A rubella virus specific fusion protein antigen, its preparation method and detection kit

Abstract

The application discloses a rubella virus specific fusion protein antigen, a preparation method and a detection kit, and the rubella virus specific fusion protein antigen comprises dominant antigens of rubella virus E1 protein and C protein, two dominant linear antigens are E1 225‑340 and C 30‑210 , and the amino acid sequence is shown in Seq ID NO. 1. The rubella virus specific fusion protein antigen has strong antigenicity and high detection accuracy, can be used for accurate and rapid identification of rubella virus, saves the conventional culture step of the rubella virus, and improves the safety of experimental operators. The rubella virus specific fusion protein antigen can be applied to a protein chip, and the detection result can be monitored by a total internal reflection ellipsometry imager. Compared with a conventional detection method, the application has the advantages of rapidness, accuracy, qualitative detection, no need of large-scale equipment and simple operation, and provides a new identification method of the rubella virus.

Description

Technical Field

[0001] This invention relates to the field of rubella virus detection technology, and in particular to a rubella virus-specific fusion protein antigen, its preparation method, and a detection kit. Background Technology

[0002] Rubella virus is the causative agent of rubella, the sole member of the genus Rubellavirus in the family Pleuroviridae. It has only one serotype and infects only humans. Rubella virus infection typically presents with mild symptoms, manifesting as an acute, mild, short-term, self-limiting rash. The greatest threat to public health from rubella virus is its teratogenic properties. Rubella virus infection in pregnant women, especially during the first trimester, can be transmitted to the fetus through the placenta, leading to various birth defects known as congenital rubella syndrome. Establishing rapid and accurate detection methods for rubella virus infection is crucial for its prevention.

[0003] Rubella virus envelope glycoproteins E1 and E2 mediate cell orientation, with E1 playing a crucial role in viral-endosome fusion. E1 is a type II viral membrane fusion protein. Following viral attachment to target cells and protein-mediated endocytosis, endosome acidification induces the dissociation of the E1 / E2 heterodimer, forming a trimer with the E1 subunit. This E1 homotrimer possesses fusion activity and, after fusion with the viral membrane in vivo, promotes the release of the viral nucleocapsid from the cytoplasm, thus facilitating viral release and transmission. However, due to the structural advantage of E1 over E2 in the extracellular space, it easily forms steric hindrance to E2, making E1 more readily recognized by antibodies. Therefore, E1 is the primary focus of research in diagnostic antigen studies.

[0004] The rubella E1 protein consists of a large extraviral domain residue (E... 1-452 ), a single transmembrane helix (E 453-468 ) and a short cytoplasmic tail (E 438-452 (It may also form a helix). E1 and E2 form non-covalent heterodimers on the surface of viral particles, making them the preferred targets for humoral immune responses. In particular, the membrane-anchored outer domain of the E1 protein has an immunogenic advantage, and large amounts of E1 antibodies are found in the serum of rubella patients. E1 has been developed in various expression and secretion systems, such as producing soluble E1 in eukaryotic hosts, producing glycosylated and soluble full-length E1 in baculovirus-infected moths, and expressing it in CHO cells and yeast expression systems.

[0005] Nucleocapsid C is associated with the E2 cytoplasmic domain of the cell membrane, enabling the host Golgi apparatus membrane to bud and form mature viral particles. Nucleocapsid C phosphorylation negatively regulates RNA-binding activity, delaying viral particle assembly during viral replication. Capsid protein C dimers and forms disulfide linkages within viral particles, regulating genomic RNA replication. It regulates subgenomic RNA synthesis through interaction with human C1QBP / SF2P32, inducing perinuclear aggregation of mitochondria and the formation of electron-dense intermitochondrial plaques—both hallmarks of rubella virus infection. Expression of C in transfected cells induces apoptosis, thus C plays a crucial role in cellular immunity. However, the preparation of rubella virus antigens using rubella nucleocapsid C protein is currently limited.

[0006] To date, there are few existing rubella virus protein expression systems, and they suffer from high production costs and low product stability. Meanwhile, E. coli systems, which have low production costs and are widely used for transcoding, are rarely used for expressing fusion proteins.

[0007] Therefore, existing technologies need further improvement. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a rubella virus-specific fusion protein antigen, its preparation method, and a detection kit. This rubella virus-specific fusion protein antigen increases the specificity of the fusion protein by preferentially selecting specific fragments of E1 and C. Furthermore, by replacing cysteine ​​residues in the specific amino acid fragments, its potential in the E. coli expression system and its stability after expression are increased, facilitating the large-scale acquisition of fusion proteins.

