CN115960213A - Preparation method and application of glycosylated hemoglobin - Google Patents

Abstract

The embodiment of the invention discloses a preparation method and application of glycosylated hemoglobin, the glycosylated hemoglobin obtained by the preparation method has good immunogenicity, and an in-vitro diagnostic kit for detecting diabetes, which is established on the basis of the preparation method, provides a quick, specific and sensitive detection method for different requirements of clinic and practice; in addition, the kit with the monoclonal antibody has the advantages of high detection speed and small dosage of blood samples, adopts a protein chip method to detect the cross reaction of the glycosylated hemoglobin and other similar antibodies, has high sensitivity of detection results, provides more accurate detection results for clinicians, and greatly improves the monitoring and management of diabetes.

Description

Preparation method and application of glycosylated hemoglobin

Technical Field

The invention relates to the field of biomedicine, in particular to a preparation method and application of glycosylated hemoglobin.

Background

Glycated hemoglobin (HbA 1 c) is a product of hemoglobin in red blood cells and saccharides (mainly glucose) in serum by a non-enzymatic reaction. The non-enzymatic reaction for forming the glycosylated hemoglobin has the characteristics of continuity, slowness and irreversible, so that the content of the glycosylated hemoglobin is determined by the past rather than the instant blood glucose concentration, and is not related to factors such as whether fasting is performed before detection, whether insulin is injected or not, whether a hypoglycemic medicament is taken or not and the like. It is generally believed that glycated hemoglobin concentration effectively reflects the average blood glucose level over the past 8 to 12 weeks. Glycated hemoglobin consists of HbA1a, hbA1b and HbA1c, wherein HbA1c accounts for about 70%, the structure of the glycated hemoglobin is stable, the glycated hemoglobin is clinically used as a monitoring index for controlling diabetes, and the concentration of the glycated hemoglobin is expressed by the percentage of hemoglobin in adult humans.

The glycosylated hemoglobin level can be used as a diagnostic test for diabetes. Chinese guidelines for the prevention and treatment of type 2 diabetes (2020 edition) formally incorporate 'glycated hemoglobin' into a diabetes diagnosis standard for the first time, and glycated hemoglobin of not less than 6.5% can be used as a basis for accurate diagnosis of diabetes. However, results below 6.5% do not exclude the possibility of diabetes and should be referred to the results of the glucose assay. If glycated hemoglobin >9.0%, it indicates that the patient is continuously hyperglycemic.

In addition, glycated hemoglobin levels can be used as a test to assess the risk of complications. Studies have shown that for every 1% reduction in mean HbA1c, the risk of mortality associated with diabetes is reduced by 21%, myocardial infarction by 14%, and microvascular complications by 37%. For each reduction in HbA1c levels by one, the risk of retinal pathology is reduced by 43% -45%. The glycosylated hemoglobin level is used as a detection means for screening high risk group of diabetes. Due to the continuity of the risk of rising HbA1 levels, those with HbA1c levels below the diabetic threshold, but > 6.0% should receive effective prophylactic intervention. However, patients with HbA1c levels below 6.0% may also be at risk. In the management of gestational diabetes, only blood sugar control is not enough, and the level of glycosylated hemoglobin should be controlled, so that the method has a positive effect on preventing eclampsia, giant children, teratocarcinosis and dead fetus.

The methods for routine measurement of HbA1c in clinical laboratories generally fall into two main categories: the type is based on the difference of charges of the glycated hemoglobin and the non-glycated blood greater protein, such as electrophoresis and ion exchange chromatography; another type is based on the structural difference between glycated hemoglobin and non-glycated hemoglobin, such as immunization, affinity chromatography. When hemoglobin variants are present, the HbA1c results measured by different detection methods may differ.

Each method of detection has advantages such as being unaffected by certain components, being reproducible, rapid, etc., but also methods are interfered by factors such as blood system diseases, hemoglobin variants, carbamylated hemoglobin, etc. However, many clinical examination workers do not have much knowledge about interference factors of the HbA1c detection method, and the accuracy of the HbA1c detection result is seriously influenced.

Disclosure of Invention

The invention provides a preparation method and application of glycosylated hemoglobin, and aims to solve the technical problem of diabetes detection in the prior art.

