CN117736278A - Kit and detection method for detecting respiratory syncytial virus infection - Google Patents
Kit and detection method for detecting respiratory syncytial virus infection Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a kit and a detection method for detecting respiratory syncytial virus infection. The invention firstly provides a recombinant protein of respiratory syncytial virus F, which is based on a wild protein sequence of respiratory syncytial virus F, and uses a Furin enzyme cleavage site between an F1 fragment and an F2 fragment of the wild protein sequence and a p27 polypeptide (Gly) 8 Flexible linker peptide sequence substitutions and optimizing codon usage. And then provides applications based on the recombinant protein.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a kit and a detection method for detecting respiratory syncytial virus infection.
Background
Respiratory Syncytial Virus (RSV) belongs to Paramyxoviridae and pneumovirus, is an important pathogen causing lower respiratory tract infection, is a main cause of hospitalization and death of infants, has the characteristics of short incubation period, easy repeated infection, rapid disease progress and the like after being infected by a host, and is easy to cause explosive epidemic. The infection of respiratory syncytial virus not only brings great pressure to the self health of the children and the family economy thereof, but also brings great economic burden to the social health system. However, despite the high infection rate, high hazard and high incidence of respiratory syncytial virus, no safe and effective prophylactic vaccine is currently available for treatment.
After infection with respiratory syncytial virus, the human body generally has a latency period of 3 to 7 days, after which the human body produces considerable IgM antibodies, and then produces a large amount of IgG antibodies, about 4 to 21 days. Although IgG antibodies are a clinically common indicator for detecting viral infection, igG antibody detection is susceptible to past infection and is not suitable as an indicator for early infection judgment. IgM antibodies are produced earlier than IgG antibodies, so specific detection of IgM antibodies raised against respiratory syncytial virus antigens can be used as markers of early respiratory syncytial virus antigen infection. It is therefore important to establish a convenient, rapid, and efficient detection method in order to be able to identify respiratory syncytial virus infection at an early stage.
Respiratory syncytial virus encodes two major surface glycoproteins: adhesion proteins (attachment protein, G) and fusion proteins (F). The adhesion G protein is a type 2 surface glycoprotein, is divided into an intracellular region, an extracellular region and a transmembrane region, and is mainly responsible for mediating the attachment of respiratory syncytial virus to host cells; the fusion F protein of the viral plasma membrane then mediates viral entry into the host cell. Meanwhile, the G protein also participates in the production process of neutralizing antibodies of organisms after infection, so that serious infection can be effectively avoided, and the neutralizing antibodies produced by the G protein can not provide cross protection effects on different subtype and genotype viruses, so that the immune escape and the repeated occurrence of RSV infection can be possibly caused. Respiratory syncytial viruses can be classified into subtypes A (RSV-A) and B (RSV-B) based on the difference in G-protein surface antigen, and further, the RSV-A subtypes can be classified into 15 genotypes and the RSV-B subtypes can be classified into 30 genotypes according to the nucleotide sequence of the 2 nd hypervariable region of the C-terminal of the G-protein gene.
The patent with the application number of CN2022106181965 and the name of "Sup>A respiratory syncytial virus antigen detection kit and application thereof" discloses Sup>A test paper and Sup>A kit for detecting RSV antigen, wherein the test paper is coated with Sup>A colloidal gold labeled RSV monoclonal antibody, and the test paper and the kit are mainly used for detecting RSV-A typing. However, the colloidal gold method has insufficient detection sensitivity, and is easy to miss for samples with low virus content.
Compared with the G protein, the F protein has Sup>A plurality of types, the F protein has higher conservation, and the corresponding F protein antibody has better cross protection effect on the RSV-A subtype and the RSV-B subtype, so the sequence for encoding the F protein is Sup>A hot spot of the current RSV antibody research. The patent with the application number of CN2014101395925 and the name of a preparation method for detecting G and F proteins of human respiratory syncytial virus antibody discloses that codons of G protein and F protein genes of respiratory syncytial virus surface antigens are optimized so as to be expressed in prokaryotic cells in high quantity. The expressed G and F protein supernatants were then Elisa coated and then used to detect patient serum. However, since prokaryotic cells have no cell nucleus, endoplasmic reticulum, golgi apparatus, etc., which are critical elements in cell trafficking and post-translational modification, the accuracy of translation of the prokaryotic expression system is insufficient, and the biological activity of the expressed protein may be lacking.
