阅读说明：本技术 基于SARS-CoV-2核蛋白的NTD多肽、编码基因、重组载体、表达方法及应用 (NTD polypeptide based on SARS-CoV-2 nucleoprotein, coding gene, recombinant vector, expression method and application ) 是由 秦小波 高华 王庆伟 张国珍 高继海 于 2020-05-22 设计创作，主要内容包括：本发明属于生物医药技术领域,涉及基于SARS-CoV-2核蛋白的NTD多肽、编码基因、重组载体、表达方法及应用；制备方法为：1)人工合成NTD多肽蛋白序列对应的核苷酸序列为模板,并克隆到大肠杆菌表达载体上,构建得到重组质粒；2)将所得的重组质粒转化大肠杆菌感受态细胞,挑选阳性克隆,获得重组大肠杆菌表达菌株；3)以获得的重组大肠杆菌表达菌株为发酵菌株进行液体发酵,发酵过程中,在添加异丙基硫代半乳糖IPTG后,产生NTD多肽,并利用组氨酸标签通过亲和层析纯化得到NTD多肽；本发明制备的NTD多肽方便高效,发酵工艺简单、耗时短、成本低,目的蛋白表达量达菌体总蛋白的20％以上。(The invention belongs to the technical field of biological medicine, and relates to NTD polypeptide based on SARS-CoV-2 nucleoprotein, coding gene, recombinant vector, expression method and application; the preparation method comprises the following steps: 1) artificially synthesizing a nucleotide sequence corresponding to an NTD polypeptide protein sequence as a template, cloning the template onto an escherichia coli expression vector, and constructing to obtain a recombinant plasmid; 2) transforming the obtained recombinant plasmid into escherichia coli competent cells, and selecting positive clones to obtain a recombinant escherichia coli expression strain; 3) carrying out liquid fermentation by taking the obtained recombinant escherichia coli expression strain as a fermentation strain, in the fermentation process, after adding isopropyl thiogalactoside IPTG, generating NTD polypeptide, and purifying by affinity chromatography by using a histidine tag to obtain NTD polypeptide; the NTD polypeptide prepared by the invention is convenient and efficient, the fermentation process is simple, the time consumption is short, the cost is low, and the expression quantity of the target protein reaches more than 20 percent of the total protein of the thallus.)

1. An NTD polypeptide based on SARS-CoV-2 nucleoprotein, wherein the NTD polypeptide is a polypeptide with an amino acid sequence as shown in any of SEQ ID Nos. 1-4.

2. An NTD polypeptide coding gene based on SARS-CoV-2 nucleoprotein, characterized in that it encodes the NTD polypeptide of claim 1.

3. The gene encoding the NTD polypeptide of claim 2, wherein the gene encoding the NTD polypeptide is a gene encoding a nucleotide sequence as shown in any one of SEQ ID nos. 5 to 8.

4. A recombinant vector of NTD polypeptide based on SARS-CoV-2 nucleoprotein, comprising the NTD polypeptide-encoding gene of claim 2.

5. A host cell comprising the recombinant vector of claim 4.

6. The host cell of claim 5, wherein the host cell is E.coli, preferably E.coli BL21(DE3), BLR (DE3), BL21(DE3) pLysS, M15, TB1, DH5 a or Top 10.

7. A method for expressing an NTD polypeptide encoding gene, comprising the steps of:

1) designing corresponding primers, amplifying by using a PCR technology and an artificially synthesized coding gene of claim 2 as a template, and inserting the amplified gene fragments into an expression plasmid by an enzyme digestion connection method to construct a recombinant vector;

2) transforming the recombinant vector into escherichia coli to obtain recombinant genetic engineering bacteria;

3) inoculating the recombinant genetic engineering bacteria into a culture medium, and inducing the expression of the NTD polypeptide by adding lactose or isopropyl thiogalactose IPTG.

8. The expression method according to claim 7, wherein the Escherichia coli is Escherichia coli BL21(DE3), BLR (DE3), BL21(DE3) pLysS, M15, TB1, DH5 a or Top 10.

9. Use of the NTD polypeptide according to claim 1, the gene encoding the NTD polypeptide according to claim 2, or the recombinant vector according to claim 4 in the preparation of a product for detecting SARS-CoV-2.

10. Use of the NTD polypeptide according to claim 1, the NTD polypeptide coding gene according to claim 2 or the recombinant vector according to claim 4 for the preparation of a SARS-CoV-2 vaccine or for the preparation of a SARS-CoV-2 nucleic acid pharmaceutical packaging polypeptide.

Technical Field

The invention relates to the technical field of biological medicine, in particular to NTD polypeptide based on SARS-CoV-2 nucleoprotein, coding gene, recombinant vector, expression method and application.

