CN115838437A - Human NT-proBNP fusion protein and preparation method and application thereof - Google Patents

Abstract

The application discloses a human NT-proBNP fusion protein, a preparation method and an application thereof. In the application, the human NT-PROBNP fusion protein is designed and developed, the preparation method of the human NT-PROBNP fusion protein based on the genetic engineering technology is developed, the polynucleotide which is optimized by the preference of the synonymous codon and used for coding the human NT-PROBNP fusion protein is introduced into a vector, the prokaryotic expression plasmid is successfully constructed, the expression quantity of the target protein is improved, and the method has the advantages of high yield, short production period, easy purification of an expression product, low cost and the like; compared with natural human NT-proBNP protein, the NT-proBNP fusion protein has better stability and higher antigen activity.

Description

Human NT-proBNP fusion protein, preparation method and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a human NT-proBNP fusion protein, a preparation method and application thereof.

Background

N-terminal pro-natriuretic peptide (NT-proBNP) is a good cardiac marker of heart failure, and is cleaved from the carboxy-terminal end of the amino acid precursor protein (pro-BNP) to yield 76-amino acid inactive NT-proBNP and 32-amino acid biologically active B-type natriuretic peptide (BNP). NT-proBNP and BNP with biological activity containing C-end 32 amino acids are released in an equimolar way, so that the NT-proBNP and the BNP with biological activity have similar clinical application in the aspects of diagnosis, treatment monitoring and prognosis of cardiovascular system diseases.

NT-proBNP is mainly produced by myocardial tissue, which cannot be purified from plasma (serum), making the preparation of the native NT-proBNP polypeptide very difficult. The direct polypeptide synthesis cost is too high, which is not beneficial to large-scale production. Low molecular weight polypeptides are prepared by genetic engineering, typically using fusion protein expression. However, a great deal of research shows that the obtained NT-proBNP protein has the defect of poor stability due to the lack of glycosylation of the protein expressed by prokaryotic recombination. Therefore, there is a need in the art to develop a method for preparing NT-proBNP protein with high stability.

Disclosure of Invention

The invention aims to provide a human NT-PROBNP fusion protein.

Another objective of the invention is to provide a preparation method of the human NT-PROBNP fusion protein.

Another object of the present invention is to provide a polynucleotide sequence encoding the human NT-PROBNP fusion protein.

It is another object of the invention to provide a vector adapted to a polynucleotide sequence encoding a human NT-PROBNP fusion protein.

It is another object of the present invention to provide kits comprising polynucleotide sequences encoding human NT-PROBNP fusion proteins.

In order to solve the above technical problems, the present invention provides, in a first aspect, a human NT-PROBNP fusion protein selected from any one of the following:

(a) A fusion protein comprising an amino acid sequence as set forth in SEQ ID No. 3;

(b) A fusion protein comprising an amino acid sequence having a homology of more than 95% with the sequence shown in SEQ ID No. 3.

In a second aspect of the invention, there is provided a polynucleotide encoding a human NT-PROBNP fusion protein, said polynucleotide being codon optimized and selected from any one of the following:

(i) A polynucleotide comprising the sequence shown in SEQ ID No. 4;

(ii) Comprises a polynucleotide having a homology of more than 95% with the sequence shown in SEQ ID NO. 4; and

(iii) Including polynucleotides complementary to the polynucleotide sequences described in (i) or (ii).

In a third aspect of the invention, there is provided an expression vector comprising a polynucleotide as provided in the first aspect of the invention.

In some preferred embodiments, the expression vector comprises a polynucleotide sequence expressing a His x 6 tag, and more preferably, the polynucleotide sequence expressing the His x 6 tag is linked to the 3' end of the expression vector.

In some preferred embodiments, the expression vector is an E.coli expression vector, more preferably pET-28a (+).

In a fourth aspect of the invention, there is provided a host cell comprising an expression vector as provided in the third aspect of the invention; or

The host cell has integrated into its genome a polynucleotide as provided in the second aspect of the invention.

