CN104292304B - With the protein bound polypeptides of gp120, polypeptide chip, its preparation method and application - Google Patents

With the protein bound polypeptides of gp120, polypeptide chip, its preparation method and application Download PDF

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CN104292304B
CN104292304B CN201310298450.9A CN201310298450A CN104292304B CN 104292304 B CN104292304 B CN 104292304B CN 201310298450 A CN201310298450 A CN 201310298450A CN 104292304 B CN104292304 B CN 104292304B
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polypeptide
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spr
spr chip
streptavidin
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CN104292304A (en
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王琛
王晨轩
王艳梅
朱劲松
杨延莲
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National Center for Nanosccience and Technology China
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National Center for Nanosccience and Technology China
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Abstract

The invention discloses one group and the protein bound polypeptides of gp120, polypeptide chip, its preparation method and application.This group of polypeptide has very strong specific binding capacity with gp120 albumen, can realize the detection in low concentration to gp120 albumen.The polypeptide chip utilizes surface plasmon resonance (Surface Plasmon Resonance, SPR) principle is prepared from, when all this group of peptide molecules are with target combination, judge that gp120 albumen is present, reduce false positive results in experimentation.Additionally, the present invention is also provided comprising this group of pharmaceutical composition of polypeptide.The present invention has significant application value in terms of the diagnosis of the disease that HIV causes.

Description

Polypeptide combined with gp120 protein, polypeptide chip, preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedicine, in particular to a polypeptide combined with gp120 protein, a polypeptide chip, a preparation method and application thereof.
Background
Acquired Immunodeficiency Syndrome (AIDS) is a Syndrome in which an Immune Deficiency is caused by infection with Human Immunodeficiency Virus (HIV), and a series of opportunistic infections and tumors are complicated. It is a disease with wide prevalence and high lethality rate. Under the condition that a universal treatment method is still lacking at present, the human immunodeficiency virus is discovered as early as possible, the disease is diagnosed in time, and the transmission path is blocked, so that the method has important significance for reducing the harm of the human immunodeficiency virus.
HIV can infect a variety of cells, including T4 lymphocytes, monocytes, macrophages, and dendritic cells, among others, that express the CD4 molecule on their surface. The CD4 molecule is a receptor for HIV invading cells, and the glycoprotein gp120 plays an important role in the process of HIV recognizing CD4 molecule. The gp120 molecule located on the envelope of HIV binds to the CD4 molecule, causing a conformational transition in the transmembrane glycoprotein gp 41. gp41 fuses HIV to the target cell membrane, so that the viral nucleocapsid enters the cell. In addition, free gp120 molecules activate excessive T cells and other major immune cells in the body, accelerating HIV virus invasion. Therefore, the gp120 molecule may be an important target molecule for diagnosing HIV related diseases. Gp120 molecules are used as drug design targets, and can be a way for effectively detecting HIV and treating immunodeficiency syndrome.
Based on the molecular structure of gp120, several related inhibitors and monoclonal antibodies have been developed, and some are based on the chip production of these antibodies. However, the monoclonal antibody chip for detecting gp120 protein has the following problems:
(1) the interaction force between the used monoclonal antibody and gp120 protein is weak, so that the detection limit of gp120 protein is low, and the detection limit of a chip prepared by the traditional gp120 monoclonal antibody is higher than 35 nM;
(2) because the judgment is carried out only on the basis of the positive results of one or a few gp120 monoclonal antibodies, more false positive results are generated in the experimental process;
(3) the gp120 monoclonal antibody chip is easy to inactivate due to the conformational change of the antibody, and the chip fails;
(4) the cost of the gp120 monoclonal antibody is high, so that the cost of the gp120 monoclonal antibody chip is difficult to reduce, and the large-scale application is limited.
Therefore, aiming at gp120 protein, a chip with low detection limit, high specificity, stable performance and lower cost is developed, and the chip has important significance for detecting HIV virus.
