CCGGAATTCGCTACCG(SEQ ID NO.2)
		EGFP region upstream primer	CCGGAATTCGTGAGTAAAG(SEQ ID NO.3)
EGFP region downstream primer	GGCCTCGAGGGCTAGCTCTTATTTG(SEQ ID NO.4)

Note: the underlined part is an enzyme cleavage site

DNA Star software is used for analyzing an amino acid sequence, a theoretical molecular weight and an isoelectric point expressed by a recombination sequence, and the recombination sequence respectively constructs the following three plasmids according to the invention conception:

(1) pET-28a (+) -C3-EGFP, wherein the nucleotide sequence of the C3-EGFP fusion protein is shown in SEQ ID NO. 5: the coded amino acid sequence is shown in SEQ ID NO. 6:

(2) the nucleotide sequence of pET-28a (+) -C3DC3-EGFP, C3DC3-EGFP fusion protein is shown in SEQ ID NO. 7: the coded amino acid sequence is shown in SEQ ID NO. 8:

(3) the nucleotide sequence of pET-28a (+) -C3DC3DC3-EGFP, C3DC3DC3-EGFP fusion protein is shown in SEQ ID NO. 9: the coded amino acid sequence is shown as SEQ ID NO. 10. (see Table 1)

The construction of the above three plasmids is a routine procedure in the art, and is briefly described as follows:

(1) the published SPG sequence and EGFP sequence were analyzed, codon optimized, followed by full-fragment synthesis, cloned into PET-28a (+) plasmid, and transformed into DH5a competent bacteria. And (5) sequencing and verifying that the glycerol strain is correctly preserved.

(2) According to the gene sequence of the C3 fragment, PCR amplifying C3, C3DC3 and C3DC3DC3 sequences from a bacterial liquid containing the C3 fragment;

(3) amplification of EGFP sequences from bacterial solutions containing EGFP sequences by PCR

(4) Firstly, connecting the amplified EGFP sequence to an expression vector after double enzyme digestion, and respectively connecting C3, C3DC3 and C3DC3DC3 fragments after identification to construct a prokaryotic expression plasmid of the recombinant protein;

(5) transferring the expression vector and the co-expression vector into an expression host for culture, and adding induced epialbumin after the activation to a logarithmic growth phase;

(6) after crushing and purification, fusion proteins C3-EGFP, C3DC3-EGFP and C3DC3DC3-EGFP are prepared.

TABLE 1 recombinant C3-EGFP sequence information

Abbreviations	Full scale	Length of sequence	Encoding amino acids	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

1.3 recombinant plasmid identification

Carrying out PCR reaction on the synthesized gene sequence, wherein the reaction system is as follows:

after the components are mixed uniformly, the mixture is placed in a PCR instrument for reaction through short-time centrifugation, and the reaction parameters are as follows:

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

Carrying out electrophoretic identification on the PCR product; the sequencing identity showed agreement with the designed sequence (FIG. 2). The size of the whole fragment of the C3-EGFP target gene is about 950 bp. The size of the C3 fragment is about 170bp, and the size of the EGFP fragment is about 750 bp. And simultaneously, sequencing and identifying the PCR product, wherein the sequencing and identifying result shows that the sequence is consistent with the designed sequence. (the C3 fragment corresponds to the upstream and downstream primers SEQ ID NO.1 and 2; the EGFP corresponds to the upstream and downstream primers SEQ ID NO.3 and 4; and the whole fragment corresponds to the upstream and downstream primers SEQ ID NO.1 and 4).

EXAMPLE 2 expression and purification of recombinant proteins

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. (FIGS. 3A, C, E)

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. (FIGS. 3B, D, F)

2.2 purification of recombinant proteins

The recombinant protein was purified using Ni-NTA Hisbind Resin (Ni-NTA SEQ ID NO: 70666-3) according to the kit instructions, and the simple procedures were 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 of 1 × Charge Buffer, 15ml of 1 × Binding Buffer solution.

(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-EGFP 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 6h induction. The three proteins are expressed in 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 body also exists at the same time. Protein hanging columns can be confirmed before and after loading, a large amount of hybrid protein is eluted in the Binding and Wash Buffer steps, and finally purified protein is obtained after Strip of Strip Buffer.

