CN114609392A - Screening method and application of HIV (human immunodeficiency virus) fully-humanized broad-spectrum neutralizing antibody - Google Patents
Screening method and application of HIV (human immunodeficiency virus) fully-humanized broad-spectrum neutralizing antibody Download PDFInfo
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Abstract
The invention provides a method for screening HIV epidemic strain antibody drugs, which can effectively screen out broad-spectrum neutralizing antibodies with high affinity to HIV domestic epidemic strains.
Description
Technical Field
The invention relates to the field of drug screening, in particular to a screening method and application of an HIV (human immunodeficiency Virus) fully-humanized broad-spectrum neutralizing antibody.
Background
The human immunodeficiency virus type I (HIV-1), the cause of the epidemic of acquired immunodeficiency syndrome (AIDS), has accumulated to cause approximately 3900 million deaths worldwide since its discovery in the early 1980 s, while approximately 120 million of all HIV-1 infected individuals are from China. Although there has been some global progress in dealing with the HIV epidemic, the number of people with AIDS continues to increase, the number of deaths worldwide also increases, and AIDS remains the most serious public health concern worldwide. Due to some characteristics of HIV-1, such as high mutability and diversity of genome, establishment of virus potential pool in resting CD4+ T lymphocyte and the like in early infection, high glycosylation and easy formation of conformation shielding on the surface of HIV-1 Envelope glycoprotein (Env), rare penetration of ENV on the surface of virus particles, and unclear induction generation mechanism of HIV-1 broad-spectrum neutralizing antibody, no thorough cure means or safe and effective vaccine is available on the market at present.
The most common treatment for AIDS is currently the well-known "cocktail" therapy, highly active antiretroviral therapy (HAART), invented by doctor, 1996. Combined antiretroviral therapy has succeeded in controlling HIV-1 infection, but it has not been possible to eliminate viral reservoirs, which leads to viral rebound after drug withdrawal, and antiviral drugs need to be taken for life, with a number of problems of poor compliance, drug resistance, etc. Therefore, after the HIV virus is fully suppressed, it is necessary to control the virus by other means. In recent years, a plurality of novel anti-HIV therapies are produced, wherein a broad-spectrum neutralizing antibody is taken as one of the forefront hot spots and is hoped by scientists, and a large number of animal experimental studies show that the anti-HIV-1 broad-spectrum neutralizing antibody can neutralize HIV-1 and prevent HIV-1 infection by specifically combining with HIV-1 envelope glycoprotein (Env), has a very high possibility of playing an important role in functional cure and radical cure of AIDS, and brings about subversive breakthrough.
The envelope glycoprotein (Env) is the only virus-encoded protein on the surface of HIV-1 virions, is the key to HIV infection, and is the only target for broadly neutralizing antibodies (bnAbs), which react against HIV-1 to target only Env on the surface of the virus. Whereas functional Env on the HIV-1 membrane is represented as a trimer of gp120-gp41 heterodimers, the highly glycosylated protein gp120 is anchored to the viral membrane by interaction with the transmembrane protein gp41, where gp120 is the primary target of broadly neutralizing antibodies. Broad spectrum neutralizing antibody epitopes on gp120 are: a CD4 binding site; the co-receptor binding site, variable 1/2 (loop V1/V2); the variable region 3(V3) homoglycan region, while the broad spectrum neutralizing antibody epitope on gp41 is predominantly the membrane proximal outer region (MPER).
The broad-spectrum neutralizing antibody can not only directly neutralize the virus strain, but also kill the virus by exciting other immune components in the body, thereby achieving the purpose of improving immunity and killing the virus. The first clinical test result of the HIV-1 broad-spectrum neutralizing antibody reported in 2015 shows that the test antibody has good safety, the continuous reduction of the plasma viral load can be observed within 28 days after single injection, and the antiviral immunity of an infected person can be obviously activated. Subsequent clinical tests of multiple phase I and phase II broad-spectrum neutralizing antibodies also show that the half-life of part of the antibodies in vivo can reach 71 +/-18 days, and the antibodies have stronger functions of activating antiviral immunity in infected persons.
At present, scientists mainly obtain purified antibodies with broad-spectrum neutralizing activity by separating from blood of infected persons, however, the apparatus for killing HIV-1 virus is very difficult to generate in naturally infected human body, only 10-15% of people can generate neutralizing antibodies after being infected with HIV-1 virus, and only 2% -5% of people have broad-spectrum neutralizing antibodies, namely more than 80% of HIV-1 strains circulating in the world can be neutralized. Studies have shown that the production of broadly neutralizing antibodies may be related to factors such as viral load, viral diversity and time to infection. In the aspect of in vitro production, most of the monoclonal antibodies on the market are humanized broad-spectrum neutralizing antibodies, have great immunological rejection to human bodies and are difficult to apply to the human bodies.
Drug development involves biological targets, genetic research, molecular biology, genetic technology and protein knowledge, and therefore, the availability of three-dimensional structures of biomolecules is an important component of the discovery of new drugs. The structure of the biomacromolecule is the core for understanding the function of the biomacromolecule, the dynamics and the behavior of the interaction of a broad-spectrum neutralizing antibody and gp120 are determined, the epitope is known, the important basis for determining the affinity of bnAbs is provided, and the biomacromolecule is also important for downstream selection and development of monoclonal antibody drug development and HIV-1 vaccine design.
