CN114805546B - T cell epitope polypeptide for detecting oral cancer and application thereof - Google Patents
T cell epitope polypeptide for detecting oral cancer and application thereof Download PDFInfo
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 181
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 53
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 34
- 208000003445 Mouth Neoplasms Diseases 0.000 title claims abstract description 23
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 title claims abstract description 23
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 21
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 13
- 241001465754 Metazoa Species 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 6
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- 230000003053 immunization Effects 0.000 claims description 10
- 102400000524 Fibrinogen alpha chain Human genes 0.000 claims description 3
- 101710137044 Fibrinogen alpha chain Proteins 0.000 claims description 3
- 239000007857 degradation product Substances 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 25
- 102000036639 antigens Human genes 0.000 abstract description 25
- 108091007433 antigens Proteins 0.000 abstract description 25
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- 238000002649 immunization Methods 0.000 description 7
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- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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Abstract
The invention discloses a T cell epitope polypeptide for detecting oral cancer and application thereof, wherein the T cell epitope polypeptide for detecting oral cancer comprises 1-40aa of short peptide, 21-60aa of short peptide, 41-80aa of short peptide, 61-100aa of short peptide, 81-120aa of short peptide, 101-140aa of short peptide, 121-160aa of short peptide and 141-178aa of short peptide, and the T cell epitope polypeptide can be obtained by a chemical synthesis method, and has simple and convenient acquisition and lower cost; the T cell epitope polypeptide for detecting oral cancer can assist in inducing and generating stronger immune response in rabbit bodies, and can be used as an immune carrier to assist in developing and producing rabbit-derived antibodies by using other antigens or hapten such as oral cancer markers-alpha peptide. Compared with the mainstream coupled KLH or BSA and other immune carriers, the epitope is small, does not basically induce antibodies except target antigens, is beneficial to synthesizing monovalent antigens, eliminates the limitation of large carrier proteins, is beneficial to improving the yield and affinity of the antibodies, and can be used for designing animal vaccines to increase the immune response of vaccine animals.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a T cell epitope polypeptide for detecting oral cancer and application thereof.
Background
When foreign antigens or pathogenic microorganisms enter the body, dendritic cells, macrophages and B cells in the blood can phagocytose these foreign substances and are digested by lysosomes of the cells to produce short peptides of 4-20 amino acid residues. Some of these short peptides bind to specific MHC II receptors of cells and are displayed on the cell surface by these receptors. Complexes of short peptides and receptors on the surfaces of dendritic cells and macrophages, if specifically bound by TCR receptors on the surface of cd4+ T cells, activate the proliferation of that specific T cell. The massive propagation of CD4+ T cells combined with the short peptide-receptor complex on the surface of B cells activates the massive propagation of B cells and the mutation of antibody genes in vivo, and the antibodies with high affinity are selected through a subsequent series of physiological reaction mutation. This short peptide digested by lysosomes and capable of binding to both MHC II and TCR receptors is a T cell epitope.
Nevertheless, when different antigens are injected into the body, some antigens induce high titer, high affinity antibodies, and some antigens induce only weak immune responses in the body, resulting in low affinity antibodies. The reasons for this phenomenon are numerous, among which the main reasons are: 1. the antigen is digested too little by the protease in the lysosome due to its nature, losing the possibility of binding to MHC II; 2. the digested short peptide with proper size is not recognized by MHC II in the cell; 3. each T cell carries a TCR, the number of which is large, but limited; not every peptide-MHC II complex can have a suitable TCR. These causes all result in the inability to activate specific T cells and thus subsequent B cells.
To address this problem, many poorly immunogenic antigens or haptens typically need to be conjugated to carrier proteins (mainly keyhole limpet hemocyanin KLH and bovine serum albumin BSA). Antigens prepared in this way have two main drawbacks: firstly, the body can generate a large amount of antibodies aiming at carrier proteins, and the amount of antibodies aiming at antigens and hapten is reduced; moreover, these antibodies have a relatively higher affinity, resulting in a lower affinity for the coupled antigen. Secondly, the coupling process to the carrier protein is complex, usually, a plurality of antigen hapten is coupled to one carrier protein, and the antibody and the coupling product can be combined in a bivalent manner, so that extremely high affinity is shown, but in fact, the original antigen hapten and the antibody have weaker binding force, so that the high-affinity antibody is not easy to obtain. Therefore, the development of better immune carriers and coupling modes has important significance for the research of antigen antibodies.
