阅读说明：本技术 新冠病毒包膜蛋白突变体侵入能力非诊断评价方法 (Non-diagnostic evaluation method for invasion capability of new coronavirus envelope protein mutant ) 是由 李星霖 陈丹瑛 赵学森 刘永梅 邱雅若 于 2021-08-26 设计创作，主要内容包括：本申请提供了一种新冠病毒包膜蛋白突变体侵入能力非诊断评价方法,其中使用了基于HIV/Luc报告载体的假病毒以及跨膜丝氨酸蛋白酶瞬时转染的稳定表达人ACE2受体的细胞。本发明还提供了相应假病毒产品及其应用。(The present application provides a non-diagnostic evaluation method of the invasive ability of a novel mutant of the envelope protein of a coronavirus, wherein pseudoviruses based on an HIV/Luc reporter vector and cells stably expressing the human ACE2 receptor transiently transfected by transmembrane serine protease are used. The invention also provides a corresponding pseudovirus product and application thereof.)

1. A method for non-diagnostic evaluation of the invasive potential of a mutant of the envelope protein of a novel coronavirus, wherein a pseudovirus expressing the mutant of the envelope protein of the novel coronavirus based on the HIV backbone is used.

2. The method of claim 1, wherein the HIV-backbone-based pseudovirus expressing a mutant of a novel coronavirus envelope protein is prepared by co-transfecting an HIV/Luc backbone plasmid with a different S protein mutant expression plasmid.

3. The method of claim 2, wherein the co-transfected subject is 293T cells.

4. The method of claim 2 or 3, wherein the HIV/Luc backbone plasmid is pNL4-3.luc.r^-E^-。

5. The method of any one of claims 1 to 4, wherein T-REx293 cells stably expressing the human ACE2 receptor are also used as target cells.

6. The method of claim 5, wherein the target cell is transiently transfected with a transmembrane serine protease.

7. A pseudovirus based on the HIV backbone and expressing a mutant of the novel coronavirus envelope protein.

8. The pseudovirus of claim 7, prepared by co-transfecting 293T cells with an HIV/Luc backbone plasmid and a different S protein mutant expression plasmid.

9. The pseudovirus of claim 8, wherein the HIV/Luc backbone plasmid is pNL4-3.luc.r^-E^-。

10. Use of the pseudovirus of any one of claims 7-9 in the preparation of a reagent set for studying the invasion ability of envelope protein mutants.

Technical Field

The present application belongs to the field of microorganisms, in particular to the field of research on viral infections. The application provides a non-diagnostic evaluation method for invasion capacity of a novel coronavirus envelope protein mutant.

Background

The envelope protein S protein of SARS-CoV-2 mediates the processes of virus adsorption and cell invasion, and is a major factor affecting the invasion ability of new coronavirus. During the new coronavirus epidemic period, a plurality of envelope protein mutant strains appear, wherein the D614G strain appears 3 months in 2020 and is a main epidemic strain at present, and the mutant strain has stronger transmission capability compared with the prior strain; furthermore, the british superstrain, which appeared in the uk at 9 months in 2020, also showed a stronger transmission capacity; a variety of different novel coronavirus envelope mutants can be found in specimens from different patients with new coronary pneumonia. Although the most direct method for evaluating the virus invasion capacity is an infection test or a plaque formation test using live viruses, firstly, the sources of different new coronavirus envelope mutants are scattered, and mutant live viruses required for research are difficult to obtain; secondly, the operation of live virus must be carried out in BSL-3 laboratory; in addition, live viruses are easy to generate gene mutation in the amplification and passage processes, the culture conditions and result interpretation standards are different, and live virus detection results in different laboratories often have large difference.

Therefore, the method for evaluating the invasion capacity of the envelope protein mutant of the new coronavirus, which is simple to operate, safe and accurate, has important significance for research, disease control and individual diagnosis of the new coronavirus.

Disclosure of Invention

The application of SARS-CoV-2 live virus in related analysis test has various limitations, and the invention establishes a pseudovirus detection system based on HIV/Luc report vector, which is used for researching the capability of different mutants of SARS-CoV-2 envelope protein to invade target cells, and has relatively safe operation and more stable detection result. The codon-optimized S protein with a 19 amino acid deletion at the C-terminus was inserted into pseudovirions and the resulting pseudoviruses would enter the cell in the same way as live viruses.

