CN109053902B

CN109053902B - Chimeric protein AQP4-CH2, preparation and application thereof

Info

Publication number: CN109053902B
Application number: CN201811056970.8A
Authority: CN
Inventors: 祖向阳; 胡志刚; 杜喆; 张晓兰; 黄磊; 付东辽; 薛云; 高伟娜; 马晓孟; 宋春辉
Original assignee: Henan University of Science and Technology
Current assignee: Henan University of Science and Technology
Priority date: 2018-09-11
Filing date: 2018-09-11
Publication date: 2021-06-15
Anticipated expiration: 2038-09-11
Also published as: CN109053902A

Abstract

The invention relates to a chimeric protein AQP4-CH2, and preparation and application thereof, and belongs to the technical field of biomedical engineering. The invention firstly utilizes the protein three-dimensional structure simulation to replace three sections of epitope (A ring, C ring and E ring) of AQP4 in a human antibody constant region CH2 with irregular curliness, so that the three sections of epitope are displayed on the surface of CH2 protein; then artificially synthesizing a chimeric protein gene with optimized codons, and constructing a prokaryotic expression vector by utilizing a molecular cloning technology; transforming escherichia coli to screen a high-efficiency expression chimeric protein strain, and purifying the chimeric protein AQP4-CH2 by Ni-affinity chromatography; the chimeric protein is used as an antigen coated enzyme label plate, and an enzyme-linked immunosorbent assay (ELISA) method is adopted to determine the AQP4 antibody in serum and cerebrospinal fluid of the patient with neuromyelitis optica for early differential diagnosis of neuromyelitis optica and multiple sclerosis.

Description

Chimeric protein AQP4-CH2, preparation and application thereof

Technical Field

The invention belongs to the technical field of biomedical engineering, and particularly relates to a chimeric protein AQP4-CH2, and preparation and application thereof.

Background

Neuromyelitis optica mainly involves the optic nerve and spinal cord, is an inflammatory demyelinating disease, and is mainly clinically manifested as vision disorders of different degrees with subacute onset, paralysis of both lower limbs, sensory and diarrheal disorders, and because it has similarity to multiple sclerosis in clinical symptoms, there is a wide debate clinically as to whether both are different subtypes of the same disease. With the increase of optic neuromyelitis cases and the progress of related researches in recent years, the optic neuromyelitis is inconsistent with the manifestation of multiple sclerosis in clinical process, neuroimaging, serology, immunopathology and the like. The spinal cord segment affected by neuromyelitis optica is longer, the length of the spinal cord segment is 3 or more than 3 vertebral body segments, the spinal cord segment is called as longitudinal expansibility transverse myelitis, and the focus of multiple sclerosis is mainly cerebral hemisphere, the focus of spinal cord is rarely more than 2 vertebral bodies, and the focus is located at the periphery of spinal cord. However, since early neuromyelitis optica may present a brain lesion, which is very different from the prognosis of multiple sclerosis, finding a biological marker that can help early diagnosis and identification will help to treat a patient.