Disclosure of Invention
The invention obtains a group of polypeptides through a large amount of experiments and creative work, finds that the group of polypeptides has strong specific binding capacity with gp120 protein, can be prepared into polypeptide chips or pharmaceutical compositions and the like, and is applied to diagnosis or auxiliary diagnosis of diseases caused by HIV infection.
The invention provides the following technical scheme:
in a first aspect, the invention provides a polypeptide that binds to a gp120 protein, said polypeptide having an amino acid sequence selected from the group consisting of SEQ id nos: 1-9, wherein the amino acid sequence is as follows:
(1)GGSRADAYRTRVDN(SEQ ID NO:1);
(2)GGTDACVPTRPNPQRV(SEQ ID NO:2);
(3)GGSLWRQSLDPCV(SEQ ID NO:3);
(4)GGVDLTPLCVSLDCTRLDN(SEQ ID NO:4);
(5)GGPDVSFRPIPIDY(SEQ ID NO:5);
(6)GGLARRRVVIDSANFTRNADTII(SEQ ID NO:6);
(7)GGNNDTIIFDQSSGGRPRIVTDS(SEQ ID NO:7);
(8) GGCDIDQIINMWQDVGDAMY (SEQ ID NO: 8); and
(9)GGSRIFDPGGGRM(SEQ ID NO:9)。
the polypeptide can be prepared by artificial chemical synthesis, and has strong specific binding capacity with gp120 protein.
In a second aspect, the present invention provides a polypeptide chip comprising a polypeptide capable of binding to gp120 protein immobilized on a solid support, said polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 9.
The polypeptide on the polypeptide chip has strong specific binding capacity with gp120 protein, and can be used for detecting gp120 protein and judging the infection condition of HIV virus according to the detection.
In one embodiment of the invention, the amino acid sequence of the polypeptide is as set forth in SEQ ID NO: 1-9, namely the polypeptide comprises the nine polypeptides.
In a third aspect, the present invention provides a method for preparing the polypeptide chip according to the second aspect, using the Surface Plasmon Resonance (SPR) principle, the method comprising:
(1) pretreating the solid phase carrier to form a spare solid phase carrier;
(2) spotting the diluted polypeptide onto the ready-to-use solid support.
In one embodiment of the present invention, the step (1) specifically comprises:
(1a) modifying a thiol molecule self-assembly layer containing carboxyl on the surface of the SPR chip through a covalent bond;
(1b) cleaning the surface of the SPR chip twice by using ethanol;
(1c) activating the surface of the SPR chip by using carbodiimide and N-hydroxysuccinimide to enable carboxyl on the alcohol molecule self-assembly layer to form unstable succinimide ester;
(1d) the SPR chip surface was washed twice with ethanol.
In one embodiment of the present invention, the step (2) further comprises pre-treating the polypeptide, and treating after spotting; specifically, the step (2) includes:
(2a) polypeptide molecules of streptavidin and N-terminal modified biotin are mixed according to a molar ratio of 1: 1, and the mass concentration of the mixed streptavidin is 1mg/mL to form a streptavidin-polypeptide mixed solution;
(2b) dripping 0.2 mu L of streptavidin-polypeptide mixed liquid on the surface of an SPR chip, taking gp120 monoclonal antibody as a positive control, and dripping 0.2 mu L of streptavidin-polypeptide mixed liquid on the surface of the SPR chip;
(2c) blocking carboxyl on the succinimide ester and the dithiol which have not participated in the reaction on the surface by ethanolamine;
(2d) cleaning the surface of the SPR chip twice by using ethanol;
(2e) packaging the SPR chip by using a microfluidic membrane, and putting the SPR chip into an SPR instrument;
(2f) repeatedly and alternately cleaning the surface of the SPR chip by 0.5 percent (volume ratio) of phosphoric acid and 1 XPBS (phosphate buffer solution) until the stationary phase SPR signal on the surface of the SPR chip is kept stable;
(2g) the SPR chip was tested for response to gp120 in 1 XPBS using 1 XPBS as the dissociation phase and 0.5% (by volume) phosphoric acid as the regenerant.
