CN113264985A - Human chorionic gonadotropin peptide aptamer and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of peptide aptamers, in particular to a human chorionic gonadotrophin peptide aptamer, which comprises any one of the following polypeptide sequences P1-P6: p1: MHLMRMKPLLLT, respectively; p2: MHPRKMLQLMLN, respectively; p3: STRLRRRSRRQT, respectively; p4: PPLRINRHILTR, respectively; p5: MKLKPMRIMINP, respectively; p6: MKSRMLPLNRRL are provided. The invention also provides a preparation method of the human chorionic gonadotropin peptide aptamer. The human chorionic gonadotropin peptide aptamer provided by the invention is good in specificity of combining with HCG, high in combining rate, and applied to HCG detection, and HCG conditions of reaction samples can be more accurate. Applications of the human chorionic gonadotropin peptide aptamer of the present invention include, but are not limited to, the preparation of a detection reagent for detecting HCG, the preparation of a biological detection sensor for detecting HCG, and the like.

Description

Human chorionic gonadotropin peptide aptamer and preparation method and application thereof

Technical Field

The invention relates to the technical field of peptide aptamers, in particular to a human chorionic gonadotropin peptide aptamer, and a preparation method and application thereof.

Background

Human Chorionic Gonadotropin (HCG) is a glycoprotein hormone secreted by trophoblast cells of placental Chorionic vesicles and consists of two distinct subunits, an alpha subunit and a beta subunit, and 244 amino acids, with a molecular weight of approximately 36.7 kDa. Structurally, the alpha subunit is similar to many hormones such as thyroid stimulating hormone, follicle stimulating hormone, etc., while the beta subunit is specific to HCG, so the clinical HCG detection mainly utilizes the specificity of the beta subunit.

The placenta can produce HCG, trophoblastomas, germ cell tumors containing trophoblastic tissue, and some non-trophoblastomas can also produce HCG. The serum of pregnant women contains mainly intact molecules of HCG, whose concentration increases exponentially in the early stages of pregnancy and is time-dependent, which plays an important role in maintaining pregnancy. If the change of the HCG value is irregular, the specific time is too high or too low, indicating that the pregnancy is abnormal. An abnormally high detection may indicate choriocarcinoma, hydatidiform mole or multiple pregnancy, and a low detection may indicate threatened/early abortion, ectopic pregnancy, gestational toxicosis or intrauterine fetal death. The detection of HCG + beta is favorable for evaluating the risk of trisomy 21 syndrome in the middle of pregnancy by combining alpha-fetoprotein detection and other accurate parameters such as gestational age and weight of pregnant women, the serum alpha-fetoprotein concentration of the trisomy 21 pregnant women is reduced, and the serum HCG + beta concentration of a mother body can reach twice of a normal median value. The monitoring of the human chorionic gonadotropin level can predict the occurrence of pregnancy-induced hypertension, and has important guiding significance for diagnosing the course of hypertensive diseases during pregnancy.

HCG is also an important serum and urine tumor marker, and elevated HCG concentrations not associated with pregnancy can also be seen in patients with germ cell, ovarian, bladder, pancreatic, gastric, lung and liver tumors. Prevalence (%) of elevated serum HCG values in various malignancies: testicular or placental choriocarcinoma (100), hydatidiform mole (97), non-seminoma (48-86), seminoma (10-22), pancreatic carcinoma (adenocarcinoma (11-80) and islet cell carcinoma (22-50)), gastric cancer (0-52), epithelial cancer (18-41), colon cancer (0-37), lung cancer (0-36), breast cancer (7-25), liver cancer (17-21), small intestine tumor (13) and kidney cancer (10).

Furthermore, HCG detection is also very important in sports medicine. Since HCG may be abused by male athletes to mask anabolic steroids or to promote testosterone production, the hormone is classified as a world anti-stimulant agency banned substance.

The detection method of HCG currently includes colloidal gold immunoassay, fluorescence immunoassay, resonance scattering spectroscopy, photoluminescence, and electrochemical immuno-electrode, among which roche's electrochemical luminescence method is the most widely used detection method of HCG.

