CN105999223B - Application of PDL1-IgGFc fusion protein in inhibiting severe malaria morbidity - Google Patents

Abstract

The invention discloses application of PDL1-IgGFc fusion protein in inhibiting severe malaria morbidity, and belongs to the technical field of preparation of antimalarial drugs. The invention constructs a fusion gene of PDL1 molecular extracellular segment and IgG molecular Fc segment, constructs the fusion gene into adenovirus or expresses in vitro, and verifies the successful expression of the fusion protein by a Western Blot method. In an in vitro cell experiment, the fusion protein can obviously inhibit CD8+ T cell activation induced by ConA, and meanwhile, in a mouse in vivo experiment, the generation of cerebral malaria caused by infection of plasmodium burgerii anka strain can be obviously relieved by injecting the recombinant adenovirus expressing the PDL1-IgGFc fusion protein into tail veins, so that the survival time of a mouse is prolonged. The invention shows that the PDL1-IgGFc fusion protein has the effect of inhibiting severe malaria, and the inhibition effect is related to the inhibition of the activation of CD8+ T cells. The fusion protein provides a new drug choice for clinically treating severe malaria such as cerebral malaria.

Description

Application of PDL1-IgGFc fusion protein in inhibiting severe malaria morbidity

Technical Field

The invention belongs to the technical field of preparation of anti-malaria drugs, and particularly relates to application of PDL1-IgGFc fusion protein in inhibiting severe malaria.

Background

Malaria is one of the most prevalent infectious diseases in the world, with 32 million people threatened worldwide, with about 2.14 million cases worldwide and 44 million deaths in 2014. There are 5 kinds of plasmodium infecting human, and severe malaria is usually caused by plasmodium falciparum, among which cerebral malaria is one of the most severe malaria, and studies have shown that the death rate can be as high as 18% even by active treatment, and most children cerebral malaria patients die within 1-2 days after central nervous system symptom onset, and even survival cases often leave nervous system sequelae. Most researches suggest that cerebral malaria is caused by adhesion of red blood cells infected by plasmodium to cerebral blood vessels, and then the blood brain barrier damage caused by the cooperation of local microcirculation disturbance of brain tissues and immunopathological injury is the most main pathological change characteristic of the cerebral malaria. However, the specific mechanism by which cerebral malaria develops blood-brain barrier injury has not been fully elucidated to date.

A large number of studies have shown that: the plasmodium component taken up by the activated cerebrovascular endothelial cells can be combined with MHC class I molecules and is presented to CD8+ T cells in a cross way, and plasmodium specific CD8+ T cells (CTL cells) kill the plasmodium antigen-presenting endothelial cells in a targeted way, so that blood brain barrier damage and subsequent pathological changes such as hemorrhage, cerebral edema and the like are caused. Therefore, it can be presumed that: if the activation degree of CD8+ CTL cells during plasmodium infection can be down-regulated in a proper way, the damage degree of a blood brain barrier can be reduced, and the incidence rate and the death rate of cerebral malaria can be reduced.

Programmed cell death-1 (PD-1) is an important negative regulation co-stimulation molecule expressed on the surface of an activated T cell, is combined with a PD-1 ligand to transduce a T cell inhibition signal, can inhibit the proliferation and activity of the T cell, and is beneficial to maintaining the self-stability of the body immunity, thereby preventing excessive immune injury and autoimmune disease. PD-1 ligands are two PDL1 and PDL 2. In recent years, breakthrough progress of PD-1/PD-L pathway research in the fields of anti-tumor, anti-autoimmune disease and the like brings new revelation to cerebral malaria immunotherapy.

Research in a non-brain type malaria model shows that PD-1 expression causes effector T cell failure, and if the PD-1/PD-L pathway is blocked, the T cell response level is further improved, and the host is more favorable for eliminating plasmodium. Meanwhile, according to the previous research of the subject group, if the PD-1/PDL channel is blocked in the cerebral malaria model, the cerebral malaria symptoms of the mouse are aggravated due to the further improvement of the response level of T cells, and the survival time is shortened, so the subject group proposes that in the cerebral malaria model, if the PD-1/PDL channel signal is artificially improved and the excessive activation of CD8+ T cells is inhibited, the cerebral malaria symptoms are expected to be relieved, and a new treatment method is provided for clinically treating severe malaria such as cerebral malaria.

Disclosure of Invention

The invention aims to provide an application of PDL1-IgGFc fusion protein in inhibiting severe malaria.

The invention is realized by the following technical scheme:

the invention discloses an application of PDL1-IgGFc fusion protein in preparation of a medicine for treating severe malaria.

The medicine is a medicine for inhibiting over activation of T cells.

The action target of the medicine is PD-1 molecule expressed on the surface of activated T cell.