Disclosure of Invention
The invention aims to provide a T cell epitope polypeptide for detecting oral cancer and application thereof, wherein the T cell epitope polypeptide for detecting oral cancer can be obtained by a chemical synthesis method, is simple and convenient to obtain, has low cost, can assist in inducing and generating stronger immune response in rabbit bodies, can be used as an immune carrier to assist other antigens or hapten such as an oral cancer marker-alpha peptide and the like in developing and producing rabbit-derived antibodies, does not induce antibodies except target antigens basically, is beneficial to synthesizing monovalent antigens, eliminates the limitation of large carrier proteins, and is beneficial to improving antibody yield and affinity.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A T cell epitope polypeptide for detecting oral cancer comprises short peptide 1-40aa, short peptide 21-60aa, short peptide 41-80aa, short peptide 61-100aa, short peptide 81-120aa, short peptide 101-140aa, short peptide 121-160aa and short peptide 141-178aa;
The nucleotide sequence of the short peptide 1-40aa is as follows:
QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNA；
The nucleotide sequence of the short peptide 21-60aa is as follows:
NFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKE；
the nucleotide sequence of the short peptide 41-80aa is as follows:
ILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWI；
the nucleotide sequence of the short peptide 61-100aa is as follows:
DKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSY；
the nucleotide sequence of the short peptide 81-120aa is as follows:
RTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAM；
the nucleotide sequence of the short peptide 101-140aa is as follows:
PGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGR；
the nucleotide sequence of the short peptide 121-160aa is as follows:
VFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLG；
the nucleotide sequence of the short peptide 141-178aa is as follows:
TKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG。
Preferably, the short peptides 1-40aa, 21-60aa, 41-80aa, 61-100aa, 81-120aa, 101-140aa, 121-160aa and 141-178aa are obtained by chemical synthesis methods, respectively.
An application of T cell epitope polypeptide for detecting oral cancer, which fuses short peptide 1-40aa, short peptide 21-60aa, short peptide 41-80aa, short peptide 61-100aa, short peptide 81-120aa, short peptide 101-140aa, short peptide 121-160aa and short peptide 141-178aa with fibrinogen alpha chain C end degradation product alpha peptide, and is used for immunizing animals to obtain alpha peptide antibody or further preparing monoclonal antibody.
Preferably the nucleotide sequence of the alpha peptide is: DEAGSEADHEGTHSTKRGHAKSRPV.
Preferably, the animal is a rabbit.
After the technical scheme is adopted, compared with the background technology, the invention has the following advantages: the short peptides 1-40aa, 21-60aa, 41-80aa, 61-100aa, 81-120aa, 101-140aa, 121-160aa and 141-178aa are suitable for being obtained by a chemical synthesis method due to shorter peptide fragments, and are simple and convenient to obtain and low in cost; the T cell epitope polypeptide for detecting oral cancer can assist in inducing and generating stronger immune response in rabbit bodies, and can be used as an immune carrier to assist in developing and producing rabbit-derived antibodies by using other antigens or hapten such as oral cancer markers-alpha peptide. Compared with the mainstream coupled KLH or BSA immune carriers, the epitope is small, does not basically induce antibodies except the target antigen, is beneficial to synthesizing monovalent antigens, eliminates the limitation of large carrier proteins, and is beneficial to improving the yield and affinity of the antibodies. In addition, the epitope can be used in the design of animal vaccines to increase the immune response in vaccine animals.
Drawings
FIG. 1 is a graph showing the titer curves after immunization with different peptide fragments according to the present invention;
FIG. 2 is a graph showing the binding of high titer serum to adjacent peptide fragments according to the present invention;
FIG. 3 is a plot of the accurate localization titer of T cell epitopes of the present invention;
FIG. 4 is a graph showing the titer of T cell epitope fusion alpha peptide of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Referring to FIGS. 1-4, a T cell epitope polypeptide for detecting oral cancer comprises short peptide 1-40aa, short peptide 21-60aa, short peptide 41-80aa, short peptide 61-100aa, short peptide 81-120aa, short peptide 101-140aa, short peptide 121-160aa and short peptide 141-178aa;
The nucleotide sequence of the short peptide 1-40aa is as follows:
QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNA；
The nucleotide sequence of the short peptide 21-60aa is as follows:
NFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKE；
the nucleotide sequence of the short peptide 41-80aa is as follows:
ILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWI；
the nucleotide sequence of the short peptide 61-100aa is as follows:
DKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSY；
the nucleotide sequence of the short peptide 81-120aa is as follows:
RTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAM；
the nucleotide sequence of the short peptide 101-140aa is as follows:
PGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGR；
the nucleotide sequence of the short peptide 121-160aa is as follows:
VFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLG；
the nucleotide sequence of the short peptide 141-178aa is as follows:
TKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG。
The short peptide 1-40aa, the short peptide 21-60aa, the short peptide 41-80aa, the short peptide 61-100aa, the short peptide 81-120aa, the short peptide 101-140aa, the short peptide 121-160aa and the short peptide 141-178aa are respectively obtained by a chemical synthesis method.
