Synthesis method of uniform sialylated AFP-L3 antigen, serum polyclonal antibody preparation and application thereof

Abstract

The invention discloses a synthesis method of a uniform sialylated AFP-L3 antigen, serum polyclonal antibody preparation and application thereof, and belongs to the field of immunology, the uniform sialylated AFP-L3 antigen comprises a sialylated AFP-L3 glycopeptide antigen or a sialylated AFP-L3 glycoprotein antigen, and the synthesis method comprises the following steps: transferring a carbohydrate chain to a starting substrate by using Endo-M N175Q transglycosylation reaction to obtain an intermediate I; wherein the starting substrate comprises a glycopeptide Fmoc-ATKVN (GlcNAc) FTEAQKAALDV-NH2 or an AFP (Alpha Fetal Protein) protein which is expressed in HEK293T and is subjected to hydrolysis treatment; modifying the intermediate I through fucose transferase FuT8 to obtain an intermediate II; the intermediate II is modified and extended through galactose transferase GalT and sialyltransferase SiaT, and an intermediate III or a sialylated AFP-L3 glycoprotein antigen is obtained; the sialylated AFP-L3 glycopeptide antigen is obtained through the steps that the sialylated AFP-L3 glycopeptide antigen is obtained, the intermediate III is subjected to Fmoc removal treatment and then conjugated with bis-succinimide glutaric acid ester and hemocyanin, and the serum polyclonal antibody generated based on the antigen has high titer and specificity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of immunology, and particularly relates to a synthesis method of uniform sialylated AFP-L3 antigen, preparation of serum polyclonal antibody and application thereof. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is a common malignant tumor. In clinical practice, the early diagnosis of HCC has always been a great challenge, which is mainly due to the high compensatory capacity of the liver and the biological characteristics of HCC itself. For a long time, as a biomarker for the diagnosis of HCC, the serum glycoprotein alpha-fetoprotein (AFP) with an N-glycosylation site has occupied an irreplaceable position in clinical practice. However, with the increasing depth and refinement of biomedical research, the limitations of AFP in the diagnosis of HCC have gradually emerged, and its sensitivity and specificity often cannot achieve the ideal diagnostic effect. According to statistical data, about 20%-30% of HCC patients do not have significantly elevated AFP levels in the body, or only have low-level expression. At the same time, AFP may also be abnormally elevated in physiological or pathological conditions such as non-cancerous liver disease and pregnancy, which further reduces the accuracy of AFP as a diagnostic indicator, leading to a large number of false positive and false negative results, which not only affects the accurate judgment of the patient's condition by the doctor, but also may mislead the treatment decision, and has an adverse effect on the prognosis evaluation of the patient.

[0003] In order to overcome these problems, researchers have been constantly looking for new biomarkers in order to improve the accuracy and sensitivity of HCC diagnosis. HCC and alpha-fetoprotein AFP produced by benign liver diseases such as hepatitis and cirrhosis have different sugar chain structures. These AFPs with different sugar chain structures are called AFP isoforms. According to the affinity of LCA, AFP can be divided into LCA non-binding type (AFP-L1, AFP-L2) and LCA binding type (AFP-L3), and the fucosylated AFP isoform (AFP-L3) produced by HCC is much higher than that of benign liver diseases. Therefore, AFP-L3 has higher specificity and sensitivity in the early diagnosis, differential diagnosis, efficacy evaluation and prognosis monitoring of HCC, and has received widespread attention from theoretical and clinical researchers in recent years. In 2005, the U.S. Food and Drug Administration (FDA) officially listed AFP-L3 as a warning marker for primary liver cancer; and in 2017, the Chinese standard for the diagnosis and treatment of primary liver cancer pointed out that for liver cancer patients with normal AFP levels, detecting AFP-L3 can improve the diagnosis rate. The existing clinical method is to separate AFP-L3 by LCA microcentrifuge column method, and then detect it by electrochemiluminescence or ELISA. The whole operation process is complicated, the reagent is expensive and time-consuming, which limits its wide application.

[0004] In summary, AFP has limitations in the diagnosis of HCC, while AFP-L3 shows higher sensitivity and specificity and can be used as a new generation of HCC specific tumor marker, but the clinical detection thereof needs to be carried out in steps, and at present, there is no simple and efficient method, SUMMARY

[0005] This section is intended to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the abstract and title of the specification of the present application in order to avoid obscuring the purpose of this section, the abstract and the title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0006] In view of the above and / or problems existing in the prior art, it is a feasible strategy to develop a new HCC detection reagent and method by preparing specific antibodies of AFP-L3.

[0007] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art and provide a synthetic method of uniform sialylated AFP-L3 antigen.

