CN115894659A - Microtubule-associated protein Tau antigen and preparation method and application thereof - Google Patents
Microtubule-associated protein Tau antigen and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a microtubule-associated protein Tau antigen and a preparation method and application thereof, wherein the microtubule-associated protein Tau antigen comprises four dominant linear epitope polypeptides, and the sequences of the dominant linear epitope polypeptides are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4. And connecting the sequence containing the Tau protein linear epitope polypeptide in series through flexible chain amino acids to obtain the amino acid sequence of the microtubule associated protein Tau antigen, wherein the amino acid sequence is shown in SEQ ID NO. 5. The recombinant Tau protein antigen is expressed and purified by prokaryotic cell Ecoli, and the expressed protein antigen has high purity. The soluble Tau protein antigen provided by the invention has good immunogenicity, and animals immunized by the recombinant Tau protein antigen can generate specific polyclonal antibody and can recognize six different Tau protein subtypes. Therefore, the antigen can be used as a high-efficiency immunogen for immune and antibody preparation and production, and is further used for scientific research detection of Tau protein and diagnosis of Tau which is a disease marker of cognitive dysfunction.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a microtubule-associated protein Tau antigen and a preparation method and application thereof.
Background
Tau protein and beta-amyloid (amyloid beta-protein, a β) are characteristic lesions of Alzheimer's Disease (AD), and are two important factors determining the occurrence and development of neurodegenerative diseases. The Tau protein is produced by monogene coding for the microtubule-associated protein Tau (MAPT) located on the 17q21.31 chromosome. Tau protein has multiple functions. Normally, the most important functions are to aggregate microtubules, including connecting, stabilizing and promoting microtubule assembly, and others include transporting nutrients in axons and dendrites and regulating the growth and development of nerve cells. A large number of microtubules are located on nerve axons, and a small number are located on dendrites and neurons.
Aberrant post-translational modifications render the Tau protein a non-functional entity, of which phosphorylation of the Tau protein is the most studied. It is now recognized that Tau pathology resulting from Tau protein increase and modification is a source of neurotoxicity. Generally, tau lesions have 3 main features: increased Tau protein levels; to an aberrant post-translational modification, such as hyperphosphorylation, sometimes associated with other post-translational modifications (e.g., truncation or acetylation); abnormal Tau protein aggregation. Once Tau protein is deposited, it is accompanied by the occurrence of neurological dysfunction. The level of Tau protein is closely related to mild cognitive dysfunction.
Characteristic cerebrospinal fluid biomarker changes in AD patients are a decrease in Α β 42, with an increase in total Tau protein (T-Tau) and phosphorylated Tau protein (p-Tau), which markers have been identified as diagnostic for dementia, mild cognitive impairment.
In humans, the MAPT gene, which encodes the Tau protein, produces six isoforms (352-441 amino acids) by alternative mRNA splicing. Based on the splicing of exons 2, 3 and 10 of human tau protein, 6 different isoforms are divided, tau352 (0N 3R), tau381 (1N 3R), tau383 (0N 4R), tau410 (2N 3R), tau412 (1N 4R) and tau441 (2N 4R), wherein exons 2 and 3 encode two 29 amino acid N-terminal insertions (0n, 1n, 2n) exon 10 leading to the generation of three isoforms with 3 or 4C-terminal repeats (3R or 4R), forming 6 different spliced isoforms by different splicing patterns. Taking the longest subtype protein of Tau (2N 4R) as an example to show the structural region, the protein mainly comprises an N-terminal projection functional region (aa 1-151), a 2N insertion sequence located in the segment, an intermediate proline enrichment region (aa 152-244), a Microtubule binding region (Microtubule-binding domain, MBD, aa 245-368), and four C-terminal repetitive sequences 4R and a C-terminal functional region (C terminal, aa 369-441).
According to the research reports of Meredith, barthe' lemy, sato, cicognola and Chen in 2013-1019, tau protein in human cerebrospinal fluid or plasma is cleaved by protease, and a large amount of Tau cleavage fragments exist in the N terminal (aa 1-151) and middle region (aa 152-244), while the content of C terminal fragments is extremely low. Therefore, how to design and synthesize the immunogen so as to include the epitope of the cutting body form antibody which can simultaneously recognize different Tau protein subtypes in plasma and cerebrospinal fluid becomes a key link for detecting the total Tau protein (t-Tau) in human tissues or body fluids. Cerebrospinal fluid T-tau level measurements have diagnostic and AD predictive value for differentiating between AD and normal controls (T-tau: 95% CI from 2.44 to 2.64, P < 0.0001) as well as stable and progressive Mild Cognitive Impairment (MCI).
