CN114316020A - BNP recombinant protein, preparation method and application thereof - Google Patents
BNP recombinant protein, preparation method and application thereof Download PDFInfo
- Publication number
- CN114316020A CN114316020A CN202111626199.5A CN202111626199A CN114316020A CN 114316020 A CN114316020 A CN 114316020A CN 202111626199 A CN202111626199 A CN 202111626199A CN 114316020 A CN114316020 A CN 114316020A
- Authority
- CN
- China
- Prior art keywords
- bnp
- recombinant protein
- recombinant
- preparing
- carrying
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention discloses a BNP recombinant protein, a preparation method and an application thereof, wherein the preparation method comprises the following steps: s1 recombinant plasmid, transformation and culture: converting a polypeptide having the sequence of SEQ ID NO: transforming the BNP recombinant plasmid with the nucleotide sequence corresponding to the amino acid sequence shown in 1 into BLR (DE3) bacteria, culturing to obtain an activated strain, then inoculating the activated strain into an LB culture medium at a ratio of 1:1000, carrying out constant-temperature shaking culture, carrying out shake flask experiment, inducing, and centrifuging to obtain thalli; s2 purification: and (4) carrying out cell lysis on the bacterial precipitation obtained by centrifugation in the step S1 to obtain a supernatant, purifying by passing through a His column, and eluting by 260mM imidazole to obtain the BNP recombinant protein. The BNP recombinant plasmid is transformed into escherichia coli to express the BNP recombinant protein, so that the BNP recombinant protein is obtained, has the same immunogenicity as the natural BNP protein, has high purity and better stability, can replace the natural BNP polypeptide, and can be used as a BNP immunodiagnosis reagent standard and a BNP detection kit.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a preparation method and application of BNP recombinant protein.
Background
Heart disease has become the number one killer in many countries, and patients are often delayed in treatment due to their hidden early symptoms. BNP (B-type natriuretic peptide, also called brain natriuretic peptide) is a neurohormone with biological activity synthesized in the ventricle, mainly expressed in the ventricle and also present in brain tissue as a quantitative marker of heart failure. When the left ventricle is not fully functional, it is rapidly synthesized and released into the blood due to myocardial expansion, which helps to regulate the heart function. BNP secreted by myocardial cells exists in the form of a precursor protein consisting of 108 amino acids, and when myocardial cells are stimulated, the precursor protein is cleaved into an inactive linear polypeptide consisting of 76 amino acids and an active cyclic polypeptide consisting of 32 amino acids under the action of an activating enzyme and released into the blood circulation, which are respectively called NT-proBNP and BNP. Thus, BNP and NT-proBNP are better than other natriuretic peptides in evaluating cardiac events, can individually predict the condition of increased end-diastolic pressure of the Left Ventricle (LV), and can better reflect and analyze the condition of patients with chronic heart failure.
At present, the detection of BNP and NT-proBNP is widely applied to the aspects of evaluating the risk of acute coronary syndrome and CHF patients and the risk of heart events of stable coronary artery disease patients, monitoring the treatment of patients with left ventricular insufficiency, exploring mild cardiac insufficiency and the like. Compared with NT-proBNP, BNP has the characteristics of being slightly influenced by age and renal function, is more specific in judging cardiac function, and has uniform cut-off value (critical value). Besides, the main advantages of BNP detection are: the heart failure can be diagnosed quickly, accurately and with high quality.
At present, quantitative detection methods of BNP are relatively extensive and are indispensable to detect standard substances. Since BNP has a molecular weight of only 3.5KD, the BNP is degraded in vivo quickly and has a half-life of about 20 minutes in blood. BNP in human body is very low, and can not be purified from blood plasma (blood serum), so that the preparation of natural polypeptide is very difficult. The direct polypeptide synthesis cost is too high, and the polypeptide has the problems of instability, difficult storage and the like.
Therefore, there is a need to develop a novel BNP recombinant protein and a preparation method thereof.
Disclosure of Invention
The invention aims to solve the technical problem of providing the BNP recombinant protein which can overcome the defects of low efficiency and easy degradation and has better activity, immunogenicity and high stability.
In order to solve the technical problems, the technical scheme adopted by the invention is that the BNP recombinant protein has the amino acid sequence shown in SEQ ID NO: 1.
SEQ ID NO: 1 is:
Met Gly Val Asp Trp Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His Trp Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His Trp Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His Trp Leu Asp His His His His His His。
another technical problem to be solved by the present invention is to provide a method for producing a polypeptide of SEQ ID NO: 1 in immunodiagnosis.
The BNP recombinant protein has the same immunogenicity as the natural BNP protein, high purity and better stability.
Preferably, the BNP recombinant protein is used as a BNP immunodiagnostic reagent standard.
Preferably, the BNP recombinant protein is used for preparing a BNP detection kit.