Background

2019 novel Coronavirus (SARS-CoV-2) is a positive strand RNA virus with envelope, belonging to genus Coronavir (Coronavir); the pneumonia caused by the infection is named as novel coronavirus pneumonia (COVID-19) by the world health organization. SARS-CoV-2 belongs to a novel coronavirus of beta genus, and the particles are mostly polymorphic, and the diameter is about 60-140 nm; the gene characteristics of the coronavirus are obviously different from those of middle east respiratory syndrome coronavirus (MERS-CoV) and acute respiratory syndrome coronavirus (SARS-CoV). The RNA can be directly translated into protein like mRNA, can also be used as a template to synthesize a negative strand, and then the negative strand is used for copying filial generation RNA, so that the mutation rate is high.

Chinese scholars firstly disclose and disclose SARS-CoV-2 genome sequence, its genome total length is about 30kb, and its predicted Open Reading Frame (ORF) is up to 11, and it can code known or unknown mature functional protein 20 more than species. The genome structure of the coronavirus has a typical coronavirus genome structure, which sequentially comprises a 5' -untranslated region, a 5' -replicase polysaccharide protein gene (orf1/ab), a Spike glycoprotein gene (Spike, S), a small envelope glycoprotein gene (envelope, E), a membrane glycoprotein gene (membrane, M), a nucleocapsid protein gene (N) and a 3 ' -untranslated region. SARS-CoV-2 is a new variant, and its epidemic rule, pathogenesis and etiology are different from the former epidemic coronavirus. The SARS-CoV-2 complete genome sequence is decoded, and shows higher sequence homology with SARS-CoV genome; and provides a very useful basis for the diagnosis and related research of COVID-19. The related research can be predicted by comparing with the protein coding characteristics of other existing coronaviruses, and powerful guidance is provided for subsequent research.

The nucleocapsid protein, N protein, is one of the major structural proteins of RNA viruses and is closely related to pathogenicity. The SARS-CoV-2N protein functions mainly in combination with viral RNA to play a role, and may be involved in RNA transcription and replication, and the more comprehensive biological function needs to be studied deeply. From the analysis of the predicted protein amino acid sequence, the protein may have new functions related to pathogenic mechanisms, and therefore has important significance in the diagnosis of COVID-19 and the development of vaccines. The recombinant N protein by utilizing genetic engineering can be applied to COVID-19 diagnosis and vaccines, but the complete recombinant N protein is completely consistent with the sequence of the virus-derived N protein, and the safety risk may exist.

Therefore, the protein sequence of SARS-CoV-2N is used to develop artificial recombinant polypeptide with partial core function, which is safer, more convenient for production and lower cost, and can be applied to COVID-19 diagnosis and vaccine development, SARS-CoV-2 genome selective package, etc.

Disclosure of Invention

Aiming at the problems, the invention aims to provide the NTD polypeptide based on the SARS-CoV-2 nucleoprotein, the coding gene, the recombinant vector, the expression method and the application thereof, the preparation of the recombinant NTD polypeptide is more convenient and efficient, the fermentation process is simple, the time consumption is short, the cost is low, and the expression quantity of the target protein reaches more than 20 percent of the total protein of the thallus.

In order to achieve the purpose, the invention adopts the technical scheme that:

an NTD polypeptide based on SARS-CoV-2 nucleoprotein, wherein the NTD polypeptide is a polypeptide with an amino acid sequence shown in any one of SEQ ID No. 1-4.

Further, based on the NTD polypeptide gene of SARS-CoV-2 nucleoprotein, it encodes NTD polypeptide of amino acid sequence as shown in SEQ ID No. 1-4; the NTD polypeptide coding gene is a coding gene of a nucleotide sequence shown in any one of SEQ ID No. 5-8; a recombinant vector of NTD polypeptide based on SARS-CoV-2 nucleoprotein, which comprises the above coding gene.

Further, the host cell comprises the recombinant vector, and the host cell is escherichia coli; the Escherichia coli is Escherichia coli BL21(DE3), BLR (DE3), BL21(DE3) pLysS, M15, TB1, DH5 alpha or Top 10.

Further, the expression method of the gene coding the NTD polypeptide comprises the following steps:

1) designing corresponding primers, amplifying by using a PCR technology and taking genes corresponding to artificially synthesized NTD polypeptides as templates, and inserting amplified gene fragments into expression plasmids by an enzyme digestion connection method to construct a recombinant vector;

2) transforming the recombinant vector into escherichia coli to obtain recombinant genetic engineering bacteria;

3) inoculating the recombinant genetic engineering bacteria into a culture medium, and inducing the expression of the NTD polypeptide by adding lactose or isopropyl thiogalactose IPTG.

Further, the Escherichia coli is Escherichia coli BL21(DE3), BLR (DE3), BL21(DE3) pLysS, M15, TB1, DH5 alpha or Top 10.

Furthermore, the NTD polypeptide coding gene or the recombinant vector of the invention are applied to the preparation of products for detecting SARS-CoV-2.