The colloidal gold immune layer test paper cannot accurately reflect the HCG amount in the body when detecting the HCG in urine. The color of the T-line deepens as HCG increases in the early stages of pregnancy, and a high-concentration hook effect appears near the peak and weakens as HCG increases. Reproductive system diseases such as hydatidiform mole, erosive hydatidiform mole, choriocarcinoma, trophoblastic tumor and the like, namely beta-HCG is abnormally increased (>100000U/L), and the color of a T line is weakened due to a high-concentration hook effect caused by overhigh concentration. After 8-9 days of ectopic pregnancy, the beta-HCG in vivo is reduced to normal, and the urine test can also be positive. After 3 months of gestation, HCG levels decline and urine tests may appear negative or weakly positive. In addition, if the test paper is stored for too long, the test paper may fail to be stored properly, resulting in false negative test results. Therefore, it is necessary to determine whether to be pregnant or not to suffer from the related diseases clinically by combining with the auxiliary diagnosis such as blood beta-HCG, clinical symptoms and imaging examination. The HCG value of the blood examination is more sensitive and more accurate than that of the early pregnancy test paper to judge whether the pregnancy or the illness is caused.

The Roche electrochemiluminescence detection range is 0.100-10000mIU/mL, and the detection of the concentration of HCG over 10000mIU/mL at 12-14 weeks of gestation cannot be met, so the detection usually needs to be diluted and then carried out for secondary detection. The method has complex instruments, poor reagent stability and harsh antibody storage conditions, and must be operated by professional laboratory personnel, and the instrument has high requirements on samples: the instrument has the defects of no blood clots and air bubbles, over 150uL sample size, uncertain dilution times and potential cross contamination, and is mainly existed in a hospital clinical laboratory or a third-party in-vitro diagnosis company, and often reports errors, so that the instrument needs an engineer to solve problems in time and cannot be popularized.

It is sufficient that the detection of the HCG in the prior art is very busy and obvious, so that the reference value of the detection result of the HCG in the prior art is limited, and the evaluation needs to be performed by means of more other detection methods.

Disclosure of Invention

Based on this, the present invention aims to provide a human chorionic gonadotropin peptide aptamer.

Another object of the present invention is to provide a method for preparing an aptamer of human chorionic gonadotropin and use thereof

The technical scheme of the invention is as follows:

a human chorionic gonadotropin peptide aptamer, wherein the polypeptide sequence of the human chorionic gonadotropin peptide aptamer is a sequence comprising any one of P1 to P6:

(SEQ ID NO.1)P1：MHLMRMKPLLLT；

(SEQ ID NO.2)P2：MHPRKMLQLMLN；

(SEQ ID NO.3)P3：STRLRRRSRRQT；

(SEQ ID NO.4)P4：PPLRINRHILTR；

(SEQ ID NO.5)P5：MKLKPMRIMINP；

(SEQ ID NO.6)P6：MKSRMLPLNRRL。

further, the polypeptide sequence of the human chorionic gonadotropin peptide aptamer preferably comprises the sequence: PPLRINRHILTR are provided.

Further, the human chorionic gonadotropin peptide aptamer comprises a modified sequence; the modifications include polar glycine modifications; the polar glycine modifications include: introducing 3 polar glycines at least one end of the sequence represented by any one of P1-P6.

Preferably, the sequence modified by polar glycine is the sequence shown in P11-P61:

(SEQ ID NO.7)P11：GGGMHLMRMKPLLLT；

(SEQ ID NO.8)P21：GGGMHPRKMLQLMLN；

(SEQ ID NO.9)P31：GGGSTRLRRRSRRQT；

(SEQ ID NO.10)P41：GGGPPLRINRHILTR；

(SEQ ID NO.11)P51：GGGMKLKPMRIMINP；

(SEQ ID NO.12)P61：GGGMKSRMLPLNRRL。

further, the sequence represented by P1 to P6 or the sequence represented by P11 to P61 may also be a sequence modified with cysteine, and the cysteine modification includes: cysteine was introduced at one end of each of the sequences represented by P1 to P6 or each of the sequences represented by P11 to P61.

