CN112920278B - Novel coronavirus specific fusion protein antigen and preparation method and application thereof - Google Patents
Novel coronavirus specific fusion protein antigen and preparation method and application thereof Download PDFInfo
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- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention discloses a novel coronavirus specific fusion protein antigen and a preparation method and application thereof, and relates to the technical field of biological engineering. The invention provides a novel coronavirus specific fusion protein antigen, the amino acid sequence of which is Seq ID No. 1; the invention provides a coding gene of a novel coronavirus specific fusion protein antigen, and the nucleotide sequence of the coding gene is Seq ID No. 2. The invention develops a novel coronavirus specific fusion protein antigen, and the novel coronavirus specific fusion protein antigen is expressed by recombinant engineering bacteria, and the novel coronavirus specific fusion protein antigen prepared by expression has better antigenicity and specificity; the detection kit prepared by the fusion protein antigen can quickly detect human serum and judge whether the human body is infected with the novel coronavirus pneumonia according to the detection kit, has good specificity, and provides technical support for quick diagnosis of the novel coronavirus pneumonia and prevention and control of the diseases.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a novel coronavirus specific fusion protein antigen and a preparation method and application thereof.
Background
The virus of the family Coronaviridae is a virus which is spherical, has protrusions on the surface and looks like a crown when observed under an electron microscope. The viral genetic material is linear single-stranded RNA with a diameter of 75-160 nm. It is widely distributed in humans, other mammals and birds and causes respiratory, intestinal, hepatic and nervous system diseases.
At present, six kinds of coronavirus are known to cause human diseases. Of these, four of these six coronaviruses, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1, are prevalent, have relatively mild symptoms, and often cause common cold symptoms in immunocompetent individuals; the other two viruses, SARS coronavirus (SARS-CoV) and "middle east respiratory syndrome" MERS coronavirus (MERS-CoV), are highly contagious and have severe symptoms.
2019 novel coronavirus, namely "2019-nCoV", which is the 7 th coronavirus now discovered, a new strain of coronavirus which has never been previously found in humans. After a person is infected with the novel coronavirus, the coronavirus is extremely harmful to the human body and even can endanger life. The study showed that 2019-nCoV belongs to the same genus of coronavirus as SARS-CoV and MERS-CoV. By gene sequence alignment, 2019-nCoV has about 80% similarity to SARS-CoV and 40% similarity to MERS-CoV. The nucleic acid sequence similarity of structural protein E was 98.68%, that of M was 93.42%, and that of N was 91.03%. And the similarity of the S protein nucleic acid sequences with the largest difference can reach 70%.
At present, a specific rapid detection method aiming at novel coronavirus needs to be established in clinical and scientific research processes. The etiological diagnosis method of the disease mainly comprises methods such as a nucleic acid detection kit, clinical symptoms, CT and the like; however, the nucleic acid detection kit method is time-consuming and not conducive to rapid diagnosis; clinical symptoms are easy to be confused by symptoms such as influenza and the like, and CT of patients with slight symptoms cannot be detected at all.
Therefore, no rapid detection method specific to the novel coronavirus exists in the market at present. How to solve the technical problems is a technical problem to be solved in the technical field of bioengineering at present.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention aims to provide a novel coronavirus specific fusion protein antigen, and a preparation method and application thereof.
The technical scheme adopted by the invention is as follows:
the invention provides a novel coronavirus specific fusion protein antigen, the amino acid sequence of which is Seq ID No. 1.
The invention provides a coding gene of a novel coronavirus specific fusion protein antigen, and the nucleotide sequence of the coding gene is Seq ID No. 2.
The invention provides a novel coronavirus specific fusion protein antigen which comprises 4 dominant linear antigens of coronavirus S protein, wherein the 4 dominant linear antigens are S respectively 12-53、S90-115、S171-203And S873-1190，S12-53From amino acid 12 to amino acid 53, S90-115From the 90 th amino acid to the 115 th amino acid, S171-203At amino acids 171 to 203, S873-1190From amino acid 873 to amino acid 1190.