Another technical problem to be solved by the present invention is to provide a polypeptide of SEQ ID NO: 1, the preparation method of the BNP recombinant protein comprises the following steps:
s1 recombinant plasmid, transformation and culture: converting a polypeptide having the sequence of SEQ ID NO: transforming the BNP recombinant plasmid of the nucleotide sequence corresponding to the amino acid sequence shown in 1 into BLR (DE3) bacteria, culturing to obtain an activated strain, then inoculating the activated strain into an LB culture medium at a ratio of 1:1000, carrying out constant-temperature shaking culture, carrying out a shaking experiment, inducing, and centrifuging to obtain thalli;
s2 purification: and (4) carrying out cell lysis on the bacterial precipitation obtained by centrifugation in the step S1 to obtain a supernatant, purifying by passing through a His column, and eluting by 260mM imidazole to obtain the BNP recombinant protein.
The BNP recombinant plasmid is transformed into escherichia coli to express the BNP recombinant protein to obtain the BNP recombinant protein, which has the same immunogenicity as the natural BNP protein, high purity, better stability, supernatant expression and simple purification, can replace the natural BNP polypeptide and can be used as a BNP immunodiagnosis reagent standard and a BNP detection kit.
Preferably, in the step S1, the temperature for culturing to obtain the activated strain is 37 ℃; the temperature of the constant temperature shaking culture was 37 ℃.
Preferably, in the step S2, the obtained BNP recombinant protein is subjected to BCA concentration measurement, and the concentration is 1.2 mg/mL; the obtained BNP recombinant protein is subjected to purity determination, the SDS electrophoresis is more than 90 percent, and the target size is 13 KD.
Preferably, in the step S2, the BNP recombinant protein obtained is subjected to activity determination to prepare the standard substance of claim 3; the diluent comprises 20mMPB, 4% BSA, 5% trehalose and 10% sorbitol, and the pH is 7.4; after the preparation, the activity is measured by using an immunochromatographic test strip.
Preferably, in step S1, the target gene expressing BNP is amplified by using PCR technique, and tryptophan W is connected to the BNP sequence, and the three repetitions are performed to obtain an amino acid sequence corresponding to the nucleotide sequence of the BNP recombinant plasmid.
PCR techniques are well known in the art.
The traditional method for extracting BNP recombinant protein has the defects of high cost, low yield, easy denaturation of target protein, limited material source and the like. The low molecular weight polypeptide is prepared by genetic engineering, and is generally expressed by adopting fusion protein, so that the defect that an expression product with low translation efficiency is easily degraded by proteolytic enzyme when the low molecular weight polypeptide is expressed independently can be overcome, but the immunogenicity of the small molecular protein is influenced to a certain extent by the larger tag protein. Therefore, some proteases are usually used to cleave the tagged protein, and the cleaved protein requires further purification and removal of the added protease, which is cumbersome and expensive.
Detailed Description
The BNP recombinant protein of the present example has a sequence selected from SEQ ID NO: 1.
And (2) mixing the amino acid sequence with a sequence selected from SEQ ID NO: 1 in immunodiagnosis, wherein the BNP recombinant protein is used as a BNP immunodiagnostic reagent standard, and is used for preparing a BNP detection kit.
The peptide of this example having SEQ ID NO: 1, the preparation method of the BNP recombinant protein with the amino acid sequence shown in the specification comprises the following steps:
s1 recombinant plasmid, transformation and culture: converting a polypeptide having the sequence of SEQ ID NO: the BNP recombinant plasmid with the nucleotide sequence corresponding to the amino acid sequence shown in 1 is transformed into BLR (DE3) bacteria (the cargo number is HG-VDN 0207; the manufacturer is Changsha Aibi vitamin science and technology Co., Ltd; the manufacturer is Takara Bio), an activated strain is obtained by culture, then the activated strain is inoculated into an LB culture medium at the ratio of 1:1000, the constant temperature shaking culture is carried out, a shake flask experiment is carried out, induction and centrifugation are carried out to obtain thalli;
s2 purification: and (4) carrying out cell lysis on the bacterial precipitation obtained by centrifugation in the step S1 to obtain a supernatant, purifying by passing through a His column, and eluting by 260mM imidazole to obtain the BNP recombinant protein.
In the step S1, the temperature for culturing to obtain the activated strain is 37 ℃; the temperature of the constant temperature shaking culture was 37 ℃.
In the step S2, the BNP recombinant protein obtained is subjected to concentration measurement by using a picnic acid BCA protein concentration measurement kit, wherein the concentration is 1.2 mg/mL; the obtained BNP recombinant protein is subjected to purity determination, the SDS electrophoresis is more than 90 percent, and the target size is 13 KD.