A method for preparing a human chorionic gonadotropin peptide aptamer comprises

Incubating HCG with M13 phage library solution, and culturing and amplifying the obtained combined phage to perform a combination experiment; repeating the screening for 3-8 times, determining the occurrence frequency of the peptide segments, and screening out phage colonies with the highest binding affinity; translating the DNA sequence of the pIII protein structural domain to obtain 6 polypeptide sequences:

(SEQ ID NO.1)P1：MHLMRMKPLLLT；

(SEQ ID NO.2)P2：MHPRKMLQLMLN；

(SEQ ID NO.3)P3：STRLRRRSRRQT；

(SEQ ID NO.4)P4：PPLRINRHILTR；

(SEQ ID NO.5)P5：MKLKPMRIMINP；

(SEQ ID NO.6)P6：MKSRMLPLNRRL。

further, before incubation of HCG with the M13 phage library solution, it is also included to add HCG to a 24-well plate for incubation in order to immobilize HCG proteins on the well walls for subsequent reactions.

Further, in the method for preparing the human chorionic gonadotropin peptide aptamer, after 6 polypeptide sequences are screened, the method further comprises the following steps: amplifying the screened 6 high-affinity polypeptide sequences, incubating with HCG, eluting, and calculating the binding rate of a single phage and HCG to obtain the polypeptide with the highest binding rate as the optimal peptide aptamer; the calculation formula of the binding rate of the single phage and HCG is as follows: binding phage titer/input phage titer × 100%; screening out the optimal polypeptide sequence as P4: PPLRINRHILTR are provided.

Further, the preparation method of the human chorionic gonadotropin peptide aptamer further comprises the step of modifying each sequence shown as P1-P6; the modifications include polar glycine modifications; the polar glycine modifications include: three polar glycines (Gly, G) are added to at least one end of each sequence shown in P1-P6 to increase the hydrophilicity and water solubility of the sequence, increase the distance from the electrode surface and reduce steric hindrance.

Preferably, the sequence modified by polar glycine is the sequence shown in P11-P61:

(SEQ ID NO.7)P11：GGGMHLMRMKPLLLT；

(SEQ ID NO.8)P21：GGGMHPRKMLQLMLN；

(SEQ ID NO.9)P31：GGGSTRLRRRSRRQT；

(SEQ ID NO.10)P41：GGGPPLRINRHILTR；

(SEQ ID NO.11)P51：GGGMKLKPMRIMINP；

(SEQ ID NO.12)P61：GGGMKSRMLPLNRRL。

furthermore, in the preparation method of the human chorionic gonadotropin peptide aptamer, in order to enable each sequence shown in P1-P6 or each modified sequence shown in P1-P6 to be subsequently and stably bound and fixed with other carriers, such as the surface of a nanogold electrode, the NH 2-end of the polypeptide sequence or each modified polypeptide sequence is subjected to cysteine (Cys, C) modification, and a cysteine (Cys, C) is introduced to provide an anchoring point with the electrode.

The human chorionic gonadotropin peptide aptamer can be applied to related detection of HCG; including but not limited to, the production of a detection reagent for detecting HCG, the production of a biosensor for detecting HCG, and the like.

The invention has the beneficial effects that:

polypeptide aptamers are a 10-20 residue length of polypeptide that as aptamers are capable of high affinity and strong specific binding to the corresponding ligand, having antibody properties. The human chorionic gonadotropin peptide aptamer provided by the invention is good in specificity of combining with HCG, high in combining rate, and applied to HCG detection, and HCG conditions of reaction samples can be more accurate.

The invention provides a human chorionic gonadotropin peptide aptamer, which is characterized in that an HCG peptide aptamer preliminarily screened from a peptide phage library is verified by an immunoblotting method (Western Blot, WB) for the first time, the binding molar ratio of the HCG peptide aptamer to HCG (HCG: peptide aptamer 1:303) is determined, theoretical support is provided for experiments, and a new thought is provided for similar research. Simultaneously, further performing cysteine modification and characterization on the peptide aptamer; the peptide aptamer has better performance, hydrophilicity and water solubility, the distance between the peptide aptamer and the surface of an electrode is increased, and the steric hindrance is reduced; glycine modification provides an anchoring point with an electrode, so that the peptide aptamer disclosed by the invention is better in application effect.

The prior art method, when detecting HCG at high concentration, destroys the hydration state of the complex due to imbalance of binding of antigen-antibody, so that precipitation occurs, showing a high concentration of hook reaction. Compared with the prior art, the invention uses different detection principles, uses the human chorionic gonadotropin peptide aptamer for detection, combines protein with peptide segment to block the transmission of electrons on the surface of an electrode, and the more the combined protein is, the larger the resistance is, thereby reflecting the amount of the combined protein according to the change of the resistance and completely avoiding the phenomenon.

Compared with protein, the peptide aptamer provided by the invention has a simpler structure, is more stable and more convenient to store, and greatly reduces the detection deviation caused by 'storage failure' in the prior art.

For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.

Drawings

FIG. 1 is a diagram showing the results of HPLC analysis of the modified P41 sequence.

FIG. 2 is a graph showing the results of mass spectrometry of the modified P41 sequence.

FIG. 3 is an image of an electrophoretic gel demonstrating the binding of peptide aptamers to HCG.

Detailed Description

The implementations described in the exemplary embodiments below are not intended to represent all implementations consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.

The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

The specific techniques or conditions not indicated in the examples of this application are performed according to the techniques or conditions described in the literature in the field or according to the product description. The reagents or instruments used in the examples of the present application are not indicated by manufacturers, and are all conventional products available from commercial sources and the like.

Example A human chorionic gonadotropin peptide aptamer of the present invention and a method for preparing the same

1. Laboratory instruments and materials

Ultra-low temperature refrigerator (Mitsubishi, of Chinese family) at-80 deg.C; precision electronic analytical balance (Mettler Toledo, germany); a constant temperature oscillator (BOYN, hang); centrifuge (michael, hunan); vortex mixer (lepert scientific instrument, beijing); pipette guns (Eppendorf, germany); electrophoresis apparatus (Bio-Rad, USA); membrane transfer apparatus (Bio-Rad, USA); magnetic stirrers (thunder magnet, shanghai); ice making machines (xuekakee, usa); gel imaging system (Bio-Rad, USA); ultra pure water meters (Heal Force, hong kong); metal bath thermostats (Kylin-Bell, haimen); HCG standards (tide organisms, shanghai); HCG- α monoclonal antibody (collar tide organism, shanghai); peptide aptamers (keptisan, shanghai); tris (2-carboxyethyl) phosphine (TCEP) (alatin, shanghai); loading Buffer (Thermo, usa); ECL color developing solution (Thermo, usa); marker (Thermo, usa); acrylamide kit (Bio-Rad, USA); PVDF membranes (Millipore, usa); BSA blocking solution (solibao, beijing); HCG standards (tide organisms, shanghai); peptide aptamers (keptisan, shanghai); tris (2-carboxyethyl) phosphine (TCEP) (alatin, shanghai); hexamercaptohexanol (MCH) (alatin, shanghai); PBS (sulibao, beijing); chloroauric acid (HAuCl4 · H2O) (alatin, shanghai); human serum albumin (alligator, shanghai); fetal bovine serum (Gibco, australia); BSA blocking solution (solibao, beijing); sodium hydroxide (alatin, shanghai); hydrochloric acid (xiangya institute of medicine); thyroid stimulating hormone (midge, beijing); follicle stimulating hormone (Zhongzhong, Beijing); luteinizing hormone (solibao, beijing); potassium ferrocyanide (AR) (national drug group, beijing); potassium ferricyanide (AR) (national drug group, beijing); phage library (Invitrogen, singapore); carbon paste (ten bars, japan); insulating ink (eimer, guan dong); PET substrate (Sihongyuan plastic, Dongguan).