[0009] To solve the above problems, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a rubella virus-specific fusion protein antigen, comprising dominant antigens of rubella virus E1 protein and C protein, wherein the two dominant linear antigens are E1 protein and C protein. 225-340 and C 30-210 Its amino acid sequence is as shown in SeqID NO.1.

[0011] Among them, E1 225-340 The amino acids located at positions 225 to 340 of the N-terminus, C 30-210 The amino acids located at positions 30 to 210 of the N-terminus are directly linked. The applicant used biological software to comprehensively analyze the amino acid sequences of the E1 and C proteins, prioritizing the sequence of the E1 protein with high antigen specificity, and supplementing it with a linear antigenic site for C. The two were then directly linked end-to-end to reconstruct an antigenic site with high specificity and sensitivity, ensuring no missed detections.

[0012] Secondly, based on this, the present invention also provides another rubella virus-specific fusion protein antigen, which is effective against the above-mentioned rubella virus-specific fusion protein antigen at the E1 level. 225-340 Replace cysteine ​​residues 225, 235, and 242 with asparagine, and change C... 30-210 The cysteine ​​at position 153 is replaced with asparagine, and its amino acid sequence is as shown in Seq IDNO.2.

[0013] The applicant's research and analysis revealed that because E. coli cannot perform protein glycosylation and post-translational modifications, the glycosylation site of E1 cannot be modified. In addition, proteins expressed in the E. coli expression system are prone to mismatched disulfide bonds due to excessive cysteine ​​residues, which makes the expressed proteins unstable for preservation and application.

[0014] In response, the applicant has E1 225-340 The 225th, 235th, 242nd and C in the middle 30-210 The cysteine ​​residue at position 153 was replaced with asparagine, thereby avoiding the formation of mismatched disulfide bonds, increasing the yield of E. coli expression, and increasing the stability of recombinant protein, which is beneficial for future preservation and application.

[0015] Thirdly, the present invention also provides a recombinant expression vector, which is formed by recombining an expression vector with the encoding gene of the aforementioned rubella virus-specific fusion protein antigen.

[0016] Preferably, the expression vector is a pET series expression vector, such as pET-32a plasmid, pET-28a or pET-41a, etc.

[0017] Fourthly, the present invention also provides a recombinant engineered bacterium, which is formed by transforming the aforementioned recombinant expression vector into a host bacterium.

[0018] Preferably, the host bacterium is Escherichia coli, such as Escherichia coli BL21(DE3), Escherichia coli Rosseta, or other mutant strains of Escherichia coli.

[0019] Fifthly, the present invention also provides a method for preparing the aforementioned rubella virus-specific fusion protein antigen, comprising the following steps:

[0020] (1) The nucleotide sequence of the rubella virus-specific fusion protein antigen was cloned into the expression vector to obtain the recombinant expression vector;

[0021] (2) The recombinant expression vector obtained in step 1 is transferred into the host bacteria to obtain recombinant engineered bacteria;

[0022] (3) Use recombinant engineered bacteria to induce the expression of rubella virus-specific fusion protein antigen;

[0023] (4) Extract and purify the rubella virus-specific fusion protein antigen obtained in step (3).

[0024] Sixthly, the present invention also provides a test kit, the diagnostic antigen of which is the aforementioned rubella virus-specific fusion protein antigen. Using this test kit, the presence of rubella virus antibodies in a sample can be detected, thereby identifying whether the subject is infected with rubella virus.

[0025] In a seventh aspect, the present invention also provides a protein chip for identifying rubella virus, wherein the protein chip has the aforementioned rubella virus-specific fusion protein antigen immobilized in a dot matrix on the surface of a solid-phase carrier; the solid-phase carrier is an agarose-modified glass slide.

[0026] Currently, diagnostic methods for rubella virus infection mainly include virus isolation, enzyme-linked immunosorbent assay (ELISA), IgG antibody affinity test, radioimmunoassay, nucleic acid hybridization, reverse transcription PCR, and quantitative real-time PCR. There are currently no reports on using recombinant rubella virus proteins to prepare protein chips for rubella virus identification. The applicant's research has found that preparing a protein chip from the aforementioned rubella virus-specific fusion protein antigen has better application prospects. Experiments have shown that the identification results of rubella virus using protein chips prepared from this rubella virus-specific fusion protein antigen and natural antigen are consistent, and batch detection of samples can be achieved with high efficiency, high accuracy, and fast speed.