The invention also provides a method for multi-parameter detection of gp120 protein in a sample, which comprises the following steps:
(1) simultaneously recording SPR signals of the polypeptides designed on the polypeptide chip when the polypeptides pass through a sample;
(2) when nine polypeptides (SEQ ID NO: 1-9) can generate adsorption peak signals, the gp120 is proved to exist in the sample.
The polypeptide sequence with strong interaction with the gp120 protein provided by the invention has lower homology in the primary structure sequence and may correspond to different binding sites on the surface of the gp120 protein. Only when nine polypeptide molecules are combined with the target, the existence of the gp120 protein is judged, which reduces the generation of false positive results in the experimental process to a certain extent.
In a fourth aspect, the present invention provides a pharmaceutical composition comprising a polypeptide according to the first aspect, optionally together with a pharmaceutically acceptable carrier.
In a fifth aspect, the present invention provides a use of the polypeptide of the first aspect, the polypeptide chip of the second aspect, or the pharmaceutical composition of the fourth aspect, in the preparation of a medicament for diagnosing or aiding in the diagnosis of diseases caused by HIV infection.
The invention has the beneficial effects that:
(1) the polypeptide provided by the invention has strong affinity with gp120 protein, realizes detection of gp120 protein at low concentration, can detect 4nM gp120 protein based on a polypeptide chip prepared from the polypeptide, has detection limit higher than 35nM of a chip prepared from a traditional gp120 monoclonal antibody, and can assist in accurately diagnosing HIV related diseases clinically;
(2) the polypeptide chip provided by the invention judges the existence of gp120 protein only when nine polypeptide molecules are combined with a target, so that the generation of false positive results in the experimental process is reduced to a certain extent;
(3) compared with a gp120 monoclonal antibody, the polypeptide provided by the invention has high stability, does not generate inactivation caused by conformational change, and has longer chip effective period;
(4) compared with a gp120 monoclonal antibody, the polypeptide provided by the invention has lower cost and can be applied in a large scale;
(5) the polypeptide provided by the invention is used for discovering and predicting the polypeptide lead compound of gp120 related diseases, provides a design model of a drug mother nucleus, and provides reference basis and standard for the existing drug structure modification;
(6) the polypeptide sequence of the target gp120 molecule provided by the invention can be applied to gp120 detection by an enzyme-linked immunosorbent assay, a surface plasmon resonance technology, a quartz vibration microbalance technology analysis, a flight time mass spectrum and isothermal titration microcalorimetry; in addition, the gp120 molecule-targeted polypeptide provided by the invention can be used for detecting gp120 molecules in enzyme-linked immunosorbent assay, fluorescence emission spectroscopy, ultraviolet absorption spectroscopy and other analysis methods through quantum dot modification, fluorescein group modification, horseradish peroxidase modification and the like.