At present, rapid specific detection methods aiming at Tau protein are required to be suggested in both diagnosis and scientific research processes. Conventional methods for detecting Tau protein are usually immunoassays, i.e., antibodies against Tau are used to detect the levels thereof, and therefore, prediction and discovery of suitable epitopes of Tau epitopes have been the focus of research. At present, no exact report aiming at the Tau protein epitope tandem antigen exists in the market, and with the development of the field of medical inspection, a soluble Tau protein epitope tandem antigen with high specificity and antigenicity is needed in diagnosis so as to be suitable for the preparation and production of high-specificity antibodies.
Therefore, a soluble Tau protein antigen is designed and obtained, and the generated antibody has good application prospect and practical significance for recognizing all six subtype proteins of Tau protein.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a multi-epitope tandem specific Tau protein antigen and a preparation method and application thereof.
In order to solve the technical problems, in a first aspect, the invention provides 4 dominant linear epitope polypeptides of microtubule-associated protein Tau, wherein the amino acid sequences of the dominant linear epitope polypeptides (1), (2), (3) and (4) are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.
The dominant linear epitope polypeptides (1), (2), (3) and (4) are Tau (2N 4R) 6-24, 111-130, 152-170 and 185-204 respectively, namely 6-24 of Tau (2N 4R) is located from 6 th amino acid to 24 th amino acid, 111-130 is located from 111 th amino acid to 130 th amino acid, 152-170 is located from 152 th amino acid to 170 th amino acid, and 185-204 is located from 185 th amino acid to 204 th amino acid.
In a second aspect, the present invention provides a microtubule-associated protein Tau antigen, the amino acid sequence of which is shown in Seq ID No. 5.
Further, the microtubule-associated protein Tau antigen is obtained by sequentially connecting the dominant linear epitope polypeptides (1), (2), (3) and (4) in series through a flexible polypeptide chain (linker), wherein the amino acid sequence of the flexible polypeptide chain is GGGGS.
Further, the nucleotide sequence of the coding gene of the microtubule-associated protein Tau antigen is shown in Seq ID No.6.
In a third aspect, the invention also provides a recombinant expression vector, wherein the recombinant expression vector is formed by recombining an expression vector and a coding gene of a microtubule-associated protein Tau antigen shown as Seq ID No.6.
Further, the expression vector is a PET28a plasmid.
In a fourth aspect, the invention also provides a recombinant engineering bacterium, which is obtained by transforming the recombinant expression vector into a host bacterium.
Further, the host bacterium is escherichia coli BL21 (DE 3).
In a fifth aspect, the present invention further provides a method for preparing a microtubule-associated protein Tau antigen, the method comprising the steps of:
(1) Cloning a coding gene of a Tau antigen shown by Seq ID No.6 into an expression vector to obtain a recombinant expression vector;
(2) Transferring the recombinant expression vector obtained in the step (1) into host bacteria to obtain recombinant engineering bacteria;
(3) Inducing and expressing Tau protein antigen by using recombinant engineering bacteria;
(4) And (4) extracting and purifying the expression protein obtained in the step (3) to obtain a recombinant Tau protein antigen.
Further, the specific steps of the step (1) are preferably: the coding gene of the Tau antigen shown in Seq ID No.6 was constructed between the multiple cloning sites NcoI and XhoI of the expression plasmid PET28a to obtain a recombinant expression vector, which was named PET28a-Tau.
Further, the specific steps of the step (2) are preferably:
(2-1) taking out BL21 (DE 3) competent cells from-80 ℃, quickly inserting the cells into ice, melting the cell mass after 5 minutes, adding 100ng PETC28a-Tau plasmid, slightly and uniformly mixing the cell mass with the bottom of an EP tube by hand, and standing the cell mass in the ice for 25 minutes;
(2-2) performing water bath heat shock at 42 ℃ for 45 seconds, quickly putting back on ice and standing for 2 minutes;
(2-3) adding 700 mu l of antibiotic-free sterile LB culture medium into a centrifuge tube, uniformly mixing, and recovering for 60 minutes at 37 ℃ and 200 rpm;
(2-4) centrifuge at 5000rpm for one minute to collect the bacteria, leave about 100. Mu.l of supernatant, gently blow and beat the resuspended bacteria block and inoculate 50mL LB medium containing 10ug/mL kanamycin, culture overnight at 37 ℃ and 150 rpm.