[0027] Eighthly, the present invention also provides a method for preparing a protein chip for identifying rubella virus, comprising the following steps:

[0028] S1. The aforementioned rubella virus-specific fusion protein antigen is spotted onto a solid-phase carrier in a certain arrangement and incubated at 37°C for 2 hours or at 4°C overnight.

[0029] S2. Blocking treatment: Take out the solid support after incubation, wash it 3 times with PBS solution for 5 minutes each time, add blocking solution, incubate at 37°C for 1 hour, take out the solid support, wash it several times and blow it dry.

[0030] Preferably, in step S1, the antigen spotting concentration is 0.05 mg / ml, and a positive sample is spotted at the corresponding spotting location as a positive control;

[0031] Preferably, the cleaning method is as follows: first, wash with PBS solution containing 0.1% Tween 1 to 3 times, then wash with PBS solution at least once, each time for 4 to 6 minutes, and finally blow dry.

[0032] Preferably, the blocking solution is a PBS solution containing 1% BSA.

[0033] Preferably, the solid support is an agarose-modified glass slide.

[0034] Preferably, the following steps are included before step S1:

[0035] S0. Preparation of agarose film-modified slides: Add 2 ml of 1% agarose solution to a clean slide and spread it evenly on the surface of the slide. After solidification, dry it at 37°C and store it in a dry environment at room temperature for later use.

[0036] In a ninth aspect, the present invention also provides a detection kit utilizing the aforementioned protein chip, which comprises the aforementioned protein chip.

[0037] In a tenth aspect, the present invention also provides a method for applying the above-mentioned protein chip, which includes the following steps:

[0038] (1) Antigen-antibody incubation: Add the serum to be tested to the prepared protein chip, rinse with deionized water and dry with nitrogen;

[0039] (2) Scanning detection: Remove the protein chip and place it on the sample plate of the total internal reflection ellipsometer, and scan the reading.

[0040] The present invention has the following beneficial effects:

[0041] 1. This invention provides a rubella virus-specific fusion protein, which can be used to accurately and rapidly identify rubella virus, eliminating the conventional rubella virus culture step and improving the safety of laboratory personnel.

[0042] 2. Advantages of this invention: It selects specific amino acid fragments, avoiding amino acid sites requiring glycosylation. Simultaneously, it replaces the easily aggregated amino acid cysteine ​​with hydrophilic asparagine, increasing the expression efficiency in *E. coli*, enhancing the stability of the fusion protein, and reducing false positives, thus facilitating its widespread application. It also reduces costs and shortens the production time of recombinant proteins.

[0043] 3. The rubella virus-specific fusion protein antigen of the present invention can be applied to protein chips, and the detection results can be monitored by a total internal reflection ellipsometer. Compared with conventional detection methods, it has the advantages of being fast, accurate, qualitative, requiring no large equipment, and simple to operate. It also provides a new identification method for rubella virus.

[0044] 4. The preparation method of this invention is simple and low in cost, and can realize large-scale detection of rubella virus samples. It has strong scalability and good application prospects. Attached Figure Description

[0045] Figure 1 For E1 225-340 and C 30-210 Amino acid sequence analysis results; Figure 1 A is E1 225-340 The results of amino acid sequence analysis; Figure 1 B is C 30-210 The results of amino acid sequence analysis;

[0046] Figure 2 SDS-PAGE results of rubella virus fusion protein purification;

[0047] Figure 3 To observe and read experimental images and grayscale values ​​of protein chips;

[0048] Figure 4 The results show the stability comparison of the fusion proteins. Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains.

[0050] In this invention, based on the E1 and C proteins of rubella virus, the amino acid sequences of the rubella virus E1 protein (NCBI sequence number NP_740664.1) and C protein (NCBI sequence number NP_740662.1) were analyzed using bioinformatics software. The selected dominant antigenic epitopes were tandemly arranged in a specific order to obtain the amino acid sequence shown in Seq ID NO.1. Further analysis of the E1 and C proteins... 225 E 235 E 242 C 153 Cysteine ​​was replaced with hydrophilic asparagine to construct and synthesize a multi-epitope fusion antigen, the amino acid sequence of which is shown in Seq ID NO.2. At the same time, the nucleic acid sequence of the protein was synthesized after codon optimization, an efficient prokaryotic expression vector was constructed, and the recombinant protein was purified to high purity.