The technical scheme adopted by the invention for solving the problems is as follows:

a method for preparing glycosylated hemoglobin comprises the following steps:

artificially synthesizing a DNA sequence shown in SEQ ID NO.1, removing an N-terminal signal peptide, designing a primer, amplifying by using a conventional PCR method (a carrier pET-28a is taken as a template) to obtain a segment of an HbA1c gene to obtain a recombinant plasmid pET-28a-HbA1c, converting a connecting product into escherichia coli DH5 alpha, selecting a clone on an LB (Langmuim-Blodgett) flat plate containing ampicillin, preparing a plasmid in a small amount, screening out a positive clone through PCR identification, and indicating that the recombinant HbA1c segment is completely consistent with the designed sequence by a sequencing result.

And (3) after sequencing verification, converting the recombinant plasmid into escherichia coli (BL 21), culturing in an LB culture medium containing ampicillin, selecting positive clones on an LB plate, carrying out plasmid enzyme digestion identification, preparing plasmids in a small amount, screening the positive clones by double enzyme digestion PCR identification, and finally obtaining the recombinant plasmid engineering bacteria containing HbA1c.

Respectively culturing the recombinant plasmid engineering bacteria in LB culture medium containing 100 mug/mL ampicillin, wherein A600 is between 0.5 and 06, then adding isoproyl beta-D-1-thiogalactopyranoside (IPTG) with the final concentration of 0.5mM to induce for 4 hours at 37 ℃, centrifuging the bacterial solution 4,000rpm after the induction is finished for 10 minutes, collecting the bacteria, and washing the precipitate with PBS; and (3) placing the PBS in an ice bath after resuspension and precipitation, performing ultrasonication and centrifugation on a large amount of thalli obtained by expression, then washing an inclusion body, and purifying the protein by using a His Trap FF purification column after washing (preparing and purifying a reagent according to a product specification). And finally, analyzing the HbA1c recombinant protein by SDS-PAGE electrophoresis, and measuring the concentration of the HbA1c recombinant protein by using a BCA protein quantification kit.

Based on the same design thought, the invention also provides a preparation method of the glycosylated hemoglobin antibody, which specifically comprises the following steps: s1, obtaining HbA1c recombinant protein according to the preparation method of the glycosylated hemoglobin; s2, expression of HbA1c recombinant protein; s3, establishing a mouse anti-HbA 1c hybridoma cell strain, mixing mouse spleen cells and mouse myeloma cells according to the cell number of 2-5, fusing the mouse spleen cells and the mouse myeloma cells by adopting a fusion promoter to obtain fused cells, culturing the fused cells in HAT selective culture solution A containing HbA1c recombinant protein, and screening out positive hybridoma cells capable of reacting with the HbA1c protein after 8-15 days; s4, secreting the glycosylated hemoglobin antibody by using the positive hybridoma obtained in S3.

The specific sequence of the SEQ ID NO.1 is

atggtgcatctgactcctgaggagaagtctgccgttactgccctgtggggcaaggtgaac gtggatgaagttggtggtgaggccctgggcaggctgctggtggtctacccttggacccagaggt tctttgagtcctttggggatctgtccactcctgatgctgttatgggcaaccctaaggtgaaggc tcatggcaagaaagtgctcggtgcctttagtgatggcctggctcacctggacaacctcaagggc acctttgccacactgagtgagctgcactgtgacaagctgcacgtggatcctgagaacttcaggc tcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaattcaccccaccagt gcaggctgcctatcagaaagtggtggctggtgtggctaatgccctggcccacaagtatcactaa；

The amino acid sequence corresponding to SEQ ID NO.1 is SEQ ID NO.2, and the details are as follows:

MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPK VKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFT PPVQAAYQKVVAGVANALAHKYH。

further, in S3, murine spleen cells and murine myeloma cells were mixed in a cell number of 4.

Furthermore, HAT selective medium A was a 25-046-CI harvesting cellgro cell culture medium available from Corning cellgro.