The invention provides a recombinant expression vector, which is formed by recombining an expression vector and a coding gene of the novel coronavirus specific fusion protein antigen.
Preferably, the expression vector is a PET32a plasmid; it may also be PET28a or PET-28 a-sumo.
The invention provides a recombinant engineering bacterium, which is formed by transforming the recombinant expression vector into host bacteria.
Preferably, the host bacterium is escherichia coli BL21(DE3), and may also be escherichia coli Rosseta or an escherichia coli mutant expression strain.
The invention provides a preparation method of a novel coronavirus specific fusion protein antigen, which comprises the following specific steps:
(1) cloning the novel coronavirus specific fusion protein antigen nucleotide sequence into an expression vector to obtain a recombinant expression vector;
(2) transferring the recombinant expression vector obtained in the step 1 into host bacteria to obtain recombinant engineering bacteria;
(3) Inducing and expressing a novel coronavirus specific fusion protein antigen by using recombinant engineering bacteria;
(4) and (4) extracting and purifying the novel coronavirus specific fusion protein antigen obtained in the step (3).
The invention provides a detection kit, and the diagnostic antigen of the detection kit is the novel coronavirus specific fusion protein antigen. The skilled in the art can use the above-mentioned novel coronavirus specific fusion protein antigen to prepare a detection kit for rapidly detecting whether the patient is a novel coronavirus according to the disclosure of the present invention and the common general knowledge in the field.
The invention provides application of a novel coronavirus specific fusion protein antigen, which is used for detecting serum of a coronavirus patient.
The beneficial effects of the invention are:
1. the invention develops a novel coronavirus specific fusion protein antigen, and the novel coronavirus specific fusion protein antigen is expressed by recombinant engineering bacteria, and the novel coronavirus specific fusion protein antigen prepared by expression has better antigenicity and specificity;
2. the detection kit prepared by the fusion protein antigen can quickly detect human serum and judge whether the human body is infected with the novel coronavirus pneumonia according to the detection kit, has good specificity, and provides technical support for quick diagnosis of the novel coronavirus pneumonia and prevention and control of the diseases.
Drawings
FIG. 1 shows the result of Bepipred line Epitope Prediction 2.0 analysis;
FIG. 2 is the SDS-PAGE result chart of the positive expression result of the recombinant engineering bacteria;
wherein the reference numeral 1 is a control group, and the reference numerals 2 to 6 are positive expression groups.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
In order to solve the above technical problems, it is important to rapidly and accurately detect novel coronavirus antibodies from patient serum. The colloidal gold test strip is established to detect the novel coronavirus antibody, so that the individual infection condition can be effectively reflected, most importantly, a specific antigen which can completely replace the novel coronavirus is searched, and the problems of high risk, insufficient timeliness, low eukaryotic expression antigen yield and the like exist because the novel coronavirus is used as a natural antigen.
It has been found that the coronavirus genome consists of a single-stranded RNA, and that no RNA polymerase required for RNA virus replication must rely on the host cell for survival. When viruses invade host cells, certain components in the cells are used for realizing self gene replication assembly. The DNA replication error repair mechanism in the host cell can repair the self DNA replication error, but can not repair the RNA of the virus, so that the error rate of the RNA virus gene in the replication process is increased. Coronavirus undergoes RNA replication and mutation during continuous passage. Such variations may cause the virus to develop new characteristics that may be good or bad, such as enhanced infectivity, reduced pathogenicity, or may infect new species.
