阅读说明：本技术 一种基于棘突蛋白基因修饰干细胞的新型冠状病毒疫苗 (Novel coronavirus vaccine based on spinous process protein gene modified stem cells ) 是由 张建立 曾琼 霍凯兵 于 2020-08-18 设计创作，主要内容包括：本发明涉及疫苗技术领域,公开了一种基于棘突蛋白基因修饰干细胞的新型冠状病毒疫苗,其活性成分为三聚体形式的棘突蛋白基因修饰的间充质干细胞,可应用于预防新型冠状病毒；同时公开了一种基于棘突蛋白基因修饰干细胞的新型冠状病毒疫苗的制备方法：构建表达棘突蛋白的腺病毒载体、制备棘突蛋白重组腺病毒颗粒、纯化富集腺病毒颗粒、获取间充质干细胞、获取棘突蛋白编码基因修饰的人脐带间充质干细胞。本发明的疫苗通过棘突蛋白的基因重组型间充质干细胞表达棘突蛋白来启动人体针对新型冠状病毒的免疫反应,产生病毒中和性抗体,从而避免病毒对人体的感染,安全、可靠、有效,具有良好的应用前景,为新型冠状病毒的预防提供了全新的选择。(The invention relates to the technical field of vaccines, and discloses a novel coronavirus vaccine based on a spinous process protein gene modified stem cell, wherein the active component of the novel coronavirus vaccine is a trimeric form spinous process protein gene modified mesenchymal stem cell, and the novel coronavirus vaccine can be applied to prevention of novel coronavirus; simultaneously discloses a preparation method of the novel coronavirus vaccine based on the spinous process protein gene modified stem cells, which comprises the following steps: the method comprises the steps of constructing an adenovirus vector for expressing the spinous process protein, preparing spinous process protein recombinant adenovirus particles, purifying and enriching the adenovirus particles, obtaining mesenchymal stem cells and obtaining the human umbilical cord mesenchymal stem cells modified by the encoding gene of the spinous process protein. The vaccine provided by the invention starts the immune reaction of a human body to the novel coronavirus through expressing the spinous process protein by the genetic recombinant mesenchymal stem cell of the spinous process protein, and generates a virus neutralizing antibody, so that the infection of the virus to the human body is avoided, and the vaccine is safe, reliable and effective, has a good application prospect, and provides a brand-new choice for the prevention of the novel coronavirus.)

1. A novel coronavirus vaccine based on a spinous process protein gene modified stem cell, which is characterized in that: the active component of the novel coronavirus vaccine is the trimeric form interspinous protein gene modified mesenchymal stem cells, and the novel coronavirus vaccine can be applied to preventing novel coronaviruses.

2. The novel coronavirus vaccine based on the modified stem cells of the spinous process protein gene as described in claim 1, characterized in that: the mesenchymal stem cell is an umbilical cord mesenchymal stem cell, is modified by a spinous process protein coding gene obtained by virtue of infection of the spinous process protein recombinant adenovirus, can express the spinous process protein, and is expressed on the surface of the cell in a trimer form.

3. The novel coronavirus vaccine based on the modified stem cells of the spinous process protein gene as described in claim 2, characterized in that: the spinous process protein is regulated and expressed by a CMV promoter of an adenovirus expression vector pAd/CMV/V5-DEST.

4. A novel coronavirus vaccine based on a spinous process protein gene-modified stem cell according to any one of claims 2 to 3, wherein: the spinous process protein is the same as the surface glycoprotein of the novel coronavirus, and the coding nucleotide sequence of the surface glycoprotein of the novel coronavirus is Seq ID No. 1.

5. A method for preparing a novel coronavirus vaccine based on a spinous process protein gene-modified stem cell according to claim 1, comprising the steps of:

(1) constructing an adenovirus vector for expressing the spinous process protein: respectively constructing adenovirus vectors for expressing the spinous process proteins by free recombination;

(2) preparing spinous process protein recombinant adenovirus particles: respectively transfecting human embryonic kidney cells HEK293T with the adenovirus vectors in the step (1) to prepare adenovirus particles;

(3) purifying and enriching adenovirus particles: purifying and enriching the adenovirus particles capable of expressing the spinous process protein in the step (2);

(4) obtaining mesenchymal stem cells: isolating mesenchymal stem cells from the umbilical cord, selecting mesenchymal stem cells in logarithmic growth phase at passage 2-3;

(5) obtaining human umbilical cord mesenchymal stem cells modified by spinous process protein coding genes: and (4) when the mesenchymal stem cells in the step (4) grow to 70-80% of confluence, infecting the mesenchymal stem cells by using the enriched adenovirus particle mixture.

Technical Field

The invention relates to the technical field of vaccines, in particular to a novel coronavirus vaccine based on a spinous process protein gene modified stem cell.

Background

The 2019-nCov virus infected person generally has symptoms of fever, hypodynamia, dry cough and headache, gradually causes dyspnea, and the severe person rapidly progresses to acute respiratory distress syndrome, septic shock, metabolic acidosis which is difficult to correct, coagulation dysfunction and even death. The virus has the shortest latent period of 1 day, the longest latent period is 14 days, the latent period has infectivity, and no specific effective treatment method for the diseases is available at present.

However, there are currently no particularly effective treatments, primarily isolation followed by aerosol inhalation of interferon-alpha, oral administration of lopinavir/ritonavir, and short-term use of glucocorticoids in patients. However, these treatments are not very effective. Pulmonary fibrosis (pulmonary fibrosis) caused by viral infection is also an important clinical manifestation in the pathogenesis, and the pulmonary fibrosis can cause irreversible damage to the lung, can cause progressive dyspnea, and can cause the damage to the lung function, and even death of serious patients.

Mesenchymal Stem Cells (MSCs) are important members of the stem cell family, originating from the early-developing mesoderm and belonging to the multipotent stem cell family, and MSCs were initially found in the bone marrow and subsequently found in many tissues during the development and development of the human body. The human body has attracted more and more attention because of its features such as multipotentiality, hematopoietic support, promotion of stem cell implantation, immune regulation and self-replication.

It was found that MSCs are a cell with differentiation potential in the early developmental stage of the larvae, and have several properties: 1) Has strong proliferation capacity and multidirectional differentiation potential, and has the capacity of differentiating into various cells such as muscle cells, liver cells, osteoblasts, fat cells, cartilage cells, stromal cells and the like under a proper in vivo or in vitro environment; 2) has the function of immunoregulation, and plays the role of immune reconstruction by inhibiting the proliferation of T cells and the immune reaction thereof through the interaction among cells and the generation of cytokines; 3) chemotaxis: MSCs can migrate to damaged tissues, tumor sites; 4) the stem cell has the advantages of convenient source, easy separation, culture, amplification and purification, and stem cell characteristics after multiple passage amplification; 5) because of no expression of major histocompatibility complex, almost no immune rejection exists, the immunologic rejection of the xenotransplantation is light, and the mating requirement is not strict. Due to the unique immunological characteristics of the mesenchymal stem cells, the mesenchymal stem cells have wide clinical application prospect in the aspect of clinical treatment.

Disclosure of Invention

Aiming at the defects in the prior art, the technical problem to be solved by the invention is to provide a novel coronavirus vaccine based on a spinous process protein gene modified stem cell, wherein the stem cell can be retained in a human body for a long time, efficiently secretes novel coronavirus spinous process proteins, starts the immune response of the human body to the novel coronavirus, generates a virus neutralizing antibody, and avoids the infection of the virus to the human body.

In order to solve the technical problem, the invention is realized by the following scheme: a novel coronavirus vaccine based on a spike protein gene modified stem cell, wherein the active ingredient of the novel coronavirus vaccine is a trimer-form spike protein gene modified mesenchymal stem cell, and the novel coronavirus vaccine can be applied to prevention of novel coronavirus.

Furthermore, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell, and the spinous process protein coding gene modification is obtained by virtue of the infection of the spinous process protein recombinant adenovirus, so that the spinous process protein can be expressed and expressed on the surface of the cell in a trimer form.

Further, the spinous process protein is regulated and expressed by a CMV promoter of an adenovirus expression vector pAd/CMV/V5-DEST.

Further, the spinous process protein is the same as the surface glycoprotein of the novel coronavirus, and the nucleotide sequence encoding the surface glycoprotein of the novel coronavirus is Seq ID No. 1.

Another object of the present invention is to provide a method for preparing a novel coronavirus vaccine based on a spinous process protein gene-modified stem cell, the method comprising the steps of:

(1) constructing an adenovirus vector for expressing the spinous process protein: respectively constructing adenovirus vectors for expressing the spinous process proteins by free recombination;

(2) preparing spinous process protein recombinant adenovirus particles: respectively transfecting human embryonic kidney cells HEK293T with the adenovirus vectors in the step (1) to prepare adenovirus particles;

(3) purifying and enriching adenovirus particles: purifying and enriching the adenovirus particles capable of expressing the spinous process protein in the step (2);

(4) obtaining mesenchymal stem cells: isolating mesenchymal stem cells from the umbilical cord, selecting mesenchymal stem cells in logarithmic growth phase at passage 2-3;

(5) obtaining human umbilical cord mesenchymal stem cells modified by spinous process protein coding genes: and (4) when the mesenchymal stem cells in the step (4) grow to 70-80% of confluence, infecting the mesenchymal stem cells by using the enriched adenovirus particle mixture.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention combines cell therapy and gene therapy, takes the spinous process protein modified mesenchymal stem cells in the form of tripolymer as the active ingredients of the vaccine, has the advantage of convenient acquisition, can supplement the number of local stem cells, efficiently secretes the tripolymer spinous process protein and human growth factors (such as EGF, bFGF and the like), improves local microenvironment and immunoregulation, stimulates an organism to generate a neutralizing antibody aiming at the novel coronavirus, is safe, reliable and effective, and has good application prospect.

(2) The invention enables the umbilical cord-derived mesenchymal stem cells to efficiently express the trimeric spinous process protein by means of gene modification, and immunizes individuals in a natural form closest to the virus spinous process protein, thereby effectively defending the virus.

(3) The invention separates the mesenchymal stem cells from the umbilical cord, can greatly expand the number of cells for treatment, overcomes the defects of less mesenchymal stem cells obtained from bone marrow, traumatic material acquisition and the like, and reduces the production and use cost.

Drawings

FIG. 1 is a schematic diagram of the domains of a novel coronavirus spike protein according to an embodiment of the present invention;

FIG. 2 is a diagram showing an RBD regional antigenicity analysis of the novel coronavirus spike protein of the present invention;

FIG. 3 is a schematic structural diagram of an adenovirus vector pAD/CMV/Spike according to an embodiment of the present invention;

FIG. 4 is a schematic diagram of a process for preparing a novel coronavirus vaccine according to an embodiment of the invention;

Detailed Description

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that the advantages and features of the invention can be more readily understood by those skilled in the art, and the scope of the invention will be more clearly and clearly defined.

Example 1

The active component of the novel coronavirus vaccine is the interspecific stem cell modified by the spinous process protein gene in the form of trimer, and the novel coronavirus vaccine can be applied to prevention of novel coronavirus.

The mesenchymal stem cells are umbilical cord mesenchymal stem cells, are subjected to spinous process protein coding gene modification through spinous process protein recombinant adenovirus infection, can express the spinous process protein, and are expressed on the surface of the cells in a trimer form.

Referring to the attached figures 1-2, the spinous process protein is Surface glycoprotein (Surface glycoprotein) of the novel coronavirus, the protein does not contain a TMPRSS2 protease cleavage site, the codon of the protein is optimized, the protein can be efficiently translated in a eukaryotic cell, the specific nucleotide sequence is shown as Seq ID No.1, and the amino acid sequence of the novel coronavirus is as follows: RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKC YGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQP TNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKF LPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVP VAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPR RARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGD STECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILP DPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMI AQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAI GKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQI DRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFP QSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEP QIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINA SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP are provided.

Referring to FIG. 3, the expression of the spinous process proteins was regulated by the CMV promoter of the adenovirus expression vector pAd/CMV/V5-DEST. Specifically, the expression sequence for expressing the spinous process protein sequentially comprises a CMV promoter, a codon-optimized spinous process protein gene open reading frame, an HBB gene 3 ' -UTR and a tailing signal (TKpA) of Thymidine Kinase (TK) from the 5 ' end to the 3 ' end.

Referring to fig. 4, the preparation method of the novel coronavirus vaccine based on the spinous process protein gene modified stem cell of the invention comprises the following steps:

(1) constructing an adenovirus vector for expressing the spinous process protein: downloading the gene sequence of the spinous process protein (MN 908947: nt21563-25384) from NCBI GenBank, performing codon optimization on the open reading frame of the spinous process protein by using a GeneOptimizer, introducing the optimized coding sequence of the spinous process protein between BamHI and XhoI enzyme cutting sites of a pENTR1A vector (A10462, ThermoFisher) by an artificial synthesis method, and respectively naming the coding sequence as pENTR1A-_COV19Spike; the spinous process proteins were isolated from pENTR1A-_COV19The Spike was transferred into the pAd/CMV/V5-DEST adenovirus expression vector (V493-20, ThermoFisher), which was designated pAd-CMV-_COV19Spike。

(2) Preparing spinous process protein recombinant adenovirus particles: 5 μ g of pAd-CMV-_COV19Spike digestion, plasmid linearization, isopropanol purification of linearized DNA fragments. Linearized pAd-CMV-_COV19Spike transfection of HEK293 cells; when the transfected HEK293 was cultured and the growth of the cells was observed, cytopathic effect (CPE) was observed after about 2 weeksNow. After transfection for 10-14 days, collecting cell precipitates, adding 2ml of sterilized PBS for suspension, repeatedly freezing and thawing cells, centrifuging, collecting supernatant, and storing at-80 ℃; taking 30-50% of supernatant, infecting 293 cells in a T25 cell culture bottle with 70% confluence, and culturing for 2-3 days to obtain obvious cytopathy; collecting cell precipitate when 1/3 cells float after 3-5 days, repeatedly freezing and thawing after PBS heavy suspension, collecting supernatant after centrifugation, and storing in-80 deg.C environment;

(3) purifying and enriching adenovirus particles: adding 5 mul of virus supernatant into 5 mul of 2 XPBS, boiling for 5 minutes, centrifuging, taking 1-2 mul as a template to perform PCR, detecting the encoding gene of the spinous process protein, and then purifying and enriching the adenovirus particles capable of expressing the spinous process protein in the step (2);

(4) obtaining mesenchymal stem cells: washing 5-10 g of normal umbilical cord tissue with serum-free Low-sugar DMEM (11885092, Low glucose DMEM, ThermoFisher) under aseptic conditions, removing blood stain, cutting umbilical cord tissue into fine particles in a sterilized culture dish, and washing with Low-sugar DMEM for 3 times until the tissue fragments become grey-white. The minced tissue was transferred to a low-sugar DMEM medium containing 1mg/ml Collagenase (C1639, Collagenase, Sigma), completely submerged, and placed in a 37 ℃ cell culture chamber for thorough digestion for 2 hours. Centrifuge at 1000rpm for 5 minutes at room temperature, discard the supernatant and resuspend the pellet in low sugar DMEM. The tissue fragment suspension was transferred to a T25 cell culture flask (430168, Corning), cultured with low-sugar DMEM containing 20% Fetal Bovine Serum (10438026, Total Bovine Serum, ThermoFisher) and placed at 37 ℃ in 5% CO₂Constant temperature cell culture case. The cells were subcultured when they were about 80% confluent by digesting the cells with 0.25% pancreatin. The cultured cells were verified to have the phenotype of mesenchymal stem cells by flow cytometry using cell surface molecular markers (CD14-, CD34-, CD45-, CD73+, CD90+, CD105+) from passage 2 to 3. The umbilical cord mesenchymal stem cells highly express CD73, CD90 and CD105, do not express CD14-, CD 34-and CD45-, so that the cultured cells are mesenchymal stem cells.

(5) Obtaining human umbilical cord mesenchymal stem cells modified by spinous process protein coding genes: using a T175 cm²Culturing human umbilical cord mesenchymal stem cells in a culture flask (431080, Corning), and adding the adenovirus particles expressing the spinous process proteins to T175 cm when the cell confluency reaches 70%²Mixing in culture flask, mixing at 37 deg.C and 5% CO₂Continuously culturing for 24-48 hours in a constant-temperature incubator, adding pancreatin for digestion, and preparing cell suspension.

Example 2

This preferred embodiment further illustrates the present invention by way of mouse experiments.

10 mice were divided into two groups, group a 5, group B5, which were placed in the same environment to live:

group a vaccine injections: handle 1X 10⁷Digesting the spinous process protein coding gene modified human umbilical cord mesenchymal stem cells by pancreatin, fully blowing and beating into single cell suspension, centrifugally collecting cell sediment at 2000rpm, and resuspending the sediment by using 2ml of medical normal saline; injecting the spinous process protein coding gene modified mesenchymal stem cells into the abdominal cavities of 5 mice respectively by using a micro-injector, wherein each injection is 100 mu l, and each injection is performed every 1-2 weeks for 3 times;

group B injection of physiological saline: injecting medical normal saline into abdominal cavities of the other 5 mice respectively by a micro-injector, wherein each injection is 100 mul, and each injection is performed every 1-2 weeks for 3 times;

finally, A, B groups of mouse sera were each collected and tested for neutralizing antibodies by ELISA, as shown in Table 1:

table 1: detection result of concentration of neutralizing antibody

The data in the table show that the novel vaccine can effectively immunize an organism by injection, induces the organism to generate a neutralizing antibody aiming at the spinous process protein, and the antibody can be stably stored in the organism for a long time.

The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.