At present, the structures of macromolecules such as broad-spectrum neutralizing antibodies and gp120 antigens are mainly characterized by experimental techniques such as X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy and a frozen electron microscope imaging technique, and the interaction between the broad-spectrum neutralizing antibodies and the gp120 antigens is obtained by obtaining the three-dimensional structure of a compound of the broad-spectrum neutralizing antibodies and the gp120 antigens, so that the antibodies are screened. The experimental techniques can obtain the real three-dimensional structures of the macromolecules of the antigens and the antibodies and provide important structural information of related proteins. However, the traditional X-ray imaging is not enough to research single protein compound, and the whole experiment needs expensive instruments and equipment and has long experiment period.
Due to the great variability of the HIV virus, the distribution of the different subtypes in the world has regional dominance, e.g., the dominant subtype of mediumafrica A, D, the subtype of B in north america and western europe, the subtype of C in south africa and india, the subtype of E in thailand, and the subtype of F in brazil. At present, the fourth investigation result of 5600 infected person samples in China shows that the types of HIV epidemic strains in China are rapidly increased, and more complex and diversified characteristics are displayed. Due to the domestic complex epidemic situation, the novel recombinant HIV virus is continuously emerging and rapidly epidemic in the population, and 4 dominant HIV-1 viruses account for 89.3% of the total HIV-infected population in 2015, namely CRF07_ BC (41.3%), CRF01_ AE (32.7%), CRF08_ BC (11.3%) and B' subtype (4.0%). However, the common bnAb medicament on the market has better curative effect mainly aiming at the foreign dominant epidemic HIV-1 strain, such as B subtype, and is blank in the aspect of treatment application aiming at the domestic dominant epidemic HIV-1 strain. Therefore, the development of a method for screening drugs for an epidemic strain in China is imminent.
Disclosure of Invention
In order to fill the blank of the prior art, the invention provides a method for screening HIV epidemic strain antibody drugs, and according to the method, broad-spectrum neutralizing antibodies with high affinity to HIV domestic epidemic strains can be effectively screened. In order to achieve the technical effects, the invention specifically provides the following technical scheme:
the invention provides a method for screening HIV epidemic strain antibody drugs, which comprises the following steps:
1) acquiring a envelope Protein sequence of an HIV epidemic strain from HIV sequence database (https:// www.hiv.lanl.gov/content/index), performing homologous modeling on the envelope Protein sequence by using HIV gp120x-ray crystal diffraction structure Data disclosed in a Protein structure database Protein Data Bank (PDB), and acquiring a gp120 three-dimensional structure of the HIV epidemic strain;
the operation process comprises the following steps:
2) obtaining a known Fab-gp120 composite three-dimensional structure of a neutralizing antibody of anti-gp120 Protein from a Protein structure database (PDB);
3) separating Fab from the Fab-gp120 composite structure obtained in the step 2) by Pymol, and processing the Fab fragment of bnAb by PDB-Tools, ANARCI Web, ProABC-2Web and other online Tools to obtain a variable region fragment (Fv) of bnAb;
4) and (3) performing molecular docking simulation by using HAADOCK 2.4 software to obtain a docking model of the bnAb variable region fragment and gp120 and a docking fraction calculated by the HAADOCK server, and selecting the bnAb with a low docking fraction as the screened antibody drug.
In one embodiment, the Fab-gp120 complex three-dimensional structure in step 2 is a complex structure of an antigen binding fragment (Fab) of bnAb, which is composed of the variable region of the antibody light chain (VL), the constant region of the light Chain (CL), the variable region of the heavy chain (VH), and the constant region 1 of the heavy chain (CH1), and HIV-gp 120.
In one embodiment, the Fv sequence in step 3 is a fragment of bnAb that is actually involved in gp120 antigen binding and consists of the variable region of the antibody light chain (VL) and the variable region of the heavy chain (VH).
In one embodiment, the circulating strain of HIV is a domestic circulating strain of HIV. Preferably, the strain is selected from CRF _01AE, CRF _07BC, CRF _08BC or B'.
In one embodiment, the known neutralizing antibody against gp120 protein is selected from N6, VRC01, 3BNC117, D5, B11, B12, 2G12, CAP256-VRC26.25, PG9, PGDM1400, or PGT 128.
In one embodiment, the method further comprises determining the affinity of the neutralizing antibody obtained from the screening for HIV strain gp 120.
In one embodiment, the affinity is determined by using a biolayer interferometry technique (BLI) to determine the affinity of the broadly neutralizing antibody for gp 120.
In one embodiment, the sensor used in the bio-layer interference technique (BLI) is regenerated first, and the regeneration buffer is prepared by: 10mM Gly-HCl, pH 1.5.
The invention achieves the following significant improvements over the prior art：
1. The technology can not only visualize the compound combined with the biological target and provide the molecular interaction related to the driving combination process by using a calculation tool, but also can score the compound in a proper and reliable mode so as to screen the broad-spectrum neutralizing antibody with high affinity to gp120, does not need expensive instruments and equipment such as a refrigeration electron microscope, only needs to utilize simple homologous modeling and molecular docking methods, can be used for easily obtaining the three-dimensional structures of the antigen and the antibody in each laboratory and quantitatively calculating the strength of the interaction, and has extremely simple and convenient operation;
2. the simulation docking method provided by the method has the advantages of rapidness and visualization, and is shorter in time compared with traditional experimental methods such as X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy and frozen electron microscope imaging technologies. At present, the time for determining the macromolecular structure and the bnAb-gp120 affinity is 1-2 weeks by a traditional experimental method, but the method established by the invention only needs 1 day of homologous modeling and 1 day of molecular docking for 2 days in total. Therefore, the detection method established by the invention is more favorable for quickly screening the bnAb and is very important for accelerating the research and development process of new drugs.