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

3.1 fluorescence Spectroscopy

Preparing different concentrations of the purified recombinant protein, and scanning an excitation spectrum (figure 4A) of the recombinant protein by using a fluorescence spectrophotometer to obtain the maximum excitation wavelength of the recombinant protein; the emission spectrum of the recombinant protein was obtained with the maximum excitation light wavelength (fig. 4B). And observing the fluorescence intensity of the three recombinant proteins. The fluorescence intensity of the three recombinant proteins is higher than that of a standard EGFP, wherein the fluorescence intensity of C3-EGFP is strongest, the fluorescence intensity of C3DC3-EGFP is second, and the fluorescence intensity of C3DC3-EGFP is weakest, which is probably related to the sizes of a C3 region and an EGFP fragment of the recombinant protein, a C3 fragment in the C3-EGFP is only one third of the size of the EGFP fragment, so that the proteins expressed by the two fragments are less structurally interfered with each other, and the C3 functional region and the EGFP functional region of the expressed protein are folded with each other to reduce the fluorescence intensity of the EGFP as the number of C3 is increased.

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) NC membranes were incubated with HRP-labeled IgG of different species (diluted with PBST 1: 2000) as antibody for 1h at room temperature.

(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 all three recombinant proteins had binding ability to donkey, mouse, rabbit, and sheep IgG (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, i.e., goat anti-mouse antibody, rabbit anti-goat antibody, mouse anti-rabbit antibody and donkey anti-goat antibody, labeled with HRP were diluted by PBST at a ratio of 1:500, 1:1000, 1:2000 and 1:4000, respectively, and incubated at 37 ℃ for 2 hours in 100. mu.l each well.

(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, 30. mu.l per well and OD450 values read。

The three recombinant proteins after dialysis were measured for concentration, and 96-well plates were coated with different concentrations as antigens, and then two antibodies diluted in multiple ratios were added to the plates to bind to the antigens. Curve fitting was performed with OD450 values as ordinate and log values of antigen concentration as abscissa, and the affinity curve is shown in fig. 6.

Substituting the fitted curves into corresponding formulas, respectively calculating affinity constants Ka, taking the average of the obtained Ka values, and obtaining the affinity constant values of the three recombinant proteins, wherein the results are shown in Table 2.

TABLE 2 affinity constants of the three recombinant proteins

	C3-EGFP	C3DC3-EGFP	C3DC3DC3-EGFP
				Goat	1.0×10⁸	1.8×10⁸	1.9×10⁸
Donkey meat	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 results show that the affinity activities of the three proteins and different species are in positive correlation with the number of C3 regions respectively. The three proteins have different IgG affinity activities with different species, and have the highest affinity activity with goats, and are donkey, mouse and rabbit respectively.

Example 4 fluorescent staining of Living Miercaria

The detection method comprises the following steps:

(1) incubating schistosoma japonicum miracidium, collecting the liquid containing miracidium in 1.5ml EP tube, centrifuging, and removing most of liquid

(2) Adding positive serum of Schistosoma japonicum to the dilution of 1:20, adding recombinant protein to the final concentration of 0.1mg/ml, and incubating at 37 deg.C for 2 h.

(3) Centrifuging at 2000rpm for 1min, discarding most of the supernatant, and adding ddH₂0 to the original volume, 2000rpm, 1min, abandoning most of the supernatant, and repeating twice;

(4) washing ddH containing miracidium₂0, sucking 10ul of the obtained product on a glass slide, covering the glass slide, and observing whether the miracidium is subjected to green fluorescence staining under a fluorescence microscope and natural light and green fluorescence excitation wavelength respectively. The results are shown in FIG. 7.

The result shows that the SPG gene and the EGFP gene fragment are reconstructed in the experiment. And (2) a C3 region which can be specifically combined with the Fc end of the antibody IgG in the SPG is reserved, and then the SPG is connected with the EGFP gene fragment, and the recombinant protein is successfully expressed by a prokaryotic expression plasmid constructed by the prokaryotic expression plasmid for constructing the rSPG-EGFP recombinant protein. The recombinant protein has the ability of combining SPG with IgG of different species and the function of EGFP emission fluorescence, and is a novel bifunctional recombinant protein.