In some preferred embodiments, the host cell is E.coli (Escherichia coli).

In some preferred embodiments, the host cell is an E.coli Rosetta (DE 3) strain.

In a fifth aspect, the present invention provides a method for preparing a human NT-PROBNP fusion protein, the method comprising the steps of: culturing the host cell of the third aspect of the invention to express the protein of interest; and

separating the target protein to obtain the human NT-PROBNP fusion protein;

wherein the target protein has an amino acid sequence shown as SEQ ID NO. 3.

In some preferred embodiments, the host cell is obtained by transformation of E.coli with a plasmid comprising a polynucleotide according to the second aspect of the invention.

In some preferred embodiments, the host cell is cultured in SB, TB or SOC media. In order to obtain a large amount of soluble expression, in a more preferred embodiment, the host cell is cultured in TB medium.

In some preferred embodiments, the host cell is cultured in a shaking environment.

In some preferred embodiments, the host cell is cultured at a temperature of 16 to 19 ℃; or culturing the host cell at a temperature of 36 to 38 ℃.

In some more preferred embodiments, higher soluble protein expression of interest is obtained by culturing the host cell in TB medium at a temperature of 16 to 19 ℃.

In some preferred embodiments, the host cell is cultured in a medium containing the kanamycin resistance gene.

In some preferred embodiments, the host cell is cultured and induced with IPTG to express the protein of interest.

In some preferred embodiments, the host cell is cultured to an OD600 of 0.6 to 0.8, followed by induction with IPTG to express the protein of interest.

In some preferred embodiments, the step of isolating the protein of interest comprises:

and (3) passing the crushed target protein supernatant through a chromatographic column, eluting, and collecting the eluent.

In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column.

In a fifth aspect, the present invention provides a kit comprising: a human NT-PROBNP fusion protein as provided in the first aspect of the invention; or

A polynucleotide as provided in the second aspect of the invention; or alternatively

An expression vector as provided in the third aspect of the invention; or

The host cell of the fourth aspect of the invention.

Compared with the prior art, the invention has at least the following advantages:

(1) The invention designs and develops human NT-PROBNP fusion protein, and develops a preparation method of the human NT-PROBNP fusion protein based on genetic engineering technology, introduces polynucleotide which is optimized by preference of synonymous codon and used for coding the human NT-PROBNP fusion protein into a vector, successfully constructs prokaryotic expression plasmid, improves the expression quantity of target protein, and has the advantages of high yield, short production period, easy purification of expression products, low cost and the like;

(2) Compared with natural human NT-PROBNP protein, the NT-PROBNP fusion protein has better stability and higher antigen activity.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

One or more embodiments are illustrated by the figures in the accompanying drawings, which correspond to and are not intended to limit the embodiments.

FIG. 1 is a SDS-PAGE identification of human NT-PROBNP fusion proteins according to an embodiment of the invention;

FIG. 2 is an electrophoretogram of the human NT-PROBNP fusion protein in the examples according to the invention.

Detailed Description

The inventor designs a human NT-proBNP fusion protein through extensive and intensive research, and the fusion protein has better stability and higher activity compared with the natural human NT-proBNP protein.

The invention also relates to a human NT-proBNP fusion protein expression system based on a prokaryotic expression system, and further obtains a polynucleotide sequence of the coding human NT-proBNP fusion protein capable of expressing a large amount of target protein in an escherichia coli expression system through preference optimization of synonymous codons, so that the expressed target protein has high yield, high activity and good stability.

Obtaining target gene/obtaining target protein related nucleic acid sequence

The full-length nucleotide sequence or a fragment thereof of the target protein or an element thereof of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

In one embodiment of the present invention, the amino acid sequence (SEQ ID NO: 1) of the target protein is analyzed by NCBI database to obtain the target gene sequence information.

In one embodiment of the present invention, the gene sequence information encoding the newly designed human NT-proBNP fusion protein is obtained by analyzing the amino acid sequence (SEQ ID NO: 3) thereof.