[0051] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0052] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0053] Experimental materials, reagents and methods:

[0054] 1. Bacterial strains and plasmids:

[0055] The host bacterium BL21(DE3) and plasmid pET-32a were purchased from Takara Bio Inc. (Dalian).

[0056] 2. Molecular biology reagents:

[0057] Restriction endonucleases EcoRI and XhoI, and T4 ligase were products of TaKaRa; plasmid purification kits and DNA fragment agarose gel extraction kits were products of QIAGEN (Germany); IPTG was a product of Promega. Other reagents were either imported or domestically produced analytical grade reagents.

[0058] 3. Gene synthesis and DNA sequence sequencing: Shanghai Sangon Biotech Co., Ltd.

[0059] 4. Gene cloning methods: DNA restriction enzyme digestion, ligation, and electrophoresis; plasmid extraction and purification; SDS-PAGE analysis of proteins, etc., should be performed according to standard procedures. Other kits should be used according to the manufacturer's instructions.

[0060] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0061] Example 1: Screening of antigenic epitopes

[0062] 1. Screening method:

[0063] Referring to the amino acid sequences of E1 and C proteins published on NCBI (NCBI sequence numbers NP_740664.1 and NP_740662.1), the secondary and tertiary domains of the proteins were obtained using online analysis tools such as ExPasy and UniProt. The linear antigen sites were then analyzed using online software such as Bepipred Linear Epitope Prediction 2.0 to screen for dominant linear antigens of rubella virus.

[0064] 2. Screening Results and Analysis:

[0065] The rubella virus E1 protein-specific linear antigen E1 was obtained through the above screening method. 225-340 It is located at amino acids 225 to 340 at the N-terminus; the C protein-specific linear antigen C 30-210 It is located at amino acid positions 30 to 210 at the N-terminus.

[0066] For detailed analysis results of Bepipred Linear Epitope Prediction 2.0, please see [link to Bepipred Linear Epitope Prediction 2.0 analysis]. Figure 1 .

[0067] Example 2: Obtaining Rubella Virus-Specific Antigenic Epitope Genes

[0068] 1. Experimental Method:

[0069] Genes encoding dominant antigenic epitopes are tandemly arranged in a specific sequence, as follows: E1 225-340 and C 30-210 A fusion antigen gene containing two multi-epitopes was constructed; at the same time, E was further investigated. 225 E 235 E 242 C 153 The cysteine ​​residues at the site were replaced with hydrophilic asparagine to obtain the fusion protein antigen with the amino acid sequence shown in Seq ID NO.2.

[0070] The rare codons of *E. coli* in the above-mentioned fusion gene were analyzed using online rare codon software. The rare codons were then replaced with nucleotides encoding the same amino acid, using *E. coli*-preferred codons, ensuring that the final synthesized multi-epitope fusion gene did not contain any rare codons from *E. coli*. Finally, the rubella virus-specific fusion protein antigen nucleotide sequence was synthesized. EcoRI and XhoI restriction endonucleases were added to both ends of the nucleotides to obtain the coding sequence of the above-mentioned rubella virus-specific fusion protein antigen. This sequence was sent to Shanghai Sangon Biotech Co., Ltd. for the synthesis of the gene sequence.

[0071] Example 3: Construction of an expression vector for rubella virus-specific antigens

[0072] 1. Extract pET-32a plasmid, which is then double-digested with EcoRI and XhoI. The large fragments digested by double enzymes are recovered by gel electrophoresis. The gene sequence encoding the rubella virus-specific fusion protein antigen is then double-digested with EcoRI and XhoI. The digested fragments are recovered by gel electrophoresis and stored at -20°C for later use.

[0073] 2. The pET-32a plasmid and the double-digested target fusion protein antigen gene fragment were ligated at a ratio of 1:3-10 using T4 ligase overnight at 16°C. The resulting recombinant expression vector was named pET32a-E1C.

[0074] Example 4: Screening and Identification of Recombinant Expression Vectors

[0075] 1. Transform the recombinant expression vector pET32a-E1C into Escherichia coli BL21(DE3) and plate it onto LB agar plates containing ampicillin (60ug / ml) and incubate overnight at 37°C. The next day, randomly select 5 transformed colonies and 2 control colonies (particle pET-32a) and extract the plasmid from each.