2. Experimental methods

2.1 screening and optimization of peptide aptamers

Adding HCG to a 24-well plate and incubating (incubation conditions: 500. mu.L of 0.1mol/L NaHCO3(pH 8.6), 4 ℃, overnight) to immobilize HCG protein on the well walls; discarding waste liquid, blocking with BSA, washing with PBS, adding M13 phage library solution, incubating (incubation system: 0.1% (v/v) TBST buffer, 50mM Tris-HCl, pH 7.5, 150mM NaCl and 0.1% (v/v) Tween-20; incubation condition: incubation at 37 deg.C for 30min), removing unbound phage, culturing and amplifying the obtained bound phage, and performing binding experiment; and (3-8) repeated screening (preferably 5) is carried out, phage colonies with the highest binding affinity are collected, and 6 polypeptide sequences are obtained by translating the DNA sequence of the pIII protein structural domain: (P1) MHLMRMKPLLLT, (P2) MHPRKMLQLMLN, (P3) STRLRRRSRRQT, (P4) PPLRINRHILTR, (P5) MKLKPMRIMINP, (P6) MKSRMLPLNRRL.

In some embodiments, the present invention provides 6 polypeptide sequences that can be further screened by: amplifying the 6 high-affinity sequences, incubating with HCG in a 24-well plate (incubation environment: 4mL PEG/NaCl solution (20%, w/v PEG-8000,2.5M NaCl), incubation condition: 4 ℃ for 2h), eluting with acid (1mL 0.2M glycine-HCl (pH 2.2) and 1mg/mL BSA), calculating the binding rate (phage binding titer/phage input titer) of a single phage to HCG, and obtaining the polypeptide with the highest binding rate as a peptide aptamer (P4: PPLRINRHILTR).

2.2 modification of peptide aptamers

Analysis of the 6 high affinity sequences revealed the presence of more positively charged amino acids (29.2%) and nonpolar hydrophobic amino acids (27.5%) and the absence of negatively charged residues, probably because HCG (pI 2.95) was negatively charged in TBST buffer (pH 7.5) during the screening process, suggesting that binding of HCG to polypeptides may include electrostatic and hydrophobic interactions. Analysis of these peptide aptamers revealed that their dissociation constants were two orders of magnitude higher than those of commercial antibodies and approached that of single-chain variable region fragments (antigen-antibody complementarity determining regions) in HCG receptors. Ascolia et al, 2002, reported the presence of a structural motif called leucine rich repeats in the extracellular binding domain of the HCG receptor, which motif is present in peptide aptamers and may be involved in HCG binding.

Based on the facts, the invention modifies the screened polypeptide sequence; namely, 3 polar glycines (Gly, G) are respectively introduced into at least one end of the polypeptide sequence. Taking the sequence P4(PPLRINRHILTR) as an example, three polar glycines (Gly, G) are added on the basis of the original polypeptide PPLRINRHILTR to obtain the sequence P41(GGGPPLRINRHILTR) which increases the hydrophilicity and water solubility, increases the distance from the electrode surface and reduces the steric hindrance.

In some embodiments, in order to enable the polypeptide sequence to be stably fixed on the surface of the nanogold electrode in subsequent applications, the NH 2-terminal of the peptide aptamer or the modified peptide aptamer is subjected to cysteine (Cys, C) modification to provide an anchoring point with the electrode; taking the sequence of P4(PPLRINRHILTR) as an example, in order to stably fix the sequence of P4(PPLRINRHILTR) or P41(GGGPPLRINRHILTR) on the surface of the nanogold electrode, the NH 2-terminal of the peptide aptamer or the modified peptide aptamer is modified with cysteine (Cys, C) to provide an anchor point with the electrode, thereby obtaining the peptide aptamer sequence: CGGG-PPLRINRHILTR or C-PPLRINRHILTR.

Example two Performance validation of the human chorionic gonadotropin peptide aptamers of the invention

2.1 basic Properties of peptide aptamers

Taking the sequence of P4(PPLRINRHILTR) as an example, a peptide calculator network tool (PepCalc https:// PepCalc. com /) is used for detecting the properties of P4 peptide such as hydrophilicity, isoelectric point and the like, and the obtained result is shown in figure 1, and the properties such as water solubility and the like meet the requirements.