Furthermore, the NTD polypeptide coding gene or the recombinant vector of the invention are applied to the preparation of SARS-CoV-2 vaccine.

Furthermore, the recombinant NTD polypeptide can be used for clinical detection of COVID-19; meanwhile, the kit is also used for the detection of SARS-CoV-2 antigen antibody and the development of vaccines; and the subsequent development of SARS-CoV-2 nucleic acid medicine packaging polypeptide and the like.

Further, the preparation method in the prokaryotic expression of the NTD polypeptide comprises the following steps:

1) after NTD polypeptide expression is induced, continuously culturing bacterial liquid, and culturing by using a shake flask method or a fermentation tank; simultaneously detecting the OD600 value of the bacterial liquid every hour, and calculating the density of the escherichia coli; culturing at OD600 ═ 2.0 or less;

2) collecting fermentation liquor, centrifuging, crushing thallus, and purifying by affinity chromatography to obtain recombinant NTD polypeptide.

Further, the culture medium of the escherichia coli is LB or PYA8, and the culture temperature is 35-38 ℃.

The invention has the beneficial effects that:

1) the NTD polypeptide of the invention reserves the N-terminal structural domain of SARS-CoV-2 nucleoprotein or the key modification site therein, and simultaneously ensures that the N-terminal structural domain has similar structures such as alpha-helix, beta-sheet layer and the like at the corresponding position of the N-terminal of SARS-CoV-2 nucleoprotein, so as to form a polypeptide analogue with similar tertiary structure, thus having higher safety;

2) the method for preparing the NTD polypeptide is more convenient and efficient, the fermentation process is simple, the time consumption is short, the cost is low, and the expression quantity of the target protein reaches more than 20 percent of the total protein of the thallus;

3) the NTD polypeptide of the invention can be applied to COVID-19 diagnosis and vaccine, SARS-CoV-2 genome selective package, and the application range is wide.

Drawings

FIG. 1 is the location of SARS-CoV-2 nucleoprotein within the SARS-CoV-2 genome;

FIG. 2 is the structure diagram of NTD polypeptide shown in SEQ ID No.1 (SEQ ID No.1 is a section of amino acid sequence at N-terminal of SARS-CoV-2 nucleoprotein, which modifies the initial amino acid and the end amino acid);

FIG. 3 is a diagram showing the structure of the NTD polypeptide shown in SEQ ID No.2 (amino acids within 5 are modified based on SEQ ID No. 1);

FIG. 4 is a diagram showing the structure of the NTD polypeptide shown in SEQ ID No.3 (the amino acids within 10 are modified based on SEQ ID No. 1);

FIG. 5 is a structural diagram of the NTD polypeptide shown in SEQ ID No.4 (30 or less amino acids are modified based on SEQ ID No. 1);

FIG. 6 is the indirect ELISA value analysis of recombinant SARS-CoV-2 nucleoprotein and NTD series polypeptide;

FIG. 7 is a SDS-PAGE result of NTD series polypeptides expressed by four pET-NTD vectors in the present invention; "Control" is a Control with pET-28(a) empty vector;

FIG. 8 is a Western test result chart of four NTD polypeptides expressed by Western test in the example of the present invention; the band corresponding to Marker and having a molecular weight of about 20kDa is the band of the detected NTD series polypeptides.

Detailed Description

In order to further illustrate the technical effects of the present invention, the present invention is specifically described below by way of examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The NTD series polypeptide refers to NTD polypeptide shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.

The invention utilizes the amino acid sequence and the nucleic acid sequence to carry out homologous comparison and analysis on sequence, and utilizes protein or polypeptide secondary structure prediction tools such as SOPMA, GOR, PROSCAN, SignalP and the like to analyze SARS-CoV-2 nucleoprotein secondary structure condition, key modification sites, core structure domain, signal peptide region, hydrophilic and hydrophobic conditions and the like. The tertiary structure condition of SARS-CoV-2 nucleoprotein, the core domain and the simulation position of key modification site, etc. are further analyzed by using a protein or polypeptide tertiary structure prediction tool such as Swiss-model, etc. Through the analysis and prediction, a segment of polypeptide at the N end of SARS-CoV-2 nucleoprotein is designed and named as NTD. The NTD series polypeptide retains the N-terminal structural domain of SARS-CoV-2 nucleoprotein or key modification sites (such as N-glycosylation site, cAMP and cGMP dependent protein kinase phosphorylation site, protein kinase C phosphorylation site, casein kinase II phosphorylation site, N-acylation site, etc.), and ensures that the N-terminal corresponding position of SARS-CoV-2 nucleoprotein has similar alpha-helix, beta-sheet layer, etc. structure, so as to form polypeptide analogs with similar tertiary structure, and the obtained NTD polypeptide sequence is shown in fig. 2-5.