Further, the specific steps of the step (3) are preferably:
according to the inoculation amount of 2 percent, 4mL of the seed liquid obtained in the step (2) is transferred into a 200mL shaking flask containing 10ug/mL of LB culture medium, the shaking flask is shaken for 2h at 37 ℃ and 150rpm in a constant temperature shaking table, IPTG is added to ensure that the final concentration is 1mmol/L, and then the efficient induction expression is carried out for 14h at 20 ℃.
Further, the specific steps of the step (4) are preferably:
centrifuging the recombinant engineering bacteria fermentation liquor with high expression in the step (3) at 12000rpm and 4 ℃ for 10min, re-suspending the precipitated thallus in 50mL of PBS buffer solution with 0.2M and pH7.4, washing for 1 time, then centrifuging at 12000rpm and 4 ℃ for 10min, re-suspending the precipitate in 50mL0.2M and pH7.4 PBS buffer solution, carrying out ice bath ultrasound for 30min, then centrifuging at 12000rpm and 4 ℃ for 15min, and collecting supernatant;
the supernatant was filtered through a 0.45um filter and purified by passing through a 5mL nickel column, and the protein sample was allowed to flow slowly through the nickel column at a flow rate of 0.5 mL/min. The foreign proteins were washed off with 50mL volumes of Buffer A, pH7.4,0.2M PBS containing 300mM NaCl and 30mM imidazole at a flow rate of 1mL/min, and the target protein was eluted with eluent Buffer B, pH7.4,0.2M PBS containing 300mM NaCl and 300mM imidazole at a flow rate of 1 mL/min.
And concentrating and desalting the obtained eluent by using a 15mL,3kDa ultrafiltration tube, replacing Buffer with pH7.4 and 0.2M PBS, centrifuging at 4000g for 10min/time, and concentrating to obtain the recombinant Tau protein antigen.
In a sixth aspect, the present invention also provides a polyclonal antibody against a Tau antigen, wherein the polyclonal antibody is prepared from a recombinant Tau protein antigen having an amino acid sequence shown as Seq ID No. 5. The recombinant Tau protein antigen can be prepared by the method.
Specifically, the polyclonal antibody can be prepared by immunizing an animal with the recombinant Tau protein antigen to obtain serum.
More specifically, after the recombinant Tau protein antigen is emulsified, a New Zealand big ear rabbit is immunized in a back multipoint immunization mode, four times of immunization are carried out, serum containing the polyclonal antibody is obtained through collection and separation, and the polyclonal antibody is prepared through purification.
In a seventh aspect, the invention provides an application of a polyclonal antibody against a Tau antigen in preparation of a Tau protein detection kit.
In an eighth aspect, the invention further provides a Tau protein detection kit, wherein the Tau protein detection kit comprises the polyclonal antibody. The Tau protein detection kit can detect and identify all six subtypes of Tau protein.
In order to solve the above technical problems, the general idea of the present invention is as follows:
tau protein is Microtubule-associated protein, and its common forms in human body are six subtypes, which are Tau352 (0N 3R), tau381 (1N 3R), tau383 (0N 4R), tau410 (2N 3R), tau412 (1N 4R), and its longest subtype Tau (2N 4R) protein is exemplified to show that its structural region mainly includes N-terminal projection functional region (aa 1-151), 2N insertion sequence is located in this segment, intermediate proline region (aa 1-244), microtubule binding region (Microtubule-binding domain, MBD, aa 245-368), which includes four C-terminal repeat sequences 4R and C-terminal functional region (C-terminal, aa 369-441).
However, according to the reports of Meredith, barthe' lemy, sato, cicognola, chen in 2013-1019, it was found that in human cerebrospinal fluid or plasma, tau protein is cleaved by protease, and largely exists as an N-terminal (aa 1-151) and a Tau cleavage fragment in the middle region (aa 152-244), but the content of C-terminal fragment is extremely low.