2.2 Synthesis and characterization of peptide aptamers

Taking the sequence P4(PPLRINRHILTR) as an example, the biosynthesis of a peptide fragment from a shanghai peptide was entrusted: CGGGPPLRINRHILTR the flow of the air in the air conditioner,

after biosynthesis, it was characterized by reverse phase high performance liquid chromatography (RP-HPLC) and Mass Spectrometry (MS):

the UPLC system used a reverse phase column (Kromasil 100-5C18, 4.6mm x 150mm,5m), both column temperature and sample temperature were 40.0 ℃, and mobile phase was solvent a (0.1% trifluoroacetic acid acetonitrile) and solvent B (0.1% trifluoroacetic acid water). A linear gradient was performed at a flow rate of 0.5 mL/min: 10% A (0-10min), 10% -70% A (10-20min), 70% A (20-25 min). The detection wavelength is 220 nm.

Referring to fig. 1 and table 1, the integration result of the high performance liquid chromatogram shows that the main peak with the retention time of 10.346min represents the target peptide segment, the area percentage is 98%, the purity of the peptide aptamer is 98.01%, and the modification requirement of the sensor is met.

In MS analysis, the parent ion types of the aptamers are [ M + H ] respectively]²⁺And [ M + H]³⁺The corresponding mass to charge ratios (M/z) are 881.25 and 587.8, respectively. The crushing voltage was 15v and the collision energy was 21 ev.

Referring to fig. 2, the results of mass spectrometry showed that the ion information of the excimer ion peak of the peptide was consistent with the corresponding amino acid sequence, and the ion peaks having mass-to-charge ratios of 587 and 881 represented three positive charges and two positive charges.

Table 1: characterization results of high performance liquid chromatography

	RT	Area	Height	％Area
						1	9.915	194260	13418	1.85
2	10.346	10296363	588286	98.01
					3	10.867	15143	5453	0.14

2.3 WB validation of binding of peptide aptamers to HCG

2.3.1 principle of the experiment

The HCG and HCG peptide fragment conjugate can be specifically combined with HCG-alpha monoclonal antibody marked by HRP, different combination ratios (1:4, 11, 20, 37, 75, 113, 189, 303 and 378) are set, ECL is catalyzed by the HRP for developing, and the brightness of the strip is observed under different combination ratios. The binding band was compared with HCG alone, and different molecular weights were reflected depending on the position of the band, thereby observing whether HCG was bound to the peptide fragment.

2.3.2 Experimental procedures

Preparing glue: mounting a clean glue comb and a clean glass plate on a glue frame, and preparing 8% of separation glue and 5% of concentrated glue according to the formulas listed in tables 2 and 3; injecting the separation gel into the glass plate by using a liquid transfer gun, reserving about 2cm of the upper layer for preparing the concentrated gel, slightly covering the upper layer with absolute ethyl alcohol, and gelling at room temperature; after about 20min, observing that the gel is indeed formed, inverting the glass plate, pouring out absolute ethyl alcohol, completely sucking the absolute ethyl alcohol by using filter paper, and adding concentrated gel; filling the rest space with concentrated glue, inserting into a 15-hole comb, and gelling at room temperature for 30 min; after the prepared concentrated gel is completely solidified and no liquid flows, the comb is taken out, the plate with the gel is fixed on an electrophoresis shelf, the electrophoresis shelf is put into an electrophoresis tank, and 1L of 1xSDS-PAGE electrophoresis buffer (diluted from 10x to 1x by pure water) is added.

Table 2: 8% separation gel ingredient list

Composition (I)	8% separation gel
		Ultrapure water	6.9mL
30% acrylamide solution	4.0mL
		1.5M Tris，pH8.8	3.8mL
10％SDS	0.15mL
		10% ammonium persulfate	0.15mL
TEMED	0.018mL

Table 3: ingredient list of 5% concentrated gum

Preparing a peptide fragment solution: taking out the vacuum-packaged peptide fragment from a refrigerator at the temperature of-20 ℃, adding 100ul PBS into 2mg of the peptide fragment to obtain a peptide fragment solution with the concentration of 20mg/ml (1 mu l is equivalent to 11 nmol); mu.l of the 20mg/ml peptide fragment solution was added to 5. mu.l of PBS to obtain a 2mg/ml peptide fragment solution (1. mu.l corresponds to 1.1 nmol).

