CN114262694B - SARS-CoV-2 vaccine candidate strain using B type influenza virus as carrier and its construction method and application - Google Patents

Abstract

A novel coronavirus vaccine candidate strain taking B type influenza virus as a vector, a construction method and application thereof relate to the field of novel coronavirus vaccines, and a recombinant plasmid NS110-RBD is constructed by truncating and retaining 110 amino acids on an NS1 gene fragment of an attenuated strain B/Yamagata/16/88 of the B type influenza virus and then inserting the fragment into a receptor binding domain gene fragment on a novel coronavirus reference strain S protein synthesized by genes; the recombinant influenza virus rescue strain rIBV-NS110-RBD which takes the attenuated strain B/Yamagata/16/88 of the influenza B virus as a framework and stably expresses a Receptor Binding Domain (RBD) gene fragment on the S protein of a new coronavirus reference strain is rescued by utilizing a reverse genetics technology. The invention lays a foundation for the research of the packaging mechanism of the type B influenza virus and the research and development of novel coronaviruses and novel influenza virus multi-linked vaccines.

Description

SARS-CoV-2 vaccine candidate strain using B type influenza virus as carrier and its construction method and application

Technical Field

The invention relates to the technical field of SARS-CoV-2 vaccine, in particular to a SARS-CoV-2 vaccine candidate strain taking influenza B virus as a carrier, a construction method and application thereof.

Background

The novel coronavirus caused severe novel coronavirus pneumonia epidemic worldwide and was declared as a pandemic in month 3 of 2020. According to World Health Organization (WHO) data, up to 10 months of 2021, more than 200 countries worldwide have reported 235,673,032 confirmed cases, including 4,814,651 deaths.

The novel coronavirus (SARS-COV-2) is the main causative agent of the pneumonia of the novel coronavirus, and belongs to the family Coronaviridae, genus beta coronavirus, with the other two coronaviruses SARS-CoV, MERS-CoV that infect humans. SARS-CoV-2 is an enveloped, forward, single stranded RNA virus with a genome of 29903nts, and is covered by an outer membrane composed of nucleocapsid protein (N), membrane protein (M), envelope protein (E) and spike protein (S). Like SARS-CoV, the S protein of SARS-CoV-2 mediates viral entry into host cells by binding to their common receptor angiotensin converting enzyme 2 (ACE 2) via the Receptor Binding Domain (RBD). During infection, the S protein is cleaved by a host protease (e.g., TMPRSS 2) into the N-terminal S1 subunit and the C-terminal S2 subunit, and converted from the pre-fusion state to the post-fusion state. The S protein is a type I fusion protein that forms a trimer on the surface of a virion, and comprises two functional subunits S1 and S2, S1 and S2 consisting of an extracellular domain (ECD) and a single transmembrane helix, mediating receptor binding and membrane fusion, respectively. Wherein S1 is responsible for binding to host cell receptors and S2 subunit is responsible for viral membrane and cell membrane fusion. S1 consists of an N-terminal domain (NTD) and a Receptor Binding Domain (RBD), which are critical for determining tissue tropism and host range. The S protein not only determines the infectivity of the virus and its transmissibility in the host, but also is the primary antigen for inducing a protective immune response. Thus, all currently under development vaccines are primarily targeted at RBD.

When the gene sequence of SARS-CoV-2 was published at the beginning of month 1 in 2020, the development of vaccines against it began and was developed at an unprecedented rate. At present, more than 200 vaccines are in different research and development stages, and the vaccine types can be mainly classified into inactivated vaccines, attenuated live vaccines, recombinant protein vaccines, mRNA vaccines and viral vector vaccines. In the severity of the epidemic, WHO successively included the list of emergency use of the gabion-Biontech, aslicon, prednisone, modenra and national drug inactivated vaccines. In the aspect of neutralizing antibodies, the inactivated vaccine is at the low end, the ChAdOx1 and mRNA candidate vaccine are in a medium range, the recombinant protein vaccine candidate vaccine is at the high end, and the generated neutralizing antibodies have the highest titer. In terms of tolerability, inactivated vaccines and recombinant protein vaccines perform relatively well, followed by mRNA vaccines and adenovirus vector vaccines, showing higher immunogenicity after the second immunization. In terms of practicality, mRNA vaccines have problems such as high cost, difficulty in transportation and storage, and the like.

To date, there is no effective treatment for SARS-CoV-2, and SARS-CoV-2 vaccine remains an effective means of controlling viruses. Clinical data of the SARS-CoV-2 vaccine at the present stage show that the SARS-CoV-2 vaccine has safety and effectiveness, but still can not meet the requirements of coping with novel coronary pneumonia epidemic situation. Thus, there is an urgent need to develop a safer and more effective SARS-CoV-2 vaccine.

Influenza a virus (InfluenzaAvirus, IAV) hosts are widespread, but Influenza B Virus (IBV) is mainly prevalent in the population and seal infection has also been reported. Like influenza a viruses, influenza B viruses also experience antigen drift continuously, but at a lower rate of evolution than influenza a viruses, and regular recombination between different influenza B strains. So far, influenza B viruses have been largely divided into two lineages, victoria and Yamagata. They are distinguished from early B/Lee/1940 influenza viruses, which have co-transmitted epidemics in the human population since the 80 s of the 20 th century, being the main epidemic strain once every three years. Furthermore, during the past decade, influenza B viruses have caused several acute respiratory disease outbreaks in schools and society, as well as secondary bacterial pneumonia infections. The effective transmission and lack of antiviral effects of influenza B viruses exacerbates people's health concerns.

Up to now, nasal spray SARS-CoV-2 vaccine based on influenza virus vector has not been reported.

Disclosure of Invention

The invention aims to provide a SARS-CoV-2 vaccine candidate strain using B type influenza virus as a carrier, and a construction method and application thereof, so as to solve the problems of the existing SARS-CoV-2 vaccine.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the constructed recombinant gene fragment is shown as SEQ ID NO. 3; the recombinant influenza virus rescue strain rIBV-NS110-RBD which takes the attenuated strain B/Yamagata/16/88 of the influenza B virus as a framework and stably expresses the receptor binding domain gene fragment on the S protein of the SARS-CoV-2 reference strain is obtained by utilizing the reverse genetics technology and the recombinant plasmid NS110-RBD.

As a preferred embodiment, after 110 amino acids are reserved in the truncated NS1 gene fragment of the attenuated strain B/Yamagata/16/88 of the type B influenza virus, the sequence of the obtained truncated fragment is shown as SEQ ID NO. 1.

As a preferred embodiment, the sequence of the receptor binding domain gene fragment on the S protein of the SARS-CoV-2 reference strain synthesized by the gene is shown in SEQ ID NO. 2.

As a preferred embodiment, the sequence of the recombinant gene fragment is shown in SEQ ID NO. 3.

The invention relates to a construction method of SARS-CoV-2 vaccine candidate strain using B type influenza virus as carrier, which mainly comprises the following steps:

step one, constructing a recombinant plasmid NS110-RBD;

step two, rescue and virus seed preparation of the recombinant influenza virus.

As a preferred embodiment, the specific operation procedure of the first step is as follows:

(1) Gene synthesis and primer design

Designing an upstream primer and a downstream primer according to an initial reference genome sequence of SARS-CoV-2Spike RBD published on GenBank, and carrying out gene synthesis on the initial reference genome sequence of SARS-CoV-2Spike RBD and the primer to obtain a RBD target gene fragment, wherein the primer information is as follows:

In Fusion-RBD-F(5’-3’)：TTTAGAGTCCAACCAACAGAAT；

In Fusion-RBD-R(5’-3’)：GAAATTGACACATTTGTTTTTAAC；

In Fusion-Vector-F(5’-3’)：AGCAGAAGCAGAGGATTTGTTT；

In Fusion-Vector-R(5’-3’)：AGTAGTAACAAGAGGATTTTTATTTTAAATTCACAA；

(2) Amplification of the Gene fragment of interest

The synthesized RBD target gene fragment is used as a template, a primer In Fusion-RBD-F/In Fusion-RBD-R is used for carrying out PCR amplification, and gel recovery and purification of the target gene fragment are carried out after 1% agarose gel electrophoresis identification is correct;

(3) Amplification of linearization vector NS110

The PBD-NS110 plasmid in the B/Yamagata/16/88 reverse genetic operation platform of the type B influenza virus is used as a template, the primer is used for PCR amplification, and after the correct identification by 1% agarose gel electrophoresis, the gel recovery and purification of the vector fragment are carried out;

(4) Construction of recombinant plasmid NS110-RBD

Mixing the amplified and purified target gene fragment and the linearized carrier fragment with deionized water, incubating together, and then taking a recombinant product to be transformed into JM109 competent cells; amplifying, purifying and sequencing monoclonal colonies containing target gene fragments through colony PCR screening;

the sequence of the amplified product of the target gene fragment is shown as SEQ ID NO. 2; the sequence of the constructed recombinant gene fragment is shown as SEQ ID NO. 3.