Therefore, the present invention uses biological software to analyze amino acid sequences, preferentially selects a sequence with stronger antigen specificity of Tau (2N 4R), and selects a sequence common to six subtypes for epitope selection based on all subtype sequences of Tau protein, i.e., excludes the 2N insertion sequence described above and retains a 3R sequence common to 6 subtypes (a subtype with a 4R sequence has a more repetitive sequence than 3R). The epitope with the high score is selected and the epitope sequence of all Tau subtypes can be identified. The Tau antigen designed in this way can be immunized to obtain specific antibody aiming at Tau, and thus the antibody has the value of recognizing different forms of Tau subtype proteins or hydrolyzed fragments in a sample.
Therefore, on the basis of a Tau protein sequence (2N 4R), a sequence of coded amino acids of a Tau (Unit prot sequence number P10636-8) protein is analyzed through bioinformatics software, dominant epitopes obtained by screening are connected in series according to a certain sequence, and a multi-epitope fusion antigen is constructed and synthesized; meanwhile, after codon optimization, a nucleic acid sequence of the protein is synthesized, a high-efficiency prokaryotic expression vector is constructed, and high-purity recombinant protein is purified.
Compared with the prior art, the invention at least has the following beneficial effects:
1. the invention develops a new recombinant Tau protein antigen, and the recombinant Tau protein antigen is expressed by recombinant engineering bacteria, the recombinant Tau protein antigen prepared by expression comprises four linear epitope polypeptides, the linear polypeptides are not overlapped with each other and are positioned in an N-terminal region and a middle proline-rich region of Tau protein (in the natural condition, the Tau protein mostly exists in the form of an N-terminal region and a middle region splicer), and the four linear epitope polypeptide sequences exist in six subtypes of Tau.
2. According to the invention, four linear epitope polypeptides are connected in series through the flexible polypeptide chain GGGGS, so that the immunogenicity and the richness of antigen epitopes are improved compared with a single polypeptide, the need of polypeptide coupling for improving the immunogenicity is also saved, the dominant linear epitopes are concentrated in one sequence by utilizing the polypeptide after being connected in series, the uncertainty of antibody epitopes after full-length protein expression is used as immunogen immunity is avoided, the risk that an antibody aiming at Tau cannot identify all Tau protein subtypes is avoided, and the antibody is favorably applied to downstream Tau protein related diagnosis or scientific research and detection.
3. The soluble Tau protein antigen provided by the invention has good immunogenicity, can stimulate an antigen immune animal to generate immune response, generates a polyclonal antibody with high titer and strong specificity, and can identify all six subtypes of Tau protein.
Drawings
FIG. 1 is a schematic diagram of Tau352 (0N 3R), tau381 (1N 3R), tau383 (0N 4R), tau410 (2N 3R), tau412 (1N 4R) and Tau441 (2N 4R) for Tau 6 protein isoforms.
FIG. 2 shows the results of analysis of the Linear Epitope of Tau protein (subtype 2N 4R) using Bepipred Linear Epitope Prediction 2.0.
FIG. 3 is a SDS-PAGE image of Tau antigen purification; wherein, the sequence number M is marker, and the sequence number R is the recombinant Tau protein antigen in a reduction state after purification.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples, but the scope of the present invention is not limited thereto.
It should be noted that the experimental methods used in the following examples are all conventional methods unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Strains and plasmids: the host bacterium BL21 (DE 3) was obtained from Shanghai Weidi Biotechnology Ltd, the plasmid PET28a was obtained from Hongxn Biotechnology Ltd, suzhou.
Molecular biological reagents: kanamycin (kan) and IPTG are products of Shanghai life. Other reagents are imported or domestic analytical pure reagents.
And (3) gene synthesis: suzhou Hongxn Biotechnology GmbH.
The gene synthesis method comprises the following steps: cloning a Tau protein antigen nucleotide sequence (an amino acid sequence corresponds to Seq ID No. 5) into an expression vector PET28a, and directly synthesizing by Suzhou Hongxn biotechnology company Limited; general molecular cloning methods such as SDS-PAGE analysis of proteins are carried out by conventional methods. Other kits were performed as described in the instructions.
