CN115876993A - Establishment of method for detecting affinity of pathogenic biological agglutinin and sialyllactose based on biomembrane interference technology - Google Patents
Establishment of method for detecting affinity of pathogenic biological agglutinin and sialyllactose based on biomembrane interference technology Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The embodiment of the invention discloses establishment of a method for detecting affinity of pathogenic biological agglutinin and sialic acid lactose based on a biomembrane interference technology (BLI), and the method is established by taking Toxoplasma gondii SABP1 protein as a research object. The method comprises the following steps: purifying His-SABP1 recombinant protein; optimizing BLI regeneration conditions; BLI measures the affinity of His-SABP1 recombinant protein for binding sialyllactose. The method provided by the invention has the advantages of few steps, simple operation and easy learning; the detection can be completed in 1 hour, and the prepared probe can be reused for more than 5 times, so that the detection time and the detection cost are greatly saved. Simultaneous batch detection of multiple samples (e.g., 8 samples) can be achieved. The method can be used for analyzing the affinity of various pathogenic biological lectins and sialyllactose, and is worthy of popularization.
Description
Technical Field
The embodiment of the invention relates to the technical field of molecular biology, in particular to establishment of a method for detecting affinity of pathogenic biological agglutinin and sialyllactose based on a biomembrane interference technology.
Background
Sialic Acid (SA) is an acidic monosaccharide of 9-carbon structure, linked to the lactose molecule primarily through α 2-3, α 2-6, α 2-8 glycosidic linkages, where the attachment of α 2-3 sialic acid to galactose residues is one of the most common modes in the vertebrate body, followed by the attachment of α 2-6 sialic acid to galactose or N-acetylgalactosamine. Sialic acid is widely present at the tail end of glycoprotein or glycolipid on the surface of a host cell, has important physiological functions, is also a receptor molecule of various pathogenic organisms, and can effectively mediate the adhesion and invasion processes of pathogens such as parasites, viruses and the like. The influenza virus HA protein can recognize host cell surface sialic acid molecules, avian influenza virus tends to combine with alpha 2-3 sialyllactose, while human influenza virus easily combines with alpha 2-6 sialyllactose; the plasmodium EBA-175/EBA-140 and toxoplasma MIC1\ SABP1 protein play an important role in the process that the parasite body depends on sialic acid pathway to invade host cells. Therefore, the affinity and preference of the agglutinative proteins of pathogenic organisms for salivary receptors are important for the study of pathogenic biology. However, the conventional research methods (including ELISA, IFA, etc.) are mainly qualitative research and cannot accurately reflect affinity values.
The Bio-layer interference technique (BLI) can monitor the binding process between molecules in real time, and calculate important data such as affinity (Kd), binding rate (Ka), dissociation rate (Kd) and the like between molecules, and has been widely used for analysis of interaction force of biomacromolecules. Related researches establish a method for detecting the affinity of polysaccharide and the pathogenic lectin protein through the technology and have made certain research progress.
Disclosure of Invention
Therefore, the embodiment of the invention provides the establishment of the method for detecting the affinity of the pathogenic biological agglutinin and the sialyllactose based on the biomembrane interference technology, and effectively solves the problems of multiple steps, complex operation, long time consumption, difficult characterization of the molar concentration of free ligand and the like in the traditional affinity detection method for binding the pathogenic biological agglutinin and the sialyllactose.
In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:
the establishment of the method for detecting the affinity of the pathogenic biological agglutinin and the sialyllactose based on the biomembrane interference technology comprises the following steps:
purifying His-SABP1 recombinant protein;
optimizing BLI regeneration conditions;
BLI measures the affinity of His-SABP1 recombinant protein for binding sialyllactose.