In a preferred embodiment, the specific operation procedure of the second step is as follows:

(1) Rescue of recombinant influenza strains

Taking 7 positive plasmids PBD-PB2, PBD-PB1, PBD-PA, PBD-HA, PBD-NA, PBD-NP, PBD-M and constructed recombinant plasmids NS110-RBD in a B/Yamagata/16/88 reverse genetic operation platform, uniformly mixing the positive plasmids PBD-PB2, PBD-PB1, PBD-PA, PBD-NP, PBD-NA and PBD-M with Lipofectamine3000 liposome transfection reagent in a centrifuge tube containing Opti-MEM, standing, dripping the mixed solution into a 6-orifice plate containing Opti-MEM, continuously culturing the mixed solution by changing Opti-MEM culture solution containing Anti-Anti and TPCK pancreatin, and collecting cell supernatant;

(2) Inoculation of chick embryo with supernatant of transfected cell

Inoculating the collected supernatant of transfected cells into SPF chick embryos of 7 days old by adopting a chick embryo allantoic cavity inoculation method, incubating the chick embryos at constant temperature, and determining hemagglutination titer, and then naming the rescued recombinant influenza virus strain as rIBV-NS110-RBD;

(3) Preparation of recombinant influenza virus rescue strain rIBV-NS110-RBD virus seed

Inoculating recombinant influenza virus rescue strain rIBV-NS110-RBD into SPF chick embryo of 7 days old by chick embryo allantoic cavity inoculation method, incubating chick embryo at constant temperature, measuring blood coagulation titer, collecting chick embryo allantoic fluid which has high blood coagulation titer and is clear and free of blood as toxic seed in centrifuge tube, centrifuging to obtain supernatant, sub-packaging, and storing in ultralow temperature refrigerator of-80 ℃.

The invention relates to an application of human nasal spray SARS-CoV-2 vaccine candidate strain taking B type influenza virus as carrier in preparing human nasal spray SARS-CoV-2 vaccine.

The beneficial effects of the invention are as follows:

the constructed recombinant gene fragment is shown as SEQ ID NO. 3; the recombinant influenza virus rescue strain rIBV-NS110-RBD which takes the attenuated strain B/Yamagata/16/88 of the influenza B virus as a framework and stably expresses a Receptor Binding Domain (RBD) gene fragment on the S protein of a SARS-CoV-2 reference strain is obtained by utilizing reverse genetics technology and recombinant plasmid NS110-RBD.

The invention uses recombinant plasmid NS110-RBD and the other 7 skeleton strain recombinant plasmids to co-transfect MDCK cells and 293T cells to rescue and express SARS-CoV-2RBD region recombinant influenza virus, and carries out morphological, molecular biological identification and virus titer and Western Blot detection on the saved recombinant influenza virus rescue strain rIBV-NS110-RBD. The result shows that the recombinant influenza virus rescue strain is successfully rescued and named rIBV-NS110-RBD; the PCR identification result shows that the RBD target gene band is correct, the gene sequence result shows that the sequence of the rescue virus strain is correct, and the virus particles of the rescue virus strain are observed to have typical characteristics of influenza virus particles under a transmission electron microscope; according to the measurement, the virus titer of the recombinant influenza virus rescue strain rIBV-NS110-RBD on MDCK cells is 105.5TCID 50/ml, the virus titer on chick embryos is 106.5EID 50/ml, and the highest hemagglutination titer can reach 25; the Western Blot experiment detects the expression of SARS-CoV-2RBD protein with the size of 35kDa. The invention is proved by experiments to successfully rescue recombinant influenza virus rescue strain rIBV-NS110-RBD expressing SARS-CoV-2RBD protein.

The recombinant influenza virus rescue strain rIBV-NS110-RBD expressing the SARS-CoV-2RBD region can stably encode and express the exogenous protein RBD on the basis of the original influenza virus attenuated strain, and can be used as a vaccine candidate strain for passage production on chicken embryo or MDCK cells compared with the existing replication-defective adenovirus vector vaccine, and has the advantages of low cost, easy production and the like; compared with adenovirus, influenza B virus is not popular in the population, and is less likely to cause anti-vector immune response; in the inoculation mode, a nasal spray inoculation mode can be adopted; the human lower respiratory tract is considered to be largely protected by IgG, the major antibody class in serum, which is delivered to the lungs; the upper respiratory tract is thought to be largely protected by secretory IgA (SIgA); natural infection of respiratory viruses can induce systemic immune response mainly comprising IgG and upper respiratory mucosa immune response mainly comprising sIgA; intramuscular or intradermal vaccination will in many cases lead to a strong induction of serum IgG but not to the production of mucosal SIgA; although some IgG may also be found at the mucosal surface of the upper respiratory tract, the lack of SIgA tends to predispose humans to upper respiratory tract infections. Nasal vaccination can be effective in inducing mucosal antibody responses, potentially providing immunity to the upper respiratory tract; compared with the traditional intramuscular injection inoculation mode, the method is more convenient and safer; not only can induce humoral immunity and cellular immunity of organisms, but also can induce mucosal immunity of organisms, so that the vaccine candidate strain and the inactivated vaccine have better immune effect under the same using dose.

Compared with vaccine strains developed by the recombinant influenza virus rescue strain rIBV-NS110-RBD expressing SARS-CoV-2RBD region, surapong koonpaew and the like, the recombinant influenza virus rescue strain has the advantage of no reassortment with influenza A virus which is popular in nature, can be rapidly produced by applying the existing mature influenza vaccine technology, and provides a new thought for developing SARS-CoV-2 vaccine by taking influenza B virus as a carrier.

Drawings

FIG. 1 shows the PCR results of the RBD gene fragment of interest and the NS110 vector fragment. Wherein a is the PCR result of the RBD target gene fragment; b is the PCR result of the NS110 vector fragment.

FIG. 2 shows the results of a hemagglutination assay for recombinant influenza virus rescue strain rIBV-NS110-RBD.

FIG. 3 shows the PCR results of 8 gene fragments of recombinant influenza virus rescue strain rIBV-NS110-RBD.

FIG. 4 shows the NS gene sequencing results of recombinant influenza virus rescue strain rIBV-NS110-RBD.

FIG. 5 is an electron micrograph (100 k X) of recombinant influenza virus rescue strain rIBV-NS110-RBD.

FIG. 6 shows Western Blot results of recombinant influenza virus rescue strain rIBV-NS110-RBD.

Fig. 7 is a safety evaluation experiment result. Wherein, A is the weight change rate of the mice after inoculation along with time, B is the survival rate of the mice after inoculation along with time, C is the tissue tropism of the mice after inoculation along with time, and D is the lung replication kinetics of the mice after inoculation along with time.

FIG. 8 is a flow chart of an immunoassay.

FIG. 9 shows the results of detection of specific antibody IgG titers by an indirect ELISA method.

FIG. 10 shows IFN-. Gamma.and IL-4ELISAPOT assay results. Wherein A is the INF-gamma detection result of the immunized mice, and B is the IL-4 detection result of the immunized mice.

Fig. 11 is a flow chart of an experiment for challenge protection of mice.

FIG. 12 shows the results of toxicity attack protection experiments for mice.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

1. Materials and methods

1 Material

1.1 cells and chick embryos

MDCK cells and 293T cells were maintained in the virology laboratory of the military medical institute of military science and veterinary research institute of the military academy of sciences; SPF chick embryos of 7-9 days old were purchased from Experimental animal technologies Inc. of Beijing vitamin Toril.

1.2 major reagents

Phusion HF DNAPolymerase from NEW ENGLAND Biolabs, inc. of America; reverseTranscriptase XL (AMV), nucleo Spin Gel and PCR Clean-up, 5 XIn-Fusion HD EnzymePremix and E.coli JM109 component Cells are available from China Bow bioengineering Co., ltd; QIAampViral RNAMini Kit and QIAprep Spin Miniprep Kit are available from QIAGEN, germany; lipofectamine3000 transmission Kit is available from Thermo Fisher SCIENTIFIC, USA; 0.25% Trypsin-EDTA, 0.05% Trypsin-EDTA, fetal Bovine Serum (FBS), DMEM medium, opti-MEM and anti-biological-Antimycic are available from Thermo Fisher SCIENTIFIC of America; 1 XPBS buffer and ampicillin were purchased from Beijing Soy Co.Ltd; TPCK trypsin was purchased from Merck Biotechnology Co., germany; BCA protein concentration assay kit was purchased from shanghai bi yun biotechnology limited, china; PVDF membranes were purchased from Bio-Rad, inc. of America.