Literature data (Chin J Biologicals August 2022, vol.35No. 8) discloses the establishment and verification of indirect immunofluorescence detection method for respiratory syncytial virus titer, which discloses that Hep-2 cells are cultured in a medium containing fetal bovine serum and are subjected to wall-attached culture by simultaneously inoculating RSV virus and cells into a 96-well plate. However, the method of adherence culture can limit the density of cell culture by adherence area, and the method of simultaneous inoculation culture of virus and cells can easily introduce pollution of other exogenous microorganisms if the operation is improper.
In view of the foregoing, there is a need for new methodology strategies that alleviate the deficiencies of the prior art.
Disclosure of Invention
In view of the above, the present invention aims to provide a preparation method of positive cells comprising a recombinant protein of respiratory syncytial virus F, a recombinant protein of transfected respiratory syncytial virus, a detection kit containing the positive cells and a detection method, and the specific technical scheme is as follows.
A recombinant protein of respiratory syncytial virus F based on the wild protein sequence of respiratory syncytial virus F, the Furin cleavage site between the F1 fragment and F2 fragment of the wild protein sequence and p27 polypeptide are described as (Gly) 8 Replacement of the flexible linker peptide sequence; the amino acid sequence of the F1 fragment is shown as SEQ ID NO. 1; the amino acid sequence of the F2 fragment is shown as SEQ ID NO. 2.
It will be appreciated that (Gly) 8 The flexible connecting peptide sequence is a polypeptide sequence with 8 continuous Gly arrays.
Further, the codon of the recombinant protein of the respiratory syncytial virus F is optimized and modified, so that the GC base proportion in the DNA sequence of the recombinant protein of the respiratory syncytial virus F is more than 50 percent.
Further, the full-length amino acid sequence of the recombinant protein of the respiratory syncytial virus F is shown as SEQ ID NO. 3.
Alternatively, the full-length nucleotide sequence of the recombinant protein of the respiratory syncytial virus F is shown as SEQ ID NO. 4.
A method of preparing and culturing positive eukaryotic cells transfected with recombinant protein of respiratory syncytial virus F, comprising the steps of:
s01: cloning the recombinant protein gene sequence of the respiratory syncytial virus F into a mammal expression plasmid to prepare a plasmid vector, wherein the plasmid carries a WPRE element (enhancing mRNA transportation);
s02: transfecting the plasmid vector prepared in the step S01 into a mammalian cell line, culturing the mammalian cell line on a culture medium containing G418, and constructing a stable cell line through G418 pressurized screening to obtain positive eukaryotic cells transfected with the respiratory syncytial virus F recombinant protein, wherein the positive eukaryotic cells stably express the respiratory syncytial virus F recombinant protein;
s03: and (3) placing the positive eukaryotic cells transfected with the respiratory syncytial virus F recombinant protein prepared in the step (S02) in a serum-free culture medium with limited chemical components, and performing suspension culture in a constant-temperature oscillator at a culture rotating speed range of 120-150rpm.
Further, the concentration of the G418 pressure screening is in the range of 400-800 mug/ml.
A kit for detecting respiratory syncytial virus infection based on an indirect immunofluorescence method comprises positive eukaryotic cells transfected with respiratory syncytial virus F recombinant protein, which are prepared by the method.
Further, the kit also comprises a fluorescein-labeled secondary antibody (into which Evan blue is added). In one embodiment of the present invention, the secondary antibody is a murine 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, and the like.
Further, the kit can also comprise normal saline, ethanol, an anti-respiratory syncytial virus monoclonal antibody and/or PBS wash.