Drawings
FIG. 1 shows the binding of gp120 monoclonal antibodies to gp120 and polypeptides 120-1 to 120-9, 24-5 developed according to the present inventionSPR curve for dissociation process. Wherein: FIG. 1a is the SPR curve of gp120 monoclonal antibody and gp120 binding dissociation process, after subtraction of SPR signal of negative control 24-5 to gp 120; FIG. 1b is an SPR curve of dissociation process of binding of 24-5 Streptavidin (SA) blending system and gp 120; FIG. 1c is the SPR curve of 120-1, SA blending system and gp120 binding dissociation process, after subtraction of SPR signal of negative control 24-5 to gp 120; FIG. 1d is the SPR curve of 120-2, SA blending system and gp120 binding dissociation process, after subtraction of SPR signal of negative control 24-5 to gp 120; FIG. 1e is the SPR curve of the dissociation process of the combination of g120-3, SA blending system and gp120, after subtraction of the SPR signal of negative control 24-5 to gp 120; FIG. 1f is the SPR curve of 120-4, SA blending system and gp120 binding dissociation process, after subtraction of SPR signal of negative control 24-5 to gp 120; FIG. 1g is SPR curve of 120-5, SA blending system and gp120 binding dissociation process, after subtraction of SPR signal of negative control 24-5 to gp 120; FIG. 1h is the SPR curve of 120-6, SA blending system and gp120 binding dissociation process, after subtraction of SPR signal of negative control 24-5 to gp 120; FIG. 1i is SPR curve of 120-7, SA blending system and gp120 binding dissociation process, after subtraction of SPR signal of negative control 24-5 to gp 120; FIG. 1j is an SPR curve of 120-8, SA blending system and gp120 association dissociation process, after subtraction of SPR signal of negative control 24-5 to gp 120; FIG. 1k is the SPR curve of 120-9, SA blending system and gp120 binding dissociation process, after subtraction of SPR signal of negative control 24-5 to gp 120. Wherein,"■" indicates a curve corresponding to a gp120 concentration of 8.7 nM;represents the curve corresponding to a gp120 concentration of 17.3 nM;curves representing gp120 concentrations at 34.7 nM;indicating a gp120 concentration of 69.3Curve in nM.
FIG. 2 shows SPR values of the adsorption amounts of the polypeptides 120-1 to 120-9 and gp120 monoclonal antibodies developed based on the present invention in equilibrium with 1. mu.g/mL gp120, gp41 and p24 dissociation processes,represents a histogram corresponding to 1. mu.g/ml gp 120;represents a bar graph corresponding to 1. mu.g/mL gp 41;represents a bar graph corresponding to 1. mu.g/mL p 24.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: detecting the dynamic process of association and dissociation of gp120 and chip surface polypeptide in solution
1. The polypeptides were synthesized according to the sequences shown in table 1 and diluted to the appropriate concentrations as required in the experiment.
TABLE 1 synthetic Polypeptides
Name (R) Sequence of
120-1 Biotin-GGSRADAYRTRVDN
120-2 Biotin-GGTDACVPTRPNPQRV
120-3 Biotin-GGSLWRQSLDPCV
120-4 Biotin-GGVDLTPLCVSLDCTRLDN
120-5 Biotin-GGPDVSFRPIPIDY
120-6 Biotin-GGLARRRVVIDSANFTRNADTII
120-7 Biotin-GGNNDTIIFDQSSGGRPRIVTDS
120-8 Biotin-GGCDIDQIINMWQDVGDAMY
120-9 Biotin-GGSRIFDPGGGRM
24-5 Biotin-GGYDTLDARQASQRVDNWMTRT
Wherein, the function of Biotin (Biotin) at the N-terminal of the polypeptide sequence is to combine with Streptavidin (SA), which belongs to the conventional technical means in the preparation of polypeptide chips. The amino acid sequence of the polypeptide 120-1-120-9 corresponds to the amino acid sequence shown in SEQ ID NO: 1 to 9. The polypeptide 24-5 is a gp 120-independent polypeptide, and is used as a negative control for investigating the gp120 adsorption capacity of SA in an experiment, and the sequence number of the amino acid sequence is SEQ ID NO: 10.
2. preparing a chip:
1) on a plexiera model KxV5 SPR standard glass substrate, 1.5-5.0nm thick Cr, 50nm thick Au was deposited using ion source assisted thermal evaporation. Plasma sputtering was used for 5 minutes. And modifying a carboxyl-containing thiol molecule self-assembly layer on the surface of the SPR chip through a covalent bond. Specifically, 5mL of a mixture of carboxyl dithiol molecules (dithiole-COOH, 1. mu.M) and hydroxyl dithiol molecules (dithiole-OH, 10. mu.M) was dropped on the surface of the SPR chip, adsorbed for 15 minutes, and then the remaining liquid was removed by suction.