[0076] 2. The extracted plasmid pET32a-E1C was double-digested with EcoRI and XhoI. The digestion products were then analyzed by electrophoresis. The electrophoresis results showed that the corresponding target fragment and vector fragment were visible in both cases, indicating that the recombinant expression vector was successfully constructed, and the positive expression bacterium pET32a-E1C was obtained. However, the target fragment was not visible after digestion of the control bacterium plasmid.

[0077] Example 5: High-efficiency expression of positive expression bacteria

[0078] 1. Inoculate the positive bacteria and control bacteria (identified as expressing positive expression) into test tubes containing 2 ml of LB medium (60 μg / ml ampicillin), and incubate at 37°C with shaking for 2 h. Add IPTG (final concentration 1 mmol / L) and continue induction at 30°C for 5 h. Collect the bacterial cells by centrifugation and perform SDS-PAGE analysis.

[0079] 2. From Figure 2 The electrophoresis results in the left image show that the target protein expressed by pET32a-E1C is approximately 30 kDa, consistent with the prediction results from the ExPASy ProtParam online software. No target protein band was observed in the control bacteria. These results demonstrate the successful acquisition of a positive recombinant engineered bacterium expressing the rubella virus-specific fusion protein antigen.

[0080] Example 6: Purification of Rubella Virus-Specific Fusion Protein Antigen

[0081] 1. Purification methods for target proteins

[0082] (1) The recombinant engineered bacteria that express the target protein efficiently were centrifuged at high speed (10000 rpm) and low temperature (4℃) for 7 min; the precipitated bacterial cells were resuspended in 0.01M PBS (pH 7.4) at 1 / 10 of the original bacterial volume, washed thoroughly once, and centrifuged at high speed (10000 rpm) and low temperature (4℃) for 15 min; the precipitate was resuspended in 0.02M PB (pH 7.4) at 1 / 10 of the original bacterial volume, sonicated on ice for 20 min, and centrifuged at 10000 rpm and 4℃ for 20 min. The pET32a-E1C expressed protein was in the form of supernatant. The supernatant was collected for purification.

[0083] (2) The supernatant was collected and filtered through a 0.22 μm filter membrane, then purified using a nickel column (Borglon Ni Bestarose FF). The protein sample was slowly passed through the nickel column at a flow rate of 0.5 mL / min, and the column was washed with 5 column volumes of sonication buffer (0.02 M PB buffer, pH 7.4). At the same flow rate, 10 column volumes of washing buffer (0.02 M PB + 0.02 M Imid pH 7.4) were used to wash away contaminating proteins. The target protein was then eluted with elution buffer (0.02 M PB + 0.25 M Imid pH 7.4) at a flow rate of 0.8 mL / min.

[0084] (3) After SDS-PAGE analysis, the eluent containing high-purity target protein was collected and dialyzed with 1000 times the volume of 20 mM Tris-HCl at pH 8.5 for 48 h. The eluent was then centrifuged at 10000 rpm / min at 4 °C for 20 min, and the supernatant was collected as the target protein (see results). Figure 2 (The image on the right).

[0085] Example 7: Immunogenicity Verification of Fusion Protein

[0086] In this embodiment, rabbits were immunized with the purified rubella virus-specific fusion protein antigen to verify whether it could induce a strong immune response, thereby indirectly verifying whether the purified fusion protein could produce rubella virus antibodies and bind to them. Rabbit antiserum was prepared and purified by immunizing New Zealand white rabbits with a combination of antigen and adjuvant, and the anti-rubella virus antibody titer was detected by indirect ELISA.

[0087] 1. Experimental Methods

[0088] (1) Five New Zealand rabbits were immunized with fusion protein as an immunogen, each receiving 200ug of antigen. The immunization method was multi-point immunization on the back.

[0089] (2) For the first immunization, the fusion protein was emulsified with complete Freund's adjuvant. Two weeks later, for the second immunization, the fusion protein was emulsified with incomplete Freund's adjuvant, and the immunization was performed at multiple sites on the back. Three days later, for the third immunization, 100 μg of the fusion protein was mixed with the same volume of 0.01 M PBS (pH 7.4), and the immunization was performed intraperitoneally. Three days later, the same immunization procedure was repeated.