A method for detecting respiratory syncytial virus infection using the above-described kit, comprising the steps of:
s01: resuspending positive eukaryotic cells transfected with respiratory syncytial virus F recombinant protein in the kit with physiological saline, and adjusting finenessThe cell density is in the range of 1 to 5 multiplied by 10 5 Preparing positive eukaryotic cell liquid by using a single/mL method, and then fixing a proper amount of the positive eukaryotic cell liquid on a glass slide for drying;
s02: putting the dried glass slide into ethanol, and performing permeation treatment on cells to expose antigen epitopes;
s03: adding a serum sample to be tested, and incubating for 30-60 minutes;
s04: further adding a fluorescein-labeled secondary antibody in the kit for co-incubation for 20-30 minutes to form a recombinant protein-sample-fluorescence secondary antibody complex;
s04: detecting the fluorescent state of the recombinant protein-sample-fluorescent secondary antibody complex, wherein green fluorescence represents positive sample (namely, the sample contains respiratory syncytial virus), and red fluorescence represents negative sample (namely, the sample does not contain respiratory syncytial virus).
Further, the volume ratio of the positive eukaryotic cell fluid, the serum sample to be tested and the fluorescein labeled secondary antibody is 10:3:3.
Beneficial technical effects
1. The invention firstly provides a recombinant protein of respiratory syncytial virus F, which is modified based on a wild protein sequence, so that the expression quantity of the recombinant protein in eukaryotic cells can be improved, and the recombinant protein has higher specificity and sensitivity when being used as an antigen. Compared with the wild protein of respiratory syncytial virus F, the recombinant protein has higher transfection efficiency, higher protein expression quantity and more stable biological activity.
2. Furthermore, the invention provides a positive eukaryotic cell transfected by the respiratory syncytial virus F recombinant protein, namely a novel cell. The cell can stably express recombinant protein of the suction path syncytial virus F. The cell is superior to the traditional method of culturing adherent cells and then inoculating viruses, the cell density is not limited by the adherent area, and the cell line is only cultured to obtain the stably expressed recombinant protein of the respiratory syncytial virus F without the need of inoculating the respiratory syncytial virus in the whole process, so that the risk of exogenous virus infection is reduced, and the cell line has better safety. In addition, the novel cell line supports a high-density serum-free suspension culture process, and the whole cell culture process adopts a chemical Component Definition (CD) culture medium, so that animal serum is not contained, the cost is low, and mycoplasma, chlamydia or animal protein pollution is not introduced; the applicable culture medium has definite components, easy preparation and convenient use. Therefore, the novel cell line is convenient for industrialization, and can be used for preparing kit products in batch production.
3. The invention is based on an indirect immunofluorescence method, adopts recombinant protein as antigen to react with corresponding antibody in a serum sample to be detected, combines a fluorescent-labeled secondary antibody with an antigen-antibody complex, and observes the specific fluorescence of the recombinant protein-sample-fluorescent secondary antibody complex under a fluorescence microscope to detect whether the serum has pathogen infection. Compared with the existing method using more colloidal gold, the method has the characteristics of high sensitivity, strong specificity and the like, and is higher in accuracy.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.
FIG. 1 is a schematic diagram of wild type respiratory syncytial virus F wild-type protein sequence modified by the method of the invention;
FIG. 2 is a graph showing the transfection efficiency of recombinant protein sequences of the present invention compared to wild-type protein sequences;
FIG. 3 shows the comparison of the fluorescence results of the recombinant protein sequences of the invention and the wild-type protein sequences 7 days after transfection;
FIG. 4 is a flow chart of a method of detecting respiratory syncytial virus infection according to the invention;
FIG. 5 shows the positive result of the indirect immunofluorescence method of the invention for detecting respiratory syncytial virus;
FIG. 6 shows the negative result of the detection of respiratory syncytial virus by the indirect immunofluorescence method of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. 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.
Herein, "and/or" includes any and all combinations of one or more of the associated listed items.
Herein, "plurality" means two or more, i.e., it includes two, three, four, five, etc.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.
In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that such a description of "within a certain range" is merely for convenience and convenienceSuccinct, and should not be construed as limiting the stiffness of the disclosed scope. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, a rangeThe description of (c) should be taken as having 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 such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.
Example 1
The present example provides a recombinant protein of respiratory syncytial virus F, and an exemplary method for transfecting positive eukaryotic cells with the protein
1. Firstly, obtaining a wild protein sequence of respiratory syncytial virus F, and combining a Furin enzyme cleavage site between an F1 fragment and an F2 fragment of the wild protein sequence with a p27 polypeptide (Gly) 8 Flexible linker peptide sequences were substituted and the F1 fragment and F2 fragment were ligated.
