CN115947797B - Monkey poxvirus recombinant antigen and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of immunodetection, and in order to realize serological diagnosis of a monkey pox virus, the invention provides a monkey pox virus recombinant antigen A29, a monkey pox virus recombinant antigen B21R and chimeric recombinant antigens A29-B21R, and also provides a monkey pox virus positive quality control product and a monkey pox virus antibody detection test strip prepared from the recombinant antigens. The recombinant antigen prepared by the invention has the advantages of high expression level, high purity and high activity. The recombinant antigen prepared by the invention is applied to a gold-labeled chromatography detection platform, has better specificity and sensitivity, and lays a foundation for serological detection of the monkey pox virus infection.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a monkey pox virus recombinant antigen and application thereof.
Background
Monkey pox is a rare, sporadic, smallpox-like zoonotic infectious disease caused by infection with a monkey pox virus (monkeypox virus). The monkey poxvirus is a member of the genus orthopoxvirus (orthopox virus) of the family Poxviridae, among which there are various viruses that can cause human diseases, such as smallpox virus, monkey poxvirus, vaccinia virus, and vaccinia virus. Monkey poxvirus is more virulent but both infectious and pathogenic than smallpox virus, and patient symptoms are similar to those observed in smallpox patients in the past, but of lesser clinical severity. Clinical symptoms of monkey pox are similar to those of smallpox, but the disease condition is lighter than that of smallpox, the death rate is 1% -10%, and the monkey pox is mainly characterized in that skin appears as blisters or pustules similar to smallpox and has precursor symptoms such as fever, headache, general discomfort, superficial lymphadenectasis and the like. Early lymphadenectasis is a characteristic of monkey pox that is distinguished from smallpox. Today, global smallpox has been destroyed for more than thirty years, while monkey pox viruses remain in nature, threatening human health. The world health organization issues early warning of outbreaks of monkey pox epidemic in recent days, and in view of the fact that cases have been found in a plurality of countries that do not have the monkey pox viruses epidemic, it is possible that more cases will be found in these countries and other countries in the future, and the monkey pox viruses will be transmitted further.
The currently internationally commonly adopted method for determining the infection of the monkey pox virus is the combination of clinical symptoms plus epidemiological background published by the American CDC officinal network and laboratory detection. The method recommended for laboratory detection is the PCR method, which has a high sensitivity, however, this method is strictly dependent on the presence of infectious virus, and also on the correct collection and storage of clinical samples. These limit the accuracy of a single PCR diagnosis, so the combined use of two or more methods is necessary to provide an accurate laboratory test diagnosis. The IgG antibodies of orthopoxviruses were found to be detectable at the earliest 2d after eruption, so that the detection window period for serum IgG antibodies can be from days after eruption to decades after recovery from infection. The selection of several orthopoxviruses encoding several other orthopoxviruses that are absent, truncated or mutated is a significant issue for the development of specific serodiagnostic methods for monkey pox viruses, which share a number of conserved surface antigens, resulting in a broad cross-reaction in standard serological assays such as ELISA, radioimmunoassays, and fluoroimmunoassay.
Disclosure of Invention
In order to realize serodiagnosis of the monkey pox virus, the invention provides a recombinant antigen for detecting the antibody of the monkey pox virus and a nucleic acid sequence for encoding the recombinant antigen, wherein the recombinant antigen can be expressed in a soluble way, and has high sensitivity and good specificity; the invention also provides a preparation method of the recombinant antigen; the invention also provides application of the recombinant antigen in a gold-labeled chromatography detection platform.
In a first aspect the present invention provides a recombinant antigen A29 for use in the detection of monkey poxvirus, said recombinant antigen A29 having the amino acid sequence shown in SEQ ID NO. 1.
In a second aspect, the invention provides a recombinant B21R antigen for detecting monkey poxvirus, wherein the recombinant B21R antigen has an amino acid sequence shown as SEQ ID NO.2, and the amino acid sequence shown as SEQ ID NO.3 is an amino acid sequence added with TRX tag and HIS tag based on SEQ ID NO. 2.
In a third aspect, the invention provides a chimeric recombinant antigen A29-B21R for use in the detection of a monkey poxvirus, wherein the recombinant antigen is prepared by ligating an A29 fragment with a B21R fragment and has the amino acid sequence shown in SEQ ID NO. 4. Further, the recombinant antigen A29-B21R is reconstructed on pGEX-4T-1 plasmid for transformation expression, the N end of the recombinant antigen A29-B21R is provided with GST tag, and the tagged recombinant antigen A29-B21R has an amino acid sequence shown in SEQ ID NO. 5.
In a fourth aspect the present invention provides the nucleotide sequences SEQ ID NO.6-8 of the recombinant antigens as described above in the first to third aspects.