An application of T cell epitope polypeptide for detecting oral cancer, which fuses short peptide 1-40aa, short peptide 21-60aa, short peptide 41-80aa, short peptide 61-100aa, short peptide 81-120aa, short peptide 101-140aa, short peptide 121-160aa and short peptide 141-178aa with fibrinogen alpha chain C end degradation product alpha peptide, and is used for immunizing animals to obtain alpha peptide antibody or further preparing monoclonal antibody.
The nucleotide sequence of the alpha peptide is as follows: DEAGSEADHEGTHSTKRGHAKSRPV.
The animals are rabbits.
Examples
1. A series of polypeptides were synthesized by chemistry:
A. Short peptides for preliminary localization of T cell epitopes:
Short peptide 1-40aa: QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNA;
short peptide 21-60aa: NFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKE;
Short peptide 41-80aa: ILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWI;
Short peptide 61-100aa: DKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSY;
short peptide 81-120aa: RTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAM;
Short peptide 101-140aa: PGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGR;
Short peptide 121-160aa: VFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLG;
Short peptide 141-178aa: TKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG.
B. Fusion peptide for fine localization of T cell epitopes:
Fusion peptide 21-40 a:
NFQDNQFQGKWYVVGLAGNADEAGSEADHEGTHSTKRGHAKSRPV；
fusion peptide 22-40 a:
FQDNQFQGKWYVVGLAGNADEAGSEADHEGTHSTKRGHAKSRPV；
fusion peptide 23-40 a:
QDNQFQGKWYVVGLAGNADEAGSEADHEGTHSTKRGHAKSRPV；
Fusion peptide 24-40 a:
DNQFQGKWYVVGLAGNADEAGSEADHEGTHSTKRGHAKSRPV；
fusion peptide 25-40 α:
NQFQGKWYVVGLAGNADEAGSEADHEGTHSTKRGHAKSRPV；
fusion peptide 21-39 a:
NFQDNQFQGKWYVVGLAGNDEAGSEADHEGTHSTKRGHAKSRPV；
fusion peptide 21-38α:
NFQDNQFQGKWYVVGLAGDEAGSEADHEGTHSTKRGHAKSRPV；
Fusion peptide 21-37 a:
NFQDNQFQGKWYVVGLADEAGSEADHEGTHSTKRGHAKSRPV；
Fusion peptide 21-36 a:
NFQDNQFQGKWYVVGLDEAGSEADHEGTHSTKRGHAKSRPV；
fusion peptide 21-35 a:
NFQDNQFQGKWYVVGDEAGSEADHEGTHSTKRGHAKSRPV。
C. Fusion peptides for validation of T cell epitope and antibody production:
Fusion peptide 23-38α:
QDNQFQGKWYVVGLAGDEAGSEADHEGTHSTKRGHAKSRPV；
Fusion peptide Biotin-alpha:
N-biotin-DEAGSEADHEGTHSTKRGHAKSRPV。
2. New Zealand white fungus immunity
During the primary immunization of New Zealand large ear white with the age of 6-8 weeks, taking synthetic peptide (short peptide 1-40aa, short peptide 21-60aa, short peptide 41-80aa, short peptide 61-100aa, short peptide 81-120aa, short peptide 101-140aa, short peptide 121-160aa and short peptide 141-178 aa) as immunogens, respectively diluting to 1mg/mL (diluting with 0.01mol/L PBS buffer solution), respectively mixing with Freund's complete adjuvant in equal volume, fully emulsifying, and subcutaneously multipoint inoculating 3 New Zealand large ear white on the back of neck, wherein the inoculation antigen dose is 2.5 mg/dose, and 1mL each; immunization was performed 2 days later, and the same volume of emulsion was performed with Freund's incomplete adjuvant and immunogen, with the same immunization dose as the first immunization dose, and the number of booster immunizations was 4.
3. Identification of immune titers
One week after the completion of the last immunization, 200. Mu.L of rabbit ears were collected, centrifuged, serum was collected, and serum titers were measured by ELISA.
1) The determination of the antiserum titer is carried out as follows:
(1) Coating:
A. The synthesized short peptides (short peptides 1-40aa, short peptides 21-60aa, short peptides 41-80aa, short peptides 61-100aa, short peptides 81-120aa, short peptides 101-140aa, short peptides 121-160aa and short peptides 141-178 aa) were directly diluted to 0.2. Mu.g/mL with 0.05mol/L carbonate buffer (pH 9.6), 100. Mu.L/well was added to the ELISA plate, and incubated in a 37℃incubator for 2 hours. The liquid in the wells was decanted, the plates were washed 3 times with PBST buffer (pH 7.2) and the wash was spun dry.