In the step S2, performing activity measurement on the obtained BNP recombinant protein to prepare the standard substance of claim 3; the diluent comprises 20mMPB, 4% BSA, 5% trehalose and 10% sorbitol, and the pH is 7.4; after the preparation, the activity is measured by using an immunochromatographic test strip.
The activity assay data are shown in table 1 below:
the data in table 1 show that the active concentration is 60% of the identified BCA concentration and that the gradient after dilution is linear.
In the step S1, the target gene expressing BNP is amplified by using PCR technique, and tryptophan W is connected to the BNP sequence, and the three repetitions are performed to obtain the amino acid sequence corresponding to the nucleotide sequence of the BNP recombinant plasmid.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Nanjing lanyu Biotechnology GmbH/Nanjing lanxuan Biotechnology GmbH
<120> BNP recombinant protein, preparation method and application thereof
<141> 2021-12-28
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 112
<212> PRT
<213> BNP recombinant protein amino acid sequence (artificial sequence)
<400> 1
Met Gly Val Asp Trp Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe
1 5 10 15
Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys
20 25 30
Val Leu Arg Arg His Trp Ser Pro Lys Met Val Gln Gly Ser Gly Cys
35 40 45
Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys
50 55 60
Lys Val Leu Arg Arg His Trp Ser Pro Lys Met Val Gln Gly Ser Gly
65 70 75 80
Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
85 90 95
Cys Lys Val Leu Arg Arg His Trp Leu Asp His His His His His His
100 105 110
Claims (9)
1. A BNP recombinant protein having the amino acid sequence of SEQ ID NO: 1.
2. Use of a BNP recombinant protein according to claim 1 in immunodiagnosis.
3. The use of a BNP recombinant protein as claimed in claim 2 for immunodiagnosis, wherein said BNP recombinant protein is used as a BNP immunodiagnostic reagent standard.
4. The use of a BNP recombinant protein according to claim 2 in immunodiagnosis, wherein the BNP recombinant protein is used for preparing a BNP detection kit.
5. A method for preparing BNP recombinant protein according to claim 1, comprising the steps of:
s1 recombinant plasmid, transformation and culture: converting a polypeptide having the sequence of SEQ ID NO: transforming the BNP recombinant plasmid with the nucleotide sequence corresponding to the amino acid sequence shown in 1 into BLR (DE3) bacteria, culturing to obtain an activated strain, then inoculating the activated strain into an LB culture medium at a ratio of 1:1000, carrying out constant-temperature shaking culture, carrying out shake flask experiment, inducing, and centrifuging to obtain thalli;
s2 purification: and (4) carrying out cell lysis on the bacterial precipitation obtained by centrifugation in the step S1 to obtain a supernatant, purifying by passing through a His column, and eluting by 260mM imidazole to obtain the BNP recombinant protein.
6. The method for preparing BNP recombinant protein according to claim 5, wherein, in the step S1, the temperature for culturing the obtained activated strain is 37 ℃; the temperature of the constant temperature shaking culture was 37 ℃.
7. The method for preparing BNP recombinant protein according to claim 5, wherein in step S2, the obtained BNP recombinant protein is subjected to BCA concentration measurement, the concentration is 1.2 mg/mL; the obtained BNP recombinant protein is subjected to purity determination, the SDS electrophoresis is more than 90 percent, and the target size is 13 KD.
8. The method for preparing BNP recombinant protein according to claim 5, wherein in step S2, the activity of the obtained BNP recombinant protein is determined, and the standard substance according to claim 3 is prepared; the diluent comprises 20mMPB, 4% BSA, 5% trehalose and 10% sorbitol, and the pH is 7.4; after the preparation, the activity is measured by using an immunochromatographic test strip.