Coronavirus particles comprise 4 structural proteins, the nucleocapsid protein (N), the envelope protein (E), the membrane protein (M) and the spike protein (S). Wherein the S protein is the clavicle-globular glycoprotein which extends out of the envelope, and plays a key role in the process that the virus is combined with a host cell surface receptor and mediates the fusion of the membrane into the cell. The S protein trimer of the novel coronavirus binds to the receptor ACE2 on the surface of the target cell and mediates subsequent viral uptake and fusion. During this process, the S protein undergoes a significant structural rearrangement, switching from the pre-fusion conformation to the post-fusion conformation. The overall structure of the S protein before and after fusion is well conserved among coronaviruses. Therefore, the S protein is a relatively important conserved surface membrane protein and a relatively conserved membrane antigen protein.
The S protein is a main structural protein of the novel coronavirus, the full length of the protein consists of 1273 amino acids, and the protein contains 2 subunits with different functions: the S1 subunit is generally spherical in shape, contains a receptor binding domain, and can bind to a host receptor; the S2 subunit facilitates the process of membrane fusion of the virus with the host cell. 319-541 amino acids in the S1 subunit are RBD regions, and the region specifically binding to the receptor is 437-508 amino acids.
However, it was found that the 319-541 amino acid region, especially 437-508 amino acid region, in the S1 subunit of the novel coronavirus was preferentially selected to generate codon mutation during multiple passages and mutation, so as to avoid the attack of human immune cells or enhance the infectivity. If the fusion protein of the region is selected, on the basis of the variation of the virus, no antibody of the region is recognized in a human body, and the fusion antigen of the region is continuously used as a diagnostic raw material, so that the missed detection of the variant virus strain is generated. Therefore, the present invention mainly avoids these above-mentioned critical areas.
Therefore, biological software is used for analyzing the amino acid sequence, a sequence with stronger antigen specificity of the S2 subunit in the conserved region is preferentially selected, and the conserved linear antigenic site of the S1 subunit is supplemented to form an antigenic site which has strong specificity, high sensitivity and no omission.
Therefore, based on the above analysis, the present invention makes the following technical solutions:
the invention takes the S protein with the largest difference between the novel coronavirus and other coronaviruses as the basis, analyzes the sequence of the amino acid coded by the structural protein S protein gene of the novel coronavirus (NCBI serial number NC-045512.2) through bioinformatics software, serially connects the dominant antigen epitopes obtained by screening according to a certain sequence, constructs and synthesizes the multi-epitope fusion antigen; meanwhile, after codon optimization, a nucleic acid sequence of the protein is synthesized, a high-efficiency prokaryotic expression vector is constructed, and high-purity recombinant protein is purified.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Strains and plasmids: host bacterium BL21(DE3), plasmid PET32a, available from Dalianbao organism (TaKaRa) engineering Co., Ltd
Molecular biological reagents: the restriction enzymes BamH I and Notl, T4 ligase are TaKaRa product; the plasmid purification kit and the DNA fragment agarose gel recovery kit are products of Germany QIAGEN; IPTG is a product of Promega. Other reagents are imported or domestic analytical pure reagents.
Synthesis and DNA sequencing of genes: shanghai Sheng Gong Ltd
The gene cloning method comprises the following steps: DNA enzyme digestion, connection and electrophoresis; extracting and purifying plasmids; general molecular cloning methods such as SDS-PAGE analysis of proteins are carried out by conventional methods. Other kits were performed as described in the instructions.
Example 1 screening of epitopes
By referring to the S protein sequence published by the novel coronavirus on NCBI, the secondary structure and the tertiary structure domain of the protein are obtained by using online analysis tools such as ExPasy and UniProt, and the common dominant Linear antigen of the novel coronavirus S protein is screened out by combining the analysis result of Bepipred Linear Epitope Prediction 2.0. Screening out specific linear antigen S of novel coronavirus S protein12-53From amino acid 12 to amino acid 53, S90-115From the 90 th amino acid to the 115 th amino acid, S171-203At amino acids 171 to 203, S873-1190From amino acid 873 to amino acid 1190. The results of the specific Bepipred line Epitope Prediction 2.0 analysis are shown in FIG. 1.