1.3 Main Equipment

The gene amplification instrument was purchased from Hangzhou Langmuir scientific instruments, inc., china; electrophoresis apparatus was purchased from Bio-Rad company, USA; agarose gel electrophoresis tanks were purchased from eastern electrophoresis apparatus limited company, beijing junyi, china; the electric heating constant temperature water tank is purchased from Shanghai-Heng technology Co., ltd; gel imager was purchased from Shanghai energy technology Co., ltd; biological safety cabinets were purchased from the company of instrument manufacturing, haar, tokyo, china; the high-speed centrifuge, 37℃incubator and 33℃incubator were purchased from Thermo Fisher SCIENTIFIC, U.S.A.; cradle (for bacteria) was purchased from Shanghai Biotechnology Co., ltd; common optical microscope was purchased from olympics corporation of japan; an ultra-low temperature refrigerator at-80 ℃ was purchased from SANYO corporation.

2 method

2.1 Gene Synthesis and primer design

Searching SARS-CoV-2Spike RBD original reference genome sequence published on GenBank, designing upstream and downstream primers by Primer Premier 5 software, designing seamless cloning primers by TAKARA functional network, and finally sending the SARS-CoV-2Spike RBD original reference genome sequence and the seamless cloning primers to the biological engineering Co-Ltd for gene synthesis, wherein the information of the designed primers is shown in Table 1.

TABLE 1 design of primers for seamless cloning

Primer name	Primer sequence (5 '-3')
		InFusion-RBD-F	TTTAGAGTCCAACCAACAGAAT
InFusion-RBD-R	GAAATTGACACATTTGTTTTTAAC
		InFusion-Vector-F	AGCAGAAGCAGAGGATTTGTTT
InFusion-Vector-R	AGTAGTAACAAGAGGATTTTTATTTTAAATTCACAA

2.2 construction of recombinant plasmid NS110-RBD

2.2.1 amplification of the Gene fragment of interest

The synthesized RBD target gene fragment is diluted to 10mM by deionized water, and PCR is performed by using the synthesized primer In Fusion-RBD-F/In Fusion-RBD-R as a template. The PCR reaction system of Phusion DNA polymerase is shown in Table 2, and the PCR reaction parameters of Phusion DNA polymerase are shown in Table 3.

TABLE 2 PCR reaction System of Phusion DNA polymerase

TABLE 3 PCR parameters of Phusion DNA polymerase

Temperature (temperature)	Time
		98℃	30s
98℃	10s
		55℃	30s
72℃	1min
		72℃	10min
4℃	-

Amplifying the target gene fragment; and (3) carrying out gel recovery and purification on the target gene fragment after the product is identified to be correct by 1% agarose gel electrophoresis.

2.2.2 amplification of linearization vector NS110

The PBD-NS110 plasmid In the preserved B-type influenza virus B/Yamagata/16/88 reverse genetic operating platform (see construction of B-type influenza virus B/Yamagata/16/88 reverse genetic operating platform, establishment of BALB/c mouse infection model, sun Weiyang; yu Zhijun; li Xue; chen Jiang; gao Xiaolong; guojiao; zhang Kun; li Yuanguo; wang Tiecheng; yang Songtao; huang Geng; zhao Yongkun; gao Yuwei; xia Xianzhu; chinese laboratory animal journal, volume 23, stage 1, 2015-02-28) is diluted to 10mM with deionized water, and the template is used for PCR amplification of Vector fragment by using the designed and synthesized primer Infusion-Vector-F/In Fusion-Vector-R, and the sequence of the truncated fragment obtained after 110 amino acids are truncated and retained for NS1 gene fragment of B-type influenza virus attenuated strain B/Yamagata/16/88 is shown In SEQ ID NO: 1. And (3) after the product is identified to be correct by 1% agarose gel electrophoresis, performing gel recovery and purification of the carrier fragment. The PCR reaction system of Phusion DNA polymerase is shown in Table 4, and the PCR reaction parameters of Phusion DNA polymerase are shown in Table 5.

TABLE 4 PCR reaction System of Phusion DNA polymerase

TABLE 5 PCR parameters of Phusion DNA polymerase

Temperature (temperature)	Time
		98℃	30s
98℃	10s
		55℃	3min30s
72℃	1min
		72℃	10min
4℃	-

100ng of each of the amplified and purified target gene fragment and the linearized vector fragment was mixed with 5 XIn-Fusion HDEnzyme Premix and deionized water to prepare a 10. Mu.l system. After co-incubation for 15min in a 50℃water bath, 2.5. Mu.l of recombinant product was transformed into JM109 competent cells. And (3) amplifying and purifying a monoclonal colony containing the target gene fragment through colony PCR screening, and finally sending to a biological engineering company for sequencing and identification.

2.2.3 construction results of recombinant plasmid NS110-RBD

The sequence of the constructed recombinant gene fragment is shown as SEQ ID NO. 3.

As shown in FIG. 1a, the RBD target gene fragment was amplified by PCR, subjected to 1% agarose gel electrophoresis and sequenced to identify a target band corresponding to the expected size, and a specific target band having a size of about 670bp (SEQ ID NO: 2) was seen in the electrophoresis chart shown in FIG. 1 a.

As shown in FIG. 1b, the NS110 vector fragment, after PCR amplification, 1% agarose gel electrophoresis and sequencing, gave a band of interest which was consistent with the expected size, and a band of specific interest of approximately 5600bp was seen in the electrophoresed pattern shown in FIG. 1 b.

2.3 rescue of recombinant influenza Virus and preparation of seed

The recombinant influenza virus rescue strain rIBV-NS110-RBD which takes the attenuated strain B/Yamagata/16/88 of the influenza B virus as a framework and stably expresses the receptor binding domain gene fragment on the S protein of the SARS-CoV-2 reference strain is obtained by utilizing the reverse genetics technology and the recombinant plasmid NS110-RBD. The specific operation is as follows:

293T cells cultured in DMEM medium containing 10% fetal bovine serum and 1% Anti-Anti were plated into 6-well cell culture plates after digestion with pancreatin containing 0.05% EDTA prior to transfection to determine cell density, about 1X 106 cells per well. After monolayer formation, transfection was performed according to Lipofectamine3000 liposome transfection reagent instructions.

2.3.1 rescue of recombinant influenza Virus strains

7 positive plasmids PBD-PB2, PBD-PB1, PBD-PA, PBD-HA, PBD-NA, PBD-NP, PBD-M and 600ng of each constructed recombinant plasmid NS110-RBD in a B-type influenza virus B/Yamagata/16/88 reverse genetic operating platform are taken. In a 1.5ml centrifuge tube containing 250. Mu.l Opti-MEM, the mixture was mixed with Lipofectamine3000 liposome transfection reagent and allowed to stand at room temperature for 20min. The plasmid and Lipofectamine3000 liposome transfection reagent mixture drop into 200ml 250 u l Opti-MEM 6 hole plate, 33 degrees C, 5% CO 2 conditions were cultured for 12 hours. After 12h of incubation, the Opti-MEM broth containing 1% Anti-Anti and 0.2. Mu.g/ml TPCK pancreatin was changed. Then, the culture was continued at 33℃under 5% CO 2 for 72 hours. After 72h, the cell supernatant was collected and the next test was continued.

2.3.2 inoculation of the supernatant of transfected cells with chick embryos

The collected supernatant of transfected cells was inoculated to 7-day-old SPF chick embryos by chick embryo allantoic cavity inoculation. The chick embryos were incubated in a 33℃incubator. After 72 hours, the sample was taken out and subjected to a Hemagglutination (HA) test to determine the hemagglutination titer. The rescued recombinant influenza strain was designated rIBV-NS110-RBD. As shown in FIG. 2, the recombinant influenza virus rescue strain rIBV-NS110-RBD was measured to have a hemagglutination titer of 25.

2.3.3 preparation of recombinant influenza Virus rescue Strain rIBV-NS110-RBD virus seed

The test method is the same as 2.3.2, and the preserved recombinant influenza virus rescue strain rIBV-NS110-RBD is inoculated to SPF chick embryos of 7 days old by adopting a chick embryo allantoic cavity inoculation method. The chick embryos were incubated in a 33℃incubator. After 72 hours, the sample was taken out and subjected to a Hemagglutination (HA) test to determine the hemagglutination titer. Collecting chick embryo allantoic fluid with high hemagglutination titer and no blood in 50ml centrifuge tube as toxic seed at 5500rpm at 4deg.C for 10min, collecting supernatant, packaging, and storing in-80deg.C ultra-low temperature refrigerator.