Optimization of synonymous codon preference

To overcome the potential problem of reduced yield when expressing heterologous proteins in E.coli, the present invention relates to polynucleotide sequences optimized for synonymous codon bias. And (3) carrying out synonymous codon preference optimization on the obtained target gene sequence, wherein the target gene sequence subjected to the synonymous codon preference optimization can express the same amino acid sequence as the target protein.

The invention relates to a polynucleotide sequence (SEQ ID NO: 4) for coding human NT-proBNP fusion protein after optimization of synonymous codon, and the sequence is used for introducing a vector, so that the stability and efficiency of an expression process are improved, and finally the obtained target protein maintains higher activity.

The invention also relates to a polynucleotide having a homology of more than 95% with the sequence shown in SEQ ID NO. 4; and a polynucleotide complementary to the sequence shown in SEQ ID NO. 4.

Vector of target gene

The present invention also relates to vectors comprising the polynucleotides of the invention. By "vector" in the present invention is meant a linear or circular DNA molecule comprising a fragment encoding a protein of interest operably linked to other fragments that provide for its transcription. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, vectors, and the like. Vector fragments may be derived from a host organism, another organism, a plasmid or viral DNA, or may be synthetic. The vector may be any expression vector which is synthetic or conveniently subjected to recombinant DNA procedures, and the choice of vector will usually depend on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. In one embodiment, the vector of the invention is an expression vector. In one embodiment of the present invention, pET-28a (+) is selected as a vector for more efficient expression.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences encoding the proteins of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Illustratively, the insertion of the foreign DNA fragment can be achieved by cohesive end ligation of single enzyme cleavage sites, directional cloning of double enzyme cleavage fragments, cohesive end ligation of different restriction enzyme cleavage sites, blunt end ligation, artificial adaptor ligation, or end ligation of oligonucleotides, using an endonuclease to cleave a vector DNA molecule into linear molecules that can be ligated to the foreign gene, and then ligating the codon-optimized gene fragment of interest to the vector.

In one embodiment of the present invention, the vector further comprises a polynucleotide sequence expressing a His x 6 tag, preferably, the polynucleotide sequence expressing the His x 6 tag is linked to the 3' end (C-terminal) of the gene sequence of interest.

Vector transformation host cell containing target gene

The invention also relates to genetically engineered host cells that have been engineered with the vector or fusion protein coding sequences of the invention. The vector containing the codon-optimized gene of interest can be inserted, transfected or otherwise transformed into a host cell by known methods to obtain a transformant containing the codon-optimized gene of interest of the present invention and capable of expressing the protein of interest. A "host cell" in the present invention is a cell into which an exogenous polynucleotide and/or vector has been introduced. The host cell may be a eukaryotic host cell or a prokaryotic host cell, the host cell preferably being a bacterium, and preferably being E.coli, more preferably E.coli ROSETTA (DE 3) species (Escherichia coli Rosetta (DE 3) strain).

Method for producing target protein

The present invention also relates to a method for preparing a protein of interest, which can express or produce a recombinant protein using the polynucleotide sequence of the present invention. Generally, the following steps are performed:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) A host cell cultured in a suitable medium;

(3) Separating and purifying protein from culture medium or cell.

Wherein, the transformation or transduction of a suitable host cell with the recombinant expression vector containing the polynucleotide of step (1) can be carried out by a conventional technique well known to those skilled in the art, and when the host is Escherichia coli, a heat shock method, an electrical transformation method, or the like can be used.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media, preferably SB, TB or SOC media, depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time. In order to promote the expression of the target protein and increase the expression level of the soluble protein, the present invention provides a preferred embodiment, wherein the host cell is cultured in TB medium, and the medium contains kanamycin resistance gene.

The protein in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof. In one embodiment of the invention, the protein of interest is molecularly imprinted using affinity chromatography.

