Oropervirus fusion protein as well as preparation method and application thereof

Abstract

The invention discloses an Olvapu virus fusion protein, which is formed by connecting and fusing an Olvapu virus Gn protein, an Olvapu virus Gc protein and an Olvapu virus N protein by using GSGSG, and the fusion protein comprises an amino acid sequence as shown in SEQ ID NO.1. The invention further discloses a preparation method of the Olvapu virus fusion protein. The invention also discloses a nucleotide sequence corresponding to the fusion protein, construction of the gene expression vector, a host cell, and a preparation method and application of the fusion protein. Colloidal gold detection test paper for detecting the Oroptovirus antibody in a human body is researched and developed by utilizing the fusion protein, and the test paper has the advantages of high sensitivity, good specificity and high detection accuracy, does not need expensive instruments and equipment at an application end, and has huge clinical significance and wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of fusion proteins, and in particular relates to an Oropouche virus fusion protein and a preparation method and application thereof. Background Art

[0002] Oropouche fever is a viral disease caused by the Oropouche virus (OROV), which is mainly transmitted by the bites of blood-sucking insects such as mosquitoes and midges. The viral genome consists of three segments of single-stranded negative-sense RNA. The virus particles are spherical with a diameter of about 80-110nm. It belongs to the Orthobunyavirus genus of the Peribunyaviridae family. The virus was first isolated from the blood of a febrile forestry worker in 1955. The name of the virus was named after the patient's residence, the village of Vega de Oropouche. The symptoms of Oropouche virus infection in humans are similar to those of dengue fever, and clinical symptoms begin to appear 4-8 days (or 3-12 days) after being bitten and infected. The patient develops the disease suddenly, usually accompanied by fever, headache, joint stiffness, pain, chills, and sometimes persistent nausea and vomiting, which lasts for up to 5-7 days. Severe cases are rare, but may lead to aseptic meningitis. Most cases will recover within seven days, but some patients may take weeks to recover. Because the symptoms of Oropouche fever are similar to those of other arbovirus infections and the infected can recover on their own, it is often misdiagnosed or ignored in clinical practice, thus underestimating its prevalence, leading to sudden outbreaks and epidemics. Currently, no specific antiviral drugs or vaccines for this virus have been developed for clinical use.

[0003] Oropouche virus (OROV) is the causative agent of oral fever and is widely disseminated in Brazil and South America, causing sporadic outbreaks. Differential diagnosis is challenging because the initial clinical symptoms caused by OROV are similar to those of other arboviruses found in overlapping geographical areas. As with most neglected tropical diseases, reagents for diagnosis and study of OROV pathogenesis are lacking. Therefore, it is very necessary to develop a method that can detect antibodies produced in humans against Oropouche virus (OROV), determine whether the virus is infected, and assess the approximate period of infection with the virus and whether it is within the window period. Summary of the invention

[0004] In order to solve one of the above problems, the present invention provides an Oropouche virus fusion protein and a preparation method and application thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides an Oropouche virus fusion protein, which is formed by connecting and fusion of Oropouche virus Gn protein, Gc protein and N protein using GSGSG, and the fusion protein includes the amino acid sequence shown in SEQ ID NO.1.

[0006] The reason for selecting these three proteins in the present invention is that it is concluded from the analysis of the virus structure that Gn protein and Gc protein are on the surface of the virus structure, and it is easier to produce antibodies against the protein, while the N protein is more contained in the virus and is more likely to produce antibodies against the protein. Therefore, these three proteins are selected to fuse to form a new fusion protein as the raw material of the colloidal gold product, and then the more suitable amino acid sequences of these three proteins are selected through the antigen epitope prediction model and connected through flexible peptides to form a fusion antigen.

[0007] After multiple designs and comparisons, the colloidal gold test strips prepared with the final purified protein as raw material were found to have better biological activity and higher sensitivity as the raw material for preparing the corresponding colloidal gold products by fusing Gn protein, Gc protein and N protein and connecting them with GSGSG.