2.4 identification of recombinant influenza Virus rescue Strain rIBV-NS110-RBD

2.4.1 PCR and sequencing identification of recombinant influenza Virus rescue Strain rIBV-NS110-RBD

And (3) extracting RNA from recombinant influenza virus rescue strain rIBV-NS110-RBD according to the instruction of a QIAamp Viral RNAMini Kit total RNA extraction kit. cDNA was reverse transcribed using Reverse Transcriptase XL (AMV), and PCR amplification was performed using 8 gene fragment-specific primers (see Table 6) of the synthesized influenza B virus, respectively. The PCR product was taken for 1% agarose gel electrophoresis identification and sequencing identification by biological engineering Co., ltd. The PCR reaction system of the Phusion DNA polymerase is shown in Table 7, and the PCR reaction parameters of the Phusion DNA polymerase are shown in Table 8.

Table 6B influenza 8 gene fragment specific primers

Primer name	Base sequence (5 '-3')
		PBD-PB2-F	CCAGCAGAAGCGGAGCGTTT
PBD-PB2-R	TTAGTAGAAACACGAGCATTTTTCACTC
		PBD-PB1-F	CCAGCAGAAGCGGAGCCTTT
PBD-PB1-R	TTAGTAGAAACACGAGCCTTTTTTCA
		PBD-PA-F	CCAGCAGAAGCGGTGCGTTT
PBD-PA-R	TTAGTAGAAACACGTGCATTTTTGATTC
		PBD-HA-F	CCAGCAGAAGCAGAGCATTTTC
PBD-HA-R	TTAGTAGTAACAAGAGCATTTTTCAATAACG
		PBD-NP-F	CCAGCAGAAGCACAGCA
PBD-NP-R	TTAGTAGAAACAACAGCATTTTT
		PBD-NA-F	CCAGCAGAAGCAGAGCATC
PBD-NA-R	TTAGTAGTAACAAGAGCATTTTTGAG
		PBD-M-F	CCAGCAGAAGCACGCACT
PBD-M-R	TTAGTAGAAACAACGCACTTTTTC
		PBD-NS110-F	CCAGCAGAAGCAGAGGA
PBD-NS110-R	TTAGTAGTAACAAGAGGATTTTTAT

TABLE 7 PCR reaction System of Phusion DNA polymerase

TABLE 8 PCR reaction parameters of Phusion DNA polymerase

Temperature (temperature)	Time
		98℃	30s
98℃	10s
		55℃	1min30s
72℃	1min
		72℃	10min
4℃	-

As shown in FIG. 3, the electrophoresis identification result shows that the size of 7 gene fragments of the recombinant influenza virus rescue strain rIBV-NS110-RBD is consistent with that of the type B influenza virus B/Yamagata/16/88 by 1% agarose gel electrophoresis.

The result of NS gene sequencing is shown in FIG. 4, and after DNA STAR comparison analysis, the sequence of 7 gene fragments of recombinant influenza virus rescue strain rIBV-NS110-RBD is consistent with that of B type influenza virus B/Yamagata/16/88, and the NS fragment is consistent with the experimental design.

The sequences of the 7 obtained gene fragments are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10 in the sequence table.

2.4.2 determination of the half tissue infection amount (TCID 50) of recombinant influenza Virus rescue Strain rIBV-NS110-RBD

MDCK cells cultured by 10% fetal bovine serum and 1% Anti-Anti DMEM medium are digested by pancreatin containing 0.25% EDTA, the cell density is measured, the cells are spread on a 96-well cell culture plate, and after culturing for about 9 hours in a 5% CO 2 cell culture box at 37 ℃, the monolayer cell density is about 80% for standby. The recombinant influenza virus rescue strain rIBV-NS110-RBD is diluted 10 times more than 8 dilutions. 100 μl of diluted virus solution was added to the 96-well plate per well, and 3 replicates were made per dilution. And a blank cell control group was established. After incubation at 33℃in a 5% CO 2 incubator for 1.5h, the virus solution was discarded. 200 μl of Opti-MEM containing TPCK pancreatin and 1% Anti-Anti at a final concentration of 2 μg/μl was added to each well. The cells were further cultured at 33℃in a 5% CO 2 incubator for 72 hours. After the culture for 72 hours, the cell supernatant was taken to determine the hemagglutination titer, and the TCID50 of the virus was calculated according to the Reed-Muench method.

The recombinant influenza virus rescue strain rIBV-NS110-RBD was inoculated with MDCK cells at different dilutions, and after 72h incubation, the cell supernatants were taken for hemagglutination assays, with the data shown in Table 9. The TCID50 of the virus was calculated according to the Reed-Muench method, and the half tissue infection amount (TCID 50) of the recombinant influenza virus rescue strain rIBV-NS110-RBD was 105.5TCID 50/ml.

TABLE 9

2.4.3 determination of the half-Length infection (EID 50) of the chick embryo of the recombinant influenza Virus rescue Strain rIBV-NS110-RBD

The recombinant influenza virus rescue strain rIBV-NS110-RBD is diluted 10 times more than 8 dilutions. Taking diluted virus liquid, and inoculating 3 SPF chick embryos of 7 days old at 100 mu l per dilution. And (3) placing the inoculated SPF chick embryo in a constant temperature incubator at 33 ℃ for incubation for 72 hours, collecting chick embryo allantoic fluid, measuring the hemagglutination titer, and calculating the EID 50 of the virus according to a Reed-Muench method.

Recombinant influenza virus rescue strain rIBV-NS110-RBD is inoculated with SPF chick embryos of 7 days old at different dilutions, and after 72 hours of incubation, chick embryo allantoic fluid is taken for a hemagglutination test, and the data are shown in Table 10. The EID 50 of the virus was calculated according to the Reed-Muench method, and the half-number of infection (EID 50) of chick embryos of the recombinant influenza virus rescue strain rIBV-NS110-RBD was 106.5EID 50/ml.

Table 10

2.4.4 electron microscope observations of recombinant influenza Virus rescue Strain rIBV-NS110-RBD

And inactivating the collected chick embryo allantoic fluid virus seed by beta-propiolactone, carrying out negative dyeing by phosphotungstic acid, and observing the basic form of the virus under a transmission electron microscope. As a result, as shown in FIG. 5, the virus particles were spherical, with a diameter of about 120nm, and had fibers on the surface. The virus particle size and morphology distribution conforms to typical characteristics of influenza virus particles.

2.4.5 Western Blot identification of recombinant influenza Virus rescue Strain rIBV-NS110-RBD

Recombinant influenza virus rescue strain rIBV-NS110-RBD and unmodified wild strain (WT) are respectively inoculated with 7-day-old SPF chick embryos, 100 mu l of each embryo is placed at 33 ℃ for 72 hours, allantoic fluid is respectively collected, and after the allantoic fluid is lysed according to the volume ratio of the allantoic fluid to the cell lysate of 1:9, western Blot verification is carried out.

As a result, as shown in FIG. 6, the recombinant influenza virus rescue strain rIBV-NS110-RBD after lysis detected a target band of 35kDa (SARS-CoV-2 Spike RBD) by Western Blot, whereas the chick embryo allantoic fluid (NC) of the negative control group inoculated with the wild-type strain (WT) and not inoculated with any virus strain did not detect RBD.

2. Safety evaluation experiment

1. Tissue virus preparation

(1) Recombinant influenza virus rescue strain rIBV-NS110-RBD was diluted to two titers of 106EID 50/ml and 105EID 50/ml with MEM medium.

(2) Two titer groups of 106EID 50/ml and 105EID 50/ml and one control group (Mock group) were set; wherein, each group is provided with 11 BALB/c mice of 4-6 weeks old; after the mice were anesthetized, the mice were infected by nasal titration, wherein mice in the two titer groups of 106EID 50/ml and 105EID 50/ml were inoculated with the above diluted virus solution, respectively, and mice in the control group (Mock group) were inoculated with MEM culture solution, and the inoculation amount of each group was 50. Mu.l/each.

(3) 5 mice were arbitrarily selected from all mice and labeled 1-5, all mice were observed 15 consecutive days from day 0 after challenge, and the weights of the labeled mice were weighed at regular intervals each day.