In the present disclosure, any exemplary or exemplary language (e.g., ") provided with respect to certain embodiments herein is used merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

If a definition or use of a term in a cited document is inconsistent or inconsistent with the definition of the term described herein, the definition of the term described herein applies and the definition of the term in the cited document does not apply.

Various terms used herein are as follows. If a term used in a claim is not defined below, the broadest definition persons in the art have given that term as reflected in a printed publication or issued patent at the time of filing.

As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component (e.g., nucleic acid or polypeptide) with which the nucleic acid or polypeptide is present in its natural source. In one embodiment, the nucleic acid or polypeptide is found only in the presence of, if any, solvents, buffers, ions or other components normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides that are present in their natural source.

As used herein, the terms "polynucleotide" and "polynucleotide sequence" may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The present invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

As used herein, the term "codon optimization" refers to a means of increasing the efficiency of gene synthesis by avoiding the use of poorly available or rare codons based on differences in codon usage exhibited by the organism actually making the protein expression or production (including E.coli, yeast, mammalian blood cells, plant cells, insect cells, etc.).

As used herein, the terms "homology" and "identity" are used interchangeably to refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by the following methods. The nucleotide or amino acid sequences of a polynucleotide or polypeptide are aligned, the number of positions in the aligned polynucleotide or polypeptide containing the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotide or polypeptide containing a different nucleotide or amino acid residue. Polynucleotides may differ at one position, for example, by comprising different nucleotides (i.e., substitutions or variations) or deletions of nucleotides (i.e., insertions or deletions of one or two nucleotides in a polynucleotide). Polypeptides may differ at one position, for example, by containing amino acids (i.e., substitutions or variations) or amino acid deletions (i.e., amino acids inserted into one or both polypeptides or amino acid deletions). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

As used herein, the terms "sequence complementary" and "reverse sequence complementary" are used interchangeably to refer to a sequence that is in the opposite direction to, and complementary to, an original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, its reverse complement is GTTCAT.

As used herein, the term "expression" includes any step involved in the production of a polypeptide in a host cell, including but not limited to transcription, translation, post-translational modification, and secretion. After expression, the host cell or the expression product can be harvested, i.e.recovered.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the present invention is further described below with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions, or according to conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are percentages and parts by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and it is to be noted that the terms used herein are merely for describing particular embodiments and are not intended to limit example embodiments of the present application.

Example 1 construction of NT-proBNP plasmid and transfection of host cells

(1) The amino acid sequence of the human NT-proBNP is obtained and is shown in SEQ ID NO. 1, and on the basis, the amino acid sequence of the human NT-proBNP fusion protein obtained by carrying out design optimization on the NT-proBNP is shown in SEQ ID NO. 3.

Sequence analysis is carried out on SEQ ID NO.3 to obtain a gene sequence for coding the gene sequence, after the gene sequence is optimized by the preference of Escherichia coli synonymous codon, a polynucleotide sequence for coding human NT-proBNP fusion protein shown in SEQ ID NO.4 is obtained, a carrier is pET-28a (+) and a C-end (His) 6 tag, and a recombinant expression plasmid is synthesized.

(2) Introduction of recombinant plasmid into host Escherichia coli

mu.L of expression plasmid was taken and added to 30. Mu.L of Escherichia coli competent Rosetta (DE 3) under ice bath conditions, and the mixture was allowed to stand on ice bath for 20min, heat-shocked for 90s, immediately kept on ice for 2min, added to 400. Mu.L of SOC medium without antibiotic, and cultured at 37 ℃ for 50min with shaking at 220 rpm. 100 μ L of the suspension was spread evenly on LB plates containing 100 μ g/mL kanamycin resistance and cultured overnight in a 37 ℃ incubator.