The pseudoviruses constructed in this study can be used to evaluate the ability of all novel variants of the envelope protein of coronavirus to invade cells, and can be further used in studies targeting the process of invasion of viruses into target cells, such as the evaluation of the effect of neutralizing antibodies and small molecule drugs designed against the S protein in preventing the viruses from entering cells. This study evaluated the level of entry of pseudoviruses by co-transfecting 293T cells with HIV/Luc backbone plasmids and different S protein mutant expression plasmids to generate pseudoviruses, while using T-REx293 cells stably expressing the human ACE2 receptor transiently transfected with transmembrane serine protease (TMPRSS2) as target cells. TMPRSS2 is a cell surface localized protease that cleaves the S protein of coronavirus during virus entry to pre-activate it and facilitate its entry into target cells. The pseudovirus constructed in the research is provided with a luciferase reporter gene, and the expression of the reporter gene can be accurately detected by a fluorescence determinator, so that the quantitative detection of the virus is realized. The invasion capacity of different S protein mutants can be researched by replacing different S protein mutant expression plasmids, and the pseudovirus detection system can also be used for researching the tissue tropism and receptor recognition mode of the virus, the effect change of a neutralizing antibody on different mutants and the effect of a small-molecule antiviral drug aiming at the S protein effect.

The realization of the new coronavirus envelope protein mutant invasion capacity evaluation technology comprises the following steps: 1. establishing a pseudovirus single-round infection system for expressing a new coronavirus envelope protein mutant based on an HIV/Luc framework; 2. construction of human ACE2(hACE2) receptor for inducible expression of target cell T-REx 293-hACE 2; 3. by simultaneously expressing TMPRSS2 in a target cell T-REx 293-hACE2, SARS-CoV-2 pseudovirus is pre-activated so as to improve the infection amount;

in one aspect, the present application provides a non-diagnostic method for assessing the invasive potential of a mutant of the envelope protein of a novel coronavirus, wherein a pseudovirus expressing the mutant of the envelope protein of the novel coronavirus based on the HIV backbone is used.

Further, the HIV skeleton-based pseudovirus expressing the mutant of the novel coronavirus envelope protein is prepared by co-transfecting an HIV/Luc skeleton plasmid with a different S protein mutant expression plasmid.

Further, the co-transfected subjects were 293T cells.

Further, the HIV/Luc skeleton plasmid is pNL4-3.Luc.R^-E^-。

Furthermore, T-REx293 cells stably expressing the human ACE2 receptor were used as target cells in the method.

Further, the target cells are transiently transfected with a transmembrane serine protease.

In another aspect, the present application provides a pseudovirus expressing a novel mutant coronavirus envelope protein based on the HIV backbone.

Further, the pseudovirus based on HIV framework expressing the mutant of the new coronavirus envelope protein is prepared by co-transfecting 293T cells with an HIV/Luc framework plasmid and a different S protein mutant expression plasmid.

Further, the HIV/Luc skeleton plasmid is pNL4-3.Luc.R^-E^-。

On the other hand, the application provides the application of the pseudovirus in preparing a reagent group for researching the invasion capacity of the envelope protein mutant.

The methods and pseudoviruses of the present application can be used for diagnostic or non-diagnostic purposes, preferably, non-diagnostic purposes such as research in science related to new coronaviruses, statistics and research and judgment of disease control data.

The S protein and the S protein mutant in the application can be protein separated from a sample or protein prepared by artificial expression; pNL4-3.Luc.R^-E^-The product is commercially available, and can also be constructed according to relevant documents.

Drawings

FIG. 1 is a schematic diagram of the structure of an HIV/Luc reporter gene and a pseudoviral package based on an HIV/Luc reporter vector;

FIG. 2 is a schematic representation of the pseudovirus titer assay, with a surrounding wrap sealed with 250 μ l DMEM or sterile water to reduce the effects of "edge effects". B11-G11 are cell control wells, B2-G10 are multiple dilution wells;

FIG. 3 is a schematic diagram of the construction of T-REx293 target cell infection platform and virus invasion system expressing hACE2 and TMPRSS 2;

FIG. 4 shows the detection of SARS-CoV-2 virus entry into host cells. Wherein the upper graph is the observation result of a fluorescent microscope for a pseudovirus invasion experiment (the pseudovirus system is provided with an EGFP reporter gene and can be detected by the fluorescent microscope): tet + indicates addition of tetracycline, Tet-indicates no tetracycline; T-REx293 cells expressing hACE2 receptor and TMPRSS2 were effective in supporting SARS-CoV and SARS-CoV-2 pseudovirus invasion, but not MERS-CoV pseudovirus invasion; the lower graph is a histogram of the invasion efficiency of three pseudoviruses.