(4) On days 3 and 6 after challenge, 3 mice (unlabeled) were sacrificed at 106EID 50/ml and 105EID 50/ml titer groups, respectively. The mice were sacrificed to dissect the organ tissue and weigh the recorded weight. Mice were sacrificed in groups to obtain turbinates (Nasal turbinates), brain (Brain), heart (Heart), lung (Lung), kidney (Kidney), liver (lever), spleen (Spleen), and Intestine (interphone).

(5) Grinding tissue viscera, centrifuging tissue grinding fluid, and freezing the tissue supernatant in an ultralow temperature refrigerator at-80deg.C.

2. Titration of tissue virus with chick embryo

(1) Taking out the frozen tissue supernatant from the ultralow temperature refrigerator at the temperature of-80 ℃, thawing by using an ice-water mixture, and rapidly placing the frozen tissue supernatant into an ice box for preservation after thawing.

(2) In a secondary biosafety cabinet, an ice box and a plurality of 1.5ml centrifuge tubes are prepared, 900 mu l of pre-prepared DMEM diluent (containing double antibody and no serum) is added into each 1.5ml centrifuge tube by a micropipette, 100 mu l of tissue supernatant is taken out by the micropipette, added into each 1.5ml centrifuge tube, and gently blown and stirred uniformly, and the tissue supernatant is sequentially diluted from 100 ten times to 106 by the method.

(3) Taking out SPF chick embryo of 7 days old in a constant temperature incubator at 37 deg.C, drawing out an air bag cavity in a darkroom with an egg candler, spraying 75% alcohol on the surface for disinfection, and placing into a secondary biosafety cabinet.

(4) And lightly punching holes at the position where the air sac cavity is drawn by using a puncher, puncturing and collecting tissue supernatant.

(5) Each dilution was repeated 3 times with an inoculum size of 100. Mu.l/chick embryo.

(6) The seed was inserted from the puncture using a 1ml syringe at 60℃and directly into the allantoic cavity.

(7) Sealing with white glue, and spraying 75% alcohol again to sterilize after inoculation of all chick embryos is completed.

(8) The cells were incubated at 33℃in a constant temperature incubator for 3 days.

(9) After the cultivation is finished, the chick embryo is placed at the temperature of minus 20 ℃ for 1 hour, and allantoic fluid is collected for a hemagglutination experiment.

(10) The EID 50 was calculated using the Reed-Muench method.

The results are shown in FIG. 7. BALB/c mice were nasal immunized with both titer groups 106EID 50 and 105EID 50 and control groups, respectively, and none of the mice died within 14 days, as shown in fig. 7B; in terms of body weight, the whole body weight of the mice was always in a rising state as shown in fig. 7 a.

To further evaluate the safety of recombinant influenza virus rescue strain rIBV-NS110-RBD, the tissue tropism of mice was analyzed, as shown in FIG. 7C, with virus titers of 3.35Log10 EID 50/g and 2.98Log10 EID 50/g, respectively, found only in the turbinates of 106EID 50 titer group 2 mice on day 3 of immunization; the virus titer was found in the lung to be 2.55log10 EID 50/g as shown in figure 7D; the other tissue virus titers were all below the lower detection limit, i.e., less than 100.5EID 50/g.

3. Immunoassay test

As shown in fig. 8, the immunization experiments are classified into a single immunization experiment (Prime) and a secondary immunization experiment (Boost). The single and secondary immunization experiments each contained the rIBV-NS110-RBD-106EID 50 and Mock groups of 9, 3 blood, 3 spleen and 3 alveolar lavage fluids. A single immunization experiment was performed to collect samples on day 21 of immunization, a second immunization was performed on day 21 after the first immunization in the two immunization groups, and the immunization dose and the immunization pattern were the same as those of the single immunization and samples were collected on day 42 of immunization.

1. Indirect ELISA method for detecting specific antibody IgG

(1) The mice serum was inactivated, metal-bathed at 56℃for 30min.

(2) Diluting commercial SARS-COV-2RBD protein (Yiqiaoshenzhou, 40592-V08H) to 5 μg/ml with coating solution concentration, coating at 4deg.C overnight;

(3) The plate was discarded, the plates were washed 3 times with 200 μl/well using a shaker, each for 1min;

(4) Preparing 5% skimmed milk as a sealing solution, and incubating at 37 ℃ for 2 hours at 150 μl/hole;

(5) The plate was discarded, the plates were washed 3 times with 200 μl/well using a shaker, each for 1min;

(6) Serum to be tested of 3 mice is taken from each group, and serum samples are diluted to 64 times, 128 times, 256 times, 512 times, 1024 times, 2048 times and 4096 times by PBST in sequence;

(7) Adding diluted serum into the wells respectively, making 3 compound wells, 100 μl/well, and incubating at 37deg.C for 2 hr;

(8) The plate was discarded, the plates were washed 3 times with 200 μl/well using a shaker, each for 1min;

(9) HRP-labeled goat anti-mouse IgG antibody was diluted with blocking solution 1:100000, 100. Mu.l/well, incubated at 37℃for 1h;

(10) The plate was discarded, the plates were washed 5 times with 200 μl/well using a shaker, each for 1min;

(11) 100 μl of TMB substrate is added into each well, incubated at room temperature for 20min in dark place, 50 μl of stop solution is added into each well after the inside of the well turns blue, the solution turns yellow, and the absorbance at the wavelength of 450mn is detected by an enzyme-labeled instrument.

The results of anti-SARS-COV-2 Spike RBD specific IgG antibody titers in the serum of mice of each experimental group on day 21 after single immunization and day 21 after double immunization are shown in FIG. 9. The results showed that both the single and double immunized mice were significantly higher than the control mice IgG antibody titers. The IgG antibody titer of the double immunized mice is obviously higher than that of the single immunized mice, and in general, the recombinant influenza virus rescue strain rIBV-NS110-RBD immunized mice can cause strong humoral immunity and mucosal immunity.

2. IFN-gamma and IL-4ELISAPOT assays

2.1 isolation of mouse spleen lymphocytes

(1) Taking immunized mice and control mice on the 21 st day after single immunization and the 21 st day after double immunization, taking 3 mice from each group, anesthetizing with isoflurane, killing the mice after neck removal, and sterilizing in 75% alcohol for later use;

(2) Fixing the mice in a biosafety cabinet, dissecting the abdomen of the mice by using sterile medical scissors and forceps, removing viscera, observing long dark red tissues which are about 3-4cm long, namely spleens, taking out carefully, and soaking the isolated spleen tissues in clean PBS solution;

(3) Placing a 100 μm cell screen on a 50ml centrifuge tube, placing a spleen on the screen, adding 8ml of RPMI1640 complete culture medium for infiltration, grinding the spleen by using a 20ml syringe handle in a pressing mode, filtering the spleen cells by using 7ml of RPMI1640 after the grinding is complete, then washing the cell screen by using 15ml of RPMI1640, and centrifuging the collected spleen cell suspension at 2000rpm for 10min;

(3) Centrifuging, removing supernatant, adding 10ml of erythrocyte lysate to resuspend cells, standing at room temperature for 10min to lyse the erythrocytes, centrifuging at 2000rpm for 10min, and repeating the steps once;

(4) The cells were resuspended in 1640 medium containing 10% serum and centrifuged at 2000rpm for 10min at room temperature;

(5) Cells were resuspended in 1640 medium containing 10% serum, diluted 10-fold for cell counting, and cell density was adjusted to 2.5X106 cells/mL for use.

2.2 IFN-gamma and IL-4ELISAPOT assays

(1) 200 μl/well of 1640 complete medium was added to IL-4 or IFN- γ coated antibody ELISAPOT plates, incubated for 30min, and discarded;

(2) Adding spleen cell suspension (2.5X106/mL), 200 μl/well, and simultaneously adding stimulator (commercial SARS-COV-2RBD protein (Yiqiaoshenzhou, 40592-V08H) with final concentration of 10 μg/mL), wherein 3 mice are selected from each group, 1 mouse has 3 compound holes, and a control hole without stimulator is set, and culturing at 37deg.C and 5% CO 2 for 48 hr;

(3) The cell suspension in the wells was discarded, washed 5 times with 200 μl/well each for 2min with sterile PBS; the antibody BVD6-24G2-biotin or R4-6A2-biotin was diluted with PBS containing 0.5% FBS to a final concentration of 1. Mu.g/ml, 200. Mu.l/well and incubated for 2h at room temperature;

(4) The wells were discarded, washed 5 times with 200. Mu.l/well in sterile PBS and incubated with PB S solution containing 0.5% FBS 1:1000 times diluted with strepavidin-ALP or strepavidin-HRP, 100. Mu.l/well for 2min at room temperature for 1h;

(5) Removing liquid in the hole, washing for 5 times with sterile PBS (phosphate buffer solution), 200 mu l/hole, filtering BCIP/NBT substrate solution with a 0.45 mu m filter for 2min each time, developing in a dark place, and after clear blue spots appear at the bottom of the hole, washing with flowing water to stop developing;

(6) The plates were air-dried at room temperature in the dark, and Spot for-minute cells (SFCs) were counted by an enzyme-linked Spot image automatic analyzer.