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> multi-species universal detection protein with green fluorescence activity and application thereof

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 19

<212> DNA

<213> C region upstream primer

<400>CGCGGATCCACCTACAAAC

<210>2

<211> 16

<212> DNA

<213> C region downstream primer

<400>CCGGAATTCGCTACCG

<210>3

<211> 19

<212> DNA

<213> EGFP region upstream primer

<400>CCGGAATTCGTGAGTAAAG

<210>4

<211> 25

<212> DNA

<213> EGFP region downstream primer

<400>GGCCTCGAGGGCTAGCTCTTATTTG

<210>5

<211>1030

<212> DNA

<213> nucleotide sequence of recombinant protein 1C3-EGFP

<400>ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCACTTACAAACTGGTTATTAATGGTAAAACCTTGAAAGGCGAAACAACTACTAAAGCAGTAGACGCAGAAACTGCACAAAAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGGTGTTTGGACTTATGATGATGCGACTAAGACCTTTAGGGTAACTGAAGGCGGTGGGGGCTCAGGAGGTGGGGGCTCAGAATTCGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAACTCGAGGCC

<210>6

<211>344

<212>protein

<213> protein sequence of recombinant protein 1C3-EGFP

<400>MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRRGSTYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVDGVWTYDDATKTFRVTEGGGGSGGGGSEFVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK-LEA

<210>7

<211>1239

<212> DNA

<213> nucleotide sequence of recombinant protein C3DC3-EGFP

<400>ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCACTTACAAACTGGTTATTAATGGTAAAACCTTGAAAGGCGAAACAACTACTAAAGCAGTAGACGCAGAAACTGCACAAAAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGGTGTTTGGACTTATGATGATGCGACTAAGACCTTTAGGGTAACTGAAAAACCAGAAGTGATCGATGCGTCTGAATTAACACCAGCCGTGACAACTTACAAACTGGTTATTAATGGTAAAACCTTGAAAGGCGAAACAACTACTAAAGCAGTAGACGCAGAAACTGCACAAAAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGGTGTTTGGACTTATGATGATGCGACTAAGACCTTTAGGGTAACTGAAGGCGGTGGGGGCTCAGGAGGTGGGGGCTCAGAATTCGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAGCTAGCC

<210>8

<211>413

<212> protein

<213> amino acid sequence of recombinant protein C3DC3-EGFP

<400>MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSTYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVDGVWTYDDATKTFRVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVDGVWTYDDATKTFRVTEGGGGSGGGGSEFVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK-ELA

<210>9

<211>1455

<212> DNA

<213> nucleotide sequence of recombinant protein C3DC3DC3-EGFP

<400>ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCACTTACAAACTTGTTATTAATGGTAAAACATTGAAAGGCGAAACAACTACTAAAGCAGTAGACGCAGAAACTGCACAAAAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGGTGTTTGGACTTATGATGATGCGACTAAGACCTTTAGGGTAACTGAAGGCAAACCAGAAGTGATCGATGCGTCTGAATTAACACCAGCCGTGACAACTTACAAACTTGTTATTAATGGTAAAACATTGAAAGGCGAAACAACTACTAAAGCAGTAGACGCAGAAACTGCACAAAAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGGTGTTTGGACTTATGATGATGCGACTAAGACCTTTAGGGTAACTGAAGGCAAACCAGAAGTGATCGATGCGTCTGAATTAACACCAGCCGTGACAACTTACAAACTTGTTATTAATGGTAAAACATTGAAAGGCGAAACAACTACTAAAGCAGTAGACGCAGAAACTGCACAAAAAGCCTTCAAACAATACGCTAACGACAACGGTGTTGATGGTGTTTGGACTTATGATGATGCGACTAAGACCTTTAGGGTAACTGAAGGCGGTGGGGGCTCAGGAGGTGGGGGCTCAGAATTCGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAACTCGAGGCC

<210>10

<211>485

<212> protein

<213> amino acid sequence of recombinant protein C3DC3DC3-EGFP

<400>MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSTYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVDGVWTYDDATKTFRVTEGKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVDGVWTYDDATKTFRVTEGKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAQKAFKQYANDNGVDGVWTYDDATKTFRVTEGGGGSGGGGSEFVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK-LEA

<210>11

<211>164

<212> DNA

<213> C3 sequence

<400>ACT TAC AAA CTT GTT ATT AAT GGT AAA ACATTG AAA GC GAA ACA ACT ACT AAA GCA GTA GAC GCA GAA ACT GCA GAA AAA GCC TTC AAA CAA TAC GCT AAC GAC AACGGT GTT GAT GGT GTT TGG ACT TAT GAT GAT GCG ACT AAG ACC TTT ACG GTA ACT GAA

<210>12

<211>45

<212> DNA

<213> D sequence:

<400>AAA CCA GAA GTG ATC GAT GCG TCT GAA TTAACA CCA GCC GTG ACA

<210>13

<211>30

<212> DNA

<213> Linker sequence

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

<210>14

<211>714

<212> DNA

<213> EGFP sequence

<400> GTG AGT AAA GGC GAG GAG CTG TTT ACC GGT GTT GTG CCG ATT CTG GTT GAG CTG GAT GGC GAT GTG AAT GGC CAC AAG TTC AGC GTG AGC GGT GAG GGT GAA GGC GATGCA ACC TAT GGC AAG CTG ACT TTA AAG TTC ATC TGC ACC ACC GGT AAA CTG CCC GTTCCG TGG CCG ACT TTA GTT ACC ACT TTA ACC TAT GGC GTT CAG TGT TTC AGC CGC TACCCG GAT CAT ATG AAA CAG CAT GAT TTT TTC AAG AGC GCC ATG CCG GAA GGC TAC GTGCAA GAA CGC ACC ATC TTT TTC AAG GAT GAT GGC AAC TAT AAA ACC CGC GCC GAA GTGAAG TTC GAA GGC GAC ACT TTA GTG AAC CGC ATT GAG CTG AAA GGC ATC GAC TTC AAAGAA GAT GGC AAT ATT TTA GGC CAT AAG CTG GAA TAT AAC TAC AAT AGC CAT AAT GTGTAT ATT ATG GCC GAC AAA CAG AAA AAT GGT ATT AAA GTG AAT TTT AAA ATT CGC CACAAT ATT GAA GAT GGC AGC GTG CAG CTGGCC GAT CAC TAC CAG CAG AAT ACC CCG ATT GGT GAT GGT CCG GTG CTG CTG CCG GAT AAT CAC TAT CTG AGC ACC CAG AGC GCT TTAAGC AAA GAT CCT AAC GAG AAG CGC GAT CAC ATG GTG CTG CTG GAG TTC GTT ACC GCAGCC GGC ATT ACT TTA GGC ATG GAT GAA CTG TAC AAA TAA GAG CTA GCC

Claims

1. A detection protein with green fluorescence activity is characterized in that an IgG binding fragment of an SPG gene is reconstructed, only a C3 region of protein G capable of being specifically bound with an Fc end of an antibody IgG is reserved, the detection protein is divided into three groups, namely C3, C3-D-C3, C3-D-C3-D-C3 are all connected with EGFP, and the prepared recombinant protein has dual activities of fluorescence and binding with antibodies of different species.

2. The detection protein of claim 1, wherein the Streptococcal Protein G (SPG) fragment is a sequence comprising a C3 segment of SPG, preferably one of C3, C3-D-C3, C3-D-C3-D-C3, and the fluorescent protein is green fluorescent protein, wherein the optimized IgG binding fragment C3 nucleotide sequence is shown in SEQ ID NO. 11; the optimized D fragment nucleotide sequence of the SPG gene is shown as SEQ ID NO. 12; the Linker nucleotide sequence between the optimized C3 and EGFP segment is shown in SEQ ID NO. 13; the nucleotide sequence of the optimized EGFP protein is shown as SEQ ID NO. 14.

3. The detection protein of claim 1, wherein the nucleotide sequence of the detection protein is shown in one of SEQ ID No.5, 7 and 9; the amino acid sequence of the detection protein is shown in one of SEQ ID NO.6, 8 and 10.

4. A method for constructing the detection protein having green fluorescence activity according to any one of claims 1 to 3, comprising:

according to the gene sequence of the C3 fragment, PCR amplifying C3, C3DC3 and C3DC3DC3 sequences from a bacterial liquid containing the C3 fragment;

amplifying an EGFP sequence from a bacterial liquid containing the EGFP sequence by PCR;

firstly, connecting the amplified EGFP sequence to an expression vector after double enzyme digestion, and respectively connecting C3, C3DC3 and C3DC3DC3 fragments after identification to construct a prokaryotic expression plasmid of the recombinant protein;

transferring the expression vector and the co-expression vector into an expression host for culture, and adding induced epialbumin after the activation to a logarithmic growth phase;

after crushing and purification, fusion proteins C3-EGFP, C3DC3-EGFP and C3DC3DC3-EGFP are prepared.

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

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

6. A product for use in an immunoassay, said product comprising the fusion protein of any one of claims 1-3.

7. The product of claim 5, which can be in the form of a probe (sensor), a test strip, a chip, a kit, etc., and when in use, the detection protein of claim 1 is mixed with other existing commercial reagents (such as enzyme-labeled antibody, fluorescence-labeled antibody, color-developing agent, substrate, etc.), and can be used for various forms of immunoassay, such as antibody detection, antibody screening, antigen detection, pathogen detection, protein interaction screening, high-throughput target protein detection, protein-nucleic acid interaction analysis, drug screening, etc.