3. The antibody screening method established by the invention has the technical advantage of high throughput, the traditional experimental method can only represent the three-dimensional structures of a plurality of antibodies and antigens at most each time, and the method established by the technology can simultaneously process dozens or even hundreds of antigen-antibody structures at one time, so that the method established by the invention is more beneficial to the research and development process of new antibody drugs, and is very important for the treatment of AIDS.
4. The invention also has the advantages that a method for determining the affinity of a plurality of HIV-1 strains which are mainly popular in China and a neutralizing antibody obtained by screening is provided, the technical obstacle that the sensor needs to be regenerated before use is innovatively discovered by the inventor, the problem of unstable detection curve of the sensor is solved by a self-prepared regeneration buffer solution, and reference is provided for HIV vaccine design, HIV-1 antibody treatment and CAR-T immune cell treatment.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is the three-dimensional structure of the Fab and Fv fragments of VRC01, N6, 3BNC 117;
FIG. 2 is a three-dimensional structure of gp120 homology modeling of 4 HIV China epidemic HIV-1 strains;
FIG. 3 is a docking model for bnAb-gp 120;
FIG. 4 is a schematic diagram of the construction of bnAb and gp 120;
FIG. 5 is a schematic representation of bnAb and gp120 expression plasmid maps;
FIG. 6 shows the restriction enzyme digestion verification of recombinant plasmid: (A) pFUSEs-bnAb-VH single-restriction enzyme agarose gel electrophoresis picture. Lane M: nucleic acid marker; lane 1: pFUSEs-VRC 01-VH; lane 2: pFUSEs-N6-VH; lane 3: pFUSEs-3 BNC 117-VH; (B) pFUSE2ss-bnAb-VL single-enzyme agarose gel electrophoresis picture. Lane M: nucleic acid marker; lane 1: pFUSE2ss-VRC 01-VL; lane 2: pFUSE2 ss-N6-VL; lane 3: pFUSE2ss-3BNC 117-VL; (C) pcDNA-gp120 double-cut agarose gel electrophoresis picture. Lane M: nucleic acid marker; lane 1: pcDNA-CRF01_ AE-gp 120; lane 2: pcDNA-CRF01_ AE-gp 120; lane 3: pcDNA-CRF01_ AE-gp 120; lane 4: pcDNA-CRF01_ AE-gp 120;
FIG. 7 shows the transfection ratio optimization of Light Chain (LC) to Heavy Chain (HC);
FIG. 8 is transfection expression time optimization;
FIG. 9 is cell density and transfection volume optimization;
FIG. 10 is a purification chromatogram for a broadly neutralizing antibody;
FIG. 11 is a purification chromatogram of gp 120;
FIG. 12 is an SDS-PAGE analysis of VRC01(A), N6(B), 3BNC117(C) after purification: figure 12A is a non-reducing and reducing SDS-PAGE analysis of purified VRC01, lane M: protein marker; lane 1: cell supernatants transfected with the blank vector plasmid DNA; lane 2: cell supernatants of transfected recombinant plasmid DNA; lane 3: a culture supernatant flowing through the column; lane 4: binding buffer washing solution; lanes 5-7: an eluted protein solution; lanes 8-14: corresponding to 1-7 reduction products, respectively. FIG. 12B is a purified N6 non-reducing SDS-PAGE analysis, lane M: protein marker; lane 1: cell supernatants of transfected recombinant plasmid DNA; lane 2: a culture supernatant flowing through the column; lane 3: binding buffer washing solution; lanes 4-7: eluted protein solution. Figure 12C is a purified 3BNC117 non-reducing SDS-PAGE analysis, lane M: protein marker; lane 1: cell supernatants of transfected recombinant plasmid DNA; lane 2: a culture supernatant flowing through the column; lane 3: binding buffer washing solution; lanes 4-11: an eluted protein solution;
FIG. 13 shows non-reducing SDS-PAGE analysis of purified CRF01_ AE (A), CRF07_ BC (B), CRF08_ BC (C)
(A) Lane M: protein marker; lane 1: cell supernatants of transfected recombinant plasmid DNA; lane 2: flowing the culture supernatant through the column; lane 3-4: binding buffer washing solution; lanes 5-7: eluted protein solution. (B) (C) lane M: protein labeling; lane 1: cell supernatants transfected with recombinant plasmid DNA; lane 2: flowing the culture supernatant through the column; lane 3: binding buffer washing solution; lanes 4-7: an eluted protein solution;
FIG. 14 is a graph comparing experiments conducted without regeneration and after regeneration of the sensor;
FIG. 15 is a bnAb-gp120 affinity assay.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1 preliminary screening for candidate bnAb stably binding to gp120 protein of HIV-1 Strain
A domestic HIV-1 strain gp120 protein model is obtained by using calculation tools such as homologous modeling and molecular docking simulation, and an optimal broad-spectrum neutralizing antibody possibly combined with the gp120 protein of a domestic epidemic HIV-1 strain is preliminarily screened, wherein the specific method comprises the following steps:
1. reading relevant documents, summarizing relevant information such as neutralizing capacity, neutralizing breadth and the like, and selecting appropriate broad-spectrum neutralizing antibodies (bnAbs) according to the summarizing situation for subsequent molecular docking.