2. And (3) further carrying out codon optimization on the sequence in the step (1) according to the preference of host Expi-293 cells (cell types: human kidney-derived epithelial cells; eukaryotic cells), and improving the GC content in the optimized recombinant protein DNA sequence from about 35% of wild type to 55% to obtain the respiratory syncytial virus F recombinant protein sequence. The respiratory syncytial virus F recombinant protein has higher specificity and sensibility as an antigen.
3. Further, the vector was ligated with a plasmid carrying the WPRE element (pcDNA3.4) to construct an expression vector. The process is schematically shown in fig. 1.
4. Plasmid was transfected into the Expi-293 cells and G418 pressure screening was performed to construct a stable cell line at a concentration ranging from 400-800. Mu.g/ml. Obtaining a cell line for stably expressing recombinant protein of respiratory syncytial virus F, which is called as Expi-293-RSV-F for short.
Transcription and translation of the F gene occurs in the cytoplasm upon replication of RSV viruses, but plasmid transcription to RNA occurs in the nucleus upon expression of recombinant proteins in mammalian cells, thus requiring out of the nucleus into the cytoplasm to complete replication of the virus. However, the original viral RNA sequence is not optimized for out-nuclear transport, so out-nuclear transport is a bottleneck for expression efficiency. The wild-type gene of virus F is enriched for A-U and may affect mRNA transport to the nucleus. The nucleotide sequence of the recombinant protein optimized by the method of the invention has the GC content increased from about 35% to 55% of the wild type compared with the wild type protein sequence, so that the expression may be increased.
Furthermore, the WPRE sequence carried with pcdna3.4 facilitates nuclear export. WPRE is a cis-acting RNA element, is a post-transcriptional regulatory sequence, and can be used for remarkably increasing the expression level and translation efficiency of mRNA before polyadenylation signals, thereby enhancing the expression of genes.
Finally, protein cleavage is removed. Wild proteins are cleaved into two peptide chains after synthesis, the two peptide chains being linked by disulfide bonds. In the modified recombinant protein, the sequence related to shearing is replaced, so that the modified recombinant protein forms only one peptide chain. Therefore, it has the effects of increasing expression level and promoting trimer.
The sequence list information related to the invention is as follows:
and (3) verifying results:
1. the recombinant protein sequence (also known as an engineered sequence) synthesized in the embodiment is compared with a wild protein sequence in transfection efficiency, and the experimental method is to prepare a cell substrate by using a direct immunofluorescence method, namely, the transfected cells are subjected to cell substrate preparation, an anti-respiratory syncytial virus antibody-FITC conjugate is added by a one-step method, incubation is carried out for 30min at 37 ℃, and after PBS washing, fluorescence intensity interpretation is carried out through a fluorescence microscope. Since 0.006% Evan blue dye was added to the antibody-FITC conjugate, red and green fluorescence was present. If positive, green fluorescence was observed under the mirror, if negative, red cells were observed under the mirror, see fig. 2 and 3. FIG. 2 shows that the modified recombinant protein sequence is more fluorescent, indicating that the transfection efficiency is higher.
2. After 7 days of transfection, the assay was performed again (FIG. 3). FIG. 3 shows that the modified sequence has more fluorescence compared with the wild sequence, which indicates that the recombinant protein obtained in the embodiment has higher expression level and more stable biological activity.
Example 2-
This example provides a method for culturing and amplifying an Expi-293-RSV-F cell
1. In the culture of stable cell line, serum-free culture medium with chemical composition limit (CD) is used, and suspension culture is carried out in a constant temperature shaker at a rotation speed of 120-150rpm and 37 deg.C of 5% -8% CO 2 。
The serum-free Medium of chemical Composition Definition (CD) used in this example was OPM-CHO CD07 Medium (Biotechnology of Orthon Pu Mai), cat# P081307-001.
2. When the cells are ready for passaging (i.e., in the logarithmic growth phase before reaching confluence), the flask is removed from the incubator and a small sample is removed from the flask using a sterile pipette.
