Preparation method of HIV-1CRF 5501B gp140 tripolymer

Abstract

The invention relates to a preparation method of an HIV-1CRF 5501B gp140 trimer in the field of protein engineering, which comprises the following steps: culturing recombinant expression cells of fusion protein, and collecting supernatant; the fusion protein comprises an HIV (human immunodeficiency virus)-1CRF 5501B gp140 as shown in SEQ ID NO. 1 and a purification tag; loading the supernatant to a purification label affinity chromatography column, balancing, eluting, and collecting the eluate; carrying out dialysis and ultrafiltration on the eluent, and collecting a concentrated solution; and purifying the concentrated solution by adopting a volume exclusion chromatography method to prepare the HIV-1CRF 5501B gp140 tripolymer. The application focuses on the unique epidemic strain HIV-1CRF 5501B, provides the preparation method of the unique epidemic strain gp140 trimer, and has important clinical value for analyzing different HIV subtypes in the aspects of epidemiology, disease treatment and the like.

Description

Technical Field

[0001] The present application relates to the field of protein engineering, and in particular to a method for preparing HIV-1CRF 55_01B gp140 trimer. Background Art

[0002] HIV enters target cells through the envelope glycoprotein (Env), which is a stable trimer formed by non-covalent binding of gp120 and gp41, mediating the entry of viral nucleic acids into host cells and is an important target for HIV treatment strategies. The CD4 binding site on gp120 is a conserved cavity formed at the interface of the glycoprotein's inner domain and outer domain. After gp120 binds to CD4, a series of conformational changes occur, leading to the generation and exposure of co-receptor binding sites (CCR5 and CXCR4 chemokine receptors). The multi-step process of HIV entry into cells provides a series of unique targets for preventing viral entry, and drugs targeting these targets are called HIV entry inhibitors. In immunological and structural studies, in order to obtain soluble Env trimers, the transmembrane (TM) region and cytoplasmic tail (CT) are often deleted from gp41 to create a gp140 protein containing gp120 and gp41 outer domains (gp41ECTO). The molecular arrangement of soluble gp140 is almost identical to that of gp120 observed in intact HIV-1 virions, and gp140 self-assembles into trimers during expression.

[0003] The recombinant HIV-1CRF 55_01B gp140 (envelope glycoprotein extracellular domain) trimer is a highly glycosylated secretory protein with high affinity to the CD4 receptor and co-receptors (CCR5 and CXCR4 chemokine receptors) on the host cell membrane. HIV-1CRF 55_01B gp140 consists of 644 amino acids with a molecular weight of 140-150KD. At present, HIVgp140 is widely used in the study of antigen-antibody or drug small molecule interactions, structural biology and other fields. Therefore, it is crucial to explore the recombinant expression method of HIV-1CRF 55_01B gp140.

[0004] In view of this, this application is hereby filed. Summary of the invention

[0005] Based on this, one or more embodiments of the present application provide a method for preparing HIV-1CRF 55_01B gp140 trimer, including the following technical solutions:

[0006] One or more embodiments of the present application provide a method for preparing HIV-1CRF 55_01B gp140 trimer, the preparation method comprising the following steps:

[0007] Cultivating recombinant expression cells of fusion protein and collecting supernatant; the fusion protein includes HIV-1CRF 5501_B gp140 shown in SEQ ID NO.1 and a purification tag;

[0008] Loading the supernatant onto a purification tag affinity chromatography column, balancing, eluting, and collecting the eluate;

[0009] dialyzing and ultrafiltration the eluate, and collecting a concentrate; and,

[0010] The concentrate was purified by size exclusion chromatography to prepare HIV-1 CRF 55_01B gp140 trimer.

[0011] In some embodiments of the present application, the balancing buffer used for balancing includes water, 45mM-55mM Tris, and 100mM-200mM NaCl, with a pH value of 8.5-8.8;

[0012] Optionally, the flow rate of the equilibration buffer is 0.5 mL / min-1.5 mL / min.

[0013] In some embodiments of the present application, the purification tag includes a Flag tag.

[0014] In some embodiments of the present application, the elution buffer used for elution includes water and 100 μg / mL-200 μg / mL 3×Flag, 45 mM-55 mM Tris, 100 mM-200 mM NaCl, and a pH value of 8.5-8.8;

[0015] Optionally, the flow rate of the elution buffer is 0.5 mL / min-1.5 mL / min.

[0016] In some embodiments of the present application, the molecular weight cut-off of the dialysis bag used for dialysis is greater than 30 kDa.