2. According to the summary of bnAb information, 11 broad-spectrum neutralizing antibodies of Anti-gp120 are selected, including VRC01, N6, 3BNC117 and the like. Their three-dimensional structures were then obtained in a Protein structure database (PDB) (http:// www.rcsb.org/PDB/home. do), which is typically a complex structure of the antigen binding domain (Fab) of bnAb and the HIV-gp120 Protein, with the Fab domain consisting of the variable region of the antibody light chain (VL), the constant region of the light Chain (CL), the variable region of the heavy chain (VH) and the constant region of the heavy chain 1(CH 1).
3. The Fab was isolated from the Fab-gp120 complex obtained in step 2 by Pymol software, and then PDB-Tools (g), (g) and (g) were combined and the like)https://wenmr.science.uu.nl/pdbtools/)、ANARCI Web(http:// opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/anarci/)、ProABC-2Web(https:// wenmr.science.uu.nl/proabc2/) The Fab fragment of bnAb was processed by an in-line tool to obtain a variable region fragment (Fv) of bnAb, which is a fragment of bnAb that is actually involved in gp120 antigen binding, consisting of the variable region of the antibody light chain (VL) and the variable region of the heavy chain (VH). The Fab and Fv fragments after treatment are shown in FIG. 1 (VRC 01, N6, 3BNC117 for example).
4. In the case of HIV sequence database (https://www.hiv.lanl.gov/content/index) Acquiring the cyst membrane protein sequences of four main epidemic HIV strains (CRF-01 AE, CRF-07 BC, CRF-08 BC, B') in China, and using the existing x-ray crystal diffraction structure data in a Swiss-Model (C/D)https://swissmodel.expasy.org/) The homologous modeling specifically comprises the following steps: entering a Swiss-Model homepage (https:// swissminor. expass. org /) → clicking "Start modeling" → uploading a gp120 amino acid sequence required to be modeled in a new page → clicking "Build Model", the Swiss-Model starts to Build a Model in a full automatic mode → generally 1-3 models are built, according to the GMQE (global Model quality estimation, the higher the number is, the higher the reliability is), the gp120 Model with the highest reliability is selected, and downloading and storing are used for subsequent molecular docking. Gp120 three-dimensional structures of 4 HIV chinese circulating strains were obtained by this procedure (fig. 2).
5. Followed by HAADOCK 2.4 (C)https://wenmr.science.uu.nl/haddock2.4/) And (5) performing molecular docking simulation, and analyzing the interaction between the candidate bnAb and the target HIV gp120 to obtain a docking model. The docking model is shown in FIG. 3 (taking docking results of VRC01, N6, 3BNC117 and domestic HIV-1-gp120 as an example).
6. The data were processed, the docking score table was collated, and bnAb was selected for the next experiment.
A bnAb with a low docking score is considered to be a potentially optimal bnAb. VRC01, N6, 3BNC117, etc. were selected for downstream experiments based on docking scores (table 1).
TABLE 1 docking scores of bnAb and 4 domestic epidemic strains
The result shows that the molecular docking model of the bnAb-gp120 is successfully obtained by the design, the interaction size of the bnAb-gp120 and the molecular docking model is calculated and obtained (reflected in the form of docking fraction), and the affinity size of the bnAb to the target HIV-1 strain is also reflected laterally.
Example 2: construction of broad-spectrum neutralizing antibody and gp120 expression plasmid
(1) Construction of broad-spectrum neutralizing antibody and gp120 expression plasmid
Taking pFUSEs-VRC 01-VH and pFUSE2ss-VRC01-VL plasmids as examples, a description of a specific experimental method is carried out:
1. the sequences VRC01-VH, VRC01-VL were obtained by amplifying the VH and VL regions of the fragment VRC from a previously constructed plasmid PLVX-EF1 alpha-VRC-strep-II-tag-G4S-3 in the laboratory by designing primers. When designing the primer, introducing homologous fragments of the expression vector at two ends of the VRC01-VH and VRC01-VL fragments respectively.
2. Antibody expression vectors pFUSEs-CHIg-hG 1 and pFUSE2ss-CLIg-hk are purchased from invivo company, and a linearized vector is obtained by double enzyme digestion, wherein the enzyme digestion sites of the vectors pFUSEs are EcoRI and NheI; the pFUSE2ss restriction sites are EcoRI and NcoI. The product was subjected to 0.8% agarose gel electrophoresis, and gel-cut and recovered in an Eppendorf tube, and the corresponding fragment was recovered using an agarose gel recovery kit from Axygen, and the purity and concentration of the product were determined.
3. Adding the recovered vector fragment and VRC01-VH and VRC01-VL fragments into Eppendorf tube at a molar ratio of 1:2 by adopting a homologous recombination technology, adding an Exnase II homologous recombinase (Vazyme) and 5 XCE II buffer, and reacting for 0.5 hour at 37 ℃; taking out 10 μ L of the connecting liquid, adding 100 μ L of DH5 α competent cells, carrying out ice bath for 30min, then carrying out heat shock at 42 ℃ for 90s, adding 500 μ L of soc culture medium at 37 ℃ and 220rpm, and culturing for 1 hour; after 1 hour, 400. mu.L of excess liquid was removed by centrifuging the Eppendorf tube 4000g for 1 min. Spreading the rest liquid on LLB plate containing bleomycin and blasticidin, and culturing at 37 deg.C for 12 hr; single colonies were picked up on each plate, and inoculated into 5mL of LB liquid medium at 37 ℃ and 220rpm for 12 hours.