Aquaporin 4 (AQP 4) is a glycoprotein, a highly selective water transmembrane transport protein distributed in the central nervous system, with a relative molecular mass of about 30000 and 301 amino acids. The molecular structure is a tetramer, wherein each monomer has independent transport water channel activity. The primary structure of the protein consists of 5 rings (A, B, C, D and E) connected with 6 transmembrane segments, and comprises 2 intracellular rings (B and D) and 3 extracellular rings (A, C and E), wherein the B and D rings are both intracellular with carboxyl and amino terminals, and A, C and the E rings are positioned outside a cell membrane and are the main epitopes of AQP 4. 6 transmembrane regions surround the B and E rings to form a barrel-shaped structure as a water molecule channel. The B and E rings are hydrophobic, any change of which causes a decrease in water channel activity, and the remaining rings are hydrophilic. The amino-and carboxy-termini of the peptide chain consist of similar repetitive sequences, each possessing an asparagine-proline-alanine (Asn-Pro-Ala, NPA) characteristic sequence, which is a characteristic structure shared by the AQP family and which is decisive for water permeability. The amino-and carboxy-termini are arranged 180 ° centrosymmetrically with respect to each other in structure, the amino acid sequence is passed through about 40% alpha helix and 42% beta sheet and turn to form its secondary structure, and the homologous tetramer reconstitutes the tertiary structure of AQP 4. AQP4 is mainly expressed on meningeal surface, ependyma and ventricles, and other parts of the brain can also be expressed in supraoptic nucleus, cerebellum, hippocampal dentate gyrus, middle and reins nucleus, choroid plexus epithelium, retina and optic nerve, and AQP4 in different parts plays respective functions, participates in secretion, distribution and reabsorption of cerebrospinal fluid together, and plays an important role in regulating the osmotic pressure in brain, the balance of K < + > and the pathological mechanism of cerebral edema.

Aquaporin 4 antibody (AQP 4 Ab), also known as neuromyelitis optica specific antibody, was discovered by Lennon in 2004. The AQP4 antibody activates the complement system, causing inflammatory demyelination and necrosis in the central nervous system, inducing damage to astrocytes. The AQP4 antibody exists in serum and cerebrospinal fluid of patients with neuromyelitis optica, and has important significance for early diagnosis and identification of neuromyelitis optica and multiple sclerosis. The clinical research on the detection method of the aquaporin 4 antibody is relatively wide, the current detection methods comprise a cell co-immunoprecipitation method, a radioimmunoprecipitation method, tissue indirect immunofluorescence detection, a cell immunofluorescence method, an enzyme-linked immunosorbent assay method and the like, and the advantages and the disadvantages of the various methods are different.

Disclosure of Invention

In view of the above, the present invention aims to provide a chimeric protein AQP4-CH2 displaying an AQP4 epitope on the surface of the human antibody constant region CH 2;

the second purpose is to provide a method for preparing the chimeric protein AQP4-CH 2;

the third purpose is to further provide application of the chimeric protein AQP4-CH2, which is used as a coating antigen for AQP4 antibody detection and can be used for early differential diagnosis of neuromyelitis optica and multiple sclerosis.

In order to achieve the purpose, the invention adopts the specific scheme that:

a chimeric protein AQP4-CH2, said chimeric protein AQP4-CH2 is displaying AQP4 epitope on human antibody IgG1 constant region CH2 protein surface; the AQP4 epitope includes the a, C and E loops; the A loop, the C loop and the E loop respectively replace irregular curly sections in a CH2 structure, so that the A loop, the C loop and the E loop are displayed on the surface of a human antibody IgG1 constant region CH2 protein.

Furthermore, the gene sequence of the A ring of the AQP4 epitope is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; the gene sequence of the AQP4 epitope C ring is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4; the gene sequence of the E loop of the AQP4 epitope is shown as SEQ ID NO. 5, and the amino acid sequence is shown as SEQ ID NO. 6; the gene sequence of the constant region CH2 of the human antibody IgG1 is shown as SEQ ID NO. 7, and the amino acid sequence is shown as SEQ ID NO. 8.

Furthermore, the amino acid sequence of the chimeric protein AQP4-CH2 is shown as SEQ ID NO: 10.

Furthermore, the gene sequence of the protein AQP4-CH2 is shown as SEQ ID NO. 9.

The invention also further protects a method for preparing the chimeric protein AQP4-CH2, and the method comprises the steps of firstly constructing a prokaryotic expression vector containing a gene sequence of SEQ ID NO. 9, transforming escherichia coli cells, expressing the chimeric protein AQP4-CH2, and separating and purifying by adopting Ni-affinity chromatography to obtain the chimeric protein AQP4-CH 2.

The invention also protects the application of the chimeric protein AQP4-CH26 in AQP4 antibody detection, and the application further refers to the application of the chimeric protein AQP4-CH2 as a coating antigen in early stage identification and diagnosis of neuromyelitis optica and multiple sclerosis.