[0017] In some embodiments of the present application, the dialysis buffer used for dialysis includes water, 45mM-55mM Tris and 100mM-200mM NaCl, and the pH value is 8.5-8.8.

[0018] In some embodiments of the present application, the molecular weight cutoff of the ultrafiltration tube used in ultrafiltration is greater than 30 kDa.

[0019] In some embodiments of the present application, the recombinant expression cells include HEK293F cells.

[0020] In some embodiments of the present application, the method of collecting the supernatant includes membrane filtration;

[0021] Optionally, the pore size of the filter membrane used in membrane filtration is 40 μm-50 μm.

[0022] In some embodiments of the present application, the size exclusion chromatography method uses Cloumn Superose 6 Increase 10 / 300GL.

[0023] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0025] Figure 1 The SDS-PAGE analysis of HIV-1CRF 55_01B gp140 affinity chromatography step-by-step purification; wherein the M lane represents the standard protein, the supernatant lane represents the supernatant before passing through the affinity column, the flow-through lane represents the solution after passing through the affinity column, and CRF 55_01B represents the eluate containing HIV-1CRF 55_01B gp140;

[0026] Figure 2 This is the size exclusion chromatography analysis of HIV-1CRF 55_01B gp140; the CRF 55_01B gp140 trimer peak represents HIV-1CRF 55_01B gp140, and the 3×Flag peak represents the Flag polypeptide used for elution in the previous affinity chromatography;

[0027] Figure 3 The curves show the binding of different concentrations of HIV-1CRF 55_01B gp140 to CD4. The curves reflect the response signals of HIV-1CRF 5501_B gp140 to CD4 at gradient concentrations of 250nM, 125nM, 62.5nM, 31.25nM, 15.625nM, and 7.813nM from top to bottom.

[0028] Figure 4 represents the binding of different test substances to CD4; T represents Temsavir, CRF 5501_B represents HIV-1CRF 55_01Bgp140, and CRF 55_01B+T represents a mixture of HIV-1CRF 55_01B gp140 and Temsavir. DETAILED DESCRIPTION

[0029] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0031] the term

[0032] Unless otherwise specified or there is a contradiction, the terms and phrases used in this application have the following meanings:

[0033] The terms "and / or", "or / and", and "and / or" used in this application include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0034] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0035] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0036] In this application, the "suitable" mentioned in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0037] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0038] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0039] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0040] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0041] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0042] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0043] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0044] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0045] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0046] One or more embodiments of the present application provide a method for preparing HIV-1CRF 55_01B gp140 trimer, the preparation method comprising the following steps:

[0047] Cultivating recombinant expression cells of fusion protein and collecting supernatant; the fusion protein includes HIV-1CRF 55_01B gp140 shown in SEQ ID NO.1 and a purification tag;

[0048] Loading the supernatant onto a purification tag affinity chromatography column, balancing, eluting, and collecting the eluate;

[0049] dialyzing and ultrafiltration the eluate, and collecting a concentrate; and,

[0050] The concentrate was purified by size exclusion chromatography to prepare HIV-1 CRF 55_01B gp140 trimer.

[0051] The distribution of different subtypes of HIV around the world is significantly different, and different subtypes also have their own characteristics in terms of disease progression, main infected populations, and drug resistance. Most of the existing studies are conducted around the subtypes that are prevalent in South Africa and China, and the purification steps for HIV envelope glycoproteins are cumbersome. This application focuses on the unique epidemic strain HIV-1CRF 55_01B, and provides a method for preparing HIV-1CRF 55_01B gp140 trimer, which has important clinical value for analyzing different HIV subtypes in epidemiology, disease treatment, etc.