4. Extracting plasmids by using an Axygen miniprep kit to obtain plasmids pFUSs-VRC 01-VH and pFUSE2ss-VRC01-VL (the map structures are shown in figure 5 and are respectively the plasmid structures shown on the left side and the middle side), sending the plasmids to a first generation sequencing verification of science and technology company of the biological engineering (Shanghai) GmbH, and performing strain conservation of DH5 alpha containing the plasmids pFUSs-VRC 01-VH and pFUSE2ss-VRC 01-VL. The construction schematic diagram of pFUSES-VRC 01-VH is shown in the upper part of FIG. 4, the complete map schematic diagram is shown in the left side of FIG. 5, the construction schematic diagram of pFUSE2ss-VRC01-VL is shown in the lower part of FIG. 4, and the complete map schematic diagram is shown in the middle part of FIG. 5.
(2) Construction of gp120 expression plasmid
The domestic 4 HIV-gp120 sequences are synthesized by Shanghai bioengineering GmbH and directly connected to the vector pcDNA3.0 to obtain 4 pcDNA3.0-gp120 expression plasmids. The construction scheme is shown in the middle of FIG. 4, and the expression plasmid is shown on the right side of FIG. 5.
Example 3: preparation and sequencing of plasmids
1. Preparation of plasmids
The DH5 alpha strain containing plasmids pFUSEs-VRC 01-VH and pFUSE2ss-VRC01-VL was inoculated into 100mL of LLB culture solution containing 100. mu.g/mL bleomycin and blasticidin, respectively, the DH5 alpha strain containing plasmid pcDNA3.0-gp120 was inoculated into 100mL of LB culture solution containing 100. mu.g/mL kanamycin, and cultured overnight at 37 ℃ and 220 rpm. The culture was centrifuged at 6000g for 20min at 4 ℃ and the supernatant was discarded.
Take out the Buffers P1 in EndoFree plasmid mega kit (Qiagen), add 120mL of precooled Buffers P1 to the E.coli pellet obtained by centrifugation, cover the centrifuge cap, and vigorously shake the centrifuge flask to completely disperse the E.coli pellet in the Buffers P1.
120mL of Buffers P2 was added to the flask, the flask was covered with a cap, and the mixture was placed on a roller mixer, slowly accelerated to 50rpm, thoroughly mixed, and then left at room temperature for 5 min.
Adding 120mL of Buffers P3 into a centrifuge bottle, covering the centrifuge bottle with a bottle cap, placing the centrifuge bottle on a roller mixer, slowly increasing the speed to the maximum rotation speed of 70rpm of the roller mixer, and thoroughly mixing until the centrifuge bottle is white non-sticky and fluffy mixed liquid. Centrifuge at 9000g for 15min at 4 ℃.
50mL of Buffer FW was poured into the QIAfilter card, and the supernatant obtained by centrifugation was poured into the QIAfilter card, and gently stirred and mixed. And pumping and filtering the mixed solution into a corresponding marked glass bottle.
20mL Buffer ER was added to each glass vial, mixed 6 times upside down and incubated at-20 ℃ for 30 min.
The labeled mega columns were placed on corresponding racks, and 35mL of Buffers QBT was added to each mega column to equilibrate and drain by gravity.
The liquid in the glass bottle is poured into the corresponding marked mega column in batches, and after the liquid in the column is drained, 200mL Buffer QC is added into each mega column in batches for washing. After the liquid in the column had run out, the waste liquid in the waste liquid collection tray was poured into a 50mL clean centrifuge tube.
40mL Buffer QN was added to each mega column, the effluent was collected using a 50mL clean centrifuge tube, mixed by inverting 6 times, and dispensed 20mL into another clean labeled 50mL centrifuge tube.
To each 50mL centrifuge tube, 14mL of isopropanol (room temperature) was added, and the mixture was mixed by inverting the mixture 6 times. Centrifuge at 15000g for 50min at 4 ℃.
The supernatant was aspirated off the clean bench, and 3.5mL of endo-free water was added to each tube to rinse without dispersing the bottom precipitate. Centrifuge at 15000g for 30min at 4 ℃. Buffer TE in an EndoFree plasma mega kit is put into an oven for preheating.
And (4) completely absorbing the centrifuged supernatant in the clean bench, and drying in the clean bench (volatilizing residual absolute ethyl alcohol for about 10 min).
Taking out the Buffer TE in the oven, adding 1mL of Buffer TE into each tube in a clean bench, blowing for 10 times by using a gun, and then putting the tube into the oven at 65 ℃, wherein the tube wall is uninterruptedly knocked to promote the precipitate to be completely dissolved. Centrifuging at 4 deg.C at 4000g for 1min to throw the liquid on the tube wall to the tube bottom, blowing, beating and mixing.
The whole liquid was transferred in a clean bench to endotoxin-free, pyrogen-free, nuclease-free EP tubes labeled accordingly. Aspirate 1. mu.L, measure plasmid concentration with a microspectrophotometer and label on the corresponding EP tube to obtain plasmids pFUSEs-VRC 01-VH, pFUSE2ss-VRC 01-VL.
Then, the single-restriction enzyme was verified by EcoRI, the pFUSs-VRC 01-VH gel electrophoresis was performed as shown in FIG. 6, and the pFUSE2ss-VRC01-VL gel electrophoresis was performed as shown in FIG. 4, with the bands being correct, followed by sequencing. The result shows that the scheme successfully constructs 3 bnAb heavy chain expression plasmids (pFUSEs-bnAb-VH), 3 bnAb light chain expression plasmids (pFUSE2ss-bnAb-VL) and 4 gp120 expression plasmids (pcDNA-gp 120).