Tau subtype protein: tau352 (0N 3R), tau381 (1N 3R), tau383 (0N 4R), tau410 (2N 3R), tau412 (1N 4R), tau (2N 4R) were all purchased from BiLegend Biotech Inc. (BiLegend agency, inc.) in Beijing.
EXAMPLE 1 screening of epitopes
By referring to a Tau protein (2N 4R) sequence published on NCBI, a secondary structure and a tertiary structure domain of the protein are obtained by utilizing an online analysis tool such as ExPasy, uniProt and the like, a Bepip line Epitope Prediction 2.0 analysis result and a sequence shared by six subtypes of the Tau protein are combined, a main fragment form of the Tau protein after in vivo hydrolysis can be identified, and four dominant Linear antigens of the Tau protein are designed and screened by combining experience. The dominant linear antigens are Tau (2N 4R) 6-24, 111-130, 152-170 and 185-204 respectively, wherein 6-24 is positioned from 6 th amino acid to 24 th amino acid, 111-130 is positioned from 111 th amino acid to 130 th amino acid, 152-170 is positioned from 152 th amino acid to 170 th amino acid, and 185-204 is positioned from 185 th amino acid to 204 th amino acid. Structural partitions, 2N insertion sequences, R1-R4 region layouts and subtype protein amino acid lengths of six subtypes of Tau are shown in figure 1.
The results of Bepipred Linear Epitope Prediction 2.0 analysis of the Linear Epitope of Tau protein (subtype 2N 4R) are shown in FIG. 2.
Example 2 Gene acquisition of specific Tau protein antigen
The genes for coding the dominant antigen epitopes are combined and connected in series according to a certain arrangement, the specific sequence is 6-24, 111-130, 152-170 and 185-204, fusion antigen genes containing 4 multi-epitopes are constructed by the serial antigens, and the antigen epitopes are connected in series through flexible chains GGGGS and correspond to the corresponding genes. And analyzing the escherichia coli rare codon in the fusion gene through rare codon online software, and replacing the escherichia coli rare codon with escherichia coli preferred codon nucleotide encoding the same amino acid, so that the finally synthesized multi-epitope fusion gene does not contain the escherichia coli rare codon.
Example 3 construction of expression vector for specific Tau protein antigen
The nucleotide sequence of the specific Tau fusion protein antigen is directly synthesized by Suzhou Hongxn biotech GmbH, and is constructed between the NcoI and XhoI multiple cloning sites of the commercialized expression plasmid PET28a, which is named as PET28a-Tau.
EXAMPLE 4 transformation and inoculation of recombinant expression vectors
1. Taking out BL21 (DE 3) competent cells from-80 ℃, quickly inserting the cells into ice, melting the cell mass after 5 minutes, adding 100ng PETC28a-Tau plasmid, stirring the EP tube bottom by hands, gently mixing the cells uniformly, and standing the cells in ice for 25 minutes;
2. heat shock in 42 ℃ water bath for 45 seconds, quickly put back on ice and stand for 2 minutes;
3. adding 700 mul of antibiotic-free sterile LB culture medium into a centrifuge tube, uniformly mixing, and recovering for 60 minutes at 37 ℃ and 200 rpm;
4. the cells were harvested by centrifugation at 5000rpm for one minute, and about 100. Mu.l of the supernatant was left to gently blow out the resuspended cell mass and inoculated into 50mL of LB medium containing kanamycin (10 ug/mL), and cultured overnight at 37 ℃ and 150 rpm.
Example 5 high Induction of expression by the expression Strain
According to the inoculation amount of 2 percent, 4mL of seed solution is transferred into a shake flask of 200mL of LB culture medium (10 ug/mL of kanamycin), the shake flask is shaken for 2h at 37 ℃ and 150rpm, IPTG (final concentration of 1 mmol/l) is added, and then the efficient induction expression is carried out for 14h at 20 ℃.
EXAMPLE 6 purification of expressed protein
The fermentation broth of the efficiently expressed recombinant engineered bacteria in example 5 was centrifuged at high speed (12000rpm, 4 ℃) for 10min, the precipitated bacteria were resuspended in 50mL of 0.2M PBS (pH 7.4), washed 1 time, centrifuged at high speed (12000rpm, 4 ℃) for 10min, the precipitate was resuspended in 50mL0.2M PBS (pH 7.4), sonicated in ice bath for 30min, and then centrifuged at 12000rpm,4 ℃ for 15min, and the supernatant was collected.