[0008] A second aspect of the present invention provides a nucleic acid encoding the Oropouche virus fusion protein of claim 1.

[0009] In some embodiments of the present invention, the sequence of the nucleic acid is shown as SEQ ID NO.2.

[0010] Since different species use different frequencies of synonymous codons, this codon preference has an impact on the translation process. If an mRNA has many clustered rare codons, this will have a negative impact on the movement speed of the ribosome and greatly reduce the protein expression level. The present invention uses Escherichia coli as an expression system. In order to obtain higher expression efficiency and higher expression amount, codon optimization that is conducive to expression in Escherichia coli is performed when performing exogenous protein expression.

[0011] The third aspect of the present invention provides an expression vector comprising the nucleic acid described in the second aspect.

[0012] In some embodiments of the present invention, the expression vector is pET32a, and the expressed fusion protein has a histidine (His) tag.

[0013] The fourth aspect of the present invention provides a host cell comprising the expression vector described in the third aspect.

[0014] In some embodiments of the present invention, the host cell is a prokaryotic host cell. Preferably, the host cell is Escherichia coli and cultured in ampicillin medium. Expression using Escherichia coli has the advantages of short cycle, low cost and high expression amount.

[0015] The present invention also discloses a method for preparing the Oropouche virus fusion protein described in the first aspect, comprising the following steps: S1. Plasmid construction and prokaryotic expression Connecting the nucleic acid sequence of the Oropouche virus fusion protein described in the second aspect of the present invention to an expression vector plasmid, and transferring the connected plasmid into a host cell for inducing expression; S2, breaking bacteria, centrifugation The expressed cells are collected by centrifugation, resuspended with Binding Buffer, disrupted by ultrasound, and the supernatant is collected after centrifugation; in some specific implementation schemes, the cells are disrupted by ultrasound 80-120 times, preferably 100 times.

[0016] S3. Purification The supernatant was passed through a Ni column and then eluted with an elution buffer to obtain the Oropouche virus fusion protein.

[0017] In some embodiments of the present invention, the expression vector plasmid is pET32a, and the host cell is Escherichia coli. Escherichia coli is cultured at 37°C in LB medium containing 50 μg / mL ampicillin. When the OD600 of the Escherichia coli culture medium is between 0.5-0.7, IPTG with a final concentration of 1 mM is used to induce expression, and the induction conditions are: 25°C, 200 rpm, 4h; using this induction condition, the fusion protein can be expressed more slowly, and there is sufficient time for the formation of a spatial conformation, which is very important for the fusion protein to function; in some preferred embodiments, induction is performed when the OD600 of the Escherichia coli culture medium is between 0.6, and the induction conditions are: 25°C, 200 rpm, 4h.

[0018] The method for disrupting bacteria of the present invention can avoid excessive disruption, which may cause loss of fusion protein.

[0019] Further, there are many ways to purify the fusion protein, such as ion exchange chromatography, gel filtration chromatography and affinity chromatography. In some embodiments of the present invention, the affinity chromatography method is selected. Since the His tag is added to the fusion protein, a one-step purification can achieve a higher purity. Preferably, the formula of the elution buffer is: 50mM Tris, 0.2M Nacl, 0.5M Imidazole, pH8.0.

[0020] The fifth aspect of the present invention provides the use of the Oropouche fever virus fusion protein described in the first aspect in the preparation of a product for detecting Oropouche fever virus antibodies.

[0021] In some embodiments of the present invention, the product includes a test strip, a test card, or a test kit.

[0022] The sixth aspect of the present invention provides a kit for Oropouche virus antibodies, comprising the Oropouche virus fusion protein described in the first aspect.

[0023] In some embodiments of the present invention, the Oropouche virus antibodies are detected using an indirect gold labeling method.

[0024] In some specific embodiments of the present invention, the detection kit includes an Oropouche virus antibody test strip or an Oropouche virus antibody test card.

[0025] Furthermore, the kit also includes mouse IgG, mouse anti-human IgG and goat anti-mouse IgG.

