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Recombinant protein as BNP (Brain Natriuretic Peptide) immunodiagnostic reagent standard as well as preparation method and application thereof

A technology of recombinant protein and recombinant plasmid, which is applied in the field of genetic engineering and can solve problems such as expensive protease, low BNP, and the impact of immunogenicity of small molecular proteins

Active Publication Date: 2012-07-25
重庆业为基生物科技集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

BNP in the human body is extremely low, and the normal peripheral blood concentration is about 100pg / ml; therefore, it cannot be purified from plasma (serum), making the preparation of natural NT-ProBNP polypeptide very difficult
However, the cost of direct peptide synthesis is too high, and the peptides are unstable and difficult to store.
Genetic engineering is used to prepare low-molecular-weight polypeptides, which are generally expressed by fusion proteins, which can overcome the defect of low translation efficiency when expressing low-molecular-weight polypeptides alone, and the expression products are easily degraded by proteolytic enzymes. Originality has a certain influence
Therefore, some proteases are usually used to remove the tagged protein. The tagged protein needs to be further purified and the added protease removed. The operation is cumbersome and the protease is very expensive.

Method used

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  • Recombinant protein as BNP (Brain Natriuretic Peptide) immunodiagnostic reagent standard as well as preparation method and application thereof
  • Recombinant protein as BNP (Brain Natriuretic Peptide) immunodiagnostic reagent standard as well as preparation method and application thereof
  • Recombinant protein as BNP (Brain Natriuretic Peptide) immunodiagnostic reagent standard as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment one Cloning of recombinant BNP gene

[0059] According to the BNP gene sequence (NM_002521.2) provided by GENEBANK, the BNP coding sequence is a total of 99 bases (SEQ ID NO: 3) encoding amino acids 77-108 in the BNP gene. Submit this sequence to the Graphical codon usage analyzer (http: / / guca.schoedl.del) and find that the codons encoded by the 2nd, 3rd, 13th, 14th, 30th, and 31st amino acids in the gene are used in E.coli BL21 The frequency is low (refer to figure 2 BNP gene codon bias analysis graph, in which the part with a column height value below 50 is a codon with a low frequency of use in Escherichia coli), after performing synonymous transformation on it, the coding sequence SEQID NO: 4 was obtained, using 2 paragraphs The Linker composed of 5 glycines connects the three modified sequences to obtain the coding sequence SEQ ID NO: 2 of recombinant BNP, and puts the coding sequence with Sal I and BamH I restriction sites, and the BamH I restrictio...

Embodiment 2

[0060] Embodiment two Construction of pET42a-BNP recombinant plasmid

[0061] The pET42a vector and the synthetic whole gene were digested with BamH I / Sal I double enzymes for 4 hours and then used T 4 DNA ligase was ligated overnight at 4°C, took a sterile centrifuge tube, added 200 μl of competent DH5α bacteria that had been prepared, put it in ice bath, pipetted 1 μl of the ligation product into the tube, transformed the DH5α bacteria, patted the tube wall to mix, and put it in an ice bath 30 minutes, 42°C water bath for 90 seconds, take out the centrifuge tube and ice-bath for 2 minutes, add 800μl room temperature 2×YT culture medium and mix well, 37°C shaker 220rpm shaking culture for 1 hour, respectively mix 50μl, 200μl and the rest Spread the transformed bacteria solution on three 2×YT culture plates containing kanamycin resistance, culture overnight in a 37°C constant temperature incubator, pick white colonies and inoculate them on LB medium for expansion, and extrac...

Embodiment 3

[0062] Embodiment Three Induced Expression and Identification of BNP Recombinant Protein

[0063] 1 Transformation of BL21 bacteria

[0064] The recombinant pET42a-BNP plasmid was used to transform BL21(DE3) bacteria, spread on LB solid medium containing kanamycin resistance, cultivate overnight in a 37°C incubator, and pick white colonies to inoculate on LB medium for expansion the next day.

[0065] Induced expression of 2BNP recombinant protein

[0066] The bacteria transformed with pET42a-BNP were expanded and cultured, and when the OD value of the bacteria reached 0.6-0.8, IPTG with a final concentration of 1 mmol / L was added to induce, and the induced bacteria were collected at time points 2, 4, 6, 8, 10, and 12 hours. The results showed that the bacteria induced by IPTG appeared a specific protein band with a molecular weight of about 40kD, which was consistent with the expected molecular weight value of pET42a / BNP, accounting for about 30% of the total bacterial pro...

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Abstract

The invention relates to a BNP (Brain Natriuretic Peptide) recombinant protein which has the amino acid sequence selected from (a) or (b): (a) an amino acid sequence represented by SEQ ID NO:1; (b) an amino acid sequence still having the activity of the BNP recombinant protein after one ore more than one amino acids are deleted, substituted or inserted into the (a). The invention also relates to an amino acid sequence for coding the BNP recombinant protein and a preparation method of the BNP recombinant protein. The BNP recombinant protein has the same immunogenicity with natural BNP protein,high purity and better stability and can be used as a BNP immunodiagnostic reagent standard to replace natural BNP polypeptide. The invention provides a basis for further researching and developing aBNP diction reagent kit.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant protein used as a BNP immunodiagnostic reagent standard, its preparation method and application. Background technique [0002] Heart disease has become the number one killer in many countries, and patients are often delayed in treatment because of its insidious early symptoms. Research reports in recent years have pointed out that when a patient's heart begins to suffer from compensatory insufficiency or failure, the concentration of a hormone in human blood—natriuretic peptide—will increase significantly. The natriuretic peptides produced in the atria, ventricles and blood vessels are also called ANP, BNP and CNP, respectively. Studies have found that in patients with heart disease, left atrium contraction weakness will cause blood retention in the heart, causing the body to secrete more of the above-mentioned hormones, which stimulate the kidneys to excrete sod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/58C12N15/16C12N15/70C12P21/02C07K1/22G01N33/74C12R1/19
Inventor 黄洪涛李鹏胡川闽易维京石延宾姚静张宪
Owner 重庆业为基生物科技集团有限公司