2. Sequencing of the target Gene
20 mu L (500ng) of plasmid DNA is respectively taken and sent out for sequencing, whether the target gene of a product produced by the plasmid is changed or not is checked according to an original seed sequence, and the target gene cannot be changed in the process of fermentation culture and amplification of working seeds under a stable process, so that the method can be used for production and correct expression of protein in the next link.
Example 4: expression of recombinant broad-spectrum neutralizing antibody and recombinant gp120 by 293F cell
The bnAbs and gp120 proteins were expressed by transient transfection of polyethyleneimine PEI (1mg/mL) and expression plasmid into 293F cells, bnAbs being a dual plasmid transfection and gp120 being a single plasmid transfection. bnAb transfection is exemplified by VRC 01.
Three days before transfection, 0.3X 106cells/mL inoculum cells were inoculated into 100mL of medium in a 250mL shake flask and incubated for 72h at 37 ℃ in a shaker incubator at 120rpm with 5% carbon dioxide concentration. Cell density on the day of transfection is expected to be 2X 106cells/ml (cells can multiply twice every 24 h). If the density is found to be less than 2X 10 during counting6cells/ml (about 1.0-2.0X 10)6cells/ml), if the cell state is good, the survival rate can reach 90 percent, and the cell can also be used for transfection.
And secondly, taking out a cell culture bottle from a shaking table in a carbon dioxide incubator on the day of transfection, using an electric pipette, and gently blowing and beating the cell suspension for about 20 times by using a 10ml pipette (cells are not blown and are easy to damage), so that inaccurate counting caused by cell agglomeration is prevented. And blowing, beating and uniformly mixing to obtain single cell suspension, taking out a small amount of single cell suspension, putting the single cell suspension into a 1.5mL EP tube for cell density counting, and calculating the cell viability and the total cell number according to the cell density counting result.
③ add 100ug of expression plasmid (antibody heavy-light chain expression plasmid in the ratio of pFUSs-VRC 01-VH: pFUSE2ss-VRC01-VL ═ 2:3, i.e. pFUSs-VRC 01-VH 40ug, pFUSE2ss-VRC01-VL 60ug) to 1ml of F culture medium, add 300. mu.L of PEI solution (1mg/ml) sterilized by filtration to 1ml of F culture medium, mix well using a pipette, and stand for 5 min.
Blowing the DNA/F culture medium mixed solution by taking an electric gun, dripping the PEI/F culture medium mixed solution into the DNA/F culture medium mixed solution by using a pipette in the right hand in a rotating way, and standing for 30min at room temperature.
Slowly dropping the DNA/PEI mixed solution into the cells.
Sixthly, after transfection, incubating for 72 hours in a shaking incubator with 37 ℃, 120rpm and 5 percent of carbon dioxide concentration.
Seventhly, adding OPM-CHO PFF06 culture medium (the addition amount is 1/20 of the transfection system) and L-Glutamine (the addition amount is 1/50 of the transfection system) for 24h of transfection.
Example 5: optimization of transfection expression conditions for broad-spectrum neutralizing antibodies
The ratio of heavy and light chains during antibody expression is critical to the yield and quality of the final antibody. Thus, we optimized the transfection ratio of Light Chain (LC) to Heavy Chain (HC), transfection expression time, cell density and transfection volume in order to improve the expression of recombinant antibodies:
1. transfection ratio optimization of Light Chain (LC) to Heavy Chain (HC)
Cells (25mL) were first used with a LC: HC ratio of 2:1, 1:1, 2:3 or 1:3.293F using only 25. mu.g of Heavy Chain (HC), Light Chain (LC) or different ratios of HC/LC DNA at a density of 200 ten thousand cells/mL, all in the pFUSEs human IgG1 expression vector. Three days after transfection, 10. mu.l of culture supernatant was analyzed by non-reducing SDS-PAGE. The results are shown in figure 7 of the drawings,HC- LC transfection ratio resulted in the highest yield of fully assembled IgGThe other HC/LC ratios all decrease to different extents. Transfection of the HC construct alone did not produce secreted products. Transfection of HC or LC constructs alone did not produce secreted products.
2. Transfection expression time optimization
To explore the optimal transfection time, the recombinant VRC01 heavy and light chain expression plasmids obtained were transfected into 293F cells (25ml) at a 2:3 ratio at a cell density of 200 ten thousand cells/ml. Culture supernatants were collected at 2, 3, 4, 5 days post-transfection and analyzed by non-reducing SDS-PAGE. As can be seen from FIG. 8, as the duration of transfection increased, the yield of recombinant antibody increased, and the expression of other proteins also increased. It is recommended to collect the supernatant 3 days (72 hours) after transfection to maximize recombinant antibody production and reduce protein contamination.
3. Cell density and transfection volume optimization
The heteroproteins increased over time in 293F cells (25ml) transiently transfected with both expression plasmids (HC + LC plasmids) at a 2:3 ratio without significant fluctuation in antibody production (FIG. 9). Our data show that antibody production per unit volume is 100ml transfection volume yielding slightly less than 25ml system. However, a transfection volume of 100ml may be a more favorable choice in terms of total expression level (fig. 9, lines 4 and 6).