[0052] Purification tag affinity chromatography utilizes the specific binding between a specific purification tag on a protein and a ligand on a chromatographic medium to achieve protein separation and purification. Common purification tags include histidine tag (His-tag), glutathione S-transferase tag (GST-tag), Flag tag (Flag-tag), etc. When a sample containing a target protein (such as HIV-1CRF 55_01Bgp140 in this application) passes through a chromatography column loaded with a specific ligand, the target protein binds to the ligand and is retained on the column, while other impurities are eluted. Further, by changing the elution conditions, such as changing the pH value, ionic strength, or adding a competitive ligand, the target protein can be eluted from the chromatography column to obtain a purified target protein. In some examples, the purification tag includes a Flag tag (DYKDDDDK). The reasons for selecting a Flag tag include: (1) Small and uniform tag: The Flag tag has only 8 amino acids, so it has little effect on the structure and function of the fusion protein. Its small size helps avoid adverse effects on the folding and function of the fusion protein, and is more suitable for maintaining the natural properties of the protein than other larger tags (such as GST). (2) High specificity and affinity: The Flag tag has a strong binding affinity with the antibody it recognizes (such as Anti-Flag antibody), so it can efficiently capture and purify the fused target protein in affinity chromatography. This high affinity ensures high purity of the target protein while reducing the possibility of nonspecific binding. (3) Versatility and wide application: As a universal expression tag, the Flag tag has been widely used and verified. Many protein expression systems commonly used in commercial and academic research support the fusion construction of the Flag tag, which has been verified and used in different research and application scenarios. Overall, the use of the Flag tag for affinity chromatography purification has many advantages, such as high efficiency, strong specificity, and little effect on the target protein. In some of the examples, the equilibration buffer used for equilibration includes water and 45mM-55mM (for example, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55mM) Tris and 100mM-200mM (for example, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200mM) NaCl, and the pH value is 8.5-8.8 (for example, 8.5, 8.6, 8.7, 8.8); Optionally, the flow rate of the equilibration buffer is 0.5mL / min-1.5mL / min (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5mL / min).In some examples, the elution buffer used for elution includes water and 100 μg / mL-200 μg / mL (e.g., 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / mL) 3×Flag, 45 mM-55 mM (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 mM) Tris, 100 mM-200 mM (e.g., 1 00, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM) NaCl, pH 8.5-8.8 (for example, 8.5, 8.6, 8.7, 8.8); Optionally, the flow rate of the elution buffer is 0.5 mL / min-1.5 mL / min (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 mL / min).

[0053] In some of these examples, the dialysis bag used for dialysis has a molecular weight cutoff greater than 30 kDa (e.g., 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 kDa).

[0054] In some of the examples, the dialysis buffer used for dialysis includes water and 45mM-55mM (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55mM) Tris and 100mM-200mM (e.g., 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200mM) NaCl, with a pH of 8.5-8.8 (e.g., 8.5, 8.6, 8.7, 8.8).

[0055] In some of these examples, the ultrafiltration tube used for ultrafiltration has a molecular weight cutoff greater than 30 kDa (e.g., 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 kDa).

[0056] The present application does not specifically limit the type of recombinant expression cells, such as CHO cells, 293T cells, and 293F cells.

[0057] CHO cells: suitable for stable long-term expression, highly resistant to most viruses, relatively low transfection efficiency, usually require long-term culture to select stable expression cell lines. Usually, the culture of CHO cells requires the use of serum, which increases costs and variability between batches.

[0058] 293T cells: suitable for transient expression, with high transfection efficiency, and can obtain high expression levels of exogenous proteins in a short time.

[0059] 293F cells: suitable for transient expression systems, high transfection efficiency, suitable for large-scale expression and production. Suitable for culture under serum-free conditions, rapid growth and easy expansion, can quickly reach high-density culture, suitable for long-term stable expression.

[0060] In some examples, the recombinant expression cells include HEK293F cells.

[0061] In some examples, the method of collecting the supernatant comprises membrane filtration;

[0062] Optionally, the pore size of the filter membrane used in membrane filtration is 40 μm-50 μm (for example, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 μm).

[0063] In some of the examples, the size exclusion chromatography was performed using Cloumn Superose 6 Increase 10 / 300GL.

[0064] The present application does not specifically limit the construction method of the recombinant expression cell. The coding gene of the fusion protein in the recombinant expression cell can be synthesized directly, which has the following advantages: (1) Precision and controllability: error-free sequence. The direct synthesis method can be designed by computer to ensure that the gene sequence is error-free. In contrast, PCR may introduce mutations or errors during the amplification process, especially when amplifying longer sequences or sequences with high GC content. (2) Flexibility and customization: customized design. The direct synthesis method allows precise control of gene design, including optimizing codon usage, adding markers or tag sequences, modifying structures, etc. This flexibility allows the gene sequence to be customized according to specific needs to optimize the expression and function of the target protein. (3) Rapidity and efficiency: Rapidly obtain the target sequence. The direct synthesis method is usually faster than PCR, especially for longer sequences or complex gene structures. This rapidity allows experiments and research to be carried out more quickly. (4) Avoiding restriction enzyme restrictions: no restriction enzyme sites are required. PCR usually relies on restriction enzyme cutting sites to construct vectors or make connections, while the direct synthesis method is not limited by restriction enzyme cutting sites and is therefore more suitable for designing and constructing complex genomes or genome fragments. Overall, the direct synthesis method has higher accuracy, flexibility and efficiency in the process of obtaining gene sequences than the PCR method, especially in application scenarios that require customized design and avoidance of sequence errors.

