Sequence of a new bunyavirus isolate M segment and its encoded Gn protein

Abstract

The application discloses a sequence of a separated new bunyavirus M segment and a Gn protein coded by the sequence, a nucleotide sequence of the M segment and an amino acid sequence coded by the nucleotide sequence, and expression, purification and activity analysis of a fusion protein formed by fusing the Gn coded by the M segment with Fc to form a Gn-Fc protein. In addition, the application also relates to cross reaction of the fusion protein with polyclonal antibodies in different patient sera.

Description

Technical Field

[0001] This application relates to the fields of molecular biology and cell biology. Specifically, it relates to the M-segment nucleotide sequence of a novel Bunyavirus (SFTSV) YTSD isolated from the Jiaodong region and its glycoprotein Gn, including the acquisition of the M-segment nucleotide sequence of YTSD, the construction, expression, purification, and activity analysis of the expression vector of the YTSD glycoprotein Gn sequence and the Gn-Fc fusion protein. Background Technology

[0002] SFTS, short for Severe Fever with Thrombocytopenia Syndrome Bunyavirus, is a tick-borne infectious disease characterized by fever, thrombocytopenia, and leukopenia. SFTSV, also known as Severe Fever with Thrombocytopenia Syndrome Bunyavirus (SFTSV), is the pathogen of SFTS. Discovered in 2009 by Chinese researchers Li Dexin et al., it is a tick-borne virus. SFTSV is an enveloped virus with a genome consisting of a single-stranded negative-sense RNA divided into L, M, and S segments. The envelope protein Gn is a spike protein on the viral envelope, responsible for binding to receptors on cells and serving as a target for neutralizing antibodies.

[0003] The incidence of SFTS is mainly concentrated in Henan, Hubei, Shandong, Anhui, Jiangsu and Liaoning provinces in China. It has also been reported in South Korea, Japan and Vietnam. The onset time is mainly concentrated in April to October. According to statistics, 131 patients were infected with SFTSV in Jiaodong area of ​​Shandong from April to October 2021. The infection rate and mortality rate are on the rise.

[0004] Currently, there are no commercially available vaccines or specific drugs for SFTS. There are also no commercially available Gn antigens for SFTSV. For the prevention and control of viral infectious diseases, vaccines and antibody drugs are both feasible strategies. This application aims to provide a Gn antigen for detecting the effectiveness of vaccines and antibody drugs, as well as for serological diagnosis.

[0005] A search revealed Chinese patent document CN113061168 B, authorized on November 9, 2021, which discloses a truncated Gn protein of fever with thrombocytopenia syndrome virus and its application. This Gn protein was obtained by truncating 24 amino acids of a natural Gn protein from a public database and is used as an antiviral vaccine. Summary of the Invention

[0006] The purpose of this invention is to solve the problems mentioned in the background art based on the sequence of the M segment of the novel Bunyavirus isolated from the Jiaodong region and its encoded Gn protein.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a sequence of the M segment of an isolated strain of new Bunyavirus and its encoded Gn protein. The present invention is based on the isolation of an SFTSV strain YTSD from the Jiaodong region, the determination of the nucleotide sequence of its M segment, and the use of the amino acid sequence of the M segment glycoprotein Gn to obtain an active recombinant protein Gn-Fc.

[0008] In this invention, the M segment of the YTSD strain has a Genebank application number of OM162020. The M segment is composed of 6 fragments. These 6 fragments are named M1, M2, M3, M4, M5, and M6, respectively, and are obtained by PCR amplification, with primers corresponding to M1-S, M1-F; M2-S, M2-F; M3-S, M3-F; M4-S, M4-F; M5-S, M5-F; and M6-S, M6-F.

[0009] In this invention, the glycoprotein Gn is derived from the M segment sequence of the YTSD strain. The nucleotide sequence of the glycoprotein Gn is shown in SEQ No. 5, and its corresponding amino acid sequence is shown in SEQ No. 6. The Gn protein includes a core sequence in which 19 amino acids are deleted from the N-terminus and 621 amino acids are deleted from the C-terminus of the M segment amino acid sequence.

[0010] The glycoprotein fusion protein Gn-Fc is a recombinant protein that is stably secreted and expressed by eukaryotic cells. It is based on the core sequence of glycoprotein Gn and expresses the Fc segment of human IgG1 antibody. The nucleotide sequence and protein sequence are respectively fused with the nucleotide sequence and amino acid sequence of Fc.

[0011] The vector used was the pHL-Fc expression vector, which was a plasmid containing amplification and expression elements and an ampicillin resistance marker. The target gene Gn-Fc was amplified in prokaryotic cells and expressed in eukaryotic 293T cells. Liposome transfection was then used to induce secretion of the gene in the 293T cell supernatant.

[0012] The eukaryotic cells used for expression are 293T cells or 293F cells, so that the target protein can be efficiently expressed in these eukaryotic cells.

[0013] Purification was performed using tags on the vector, either His or Fc tags, nickel column purification, or affinity purification.

[0014] Activity analysis was performed using an enzyme-linked immunosorbent assay (ELISA). Gn-Fc was co-incubated with polyclonal antibodies from the serum of different patients to detect its binding activity and cross-reactivity with different antibodies.

[0015] This invention provides an optimized Gn antigen, which truncates the Gn sequence to 433 amino acids, preserving both the conserved sequence of the Gn protein and ensuring its secretion and extracellular expression. It can be used as a detection method for the binding activity of recombinant vaccines and antibody drugs, as well as for the serological diagnosis of SFTSV patients, and is a commercially available Gn antigen.