[0090] (3) Three days after the last immunization, blood was collected from the marginal ear vein of the rabbit and the rabbit antiserum was obtained by centrifugation at 4000 rpm for 10 min.

[0091] (4) Take the fusion protein and dilute it with carbonate buffer (50mM, pH 9.6) to coat the ELISA plate with 100ul / well and 100ng / well of protein, and incubate at 4°C overnight. The next day, block with 20% fetal bovine serum and incubate at 37°C for 2 hours. Use the rabbit antiserum obtained in the previous step as the primary antibody by serial dilution and incubate at 37°C for 1 hour. Then add 1:5000 secondary antibody goat anti-rabbit and then add chromogenic solution for color development.

[0092] 2. Experimental Results

[0093] The results showed that the antibody titer was as high as 1:50000 or higher, indicating that the fusion protein had good immunogenicity.

[0094] Example 8: Antigenicity Comparison of Fusion Proteins

[0095] In this embodiment, the rubella virus-specific fusion protein antigen provided by the present invention is compared and analyzed with other similar fusion proteins. The specific information of the fusion proteins of each experimental group used for comparison is shown in Table 1 below.

[0096] 1. Group settings:

[0097] The amino acid sequence characteristics of the fusion proteins in each pair are as follows:

[0098] Experimental group 1: Using E1 225-340 and C 30-210 The only difference from Example 2 is that no mutations were performed at the four sites;

[0099] Experimental group 2: Using E1 225-340 The only difference from Example 2 is the absence of C. 30-210 .

[0100] Experimental group 3: Using C 30-210 The only difference from Example 2 is the absence of E1. 225-340 .

[0101] Experimental group 4: Using E1 225-295 and C 30-210 The only difference from Example 2 is that the sequence of the E1 fragment is different;

[0102] Experimental group 5: Using E1 225-340 and C 3-123 The only difference from Example 2 is that the sequence of fragment C is different.

[0103] 2. Experimental methods:

[0104] (1) The coding sequences of each recombinant protein in Example 2 and Experimental Groups 1-5 were respectively constructed with the pET32a vector to form recombinant expression vectors and transformed into Escherichia coli expression strains. After each expression strain was cultured and the target protein was induced to express, the expressed proteins were extracted and purified respectively.

[0105] (2) The purified recombinant proteins from each group were diluted with carbonate buffer (50mM, pH 9.6) and coated with ELISA plates at 100 μL / well and 100 ng / well, respectively, and incubated overnight at 4°C. After washing, 200 μL / well of PBS containing 10% fetal bovine serum was added and the plates were blocked in a 37°C water bath for 2 h. After washing, the plates were stored at 4°C for later use.

[0106] (3) 900 positive serum samples and 100 negative serum samples were tested by indirect enzyme-linked immunosorbent assay.

[0107] Table 1. Fusion antigen sequences and treatment methods for Example 2 and the experimental group.

[0108]

[0109] 3. Experimental Results and Analysis

[0110] (1) As can be seen from the results in Table 2, compared with each experimental group, the fusion protein antigen prepared in Example 2 had the lowest false positive rate, the highest positive detection rate, and the highest detection accuracy; among the experimental groups, Experimental Group 1 had a high positive detection rate, but a false positive rate of 5%. This indicates that for E 225 E 235 E 242 C 153 Mutation treatment that replaces cysteine ​​with asparagine can effectively avoid false positives in test results.

[0111] (2) The false negative rate of recombinant protein antigens in experimental groups 2-5 was relatively high, and the positive detection rate was significantly lower than that in Example 2 and experimental group 1.

[0112] A comprehensive analysis of the above results shows that the antigenic site in Example 2 is optimal; and even if the proteins selected for the specific sites are the same, the differences in the specific site sequences can lead to significant differences in the positive detection rate of the test results.

[0113] Table 2. Fusion antigen sequences and treatments for the examples and comparative examples.

[0114] Example 2 100 900 0 0 0 0 100％ Experimental group 1 100 900 0 0 5 5％ 100％ Experimental group 2 100 900 48 5.3％ 4 4％ 94.7％ Experimental group 3 100 900 848 94.2％ 1 1％ 5.8％ Experimental group 4 100 900 26 2.9％ 2 2％ 97.1％ Experimental group 5 100 900 11 1.2％ 4 4％ 98.8％

[0115] Example 9: Comparison of the stability of fusion proteins

[0116] In this embodiment, the fusion proteins of Example 2 and Experimental Groups 1-5 in Table 1 of Example 8 were coated with enzyme-linked plates for stability verification and comparison.