Amino acid sequence SEQ ID NO 1 of human NT-proBNP

HPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPR

Escherichia coli synonymous codon preference optimized polynucleotide sequence SEQ ID NO 2

CACCCCCTAGGAAGTCCAGGGTCAGCTAGCGACCTGGAAACCAGCGGTTTACAAGAGCAGCGTAACCACCTCCAAGGCAA

GCTGAGCGAACTGCAGGTTGAACAGACTTCTTTGGAGCCGTTGCAAGAGTCCCCGAGACCAACGGGTGTTTGGAAAAGCC

GTGAGGTGGCGACCGAAGGCATCCGCGGTCATCGTAAGATGGTGCTGTACACCCTGCGTGCTCCGCGC

Amino acid sequence SEQ ID NO 3 of optimized human NT-proBNP fusion protein

MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPED

LDMEDNDIIEAHREQIGGATYHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATE

GIRGHRKMVLYTLRAPR

Optimized polynucleotide sequence SEQ ID NO 4 of human NT-proBNP fusion protein

ATGAGTGATTCAGAAGTAAATCAAGAGGCTAAGCCAGAGGTCAAGCCGGAGGTGAAGCCGGAAACCCATATCAACCTGAA

AGTTTCTGATGGCTCCAGCGAAATTTTTTTCAAGATCAAAAAGACTACGCCGCTGCGCCGTCTGATGGAAGCGTTTGCAA

AACGTCAGGGTAAAGAAATGGATAGCCTGCGTTTCTTGTACGACGGCATTCGTATTCAAGCGGATCAAACCCCGGAGGAC

TTGGACATGGAGGATAATGACATCATCGAGGCCCACCGCGAACAGATTGGTGGTGCTACCTATCACCCCCTAGGAAGTCC

AGGGTCAGCTAGCGACCTGGAAACCAGCGGTTTACAAGAGCAGCGTAACCACCTCCAAGGCAAGCTGAGCGAACTGCAGG

TTGAACAGACTTCTTTGGAGCCGTTGCAAGAGTCCCCGAGACCAACGGGTGTTTGGAAAAGCCGTGAGGTGGCGACCGAA

GGCATCCGCGGTCATCGTAAGATGGTGCTGTACACCCTGCGTGCTCCGCGC

Example 2 expression of the Gene of interest

Selection procedure the monoclonal prepared in example 1 was aseptically inoculated into 100. Mu.g/mL Carna resistant TB, SB, SOC media, cultured with shaking at 220rpm at 37 ℃ until OD600 was between 0.6 and 0.8, induced with IPTG, and incubated overnight with shaking at 37 ℃ and 18 ℃ respectively. Sampling, carrying out ultrasonic disruption and carrying out SDS-PAGE identification, wherein the predicted molecular weight of the human NT-proBNP fusion protein is 22.80KD, and the identification result is shown in figure 1.

As shown in fig. 1, columns 1-2: expressing the crushed supernatant and precipitate in TB culture medium at 37 ℃;3-4 columns: supernatant and precipitate from SB culture medium disruption at 37 deg.C; 5-6 columns: expressing the crushed supernatant and precipitate in SOC culture medium at 37 deg.c; 7-8 columns: expressing the crushed supernatant and precipitate in TB culture medium at 18 ℃; columns 9-10: the crushed supernatant and precipitate are finally expressed in SB culture medium at 18 ℃; columns 11-12: the crushed supernatant and precipitate were expressed in SOC medium at 18 ℃.

The results show that the soluble expression of the human NT-proBNP fusion protein can be seen in SB, TB and SOC culture media at 37 ℃ and 18 ℃; the highest soluble expression ratio of the supernatant in the SOC culture medium at 18 ℃ is achieved, and the expression amount accounts for more than 80% of the total protein of the thallus.

Example 3 purification of expression product

Shake-flask culturing 1.5L of bacterial liquid with 18 ℃ SOC culture medium, and centrifugally collecting the wet weight of the bacterial strain: 30g. About 4g of the cells were weighed and resuspended in 35ml of lysine Buffer on ice. Centrifuging after ultrasonic crushing, centrifuging at 20000rpm and 4 ℃ for 30min, taking the supernatant, and filtering with a 0.22-micron needle filter to obtain the filtered bacterial liquid. Filtering, performing Ni-column affinity chromatography, eluting with 50mM Tris-HCl,50mM NaCl,200mM imidazole pH 7.0 to obtain the target protein, and eluting to obtain 22.7mg protein, wherein the electrophoretogram is shown in FIG. 2.