2) The surface of the chip is washed twice with ethanol (such as 95% ethanol) and dried with high-purity nitrogen.
3) The surface was activated using carbodiimide (EDC) with N-hydroxysuccinimide (NHS). Specifically, 75mg/mL EDC and 11.5mg/mL NHS were mixed in equal volume, 5mL was placed on the surface of the SPR chip modified with dithiol, the reaction was carried out for 20 minutes, and the remaining liquid was removed.
4) The surface of the chip is washed twice with ethanol (such as 95% ethanol) and dried with high-purity nitrogen.
5) Polypeptide molecules (120-1, 120-2, 120-3, 120-4, 120-5, 120-6, 120-7, 120-8, 120-9 and 24-5) of SA and N-terminal modified biotin are mixed according to a molar ratio of 1: 1, the mass concentration of SA in the mixture is 1mg/mL, and the solution or suspension of the SA and the SA is mixed for 10 minutes for standby. Here, the blending group of 24-5 with SA, as a negative control for gp120 adsorption ability of SA was examined, regardless of gp 120.
6) 0.2 mu of LSA-polypeptide mixed liquid is dripped on the surface of an SPR chip, 0.2 mu L of gp120 monoclonal antibody is used as a positive control, 0.2 mu L of the mixed liquid is dripped on the surface of the SPR chip, gp41 monoclonal antibody inactivated by high-temperature treatment is used as a negative control of background nonspecific adsorption, 0.2 mu L of the mixed liquid is dripped on the surface of the SPR chip, and the reaction time is 2 hours or more than 2 hours. During the reaction process, a humidifier is used to keep the surface of the chip in a humid environment.
7) Ethanolamine is used for blocking active sites which do not participate in the reaction on the surface. Specifically, 5mL of ethanolamine was dropped on the surface of the chip, and after 20 minutes of reaction, the remaining liquid was aspirated away.
8) The surface of the chip is washed twice with ethanol (such as 95% ethanol) and dried with high-purity nitrogen.
9) The chip is packaged by the microfluidic membrane and is arranged in an SPR instrument.
10) The chip surface was washed with 0.5% (by volume) phosphoric acid and 1 × PBS alternately and repeatedly, specifically, 1 × PBS (prepared to 0.4g NaCl, 0.01g KCl, 182mg Na2HPO4·12H2O、12mgKH2PO4、0.01gNaN3Dissolving with 50ml of secondary water, filtering with a 0.22 mu m water-phase filter membrane), fixing the flow velocity of the mobile phase to 1 mu L/s, cleaning the surface of the chip by using a micro-fluidic pipeline of an SPR instrument, wherein the period of each mobile phase replacement is 240s, and continuously cleaning the surface of the chip until the SPR signal curve of the used stationary phase does not decrease any more, namely the stationary phase (SA and polypeptide) on the surface is specifically adsorbed on the surface of the chip.
3. The kinetic constants of the association and dissociation of each polypeptide and gp120 molecule are calculated by using the chip:
1) gp120 solutions were prepared at 4.3nM, 8.7nM, 17.3nM, 34.7nM, 69.3nM in 1 XPBS.
2) The gp120 solution was passed sequentially from low to high concentration across the chip surface at a flow rate of 1. mu.L/s. Binding time 150s, dissociation time 150s, and regeneration time 250 s. 1 XPBS was used as a dissociation solution, and 0.5% (by volume) phosphoric acid was used as a regeneration solution. Binding dissociation SPR curves were recorded for each polypeptide, monoclonal antibody and gp 120. The inactivated gp41 monoclonal antibody was used as background for nonspecific adsorption on the chip surface, and the background was uniformly subtracted from the SPR of each data point. As shown in FIG. 1, the monoclonal antibody against gp120 and the polypeptide probe molecules designed from 120-1 to 120-9 have strong binding ability against gp 120. As shown in FIG. 1a, the detection limit of gp120 by the monoclonal antibody against gp120 was 34.7nM, which is 4.3nM higher than that of other polypeptide molecules (FIGS. 1 c-k). As shown in FIG. 1b, the effective binding peak of gp120 cannot be detected by the gp 120-independent polypeptide molecules 24-5, i.e., the SA does not contribute much to the binding of gp 120.