The term "chimeric protein" refers to the expression product of two different genes combined using DNA recombination techniques, i.e., the expression product of a chimeric gene.

Has the advantages that:

the innovation of the invention is that three peptide segments (A ring, C ring and E ring) of AQP4 epitope are respectively substituted for amino acids in irregular coil structure in human antibody constant region CH2 protein, so that the three peptide segments are displayed on the surface of CH2 protein to construct chimeric protein AQP4-CH 2. The chimeric protein has the following advantages: firstly, AQP4 is a multi-transmembrane glycoprotein, active protein cannot be obtained by adopting conventional prokaryotic expression, and AQP4 protein can be obtained by adopting a eukaryotic expression system, but the yield is low, the cost is high, and the method is not suitable for large-scale production and use, and three antigen epitopes of AQP4 are embedded into human antibody CH2 protein, so that the large-scale expression of the protein by using the prokaryotic expression system becomes possible; secondly, a human antibody CH2 is selected as a scaffold protein, so that false positive caused by cross reaction between the scaffold protein and the human antibody in the detection process can be avoided; the chimeric protein can simultaneously display three independent epitopes of AQP4, can simulate the spatial position of the AQP4 epitope, and improves the binding capacity with the AQP4 antibody.

The AQP4-CH2 chimeric protein enables an AQP4 epitope to be displayed on the surface of CH2 protein so as to be combined with an AQP4 antibody, is used as an envelope antigen for detecting the AQP4 antibody by an ELISA method, and is used for early diagnosis and differential diagnosis of neuromyelitis optica and multiple sclerosis.

Drawings

FIG. 1 is a diagram showing an amino acid sequence alignment of a human antibody constant region CH2 and a chimeric protein AQP4-CH 2; wherein, the underlined parts in the figure are the substitution sites of AQP4 epitope A ring, C ring and E ring for the amino acid in CH 2;

FIG. 2 is a three-dimensional structure mimic structure diagram of the chimeric protein AQP4-CH2, wherein the green part (part I) is the scaffold protein CH2, the yellow part (part II) is the AQP4 epitope A ring, the red part (part III) is the AQP4 epitope C ring, and the blue part (part IV) is the AQP4 epitope E ring;

FIG. 3 is a schematic representation of the surface display of the epitope of AQP4 on the chimeric protein AQP4-CH 2;

FIG. 4 is an electrophoresis diagram of the agarose gel electrophoresis detection vector pET-22b (+) -AQP4-CH2 and the PCR identification recombinant vector pET-22b (+) -AQP4-CH 2; in the figure, M: DNA molecular weight standards (bp); 1: plasmid vector pET-22b (+) -AQP4-CH 2; 2: PCR amplification products;

FIG. 5 is an electrophoresis chart of SDS-PAGE for detecting the expression of the chimeric protein; in the figure, M: protein molecular weight standards (kDa); 0: bacterial negative control before addition of inducer; 2-4: respectively showing the bacterial samples expressing the chimeric protein after 2, 3 and 4 hours of adding the inducer;

FIG. 6 is an electrophoretogram of SDS-PAGE identifying Ni-affinity chromatography purification of chimeric proteins; in the figure, M: protein molecular weight standards (kDa); 1: a bacterial negative control; 2: inducing the sample after the disruption of the bacteria expressing the chimeric protein; 3: a 100mM imidazole-eluted chimeric protein sample; 4: a 250mM imidazole-eluted chimeric protein sample; 5: a 500mM imidazole-eluted chimeric protein sample;

FIG. 7 is a comparison graph of AQP4 antibody content in serum and cerebrospinal fluid of a patient with neuromyelitis neurosis detected by ELISA method with chimeric protein AQP4-CH2 as coating antigen.