Example 6: broad-spectrum neutralizing antibodies and purification of gp120
Purification of bnAb was performed using Protein a affinity chromatography pre-packed column targeted to the Fc-fragment of immunoglobulin (IgG), and gp120 was purified using Ni metal chelating column targeted to History tag. The method comprises the following specific steps:
1. sample preparation
Collecting transfected cell suspension 72h after transfection, centrifuging for 300g 5min and 4000g 30min, filtering with 0.22um filter membrane to remove impurities to obtain relatively pure supernatant (sample), and purifying protein on NGC Scout 10Plus purifier
2. Purification process
Note: all steps were performed using the a pump, except for the eluent (elution using the B pump) and the sample applicator using the sample pump.
Starting up: the collector, the instrument and the computer are sequentially started.
Cleaning the system: the whole system was purged with distilled water at a flow rate of 5 ml/min.
Thirdly, exhausting: the system is exhausted in the order of the system after the pump and the A/B pump.
And fourthly, in order to ensure that the solution A and the solution B are mixed at the beginning of the elution step, a pump B is filled with buffer B (eluent) in advance, and the volume of the pump is about 5 ml. All steps were performed using the a pump, except for the eluent (elution using the B pump) and the sample applicator using the sample pump.
Connecting the column: a pump slowly distilled water (0.5ml/min) and connect the column first up and then down to the NGC Scout 10Plus purifier.
Sixthly, washing the mixture by 10ml of distilled water to remove the ethanol, and obtaining the product with the concentration of 6 ml/min.
Seventhly, using 10mL of binding buffer for balancing.Ensure the stability of the ultraviolet baseline, pH and conductivity.6ml/min。
Loading samples: the flow rate of the sample is slowed down to ensure that the sample and the matrix have sufficient contact time to perform adsorption, or the flow is stopped after the sample is loaded, so that the sample reacts in the chromatographic column for a period of time, 4 ml/min.
Ninthly, washing with 6mL of binding buffer. Ensure the return of ultraviolet raysBack to near baseline。3ml/min。
The fraction in the red (R) is eluted in a linear gradient with 5mL of elution buffer and finally in one step with 1mL, 3 mL/min. The eluted fractions are neutralized by adjusting the pH after elution or eluted into a basic buffer, such as 1M Tris-HCl, pH 9.0, plus 1/10 volumes of the antibody eluate volume.
Purification chromatograms are shown in fig. 10 and fig. 11, the upper half of fig. 10 is a purification chromatogram of VRC01, including the whole purification process and an enlarged view of an elution peak, and the lower half is an enlarged view of an elution peak of N6 and 3BNC 117. FIG. 11 is a large peak elution chart for gp120 purification. The results showed that all the target proteins were eluted, indicating that the target proteins could be purified and eluted, and further protein size verification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was required.
3. Whether the bnAb and gp120 are successfully purified is verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
The gel concentration was 10%, and the formulations of the separation gel and the concentrated gel for protein electrophoresis are shown in the following table:
TABLE 2 Release glue formulation
TABLE 3 concentrated gum formulation
| Concentration of |
5％ |
| H2O/mL | 1.75 |
| 30% acrylamide/mL | 0.5 |
| 4 × Tris-SDS concentrated gel buffer/mL | 0.75 |
| AP (ammonium persulfate)/uL | 30 |
| TEMED/ |
4 |
| Total volume/ |
3 |
The specific experimental method is as follows:
the collected purified bnAb is mixed with electrophoresis 5 Xcloning buffer in proportion to prepare a sample.
And secondly, performing electrophoresis sample loading operation on the prepared sample, connecting a power supply after sample loading is finished, initially setting the voltage to be 80V, and replacing the voltage to be 120V after the sample strip pressure reaches the boundary line between the concentrated gel and the separation gel.
Taking down the protein gel after electrophoresis, placing the protein gel in Coomassie brilliant blue staining solution, and shaking and staining the protein gel for about 30min at room temperature.
And fourthly, discarding the dyeing liquid, replacing a proper amount of decoloring liquid, and shaking at room temperature to decolor the glue.
The results are shown in FIGS. 12-13, and FIG. 12 shows that non-reducing SDS-PAGE has a band at 180KD, indicating that the bnAb purification was successful; reduced SDS-PAGE had bands at 50kD and 25kD, respectively for the heavy (50kD) and light (25kD) chains of bnAb, which also reflected successful bnAb purification. FIG. 13 shows a band around 120kD, indicating the success of gp120 purification.
Example 7: affinity determination of bnAb with several major domestic HIV-1-gp120
The affinity (KD) of the broadly neutralizing antibody and gp120 was determined using the biolayer interferometry (BLI) with the Octet QK2 (forte' Bio). The specific experimental method is as follows:
dilution of gp120 antigen/broadly neutralizing antibody: diluting the expression purified broad-spectrum neutralizing antibody to 10 mug/ml; gp120 antigen was diluted in 2-fold gradients as needed for a total of 4 concentrations (with zero concentration).
Pre-wetting the sensor: PBST (200 ul/well) was added to the pre-wetted plate, and the sensor was placed in the PBST-added well and pre-wetted for 10 min.
Checking whether non-specific binding exists or not: baseline (15s) → association (time as the case may be, observed to stop without significant non-specific binding) → stop.
Regenerating the sensor: according to our earlier experiments, if the AHC sensor used for the first time is used for detection without regeneration, the real-time curve during the experiment is very unstable, a serpentine curve appears, the experiment result is unstable, the system cannot be fitted, and the affinity cannot be calculated, and the curve run out by the regenerated sensor is obviously more stable, which is beneficial to the accuracy of the calculation of the affinity KD value (as shown in fig. 14). Therefore, after determining no non-specific binding, we first regenerate the first-used AHC sensor by: baseline (15s) → regeneration (30s) → stop. The preparation method of Regeneration buffer comprises the following steps: 10mM Gly-HCl, pH 1.5.