[0008] To solve the above technical problems, the present application provides the following technical solutions: the uniform sialylated AFP-L3 antigen includes sialylated AFP-L3 glycopeptide antigen or sialylated AFP-L3 glycoprotein antigen, and the synthetic method is,

[0009] The Endo-M N175Q transglycosylation reaction is used to transfer the sugar chain to the starting substrate to obtain intermediate I;

[0010] The starting substrate includes glycopeptide Fmoc-ATKVN(GlcNAc)FTEAQKAALDV-NH2 or AFP protein expressed in HEK293T and treated by hydrolysis;

[0011] The intermediate I is modified by fucose transferase FuT8 to obtain intermediate II;

[0012] The intermediate II is modified and extended by galactose transferase GalT and sialic acid transferase SiaT to obtain intermediate III or sialylated AFP-L3 glycoprotein antigen;

[0013] When the starting substrate is a glycopeptide, the intermediate III is obtained after modification and extension, and when the starting substrate is a hydrolyzed AFP protein, the sialylated AFP-L3 glycoprotein antigen is obtained after modification and extension;

[0014] The intermediate III is subjected to Fmoc removal treatment, and then conjugated with bisuccinimidyl glutarate and hemocyanin to obtain the sialylated AFP-L3 glycopeptide antigen.

[0015] As a preferred scheme of the method for synthesizing the uniform sialylated AFP-L3 antigen according to the present application, wherein: the transfer of the sugar chain to the starting substrate by the Endo-M N175Q transglycosylation reaction comprises,

[0016] 1 mM of the starting substrate, 30 mM of the glycosyl donor GlcNAc2Man3GlcNAc-oxa, and 1.5 μg of Endo-M N175Q are reacted in 10 μL of Tris-HCl at 30°C for 10 min, and the reaction is quenched by heating to obtain the intermediate I.

[0017] As a preferred scheme of the method for synthesizing the uniform sialylated AFP-L3 antigen according to the present application, wherein: the preparation method of the AFP protein expressed in HEK293T and treated by hydrolysis comprises,

[0018] The mammalian cell expression plasmid pME-His-AFP is constructed, transiently transfected into HEK293T, and after incubation and culture in a carbon dioxide incubator for 1 d, the transfection liquid is poured out, and after being replaced with complete culture medium, the culture is continued, and after 3-4 d, the secreted protein in the culture medium is collected, Western Blot analysis is performed on the protein expression, and after confirming the protein expression, the protein is cultured in large quantities and purified to obtain the AFP protein;

[0019] The AFP protein is added with 20 μg of Endo-M WT in Tris-HCl, and reacted at 30°C for 2 h to realize the hydrolysis of the AFP protein by Endo-M WT, i.e., to obtain the AFP protein expressed in HEK293T and treated by hydrolysis.

[0020] As a preferred scheme of the method for synthesizing the uniform sialylated AFP-L3 antigen according to the present application, wherein: the modification of the intermediate I by the fucose transferase FuT8 comprises,

[0021] 1 mM of the intermediate I, 2 mM of GDP-Fuc, and 5 μg of FuT8 are reacted in 50 μL of MES-NaOH at 30°C overnight, and the reaction progress is detected by HPLC or SDS-PAGE until the conversion rate reaches 100%, i.e., to obtain the intermediate II.

[0022] As a preferred scheme of the method for synthesizing the uniform sialylated AFP-L3 antigen according to the present application, wherein: the modification and extension of the intermediate II by the galactosyltransferase GalT and the sialyltransferase SiaT comprise,

[0023] To the intermediate II system, 15 mM of UDP-Gal, 10 mM of CMP-Neu5Ac, 1 μg of GalT and 1 μg of SiaT are added, and the reaction is carried out at 37°C for 1 h, and the reaction is detected by HPLC or SDS-PAGE until completion, to obtain the intermediate III or the sialylated AFP-L3 glycoprotein antigen.

[0024] As a preferred scheme of the method for synthesizing the sialylated AFP-L3 antigen according to the present application, wherein: the method for de-Fmoc treating the intermediate III and then conjugating the bisuccinimidyl glutarate and the hemocyanin comprises,

[0025] The intermediate III is dissolved in a DMF solution containing 10% piperidine to remove the Fmoc group, the solvent of the mixture is removed by rotary evaporation under reduced pressure, the mixture is dissolved in a phosphate buffer / DMF mixed solvent with a volume ratio of 1:4 of 0.1 M phosphate buffer / DMF and pH 8.0, 15 eq. of DSG is then added, and the reaction is carried out at room temperature for 3-6 h to obtain DSG-ATKVN(Sia2Gal2GlcNAc2Man3GlcNAcFucGlcNAc)FTE AQKAALDV-NH2.

[0026] The solvent of the reaction mixture is removed by rotary evaporation under reduced pressure, the mixture is dissolved in 0.1 M phosphate buffer with pH 8.0 in the reaction container, and then 1:30 molar ratio of the carrier protein KLH with the sugar chain is added, and the reaction is carried out at room temperature under slow stirring for 2.5-3 d to obtain the sialylated AFP-L3 glycopeptide antigen.

[0027] Another object of the present application is to provide a sialylated AFP-L3 antigen.

[0028] Another object of the present application is to provide a serum polyclonal antibody produced by immunological test of the sialylated AFP-L3 glycopeptide antigen or the sialylated AFP-L3 glycoprotein antigen.

[0029] Another object of the present application is to provide an application of the serum polyclonal antibody in specifically recognizing and combining the sialylated AFP-L3 antigen.

[0030] Another object of the present application is to provide a kit for detecting the sialylated AFP-L3 antigen, which is composed of the serum polyclonal antibody and a secondary antibody; wherein the serum polyclonal antibody and the secondary antibody are provided with detectable labels.

[0031] The present application has the following beneficial effects:

[0032] (1) The present application provides the first in vitro synthesis method of uniform sialylation AFP-L3 antigen, the reaction raw material of the method is simple and easy to obtain, the operation is simple, the reaction condition is mild, and the method has important value in the physical and chemical research of HCC.

[0033] (2) The serum polyclonal antibody of the present application can specifically recognize and bind sialylated AFP-L3 antigen, and the antibody of the present application has great clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor. Among them:

[0035] Figure 1 The flow chart of the synthesis method of sialylated AFP-L3 glycopeptide antigen (a) and glycoprotein antigen (b) in the present application embodiment 1 and 2.

[0036] Figure 2 The result chart of ELISA method for detecting serum polyclonal antibody titer in the present application embodiment 3.

[0037] Figure 3 The specificity result chart of ELISA method for detecting serum polyclonal antibody in the present application embodiment 3.

[0038] Figure 4 The result chart of antibody type in the serum polyclonal antibody produced by ELISA method for detecting glycopeptide antigen in the present application embodiment 3.

[0039] Figure 5 The specificity result chart of Western Blot for detecting serum polyclonal antibody produced by glycopeptide antigen in the present application embodiment 3. DETAILED DESCRIPTION

[0040] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail in combination with the description.

[0041] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the connotation of the present application, therefore the present application is not limited to the specific embodiments disclosed below.

[0042] Second, the term "one embodiment" or "an embodiment" as may appear in various places of the specification is not necessarily referring to the same embodiment, nor is it excluding other embodiments. It is also not necessary for all objects, features or structures to be present and / or to work, in every embodiment.

[0043] The raw materials / chemical reagents used in the present application are commercially available in the art unless otherwise specified.

[0044] Example 1

[0045] Reference Figure 1 a, the present embodiment provides a method for synthesizing sialylated AFP-L3 glycopeptide antigen, specifically:

[0046] The substrate glycopeptide Fmoc-ATKVN(GlcNAc)FTEAQKAALDV-NH2 was dissolved in DMSO to prepare a 50 mM solution;

[0047] 0.5 μL of the substrate, 30 mM of GlcNAc2Man3GlcNAc2-oxa donor, 1.5 μg of Endo-MN175Q were added to the system, and the reaction was carried out at 30°C for 30 min. The reaction progress was detected by liquid chromatography until the reaction conversion rate reached 100%, i.e., the intermediate I was generated;

[0048] 1 mM of intermediate I was added to MES-NaOH (pH 6.0) until the pH decreased to 6.5, then GDP-Fuc (2 mM) and FuT8 (5 μg) were added, and the reaction was carried out at 37°C overnight. The reaction progress was detected by liquid chromatography until the conversion rate reached 100%, and the intermediate II was obtained;

[0049] On the basis of the system of intermediate II, the donor UDP-Gal (15 mM), CMP-NANA (10 mM) and GalT (1 μg), SiaT (1 μg) were added, and the reaction was carried out at 37°C for 1 h. The reaction progress was detected by liquid chromatography until the complete reaction, i.e., the intermediate III was obtained. The product was purified and desalted by C18 ODS column, respectively;

[0050] Characterization data of intermediate III: mass spectrometry data (MALDI-TOF) m / z: calculated value C 167 H 376 N 26 O 100 , 4277.52; found 4368.829 [M+4Na] + .

[0051] The intermediate III was dissolved in DMF, then 10% piperidine was added, and the reaction was stirred at room temperature for 1 h. The reaction progress was monitored by TLC. After the Fmoc group was removed, the solvent was removed by rotary evaporation under reduced pressure. The product obtained was used without purification.

[0052] The reaction mixture in which the Fmoc protecting group was removed was dissolved in a mixed solvent of 0.1 M pH 8.0 phosphate buffer / DMF (1:4, v / v), and DSG (15 eq.) was added. The reaction was carried out at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the product DSG-ATKVN(Sia2Gal2GlcNAc2Man3GlcNAcFucGlcNAc)FTEAQKAALDV-NH2 was obtained by rotary evaporation under reduced pressure. The product obtained was used without purification.

[0053] The reaction mixture of the previous step was dissolved in phosphate buffer (0.1 M, pH 8.0), and the carrier protein KLH was added at a molar ratio of 1:30 with respect to the sugar chain. The reaction was slowly stirred for 3 d.

[0054] After the reaction was completed, the desalination and purification were carried out on a dextran gel column to obtain the sialylated AFP-L3 glycopeptide antigen and the detection glycoconjugate BSA-ATKVN(Sia2Gal2GlcNAc2Man3GlcNAcFucGlcNAc)FTEAQKAALDV-NH 2， The mass of the oligosaccharide in the glycopeptide antigen was detected by the phenol sulfuric acid method, the mass of the protein in the glycopeptide antigen was detected by the BCA method, and the sugar loading amount was calculated to be 22.9% by the sugar loading amount calculation formula [Sugar loading amount (%) = (mass of sugar in sample / total mass of sample) x 100%].

[0055] Example 2

[0056] Reference Figure 1 b, the present embodiment provides a method for synthesizing a sialylated AFP-L3 glycoprotein antigen, in particular:

[0057] HEK293 cells were inoculated in a 6 cm plate, 5 mL of DMEM basic medium was added, and the plate was incubated at 37°C in 5% CO2. When the cells grew to 90%-100% (S / S) cell density (cell coverage area in the field of view / field of view area), the supernatant medium was removed, and the residual medium was washed with D-PBS slowly. After washing, 300 μL of trypsin / EDTA was added, and the trypsin and cells were allowed to contact for 1 min. The plate was gently tapped to make the adherent cells fall off. Then, 1.5 mL of DMEM basic medium was added, and the cells were mixed by gently blowing and sucking with a gun head or a pipette gun until the cells were in a single cell state.

[0058] Discard 60%-70%(v / v) of the cells, and finally add an appropriate amount of DMEM basic medium, and incubate at 37°C, 5% CO2, and then inoculate the digested cells into a 6-well plate at a cell density of about 80% of the cell number required to cover the 6-well plate. Incubate at 37°C, 5% CO2 for 12 hours until the cells adhere to the wall. At this time, the cell state can be used for plasmid transfection.

[0059] Add 4 μg of plasmid to 250 μL of Opti-MEM reduced serum medium, mix gently, and add 5 μL of lipofectamine 2000 to 250 μL of Opti-MEM, mix, and incubate at room temperature for 5 minutes. Mix the two solutions and incubate at room temperature for 20 minutes.

[0060] Slowly add the mixed solution to the cell culture solution in the 6-well plate, shake gently, and incubate at 37°C, 5% CO2. After 12 hours of transfection, replace the fresh DMEM basic medium and continue to culture the cells. After 12 hours of transfection, collect the culture solution for immunoblotting analysis of protein expression. After confirming protein expression, culture and purify a large amount of AFP protein.

[0061] Subsequently, purify the AFP protein, add 20 μg of Endo-M WT per milligram of protein for enzymatic digestion, and incubate at 30°C for 4 hours. Detect the reaction progress by Coomassie brilliant blue staining until the protein bands are completely small. Quench the reaction at 100°C for 2 minutes to obtain the AFP protein expressed in HEK293T and treated by hydrolysis.

[0062] Add 30-fold equivalents of the sugar donor GlcNAc2Man3GlcNAc-oxa and Endo-M N175Q (1.5 μg) to the above reaction system for transglycosylation. Detect the reaction progress by Coomassie brilliant blue staining until the protein bands are completely large. Purify and hydrolyze the N-glycan under PNGase F enzyme digestion to further verify that the N-glycan connected to AFP is GlcNAc2Man3GlcNAc2, and obtain intermediate I.

[0063] Add 1 mM of intermediate I to 50 μL of MES-NaOH (pH 6.0) to reduce the pH to 6.5, then add GDP-Fuc (2 mM) and FuT8 (5 μg), and incubate at 37°C overnight. Detect the reaction progress by SDS-PAGE until the conversion rate reaches 100%. Purify and hydrolyze the N-glycan under PNGase F enzyme digestion to further verify that the N-glycan connected to AFP is GlcNAc2Man3GlcNAcFucGlcNAc, and obtain intermediate II.