Example 2 epitope Gene acquisition of novel coronavirus S protein-specific antigens
The genes for coding the dominant antigen epitope are combined and connected in series according to a certain arrangement, and the specific sequence is S 12-53、S90-115、S171-203、S873-1190Constructing fusion antigen gene containing 4 multi-epitopes, analyzing the escherichia coli rare codon in the fusion gene by rare codon online software, and replacing the escherichia coli rare codon by escherichia coli preferred codon nucleotide encoding the same amino acid, so that the finally synthesized multi-epitope fusion gene does not contain the escherichia coli rare codon. Finally, synthesizing the antigen nucleotide sequence of the novel coronavirus specific fusion protein, and adding BamH and Not I restriction enzymes at two ends of the nucleotide.
EXAMPLE 3 construction of expression vectors for novel coronavirus S protein-specific antigens
Extracting PET32a plasmid, double-digesting the plasmid with BamH I and Not I, and recovering the double-digested large fragment after electrophoresis. The new coronavirus specific fusion protein antigen is double digested with BamH I and Not I, gel is recovered after electrophoresis, and the gel is kept at-20 ℃ for later use.
The double-restriction plasmid and the double-restriction target fragment are connected by T4 ligase overnight at 16 ℃ according to the proportion of 1: 3-10. The recombinant expression vector is obtained after connection and is named as PET32 a-S.
EXAMPLE 4 screening and identification of recombinant expression vectors
The recombinant expression vector was transformed into E.coli BL21(DE3), spread on LB plates containing ampicillin (60ug/ml), and incubated overnight at 37 ℃. The next day, 5 transformed colonies and 2 control colonies (plasmid PET32a) were randomly picked and plasmids were extracted, respectively. PET32a-S is subjected to double enzyme digestion by BamH I and Not I, electrophoresis is carried out after enzyme digestion, corresponding target fragments and vector fragments can be seen, and the result shows that the construction of the recombinant expression vector is successful, namely the recombinant expression strain is a positive expression strain. The objective fragment was not observed after the control plasmid had been digested.
Example 5 high expression after identification of Positive-expressing bacteria
The positive expression-identified bacteria and the control bacteria were inoculated into 2ml of LB medium (60ug/ml ampicillin) in a test tube, shaken on a constant temperature shaker at 37 ℃ for 2 hours, added with IPTG (final concentration of 1mmol/l), and induced at 30 ℃ for 6 hours. The cells were collected by centrifugation and examined by SDS-PAGE. The size of the target protein expressed by PET32a-S is found to be about 66KD, which is consistent with the prediction result of ExPSY ProtParam online software. The control bacteria had no band of the target protein. The recombinant engineered bacteria expressing the novel coronavirus specific fusion protein antigen are obtained (the result is shown in figure 1).
Example 7 purification of expressed protein
Centrifuging the efficiently expressed recombinant engineering bacteria at high speed (12000rpm) and low temperature (4 ℃) for 15min, re-suspending the precipitated bacteria in 1/10 0.01M PBS (PH7.4) in the volume of the original bacteria liquid, fully washing for 1 time, centrifuging the bacteria at high speed (12000rpm) and low temperature (4 ℃) for 15min, re-suspending the precipitate in 1/10 0.01M PBS (PH7.4) in the volume of the original bacteria liquid, centrifuging the bacteria at 4 ℃ for 20min at ice bath ultrasound for 30min, 12000rpm for 32 min, allowing the PET32a-S expression protein to exist in an inclusion body form, and collecting the precipitate as the inclusion body protein.
The inclusion bodies were washed 1 time with 0.01M PBS (pH7.4) and then solubilized with an inclusion body solubilization solution of 50mM NaH 2PO4·2H2O, 500mM NaCl, 8M Urea, 6h of dissolution at 4 ℃, 12000rpm/min, 20min of centrifugation at 4 ℃, collecting supernatant, filtering the supernatant by a 0.22um filter membrane, purifying the supernatant by a nickel column, enabling the protein sample to slowly flow through the nickel column at the flow rate of 0.5ml/min, and washing the chromatographic column by inclusion body dissolving liquid with 5 times of the volume of a bed. The flow rate was constant, the column volume of the washing Buffer solution was 10 times the column volume to wash off the contaminating proteins, and the elution Buffer was used to elute the target proteins at a flow rate of 0.8 ml/min.