3. The total number of cells and the percentage of viable cells in the samples were determined using an automated cytometer and trypan blue exclusion. Diluting the cells with serum-free medium to an inoculation density in the range of 0.5-2.0X10 6 And each mL.
4. Suspension culture of cells requires no medium change. Sufficient medium is added to dilute the culture to a density in the range of 0.5-2.0X10 6 And each mL. The addition of fresh medium is sufficient to supplement the cell nutrients.
Culture results: culturing in 30mL culture system for 3 days to obtain cell amount of 4.0-10.0X10 6 And each mL. When observed under a microscope, the cells grow in a single dispersion way, are full in shape and uniform in size. The embodiment can realize the gradual amplification of 6-orifice plates-T125 ml shake flasks-1L shake flasks-fermentation tanks, and meets different production and application scenes.
Example 3
The present embodiment provides a method for detecting respiratory syncytial virus infection
1. Positive cells transfected with recombinant proteins of respiratory syncytial virus (primary antibody) were resuspended in physiological saline, counted and the density was adjusted to 5X 10 5 The cells were attached to a slide glass (solid phase carrier) by drying the solution, and the empty cells of the Expi-293 were fixed in the same manner. And (3) putting the dried positive cell slide covered with the recombinant protein (primary antibody) of the respiratory syncytial virus into ethanol, fixing cell permeation treatment, exposing the epitope on the recombinant protein, naturally drying, and respectively adding the slide to be detected into 15uL positive control, 15uL negative control and 15uL serum sample to be detected. Wherein, the positive control is anti-respiratory syncytial virus monoclonal antibody, and the negative control is serum sample diluent to be tested.
2. The slide to be examined is put into a wet box and incubated at 37 ℃ for 60min, then the slide is soaked in PBS (0.01M, pH 7.4), and the slide is placed on a horizontal shaking table to shake for 3 min, repeated three times and naturally dried.
3. 15uL of fluorescein-labeled secondary antibody was added to each well, and this example was incubated at 37℃for 30min using a murine anti-human IgM-FITC conjugate (0.006% Evan blue dye was added to the conjugate). Then the glass slide is soaked in the washing liquid, the horizontal shaking table is placed for 3 minutes to shake lightly, the three times of repetition are carried out, and the glass slide is naturally dried. Fluorescence was observed under a fluorescence inversion microscope. If positive, green fluorescence is observed under the mirror, and if negative, red cells are observed under the mirror.
In the embodiment, positive cells transfected with the recombinant cytovirus F are used as specific antigens and spread on a glass slide based on an indirect immunofluorescence method, a clinical sample to be detected is added and then combined with FITC labeled secondary antibodies, and visual observation is carried out after color development. See fig. 4-6.
Example 4
Actual sample detection
Using the recombinant protein prepared in example 1 and the detection method of example 3, specificity and sensitivity tests were performed on 60 clinical respiratory syncytial virus IgM antibody positive serum samples and 100 clinical respiratory syncytial virus IgM antibody negative serum samples. And meanwhile, cross reaction verification is carried out on positive samples of common pathogens of other respiratory diseases. And secondly, comparing the positive detection rate of the colloidal gold product of the IgM antibody of the respiratory syncytial virus on the same platform.
TABLE 1 specificity and sensitivity detection results of recombinant proteins (antigens) of the invention
The results in Table 1 show that the recombinant protein of the present invention has a sensitivity (accuracy of detection of positive samples) of up to 95% and a specificity (accuracy of detection of no negative samples) of up to 97%.
TABLE 2 comparison of detection rate results of colloidal gold product positive samples of IgM antibodies of respiratory syncytial virus according to the invention
| Project | Number of positive samples | Negative number of samples | Totals to |
| The product of the invention | 57 | 3 | 60 |
| Colloidal gold method product | 54 | 6 | 60 |
The results in Table 2 show that the detection rate of positive samples of the recombinant protein of the invention as an antigen can reach 95%, and the detection rate of positive samples of IgM antibody colloidal gold products of the same platform respiratory syncytial virus is 90%. The recombinant protein of the invention has higher detection accuracy as an antigen.