The supernatant was filtered through a 0.45um filter and purified by passing through a 5mL nickel column (Ni NTA Beads, hezhou Tiandi and Biotech Co., ltd.) and the protein sample was passed through the nickel column slowly at a flow rate of 0.5 mL/min. The impure protein was washed off with a volume of 50mL of Buffer A (pH 7.4,0.2M PBS containing 300mM NaCl and 30mM imidazole) at a flow rate of 1mL/min, and the target protein was eluted with an eluent Buffer B (pH 7.4,0.2M PBS containing 300mM NaCl and 300mM imidazole) at a flow rate of 1 mL/min.
Concentration and desalting were carried out using a Millipore 15mL,3kDa ultrafiltration tube, with a displacement Buffer of pH7.4,0.2M PBS, and centrifugation conditions of 4000g, 10min/time. The final protein was >95% pure and 6.5mg yield as determined by SDS-PAGE, see FIG. 3.
Example 7 preparation of a Multi-antiserum to recombinant Tau protein
Preparing multi-antiserum from the purified Tau protein antigen, preparing and purifying the obtained rabbit antiserum in a mode of immunizing a New Zealand big ear white rabbit by combining the antigen and an adjuvant, and detecting the titer of the anti-Tau protein antigen antibody by an indirect ELISA method.
1. The main reagents are as follows: complete Freund's adjuvant and incomplete Freund's adjuvant are available from SIGMA corporation. Other reagents are domestic or imported analytical pure reagents.
2. Immunization procedure
The purified recombinant Tau protein prepared in example 6 was used as an immunogen to immunize 4 New Zealand big ear rabbits (accession numbers K0001-K0004) with 200ug of antigen per immunization in a dorsal multilateration. The first immunization is carried out by mixing and emulsifying complete Freund adjuvant and purified Tau protein, the second immunization is carried out after two weeks, the incomplete Freund adjuvant and the purified Tau protein are mixed and emulsified, and the immunization mode is back multipoint immunization. The third immunization is carried out after two weeks, incomplete Freund adjuvant and purified Tau protein are mixed and emulsified, the immunization mode is back multipoint immunization, and the fourth immunization is carried out after two weeks by the same immunization program.
3. Indirect ELISA for rabbit antiserum titer
Five days after the fourth immunization, blood is taken from the ear vein of the rabbit, and the rabbit antiserum is obtained by centrifugation at 4000rpm for 10 min. The fusion protein was diluted with carbonate buffer (50mM, PH9.6) and then coated on an ELISA plate at 100 ul/well and 100 ng/well of recombinant Tau antigen protein, overnight at 4 ℃. The following day 1% bovine serum albumin blocking, 37 ℃ blocking for 2h, rabbit antiserum diluted four times in gradient as primary antibody (first well 1 dilution, then dilution by multiple ratio, each concentration measured in parallel in two wells), negative control using 1% bsa TBS solution as primary antibody, 37 ℃ water bath incubation for 1h, adding 1. The OD450 signal value of the enzyme linked plate is detected, and the result shows that the titer (shown in the table I) is as high as 1.
TABLE 1 results of the experiment for detecting rabbit antiserum titer by indirect enzyme-linked immunization (A450 value)
The enzyme conjugate plates were coated with a purchased amount of 100 ng/well of 6 different isoforms of Tau protein, while a non-related protein (No. NSP) was used as a negative control to demonstrate specificity, all coated in the same pattern and left overnight at 4 ℃. The next day, 1% bovine serum leucorrhea was blocked for 2h at 37 ℃, and rabbit antiserum 1:20000 dilution as primary antibody, 1% BSA TBS as serum negative control, 37 ℃ water bath incubation for 1h, adding 1. And the result shows that rabbit antiserum can specifically recognize Tau proteins of different subtypes, the epitope of an antibody generated by an immunogen is positioned in a sequence shared by the six subtype proteins, the favorable immunogenicity and specificity of the Tau recombinant protein are proved, and the generated polyclonal antibody can recognize the Tau proteins of different subtypes, so that the kit has higher value and significance in future diagnosis and detection applications.