In some preferred embodiments, the method of BLI detecting affinity of His-SABP1 recombinant protein for binding sialyllactose comprises:
sensor activation: pre-wetting a Streptavidin (SA) biosensor in PBS buffer for at least 10 minutes;
sialyllactose immobilisation: immersing the SA biosensor in a sialyllactose solution diluted to a concentration of 2.4 mu M by using a PBS buffer solution, and solidifying for 180-900 seconds;
and (3) sealing: immersing the cured SA biosensor in a PBST-BSA solution for blocking for 90-120 seconds;
sample detection and result judgment: and (3) immersing the sealed SA biosensor into a sample to be detected to perform affinity detection of His-SABP1 recombinant protein combined with sialyllactose, and reading a combined signal in real time in the detection process.
In some preferred embodiments, the sialyllactose is a 3 '-sialylgalactose polyacrylamide biotin conjugate or a 6' -sialylgalactose polyacrylamide biotin conjugate.
In some preferred embodiments, the method of purifying the His-SABP1 recombinant protein comprises:
and (2) re-shaking the His-SABP1 recombinant protein expression strain, carrying out induction expression at the temperature of 0.1MIPTG and 22 ℃, collecting the crushed bacteria, then carrying out induction on the supernatant and an affinity chromatography nickel column, and then eluting by using 250mM imidazole eluent to obtain the His-SABP1 recombinant protein with soluble expression.
In some preferred embodiments, the method of optimizing the BLI regeneration conditions comprises:
the sialyllactose is fixed on a streptavidin biosensor, after the target protein with the same concentration is combined, different biosensor regeneration reagents are used, the steps of combining, dissociating and regenerating are repeated, and the optimal regeneration reagent is determined.
In some preferred embodiments, the optimal regeneration agent is a hydrochloric acid solution of pH = 0.5.
The embodiment of the invention has the following advantages:
the research focuses on the size of interaction force between artificial oligosaccharide-sialyllactose and pathogenic organism lectin protein, can better reflect the interaction relationship between pathogenic organisms and host cell surface sialic acid receptors, and simultaneously, the key reagent selected in the research, namely sialyllactose, is a commercial reagent, has high purity and clear structure, and is more favorable for researchers to analyze test results. Moreover, the BLI-based detection method established by the research can complete detection in 1 hour, and the prepared probe can be reused for more than 5 times, so that the detection time and the detection cost are greatly saved. Therefore, the method can be used for analyzing the affinity of various pathogenic biological lectins and sialyllactose, and is worth popularizing.
The pathogen lectin protein selected in the project is derived from the preliminary research results of the laboratory, and the experiment proves that the protein (SABP 1) coded by the Toxoplasma gondii TGME49-225940 gene can specifically identify host cell surface sialic acid to mediate insect invasion, so that the project takes the protein as a research object to establish a detection method.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary and that other implementation drawings may be derived from the provided drawings by those of ordinary skill in the art without inventive effort.
FIG. 1 shows the results of mass purification and identification of His-SABP1 recombinant proteins provided in the examples of the present invention;
fig. 2 shows the results of regeneration of hydrochloric acid (pH = 0.5) regeneration reagent provided by the embodiment of the present invention;
FIG. 3 shows the result of affinity data (ProcessedData) of the BLI for detecting the binding of His-SABP1 recombinant protein to (3' -SL) sialyllactose;
FIG. 4 shows the result of affinity data (ProcessedData) of the BLI provided in the present invention for detecting the binding of His-SABP1 recombinant protein to (6' -SL) sialyllactose.
Detailed Description
The present invention is described in terms of specific embodiments, and other advantages and benefits of the present invention will become apparent to those skilled in the art from the following disclosure. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The information on the main reagents and consumables used in the following examples is shown in Table 1; the main instrument information is shown in table 2.
TABLE 1 Main reagent consumables information table
TABLE 2 Main Instrument information Table
Example 1 Mass purification by affinity chromatography to obtain SABP1 recombinant protein
And (3) rereeling the His-SABP1 recombinant protein expression strain, carrying out induction expression at the temperature of 0.1MIPTG and 22 ℃, collecting the crushed strain, then carrying out induction on the supernatant and an affinity chromatography nickel column, and then eluting by using 250mM imidazole eluent to obtain the His-SABP1 recombinant protein with soluble expression. The purified recombinant protein was verified by SDS-PAGE, and the purification results are shown in FIG. 1.