B. For the fusion peptides (fusion peptides 21-40. Alpha., fusion peptides 22-40. Alpha., fusion peptides 23-40. Alpha., fusion peptides 24-40. Alpha., fusion peptides 25-40. Alpha., fusion peptides 21-39. Alpha., fusion peptides 21-38. Alpha., fusion peptides 21-37. Alpha., fusion peptides 21-36. Alpha., fusion peptides 21-35. Alpha., fusion peptides 23-38. Alpha., and fusion peptides Biotin-alpha.), streptavidin was diluted to 10. Mu.g/mL with 0.05mol/L carbonate buffer (pH 9.6), 100. Mu.L/well was added to an ELISA plate, and incubated in a 37℃incubator for 2 hours. The liquid in the wells was decanted, the plates were washed 1 time with PBST buffer (pH 7.2) and the wash was spun dry. 100 uL/well of 20mM PBS containing 5% BSA and 0.1. Mu.g/mL biotin-labeled alpha peptide was added and incubated in an incubator at 37℃for 2 hours. The liquid in the wells was decanted, the plates were washed 3 times with PBST buffer (pH 7.2) and the wash was spun dry.
(2) Closing: 150. Mu.L of blocking solution (5% BSA) was added to each well, the wells were blocked at 37℃for 1 hour, the liquid in the wells was dried, the plate was washed 3 times with PBST buffer (pH 7.2), and the washing solution was patted dry.
(3) Adding antiserum: antisera were diluted starting at 1:1000 with 2 gradient and starting with 20mM PBS containing 5% BSA for a total of 8 gradients. The sample was added at 100. Mu.L per well, incubated at 37℃for 30min, washed 3 times with PBST buffer (pH 7.2), and patted dry. Incubation was carried out for 30min, washed 3 times with PBST buffer (pH 7.2) and patted dry. The serum from the immunized rabbits was also used as a negative control.
(4) Adding enzyme-labeled secondary antibodies: mu.L of HRP-goat anti-rabbit IgG (5000-fold dilution in 20mM PBS containing 5% BSA) was added to each well, incubated at 37℃for 30min, washed 3 times with PBST buffer (pH 7.2), and patted dry.
(5) Color development: horseradish peroxidase substrate 3,3', 5' -tetramethylbenzidine solution and 30% hydrogen peroxide by mass were mixed in a volume ratio of 1:1, and incubated at 37℃for 15min at 100. Mu.L per well. Then 50. Mu.L of stop solution (2 mol/L H.sup.2SO.sup.4) was added to each well.
(6) Reading and measuring: the absorbance (OD) was read with a microplate reader at a wavelength of 450 nm.
The identification result of the immune titer shows that: the immune serum titers of short peptides 1-40aa and 21-60aa were highest. Wherein, the serum of the short peptide 1-40aa only has an immune response to the serum and does not react with the short peptide 21-60aa, which indicates that the generated antibody only acts on the short peptide 1-20aa; serum from short peptide 21-60aa was equivalent in potency to the synthesized short peptides 21-60aa and 41-80aa, and did not react with the peptides from short peptide 1-40aa, indicating that antibodies raised from short peptide 21-60aa were directed primarily to the region overlapping short peptide 41-80aa (i.e., short peptide 41-60aa range).
4. T cell epitope fine localization
T cell epitope size is generally 10-17 amino acid residues, so that peptide fragments with different sizes within 24-45aa are intercepted and fused at the N end of alpha peptide (DEAGSEADHEGTHSTKRGHAKSRPV) for immune titer analysis, and the immune titer results are shown in Table 1:
TABLE 1 results of immunotitres
| N-terminal fused peptide fragment | Immune titers | N-terminal fused peptide fragment | Immune titers |
| NFQDNQFQGKWYVVGLAGNA | 128000 | NFQDNQFQGKWYVVGLAGN | 128000 |
| FQDNQFQGKWYVVGLAGNA | 128000 | NFQDNQFQGKWYVVGLAG | 128000 |
| QDNQFQGKWYVVGLAGNA | 128000 | NFQDNQFQGKWYVVGLA | 64000 |
| DNQFQGKWYVVGLAGNA | 64000 | NFQDNQFQGKWYVVGL | 32000 |
| NQFQGKWYVVGLAGNA | 0 | NFQDNQFQGKWYVVG | 0 |
| QDNQFQGKWYVVGLAG | 128000 | ||
| Alpha peptide without N-terminal fusion | 0 |
Through immune data analysis, the immune titer of the alpha peptide fused with QDNQFQGKWYVVGLAGNA and NFQDNQFQGKWYVVGLAG at the N end is highest, and 128000 is achieved; the immune titer of the alpha peptide fused with DNQFQGKWYVVGLAGNA and NFQDNQFQGKWYVVGLA is higher, and the immune titer reaches 64000, and the core sequence of the epitope is optimally QDNQFQGKWYVVGLAG.