The expression content of the target protein is calculated to be 170.25mg/L, and the purity reaches 90 percent.

Example 4 protein stability assay

Natural human NT-proBNP (SEQ ID NO: 1) was prepared by reference to the method in example 2, and the stability of natural human NT-proBNP and the present inventors NT-proBNP was compared.

The stability of natural NT-proBNP and the NT-proBNP fusion protein of the invention is detected by using a rifu kit, and 4 tubes of target antigen are placed in an oven at 37 ℃ and 20 mu L of target antigen is placed in each tube. Sampling is carried out for 0 day, 3 days, 5 days and 7 days respectively, the luminous value is detected, and the stability of the target antigen is verified.

As shown in Table 1, the recombinant expressed human NT-proBNP fusion protein has no decrease in luminescence value after being placed at 37 ℃ for 3 and 5 days, and has some decrease in luminescence value after 7 days, but has better overall stability, and the stability can be improved by adding a stabilizer in the later period.

As is clear from Table 2, the stability of the natural NT-proBNP protein was significantly reduced after 3 days under the same conditions. The fusion protein of the invention is shown to have stability obviously higher than that of natural NT-proBNP.

TABLE 1

TABLE 2

Example 5 Elisa assay for identifying immunological Activity

The Darriy finished product kit is used for testing the antigen activity of the human NT-proBNP fusion protein, and the result shows that the highest luminous value of the protein can reach 323 ten thousands, which shows that the antigen activity is higher. The linear regression equation for concentration versus RLU value is: y =93.332x +15972 ² =0.9947, indicating good linearity (R) ² ＞0.99)。

As can be seen from Table 3 below, the recombinantly expressed human NT-proBNP fusion protein binds well to antibodies and is immunogenic.

TABLE 3

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of practicing the invention, and that various changes in form and detail may be made therein without departing from the spirit and scope of the invention in practice.

Claims (10)

1. A human NT-proBNP fusion protein selected from any one of the following:

(a) A fusion protein having an amino acid sequence shown as SEQ ID No. 3;

(b) A fusion protein having an amino acid sequence having a homology of more than 95% with the sequence shown in SEQ ID No. 3.

2. An isolated polynucleotide encoding a human NT-proBNP fusion protein, wherein the polynucleotide is codon optimized and the polynucleotide is selected from any one of:

(i) A polynucleotide having a sequence as shown in EQ ID No. 4;

(ii) A polynucleotide having a homology of greater than 95% with the sequence shown in SEQ ID No. 4; and

(iii) (iii) a polynucleotide having a sequence complementary to the polynucleotide sequence described in (i) or (ii).

3. An expression vector comprising the polynucleotide of claim 2.

4. The expression vector of claim 3, wherein the expression vector comprises a polynucleotide sequence expressing a His x 6 tag.

5. The expression vector of claim 3, wherein the expression vector is an E.coli expression vector, preferably pET-28a (+).

6. A host cell comprising the expression vector of any one of claims 3 to 5; or

The host cell having integrated into its genome the polynucleotide of claim 2.

7. A method of preparing a human NT-proBNP fusion protein, comprising the steps of:

transforming a host cell with a vector comprising the polynucleotide of claim 1;

culturing the host cell to express the human NT-proBNP fusion protein.

8. The method of claim 6, wherein the host cell is cultured in SB, TB or SOC media;

and/or, when the host cell is cultured, the culture medium contains a kanamycin resistance gene.

9. The method of claim 6, wherein the host cell is cultured and induced to express the protein of interest by IPTG.

10. A kit, comprising: a human NT-proBNP fusion protein of claim 1; or

The polynucleotide of claim 2; or

The expression vector of any one of claims 3 to 5; or

The host cell of claim 6.

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