Further, S4 comprises the steps of: s4.1, injecting the pristanane into the abdominal cavity of the mouse, wherein the injection amount is 0.5ml per mouse; 4.2-10 days after S, 1X 10 injections were administered to the abdominal cavity of mice ⁶ ～5×10 ⁶ (ii) positive hybridoma cells; s4.3, collecting ascites of the mouse, centrifuging, removing cell components, precipitates, fat and an oil layer in the ascites, and collecting a middle layer; s4.4, precipitating the solution in the middle layer by using saturated ammonium sulfate, re-suspending, and collecting a liquid layer, wherein the collected solution is ascites re-suspension; s4.5, adopting purification columns corresponding to different diabetes subtypes to separate the glycosylated hemoglobin antibody in the ascites resuspension to obtain the glycosylated hemoglobin monoclonal antibody.

Further, the specific operations of S4.4 are: 5.0ml PBS was added drop-wise to 5ml ascites resuspension; after mixing evenly, adding 10ml of saturated ammonium sulfate solution drop by drop, and continuing to stir slowly for 30min; after standing for 2h, centrifugation was carried out for 15min, the supernatant was discarded, the pellet was resuspended in PBS, and the resuspension was then filtered through a 0.22 μm filter.

Further, the specific operation of S4.5 is: balancing the selected purification column by using a binding buffer solution, an elution buffer solution and a regeneration buffer solution, then loading the ascites resuspension at the speed of 1ml/min, balancing by using the binding buffer solution after loading, washing to a baseline position by using an eluent, and collecting an antibody peak solution which contains a glycosylated hemoglobin monoclonal antibody; the antibody peak solution was dialyzed against PBS buffer, and the glycated hemoglobin monoclonal antibody thus obtained was dispensed and stored at-70 ℃. Based on the same design concept, the invention also provides a glycosylated hemoglobin antibody, and the glycosylated hemoglobin antibody is obtained by the preparation method.

Based on the same design concept, the invention also provides a kit which comprises an ELISA micropore plate, wherein the ELISA micropore plate is coated with the glycosylated hemoglobin antibody.

Further, the glycated hemoglobin antibody was blocked with PBS buffer, and the PBS buffer contained 5% BSA and 0.2% sucrose.

Further, the kit also comprises a detection antibody, wherein the detection antibody is HRP-labeled anti-HbA 1c detection antibody freeze-dried powder.

Further, the solution also comprises 10ml of stop solution, wherein the stop solution contains sulfuric acid, and the concentration of the stop solution is 2mol/L.

In summary, compared with the prior art, the method for preparing a glycated hemoglobin antibody and the kit having the same provided by the present invention have at least the following technical effects:

1) The monoclonal antibody secreted by the hybridoma cell strain glycosylated hemoglobin has good specificity, and the in-vitro diagnostic kit for detecting the diabetes, which is established on the basis of the monoclonal antibody, provides a quick, specific and sensitive detection method for different requirements of clinic and practice;

2) The kit for quantitatively detecting the diabetes mellitus has the advantages of high detection speed and small dosage of blood samples, adopts a protein chip method to detect the cross reaction of the glycosylated hemoglobin antibody and other similar antibodies, has high sensitivity of a detection result, provides a more accurate detection result for a clinician, and greatly improves the monitoring and management of the diabetes mellitus.

Detailed Description

For better understanding and implementation, the technical solutions in the embodiments of the present invention are clearly and completely described below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example one

In the technical solution of this embodiment, the method for preparing glycated hemoglobin includes the steps of:

1. preparation of antigen (HbA 1c recombinant protein)

The method comprises the following steps:

1) Construction of pET28a-HbA1c recombinant vector

Artificially synthesizing a DNA sequence shown in SEQ ID NO.1, removing a signal peptide at the N tail end, designing a primer, amplifying by using a conventional PCR method (a carrier pET-28a is taken as a template) to obtain a segment of an HbA1c gene to obtain a recombinant plasmid pET-28a-HbA1c, converting a connecting product into escherichia coli DH5 alpha, selecting a clone on an LB (Langmuim) flat plate containing ampicillin, preparing a plasmid in a small amount, screening out a positive clone through PCR identification, and indicating that the recombinant HbA1c segment is completely consistent with the designed sequence by a sequencing result.

2) Expression of HbA1c recombinant protein

And (3) after sequencing verification, converting the recombinant plasmid into escherichia coli (BL 21), culturing in an LB culture medium containing ampicillin, selecting positive clone on an LB plate, carrying out plasmid enzyme digestion identification, preparing the plasmid in a small amount, screening the positive clone by using double enzyme digestion PCR identification, and finally obtaining the recombinant plasmid engineering bacterium containing HbA1c.