[0026] In some specific embodiments of the present invention, the kit includes an indirect gold labeling test strip, and the reagent method of the test strip is as follows: S1, prepare fusion protein colloidal gold complex and mouse IgG colloidal gold complex respectively; S2, diluting the fusion protein colloidal gold complex and the mouse IgG colloidal gold complex with a gold label diluent, adding 0.2 g / ml sucrose, and drying to prepare a gold label pad; S3, using mouse anti-human IgG diluted in the membrane spotting solution as the test line, and using goat anti-mouse IgG diluted to 1.5 mg / mL in the same diluent as the quality control line, streaked on the nitrocellulose membrane; S4, install the above gold label pad, coated raw materials on nitrocellulose membrane, absorbent paper, sample pad, etc. on the PVC backing plate.

[0027] The sample pad is partially overlapped and pressed on the gold label pad, the gold label pad is partially overlapped and pressed on the nitrocellulose membrane, and the absorbent paper is partially overlapped and pressed on the nitrocellulose membrane. The nitrocellulose membrane is divided into a test area and a quality control area. The test area is set with a test line (T line), and the quality control area is set with a quality control line (C line). The test line is close to the gold label pad, and the quality control line is close to the absorbent paper, so that the test strip is prepared.

[0028] The present invention also provides a method for preparing an indirect method test strip. The assembled test strip is cut into 3 mm strips by a strip cutter and then loaded into a specially prepared plastic card to form a mature test reagent card.

[0029] In some specific embodiments of the present invention, when the test strip is used, the biological sample is dripped onto the sample pad and the test result is determined after being placed at room temperature for 10 minutes. The determination criteria are as follows: ① Two bands appear, one in the quality control area and the other in the T test area, which is positive, indicating that there are antibodies to the virus in the body, and the person may be infected with the virus or have been infected in the past; ② A band appears, located in the quality control area, which is negative; ③ If no strip appears on the quality control line, it indicates that the test strip is damaged. Regardless of whether a strip appears on the detection line, a new test strip should be replaced and retested.

[0030] In some embodiments of the present invention, a positive result of an Oropouche virus antibody test indicates that the biological sample contains Oropouche virus antibodies, which means that the biological sample is infected with Oropouche virus or has been infected with Oropouche virus in the past.

[0031] In some embodiments of the present invention, the biological sample is serum or plasma, or any other body fluid that may contain antibodies.

[0032] Beneficial Effects of the Invention Compared with the prior art, the present invention has the following beneficial effects: the method of the present invention is convenient and quick, and can identify whether the body produces Oropouche virus antibodies or whether it is a past infection in a relatively short period of time. IgG antibodies will be produced after the body is infected with the virus. By detecting these specific antibodies in the serum, it can be determined whether the subject is infected with the virus. And no expensive instruments and equipment are required; the sensitivity is high and the specificity is good. The present invention has a high reference value for the diagnosis and clinical medication of Oropouche virus antibody infection and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The gel electrophoresis result of the purification of the fusion protein in Experiment 2 of Example 1 of the present invention is shown; wherein, Lane 1 Protein Marker 2: loading after cell disruption; Lane 3: flow-through after cell disruption; Lane 4: washing of impurities with 0.05M Imidazole; Lane 5: elution with 0.5M Imidazole; Figure 2 The gel electrophoresis result of the purification of the fusion protein in Experiment 3 in Example 1 of the present invention is shown; wherein, Lane 1 Protein Marker 2: 0.5M Imidazole elution; Figure 3 The gel electrophoresis result of the purification of the fusion protein in Experiment 4 of Example 1 of the present invention is shown; wherein, Lane 1 Protein 2: loading after cell disruption; Lane 3: flow-through; Lane 4: elution with 0.5M Imidazole; Figure 4The sensitivity results of the fusion protein in Experiments 2 to 4 in Example 1 of the present invention are shown; Figure 5 The color chart for reference of the test result readings in Example 1 of the present invention is shown; Figure 6 The structure diagram of the detection reagent strip of the present invention is shown; Figure 7 A standard diagram for interpreting test strip results of the present invention is shown. DETAILED DESCRIPTION

[0034] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0036] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0037] Example 1 Construction and screening of the Oropouche virus fusion protein gene expression vector The three proteins, N protein, Gn protein and Gc protein, were selected for fusion to obtain the Oropouche virus fusion protein, and the proteins were connected and fused with GSGSG.