[0064] To the above intermediate II system, add donor UDP-Gal (15 mM), CMP-NANA (10 mM) and GalT (1 μg), SiaT (1 μg), 37°C for 1 h, detect the reaction process by SDS-PAGE, and further verify that the N-glycan linked to AFP is Sia2Gal2GlcNAc2Man3GlcNAcFucGlcNAc, i.e. to obtain the sialylated AFP-L3 glycoprotein antigen.

[0065] PNGase F enzyme cleavage of the sugar chain Sia2Gal2GlcNAc2Man3GlcNAcFucGlcNAc, characterization data: mass spectrometry data (MALDI-TOF) m / z: calculated value C 78 H 145 N5O 75 2412.128 [M+4Na] Found 2412.128 [M+4Na] + .

[0066] Example 3

[0067] This example is based on the sialylated AFP-L3 glycopeptide antigen or sialylated AFP-L3 glycoprotein antigen to produce sialylated AFP-L3 antibody by immunological test, specifically:

[0068] The experimental animals selected for the immunological experiment are C57BL / 6 mice 5 weeks old, and the experiment includes a blank group, an experimental group and a control group, a total of 7 groups, five mice in each group, and the antigens are immunized by subcutaneous injection.

[0069] Control group 1 (GlcNAc-P): each mouse is injected with 12 μg of unmodified glycopeptide ATKVN (GlcNAc) FTEAQKAALDV-NH2;

[0070] Control group 2 (KLH): each mouse is injected with 80 μg of carrier protein KLH;

[0071] Experimental group 3 (SiaF-P-KLH): each mouse is injected with 80 μg of sialylated AFP-L3 glycopeptide antigen (SiaF-P);

[0072] Experimental group 4 (SiaF-P-KLH-AA): each mouse is injected with a mixture containing 80 μg of sialylated AFP-L3 glycopeptide antigen (SiaF-P) and aluminum hydroxide adjuvant;

[0073] Experimental group 5 (SiaF-P-KLH-FA): each mouse is injected with a mixture containing 80 μg of sialylated AFP-L3 glycopeptide antigen (SiaF-P) and Freund's adjuvant;

[0074] Group 6 (AFP-FA) each mouse was injected with 75 μg of AFP protein expressed in HEK293T (Gn-AFP) mixed with Freund's adjuvant;

[0075] Group 7 (SiaF-AFP-FA) each mouse was injected with 75 μg of sialylated AFP-L3 glycoprotein antigen (SiaF-AFP) mixed with Freund's adjuvant.

[0076] The immunization was boosted with the same dose on day 1, day 14, day 28, day 42, and the serum was collected on day 0, day 13, day 20, day 34, day 47, day 52 for detection by ELISA.

[0077] Detection of serum polyclonal antibody titer by ELISA

[0078] The sialylated AFP-L3 glycoprotein antigen was diluted to 20 ng / mL of protein solution in PBS (pH 7.4) buffer, 100 μL per well coated in polystyrene 96-well plates, and blocked at 37°C for 1 h, the solution in the well was aspirated, washed with PBST (phosphate buffer containing 0.05% Tween-20, pH 7.4) for three times, 100 μL of 10% BSA-PBST solution was added for 1 h at room temperature, the solution in each well was aspirated, and washed with PBST for three times.

[0079] The serum of all groups was diluted from 1:2700 to 1:656100, 100 μL per well, reacted at 37°C for 2 h, the liquid in each well was aspirated, washed with PBST for three times; goat anti-mouse Ig (G+M) labeled with alkaline phosphatase (AP) was diluted 1:1000, 100 μL per well, reacted at room temperature for 1 h, washed with PBST for three times. Finally, para-nitrophenyl phosphate (PNPP) liquid substrate was added, 100 μL per well, reacted at room temperature for 30 min in the dark. The absorbance value at 405 nm was read by a microplate reader. The dilution factor of the serum and the corresponding optical density (OD) value were plotted to obtain the best fitting line. The dilution value when the OD reached 0.2 was calculated using the linear equation, and the antibody titer was obtained as the inverse of the dilution value, and three parallel results were measured for each group.

[0080] The results are shown in Figure 2 As shown, the serum polyclonal antibody of each group was used as a primary antibody to incubate and detect the titer of the polyclonal antibody. The antibody titer produced by the mice injected with unmodified glycopeptide, KLH, and glycopeptide-KLH conjugate without adjuvant was lower than 40,000, and there was no significant difference, indicating that the glycopeptide alone or the glycopeptide-KLH conjugate without adjuvant cannot stimulate the mouse to produce an effective immune response.