③ sample loading: mixing as shown in Table 4, decocting at 100 deg.C for 15min for denaturation, and loading 10ul per well.

Table 4: summary of each component

And fourthly, electrophoresis: concentrating gel electrophoresis, performing 80V electrophoresis for 30min, separating gel electrophoresis, adjusting voltage to 120V, and stopping electrophoresis when bromophenol blue electrophoresis is near the bottom of gel (about 1h, 1cm from the bottom).

Turning the film: 1L of 1x membrane transfer solution (diluted from 80ml to 800ml, added with 200ml methanol, total 1L) is prepared, and filter paper, sponge and PVDF membrane are prepared continuously, and the PVDF membrane is cut into a proper size and is soaked in methanol for about 30s to activate the positive charge group on the membrane, so that the PVDF membrane can be combined with the negatively charged protein more easily. Taking out and soaking the mixture and other two solutions in the precooled membrane transferring solution for later use; prying the glass plates by using a plastic plate, flatly paving the whole glue on one of the glass plates, and cutting off the part which is not needed by the glue according to the molecular weight indicated by a marker; opening the film transferring clamp, placing the black panel below, placing the sponge, the filter paper, the gel, the PVDF film, the filter paper and the sponge in sequence from bottom to top, carefully paving each layer by using a glass rod, removing air bubbles between each layer (not damaging the gel and the PVDF film), and fixing the transferring clamp; and (3) putting the transfer clip into an electrophoresis tank, adding a proper amount of precooled membrane transfer liquid into the immersion transfer clip, covering an electrode cover, putting the electrophoresis tank under the ice-water bath condition, and transferring the membrane for 2 hours at 250 mA.

Closing: and after the membrane transferring is finished, taking out the PVDF membrane from the membrane transferring clamp, dyeing the PVDF membrane by ponceau red dye, checking the membrane transferring effect, cutting the PVDF membrane into strips with corresponding sizes according to the size of the needed protein, placing the PVDF membrane in 5% milk sealing liquid, sealing the strips on a shaking table for 2 hours at room temperature, and then washing the strips by PBST to be clean and sealing the strips by milk.

Seventhly, incubation antibody: putting the PVDF membrane into a hybridization bag, adding a proper amount of corresponding antibody diluent (TBST diluent, the concentration of the antibody is 1:500-1:1000) to ensure that the PVDF membrane can be completely immersed, removing bubbles, sealing, and shaking overnight in a refrigerator at 4 ℃; the membrane was rinsed in TBST 3 times for 10min each.

(viii) chemiluminescence, development, and fixation: the liquid transferring gun sucks equivalent and proper luminescent liquid A (luminal) and luminescent liquid B (hydrogen peroxide), the luminescent liquid A and the luminescent liquid B are uniformly mixed, the mixed liquid is uniformly dripped on the membrane as much as possible, the membrane is placed into an imager for development, and the whole process is carried out under the condition of keeping out of the sun.

Ninthly, analyzing gel images: relative expression levels of each protein were expressed by image scanning using Quantity one (bio. rad) software (see fig. 4), semiquantitative determination using image J software, and calculation of gray scale values for each protein (see table 5).