TABLE 2 results of experiments on rabbit antiserum identification of Tau subtype protein by indirect enzyme-linked immunosorbent assay (A450 value)
Claims (13)
1. The 4 dominant linear epitope polypeptides of the microtubule-associated protein Tau are characterized in that the amino acid sequences of the dominant linear epitope polypeptides (1), (2), (3) and (4) are respectively shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
2. A microtubule-associated protein Tau antigen which is obtained by serially connecting the dominant linear epitope polypeptides (1), (2), (3), and (4) according to claim 1 in sequence via a flexible polypeptide chain having an amino acid sequence of GGGGS.
3. The microtubule-associated protein Tau antigen of claim 2, having the amino acid sequence shown in Seq ID No. 5.
4. The gene encoding the microtubule-associated protein Tau antigen as claimed in claim 2 or 3, wherein the nucleotide sequence thereof is Seq ID No.6.
5. A recombinant expression vector, which is obtained by recombining an expression vector with the gene encoding the microtubule-associated protein Tau antigen of claim 4.
6. The recombinant expression vector of claim 5, wherein the expression vector is a PET28a plasmid.
7. A recombinant engineered bacterium obtained by transforming the recombinant expression vector of claim 5 or 6 into a host bacterium.
8. The recombinant engineered bacterium of claim 7, wherein said host bacterium is Escherichia coli BL21 (DE 3).
9. The method of producing a microtubule-associated protein Tau antigen as claimed in claim 2 or 3, comprising the steps of:
(1) Cloning the coding gene of the microtubule-associated protein Tau antigen shown in Seq ID No.6 into an expression vector to obtain a recombinant expression vector;
(2) Transferring the recombinant expression vector obtained in the step (1) into host bacteria to obtain recombinant engineering bacteria;
(3) Inducing and expressing Tau protein antigen by using recombinant engineering bacteria;
(4) And (4) extracting and purifying the expression protein in the step (3) to prepare the recombinant Tau protein antigen.
10. A polyclonal antibody aiming at a Tau antigen is characterized in that the polyclonal antibody is prepared from a recombinant Tau protein antigen with an amino acid sequence shown as Seq ID No. 5.
11. Polyclonal antibodies against Tau antigens according to claim 10, characterized in that said recombinant Tau protein antigens are prepared according to the method of claim 9.
12. Use of a polyclonal antibody against a Tau antigen as defined in claim 10 or 11 for the preparation of a kit for the detection of Tau protein.
13. A Tau protein detection kit comprising the polyclonal antibody of claim 12.
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| WO2011013034A1 (en) * | 2009-07-30 | 2011-02-03 | Pfizer Vaccines Llc | Antigenic tau peptides and uses thereof |
| CN103827311A (en) * | 2011-07-04 | 2014-05-28 | 北欧生物科技公司 | Biochemical markers for neurodegenerative conditions |
| CN107586322A (en) * | 2017-08-28 | 2018-01-16 | 黑龙江八农垦大学 | Infectious bovine rhinotrachetis virus gD Protein Epitopes polypeptide and its inhibitor and monoclonal antibody and its application |
| CN112105630A (en) * | 2018-02-15 | 2020-12-18 | T·比约尔克隆德 | Modified viral capsids |
| TW202221022A (en) * | 2020-08-07 | 2022-06-01 | 愛爾蘭商普羅佘納生物科技有限公司 | Tau vaccine for the treatment of alzheimer's disease |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011013034A1 (en) * | 2009-07-30 | 2011-02-03 | Pfizer Vaccines Llc | Antigenic tau peptides and uses thereof |
| CN103827311A (en) * | 2011-07-04 | 2014-05-28 | 北欧生物科技公司 | Biochemical markers for neurodegenerative conditions |
| CN107586322A (en) * | 2017-08-28 | 2018-01-16 | 黑龙江八农垦大学 | Infectious bovine rhinotrachetis virus gD Protein Epitopes polypeptide and its inhibitor and monoclonal antibody and its application |
| CN112105630A (en) * | 2018-02-15 | 2020-12-18 | T·比约尔克隆德 | Modified viral capsids |
| TW202221022A (en) * | 2020-08-07 | 2022-06-01 | 愛爾蘭商普羅佘納生物科技有限公司 | Tau vaccine for the treatment of alzheimer's disease |
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