Respectively culturing the recombinant plasmid engineering bacteria in LB culture medium containing 100 mu g/mL ampicillin, wherein A600 reaches 0.5-06, then adding isoproyl beta-D-1-thiogalactopyraside (IPTG) with final concentration of 0.5mM, inducing for 4h at 37 ℃, centrifuging for 10min at 4,000rpm of bacterial solution after induction is finished, collecting bacteria, and washing precipitates by PBS; placing the PBS in an ice bath after resuspension and precipitation, centrifuging at 12000rpm for 20min after ultrasonic bacteria breaking, and respectively performing SDS-PAGE electrophoresis on the supernatant and the precipitate, wherein the result shows that: the expressed HbA1c recombinant protein is expressed in a cytoplasm insoluble way.

3) Purification and quantification of HbA1c recombinant proteins

The cells obtained by mass expression are subjected to ultrasonication and centrifugation, inclusion body washing is performed, and after washing is completed, protein is purified by a His Trap FF purification column (reagent preparation and purification are performed according to product instructions). And analyzing the HbA1c recombinant protein finally obtained by SDS-PAGE electrophoresis, and measuring the concentration of the HbA1c recombinant protein by using a BCA protein quantification kit.

2. Establishment of mouse anti-HbA 1c hybridoma cell strain

The method comprises the following steps:

a. 2 healthy female BALB/c mice (1 #, 2#,3 #) which are 6-8 weeks old and 18g in weight are selected and are bred adaptively for 1 week, and then negative blood is collected to be used as a control;

b. the immunization program used 4 basic immunizations and 1 booster immunization. A medium-range immunization scheme (0.3 mL/mouse, 2 weeks/mouse) is adopted, and when the vaccine is used for the first time (50 mu g/mouse), the immunogen is stirred and emulsified with an equal volume of Freund's complete adjuvant, and the vaccine is injected into the back part of a patient at multiple points subcutaneously. Then, conventional immunization is carried out for 3 times according to stirring and emulsification of the immunogen and equivalent volume of Freund incomplete adjuvant; the titer is measured 7 days after the 4 th conventional immunization, the result is shown in table 1, and the titer of the immunized mice obviously reaches 1:320000 or above, and preparing for booster immunization;

c. boosting the immunity without adding an adjuvant, wherein the boosting dose is 50 mu g/mouse, 3 days after boosting the immunity, picking eyeballs, collecting blood, separating serum and storing, and simultaneously taking spleens;

d. mixing spleen cells and myeloma cells according to the cell number of about 4, fusing under the fusion promoting effect of polyethylene glycol (PEG, molecular weight of 1450), culturing the fused cells in HAT selective culture solution (25-046-CI harvesting cell gro), screening positive hybridoma capable of reacting with HbA1c protein by an indirect ELISA method after 10 days, carrying out expanded culture on the preliminarily screened positive hybridoma, and eliminating a labeled protein (His-tag) hybridoma after two days to re-screen out hybridoma not labeled with the HbA1c protein;

e. and continuously subcloning the obtained positive hybridoma cells at least twice by using a limiting dilution method, culturing the subclones by using an HT selective medium (25-047-CI cloning cellgro) each time, and performing ELISA screening after 8-10 days of subcloning until the positive rate of the monoclonal cells is 100 percent to obtain the hybridoma cell strain capable of stably secreting the monoclonal antibody.

3. Preparation of mouse anti-HbA 1c monoclonal antibody and identification of monoclonal antibody subtype

60ul of culture supernatant of the hybridoma cell strain HbA1c is taken, the subtype of the monoclonal antibody secreted by the hybridoma cell strain HbA1c is identified according to the specification of the mouse monoclonal antibody subtype identification reagent strip, and the result shows that: the antibody subtype of the supernatant of hybridoma HbA1c is IgG1.