Detailed Description

The present invention will be described in detail with reference to the following examples and the accompanying drawings, wherein the following examples are implemented on the premise of the technical scheme of the present invention, and detailed embodiments and specific operation procedures are provided, but the scope of the present invention is not limited to the following examples.

Example 1 chimeric protein design based on three-dimensional Structure modeling

The three-dimensional structure of the human antibody constant region CH2 (NO. 3DJ 9) registered in the GeneBank protein three-dimensional structure database, wherein the antibody constant region CH2 has a framework structure composed of beta sheets and is connected by irregular coils and alpha helix to form a structure similar to a barreled structure, and the irregular coils are displayed on the upper surface of the barreled domain. According to the amino acid sequence and the three-dimensional structure of a human antibody constant region CH2, the amino acid sequence of an irregular coil part in a CH2 structure is replaced by an A loop, a C loop and an E loop in three epitopes of AQP4, wherein the A loop is used for replacing amino acids from 89 th to 96 th, the C loop is used for replacing amino acids from 29 th to 38 th, and the E loop is used for replacing amino acids from 59 th to 64 th, and specific amino acid replacement information is shown in figure 1. Using protein structure simulation software, the distribution of the polypeptide on CH2 was analyzed, and the substituted AQP4 epitope a-loop, C-loop and E-loop polypeptides were all displayed on the surface of CH2 protein, as shown in fig. 2 and 3.

Wherein, the gene sequence of the A ring of the AQP4 epitope is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; the gene sequence of the AQP4 epitope C ring is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4; the gene sequence of the E loop of the AQP4 epitope is shown as SEQ ID NO. 5, and the amino acid sequence is shown as SEQ ID NO. 6; the gene sequence of the constant region CH2 of the human antibody IgG1 is shown as SEQ ID NO. 7, and the amino acid sequence is shown as SEQ ID NO. 8.

Example 2 Artificial Synthesis of chimeric protein DNA

Selecting codon with highest codon utilization rate of Escherichia coli according to amino acid sequence of chimeric protein, referring to codon utilization rate database, designing optimal chimeric protein nucleic acid sequence, entrusting organism company to artificial synthesis of gene, and adding gene at two ends of the synthesized geneNcoI andXhoi restriction enzyme site. Wherein, the gene sequence of the protein AQP4-CH2 is shown as SEQ ID NO. 9, and the amino acid sequence is shown as SEQ ID NO. 10.

Example 3 construction of chimeric Gene prokaryotic expression vectors

According to molecular cloning methods, usingNcoI andXhoi, double enzyme digestion of artificially synthesized chimeric gene and a vector pET-22b (+), and after recovery, the chimeric gene is connected with the vector pET-22b (+), so as to construct a vector pET-22b (+) -AQP4-CH2 and transform escherichia coli Top 10. Ampicillin resistance was used to screen positive clones and sequencing was used to identify correct vector sequences, as shown in FIG. 4.

Example 4 screening of strains highly expressing chimeric proteins

An expression vector pET-22b (+) -AQP4-CH2 plasmid is prepared by an alkaline-SDS lysis method, escherichia coli BL21(DE3) is transformed, Ampicillin (AMP) resistance is utilized to screen positive clones, different monoclonals are selected and inoculated into 100 ml LB-AMP culture medium, and the mixture is kept still at 25 ℃ for overnight. The next day, at 37 deg.C, 220 r/min, culturing the strain to OD600=0.6, taking out the shake flask, rapidly cooling the culture medium in ice bath, adding inducer IPTG to induce the expression of the target protein, and detecting the expression level of the chimeric protein by SDS-PAGE, as shown in FIG. 5. Wherein, after the inducer is added, the chimeric protein AQP4-CH2 starts to express, and the expression quantity gradually increases along with the prolonging of the induction time.