[0117] 1. Experimental Method:

[0118] The fusion protein antigens from Example 2 and Experimental Groups 1-6 were coated onto ELISA plates (coating method as in Example 8), and incubated at 37°C for 7 days. The coated ELISA plates were removed daily, and the changes in OD values ​​of the same positive control serum were measured under identical temperature and humidity conditions. A decrease in OD value indicates a reduction in the amount of fusion protein coated on the ELISA plate, further demonstrating decreased stability of the fusion protein. The results are shown below. Figure 4 .

[0119] 2. Experimental Results and Analysis

[0120] The experiment showed that the fusion antigens of Example 2 and the experimental group did not undergo significant changes after being placed at 37°C for 3 days. After 3 days, the recombinant antigen of Example 2 had the best liquid stability at 37°C, basically maintaining the detection value of the first day at 37°C, while the stability of the recombinant antigens of Experimental Groups 2 to 5 gradually decreased with the extension of time.

[0121] Example 10: Preparation and Detection Analysis of Protein Chips

[0122] This embodiment, based on the foregoing, provides a protein chip using the recombinant protein antigen prepared in Example 2. The preparation method, application, and detection analysis of this protein chip are described in detail below.

[0123] 1. Methods for preparing protein chips

[0124] (1) Preparation of agarose film modified glass slides

[0125] Add 2 ml of 1% agarose solution to a clean glass slide, spreading it evenly on the substrate surface. After solidification, dry the slide at 37°C and store it in a dry environment at room temperature for later use. Before use, place the slide in a 0.1 mol / L sodium periodate solution and oxidize it at room temperature for 30 min. Then wash the slide three times with distilled water for 5 min each time, and air dry it for later use.

[0126] (2) Fabrication of protein chips

[0127] The recombinant protein antigen was spotted onto an agarose slide in a specific arrangement and incubated at 37°C for 2 hours or overnight at 4°C. The antigen concentration was 0.05 mg / ml, and a positive sample was spotted at the corresponding location as a positive control. For blocking, the incubated slide was removed and washed three times with PBS solution for 5 minutes each time. Blocking buffer (PBS solution containing 1% BSA) was added, and the slide was incubated at 37°C for 1 hour. The slide was then washed three times with PBST (PBS containing 0.1% Tween) and once with PBS for 5 minutes each time, and then dried.

[0128] 2. Detection and Analysis

[0129] (1) Testing and identification of standard samples:

[0130] The standard sample was tested using a 2×2 point chip. Two points were selected in the first row as control points: control point 1 was a blank control and control point 2 was a negative control. Two points were selected in the second row as test points: test point 1 was clinically positive serum and test point 2 was clinically negative serum.

[0131] (2) Reading the protein chip:

[0132] Remove the chip, rinse it with deionized water and dry it with nitrogen. Then place the chip on the sample plate of the total internal reflection ellipsometer to identify the thickness change of the molecular film at each detection site on the chip. This change will be displayed on the computer monitor. The film thickness and the gray value on the monitor will change in a consistent manner. The greater the film thickness, the higher the gray value. Observe and read the gray value of the experimental image.

[0133] (3) Results and Analysis

[0134] from Figure 3 The results showed that the gray values ​​of the blank control, negative control, and clinical negative serum were similar (34-38), which was in line with expectations. However, the gray value of the clinical positive serum (reaching 87) was much higher than that of the negative serum, indicating that the test results were accurate.

[0135] Example 11 A new method for identifying rubella virus

[0136] This embodiment provides a novel method for identifying rubella virus in a sample using a recombinant protein antigen protein chip from Example 10, as detailed below:

[0137] 1. Test method:

[0138] ① The recombinant protein chip prepared in Example 10 was used to detect rubella virus in 100 random serum samples;

[0139] ② The 100 random serum samples were simultaneously detected using the conventional ELISA method (using the recombinant fusion protein prepared in Example 2) and used as control group 1 to analyze the difference in detection results between the new protein chip method and the conventional ELISA method;

[0140] ③ A protein chip prepared using the natural antigen (Meridian 6076 Rubella Grade IV) according to the method of Example 10 was used to detect 100 random serum samples, which were used as control group 2 to analyze the difference between the recombinant protein antigen of the present invention and the natural antigen used in the new method.