TABLE 1 potency test results after four conventional immunizations of mice

Efficacy value	HbA1c	NC
			1w	1.576	0.311
2w	1.427
			4w	1.334
8w	1.135
			16w	0.804
32w	0.539
			64w	0.338
128w	0.284

Example two

The preparation and purification of the mouse anti-SCCA ascites monoclonal antibody specifically comprises the following steps:

a. selecting female healthy BALB/c mice with the age of 8-12 weeks, and injecting 0.5mL per mouse of pristane into the abdominal cavity; 7-10 days later, each mouse was injected with 1 × 106-5 × 106 monoclonal hybridoma cell lines of the first example, and the cells were blown down from the culture dish or diluted with PBS or serum-free medium;

b. centrifuging ascites at 10000r/min for 15min, removing cell components and other precipitates, fat, oil layer and the like, and collecting the middle layer;

c. precipitation of saturated ammonium sulfate: sucking 5mL of the middle layer, transferring the middle layer into a small beaker, and dropwise adding 5.0mL of PBS (PBS) which passes through a 0.22 mu m filter membrane under stirring; after mixing evenly, 10mL of saturated ammonium sulfate solution (pH7.4) is added drop by drop, and the mixture is continuously and slowly stirred for 30min; standing for 2h, centrifuging at 10000r/min for 15min, discarding the supernatant, resuspending the precipitate with PBS (phosphate buffer solution) with a filter membrane of 0.22 μm, and filtering the resuspension solution with a filter membrane of 0.22 μm;

d. according to different subtypes of the antibody, different purification columns of GE Healthcare company are selected, igG and IgM columns have corresponding instructions, and different buffers are configured according to the instructions for purification: taking IgG antibody purification as an example, firstly, balancing the column by using a binding buffer solution, an elution buffer solution and a regeneration buffer solution, generally balancing 5 column volumes, then loading the ascites resuspension obtained in the step c at the speed of 1mL/min, balancing by using the binding buffer solution after loading is finished, then washing to a baseline position by using an eluent, and collecting an antibody peak; the antibodies were dialyzed against PBS buffer, the antibody concentration was determined using BCA Protein quantification Kit (Pierce BCA Protein Assay Kit 23225Thermo Scientific), and the antibodies were dispensed and frozen at-70 ℃.

EXAMPLE III

The specificity identification of the monoclonal antibody specifically comprises the following steps:

in this example, the HbA1c recombinant antigen and VIS, NPY, ANGII, RES, ADPN, RBP4, VAP, CART, APC, GHR, NES proteins (see table 2) described in example one were used as coating antigens, respectively, and the monoclonal antibody prepared in example two was used as a recognition antibody to detect HbA1c by indirect ELISA.

TABLE 2 HbA1c data results for cross antigen detection samples

1) Enzyme label plate coating

The coated antigen was diluted to 1. Mu.g/mL with a coating solution (Na 2CO 3.5 g, naHCO 3.2 g, na2N3.2 g, ddH2O to 1L, pH 9.6), mixed well and added to a 96-well microplate at 100. Mu.L per well, and the plate was sealed at 4 ℃ overnight.

2) Enzyme label plate sealing

PBS containing 5% skim milk was used as a blocking solution. Firstly, the enzyme label plate coated overnight is patted dry, 200 mu L/well of sealing liquid is added, the sealing is carried out for 2h at 37 ℃, the plate is washed for 6 times by a plate washing machine, then the enzyme label plate is patted dry and is reserved at 4 ℃ or stored for a long time at-20 ℃.

3) Indirect ELISA detection method

Adding the monoclonal antibody prepared in the second embodiment into the closed enzyme label plate, adding 100 mu L/well, incubating for 1h at 37 ℃, washing the plate for 6 times by using a plate washing machine, then beating the enzyme label plate to be dry, adding a certain concentration of biotin-labeled goat IgG antibody (purchased from Raybiotech in USA), adding 100 mu L/well, and incubating for 1h at 37 ℃; after washing the plate, streptavidin-labeled Horse Radish Peroxidase (HRP) (available from Raybiotech, USA) was added at 100. Mu.L/well, and incubated at 37 ℃ for 1h; after washing the plate, adding TMB color developing solution, after the color development is completed, adding 2M concentrated sulfuric acid to stop the color development, measuring OD450 by using an enzyme labeling instrument (American Biotek), and comparing and analyzing the cross reaction, wherein the final result shows that the monoclonal antibody prepared in the second embodiment only recognizes HbA1c.

Example four

The enzyme-linked immunoassay kit for detecting the diabetes comprises the following components:

1. ELISA microplate: the capture antibody-monoclonal antibody was coated and blocked with 5% BSA,0.2% sucrose in PBS, and the monoclonal antibody was secreted from the hybridoma cell line HbA1c.