In a fifth aspect the present invention provides the primer sequences SEQ ID NO.9-12 required for amplifying the nucleotide sequence according to the third aspect.
In a sixth aspect, the present invention provides an E.coli BL21 (Rosetta) strain transformed with the expression vector pGEX-4T-1 for constructing the above recombinant antigen.
The seventh aspect of the present invention also provides a method for preparing the recombinant antigen, comprising:
(1) The nucleotide sequence of SEQ ID NO.6-7 is synthesized after codon optimization, and is respectively constructed on pET28a and pET32a vectors, and is named pET28a-A29 and pET32a-B21R;
(2) Amplifying the nucleotide sequence shown in SEQ ID NO.8 by nested PCR (polymerase chain reaction) with SEQ ID NO.6-7 as a template and SEQ ID NO.9-12 as a primer;
(3) Ligating the amplified nucleotide sequence of SEQ ID NO.8 to the vector pGEX-4T-1, forming the recombinant plasmid pGEX-4T-1-A29-B21R;
(4) Transforming the recombinant plasmids in the steps (1) and (3) into competent cells of escherichia coli, and carrying out recombinant antigen expression;
(5) Separating and purifying the recombinant antigen by means of affinity chromatography, etc.
In an eighth aspect, the present invention provides a monkey poxvirus positive quality control article, and a method for preparing the same: the purified recombinant proteins pET28a-A29 and pET32a-B21R are respectively used as immunogens to immunize rabbits, and the rabbit polyclonal antiserum obtained after immunization is used as a monkey pox virus positive quality control product after gradient dilution.
In a ninth aspect, the invention provides a monkey pox virus antibody test strip prepared from the recombinant antigen.
The monkey pox virus detection test strip comprises a coating antigen, a labeled antigen, a GST monoclonal antibody and a marker; the marked antigen is a monkey pox recombinant antigen A29-B21R with GST tag added at the N end shown in SEQ ID NO. 5;
The coating antigen is a mixture of at least two monkey pox recombinant antigens in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4;
the label is indirectly bound to the labeled antigen by a GST monoclonal antibody.
The beneficial effects are that: the recombinant antigen provided by the invention can be expressed in a soluble way, and has high sensitivity and good specificity; provides a basis for serodiagnosis of monkey poxvirus.
Drawings
FIG. 1 is a flow chart of PCR of the A29-B21R gene sequence of the present invention;
FIG. 2 shows the results of polyacrylamide gel electrophoresis of recombinant antigens A29, B21R, A29-B21R of the invention;
FIG. 3 shows the results of ELISA titers of the rabbit polyclonal antibody of the present invention;
FIG. 4 shows the results of the quality control product titer detection of the monkey poxviruses prepared in the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1: recombinant plasmid construction
(1) Recombinant plasmid construction
Monkey Poxvirus (MPV) was synthesized by codon optimization into A29 (1-110 aa), B21R (1781-1810 aa-linker (G3S) 3 -1841-1870 aa) (by the company of the Rhagophthalmus ohba Co., ltd.) with reference to GenBank database NC_003310.1 sequence, the nucleotide sequences were SEQ ID NO.6, SEQ ID NO.7, respectively, and they were constructed on pET28a, pET32a vectors by the synthesis company, respectively, to prepare recombinant plasmids pET28a-A29, pET32a-B21R, the amino acid sequences obtained after translation of the two recombinant plasmids were SEQ ID NO.1, SEQ ID NO.3, respectively.
And (3) utilizing primer5 primer design software, combining pGEX-4T-1 vector enzyme cutting site patterns according to primer design and enzyme cutting site design principles, respectively adding enzyme cutting sites and protecting bases in front of upstream and downstream primers to design upstream and downstream primers, simultaneously designing complementary primers for connection, and connecting the A29 fragment and the B21R fragment in a nested PCR mode. The primers were designed as follows:
P1：5’-CGGGATCCCCGGCTACCGAATTTTTCT-3’
P2：5’-CACCGCCTTCGCCGCCGCTTCCGCACGCTGTTTCAGGGTTTCTTCGT-3’
P3：5’-AAGCGGCGGCGAAGGCGGTGGCGGAAGCGCTGAATGAT-3’
P4：5’-CCCTCGAGTTAAACTGTTTCCAGGCTATCATGATGT-3’
Primer P1 is shown in a sequence table SEQ ID NO.9, primer P2 is shown in a sequence table SEQ ID NO.10, primer P3 is shown in a sequence table SEQ ID NO.11, primer P4 is shown in a sequence table SEQ ID NO.12, and an amplification sequence is shown in a sequence table SEQ ID NO. 8.
The reaction system was added as shown in Table 1, and amplification was performed by the amplification procedure shown in Table 2.