Then synthesizing the alpha peptide with the N-terminal fused QDNQFQGKWYVVGLAG, and through immune verification, the immune titer of the fused peptide also reaches 128000. QDNQFQGKWYVVGLAG is thus a T cell epitope suitable for use as an immune vector.
In conclusion, the T cell epitope polypeptide for detecting oral cancer can assist in inducing stronger immune response in rabbit bodies, and can be used as an immune carrier to assist in development and production of rabbit-derived antibodies by other antigens or hapten such as oral cancer marker-alpha peptide. Compared with the mainstream coupled KLH or BSA immune carriers, the epitope is small, does not basically induce antibodies except the target antigen, is beneficial to synthesizing monovalent antigens, eliminates the limitation of large carrier proteins, and is beneficial to improving the yield and affinity of the antibodies. In addition, the epitope can be used in the design of animal vaccines to increase the immune response in vaccine animals.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.
Claims (4)
1. The T cell epitope polypeptide for detecting the oral cancer is characterized by being prepared by fusing a short peptide 21-40aa with an alpha peptide; wherein, the amino acid sequence of the T cell epitope polypeptide for detecting oral cancer is as follows:
NFQDNQFQGKWYVVGLAGNADEAGSEADHEGTHSTKRGHAKSRPV。
2. A T cell epitope polypeptide for detecting oral cancer as claimed in claim 1, wherein: the short peptide 21-40aa is obtained by a chemical synthesis method.
3. Use of a T cell epitope polypeptide for detecting oral cancer according to any one of claims 1-2, wherein: the short peptide 21-40aa is fused with alpha peptide which is a degradation product of C end of fibrinogen alpha chain, and is used for immunizing animals to obtain alpha peptide antibody or further preparing monoclonal antibody.
4. The use of a T cell epitope polypeptide for detecting oral cancer according to claim 3, wherein: the animals are rabbits.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776153A (en) * | 2012-07-05 | 2012-11-14 | 南京基蛋生物科技有限公司 | Mouse anti-human neutrophil gelatinase-associated lipocalin (NGAL) monoclonal antibodies and hybridoma cell strains and application thereof |
| CN108610425A (en) * | 2018-05-03 | 2018-10-02 | 中国药科大学 | A kind of single B cell epitope of energy coupling or haptens are used to prepare linear 23 peptide and application thereof of immunomodulatory peptides and antibody |
| CN109836505A (en) * | 2017-11-24 | 2019-06-04 | 中国药科大学 | One kind is used to connect the carrier peptides of B epitope or haptens and the immunogene containing carrier peptides in medicine and immunologic purposes |
| CN110003321A (en) * | 2019-04-02 | 2019-07-12 | 青岛中科爱博生物科技有限公司 | A kind of soluble recombination NGAL Argine Monohydrochloride, gene order and protein preparation method |
| CN113621050A (en) * | 2014-05-22 | 2021-11-09 | 皮里斯制药有限公司 | Novel specific binding polypeptides and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776153A (en) * | 2012-07-05 | 2012-11-14 | 南京基蛋生物科技有限公司 | Mouse anti-human neutrophil gelatinase-associated lipocalin (NGAL) monoclonal antibodies and hybridoma cell strains and application thereof |
| CN113621050A (en) * | 2014-05-22 | 2021-11-09 | 皮里斯制药有限公司 | Novel specific binding polypeptides and uses thereof |
| CN109836505A (en) * | 2017-11-24 | 2019-06-04 | 中国药科大学 | One kind is used to connect the carrier peptides of B epitope or haptens and the immunogene containing carrier peptides in medicine and immunologic purposes |
| CN108610425A (en) * | 2018-05-03 | 2018-10-02 | 中国药科大学 | A kind of single B cell epitope of energy coupling or haptens are used to prepare linear 23 peptide and application thereof of immunomodulatory peptides and antibody |
| CN110003321A (en) * | 2019-04-02 | 2019-07-12 | 青岛中科爱博生物科技有限公司 | A kind of soluble recombination NGAL Argine Monohydrochloride, gene order and protein preparation method |
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