Pre-experiment: the single concentration analyte was used for the experiment and the highest concentration point was adjusted according to the binding dissociation curve, the highest concentration being significantly curved within the binding time.
Sixthly, formal experiment: the experiment is carried out by adopting 3 concentration points and 0 concentration, and a more accurate affinity KD value can be obtained. The highest concentration is required to be curved, each concentration point is uniformly dispersed, and the dissociation is more than 5%.
TABLE 4BLI Experimental procedure setup
| Step (ii) of | Time(s) |
| baseline | 90 |
| loading | 120-150 ℃ with a curing height of 1-1.5nm |
| baseline2 | 60 |
| |
600 |
| |
600 |
| regeneration | 30 |
Processing BLI data: opening the Analysis software octaanalysis Studio 12.2, selecting the data to be analyzed in the upper left corner of the "Home" interface → clicking "preprcessed data" → setting a blank (selecting a sensor of 0 concentration, setting a mouse right button to "reference sensor") → subtracting a blank (selecting an entire column of sensors, and selecting a mouse right button "background reference in column") → reference sample (this step may modify the sample name, concentration, etc., if set at the time of the experiment, skipping directly) → clicking data correlation → Align YAxis (selecting Average of base step, zeroing the baseline) → inter step correlation (selecting separation, aligning the dissociation step with the binding step curve) → matching (checking the Analysis result of dynamics → smoothing the curve) Analysis.
According to the results shown in table 5, the actual affinities of the three bnAb screened by the molecular docking simulation to the gp120 proteins of the three strains are all better (the smaller the KD value is, the better the affinity is, the lower the KD value is, the affinity is less than 100nM, which meets the requirement for the affinity of antibody drugs), wherein the affinity value of the N6 antibody to the antibodies of the three strains is even lower than 10nM, which indicates that the antibody has excellent affinity to the gp120 proteins of the three strains. And the combination and dissociation can be dynamically monitored in real time through OctetAnalysis Studio 12.2 software in the process of affinity determination (figure 14), and the real-time monitoring can lead the experimenters to visually observe the combination and dissociation process of the antigen-antibody, thus being convenient for finding, analyzing and solving problems in the experimental process more timely and coping with unexpected conditions in the experiment. For example, if binding is not observed in the binding step, the experiment can be stopped immediately, and no problem is found until the analysis results are finished.
TABLE 5 bnAb-gp120 affinity (KD) summary Table
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A method for HIV circulating strain antibody drug screening, comprising the steps of:
1) acquiring a envelope Protein sequence of an HIV epidemic strain from HIV sequence database (https:// www.hiv.lanl.gov/content/index), performing homologous modeling on the envelope Protein sequence by using HIV gp120x-ray crystal diffraction structure Data disclosed in a Protein structure database Protein Data Bank (PDB), and acquiring a gp120 three-dimensional structure of the HIV epidemic strain;
the operation process comprises the following steps:
2) obtaining a known Fab-gp120 composite three-dimensional structure of a neutralizing antibody of anti-gp120 Protein from a Protein structure database (PDB);
3) separating Fab from the Fab-gp120 composite structure obtained in the step 2) by Pymol, and processing the Fab fragment of bnAb by PDB-Tools, ANARCI Web, ProABC-2Web and other online Tools to obtain a variable region fragment (Fv) of bnAb;
4) and (3) performing molecular docking simulation by using HAADOCK 2.4 software to obtain a docking model of the bnAb variable region fragment and gp120 and a docking fraction calculated by the HAADOCK server, and selecting the bnAb with a low docking fraction as the screened antibody drug.
2. The method of claim 1, wherein the Fab-gp120 complex three-dimensional structure in step 2) is a complex structure of an antigen binding fragment (Fab) of bnAb consisting of a variable region of an antibody light chain (VL), a constant region of a light Chain (CL), a variable region of a heavy chain (VH) and a constant region of a heavy chain 1(CH1) and HIV-gp 120.
3. The method of claim 1, wherein the Fv sequence of step 3) is a fragment of bnAb involved in binding to the gp120 antigen and consists of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) of an antibody.
4. The method of claim 1, wherein the circulating strain of HIV is a domestic circulating strain of HIV.
5. The method of claim 4, wherein the HIV pandemic strain is selected from CRF _01AE, CRF _07BC, CRF _08BC, or B'.
6. The method of claim 1, wherein the known neutralizing antibodies against gp120 protein are selected from N6, VRC01, 3BNC117, D5, B11, B12, 2G12, CAP256-VRC26.25, PG9, PGDM1400, or PGT 128.
7. The method of any one of claims 1 to 6, further comprising the step of determining the affinity of the neutralizing antibody obtained from the screening for HIV strain gp 120.
8. The method of claim 7, wherein the affinity is determined by using biolayer interferometry (BLI) to determine the affinity of broadly neutralizing antibodies to gp 120.
9. The method according to claim 8, wherein the sensor used in the biolayer interferometry technique (BLI) is regenerated first, and the regeneration buffer is formulated as: 10mM Gly-HCl, pH 1.5.
10. A pharmaceutical composition for treating HIV diseases caused by HIV domestic circulating strains, wherein the active ingredient of the pharmaceutical composition is VRC01, N6 or 3BNC117 antibody.
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