3) The binding dissociation constants between the respective polypeptide molecules and the gp120 monoclonal antibodies and gp120 were calculated as shown in Table 2 (E stands for scientific notation, e.g. 4.79E +04 stands for 4.79 × 104And the like), equilibrium dissociation constant (K) of 120-1 to 120-9D) Value (10)-9~10-10M) monoclonal antibodies (10) all less than gp120-8M). Namely, the designed polypeptide molecules 120-1 to 120-9 have stronger binding capacity with gp120 molecules compared with monoclonal antibodies.
TABLE 2 binding dissociation constants
gp120 monoclonal antibody 4.79E+04 1.03E-03 4.63E+07 2.16E-08
120-1 3.51E+05 3.93E-04 8.92E+08 1.12E-09
120-2 1.31E+05 5.00E-04 2.62E+08 3.81E-09
120-3 3.17E+05 4.77E-04 6.65E+08 1.50E-09
120-4 3.21E+05 5.09E-04 6.30E+08 1.59E-09
120-5 3.74E+05 3.46E-04 1.08E+09 9.24E-10
120-6 4.77E+05 3.34E-04 1.43E+09 6.99E-10
120-7 3.32E+05 2.77E-04 1.20E+09 8.35E-10
120-8 3.33E+05 1.83E-04 1.82E+09 5.49E-10
120-9 3.23E+05 4.58E-05 7.06E+09 1.42E-10
24-5 Is free of Is free of Is free of Is free of
Example 2: evaluation of interference Effect of gp41 and p24 in solution on detection of chip surface polypeptide molecules
1. The polypeptides used were: the same as in example 1.
2. Preparing a chip: the same as in example 1.
3. The chip is used for evaluating the binding capacity of each polypeptide and gp41 molecules:
1) gp41 solution was prepared at 1. mu.g/mL in 1 XPBS.
2) Gp41 solution was passed over the chip surface at a flow rate of 1. mu.L/s, with an association time of 150s, a dissociation time of 150s, and a regeneration time of 250s, using 1 XPBS as the dissociation solution, and 0.5% (by volume) phosphoric acid as the regeneration solution.
3) The SA-24-5 data set was used as background non-specific adsorption on the chip surface, and the background was subtracted from each data point.
4) And counting the SPR value and standard deviation of each polypeptide molecule and gp120 monoclonal antibody in the desorption stable state.
4. The chip is used for evaluating the binding capacity of each polypeptide and p24 molecules:
1) a1. mu.g/mL solution of p24 was prepared in 1 XPBS.
2) The p24 solution was passed over the chip surface at a flow rate of 1. mu.L/s for a binding time of 150s, a dissociation time of 150s, and a regeneration time of 250s, using 1 XPBS as the dissociation solution, and 0.5% (by volume) phosphoric acid as the regeneration solution.
3) Background was subtracted from each data point using the inactivated gp41 monoclonal antibody data set as background non-specific adsorption to the chip surface.
4) And counting the SPR value and standard deviation of each polypeptide molecule and gp120 monoclonal antibody in the desorption stable state.