FIG. 5 is a graph showing the results of the evaluation method of the present application for evaluating various S protein mutants.

Detailed Description

In order to make the present invention more clear and intuitive for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.

Example 1 construction of a pseudoviral Single round infection System expressing a novel mutant of the coronavirus envelope protein based on the HIV backbone

(1) Construction of S protein expression plasmid:

the S protein is used as an envelope protein of SARS-CoV-2, the expression level of the S protein in 293T cells is low, therefore, under the premise of not changing the amino acid sequence of SARS-CoV-2(MN908947) S protein, we perform codon optimization, delete 17 amino acids at the N terminal and 19 amino acids at the C terminal, and then design various S protein mutations (including P26H, H146Q, R246E + S247N, V407L, N422K, L452Q, E471K, V35 483F, G485V, Q493H, Q498H, Y505C, L518V, D614G, R682G, R682H, R685G, R685H, A694D, A771V and I1130L) on the basis, clone the sequence into pSecTag2/HygroA vector, and perform amplification and purification.

(2) Combining the eukaryotic expression plasmid containing S protein with Env-deleted skeleton plasmid pNL4-3. Luc.R. with luciferase reporter gene^-E^-Co-transfecting 293T cells to obtain the recombinant pseudovirus, wherein the specific method comprises the following steps:

a)293T cells were inoculated into 6-well cell culture dishes, after overnight culture, the confluency reached 80%, and DMEM medium containing 10% FBS in each well of the dish was supplemented to 4ml before transfection.

b) 1 mu g of expression plasmid of S protein mutant and a skeleton plasmid pNL4-3.Luc.R^-E^-3 μ g as per 1: 3(w/w) into a 1.5ml centrifuge tube containing 400. mu.l of antibiotic-free serum-free DMEM medium and mixed.

c) Add 6. mu.l Turbofect transfection reagent (DNA and transfection reagent 1:3, w/v), mix well and let stand 20min at room temperature. The plasmid-transfection reagent mixture is added dropwisePut into a 6-hole cell culture dish and gently shaken and mixed evenly. Standing at 37 deg.C for CO₂Culturing in an incubator.

d) After 24h, replacing the original 4ml of culture medium in the culture dish with a DMEM culture medium containing 10% FBS, then respectively collecting the virus liquid for 48h and 72h, supplementing the culture medium with the same volume after collecting the virus liquid each time to obtain 8ml of virus stock solution, centrifuging at 3000rpm for 5 minutes, filtering the supernatant through a 0.45 mu m sterile filter, subpackaging with 1.5ml centrifuge tubes, and freezing and storing in a refrigerator at-80 ℃ for later use.

The structure of the HIV/Luc reporter gene and the pseudoviral package based on the HIV/Luc reporter vector are shown in FIG. 1.

(3) Pseudovirus titer assay (test methods as shown in FIG. 2)

T-REx 293-hACE2 cells which are transfected with pCAGGS-TMPRSS2 and stably express hACE2 are inoculated into a 96-well plate, and are induced by tetracycline with the final concentration of 1 mu g/ml for 24 hours, and then the pseudovirus of the SARS-CoV-2S protein mutant to be detected is titrated. Firstly, 10-fold dilution is carried out on virus stock solution, then 3-fold continuous dilution is carried out, 9 gradients (the dilution range is 1:10-1:65610) are totally arranged, and each gradient is provided with 6 compound holes; meanwhile, setting a cell control without pseudovirus; one circle of the peripheral holes are sealed by 250 mu l of culture medium, so that the test result is prevented from being influenced by factors such as marginal culture medium evaporation and the like. At 37 ℃ with 5% CO₂And (3) incubating in an incubator for 48-72h, discarding all culture media, adding 30 mu l of cell lysate into each hole to lyse cells, incubating at room temperature for 10min, and adding 50 mu l of luciferase substrate into each hole to perform chemiluminescence detection. 50% Tissue Culture Infectious Dose (TCID) of pseudovirus was calculated according to the Reed-Muench method₅₀)。