[0081] The immunization effect was significantly improved after the glycoprotein conjugate was mixed with aluminum hydroxide adjuvant or Freund's adjuvant and injected into mice, and the effect of Freund's adjuvant was better than that of aluminum hydroxide adjuvant. For the AFP protein injection experimental group, the AFP protein expressed in HEK293T and the sialylated AFP-L3 glycoprotein antigen both produced strong immune responses after being mixed with Freund's adjuvant.

[0082] Specificity of serum polyclonal antibodies detected by ELISA

[0083] The AFP protein expressed in HEK293T was diluted to a protein solution of 20 ng / mL in PBS (pH 7.4) buffer, 100 μL per well was coated in a polystyrene 96-well plate, and was blocked at 37°C for 1 h, the solution in the well was aspirated, and was washed with PBST three times, 100 μL of 10% BSA-PBST solution was added for room temperature blocking for 1 h, the solution in each well was aspirated, and was washed with PBST three times, the serum produced by control group 2 and experimental groups 4-7 was diluted from 1:2700 to 1:656100, 100 μL per well, and was reacted at 37°C for 2 h, the liquid in each well was aspirated, and was washed with PBST three times; the AP-labeled goat anti-mouse Ig (G+M) was diluted at 1:1000, 100 μL per well, and was reacted at room temperature for 1 h, and was washed with PBST three times. Finally, the PNPP liquid substrate was 100 μL per well, and was reacted at room temperature for 30 min in the dark. The absorbance value at 405 nm was read by an enzyme-labeled instrument. The dilution multiple of the serum and the OD value were plotted to obtain a logarithmic curve, and the best fitting line was obtained. The dilution value when the OD reached 0.2 was calculated using the linear equation, and the antibody titer was obtained as the inverse of the dilution value, and three parallel results were measured for each group.

[0084] The serum polyclonal antibodies produced by control group 2 and experimental groups 4-7 were used as a primary antibody to incubate and detect the polyclonal antibody titer, and the results are shown in Figure 3 Compared with the KLH group, all experimental groups had certain recognition for the AFP protein expressed in HEK293T, but the recognition ability of the sialylated glycopeptide group: groups 4 and 5 was weak, and the serum polyclonal antibodies produced by the sialylated AFP-L3 glycoprotein antigen had strong recognition for non-homogeneous glycoforms AFP, and there was no obvious difference with the intermediate (AFP protein expressed in HEK293T) experimental group, and the titer was more than 150,000. Combined with the experimental results, it is indicated that the antibodies produced by the sialylated AFP-L3 glycopeptide antigen and the sialylated AFP-L3 glycoprotein antigen have certain recognition specificity, and the glycopeptide group has strict recognition for glycoforms, and the specificity is stronger than that of the glycoprotein group. Figure 2

[0085] Detection of antibody types in serum polyclonal antibodies produced by glycopeptide antigens by ELISA

[0086] ​Sialylated AFP-L3 glycopeptide antigen was diluted to 20 ng / mL of protein solution in PBS (pH 7.4) buffer, 100 μL per well coated in a 96-well plate, blocked at 37°C for 1 h, the solution in the well was aspirated, washed with PBST for three times, 100 μL of 10% BSA-PBST solution was added and blocked at room temperature for 1 h, the solution in each well was aspirated and washed with PBST for three times, the serum of experimental group 5 (containing sialylated AFP-L3 glycopeptide antigen) was diluted from 1:2700 to 1:656100, 100 μL per well, reacted at 37°C for 2 h, the liquid in each well was aspirated and washed with PBST for three times, AP-labeled goat anti-mouse Ig (G+M), IgG, IgM was diluted at 1:1000, 100 μL per well, reacted at room temperature for 1 h, washed with PBST for three times, PNPP liquid substrate, 100 μL per well, reacted at room temperature for 30 min in the dark. The absorbance value at 405 nm was read by a microplate reader. The titer was calculated by a standard curve, and each sample was measured three times.

[0087] The results are shown in Table 1. Figure 4 The titer of IgG was significantly higher than that of IgM, which was produced by sialylated AFP-L3 glycopeptide antigen mixed with Freund's adjuvant. The experimental results showed that the conjugate mixed with Freund's adjuvant produced an immune response with long-term memory.