[0038] During the protein fusion process, we designed the following experiments: Experiment 1: Choose to fuse the two proteins Gn and Gc, and connect them with GSGSG; Experiment 2: Gn protein and N protein were selected for fusion, and the proteins were connected with GSGSG; Experiment 3: Choose to fuse the two proteins Gc and N, and connect them with GSGSG. Experiment 4: Choose to fuse Gc protein, Gn protein and N protein, and connect the proteins with GSGSG.

[0039] The above four designed fusion proteins were connected to the pET-32a vector or the pET-30a vector. Among them, the fusion protein in Experiment 1 had no obvious expression band after being connected to the pET-30a vector and the pET-32a vector.

[0040] The three fusion proteins in Experiment 2, Experiment 3, and Experiment 4 showed obvious target band expression through SDS-PAGE electrophoresis, such as Figures 1 to 3 shown.

[0041] We initially used these three fusion proteins for the preparation of colloidal gold products. The results of colloidal gold sensitivity verification are shown in Table 1 and Figure 4 shown.

[0042] Table 1 Colloidal gold sensitivity verification results

[0043] The results refer to Figure 5 The color card and its readings are used to interpret the results. G is used to represent the signal in the table. The larger the number, the stronger the signal.

[0044] The results showed that the sensitivity of the fusion protein in experiment 4 was significantly better than that of the other two fusion proteins.

[0045] Finally, it was determined that the final fusion protein of the present invention is a protein obtained by fusion of three proteins, namely, Oropouche virus Gc protein, Gn protein and N protein, which are connected by GSGSG.

[0046] The amino acid sequence of the Oropouche virus fusion protein is as follows (SEQ ID NO. 1): RYGQVLNAGVVRVFFLNGSGSGFRATVVVPLAEVKGGSGSGRYWTILIYSIIGVVIIVILVYILLPIGRLLGSGSGHIIPKMYTCRAACDI The nucleic acid sequence of the gene encoding the Oropouche virus fusion protein is as follows (SEQ ID NO. 2): CGCTATGGCCAGGTGCTGAACGCGGGCGTGGTGCGCGTGTTTTTTCTGAACGGCAGCGGCAGCGGCTTTCGCGCGACCGTGGTGGTGCCGCTGGCGGAAGTGAAAGGCGGCAGCGGCAGCGGCCGCTATTGGACCA TTCTGATTTATAGCATTATTGGGCTGGTGATTATTGTGATTCTGGTGTATATTCTGCTGCCGATTGGCCGCCTGCTGGGCAGCGGCAGCGGCCATATTATTCCGAAATGTATACCTGCCGCGCGGCGTGCGATATT The gene sequences of the above recombinant fusion proteins have been codon-optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The synthesized genes were inserted into the E. coli expression vector pET32a (+) through genetic engineering methods to form a recombinant expression vector.

[0047] A His tag is added to the fusion protein, which enables one-step purification to achieve a higher purity.

[0048] Example 2: Induced expression and purification of fusion protein The recombinant expression vector obtained in Example 1 was transformed into Escherichia coli, and the Escherichia coli was cultured at 37°C in LB medium containing 50 μg / mL ampicillin. When the OD600 of the Escherichia coli culture solution reached 0.6, IPTG with a final concentration of 1 mM was used to induce expression. The induction conditions were: 25°C, 200 rpm, and 4 h. Using this induction condition, the fusion protein can be expressed more slowly, and there is sufficient time for the formation of a spatial conformation, which plays a very important role in the function of the fusion protein.