After SDS-PAGE detection of the eluent, collecting the eluent containing high-purity target protein, dialyzing and renaturing through Urea gradient, wherein the renaturation solution is 20mM Tris-HCl, 6M Urea and PH8.5, the concentration of Urea is gradually reduced to 4M, 2M and 0M, the dialysis time of each gradient is 2h, finally dialyzing for 48h by using 20mM Tris-HCl with the volume of 1000 times of the target protein and PH8.5, 12000rpm/min, centrifuging for 20min at 4 ℃, and collecting supernatant, namely the target protein.
EXAMPLE 8 preparation of multiple antisera to fusion proteins
Preparing polyclonal antiserum from the purified novel coronavirus specific fusion protein antigen, preparing and purifying the obtained rabbit antiserum in a mode of immunizing a New Zealand big ear white rabbit by combining the antigen and an adjuvant, and detecting the titer of the anti-rubella virus antibody by an indirect ELISA method.
1. The main reagents are as follows: complete Freund's adjuvant and incomplete Freund's adjuvant are available from SIGMA corporation. Other reagents are domestic or imported analytical pure reagents.
2. Immunization procedure
The fusion protein is used as immunogen to immunize 5 New Zealand big ear rabbits, each rabbit is immunized with 200ug of antigen, and the immunization mode is back multipoint immunization. The first immunization uses complete Freund's adjuvant and fusion protein to mix and emulsify, the second immunization after two weeks, uses incomplete Freund's adjuvant and fusion protein to mix and emulsify, the immunization mode is back multipoint immunization. Three days later, the third immunization was performed by intraperitoneal immunization with 100ug of the fusion protein mixed with the same volume of 0.01MPBS (pH 7.4). The same immunization program was used once three days later.
3. Indirect ELISA for rabbit antiserum titer
Three days after the last immunization, blood is taken from the ear vein of the rabbit, and the rabbit antiserum is obtained by centrifuging at 4000rpm for 10 min. The fusion protein was diluted with carbonate buffer (50mM, pH9.6) and coated onto an ELISA plate at 100 ul/well and 100 ng/well protein, overnight at 4 ℃. Sealing with 20% calf serum the next day, sealing with 37 deg.C water bath for 2 hr, diluting rabbit antiserum with gradient to obtain primary antibody, incubating in 37 deg.C water bath for 1 hr, adding 1:5000 secondary antibody to goat anti-rabbit, and adding color developing solution for developing. The potency is as high as 1:50000, which shows that the fusion protein has better antigenicity and specificity.
Example 9 detection of novel coronavirus Using purified fusion protein
The purified recombinant protein was diluted with carbonate buffer (50mM, pH9.6) and then coated on an enzyme-linked plate at a protein content of 100 ul/well and 100 ng/well, overnight at 4 ℃. After washing, 200 ul/hole 10% calf serum PBS (phosphate buffer solution) is added in a water bath at 37 ℃ for sealing for 2h, and after washing, 4 ℃ is reserved.
The indirect enzyme-linked immunosorbent assay detects 5 parts of nucleic acid to detect anti-novel coronavirus IgG positive serum and 5 parts of normal human serum. OD was measured using a wavelength of 450 nm. The results show (see table 1) that the fusion protein can react with 5 parts of anti-novel coronavirus positive serum and does not react with 5 parts of normal human serum, which indicates that the fusion protein has better antigenicity and specificity.