[0088] Western Blot detection of the specificity of serum polyclonal antibodies produced by glycopeptide antigen

[0089] To detect the specificity of serum polyclonal antibodies produced by glycopeptide antigen, AFP protein expressed in HEK293T, sialylated AFP-L3 glycoprotein antigen, human serum albumin (HSA) and human transferrin (HTF) were loaded into the protein gel lane, respectively. After protein electrophoresis, Coomassie brilliant blue staining was performed, and water was decolorized. In the WB part, the gel was transferred to the PVDF membrane, the first antibody was the collected serum polyclonal antibodies produced by glycopeptide antigen, the second antibody was Anti-Mouse, and the color development liquid was used for color development analysis.

[0090] The results are shown in Table 1. Figure 5As shown, the two AFP proteins in the first two lanes are both about 71 kDa in size, and the protein concentration is approximately. The proteins in the rear lanes are HSA 66 kDa and HTF 70 kDa, respectively, and both HAS and HTF are human-derived proteins, and HTF usually has a double antenna complex type N-glycan, which is similar to the sialylated AFP-L3 glycoprotein antigen glycoform structure. After incubation with the serum polyclonal antibody, it can be seen that the serum does not recognize other types of proteins, and shows different degrees of binding ability to AFP of different glycoforms. The sialylated AFP-L3 glycoprotein antigen band is the deepest, and the AFP protein band expressed in HEK293T is lighter. It is proved that the serum polyclonal antibody produced has certain recognition ability to AFP protein, and the recognition ability to sialylated AFP-L3 glycoprotein antigen is the strongest, and HTF with the same complex type N-glycan is not recognized by the serum polyclonal antibody, which proves the specificity of the obtained serum polyclonal antibody.

[0091] From the results, it can be seen that the serum polyclonal antibody of the application has the value of being used as an in vitro clinical diagnostic reagent for HCC related diseases.

[0092] In summary, the application discloses a synthesis method of uniform sialylated AFP-L3 antigen, a glycosyl transfer reaction is performed on a starting substrate by Endo-MN175Q to obtain intermediate I; wherein the starting substrate includes a glycopeptide Fmoc-ATKVN(GlcNAc)FTEAQKAALDV-NH2 or AFP protein expressed in HEK293T and treated by hydrolysis; intermediate I is modified by a fucose transferase FuT8 to obtain intermediate II; intermediate II is modified and extended by a galactose transferase GalT and a sialic acid transferase SiaT to obtain intermediate III or sialylated AFP-L3 glycoprotein antigen; intermediate III is subjected to Fmoc removal treatment, and then is conjugated with bisuccinimidyl glutarate and hemocyanin, to obtain sialylated AFP-L3 glycopeptide antigen. Initially, the application selects a natural glycopeptide sequence Fmoc-FTKVN(GlcNAc)FTEIQKLVLDV-NH2 near the N-glycosylation site of AFP protein as a starting substrate, because the proportion of hydrophobic amino acids in the peptide segment is high, the water solubility is poor, and the first glycosyl transfer reaction is difficult to perform, therefore, part of the hydrophobic amino acids are mutated into alanine which has stronger hydrophilicity, and finally the optimized glycopeptide substrate Fmoc-ATKVN(GlcNAc)FTEAQKAALDV-NH2 is adopted.

[0093] The serum polyclonal antibody produced based on the antigen of the application has high titer and specificity. The method of the application has simple and easily available reaction raw materials, simple operation, and mild reaction conditions, and has important value in the physicochemical research of HCC.

[0094] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced, without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A method for the synthesis of homogeneous sialylated AFP-L3 antigen, characterized by: The uniform sialylation AFP-L3 antigen includes a sialylation AFP-L3 glycopeptide antigen or a sialylation AFP-L3 glycoprotein antigen, and a synthesis method is, The glycosyl transfer reaction of Endo-M N175Q is used to transfer a sugar chain to a starting substrate to obtain an intermediate I; The starting substrate includes a glycopeptide Fmoc-ATKVN(GlcNAc)FTEAQKAALDV-NH2 or an AFP protein expressed in HEK293T and treated by hydrolysis; The intermediate I is modified by a fucose transferase FuT8 to obtain an intermediate II; The intermediate II is modified and extended by a galactose transferase GalT and a sialic acid transferase SiaT to obtain an intermediate III or a sialylation AFP-L3 glycoprotein antigen; When the starting substrate is a glycopeptide, the intermediate III is obtained after modification and extension, and when the starting substrate is the hydrolyzed AFP protein, the sialylation AFP-L3 glycoprotein antigen is obtained after modification and extension. The intermediate III is subjected to Fmoc removal treatment, and then is conjugated with bis-succinimidyl glutarate and hemocyanin to obtain the sialylation AFP-L3 glycopeptide antigen.