Example 5 expression and purification of chimeric proteins

The expression strain obtained by screening is used for enlarging the culture scale and expressing the chimeric protein in large quantity. The cultured bacteria were collected, disrupted by sonication, centrifuged at 10000 rpm for 30 minutes, the supernatant was separated, and the chimeric protein was purified by Ni-affinity chromatography, as shown in FIG. 6, whereby the purified chimeric protein was obtained in a yield of 1.84 mg/g of cells. When the protein is eluted, the eluent contains imidazole with a certain concentration, when the imidazole concentration is low, the chimeric protein is already eluted, the purity is lower, and when the imidazole concentration is high, the concentration of the eluted chimeric protein is slightly lower, but the purity is higher.

Example 6 detection of AQP4 antibody by ELISA

0.1M pH8.6 Na was used₂CO₃Dialyzing the purified chimeric protein, diluting the chimeric protein to 0.1 mg/ml as a coating solution, adding 100ul of the chimeric protein into each hole of an enzyme-labeled plate, storing the chimeric protein overnight at 4 ℃ to enable the chimeric protein to be combined on the enzyme-labeled plate, and simultaneously adding 100ul of 5% bovine serum albumin into each hole as negative control; discarding the coating solution, adding 200ul of 5% bovine serum albumin into each well, and sealing at 37 ℃ for 2 hours; taking serum and cerebrospinal fluid of a patient with neuromyelitis optica and negative control serum and cerebrospinal fluid, adding 100ul of serum and cerebrospinal fluid into each hole, and incubating for 1 hour at 37 ℃; discarding the sample solution, and washing the plate 6 times by using a washing solution; adding secondary HRP labeled antibody into each hole, and incubating for 1 hour at 37 ℃; discarding the secondary antibody, and washing the plate 6 times by using a washing solution; the reaction was carried out at room temperature for 10 minutes by adding a developing solution, and absorbance was measured at 405nm, and the results are shown in FIG. 7. The result shows that the AQP4 antibody in the serum and cerebrospinal fluid of the neuromyelitis patient is sensitively detected by adopting an ELISA method by taking the chimeric protein AQP4-CH2 as a coating antigen.

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Claims

1. A chimeric protein AQP4-CH2, characterized in that: the chimeric protein AQP4-CH2 is a human antibody IgG1 constant region CH2 protein surface display AQP4 epitope; the AQP4 epitope includes the a, C and E loops; the A loop, the C loop and the E loop respectively replace irregular curly sections in a CH2 structure, so that the A loop, the C loop and the E loop are displayed on the surface of a human antibody IgG1 constant region CH2 protein.

2. The chimeric protein AQP4-CH2 of claim 1, wherein: the gene sequence of the A ring of the AQP4 epitope is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; the gene sequence of the AQP4 epitope C ring is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4; the gene sequence of the E loop of the AQP4 epitope is shown as SEQ ID NO. 5, and the amino acid sequence is shown as SEQ ID NO. 6; the gene sequence of the constant region CH2 of the human antibody IgG1 is shown as SEQ ID NO. 7, and the amino acid sequence is shown as SEQ ID NO. 8.

3. The chimeric protein AQP4-CH2 of claim 1, wherein: the amino acid sequence of the chimeric protein AQP4-CH2 is shown as SEQ ID NO: 10.

4. The chimeric protein AQP4-CH2 of claim 3, wherein: the gene sequence of the protein AQP4-CH2 is shown in SEQ ID NO. 9.

5. A method of making the chimeric protein AQP4-CH2 according to claim 4, characterized in that: firstly, a prokaryotic expression vector containing a gene sequence of SEQ ID NO. 9 is constructed, an escherichia coli cell is transformed, a chimeric protein AQP4-CH2 is expressed, and the chimeric protein AQP4-CH2 is obtained by adopting Ni-affinity chromatography separation and purification.

6. The use of the chimeric protein AQP4-CH2 as claimed in any one of claims 1 to 4 in the preparation of a medicament for the detection of the AQP4 antibody.

7. The use of claim 6, wherein: the chimeric protein AQP4-CH2 is used as a coating antigen in the preparation of early stage identification and diagnosis medicines for neuromyelitis optica and multiple sclerosis.