Table 5: grey value of each protein

Numbering

1

2

3

4

5

6

Binding ratio

Pure HCG

1：75

1：113

1：189

1：303

1：378

Grey scale value (n is 5)

212±6

180±7

135±5

91±1

72±2

69±3

2.3.3 Experimental conclusion analysis

The gray scale of the HCG band with the molecular weight of 36kd becomes lighter along with the increase of the combination proportion, which indicates that the protein and the peptide fragment are combined into a compound, the molecular weight is larger, and the compound and the HCG protein do not appear on the same band; the compound has no band in the experiment, on one hand, the peptide aptamer and the action site of HCG are in an alpha chain and compete with HCG-alpha monoclonal antibody for the action site, so that the compound cannot be developed; on the other hand, the protein with larger molecular weight can not be separated out due to the concentration of the prepared gel. The binding ratio of HCG to the peptide aptamer is about 1:303 (molar ratio), and the peptide aptamer of the invention can be formulated with reference to this binding ratio when detecting HCG.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

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1 5 10 15

<210> 10

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Gly Gly Pro Pro Leu Arg Ile Asn Arg His Ile Leu Thr Arg

1 5 10 15

<210> 11

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gly Gly Gly Met Lys Leu Lys Pro Met Arg Ile Met Ile Asn Pro

1 5 10 15

<210> 12

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gly Gly Gly Met Lys Ser Arg Met Leu Pro Leu Asn Arg Arg Leu

1 5 10 15

Claims (10)

1. A human chorionic gonadotropin peptide aptamer, wherein a polypeptide sequence of the human chorionic gonadotropin peptide aptamer comprises a sequence shown in any one of SEQ ID No.1 to SEQ ID No. 6.

2. The human chorionic gonadotrophin peptide aptamer according to claim 1, wherein said human chorionic gonadotrophin peptide aptamer comprises a sequence that is modified in the following way: introducing 3 polar glycines at least one end of a sequence comprised by said human chorionic gonadotrophin peptide aptamer.

3. The human chorionic gonadotropin peptide aptamer according to claim 2, wherein said modified sequence is a sequence according to any one of SEQ ID No.7 to SEQ ID No. 12.

4. The human chorionic gonadotrophin peptide aptamer according to any one of claims 1 to 3, wherein said human chorionic gonadotrophin peptide aptamer comprises a sequence which is a polypeptide sequence modified by cysteine.

5. A method of preparing a human chorionic gonadotropin peptide aptamer, comprising:

incubating human chorionic gonadotropin with M13 bacteriophage, culturing and amplifying the obtained combined bacteriophage, and performing a combination experiment; repeatedly screening for 3-8 times to find out phage colonies with the highest binding affinity; the DNA sequence of the pIII protein structural domain is translated to obtain the sequences shown in SEQ ID NO.1 to SEQ ID NO. 6.

6. The method of claim 5, wherein the incubation is performed by adding human chorionic gonadotropin to a 24 well plate prior to the incubation of human chorionic gonadotropin with M13 bacteriophage.

7. The method of claim 5, wherein the sequence of SEQ ID No.1 to SEQ ID No.6 is selected, and further comprising: incubating the sequences shown in SEQ ID No.1 to SEQ ID No.6 with human chorionic gonadotropin, calculating the binding rate of a single phage and the human chorionic gonadotropin, and screening to obtain a preferred sequence; the calculation formula of the binding rate of the single bacteriophage and the human chorionic gonadotropin is as follows: binding phage titer/input phage titer × 100%.

8. The method of any one of claims 5-7, wherein the screening for the sequences set forth in SEQ ID No. 1-6 further comprises: modifying the screened polypeptide sequence with glycine; the operation method comprises the following steps: three polar glycines are added to at least one end of any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO. 6.

9. The method for preparing a human chorionic gonadotropin peptide aptamer according to any one of claims 4 to 7, wherein the method further comprises: a cysteine modification of the NH 2-terminus of a sequence obtained from the method of preparing a human chorionic gonadotropin peptide aptamer according to any one of claims 4 to 7.

10. The human chorionic gonadotropin peptide aptamer according to any one of claims 1 to 3, or the human chorionic gonadotropin peptide aptamer prepared by the method for preparing the human chorionic gonadotropin peptide aptamer according to any one of claims 4 to 9, which is used for preparing a detection reagent for detecting human chorionic gonadotropin or preparing a biological detection sensor for detecting human chorionic gonadotropin; in the detection reagent for detecting human chorionic gonadotropin or the biological detection sensor for detecting human chorionic gonadotropin, the bonding molar ratio of the human chorionic gonadotropin to the human chorionic gonadotropin peptide aptamer is 1: 303.

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