9. The method for preparing BNP recombinant protein according to claim 5, wherein in step S1, the PCR technique is adopted to amplify the target gene expressing BNP, tryptophan W is connected to the BNP sequence, and the three repetitions are performed to obtain the amino acid sequence corresponding to the BNP recombinant plasmid nucleotide sequence.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111626199.5A CN114316020A (en) | 2021-12-28 | 2021-12-28 | BNP recombinant protein, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111626199.5A CN114316020A (en) | 2021-12-28 | 2021-12-28 | BNP recombinant protein, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114316020A true CN114316020A (en) | 2022-04-12 |
Family
ID=81014240
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111626199.5A Pending CN114316020A (en) | 2021-12-28 | 2021-12-28 | BNP recombinant protein, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114316020A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1273248A (en) * | 1999-11-24 | 2000-11-15 | 刘建宁 | Efficient gene engineering process for preparing polypeptide medicines |
| CN1741814A (en) * | 2002-11-26 | 2006-03-01 | 诺贝克斯公司 | Modified naturetic compounds, conjugates, and uses thereof |
| CN102040660A (en) * | 2009-10-16 | 2011-05-04 | 重庆紫禾医药技术开发有限公司 | Recombinant protein as BNP (Brain Natriuretic Peptide) immunodiagnostic reagent standard as well as preparation method and application thereof |
| CN103923937A (en) * | 2014-04-09 | 2014-07-16 | 石家庄沃泰生物科技有限公司 | Method for soluble expression of recombinant protein of human brain natriuretic peptide and application |
| CN111527410A (en) * | 2017-12-28 | 2020-08-11 | 盐野义制药株式会社 | BNP assay standard |
-
2021
- 2021-12-28 CN CN202111626199.5A patent/CN114316020A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1273248A (en) * | 1999-11-24 | 2000-11-15 | 刘建宁 | Efficient gene engineering process for preparing polypeptide medicines |
| CN1741814A (en) * | 2002-11-26 | 2006-03-01 | 诺贝克斯公司 | Modified naturetic compounds, conjugates, and uses thereof |
| CN102040660A (en) * | 2009-10-16 | 2011-05-04 | 重庆紫禾医药技术开发有限公司 | Recombinant protein as BNP (Brain Natriuretic Peptide) immunodiagnostic reagent standard as well as preparation method and application thereof |
| CN103923937A (en) * | 2014-04-09 | 2014-07-16 | 石家庄沃泰生物科技有限公司 | Method for soluble expression of recombinant protein of human brain natriuretic peptide and application |
| CN111527410A (en) * | 2017-12-28 | 2020-08-11 | 盐野义制药株式会社 | BNP assay standard |
Non-Patent Citations (2)
| Title |
|---|
| 任运生,李恩民,黄革,刘颖,温博贵: "原核表达的多串重复人脑钠素蛋白的裂解(英文)", 汕头大学医学院学报, no. 01, pages 1 - 3 * |
| 饶贤才;胡福泉;: "基因串联体的构建策略及其表达模式", 医学研究生学报, no. 06, pages 557 - 560 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0322094B1 (en) | N-terminal fragments of human serum albumin | |
| JP4504014B2 (en) | Methods for producing insulinotropic GLP-1 (7-36) polypeptides and / or GLP-1 analogs | |
| JP2002504494A (en) | Method for producing mouse and human endostatin | |
| CN103923937A (en) | Method for soluble expression of recombinant protein of human brain natriuretic peptide and application | |
| Kadonookuda et al. | Baculovirus-mediated production of the human growth hormone in larvae of the silkworm, Bombyx mori | |
| CN100432230C (en) | Fusion expression method for metallothionein and use thereof | |
| US4714674A (en) | Chemotactic assay for immunogenicity | |
| CN114316020A (en) | BNP recombinant protein, preparation method and application thereof | |
| KR0161656B1 (en) | Method for the production of glucagon | |
| WO2022089340A1 (en) | Method for expressing human chymotrypsinogen and preparing recombinant human chymotrypsin by using genetically engineered rice | |
| CN104342423B (en) | High activity recombinant human chymotrypsin preparation method and application | |
| CN109776653B (en) | Human serum albumin adhesion peptide and application thereof | |
| KR102006101B1 (en) | Method for soluble overexpression of active recombinant human fibroblast growth factor 21 | |
| HU219544B (en) | Process for removal of aminoterminal dipeptides or dipeptides from polypeptid precursors | |
| RU2453604C1 (en) | Hybrid protein (versions), bacterial strain escherichia coli - hybrid protein producer (versions) and method for producing methionine-free human interferon alpha-2 | |
| CN112142848A (en) | Recombinant human insulin and purification preparation method thereof | |
| JPH02124092A (en) | Production of batroxobin by genetic recombination | |
| CN114349848A (en) | SAA recombinant protein and preparation method and application thereof | |
| Zhang et al. | Soluble expression and purification of recombinant bovine ferritin H-chain | |
| JP2845558B2 (en) | DNA sequence of methionine aminopeptidase | |
| JP4252299B2 (en) | Novel disulfide oxidoreductase and protein activation method using the enzyme | |
| CN108929365B (en) | Renaturation method of recombinant protein | |
| Wang et al. | An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase | |
| CN117467024A (en) | Fusion tag recombinant collagen and expression and application thereof | |
| JP2650856B2 (en) | Method for producing C-terminal amidating enzyme |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 210000 No. 2, Qiande Road, Jiangning District, Nanjing, Jiangsu Province Applicant after: LANSION BIOTECHNOLOGY Co.,Ltd. Applicant after: Jiangsu Huakong Biotechnology Co., Ltd Address before: 210000 No. 2, Qiande Road, Jiangning District, Nanjing, Jiangsu Province Applicant before: LANSION BIOTECHNOLOGY Co.,Ltd. Applicant before: Nanjing lanxuan Biotechnology Co., Ltd |
|
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |