A kit and detection method for detecting respiratory syncytial virus infection.

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a kit and a detection method for detecting respiratory syncytial virus infection. The present invention first provides a respiratory syncytial virus F recombinant protein, which is based on the respiratory syncytial virus F wild protein sequence, replaces the Furin restriction site and p27 polypeptide between the F1 fragment and the F2 fragment of the wild protein sequence with a (Gly)8 flexible connecting peptide sequence, and optimizes the codons. Then, an application based on the recombinant protein is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a kit and a detection method for detecting respiratory syncytial virus infection. Background Art

[0002] Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and the genus Pneumovirus. It is an important pathogen that causes lower respiratory tract infections. RSV infection is the main cause of hospitalization and death in infants and young children. After infecting the host, it has the characteristics of a short incubation period, easy repeated infection, and rapid disease progression, which can easily cause an explosive epidemic. Infection with RSV not only puts great pressure on the health of the children themselves and their family finances, but also brings a large economic burden to the social health system. However, despite the high infection rate, high harm and high incidence of RSV, there is currently no safe and effective vaccine to prevent it.

[0003] After the human body is infected with respiratory syncytial virus, there is generally an incubation period of 3 to 7 days. After about 4 to 21 days, the human body will produce considerable IgM antibodies, followed by a large amount of IgG antibodies. Although IgG antibodies are a common clinical indicator for detecting viral infection, IgG antibody detection is easily affected by previous infection and is not suitable as an indicator for early infection judgment. IgM antibodies are produced earlier than IgG antibodies, so the specific detection of IgM antibodies produced against respiratory syncytial virus antigens can be used as a sign of early respiratory syncytial virus antigen infection. Therefore, in order to be able to identify respiratory syncytial virus infection at an early stage, it is crucial to establish a convenient, rapid and effective detection method.

[0004] Respiratory syncytial virus encodes two main surface glycoproteins: adhesion protein (G) and fusion protein (F). The adhesion G protein is a type 2 surface glycoprotein, which is divided into intracellular region, extracellular region and transmembrane region. It is mainly responsible for mediating the attachment of respiratory syncytial virus to host cells; the fusion F protein of the viral plasma membrane mediates the entry of the virus into host cells. At the same time, the G protein is also involved in the production of neutralizing antibodies in the body after infection, which can effectively avoid severe infection. Since the neutralizing antibodies produced by the G protein cannot provide cross-protection against different subtypes and genotypes of viruses, it may lead to immune escape and repeated RSV infection. According to the differences in the surface antigens of the G protein, respiratory syncytial virus can be divided into subtype A (RSV-A) and subtype B (RSV-B). Further, according to the different nucleotide sequences of the second hypervariable region at the C-terminus of the G protein gene, the RSV-A subtype can be subdivided into 15 genotypes, and the RSV-B subtype can be divided into 30 genotypes.

[0005] The patent with application number CN2022106181965 and invention name "A respiratory syncytial virus antigen detection kit and its application" discloses a test strip and a kit for detecting RSV antigen. The test strip is coated with RSV monoclonal antibodies labeled with colloidal gold. The test strip and the kit are mainly used to detect RSV-A typing. However, the colloidal gold method is not sensitive enough and is prone to miss detection of samples with low virus content.

[0006] Compared with the numerous typing of G protein, F protein has higher conservatism, and the corresponding antibody of F protein has better cross-protection between RSV-A subtype and RSV-B subtype, so the sequence encoding F protein is currently a hot spot in RSV antibody research. The patent with application number CN2014101395925 and invention name "A method for preparing G and F proteins for detecting antibodies to human respiratory syncytial virus" discloses the optimization of the codons of the G protein and F protein genes of the surface antigen of respiratory syncytial virus, so that high expression is achieved in prokaryotic cells. The expressed G and F protein supernatants are then coated with Elisa and then used to detect patient serum. However, since there are no nuclei, endoplasmic reticulum, Golgi bodies, etc. in prokaryotic cells, which are key elements in cell transport and post-translational modification, the translation accuracy of the prokaryotic expression system is insufficient, and the biological activity of the expressed protein may be lacking.

[0007] The literature "Establishment and Validation of Indirect Immunofluorescence Detection Method for Respiratory Syncytial Virus Titer" (Chin J Biologicals August 2022, Vol. 35 No. 8) discloses a method of culturing Hep-2 cells with a culture medium containing fetal bovine serum, and simultaneously inoculating RSV virus and cells in a 96-well plate for adherent culture. However, the adherent culture method will limit the density of cell culture by the adherent area, and the simultaneous inoculation and culture of viruses and cells is prone to contamination by other exogenous microorganisms if the operation is not done properly.

[0008] In summary, it is necessary to propose new methods and strategies to alleviate the shortcomings of existing technologies. Summary of the invention

[0009] In view of this, the object of the present invention is to provide a respiratory syncytial virus F recombinant protein, a preparation method of positive cells transfected with respiratory syncytial virus recombinant protein, a detection kit containing the positive cells and a detection method. The specific technical scheme is as follows.

[0010] A respiratory syncytial virus F recombinant protein, the respiratory syncytial virus F recombinant protein is based on the respiratory syncytial virus F wild protein sequence, the Furin restriction site and the p27 polypeptide between the F1 fragment and the F2 fragment of the wild protein sequence are replaced with a (Gly)8 flexible connecting peptide sequence; the amino acid sequence of the F1 fragment is shown in SEQ ID NO.1; the amino acid sequence of the F2 fragment is shown in SEQ ID NO.2.

[0011] It can be understood that the (Gly)8 flexible linker peptide sequence is a polypeptide sequence consisting of 8 consecutive Gly groups.

[0012] Furthermore, the codons of the respiratory syncytial virus F recombinant protein are optimized and modified so that the GC base ratio in the DNA sequence of the respiratory syncytial virus F recombinant protein is greater than 50%.

[0013] Furthermore, the full-length amino acid sequence of the respiratory syncytial virus F recombinant protein is shown in SEQ ID NO.3.

[0014] Alternatively, the full-length nucleotide sequence of the respiratory syncytial virus F recombinant protein is shown as SEQ ID NO.4.

[0015] A method for preparing and culturing positive eukaryotic cells transfected with respiratory syncytial virus F recombinant protein comprises the following steps:

[0016] S01: The above-mentioned respiratory syncytial virus F recombinant protein gene sequence is cloned into a mammalian expression plasmid to prepare a plasmid vector, wherein the plasmid carries a WPRE element (to enhance mRNA transport);

[0017] S02: transfecting the plasmid vector prepared in S01 into a mammalian cell line, culturing the cell line in a medium containing G418, and constructing a stable cell line by G418 pressure screening to obtain positive eukaryotic cells transfected with respiratory syncytial virus F recombinant protein, wherein the positive eukaryotic cells stably express the respiratory syncytial virus F recombinant protein;

[0018] S03: The positive eukaryotic cells transfected with the respiratory syncytial virus F recombinant protein prepared in S02 are placed in a chemically defined serum-free culture medium, and suspended and cultured in a constant temperature oscillator at a culture speed range of 120-150 rpm.

[0019] Furthermore, the concentration range of the G418 pressure screening is 400-800 μg / ml.

[0020] A kit for detecting respiratory syncytial virus infection based on indirect immunofluorescence method, the kit comprising positive eukaryotic cells transfected with respiratory syncytial virus F recombinant protein prepared by the above method.

[0021] Further, the kit also includes a fluorescein-labeled secondary antibody (in which Evans blue is added). In one embodiment of the present invention, the secondary antibody is a mouse anti-human IgM-FITC conjugate, but the secondary antibodies listed in the present invention are not limited thereto, and may also include a goat anti-human IgM-FITC conjugate, a rabbit anti-human IgM-FITC conjugate, etc.

[0022] Furthermore, the kit may also include physiological saline, ethanol, anti-respiratory syncytial virus monoclonal antibody and / or PBS washing solution.

[0023] A method for detecting respiratory syncytial virus infection using the above kit comprises the following steps:

[0024] S01: Resuspend the positive eukaryotic cells transfected with the respiratory syncytial virus F recombinant protein in the kit with physiological saline and adjust the cell density to 1-5×10 5 / mL was prepared as a positive eukaryotic cell solution, and then an appropriate amount of the positive eukaryotic cell solution was fixed on a glass slide and dried;

[0025] S02: Place the dried slides in ethanol to permeabilize the cells and expose the antigen epitopes;

[0026] S03: Add the serum sample to be tested and incubate for 30-60 minutes;

[0027] S04: further adding the fluorescently labeled secondary antibody in the kit and incubating for 20-30 minutes to form a recombinant protein-sample-fluorescent secondary antibody complex;

[0028] S04: Detect the fluorescence state of the recombinant protein-sample-fluorescent secondary antibody complex, green fluorescence represents a positive sample (ie, the sample contains respiratory syncytial virus), and red fluorescence represents a negative sample (ie, the sample does not contain respiratory syncytial virus).

[0029] Furthermore, the volume ratio of the positive eukaryotic cell fluid, the serum sample to be tested and the secondary antibody labeled with fluorescein is 10:3:3.

[0030] Beneficial technical effects

[0031] 1. The present invention first provides a respiratory syncytial virus F recombinant protein, which is modified based on the wild protein sequence so that its expression in eukaryotic cells can be increased, and it has higher specificity and sensitivity when used as an antigen. Experiments have shown that compared with the respiratory syncytial virus F wild protein, the recombinant protein has higher transfection efficiency, higher protein expression, and more stable biological activity.

[0032] 2. Further, the present invention provides a positive eukaryotic cell transfected with the respiratory syncytial virus F recombinant protein, that is, a new cell. The cell can stably express the respiratory syncytial virus F recombinant protein. The cell uses a suspension culture method, which is superior to the traditional adherent cell culture followed by virus inoculation. The cell density will not be limited by the adherent area, and since there is no need for respiratory syncytial virus inoculation throughout the process, only the cell line needs to be cultured to obtain stably expressed respiratory syncytial virus F recombinant protein, thereby reducing the risk of exogenous virus infection and having better safety. In addition, the new cell line supports high-density, serum-free suspension culture technology, and the cell culture process uses a chemically defined (CD) culture medium, which does not contain animal serum, has low cost, and does not introduce contamination by mycoplasma, chlamydia or animal protein; this applicable culture medium has clear components, is easy to prepare and is easy to use. Therefore, the new cell line is convenient for industrialization and can be mass-produced for the preparation of kit products.

[0033] 3. The present invention is based on indirect immunofluorescence method, using recombinant protein as antigen to react with corresponding antibody in the serum sample to be tested, combining fluorescently labeled secondary antibody with antigen-antibody complex, and observing the specific fluorescence of recombinant protein-sample-fluorescent secondary antibody complex under fluorescence microscope to detect whether the serum is infected with pathogens. This method has the characteristics of high sensitivity and strong specificity. Compared with the colloidal gold method currently used more, the method of the present invention has higher accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0035] Figure 1 This is a schematic diagram of the sequence of wild-type respiratory syncytial virus F wild protein modified by the method of the present invention;

[0036] Figure 2 Comparison of transfection efficiency between the recombinant protein sequence of the present invention and the wild protein sequence;

[0037] Figure 3 Comparison of fluorescence results of the recombinant protein sequence of the present invention and the wild protein sequence 7 days after transfection;

[0038] Figure 4 This is a flow chart of the method for detecting respiratory syncytial virus infection of the present invention;

[0039] Figure 5 The positive result of the indirect immunofluorescence method for detecting respiratory syncytial virus of the present invention;

[0040] Figure 6 This is a negative result of the indirect immunofluorescence method for detecting respiratory syncytial virus of the present invention. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0043] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0044] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0045] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0046] In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the range The description of should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0047] Example 1

[0048] This embodiment provides a respiratory syncytial virus F recombinant protein and an example of a method for transfecting positive eukaryotic cells with the protein

[0049] 1. First, obtain the wild protein sequence of respiratory syncytial virus F, replace the Furin cleavage site and p27 polypeptide between the F1 fragment and the F2 fragment of the wild protein sequence with a (Gly)8 flexible connecting peptide sequence, and connect the F1 fragment and the F2 fragment.

[0050] 2. According to the codon preference of the host Expi-293 cells (cell type: human kidney-derived epithelial cells; eukaryotic cells), the sequence of step 1 was further codon optimized, and the GC content in the optimized recombinant protein DNA sequence was increased from about 35% of the wild type to 55%, thereby obtaining a recombinant protein sequence of respiratory syncytial virus F. The recombinant protein of respiratory syncytial virus F has high specificity and sensitivity as an antigen.

[0051] 3. Further connect with the plasmid (pcDNA3.4) carrying the WPRE element to construct an expression vector. The schematic diagram of this process is shown in Figure 1 .

[0052] 4. The plasmid was transfected into Expi-293 cells, and a stable cell line was constructed by G418 pressure screening, and the concentration range of pressure screening was 400-800 μg / ml. A cell line stably expressing the recombinant protein of respiratory syncytial virus F was obtained, referred to as Expi-293-RSV-F.

[0053] When RSV virus replicates, the transcription and translation of the F gene occurs in the cytoplasm, but when the recombinant protein is expressed in mammalian cells, the plasmid transcribes and produces RNA in the nucleus, so it is necessary to export the nucleus to the cytoplasm to complete the replication of the virus. However, the original viral RNA sequence is not optimized for nuclear transport, so nuclear transport is the bottleneck of expression efficiency. The wild F gene of the virus is rich in AU, which may affect the transport of mRNA to the nucleus. The nucleotide sequence of the recombinant protein optimized by the method of the present invention has a GC content increased from about 35% of the wild type to 55% compared to the wild protein sequence, so it may increase expression.

[0054] In addition, the WPRE sequence carried by pcDNA3.4 facilitates nuclear export. WPRE is a cis-acting RNA element and a post-transcriptional regulatory sequence. When placed before the polyadenylation signal, it can significantly increase the expression level and translation efficiency of mRNA, thereby enhancing gene expression.

[0055] Finally, protein shearing is removed. After synthesis, wild proteins are sheared into two peptide chains, which are connected by disulfide bonds. In the modified recombinant protein, the sequence related to shearing is replaced, so the modified recombinant protein only forms one peptide chain. Therefore, it has the effect of increasing expression and promoting trimerization.

[0056] The sequence listing information involved in the present invention is as follows:

[0057]

[0058]

[0059] Verification results:

[0060] 1. The transfection efficiency of the recombinant protein sequence (also called modified sequence) synthesized in this example is compared with that of the wild protein sequence. The experimental method is to use a direct immunofluorescence method, that is, to prepare a cell substrate for the transfected cells, add an anti-respiratory syncytial virus antibody-FITC conjugate in a one-step method, incubate at 37°C for 30 minutes, wash the slide with PBS, and read the fluorescence intensity under a fluorescence microscope. Since 0.006% Evans blue dye is added to the antibody-FITC conjugate, red and green fluorescence will be present. If positive, green fluorescence will be observed under the microscope. If negative, red cells will be observed under the microscope. Figure 2 and Figure 3 . Figure 2 It can be seen that the modified recombinant protein sequence has more fluorescence, indicating that its transfection efficiency is higher.

[0061] 2. 7 days after transfection, test again ( Figure 3 ). Figure 3 It can be seen that the fluorescence of the modified sequence is still greater than that of the wild sequence, indicating that the recombinant protein obtained in this example has a higher expression level and more stable biological activity.

[0062] Example 2-

[0063] This embodiment provides a method for culturing and amplifying Expi-293-RSV-F cells

[0064] 1. In the culture of stable cell lines, a chemically defined (CD) serum-free medium was used, and the cells were suspended and cultured in a constant temperature oscillator at a speed of 120-150 rpm, 37°C, and 5%-8% CO2.

[0065] The chemically defined (CD) serum-free culture medium used in this example is OPM-CHO CD07 Medium (OPM Biotech), catalog number P081307-001.

[0066] 2. When the cells are ready to be passaged (i.e., in the logarithmic growth phase before reaching confluence), remove the flask from the incubator and use a sterile pipette to remove a small sample from the flask.

[0067] 3. Use an automated cell counter and trypan blue exclusion to determine the total number of cells and percentage of live cells in the sample. Dilute the cells with serum-free medium to a seeding density ranging from 0.5 to 2.0 × 10 6 Pieces / mL.

[0068] 4. For suspension culture cells, there is no need to change the medium. Add enough medium to dilute the culture to a density range of 0.5-2.0×10 6 Adding fresh culture medium is sufficient to replenish cell nutrients.

[0069] Culture results: In a 30 mL culture system, the cell count was 4.0-10.0×10 6 pcs / mL. Under the microscope, the cells grew individually and dispersedly, with full morphology and uniform size. This embodiment can achieve step-by-step amplification from 6-well plate-T125ml shake flask-1L shake flask-fermenter to meet different production application scenarios.

[0070] Example 3

[0071] This embodiment provides a method for detecting respiratory syncytial virus infection

[0072] 1. Resuspend the positive cells transfected with respiratory syncytial virus recombinant protein (primary antibody) in normal saline, count the cells, and adjust the density to 5×10 5 / mL is prepared as a positive eukaryotic cell solution, 50uL of positive eukaryotic cell solution is added to each well and added to a blank glass slide, dried to make the cells adhere to the glass slide (solid phase carrier), and the Expi-293 blank cells are fixed in the same way. The dried positive cell slide covered with respiratory syncytial virus recombinant protein (primary antibody) is placed in ethanol, and the fixed cells are permeabilized to expose the antigenic epitope on the recombinant protein. After natural drying, 15uL of positive control, 15uL of negative control and 15uL of serum sample to be tested are added to the glass slide to be tested. Among them, the positive control is anti-respiratory syncytial virus monoclonal antibody, and the negative control is the dilution of the serum sample to be tested.

[0073] 2. Place the slide to be tested in a wet box at 37°C and incubate for 60 minutes. Then soak the slide in PBS washing solution (0.01M, pH 7.4), place it on a horizontal shaker and shake it gently for 3 minutes. Repeat three times and let it dry naturally.

[0074] 3. Add 15uL of fluorescein-labeled secondary antibody to each well. In this example, mouse anti-human IgM-FITC conjugate (0.006% Evans blue dye is added to the conjugate) is used, and incubated at 37°C for 30 minutes. Then soak the slide in the wash solution, place it on a horizontal shaker and shake it gently for 3 minutes, repeat three times, and dry it naturally. Observe the fluorescence under an inverted fluorescence microscope. If positive, green fluorescence is observed under the microscope, and if negative, red cells are observed under the microscope.

[0075] This example is based on the indirect immunofluorescence method. The positive cells transfected with recombinant cellular virus F are used as specific antigens and spread on a glass slide. The clinical sample to be tested is added and then combined with a FITC-labeled secondary antibody. After color development, the cells are observed with the naked eye. Figure 4-6 .

[0076] Example 4

[0077] Actual sample testing

[0078] The recombinant protein prepared in Example 1 and the detection method of Example 3 were used to test the specificity and sensitivity of 60 clinical serum samples positive for respiratory syncytial virus IgM antibodies and 100 serum samples negative for clinical respiratory syncytial virus IgM antibodies. At the same time, cross-reaction verification was performed on positive samples of other common pathogens of respiratory diseases. Secondly, the positive detection rate of colloidal gold products of respiratory syncytial virus IgM antibodies on the same platform was compared.

[0079] Table 1 Specificity and sensitivity test results of the recombinant protein (antigen) of the present invention

[0080]

[0081]

[0082] The results in Table 1 show that the sensitivity (accuracy of detecting positive samples) of the recombinant protein of the present invention as an antigen can reach 95%, and the specificity (accuracy of not detecting negative samples) can reach 97%.

[0083] Table 2 The positive sample detection rate results of the present invention compared with the same platform respiratory syncytial virus IgM antibody colloidal gold product

[0084] project Number of positive samples Number of negative samples total Product of the present invention 57 3 60 Colloidal gold products 54 6 60

[0085] The results in Table 2 show that the detection rate of positive samples of the recombinant protein of the present invention as an antigen can reach 95%, and the detection rate of positive samples of the colloidal gold product of respiratory syncytial virus IgM antibody on the same platform is 90%. The detection accuracy of the recombinant protein of the present invention as an antigen is higher.

[0086] Table 3 Validation results of cross-reactions of the detection kit of the present invention with positive samples of other common pathogens of respiratory diseases

[0087]

[0088] The results in Table 3 show that the recombinant protein of the present invention is used as an antigen to detect positive samples of other common pathogens of respiratory diseases, and the positive rate is 0. This shows that the recombinant protein of the present invention has good specificity for detecting respiratory syncytial virus as an antigen.

[0089] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A kit for detecting respiratory syncytial virus infection based on indirect immunofluorescence assay, characterized in that: The kit contains a specific antigen, which is a positive eukaryotic cell that stably expresses the recombinant protein of respiratory syncytial virus F; the positive eukaryotic cell that stably expresses the recombinant protein of respiratory syncytial virus F is expressed at a rate of 5×10 5 / mL is prepared and set to a working volume of 50μL; the kit also contains a fluorescein-labeled secondary antibody; during detection, the positive eukaryotic cells stably expressing the respiratory syncytial virus F recombinant protein are set to form a positive eukaryotic cell-sample-fluorescent secondary antibody complex with the fluorescein-labeled secondary antibody and the serum sample to be tested; the full-length amino acid sequence of the respiratory syncytial virus F recombinant protein is shown in SEQ ID NO.

3.

2. The kit according to claim 1, characterized in that The kit also includes physiological saline, ethanol, anti-respiratory syncytial virus monoclonal antibody and / or PBS washing solution.

3. The kit according to claim 1, characterized in that The positive eukaryotic cells stably expressing the respiratory syncytial virus F recombinant protein are prepared by the following steps: S01: Cloning the respiratory syncytial virus F recombinant protein sequence into a mammalian expression plasmid to prepare a plasmid vector, wherein the plasmid carries a WPRE element, and the full-length amino acid sequence of the respiratory syncytial virus F recombinant protein is shown in SEQ ID NO.3; S02: transfecting the plasmid vector prepared in S01 into a mammalian cell line, culturing the cell line in a medium containing G418, and constructing a stable cell line by G418 pressure screening to obtain positive eukaryotic cells transfected with respiratory syncytial virus F recombinant protein; the positive eukaryotic cells stably express the respiratory syncytial virus F recombinant protein; S03: The positive eukaryotic cells stably expressing the respiratory syncytial virus F recombinant protein prepared in S02 are placed in a chemically defined serum-free culture medium, and suspended and cultured in a constant temperature oscillator at a culture speed range of 120-150 rpm.

4. The kit according to claim 3, characterized in that The concentration range of the G418 pressure screening is 400-800 μg / ml.

Images