[0016] In practice, the Gn sequence was optimized to facilitate expression in eukaryotic cells (293F or 293T cells) and secretion into the cell supernatant. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0018] Figure 1 The results are PCR results for the six fragments in segment M.

[0019] Figure 2 The result of constructing the glycoprotein Gn vector;

[0020] Figure 3 The results are from a Western blot analysis of the glycoprotein Gn.

[0021] Figure 4 The results show the cross-reactivity of the glycoprotein Gn-Fc fusion protein with polyclonal antibodies in different patients.

[0022] Figure 5 The results show the reaction of the glycoprotein Gn-Fc fusion protein with serially diluted serum.

[0023] Figure 6 The binding activity of the glycoprotein Gn-Fc fusion protein with serum from different patients was measured. Detailed Implementation

[0024] The following describes the specific implementation process of the present invention. The methods and techniques used are known to those skilled in the art, and the reagents or instruments used are all conventional products that can be obtained through commercial purchase.

[0025] Example 1. The strains and plasmids involved in this invention

[0026] 1. The YTSD strain was isolated from the acute phase serum of SFTSV inpatients at Qishan Hospital in Yantai City and stored in Trizol for later use.

[0027] 2.293T cells are cell lines preserved in our laboratory.

[0028] 3. The pHL-Fc vector was purchased from addgene.

[0029] 4. The serum and polyclonal antibodies were obtained from the acute or convalescent serum of SFTSV patients at Qishan Hospital in Yantai City.

[0030] Example 2. Obtaining the M segment of the SFTSV strain

[0031] RNA was extracted from serum collected from patients in the acute phase using the Trizol method and reverse transcribed into cDNA. Using cDNA as a template, PCR was performed with high-fidelity polymerase (NEB) using six primer pairs: M1-S, M1-F; M2-S, M2-F; M3-S, M3-F; M4-S, M4-F; M5-S, M5-F; and M6-S, M6-F. Six DNA bands (M1, M2, M3, M4, M5, and M6) were obtained, and after sequencing, they were assembled into the M segment. The nucleotide sequence of the M segment is shown in SEQ NO 1, and the corresponding amino acid sequence is shown in SEQ NO 2.

[0032] Based on the codon bias of human cells, the nucleotide sequences of the M segment encoding glycoproteins Gn and Gc were optimized and synthesized. The optimized nucleotide sequences of the M segment encoding glycoproteins Gn and Gc (SEQ NO 3) and the corresponding amino acid sequences (SEQ NO 4) were obtained.

[0033] Example 3. Construction of Gn-Fc recombinant plasmid

[0034] The amino acid sequence used in this embodiment is the Gn nucleotide sequence (SEQ NO5) optimized according to human cell preferences, and the corresponding Gn amino acid sequence (SEQ NO 6). A Gn-Fc expression vector (e.g., pHL-Fc vector and the signal peptide carried by the vector itself) was constructed. Figure 2 The Gn sequence in Gn-Fc is the natural Gn with 19 amino acids removed from the N-terminus and 54 amino acids shortened from the C-terminus, and the Fc segment of the IgG1 antibody (240 amino acids) added. The nucleotide sequence of Gn-Fc is (SEQ NO 7), and the amino acid sequence of Gn-Fc is (SEQ NO 8).

[0035] Example 4. Detection of Gn-Fc protein expression level

[0036] 1. Cell preparation and transfection: Recombinant plasmids were transfected into 293T cells. Taking a 6-well cell culture plate as an example, PEI transfection reagent was used. The ratio of plasmid to PEI was added according to the instructions. GFP was transfected as a negative control. Opti-MEM serum-free medium was used for transfection. Cell supernatant was collected 36-48 hours after transfection.

[0037] 2. Gn-Fc purification: Gn-Fc was purified using protein A affinity purification and Ni column purification, followed by ultrafiltration concentration, with a final yield of 1 μg / mL. Western blot analysis was performed to detect Gn-Fc expression; the molecular weight was approximately 90 kDa (e.g., ...). Figure 3 ).

[0038] Example 4. Assay of the reactivity of Gn-Fc with polyclonal antibodies

[0039] Convalescent serum was collected from SFTSV patients. To avoid complement interference, polyclonal antibodies from different patients' serum were purified via protein A affinity assay and then bound to Gn-Fc by ELISA to detect their binding activity. It was found that different polyclonal antibodies exhibited different reactivity with Gn-Fc (e.g., ...). Figure 4 ).

[0040] Example 5. Determination of cross-reactivity between Gn-Fc and patient serum

[0041] Convalescent serum was collected from SFTSV patients. Complement was inactivated at 56°C for 30 minutes. The serum was then serially diluted at 1:10, 1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000, and 1:4000. Standard serum dilutions were determined by ELISA. Figure 5 Using a serum dilution of 1:500 as a baseline, the cross-reactivity of Gn-Fc with serum from different patients was determined, with a positive rate of 93% (e.g., ...). Figure 6 ).

[0042] Nucleotide or amino acid sequence listing:

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[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features, and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant protein Gn-Fc of an isolated strain of neo Bunyavirus, wherein the nucleotide sequence of the recombinant protein Gn-Fc is SEQ NO 7 and the amino acid sequence is SEQ NO 8.

2. The use of the recombinant protein Gn-Fc according to claim 1 in the preparation of a diagnostic reagent for SFTSV patients' serum.

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