TABLE 1 PCR reaction system
TABLE 2 amplification procedure
Amplifying the fragment A29 (15-50 aa) by taking the synthetic gene A29 as a template, P1 as an upstream primer and P2 as a downstream primer; the method comprises the steps of taking a synthetic gene B21R as a template, taking P3 as an upstream primer and P4 as a downstream primer to amplify a second section of target gene sequence B21R (B21R (1781-1810 aa-linker (G3S) 3 -1841-1870 aa)), recovering amplified products, mixing the amplified products according to a molar ratio of 1:1, taking the amplified products as the template, taking P1 as the upstream primer and P4 as the downstream primer to amplify A29-B21R fragments, specifically referring to figure 1, recovering the amplified products of the A29-B21R, carrying out double digestion by BamHI and XhoI (various enzymes for molecular biology are purchased from Dabao bioengineering Co., ltd.), recovering digested products, connecting the digested products to a carrier pGEX-4T-1 after digestion of BamHI and XhoI, carrying out DH5 alpha conversion, PCR identification of recombinant plasmids, obtaining positive transformants, carrying out sequencing, and comparing sequencing results, wherein the coincidence rate is 100%.
Extracting the recombinant plasmid, and transforming an escherichia coli expression strain Rosetta to obtain A29 and B21R, A-B21R recombinant antigen expression strains: R/pET28a-A29, R/pET32a-B21R, R/pGEX-4T-1-A29-B21R.
Example 2: recombinant antigen expression purification
Induction of expression:
Inoculating 10 μl of recombinant plasmid expression strain into 5ml LB culture medium containing antibiotics, shaking culturing at 37deg.C and 200rpm for about 16 hours, adding 200 μl of strain solution into 60ml LB culture medium containing AMP with the same concentration, shaking culturing at 37deg.C and 200rpm for overnight, inoculating all strain solution into 1L LB culture medium containing AMP with the same concentration, shaking culturing at 28deg.C and 150rpm for 2-2.5 hours, measuring OD 600 between 0.5-0.8, adding inducer IPTG (purchased from AMRESCO) with final concentration of 0.5mM, continuing shaking culturing at 28deg.C and 150rpm for 4.5 hours, centrifuging at 6000rpm for 5 minutes, and collecting strain precipitate; the bacterial pellet was resuspended in 50ml 20mM PBS (pH 7.4), centrifuged at 6000rpm for 15min and the pellet was collected.
Protein purification: bacterial pellet was resuspended in 20mM PBS (pH 7.4) and subjected to low temperature pressure disruption, high pressure homogenizer parameters set: pressure: 850Bar, temperature 4 ℃. Three cycles were performed, and after the pressure disruption was completed, the disrupted product was centrifuged at 7500rpm at low temperature for 30min, and the supernatant was collected. SDS-PAGE identifies recombinant protein expression. The recombinant protein N-terminal carried 6×HIS tag is purified by nickel ion chelation, the recombinant protein N-terminal carried GST tag is purified by GST affinity chromatography column, and the collected primary pure protein is purified by Q column.
After purification, the recombinant protein is subjected to SDS-PAGE detection, and the recombinant protein gel electrophoresis diagram is shown in the accompanying figure 2, wherein the size of the A29 protein is about 13.6kDa, the size of the B21R protein is about 26.8kDa, and the size of the A29-B21R protein is about 39.0kDa. The purity of the recombinant protein after analysis and purification from the results of FIG. 2 is more than or equal to 95%.
Example 3: preparation of monkey pox virus positive quality control
(1) Selecting two healthy New Zealand white rabbits with a weight of about 2kg, and firstly taking blood as a negative control;
(2) Antigen A29 and B21R were diluted to 1. Mu.g/. Mu.L (2-fold final concentration) with physiological saline, respectively;
(3) Mixing the adjuvant fully, taking out the required dosage under the aseptic condition, and mixing the dosage with the diluted antigen in the step (2) rapidly according to the volume ratio of 1:1 to obtain the injection with the antigen concentration of 0.5 mug/mu L;
(4) Immunization was performed by subcutaneous injection at four locations, 200 μl each, on the back or thigh root.
(5) Immunization was boosted 1 time in the same manner on days 21 and 42, respectively.
(6) Micro blood is collected on the 52 th to 56 th days, ELISA measurement is carried out on A29 and B21R according to 1ug/ml coating, the antibody titer is in the range of 1:10000-1:1000000, the result is shown in figure 3, the detection result shows that the titer can reach 1:100000, and a large amount of blood collection is carried out.