TABLE 3 Cross-reaction test results of the test kit of the invention with samples positive for common pathogens of other respiratory diseases
The results in Table 3 show that the positive rate of the recombinant protein of the invention used as an antigen for detecting common pathogens of other respiratory diseases is 0. The recombinant protein provided by the invention has better specificity for detecting respiratory syncytial virus as an antigen.
The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.
Claims (10)
1. A recombinant protein of respiratory syncytial virus F, which is characterized in that the recombinant protein of respiratory syncytial virus F is based on a wild protein sequence of respiratory syncytial virus F, and a Furin enzyme cleavage site between an F1 fragment and an F2 fragment of the wild protein sequence and a p27 polypeptide are combined into a whole (Gly) 8 Flexible linker peptide sequence substitutionsThe method comprises the steps of carrying out a first treatment on the surface of the The amino acid sequence of the F1 fragment is shown as SEQ ID NO. 1; the amino acid sequence of the F2 fragment is shown as SEQ ID NO. 2.
2. The recombinant respiratory syncytial virus F protein according to claim 1, wherein the codons of the recombinant respiratory syncytial virus F protein are optimally engineered such that the GC base ratio in the DNA sequence of the recombinant respiratory syncytial virus F protein is greater than 50%.
3. The recombinant protein of respiratory syncytial virus F according to claim 2, wherein the full-length amino acid sequence of the recombinant protein of respiratory syncytial virus F is shown in SEQ ID No. 3.
4. A method for preparing and culturing positive eukaryotic cells transfected with recombinant protein of respiratory syncytial virus F, comprising the steps of:
s01: cloning the respiratory syncytial virus F recombinant protein sequence of any one of claims 1-3 into a mammalian expression plasmid carrying a WPRE element to produce a plasmid vector;
s02: transfecting the plasmid vector prepared in the step S01 into a mammalian cell line, culturing the plasmid vector on a culture medium containing G418, and constructing a stable cell line through G418 pressurized screening to obtain positive eukaryotic cells transfected with recombinant proteins of respiratory syncytial virus F; the positive eukaryotic cell stably expresses recombinant protein of respiratory syncytial virus F;
s03: and (3) placing the positive eukaryotic cells transfected with the respiratory syncytial virus F recombinant protein prepared in the step (S02) in a serum-free culture medium with limited chemical components, and performing suspension culture in a constant-temperature oscillator at a culture rotating speed range of 120-150rpm.
5. The method of claim 4, wherein the concentration of G418 pressure screening is in the range of 400-800 μg/ml.
6. A kit for detecting respiratory syncytial virus infection based on an indirect immunofluorescence method, which is characterized by comprising positive eukaryotic cells transfected with respiratory syncytial virus F recombinant protein prepared by the method of claim 4 or 5.
7. The kit of claim 6, further comprising a fluorescein-labeled secondary antibody.
8. The kit of claim 6, wherein the kit further comprises physiological saline, ethanol, anti-respiratory syncytial virus monoclonal antibodies, and/or PBS wash.
9. A method of detecting respiratory syncytial virus infection using the kit of any one of claims 6-8, comprising the steps of:
s01: resuspending positive eukaryotic cells transfected with respiratory syncytial virus F recombinant protein in the kit with physiological saline, and adjusting the cell density to be 1-5 multiplied by 10 5 Preparing positive eukaryotic cell liquid by using a single/mL method, and then fixing a proper amount of the positive eukaryotic cell liquid on a glass slide for drying;
s02: putting the dried glass slide into ethanol, and performing permeation treatment on cells to expose antigen epitopes;
s03: adding a serum sample to be tested, and incubating for 30-60 minutes;
s04: further adding a fluorescein-labeled secondary antibody in the kit for co-incubation for 20-30 minutes to form a recombinant protein-sample-fluorescence secondary antibody complex;
s04: detecting the fluorescence state of the recombinant protein-sample-fluorescent secondary antibody complex, wherein green fluorescence represents positive sample, and red fluorescence represents negative sample.
10. The method of claim 9, wherein the volume ratio of positive eukaryotic cell fluid, test serum sample, and fluorescein labeled secondary antibody is 10:3:3.
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