[0141] 2. Experimental Results:

[0142] The gray values ​​of 100 random serum samples were detected by protein chip prepared from recombinant proteins, as shown in Table 3. The results of ELISA detection of control group 1 are shown in Table 4. The results of natural antigen control group 2 are shown in Table 5.

[0143] (1) Comparing the results in Tables 3 and 4, it can be seen that among the 100 random serum samples tested, there were 96 positive serum samples and 4 negative serum samples. Among the 96 positive serum samples, 2 were weakly positive (samples No. 62 and No. 64). These samples showed obvious grayscale changes in the prepared protein chip detection. However, after the first ELISA test was negative, these two serum samples were retested and then judged as positive. This indicates that the protein chip prepared using the recombinant protein as a probe is more sensitive to the detection of rubella virus than the ELISA test.

[0144] (2) As can be seen from the comparison of Table 3 and Table 5, the detection results of the recombinant protein chip prepared by the present invention are consistent with the detection results of the protein chip prepared from natural protein, with no missed or false detections. The recombinant protein chip can be used as a substitute for the natural protein chip.

[0145] Table 3. Gray values ​​of 100 random serum samples detected by recombinant protein microarray.

[0146]

[0147] Note: The first, second, third, and fourth test points in the first row represent the blank control, negative control, negative control, and positive control, respectively.

[0148] Table 4. ELISA results of 100 random serum samples

[0149]

[0150] Note: The first, second, third, and fourth test points in the first row represent the blank control, negative control, negative control, and positive control, respectively.

[0151] Table 5. Gray values ​​of 100 random serum samples detected by natural antigen protein chip.

[0152]

[0153] Note: The first, second, third, and fourth test points in the first row represent the blank control, negative control, negative control, and positive control, respectively.

[0154] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A rubella virus-specific fusion protein antigen, characterized in that, This includes the dominant antigens of the linked rubella virus E1 and C proteins; the two dominant linear antigens are E1 and C2. 225-340 and C 30-210 The amino acid sequence of the fusion protein antigen is as shown in SEQ ID NO.

1.

2. A rubella virus-specific fusion protein antigen, characterized in that, The E1 of the fusion protein antigen according to claim 1 225-340 Replace cysteine ​​residues 225, 235, and 242 with asparagine, and change C... 30-210 The cysteine ​​at position 153 is replaced with asparagine, and the amino acid sequence of the fusion protein antigen is as shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, The recombinant expression vector is formed by recombining an expression vector with the encoding gene of the rubella virus-specific fusion protein antigen as described in claim 1.

4. The recombinant expression vector according to claim 3, characterized in that, The expression vector is a pET series expression vector.

5. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria are formed by transforming the host bacteria into the recombinant expression vector as described in claim 3.

6. A method for preparing the rubella virus-specific fusion protein antigen as described in claim 1 or 2, characterized in that, Includes the following steps: (1) The nucleotide sequence of the rubella virus-specific fusion protein antigen was cloned into the expression vector to obtain the recombinant expression vector; (2) The recombinant expression vector obtained in step 1 is transferred into the host bacteria to obtain recombinant engineered bacteria; (3) Use recombinant engineered bacteria to induce the expression of rubella virus-specific fusion protein antigen; (4) Extract and purify the rubella virus-specific fusion protein antigen obtained in step (3).

7. A test kit, characterized in that, Its diagnostic antigen is the rubella virus-specific fusion protein antigen as described in claim 1 or 2.

8. A protein chip for identifying rubella virus, characterized in that, The protein chip has rubella virus-specific fusion protein antigens as described in claim 1 or 2 fixed in a dot matrix on the surface of a solid support; the solid support is an agarose-modified glass slide.

9. The method for preparing a protein chip for identifying rubella virus as described in claim 8, characterized in that, Includes the following steps: S1. The rubella virus-specific fusion protein antigen described in claim 1 is spotted onto a solid-phase carrier in a certain arrangement and incubated at 37°C for 2 hours or at 4°C overnight. S2. Blocking treatment: Take out the solid support after incubation, wash it 3 times with PBS solution for 5 minutes each time, add blocking solution, incubate at 37°C for 1 hour, take out the solid support, wash it several times and blow it dry.

10. A protein chip detection kit, characterized in that, It includes the protein chip as described in claim 8.

Images