TABLE 1 Indirect enzyme-linked immunosorbent assay test results (A450 values)
| 1 | 2 | 3 | 4 | 5 | |
| Positive serum | 1.265 | 1.898 | 0.665 | 0.438 | 0.699 |
| Normal human serum | 0.016 | 0.005 | 0.028 | 0.018 | 0.007 |
Example 10 detection kit for coronavirus IgM/IgG total antibody by fluorescence immunochromatography
The kit adopts a dry fluorescence chromatography technology and a double-antigen sandwich method principle to detect the concentration of the new coronavirus IgM/IgG total antibody in a sample. The structure of the kit is the same as that of the kit in the prior art, the kit comprises a nitrocellulose membrane coated with an antigen, a release pad of the antigen marked by fluorescein and other reagents, and the coated and marked antigens are fusion proteins purified in the embodiment 8.
During detection, a sample is added into a sample adding hole of a detection card, a human anti-novel coronavirus IgM/IgG antibody and fluorescence-labeled novel coronavirus fusion protein in the sample are subjected to immunoreaction to form an immunoconjugate, the immunoconjugate is captured by the novel coronavirus fusion protein of a T line when the conjugate is chromatographed to a T line of the detection card, and the human anti-novel coronavirus IgM/IgG antibody and the fluorescence-labeled novel coronavirus fusion protein immunoconjugate are captured by goat anti-human IgG of the C line when the sample is chromatographed to a C line to serve as a quality control line. The intensity of the fluorescence signal at the T-line is positively correlated with the concentration of the anti-novel coronavirus antibody in the sample. Therefore, the concentration of total anti-novel coronavirus IgM/IgG antibodies in the sample can be detected by using a suitable dry fluorescence instrument.
After the detection card detects 10 serum samples of patients with new coronavirus positive nucleic acid detection, 10 samples are positive, and the sensitivity is 100%. All of the 100 healthy control serum samples were negative with a specificity of 100%.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Sequence listing
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405 410 415
Glu Val Ala
Claims (7)
1. A novel coronavirus specific fusion protein antigen is characterized in that the amino acid sequence of the fusion protein antigen is Seq ID No. 2.
2. A recombinant expression vector, which is prepared by recombining an expression vector and the encoding gene of the novel coronavirus specific fusion protein antigen according to claim 1.
3. The recombinant expression vector of claim 2, wherein the expression vector is the PET32a plasmid.
4. A recombinant engineered bacterium obtained by transforming the recombinant expression vector according to claim 2 or 3 into a host bacterium.
5. The recombinant engineered bacterium of claim 4, wherein the host bacterium is Escherichia coli BL21(DE 3).
6. The method for preparing the novel coronavirus specific fusion protein antigen according to claim 1, which comprises the following steps:
(1) Cloning the novel coronavirus specific fusion protein antigen nucleotide sequence of claim 1 into an expression vector to obtain a recombinant expression vector;
(2) transferring the recombinant expression vector obtained in the step 1 into host bacteria to obtain recombinant engineering bacteria;
(3) inducing and expressing a novel coronavirus specific fusion protein antigen by using recombinant engineering bacteria;
(4) and (4) extracting and purifying the novel coronavirus specific fusion protein antigen obtained in the step (3).
7. A test kit characterized in that its diagnostic antigen is the novel coronavirus-specific fusion protein antigen of claim 1.
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| CN113624965B (en) * | 2021-08-05 | 2024-02-09 | 中国人民解放军军事科学院军事医学研究院 | Application of N-protein specific IgG4 in screening novel coronavirus infected person and vaccinated person |
| CN115785263A (en) * | 2021-09-10 | 2023-03-14 | 广东菲鹏生物有限公司 | Novel crown antibody or antigen binding fragment thereof and application thereof |
| CN113999819B (en) * | 2021-11-10 | 2023-11-28 | 青岛硕景生物科技有限公司 | Hybridoma cell strain secreting anti-Trx protein monoclonal antibody and anti-Trx protein monoclonal antibody |
| CN116751305A (en) * | 2022-02-08 | 2023-09-15 | 苏州方舟生物科技有限公司 | Beta genus coronavirus fusion recombinant protein and preparation method and application thereof |
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