2. The method of synthesizing homogeneous sialylated AFP-L3 antigen as claimed in claim 1 wherein: The glycosyl transfer reaction of Endo-M N175Q is used to transfer a sugar chain to a starting substrate to obtain an intermediate I; 1 mM of the starting substrate, 30 mM of a glycosyl donor GlcNAc2Man3GlcNAc-oxa and 1.5 μg of Endo-M N175Q are reacted in 10 μL of Tris-HCl at 30°C for 10 min, and the reaction is quenched by heating to obtain the intermediate I.

3. The method of synthesizing homogeneous sialylated AFP-L3 antigen as claimed in claim 2 wherein: The preparation method of the AFP protein expressed in HEK293T and treated by hydrolysis includes, A mammalian cell expression plasmid pME-His-AFP is constructed, and is transiently transfected into HEK293T, and the transfection liquid is poured out after incubation and culture in a carbon dioxide incubator for 1 d, and complete culture medium is replaced to continue culture, and secreted proteins in the culture medium are collected after 3-4 d, Western Blot analysis is performed on protein expression, and after confirmation of protein expression, large-scale culture and purification are performed to obtain the AFP protein; 20 μg of Endo-M WT is added to the AFP protein in Tris-HCl, and the AFP protein is hydrolyzed by the Endo-M WT at 30°C for 2 h to obtain the AFP protein expressed in HEK293T and treated by hydrolysis.

4. The method of synthesizing homogeneous sialylated AFP-L3 antigen as claimed in claim 1 wherein: The intermediate I is modified by a fucose transferase FuT8 to obtain an intermediate II; 1 mM of the intermediate I, 2 mM of GDP-Fuc and 5 μg of FuT8 are reacted in 50 μL of MES-NaOH at 30°C overnight, and the reaction progress is detected by HPLC or SDS-PAGE until the conversion rate reaches 100% to obtain the intermediate II.

5. The method of synthesizing homogeneous sialylated AFP-L3 antigen as claimed in claim 1 wherein: The intermediate II is modified and extended by a galactose transferase GalT and a sialic acid transferase SiaT to obtain an intermediate III or a sialylation AFP-L3 glycoprotein antigen; 1 mM of the intermediate I, 2 mM of GDP-Fuc and 5 μg of FuT8 are reacted in 50 μL of MES-NaOH at 30°C overnight, and the reaction progress is detected by HPLC or SDS-PAGE until the conversion rate reaches 100% to obtain the intermediate II. To the intermediate II system, 15 mM of UDP-Gal, 10 mM of CMP-Neu5Ac, 1 μg of GalT and 1 μg of SiaT are added, and the reaction is carried out at 37°C for 1 h, and the reaction is detected by HPLC or SDS-PAGE until completion, to obtain the intermediate III or the sialylated AFP-L3 glycoprotein antigen.

6. The method of synthesizing homogeneous sialylated AFP-L3 antigen as claimed in claim 5 wherein: The method for de-Fmoc treating the intermediate III and then conjugating the bis-succinimidyl glutarate and the hemocyanin comprises, The intermediate III is dissolved in a DMF solution containing 10% piperidine to remove the Fmoc group, the solvent of the mixture is removed by rotary evaporation under reduced pressure, the mixture is dissolved in a 0.1 M phosphate buffer / DMF mixed solvent with a volume ratio of 1:4 and pH 8.0, 15 eq. of DSG is added, and the reaction is carried out at room temperature for 3-6 h to obtain DSG-ATKVN(Sia2Gal2GlcNAc2Man3GlcNAcFucGlcNAc)FTEAQKAALDV-NH2. The solvent of the reaction mixture is removed by rotary evaporation under reduced pressure, the mixture is dissolved in 0.1 M phosphate buffer with pH 8.0, and then 1:30 molar ratio of the carrier protein KLH with respect to the sugar chain is added, and the reaction is carried out at room temperature under slow stirring for 2.5-3 d to obtain the sialylated AFP-L3 glycopeptide antigen.

7. The homogeneous sialylated AFP-L3 antigen synthesized by the synthetic method according to any one of claims 1 to 6, characterized in that: The uniform sialylated AFP-L3 antigen comprises the sialylated AFP-L3 glycopeptide antigen or the sialylated AFP-L3 glycoprotein antigen.

8. A serum polyclonal antibody characterized in that: The sialylated AFP-L3 glycopeptide antigen or the sialylated AFP-L3 glycoprotein antigen of claim 7 is produced by immunological tests.

9. The serum polyclonal antibody of claim 8 for specifically recognizing and binding the sialylated AFP-L3 antigen.

10. A kit for detecting sialylated AFP-L3 antigen, characterized by: The serum polyclonal antibody of claim 8 and a second antibody constitute; wherein the serum polyclonal antibody and the second antibody are provided with a detectable label.

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