[0065] This application focuses on the unique epidemic strain HIV-1CRF 55_01B in Shenzhen. There is currently a lack of research on its own structure and function and the differences with other major epidemic subtypes. In some examples, the HEK293F expression system is used to fuse the Flag-tag at the C-terminus of the protein to help protein purification. It is simple, efficient, and low-cost, achieving higher expression efficiency and stability, and the product has strong biological activity and retains most of the biological functions of HIV envelope glycoproteins. In addition, the purity is high, for example, the purity of Example 1 is 95%.

[0066] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0067] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0068] Example 1

[0069] 1. HIV-1CRF 5501_B gp140 vector construction

[0070] The gp140 sequence SEQ ID NO.1 (33-678) of 55_01B.CN.10.HNCS102056 (Genebank record: AFV52236.1) was obtained from the HIV database, and the nucleotide (sequence information came from the Los Alamos HIV sequence database www.hiv.lanl.gov; ACCESSION: JX574661) fused with Flag-tag (SEQ ID NO.2: DYKDDDDK) was commissioned to Universal Biosynthesis and cloned into the Escherichia coli expression vector pcmv, and transformed into Escherichia coli TOP10. The recombinant expression vector pcmv-gp140 was obtained while ensuring that the reading frame was not shifted. After DNA sequencing and comparison, the recombinant expression vector pcmv-gp140 was successfully constructed.

[0071] SEQ ID NO.1 is as follows:

[0072] LWVTVYYGVPVWRDAETTLFCASDAKAHVTEMHNVWATHACVPTDPNPQEIYLENVTEN

[0073] FKWKNNMVDQMHEDVISLWEQSLKPCVKLTPLCVTLKCTNAKFNNTTGGPDTPNITDEVR

[0074] NCTFNMTTEIRDKKQQVHALFYRLDIVSIGNENNSEYRLINCNTSVIKQACPKVSFDPIPIHY

[0075] CTPAGYAILKCNDKNFNGTGPCKNVSSIQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNA

[0076] KTIIVHLNNSVTINCTRPANNTRTGITIGPGQVFYRTGDIIGDIRKAFCEINRTEWKEVLKQVT

[0077] SKLKGHFNTEKIILQPPSGGDVEITMHHFNCRGEFFYCNTTKLFNSTWIMENDTWTDNKTM

[0078] EEGVNGTMGNNSIFIIPCRIKQIVNMWQGTGQAMYAPPIRGPINCVSNITGILLTRDGGNSNG

[0079] SNETFRPGGGDIKDNWRSELYKYKVVQLEPLGVAPTRAKRRVVEREKRAVGIGAMIFGFLG

[0080] AAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQARLLAVER

[0081] YLKDQKFLGLWGCSGKIICTTNVPWNSTWSNKSFEEIWQNMTWIEWEREISNYTNTIYDLLMESQNQQDKNEQELLELDK.

[0082] 2. Expansion of recombinant bacteria and extraction of pCMV-gp140 plasmid

[0083] 10 μL pcmv-gp140 (E. coli TOP10) and 100 μL ampicillin were added to 100 mL LB, and the culture was incubated overnight at 37°C, 200 rpm, and shaken for 16 h. The plasmid was extracted using the TIANGEN endotoxin-free plasmid extraction kit.

[0084] 3. Expression and purification of recombinant HIV-1CRF 5501_B gp140

[0085] 3.1 Plasmid transfection into HEK293F and protein expression

[0086] (1) Before transfection, adjust the cell concentration in the HEK293F cell suspension to 2×10 6 Pieces / mL.

[0087] (2) Plasmid dosage: Taking 200 mL of cells as an example, 200 μg of plasmid is required.

[0088] (3) Plasmid working solution: The plasmid was suspended in PBS buffer and made up to 8 mL with PBS buffer. The solution was mixed by inverting the tube upside down to prevent the plasmid from breaking.

[0089] (4) PEI (EZ-Trans) dosage: PEI: plasmid = 5:1 (weight ratio); PEI: 1000 μg is required for a specification of 1 mg / mL.

[0090] (5) PEI working solution: PEI:PBS=1:7 (weight ratio), mix well with a vortex mixer to prepare PEI working solution.

[0091] (6) Add 8 mL of PEI working solution to the plasmid working solution, shake well while adding, and let stand for 20 minutes to form an EZ-Trans-DNA complex to prepare a mixed transfection solution.

[0092] (7) Slowly add the mixed transfection solution (16 mL) into the cell culture flask (the cell concentration is adjusted to 2 × 10 6 / mL), and gently shake the culture bottle during the process.

[0093] (8) After transfection, the cells were expressed in a suitable culture medium (Sino Biological SMM 293-TII) at 37°C, 120 rpm, and 5% CO2. After 2 days, feed was added once (35 mL of 293-specific feed, Sino Biological SMS293-SUPI, was added per 1 L of cell culture medium). After 5 days, the supernatant containing the expression product was collected.

[0094] In this step, the composition of PBS buffer is: Na2HPO4 8mM, NaCl 136mM, KH2PO4 2mM, KCl2.6mM, pH 7.0-7.2, isotonic with human blood.

[0095] 3.2 Purification of recombinant HIV-1CRF 5501_B gp140

[0096] 1) Using the NGC quest 10 purification system of BIO-RAD, the supernatant after filtration through a 45 μm filter membrane was loaded onto a Flag affinity chromatography column at a flow rate of 1 mL / min, and the flow-through was collected.

[0097] 2) After loading, use solution A (50mM Tris, 150mM NaCl, pure water, pH8.5) to balance and wash impurities at a flow rate of 2mL / min, and use solution B (150μg / mL 3×Flag, 50mM Tris, 150mM NaCl, pure water, pH8.5) to elute the target protein at a flow rate of 1mL / min to obtain about 16mL of eluate.

[0098] 3) Take the supernatant, flow-through and eluate containing the target protein after filtration, denature them and perform SDS-PAGE electrophoresis analysis. The results are as follows: Figure 1 .according to Figure 1 It can be seen that in lanes 4 and 5, i.e. lanes corresponding to 5501_B, the eluate of the sample analysis contains a high concentration of recombinant HIV-1CRF 5501_B gp140 protein with a molecular weight of 140-150Kda, while the concentration of recombinant HIV-1CRF 5501_B gp140 protein in the supernatant and flow-through is relatively low.

[0099] 4) The eluate was transferred to a 30 kDa dialysis bag and dialyzed overnight in a solution (50 mM Tris, 150 mM NaCl, pure water, pH 8.5). After dialysis, the eluate was concentrated using a 30 kDa ultrafiltration tube and the concentrate was collected.

[0100] 5) The concentrate was further purified by size exclusion chromatography (SEC), and the solution with a molecular weight of about 420-450 kDa was collected using Cloumn Superose 6 Increase 10 / 300 GL from Citva to obtain the recombinant HIV-1 CRF5501_Bgp140 trimer. The results are as follows: Figure 2 . Figure 2 It was demonstrated that the concentrate contained 3×Flag in addition to the recombinant HIV-1CRF 5501_B gp140 trimer, which could be removed after further purification by SEC.

[0101] The purification conditions include: the column model is Cloumn Superose 6Increase 10 / 300GL; the flow rate is 0.650mL / min; the buffer is 50mM Tris, 150mM NaCl pH8.5; and the solution with a 280nm UV absorption exceeding 5mAu and a molecular weight of 420-450KDa in the collection pipeline.

[0102] 4. BLI method to verify the interaction between recombinant HIV-1CRF 5501_B gp140 trimer and CD4

[0103] The Gator Plus instrument was used, the temperature was set to 30°C, the fixed buffer was PBST (phosphate buffer), and the binding buffer was PBST + 1% DMSO. Human CD4 was fixed at a concentration of 10 μg / mL through a Ni-NTA probe, and the recombinant HIV-1CRF 5501_B gp140 trimer gp140 was set at 6 concentration gradients (250nM, 125nM, 62.5nM, 31.25nM, 15.625nM, 7.813nM). The test results are as follows Figure 3 , Table 1.

[0104] Table 1. Binding parameters of CD4 and HIV-1CRF 5501_B gp140

[0105]

[0106]

[0107] Figure 3 The dynamic process of binding and dissociation between CD4 and recombinant HIV-1CRF 5501_B gp140 at gradient concentrations is shown. CD4 binds to recombinant HIV-1CRF 5501_B gp140 and then slowly dissociates. Table 1 provides the binding constant kon, dissociation constant koff, and dissociation equilibrium constant KD of CD4 and recombinant HIV-1CRF 5501_B gp140 at gradient concentrations. The data show that the dissociation equilibrium constant of CD4 and recombinant HIV-1CRF 5501_B gp140 is 8.56×10 -9 M, there is a strong affinity between the two.

[0108] 5. Effect of Temsavir on the binding of recombinant HIV-1CRF 55_01B gp140 trimer to CD4

[0109] Fostemsavir is an HIV-1 gp120 directional attachment inhibitor and is currently the only FDA-approved drug targeting the membrane adhesion process. Temsavir is the active form of Fostemsavir after hydrolysis after oral administration into the body, and directly binds to the gp120 subunit present in the HIV-1 envelope glycoprotein gp140 on the virus surface.

[0110] Similarly, 10 μg / mL human CD4 was fixed and the binding of CRF 5501_B gp140+Temsavir mixture (gp140 200 nM, Temsavir 50 μM) to CD4 was detected. The other conditions were the same as those described in item 4. At the same time, parallel experiments with Buffer, Temsavir, and CRF 5501_B were added as controls. Results: Temsavir did not affect the binding of gp140-5501B to CD4. Figure 4 shown.

[0111] The present application example focuses on the unique epidemic strain HIV-1CRF 55_01B in Shenzhen. Currently, there is a lack of research on its own structure and function and the differences with other major epidemic subtypes. The HEK293F expression system is used to fuse the Flag-tag at the C-terminus of the protein to help protein purification. It is simple, efficient, and low-cost, achieving higher expression efficiency and stability. The product has strong biological activity and retains most of the biological functions of HIV envelope glycoproteins. In addition, the purity is high.

[0112] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0113] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for preparing HIV-1CRF 55_01B gp140 trimer, characterized in that: The preparation method comprises the following steps: Cultivating recombinant expression cells of fusion protein and collecting supernatant; the fusion protein includes HIV-1CRF 55_01B gp140 shown in SEQ ID NO.1 and a purification tag; Loading the supernatant onto a purification tag affinity chromatography column, balancing, eluting, and collecting the eluate; The eluate is dialyzed and ultrafiltered, and a concentrated solution is collected; as well as, The concentrate was purified by size exclusion chromatography to prepare HIV-1 CRF 55_01B gp140 trimer.

2. The method for preparing HIV-1CRF 55_01B gp140 trimer according to claim 1, characterized in that: The equilibration buffer used for equilibration includes water, 45mM-55mM Tris, and 100mM-200mM NaCl, with a pH of 8.5-8.8; Optionally, the flow rate of the equilibration buffer is 0.5 mL / min-1.5 mL / min.

3. The method for preparing HIV-1CRF 55_01B gp140 trimer according to claim 1, characterized in that: The purification tag includes a Flag tag.

4. The method for preparing HIV-1CRF 55_01B gp140 trimer according to claim 3, characterized in that: The elution buffer used for elution included water and 100 μg / mL-200 μg / mL 3×Flag, 45 mM-55 mM Tris, 100 mM-200 mM NaCl, pH 8.5-8.8; Optionally, the flow rate of the elution buffer is 0.5 mL / min-1.5 mL / min.

5. The method for preparing HIV-1CRF 55_01B gp140 trimer according to claim 1, characterized in that: The molecular weight cut-off of the dialysis bag used for dialysis is more than 30 kDa.

6. The method for preparing HIV-1CRF 55_01B gp140 trimer according to claim 1, characterized in that: The dialysis buffer used for dialysis includes water, 45mM-55mM Tris and 100mM-200mM NaCl, with a pH value of 8.5-8.

8.

7. The method for preparing HIV-1CRF 55_01B gp140 trimer according to claim 1, characterized in that: The ultrafiltration tube used in ultrafiltration has a molecular weight cutoff of more than 30 kDa.

8. The method for preparing HIV-1CRF 55_01B gp140 trimer according to any one of claims 1 to 7, characterized in that: The recombinant expression cells include HEK293F cells.

9. The method for preparing HIV-1CRF 55_01B gp140 trimer according to any one of claims 1 to 7, characterized in that: The method of collecting the supernatant includes membrane filtration; Optionally, the pore size of the filter membrane used in membrane filtration is 40 μm-50 μm.

10. The method for preparing HIV-1CRF 55_01B gp140 trimer according to any one of claims 1 to 7, characterized in that: The size exclusion chromatography method used Cloumn Superose 6 Increase 10 / 300GL.

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