To evaluate the in vitro memory effect of spleen lymphocytes, INF-gamma and IL-4 secreted by spleen lymphocytes after in vitro stimulation were measured at the cellular level using ELISPot method, and the results are shown in FIG. 10. After spleen cells are stimulated by commercial SARS-COV-2RBD protein (Yinqiao Shenzhou, 40592-V08H), the number of INF-gamma and IL-4 spots formed by immunized mice is different from that of a control group, and the difference has statistical significance (p < 0.05). INF-gamma is a Th1 type cytokine, involved in the antiviral action of cellular immune responses, and IL-4 is a cytokine produced by Th2 cells, associated with humoral immune responses. In conclusion, recombinant influenza virus rescue strain rIBV-NS110-RBD can produce Th1 and Th2 cytokines after immunization of mice, thereby enhancing cellular and humoral immune responses and regulating overall immune responses.

4. Toxicity attack protection experiment for mice

As shown in FIG. 11, the immunoprotection experiments of the mice were performed in a biosafety tertiary laboratory after single and double immunization with rIBV-NS110-RBD-106EID 50, using 6-8 month old BALB/c female mice.

The immunoprotection experiments were divided into 4 groups of 8 mice each. The mice were observed continuously for 14 days after challenge and body weights were recorded. On day 3 after challenge, 3 mice from each group were dissected and lung removed. The obtained lung tissue is used for measuring the virus load and pathological sections of the lung respectively.

The results are shown in FIG. 12. In immunoprotection experiments, there was a significant difference in viral load in the lungs of immunized mice versus control; after continuous observation for 7 days, the weight of the mice is slowly reduced; mice in the control group all died on day 4 post challenge, with the lethal model control established.

In the double immunoprotection experiments, immunized mice survived, with 100% protection. Mice in the control group all died on day 4 post challenge, with the lethal model control established.

The invention discloses a SARS-CoV-2 vaccine candidate strain using B type influenza virus as carrier, its construction method and application, and the person skilled in the art can use this content to properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the invention has been described with reference to preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the invention described herein without departing from the spirit or scope of the invention.

Sequence listing

<110> military medical and veterinary institute of the military academy of sciences

<120> novel coronavirus vaccine candidate strain using influenza B virus as vector, construction method and application thereof

<160> 10

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attttggagt gctacgaaag gctttcatgg caaagagccc ttgactaccc tggtcaagac 180

cgcctaaaca gactaaagag aaaattagaa tcaagaataa agactcacaa caaaagtgag 240

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aacatattgt tccacaaaac agtaatagcc aacagctcca taatagctga catgattgta 900

tcattatcat tattggaaac attgtatgaa atgaaggatg tggttgaagt gtacagcagg 960

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attttggagt gctacgaaag gctttcatgg caaagagccc ttgactaccc tggtcaagac 180

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cctgaaagta aaaggatgtc tcttgaagag agaaaagcta ttggagtaaa aatgatgaaa 300

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gctttactaa tgtctatgca gattcatttg taattagagg tgatgaagtc agacaaatcg 660

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gctgcgttat agcttggaat tctaacaatc ttgattctaa ggttggtggt aattataatt 780

acctgtatag attgtttagg aagtctaatc tcaaaccttt tgagagagat atttcaactg 840

aaatctatca ggccggtagc acaccttgta atggtgttga aggttttaat tgttactttc 900

ctttacaatc atatggtttc caacccacta atggtgttgg ttaccaacca tacagagtag 960

tagtactttc ttttgaactt ctacatgcac cagcaactgt ttgtggacct aaaaagtcta 1020

ctaatttggt taaaaacaaa tgtgtcaatt tctaaaaata gtattaaggg acatgaacaa 1080

caaagatgca aggcaaaaga taaaagagga agtaaacact cagaaagaag ggaaattccg 1140

tttgacaata aaaagggata tacgtaatgt gttgtccttg agagtgttgg taaacggaac 1200

attcatcaag caccctaatg gatacaagtc cttatcaact ctgcatagat tgaatgcata 1260

tgaccagagt ggaagacttg ttgctaaact tgttgctact gatgatctta cagtggagga 1320

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taagaaaatt caatacatca agaattgaaa agaacccttc attaaggatg aagtgggcaa 180

tgtgttctaa ttttcccttg gctctgacca agggtgatat ggcaaacaga atccccttgg 240

aatacaaggg aatacaactt aaaacaaatg ctgaagacat aggaaccaaa ggccaaatgt 300

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taatgggggc taatgacgta agtgaattag aatcacaagc tcagctaatg ataacatatg 1620

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accagttcat aaagttgttg cccttttgtt tctcaccacc aaaattaagg agcaatgggg 1920

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acgggaacag gccacacaat agacaccgtg atcagaacac atgagtactc gaacaaggga 180

aaacagtacg tttctgacgt cacaggatgt acaatggtag atccaacaaa tgggccatta 240

cccgaagaca atgagccgag tgcctatgca caattagatt gcgttctgga ggctttggat 300

agaatggatg aagaacatcc aggtctgttt caagcagcct cacagaatgc catggaggca 360

ctaatggtca caactgtaga caaattaacc caggggagac agacttttga ttggacagta 420

tgcagaaacc agcctgctgc aacggcacta aacacaacaa taacctcctt taggttgaat 480

gatttgaatg gagctgacaa gggtggattg gtaccctttt gccaagatat cattgattca 540

ttggacaaac ctgaaatgac tttcttctca gtaaagaata taaagaaaaa attgcctgct 600

aaaaacagaa agggtttcct cataaagaga ataccaatga aagtaaaaga cagaataacc 660

agagtggaat acatcaaaag agcattgtca ttaaacacaa tgacaaaaga tgctgaaagg 720

ggcaaactaa aaagaagagc gattgcaacc gctggaatac aaatcagagg gtttgtaata 780

gtagttgaaa acttggctaa aaatatctgt gaaaatctag aacaaagtgg tttgcccgta 840

ggtggaaacg aaaagaaggc caaactgtca aatgcagtgg ccaaaatgct cagtaactgc 900

ccaccaggag ggatcagcat gacagtaaca ggagacaata ctaaatggaa tgaatgctta 960

aatccaagaa tctttttggc tatgactgaa agaataacca gagacagccc aatttggttc 1020

cgggattttt gtagtatagc accggtcttg ttctccaata aaatagccag attgggaaaa 1080

ggatttatga taacaagcaa aacaaaaaga ctgaaggctc aaataccttg tcctgatctg 1140

tttagcatac cattagaaag atataatgaa gaaacaaggg caaaattgaa aaagctgaaa 1200

ccattcttca atgaagaagg aacggcatct ttgtcgcctg ggatgatgat gggaatgttt 1260

aatatgctat ctaccgtgtt gggagtagcc gcactaggta tcaaaaacat tggaaacaaa 1320

gaatacttat gggatggact gcaatcttct gatgattttg ctctgtttgt taatgcaaaa 1380

gatgaagaga catgtatgga aggaataaac gacttttacc gaacatgtaa attattggga 1440

ataaacatga gcaaaaagaa aagttactgt aatgaaactg gaatgtttga atttacaagc 1500

atgttctata gagatggatt tgtatctaat tttgcaatgg aaattccttc atttggagtt 1560

gctggagtaa atgaatcagc agatatggca ataggaatga caataataaa gaacaatatg 1620

atcaacaatg ggatgggtcc agcaacagca caaacagcca tacaattatt catagctgat 1680

tataggtaca cctacaaatg ccacagggga gattccaaag tggaaggaaa aagaatgaaa 1740

attataaagg agctatggga aaacactaaa ggaagagatg gtctgttagt agcagatggt 1800

gggcctaaca tttacaattt gagaaactta catatcccag aaatagtatt gaagtacaac 1860

ctaatggacc ctgaatacaa agggcgatta cttcatcctc aaaatccctt tgtaggacat 1920

ttgtctattg agggcatcaa agaagcagat ataaccccag cacatggtcc cgtaaagaaa 1980

atggattatg atgcagtgtc tggaactcat agttggagaa ccaaaaggaa cagatctata 2040

ctaaatac 2048

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<211> 2048

<212> DNA

<213> human (Fragment)

<400> 6

agcagaagcg gtgcgtttga tttgccataa tggatacttt tattacaaga aacttccaga 60

ctacaataat acaaaaggcc aaaaacacaa tggcagaatt tagtgaagat cctgaattac 120

aaccagcaat gctattcaac atctgcgtcc atctagaggt ttgctatgta ataagtgaca 180

tgaattttct tgacgaagaa ggaaaagcat atacagcatt agaaggacaa ggaaaagaac 240

aaaacttgag accacaatat gaagtaattg agggaatgcc aagaaacata gcatggatgg 300

tccaaagatc cttagctcaa gagcatggaa tagagactcc aaagtatctg gctgatttgt 360

ttgattataa aaccaagagg tttatagaag ttggaataac aaaaggattg gctgatgatt 420

acttttggaa aaagaaagaa aagctgggaa atagcatgga actgatgata ttcagctaca 480

atcaagacta ttcgttaagt aatgaatcct cattggatga ggaagggaaa gggagagtgc 540

taagcagact cacagaactt caggctgaat taagtctgaa aaacctatgg caagttctca 600

taggagaaga agatgttgaa aagggaattg actttaaact tggacaaaca atatctagac 660

taagggatat atctgttcca gctggtttct ccaattttga aggaatgagg agctacatag 720

acaatataga tcctaaagga gcaatagaga gaaatctagc aaggatgtct cccttagtat 780

cagccacacc taaaaagttg aaatgggagg acctaagacc aatagggcct cacatttaca 840

accatgagct accagaagtt ccatataatg cctttcttct aatgtctgat gaattggggc 900

tggccaatat gactgaggga aagtccaaaa aaccgaagac attagccaaa gaatgtctag 960

aaaagtactc aacactacgg gatcaaactg acccaatatt aataatgaaa agcgaaaaag 1020

ctaacgaaaa cttcctatgg aagctgtgga gggactgtgt aaatacaata agtaatgagg 1080

aaatgagtaa cgagttacag aaaaccaatt atgccaagtg ggccacagga gatggattaa 1140

cataccagaa aataatgaaa gaagtagcaa tagatgacga aacaatgtgc caagaagagc 1200

ctaaaatccc taacaaatgt agagtggctg cttgggttca aacagagatg aatctattga 1260

gcaatctgac aagtaaaaga gctctggacc taccagaaat agggccagac gtagcacccg 1320

tggagcatgt agggagtgaa agaaggaaat actttgttaa tgaaatcaac tactgtaagg 1380

cctccacagt tatgatgaag tatgtgcttt ttcacacttc attgttgaat gaaagcaatg 1440

ccagcatggg aaaatataaa gtaataccaa taaccaatag agtagtaaat gaaaaaggag 1500

aaagtttcga catgctttat ggtctggcgg ttaaaggaca atctcatctg aggggagata 1560

ctgatgttgt aacagttgtg actttcgaat ttagtagtac agatcccaga gtggactcag 1620

gaaagtggcc aaaatatact gtgtttagga ttggctccct atttgtgagt gggagggaaa 1680

aatctgtgta cctatattgc cgagtgaatg gcacaaataa gatccaaatg aaatggggaa 1740

tggaagctag aagatgtctg cttcaatcaa tgcaacaaat ggaagcaatt gttgaacaag 1800

aatcatcgat acaaggatat gacatgacca aagcttgttt caagggagac agagtaaata 1860

gccccaaaac tttcagtatt gggactcaag aaggaaaact agtaaaagga tcctttggga 1920

aagcactaag agtaatattt accaaatgtt tgatgcacta tgtatttgga aatgcccaat 1980

tggaggggtt tagtgccgag tctaggagac ttctactgtt aattcaagca ttaaaggaca 2040

gaaagggc 2048

<210> 7

<211> 1879

<212> DNA

<213> human (Fragment)

<400> 7

agcagaagca gagcattttc taatatccac aaaatgaagg caataattgt actactcatg 60

gtagtaacat ccaacgcaga tcgaatctgc actgggataa catcttcaaa ctcacctcat 120

gtggtcaaaa cagctactca aggggaagtt aatgtgactg gtgtgatacc actgacaaca 180

acaccaacaa aatctcattt tgcaaatctc aaaggaacaa agaccagagg gaaactatgc 240

ccaaactgtc tcaactgcac agatctggat gtggccttgg gcagaccaat gtgtatgggg 300

accatacctt cggcaaaagc ttcaatactc cacgaagtca gacctgttac atccgggtgc 360

tttcctataa tgcacgacag aacaaaaatc agacagctac ccaatcttct cagaggatat 420

gaaaatatca gattatcaac ccataacgtt atcaacgcag aaagggcacc aggaggaccc 480

tacagacttg gaacctcagg atcttgccct aacgttacca gtagaaacgg attcttcgca 540

acaatggctt gggctgtccc aagggacaac aaaacagcaa cgaatccact aacagtagaa 600

gtaccataca tttgcacaaa aggagaagac caaattactg tttgggggtt ccattctgat 660

gacaaaaccc aaatgaaaaa cctctatgga gactcaaatc ctcaaaagtt cacctcatct 720

gccaatggag taaccacaca ttatgtttct cagattggtg acttcccaaa tcaaacagaa 780

gacggagggc taccacaaag cggcagaatt gttgttgatt acatggtgca aaaacctggg 840

aaaacaggaa caattgtcta tcaaagaggt gttttgttgc ctcaaaaggt gtggtgcgca 900

agtggcagga gcaaggtaat aaaagggtcc ttgcctttaa ttggtgaagc agattgcctt 960

cacgaaaaat acggtggatt aaacaaaagc aagccttact acacaggaga acatgcaaaa 1020

gccataggaa attgcccaat atgggtgaaa acacctttga agcttgccaa tggaaccaaa 1080

tatagacctc ctgcaaaact attaaaggaa aggggtttct tcggagctat tgctggtttc 1140

ttagagggag gatgggaagg aatgattgca ggttggcacg gatacacatc tcatggagca 1200

catggagtgg cagtggcagc agaccttaag agcacgcaag aagccataaa caagataaca 1260

aaaaatctca attctttgag tgagctagaa gtaaagaatc ttcaaagact aagtggtgcc 1320

atggatgaac tccacaacga aatactcgag ctggatgaga aagtggatga tctcagagct 1380

gacacaataa gctcgcaaat agagcttgca gtcttgcttt ccaacgaagg aataataaac 1440

agtgaagatg agcatctatt ggcacttgag agaaaactaa agaaaatgct gggtccctct 1500

gctgtagaca tagggaatgg atgcttcgaa accaaacaca agtgcaacca gacctgctta 1560

gacaggatag ctgctggcac ctttaatgca ggagaatttt ctcttcccac ttttgattca 1620

ctgaatatta ctgctgcatc tttaaatgat gatggattgg ataatcatac tatactgctc 1680

tactactcaa ctgctgcttc tagtttggcc gtaacattga tgatagctat ttttattgtt 1740

tatatggtct ccagagacaa tgtttcttgc tccatctgtc tataaggaaa attaagccct 1800

gtattttcct ttattgtagt gcttgtttgc ttgttaccat tacaaagaaa cgttattgaa 1860

aaatgctctt gttactact 1879

<210> 8

<211> 1557

<212> DNA

<213> human (Fragment)

<400> 8

agcagaagca gagcatcttc tcaaaactga ggcaaatagg ccaaaaatga acaatgctac 60

cttcaactat acaaacgtta accctatttc tcacatcagg gggagtgtta ttatcactat 120

atgtgtcagc ttcactgtca tacttactgt attcggatat attgctaaaa ttttcaccaa 180

cagaaataac tgcaccaaaa gtgccattgg attgtgcaaa cgcatcaaat gttcaggctg 240

tgaaccgttc tgcaacaaaa gggatgacac ttcttctctc agaaccggag tggacatacc 300

ctcgtttatc ttgccagggc tcaacctttc agaaagcact cctaattagc cctcatagat 360

tcggagaaac cagaggaaac tcagctccct tgataataag ggaacctttt attgcttgtg 420

gaccaaagga atgcaaacac tttgctctaa cccattatgc agctcaacca gggggatact 480

acaatggaac aagagaggac agaaacaagc tgaggcatct gatttcagtc aaattgggca 540

aaatcccaac agtagaaaac tccattttcc acatggcagc ttggagcggg tccgcatgcc 600

atgatggtag agaatggaca tatatcggag ttgatggccc tgacagtaat gcattgatca 660

aaataaaata tggagaagca tatactgaca cataccattc ctatgcaaac aacatcctaa 720

gaacacaaga aagtgcctgc aattgcatcg ggggagattg ttatcttatg ataactgatg 780

gctcagcttc aggaattagt aaatgcagat ttcttaagat tcgagagggt cgaataataa 840

aagaaatatt tccaacagga agagtagaac atactgaaga atgcacatgc ggatttgcca 900

gcaataaaac catagaatgt gcctgtagag ataacagtta cacagcaaaa agaccctttg 960

tcaaattaaa tgtggagact gatacagctg aaataagatt gatgtgcaca gagacttatt 1020

tggacacccc cagaccagat gatggaagca taacagggcc ttgcgaatct aatggggaca 1080

aagggcgtgg aggcatcaag ggaggatttg ttcatcaaag aatggcatcc aagattggaa 1140

gatggtactc tcgaacgatg tctaaaactg aaagaatggg gatggaactg tatgtcaagt 1200

atgatggaga cccatggact gacagtgacg cccttgctcc tagtggagta atggtttcaa 1260

tgaaagaacc tggttggtat tcctttggct tcgaaataaa agataagaaa tgtgatgtcc 1320

cctgtattgg gatagagatg gtacatgatg gtggaaaaaa gacttggcac tcagcagcaa 1380

cagccattta ctgtttaatg ggctcaggac aattgctatg ggacactgtc acaggtgttg 1440

atatggctct gtaatggagg aatggttgag tctgttctaa accctttgtt cctattttgt 1500

ttgaacaatt gtccttactg aacttaattg tttctcaaaa atgctcttgt tactact 1557

<210> 9

<211> 1844

<212> DNA

<213> human (Fragment)

<400> 9

agcagaagca cagcattttc ttgtgaactt caagtaccaa caaaagaact gaaaatcaaa 60

atgtccaaca tggatattga cggtatcaac actgggacaa ttgacaaaac accggaagaa 120

ataacttctg gaaccagtgg gacaaccaga ccaatcatca gaccagcaac ccttgcccca 180

ccaagcaaca aacgaacccg gaacccatcc ccggaaagag caaccacaag cagtgaagct 240

gatgtcggaa ggaaaaccca aaagaaacag accccgacag agataaagaa gagcgtctac 300

aatatggtag tgaaactggg tgaattctat aaccagatga tggtcaaagc tggactcaac 360

gatgacatgg agagaaacct aatccaaaat gcgcatgctg tggaaagaat tctattggct 420

gccactgatg acaagaaaac tgaattccag aagaaaaaga atgccagaga tgtcaaagaa 480

gggaaagaag aaatagacca caacaaaaca ggaggcacct tttacaagat ggtaagagat 540

gataaaacca tctacttcag ccctataaga attacctttt taaaagaaga ggtgaaaaca 600

atgtacaaaa ccaccatggg gagtgatggt ttcagtggac taaatcacat aatgattggg 660

cattcacaga tgaatgatgt ctgtttccaa agatcaaagg cactaaaaag agttggactt 720

gacccttcat taatcagtac ctttgcagga agcacactcc ccagaagatc aggtgcaact 780

ggtgttgcga tcaaaggagg tggaacttta gtggctgaag ccattcgatt tataggaaga 840

gcaatggcag acagagggct attgagagac atcaaagcca agactgccta tgaaaagatt 900

cttctgaatc taaaaaacaa gtgctctgcg ccccaacaaa aggctctagt tgatcaagtg 960

atcggaagta gaaatccagg gattgcagac attgaagacc taaccctgct tgctcgtagt 1020

atggtcgttg ttaggccctc tgtggcgagc aaagtagtgc ttcccataag catttatgct 1080

aaaatacctc aactagggtt caatgttgaa gaatactcta tggttgggta tgaagccatg 1140

gctctttaca atatggcaac acctgtttcc atattaagaa tgggagatga tgcaaaagat 1200

aaatcgcaat tattcttcat gtcttgcttc ggagctgcct atgaagacct gagagttttg 1260

tctgcattaa caggcacaga attcaagcct agatcagcat taaaatgcaa gggtttccat 1320

gttccagcaa aggaacaggt ggaaggaatg ggggcagctc tgatgtccat caagctccag 1380

ttttgggctc caatgaccag atctgggggg aacgaagtag gtggagacgg agggtctggc 1440

caaataagtt gcagcccagt gtttgcagta gaaagaccta ttgctctaag caagcaagct 1500

gtaagaagaa tgctgtcaat gaatattgag ggacgtgatg cagatgtcaa aggaaatcta 1560

ctcaagatga tgaatgactc aatggctaag aaaaccaatg gaaatgcttt cattgggaag 1620

aaaatgtttc aaatatcaga caaaaacaaa accaatcccg ttgaaattcc aattaagcag 1680

accatcccca atttcttctt tgggagggac acagcagagg attatgatga cctcgactat 1740

taaagcaaca aaatagacac tatgactgtg attgtttcag tacgtttgga atgtgggtgt 1800

ttactcttat tgaaataaat ataaaaaatg ctgttgtttc tact 1844

<210> 10

<211> 1190

<212> DNA

<213> human (Fragment)

<400> 10

agcagaagca cgcactttct taaaatgtcg ctgtttggag acacaattgc ctacctgctt 60

tcattgacag aagatggaga aggcaaagca gaactagcag aaaaattaca ctgttggttc 120

ggtgggaaag aatttgacct agactctgcc ttggaatgga taaaaaacaa aagatgctta 180

actgatatac agaaagcact aattggtgcc tctatctgct ttttaaaacc caaagaccaa 240

gaaagaaaaa gaagattcat cacagagccc ctatcaggaa tggggacaac agcaacaaaa 300

aagaaaggcc tgattctagc tgagagaaaa atgagaagat gtgtgagttt tcatgaagca 360

tttgaaatag cagaaggcca tgaaagctca gcgctactat attgtctcat ggtcatgtac 420

ctgaaccctg gaaattattc aatgcaagta aaactaggaa cgctctgtgc tttgtgcgag 480

aaacaagcat cacattcaca cagggctcat agcagagcag caagatcttc agtgcctgga 540

gtgaggcgag aaatgcagat ggtctcagct atgaacacag caaaaacaat gaatggaatg 600

ggaaagggag aagacgtcca aaaactggca gaagagctgc aaagcaacat tggagtattg 660

agatctcttg gggcaagtca aaagaatggg gaaggaattg caaaggatgt gatggaagtg 720

ctaaagcaga gctctatggg aaattcagct cttgtgaaga aatacctata atgctcgaac 780

catttcagat tctttcaatt tgttctttca tcttatcagc tctccatttc atggcttgga 840

caatagggca tttgaatcaa ataaaaagag gagtaaacat gaaaatacga ataaaaaatc 900

caaataaaga gacaataaac agagaggtat caattttgag acacagttac caaaaagaaa 960

tccaggccaa agaaacaatg aaggaagtac tctctgacaa catggaggta ttgagtgacc 1020

acatagtaat tgaggggctt tctgctgaag agataataaa aatgggtgaa acagttttgg 1080

aggtagaaga attgcattaa attcaatttt tactgtattt cttgctatgc atttaagcaa 1140

attgtaatca atgtcagcaa ataaactgga aaaagtgcgt tgtttctact 1190

Claims (4)

1. A SARS-CoV-2 vaccine candidate strain using B type influenza virus as carrier is characterized by constructing recombinant gene fragment as shown in SEQ ID NO. 3; the recombinant influenza virus rescue strain which takes the attenuated strain B/Yamagata/16/88 of the influenza B virus as a framework and stably expresses the receptor binding domain gene fragment on the S protein of the SARS-CoV-2 reference strain is obtained by utilizing the reverse genetics technology and the recombinant plasmid NS110-RBD rescue;

the recombinant plasmid NS110-RBD represents a recombinant plasmid containing a recombinant gene fragment shown as SEQ ID NO. 3.

2. The SARS-CoV-2 vaccine candidate strain using influenza B virus as the carrier of claim 1, wherein the sequence of the receptor binding domain gene fragment on the S protein of the SARS-CoV-2 reference strain is as shown in SEQ ID NO. 2.

3. The method for constructing a candidate strain of SARS-CoV-2 vaccine using influenza B virus as a vector according to claim 1 or 2, comprising the steps of: step one, constructing a recombinant plasmid NS110-RBD;

step two, rescue and virus seed preparation of the recombinant influenza virus.

4. Use of a SARS-CoV-2 vaccine candidate strain with influenza B virus as vector according to claim 1 or 2 for the preparation of a nasal spray SARS-CoV-2 vaccine for humans.