5. The designed polypeptide molecule is compared with the ability of gp120, gp41 and p24 to bind with 1 mu g/mL
As shown in FIG. 2, for 1. mu.g/mL gp120 molecular solution (i.e., 8.7nM), the nine designed polypeptide probes 120-1 to 120-9 all gave effective binding signals greater than 4 times the SPR noise, while for gp120 monoclonal antibodies, the noise was comparable to the binding amount signal and it was not possible to determine whether the signals were true SPR binding signals. For 1. mu.g/mL gp41 molecular solution, only 120-2 of 120-1 to 120-9 had binding averages greater than 4 times the noise. Meanwhile, the SPR response signals of 120-1 to 120-9 to gp41 are much smaller than those caused by gp 120. Therefore, the gp41 molecule does not affect the detection of the gp120 molecule. Whereas for the monoclonal antibody against gp120, the SPR response signal is higher than the signal caused by gp120 at the same mass concentration. For 1. mu.g/mL of p24 molecular solution, the mean value of the binding of each of the groups 120-1 to 120-9 was less than 4 times the noise value. While the SPR response signals of 120-1 to 120-9 to p24 are much smaller than those induced by gp 120. Therefore, the molecule p24 has no influence on the detection of the gp120 molecule. For monoclonal antibodies to gp120, no SPR binding dissociation signal on p24 was detected.
Therefore, the detection of gp120 molecules can be realized by using the nine designed polypeptide probe molecules as a combination of multiple indexes. At the same time, the probability of detecting false positives can also be reduced.
Those skilled in the art will understand that: although specific embodiments of the invention have been described in detail, it will be appreciated that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure, and that such variations are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (6)

1. A polypeptide that binds to gp120 protein, wherein the amino acid sequence of said polypeptide is as set forth in SEQ ID NO: 1 to 9.
2. A polypeptide chip is characterized by comprising a polypeptide which is fixed on a solid phase carrier and can be combined with gp120 protein, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 to 9.
3. A method of preparing the polypeptide chip of claim 2, comprising:
(1) pretreating the solid phase carrier to form a spare solid phase carrier;
(2) spotting the diluted polypeptide onto the ready-to-use solid support.
4. The preparation method according to claim 3, wherein the step (1) specifically comprises:
(1a) modifying a thiol molecule self-assembly layer containing carboxyl on the surface of the SPR chip through a covalent bond;
(1b) cleaning the surface of the SPR chip twice by using ethanol;
(1c) activating the surface of the SPR chip by using carbodiimide and N-hydroxysuccinimide to enable carboxyl on the alcohol molecule self-assembly layer to form unstable succinimide ester;
(1d) the SPR chip surface was washed twice with ethanol.
5. The method of claim 3, wherein the step (2) further comprises a pretreatment of the polypeptide and a treatment after spotting; specifically, the step (2) includes:
(2a) polypeptide molecules of streptavidin and N-terminal modified biotin are mixed according to a molar ratio of 1: 1, and the mass concentration of the mixed streptavidin is 1mg/mL to form a streptavidin-polypeptide mixed solution;
(2b) dripping 0.2 mu L of streptavidin-polypeptide mixed liquid on the surface of an SPR chip, taking gp120 monoclonal antibody as a positive control, and dripping 0.2 mu L of streptavidin-polypeptide mixed liquid on the surface of the SPR chip;
(2c) blocking carboxyl on the succinimide ester and the dithiol which have not participated in the reaction on the surface by ethanolamine;
(2d) cleaning the surface of the SPR chip twice by using ethanol;
(2e) packaging the SPR chip by using a microfluidic membrane, and putting the SPR chip into an SPR instrument;
(2f) repeatedly and alternately cleaning the surface of the SPR chip by using phosphoric acid and 1 × PBS according to the volume ratio of 0.5% until the stationary phase SPR signal on the surface of the SPR chip is kept stable;
(2g) the SPR chip was tested for response to gp120 in 1 XPBS using 1 XPBS as the dissociation phase and 0.5% by volume phosphoric acid as the regeneration solution.
6. Use of a set of polypeptides according to claim 1 for the preparation of a medicament for the diagnostic or adjuvant diagnosis of a disease caused by HIV infection.
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