2. 15ml of specimen diluent: BSA 0.5% and Tween 20 0.05% in PBS buffer were added.

3. 15ml of detection antibody diluent: adding 0.5% BSA,0.05% Tween 20 in PBS buffer.

4. Detecting an antibody: HRP-labeled anti-HbA 1c detection antibody lyophilized powder (Santa Cruz, sc-130320).

5. And (3) standard substance: hbA1c recombinant protein standard-dry powder recombinant protein HbA1c-4.

6. 10X Wash 30ml:0.2M Tris-HCl,5M NaCl,0.5% Tween 20.

7. 20ml of substrate: TMB solution.

8. 10ml of stop solution: 2M h2so4.

9. Kit product instruction, packing box, plastic film.

The operation flow of the enzyme linked immunosorbent assay kit for detecting the cervical cancer in the embodiment is as follows:

1) Adding HbA1c protein standard samples subjected to gradient dilution (1000 ng/ml,333ng/ml,111ng/ml, 37.1ng/ml,12.3ng/ml,41ng/ml,13ng/ml and 0 ng/ml) and 30 blood samples to be detected into a micro-porous plate, performing two repetitions on each sample, adding 100ul to each well, and reacting at 37 ℃ for 40 minutes;

2) Preparing 1X washing liquid to wash the plate for 5 times for 10 minutes on a plate washing machine;

3) Adding a detection antibody diluent into a microporous plate, uniformly mixing, and adding the mixture into the microporous plate for incubation for 40 minutes;

4) And washing again, adding a substrate, reacting for 10 minutes, adding a stop solution, developing, and reading on an enzyme label plate.

And calculating a standard curve according to the reading, obtaining a linear relation between the reading and the HbA1c protein standard, and substituting the OD value of the sample into a linear formula to obtain the content of the sample. The whole process does not exceed 1 and a half hour.

EXAMPLE five

VIS, NPY, ANGII, RES, ADPN, RBP4, VAP, CART, APC, GHR and NES antigens are diluted to certain concentration and then detected by using the kit of the fourth embodiment, the data results are shown in tables 3 and 4, wherein 1,2 is listed as a standard curve, 3,4,5 is listed as data for detecting human serum samples by using cross antigens, and the result shows that no obvious cross reaction is found under the condition of high concentration of each cross antigen.

TABLE 3 HbA1c data results for cross antigen detection samples

TABLE 4 HbA1c data results for cross antigen detection samples

Cross antigens	Concentration of	Test value
			HbA1c	1mg/ml	2.5
VIS	1mg/ml	0.4
			NPY	1mg/ml	0.4
ANGⅡ	1mg/ml	0.5
			RES	1mg/ml	0.5
ADPN	1mg/ml	0.6
			RBP4	1mg/ml	0.6
VAP	1mg/ml	0.5
			CART	2mg/ml	0.3
APC	1mg/ml	0.6
			GHR	1mg/ml	0.6
NES	1mg/ml	0.5

The technical means disclosed in the invention scheme are not limited to the technical means disclosed in the above embodiments, but also include the technical scheme formed by any combination of the above technical features. It should be noted that modifications and adaptations can be made by those skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

SEQUENCE LISTING

<110> Rebo-ao (Guangzhou) Biotechnology Ltd

<120> preparation method and application of glycosylated hemoglobin

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 444

<212> DNA

<213> Artificial sequence

<400> 1

atggtgcatc tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac 60

gtggatgaag ttggtggtga ggccctgggc aggctgctgg tggtctaccc ttggacccag 120

aggttctttg agtcctttgg ggatctgtcc actcctgatg ctgttatggg caaccctaag 180

gtgaaggctc atggcaagaa agtgctcggt gcctttagtg atggcctggc tcacctggac 240

aacctcaagg gcacctttgc cacactgagt gagctgcact gtgacaagct gcacgtggat 300

cctgagaact tcaggctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc 360

aaagaattca ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat 420

gccctggccc acaagtatca ctaa 444

<210> 2

<211> 147

<212> PRT

<213> Artificial sequence

<400> 2

Met Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp

1 5 10 15

Gly Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu

20 25 30

Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp

35 40 45

Leu Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His

50 55 60

Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp

65 70 75 80

Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys

85 90 95

Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val

100 105 110

Cys Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln

115 120 125

Ala Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His

130 135 140

Lys Tyr His

145

Claims (10)

1. A method for preparing glycosylated hemoglobin, which is characterized by comprising the following steps: amplifying a DNA sequence shown by artificially synthesized SEQ ID NO.1 to obtain a HbA1c gene fragment to obtain a recombinant plasmid pET-28a-HbA1c, respectively culturing the recombinant plasmid in LB culture media containing 100 mu g/mL ampicillin after sequencing verification, and washing the precipitate with PBS; and (3) centrifuging after ultrasonication, and washing an inclusion body to finally obtain the HbA1c recombinant protein.