EXAMPLE 2 optimization of BLI regeneration conditions
In the "DataAcquisition" software, a corresponding program is set. The sialyllactose-immobilized sample layout is shown in table 3, the sialyllactose immobilization method is shown in table 4, the regeneration condition test sample layout is shown in table 5, and the regeneration condition test determination method is shown in table 6.
(1) Pre-wetting the sensor: SA biosensors were immersed in PBS buffer (140mM NaCl,2.7mM KCl,10mM Na; K.M.M. 2 HPO 4 ，1.8mMKH 2 PO 4 ) Pre-wetting for 10 minutes;
(2) Curing of sialic acid: the SA biosensor was immersed in a 2.4. Mu.M sialyllactose solution (the diluent was PBS buffer) and allowed to solidify for 600 seconds;
(3) And (3) sealing: the cured SA biosensor was immersed in PBST-BSA solution (0.1 g BSA and 10. Mu.l Tween 20 in 20ml PBS buffer) and blocked for 90 seconds;
(4) Association (Association): his-SABP1 protein was diluted to 5. Mu.M (protein dilution PBST-BSA), and the SA sensor was immersed in the protein sample and bound for 90 seconds;
(5) Dissociation (Dissociation): immersing the SA biosensor in the PBST-BSA solution again, and dissociating for 60 seconds;
(6) Regeneration (Regeneration): the SA biosensors were immersed in different biosensor regeneration reagents: regeneration in sodium citrate (pH = 3), glycine (pH = 1), hydrochloric acid (pH = 0.5) for 900 seconds;
(7) And (5) judging a result: reading the binding signal in real time in the detection process, and detecting that the attenuation of the binding signal of the same analyte is less than or equal to 10 percent after regeneration so as to judge that the biosensor is successfully regenerated. Using the "Data Analysis" software for fitting curve Analysis, the hydrochloric acid (pH = 0.5) reagent had a high level of reproducibility of protein binding throughout the assay, with a binding signal decay of ≦ 1%. It was thus confirmed that the biosensor was regenerated using a hydrochloric acid (pH = 0.5) reagent, and the detection results are shown in fig. 2.
Table 3 sialyllactose cured sample layout
| 1 | 2 | |
| G | PBS | 3'-SL or 6' -SL |
| H | PBS | 3'-SL or 6' -SL |
TABLE 4 sialyllactose solidification method TABLE
| Step | (SA)Biosensors | Reagent | Time(sec) | StepType | |
| 1 | G1、 | PBS | 60 | Baseline | |
| 2 | G2、H2 | 3'-SL or 6' -SL | 600 | Loading | |
| 3 | G1、 | PBS | 60 | Baseline |
Table 5 regeneration conditions experimental sample layout table
| 3 | 4 | 5 | 6 | |
| G | PBST-BSA | 5μM-SABP1 | Regeneration reagent | PBS |
| H | PBST-BSA | PBST-BSA | Regeneration reagent | PBS |
TABLE 6 Experimental determination method of regeneration conditions
Example 3BLI detection of affinity of His-SABP1 recombinant protein for binding to sialic acid sugar
In the software of "DataAcquisition", a corresponding program is set up to input the corresponding concentration of the protein. Among them, sialic acid-immobilized samples are shown in Table 3, sialic acid immobilization method is shown in Table 4, recombinant protein-bound sialic acid sample is shown in Table 7, and recombinant protein-bound sialic acid measurement method is shown in Table 8.