(7) Mixing collected rabbit anti-A29 and B21R according to a ratio of 1:1, respectively diluting according to a ratio of 1:100,1:1000,1:10000,1:100000 and 1:1000000 to prepare a positive quality control product, and simultaneously using pre-immune rabbit serum as a negative quality control product. Meanwhile, the A29 and the B21R, A-B21R are respectively used as coating antigens for ELISA detection, the detection results are shown in figure 4, and the results show that the prepared positive quality control product has strong reactivity with the three recombinant antigens in the ratio of 1:100-1:100000 according to different dilution gradients.
Example 4: preparation of gold-labeled test strip for detecting monkey pox virus antibody by double-antigen sandwich method
(1) Colloidal gold mark
Adding 1mL of colloid Jin Fang into a glass cup, stirring, adding a proper amount of 0.2MK 2CO3, adjusting pH to 7.6, and stirring for 1min; adding 20 μg of anti-GST-tag monoclonal antibody (Shandong Shuoguo biological), stirring for 1min; 100 mu L of 1% PEG20000 is added and stirred for 1min; centrifuge at 5000g for 10min, discard supernatant, and resuspend the pellet with 100ul of colloidal Jin Xishi (20mM PB,150mM NaCl,0.1%PEG20000,0.1%TritonX-100,2% sucroses, 0.01% NaN 3); finally, 5 mu g A29+B21R was added to the 100. Mu.L colloidal gold-labeled GST monoclonal antibody complex, and the mixture was thoroughly mixed and stored at 4 ℃. The gold-labeled compound is diluted by colloid Jin Xishi liquid 10 times and then uniformly coated on glass fiber, and is dried at 37 ℃ to prepare the gold-labeled pad.
(2) Nitrocellulose membrane (NC membrane) coating
The prepared monkey pox virus recombinant antigen A29 and B21R are mixed according to the concentration ratio of 1:1, then diluted to the working concentration by a coating liquid (20 mM PBS5% sucrose pH 7.4) to be used as a detection line (T line) coating raw material, and a quality control line (C line) coating raw material (goat anti-mouse IgG purchased from Shandong Shuogong) is streaked on an NC film by using a streaking film machine, and then baked for 30min at 37 ℃ to complete the raw material immobilization.
(3) Assembly
The immobilized NC film, a sample pad, a gold Mark pad, a PVC bottom plate, water absorbing paper and Mark paste are assembled (the sample pad, the gold Mark pad, the NC film, the PVC bottom plate, the water absorbing paper and the Mark paste are all purchased from Kang Hua organisms in Shandong province), and a strip cutting machine is used for cutting into test strips with the thickness of 3-4 mm.
Before use, the test strip and sample are returned to room temperature, and the test strip should be used within 1 hour of being placed at room temperature.
And (3) putting the test strip on a table top, adding 70ul of serum sample to be tested, and observing the experimental result about 20 min. And only the color development detection result of the T line is negative, the color development of the T line and the C line shows that the result is positive, and neither the color development shows that the test strip is invalid, and the detection result is invalid.
Example 5: application of monkey pox virus double-antigen sandwich method colloidal gold test strip
The prepared monkey pox virus positive quality control product and 10 healthy volunteer serum without smallpox vaccine and 10 healthy volunteer serum with smallpox vaccine are detected by using the colloidal gold test strip prepared in the example 4, the detection results are shown in the table 3, the positive quality control can be detected, and the detection sensitivity can reach 1:100000; the results preliminarily demonstrate the specificity of A29, B21R as coating antigen and A29-B21R as labeled conjugate antigen for use as a raw material for colloidal gold applications.
Table 3: sample detection result of monkey pox virus antibody detection colloidal gold test strip
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (3)
1. A monkey pox recombinant antigen has an amino acid sequence shown in SEQ ID NO. 3.
2. A recombinant vector comprising the amino acid sequence of the monkey pox recombinant antigen according to claim 1 and capable of expressing the recombinant antigen.
3. A rabbit multi-antibody monkey pox property control product, which is characterized in that the rabbit multi-antibody monkey pox property control product is obtained by immunization by taking the recombinant antigen as an immunogen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210921306.5A CN115947797B (en) | 2022-08-02 | 2022-08-02 | Monkey poxvirus recombinant antigen and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN102839224A (en) * | 2012-06-21 | 2012-12-26 | 浙江国际旅行卫生保健中心 | Reagent and method for detecting monkeypox virus through isothermal amplification |
| CN113661246A (en) * | 2018-12-21 | 2021-11-16 | 渥太华医院研究所 | Modified orthopoxvirus vector |
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| CN102839224A (en) * | 2012-06-21 | 2012-12-26 | 浙江国际旅行卫生保健中心 | Reagent and method for detecting monkeypox virus through isothermal amplification |
| CN113661246A (en) * | 2018-12-21 | 2021-11-16 | 渥太华医院研究所 | Modified orthopoxvirus vector |
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