[0049] The culture solution was centrifuged at 4°C and 7000rpm for 10min to collect the cells; the cells were broken with a buffer solution Binding Buffer; the cells were broken with ultrasound at 600w, ultrasound for 2s, intervals of 5s, for a total of 100 times; the supernatant was collected by centrifugation at 4°C and 12000rpm for 30min, and the fusion protein was in the supernatant. The breaking method of the present invention avoids the situation where the breaking is too intense and causes the loss of the fusion protein.

[0050] The fusion protein is further purified. There are many ways to purify the fusion protein, such as ion exchange chromatography, gel filtration chromatography and affinity chromatography. In this embodiment, the affinity chromatography method is selected. Since the His tag is added to the fusion protein, a one-step purification can achieve a higher purity: the supernatant containing the fusion protein is passed through a Ni column, and then eluted with an elution buffer to obtain the target protein. The formula of the elution buffer is: 50mM Tris, 0.2M Nacl, 0.5M Imidazole, pH8.0.

[0051] Example 3: Application of fusion protein in detecting Oropouche virus antibodies 1. Preparation of gold label test strips 1.1. Firing of colloidal gold Add 1000 mL of ultrapure water to a conical flask, heat to boiling on a magnetic heating stirrer, then add 4 mL of 10% chloroauric acid (sigma), then add 4.8 mL of 10% trisodium citrate solution, continue heating and boiling for 5 minutes, then cool to room temperature, filter the colloidal gold with a 0.22 μm filter, and place at 4°C for use.

[0052] 1.2. Labeling of Oropouche virus fusion protein Take 100mL of colloidal gold solution and put it into a beaker, stir and add 0.2MK 2 CO 3 Adjust the pH of the gold water to 9.5, add 1 mg of purified Oropouche virus fusion protein after stirring, stir at room temperature for 15 minutes, add 1 mL of 10% BSA solution, stir at room temperature for 15 minutes, centrifuge at 12000 rpm for 10 minutes, carefully aspirate and discard the supernatant, and dilute the precipitate to 1 mL with gold label diluent (Tris 2.5 g / L, casein 2.5 g / L, 0.03% Proclin 300). This is the labeled Oropouche virus fusion protein colloidal gold complex.

[0053] 1.3. Mouse IgG labeling Take 100mL of colloidal gold solution and put it into a beaker, stir and add 0.2MK 2 CO 3Adjust the pH of the gold water to 7.0, add 1 mg of mouse IgG (Hangzhou Longji Biotechnology Co., Ltd., product number: AS00901) after stirring, stir at room temperature for 15 minutes, add 1 mL of 10% BSA solution, stir at room temperature for 15 minutes, and centrifuge at 12000 rpm for 10 minutes. Carefully aspirate and discard the supernatant, and dilute the precipitate to 1 mL with gold label diluent (Tris 2.5 g / L, casein 2.5 g / L, 0.03% Proclin 300). This is the labeled mouse IgG colloidal gold complex.

[0054] The gold-labeled complex was diluted to a certain concentration with a gold-labeled diluent, and then 0.2 g / ml of sucrose was added. The mixture was dried at 37° C. for 8-12 hours to prepare a gold-labeled pad.

[0055] 1.4. Mouse anti-human IgG spotting Use the membrane diluent (2.9 g / L Na 2 HPO 4 .12H 2 O, 0.282 g / L NaH 2 PO 4 .2H 2 Dilution of mouse anti-human IgG (Hangzhou Longji Biotechnology Co., Ltd.) with 1% 4% PBS (pH 7.0, 8.775 g / L Nacl, 200 ul / L Proclin300) was used as the test line (Test-Line) of the colloidal gold test strip. Sheep anti-mouse IgG (Hangzhou Longji Biotechnology Co., Ltd.) was diluted to 1.5 mg / mL with the same dilution as the control line (Control-Line, C line) of the colloidal gold test strip. The above two diluted solutions were streaked onto nitrocellulose membrane and dried at 37°C overnight.