2. A method for preparing a glycated hemoglobin antibody, comprising the steps of:

s1, obtaining the HbA1c recombinant protein according to the preparation method of the glycated hemoglobin according to claim 1;

s2, expression of HbA1c recombinant protein;

s3, establishing a mouse anti-HbA 1c hybridoma cell strain, mixing mouse spleen cells and mouse myeloma cells according to the cell number of 2-5, fusing the mouse spleen cells and the mouse myeloma cells by using a fusion promoter to obtain fused cells, culturing the fused cells in HAT selective culture solution A containing the HbA1c recombinant protein, and screening out positive hybridoma cells capable of reacting with the HbA1c protein after 8-15 days;

s4, secreting the glycosylated hemoglobin antibody by using the positive hybridoma obtained through the S3.

3. The method for producing a glycated hemoglobin antibody according to claim 2, wherein: in S3, the murine spleen cells are mixed with the murine myeloma cells by cell number 4.

4. The method for producing a glycated hemoglobin antibody according to claim 3, wherein: the HAT selective culture solution A is a cell culture solution which is available from Corning cellgro and has the model number of 25-046-CIcorning cellgro.

5. The method for producing a glycated hemoglobin antibody as set forth in claim 2, wherein the S4 comprises the steps of:

s4.1, injecting the pristanane into the abdominal cavity of the mouse, wherein the injection amount is 0.5ml per mouse;

s4.2-10 days later, 1X 10 injections were given to the abdominal cavity of the mice ⁶ ～5×10 ⁶ (ii) each of said positive hybridoma cells;

s4.3, collecting ascites of the mouse, centrifuging, removing cell components, precipitates, fat and an oil layer in the ascites, and collecting a middle layer;

s4.4, carrying out precipitation treatment on the solution in the middle layer by adopting saturated ammonium sulfate, carrying out heavy suspension, and collecting a liquid layer, wherein the collected solution is used as an ascites heavy suspension;

and S4.5, separating the glycosylated hemoglobin antibody in the ascites resuspension by adopting purification columns corresponding to different diabetes subtypes to obtain the glycosylated hemoglobin monoclonal antibody.

6. The method for producing a glycated hemoglobin antibody according to claim 5, wherein the specific operation of S5.4 is:

adding 5.0ml PBS dropwise into 5ml of the ascites resuspension;

after mixing evenly, adding 10ml of saturated ammonium sulfate solution drop by drop, and continuing to stir slowly for 30min;

after standing for 2 hours, the mixture was centrifuged for 15 minutes, the supernatant was discarded, and the pellet was resuspended in PBS, and then the resuspension solution was passed through a 0.22 μm filter.

7. The method for producing a glycated hemoglobin antibody according to claim 5, wherein S5.5 comprises the steps of:

balancing the selected purification column by using a binding buffer solution, an elution buffer solution and a regeneration buffer solution, then loading the ascites resuspension at the speed of 1ml/min, balancing by using the binding buffer solution after loading, washing to a baseline position by using an eluent, and collecting an antibody peak solution, wherein the antibody peak solution contains the glycosylated hemoglobin monoclonal antibody;

dialyzing the antibody peak solution with PBS buffer, dispensing the glycated hemoglobin monoclonal antibody obtained thereby, and freezing at-70 ℃.

8. A glycated hemoglobin antibody, comprising: the glycated hemoglobin antibody is obtained by the production method according to any one of claims 2 to 7.

9. A kit comprising an ELISA plate coated with the glycated hemoglobin antibody of claim 8.

10. The kit of claim 9, wherein the glycohemoglobin antibodies are blocked with PBS buffer containing 5% bsa and 0.2% sucrose.