(1) Pre-wetting of a sensor: immersing the SA biosensor in PBS buffer solution for prewetting for 10 minutes;
(2) Curing of sialic acid: the SA biosensor was immersed in a 2.4. Mu.M sialyllactose solution (the diluent was PBS buffer) and allowed to solidify for 600 seconds;
(3) And (3) sealing: immersing the cured SA biosensor in a solution containing PBST-BSA for blocking for 90 seconds;
(4) Association (Association): his-SABP1 protein is respectively diluted to 0.625 μ M, 1.25 μ M, 2.5 μ M, 5 μ M and 10 μ M (protein diluent is PBST-BSA), and the SA sensor is immersed into the protein sample according to the dilution concentration sequence and combined for 90 seconds;
(5) Dissociation (Dissociation): the SA biosensor was then immersed in the solution containing PBST-BSA and dissociated for 60 seconds.
(6) Regeneration (Regeneration): the SA sensor was immersed in a hydrochloric acid (pH = 0.5) regeneration reagent to regenerate the sensor for 900 seconds. Repeating the steps (3), (4), (5) and (6) in the examples according to different dilution concentrations;
(7) Sample detection and result judgment: and reading the binding signal in real time in the detection process, and analyzing the detection result according to the binding signal. The results of affinity assays for His-SABP1 recombinant proteins binding to sialyl saccharides are shown in FIGS. 3, 4 and Table 9. Fitting analysis is carried out by using a software of DataAnalysis, the binding affinity (KD) of the Toxoplasma gondii His-SABP1 recombinant protein and 3' -SL is 1.68 mu M, and the fitting degree reaches 0.9639; the binding affinity (KD) of the Toxoplasma gondii His-SABP1 recombinant protein and 6' -SL is 0.512 mu M, and the fitting degree reaches 0.9443.
TABLE 7 recombinant protein-bound sialic acid samples are plated
TABLE 8 determination of sialic acid binding by recombinant proteins
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (6)
1. The establishment of the method for detecting the affinity of pathogenic biological agglutinin and sialyllactose based on the biomembrane interference technology (BLI) is characterized by comprising the following steps:
purifying His-SABP1 recombinant protein;
optimizing BLI regeneration conditions;
BLI measures the affinity of His-SABP1 recombinant protein for binding to sialyllactose.
2. Establishment of the method according to claim 1, wherein the method for BLI to detect affinity of His-SABP1 recombinant protein for binding sialyllactose comprises:
sensor activation: pre-wetting a Streptavidin (SA) biosensor in PBS buffer for at least 10 minutes;
sialyllactose immobilisation: immersing the SA biosensor in a sialyllactose solution diluted to a concentration of 2.4 mu M by using a PBS buffer solution, and solidifying for 180-900 seconds;
and (3) sealing: immersing the cured SA biosensor in a PBST-BSA buffer solution for blocking for 90-120 seconds;
sample detection and result judgment: and (3) immersing the sealed SA biosensor into a sample to be detected to perform affinity detection of His-SABP1 recombinant protein combined with sialyllactose, and reading a combined signal in real time in the detection process.
3. Establishment of the method according to claim 2,
the sialyllactose is a 3 '-sialylgalactose polyacrylamide biotin conjugate or a 6' -sialylgalactose polyacrylamide biotin conjugate.
4. Establishment of the method according to claim 1, wherein the method for purification of His-SABP1 recombinant protein comprises:
and (3) rereeling the His-SABP1 recombinant protein expression bacteria, carrying out induction expression at the temperature of 0.1MIPTG and 22 ℃, collecting supernatant after the bacteria are crushed, carrying out induction on the supernatant and an affinity chromatography nickel column, and then eluting by using 250mM imidazole eluent to obtain the His-SABP1 recombinant protein with soluble expression.
5. The method of establishing of claim 1, wherein said method of optimizing of BLI regeneration conditions comprises:
the sialyllactose is fixed on a streptavidin biosensor, after target protein with the same concentration is combined, different biosensor regeneration reagents are used, the steps of combining, dissociating and regenerating are repeated, and the optimal regeneration reagent is determined.
6. Establishment of the method according to claim 5, characterized in that the optimal regeneration reagent is hydrochloric acid solution with pH = 0.5.
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