EXAMPLE 2 establishment and characterization of hACE2 receptor inducible expression System (the basic procedure is shown in FIG. 3)

(1) SARS-CoV-2 pseudovirus infects target cell T-REx 293-hACE2+ TMPRSS2

A myc tag is introduced at the N-terminal end of the hACE2 receptor, and then the myc tag is cloned into an inducible expression vector pCDNA5, so that subsequent expression identification is facilitated. A human embryonic kidney 293 cell line (T-REx 293-ACE2) expressing ACE2 is established, and the expression of ACE2 is induced by tetracycline. The expression level of ACE2 can be indirectly regulated by controlling the concentration of tetracycline. Transient expression of TMPRSS2 protease is simultaneously induced and expressed at ACE2 receptor, thereby pre-activating SARS-CoV-2 virus to improve its infection capacity.

(2) Identification of hACE2 receptor-induced expression System

T-REx 293-ACE2 cells were treated with tetracycline (final concentration 1. mu.g/ml) for 24h, total protein was extracted by lysing the cells, and expression of hACE2 receptor molecule was identified by Western Blot using the myc tag added at the N-terminus.

EXAMPLE 3 establishment of SARS-CoV-2S protein pseudovirus in vitro infection model and pseudovirus invasion assay (FIG. 4)

(1) T-REx 293-hACE2 induced expression cells to be seeded into 6-well plates one day before transfection of TMPRSS2, resulting in a cell abundance of about 80% at the time of transfection.

(2) Transfecting pCAGGS-TMPRSS2 plasmid 4 mu g to T-REx 293-hACE2 cells in a 6-well plate, and inoculating to a 96-well cell culture plate after transfecting for 24 h; the next day, tetracycline was used to induce expression of the hACE2 receptor; after 24h induction with tetracycline (final concentration 1. mu.g/ml), pseudoviruses were formulated to contain 1000TCID per 100. mu.l₅₀The infection dosage of the mutant pseudovirus is 100 mul of virus liquid with the infection dosage is added into each hole, 3-6 auxiliary holes can be arranged for each mutant pseudovirus, and the luciferase activity of the cells is detected 48-72h after infection.

EXAMPLE 4 evaluation of the Change in the invasion Capacity of 21 mutants such as SARS-CoV-2S protein mutant D614G by the technique

Taking S protein P26H, H146Q, R246E + S247N, V407L, N422K, L452Q, E471K, V483F, G485V, Q493H, Q498H, Y505C, L518V, D614G, R682G, R682H, R685G, R685H, A694D, A771V and I1130L mutants as examples, the change of the capability of invading host cells relative to the capability of invading pseudoviruses of SARS-CoV-2 reference strain is evaluated by using the technology.

The method comprises the following steps: (1) the pseudovirus comprising SARS-CoV-2 and its mutant strains were prepared as described above. Mu.g of pCAGGS-SARS-CoV-2S and 3. mu.g of pNL4-3.Luc. R^-E^-Plasmid cotransfection of 293T cells, harvesting of viral supernatants 48h and 72h after transfection, centrifugation to remove cell debris, filtration through a 0.45 μm sterile membrane, packaging, and determination of TCID₅₀. (2) SARS-CoV-2S protein mutant pseudopathyToxicity test for the ability to invade host cells: T-REx 293-hACE2 cells were plated in 6-well plates, transfected with pCAGGS-TMPRSS2 plasmid at 24h after transfection at 2X 10⁴One well was inoculated to a 96-well plate, and tetracycline was added to each well at a final concentration of 1. mu.g/ml after its adherence, and induced to express hACE2 for 24 h. Containing 1000TCID₅₀100 μ l of virus solution of pseudovirus was transferred to 96-well cell plates pre-plated with target cells. CO at 37 deg.C₂The incubator is used for culturing for 48-72 h. The supernatant was discarded, 30. mu.l of cell lysate was added to each well, and after incubation at room temperature for 10min, 50. mu.l of luciferase substrate was added to each well, and luciferase activity was measured using a GloMax96 microplate luminometer (Promega). The ability of the different S protein mutants to invade cells was evaluated based on the invasion activity of the reference strain (as shown in FIG. 5).

The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications to the present invention based on the disclosure of the present invention within the protection scope of the present invention.