[0056] 1.5. Assembly of indirect test strips for Oropouche virus antibody detection Install the gold label pad, coated raw nitrocellulose membrane (NC membrane), absorbent paper, sample pad, etc. on the PVC back plate to assemble the Oropouche virus indirect test strip. The structure of the test strip is as follows Figure 6 As shown: the sample pad, gold label pad, NC membrane and absorbent paper are assembled on the PVC back plate respectively. The sample pad is partially overlapped and pressed on the gold label pad, the gold label pad is partially overlapped and pressed on the NC membrane, and the absorbent paper is partially overlapped and pressed on the NC membrane. The NC membrane is divided into a test area and a quality control area. The test area is set with a detection line (T line), and the quality control area is set with a quality control line (C line). The detection line is close to the gold label pad, and the quality control line is close to the absorbent paper.

[0057] 2. Preparation of test reagent card The assembled test strips are cut into 3mm wide strips using a strip cutter and then placed into specially made plastic cards to form test reagent cards.

[0058] 3. Oropouche virus antibody test Add 10 μL of the sample to be tested (human serum, plasma) to the sample injection point (S), then add 90 μL of sample diluent to the sample injection point (S), and let it stand at room temperature for 10 minutes before determining the result. The result determination criteria are as follows: Figure 7 As shown: (1) Two bands appear, one in the quality control area and the other in the T detection area, which is positive, indicating that there are antibodies to the virus in the body, which may indicate infection with the virus or a previous infection; (2) A band appears in the quality control area, indicating negative; (3) If no band appears on the quality control line, it indicates that the test strip is damaged. Regardless of whether a band appears on the test line, a new test strip should be replaced and retested.

[0059] 4. Results of indirect detection of Oropouche virus antibody test strips / cards A total of 20 sera from patients with Oropouche disease (sample numbers 1 to 20) and 50 sera from healthy subjects (sample numbers 21 to 70) were tested. The antibody test results are shown in Table 2. Some of the test results are shown in Figure 3 shown.

[0060] Table 2 Oropouche virus antibody test results

[0061] From the above results, we can see that the sensitivity of sample detection reached 95%, the specificity was 98%, and the overall compliance rate was 97.1%.

[0062] The above results show that the Oropouche virus fusion protein of the present invention has very high sensitivity and specificity in detecting human Oropouche virus antibodies, can be used as a raw material for making Oropouche virus antibody detection test strips, and can be widely used in clinical detection.

[0063] After reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in this application.

Claims

1. An Oropouche virus fusion protein, characterized in that: The protein is formed by connecting and fusion of the Oropouche virus Gn protein, Gc protein and N protein by using GSGSG. The fusion protein includes the amino acid sequence shown in SEQ ID NO.

1.

2. A nucleic acid, characterized in that: The nucleic acid encodes the Oropouche virus fusion protein of claim 1.

3. A nucleic acid according to claim 2, characterized in that: The sequence of the nucleic acid is shown in SEQ ID NO.

2.

4. An expression vector, characterized in that: Comprising the nucleic acid according to claim 2 or 3.

5. A host cell, characterized in that Containing the expression vector according to claim 4.

6. Use of the Oropouche virus fusion protein according to claim 1 in the preparation of a product for detecting Oropouche virus antibodies.

7. The use according to claim 6, characterized in that: The products include test strips, test cards, and test kits.

8. A kit for Oropouche virus antibodies, characterized in that: Comprising the Oropouche virus fusion protein of claim 1.

9. A method for preparing the Oropouche virus fusion protein of claim 1, comprising the following steps: S1. Plasmid construction and prokaryotic expression Connecting the nucleic acid sequence of the Oropouche virus fusion protein of claim 1 to an expression vector plasmid, and transferring the recombinant expression vector plasmid into a host cell for inducing expression; S2, breaking bacteria, centrifugation Collect the expressed cells by centrifugation, resuspend them with Binding Buffer, disrupt the cells by ultrasonication 80-120 times, and collect the supernatant after centrifugation; S3. Purification The supernatant is passed through an affinity chromatography column, and then eluted with an elution buffer to obtain the Oropouche virus fusion protein, wherein the elution buffer comprises: 50mM Tris, 0.2M Nacl, 0.5M Imidazole, pH8.0.

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