The medicine is a medicine for inhibiting CD8+ T cell over-activation through a PD-1/PD-L pathway.

The medicine is used for inhibiting cerebral malaria.

The PDL1-IgGFc fusion protein is a fusion protein consisting of a PDL1 molecular extracellular domain and an IgG molecular Fc segment.

The nucleotide sequence of the PDL1-IgGFc fusion protein is shown in SEQ ID No. 1.

The cell administration amount of the PDL1-IgGFc fusion protein is 1-100 mu g/ml.

The animal dosage of the PDL1-IgGFc fusion protein is 1-100 mg/kg.

Compared with the prior art, the invention has the following beneficial technical effects:

the fusion gene of PDL1 molecular extracellular domain and IgG molecular Fc fragment is constructed by a molecular biology method, the fusion gene is constructed into adenovirus or is expressed in vitro, and the successful expression of the fusion protein is verified by a qPCR and Western Blot method. In vitro cell experiments, the fusion protein can obviously inhibit CD8+ T cell activation induced by ConA, and meanwhile, in mouse in vivo experiments, the recombinant adenovirus expressing the PDL1-IgG1Fc fusion protein through tail vein injection can obviously relieve the occurrence of cerebral malaria caused by infection of plasmodium burgerii anka strain and prolong the survival time of mice. Therefore, the PDL1-IgGFc fusion protein has the effect of inhibiting severe malaria occurrence, and the inhibition effect is related to the inhibition effect of CD8+ T cell activation. The fusion protein provides a new drug choice for clinically treating severe malaria such as cerebral malaria.

Drawings

FIG. 1 is a result chart of cloning mouse PDL1 extracellular domain and IgG1Fc segment genes; wherein, (a) is PDL1 extracellular domain PCR result, and (b) is IgG1Fc fragment PCR result;

FIG. 2 is a pIRES2-EGFP plasmid map;

FIG. 3 shows construction results of IgG1Fc-pIRES2, results of EcoR I and Sac II double enzyme digestion identification, and a small fragment is IgG1 Fc;

FIG. 4 shows the construction result of PDL1-IgG1Fc-pIRES 2; wherein, (a) is a Bgl II and EcoR I double-enzyme digestion identification result, and the small fragment is PDL 1; (b) to construct a successful plasmid schematic;

FIG. 5 shows the result of expression of green fluorescent protein observed under a microscope by transfecting 293 cells with PDL1-IgG1Fc-pIRES 2;

FIG. 6 is a schematic diagram of the construction principle of recombinant adenovirus;

FIG. 7 shows PCR-verified PDL1-IgG1Fc/IgG1Fc-PMT85 shuttle plasmid vector results; wherein (a) is PCR validated PDL1-IgG1 Fc; (b) IgG1Fc was validated for PCR;

FIG. 8 is a PDL1-IgG1Fc/IgG1Fc-PMT85 shuttle plasmid vector map; wherein (a) is PDL1-IgG1Fc-PMT 85; (b) is IgG1Fc-PMT 85;

FIG. 9 shows HEK293 cells infected with recombinant adenovirus;

FIG. 10 shows Western Blot to detect the expression of target protein;

FIG. 11 shows the result of Western Blot detection of in vitro expressed target protein;

FIG. 12 shows the results of PDL1-IgG1Fc inhibiting spleen cell proliferation; wherein (a) is ConA stimulating spleen cell proliferation, cell number is 1 × 10⁶Cells/well, stimulation for 72 hours; (b) as a result of the inhibition of spleen cell proliferation by PDL1-IgG1 Fc;

FIG. 13 shows the results of PDL1-IgG1Fc inhibiting CD8+ T cell proliferation; wherein (a) is ConA concentration of 2 μ g/ml, and stimulation is carried out for 72 hours; (b) PDL1-IgG1Fc inhibited CD8+ T cell proliferation results;

FIG. 14 shows the results of in vivo experiments with PDL1-IgG1Fc recombinant adenovirus.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The results of preliminary experiments in the PD-1 gene knockout brain type malaria mouse model prove that: the regulation and control of PD-1/PD-L pathway has obvious influence on cerebral malaria outcome, which makes possible the immunotherapy strategy of alleviating the symptoms of severe malaria such as cerebral malaria by properly up-regulating PD-1/PD-L pathway.

The present invention proposes the following assumptions based on the above research results: if in a mouse cerebral malaria model, the vascular endothelial cells can specifically over express PD-L ligand, CD8+ CTL cells receive more PD-L signals from the endothelial cells, and more PD-1 receptors on the surface of the endothelial cells are activated, so that more inhibition signals are transduced into the cells, the activation degree of the CD8+ CTL cells is further reduced, the damage degree of the endothelial cells and the blood brain barrier is finally reduced, and the incidence rate and the death rate of cerebral malaria are reduced.

The invention constructs PDL1-IgG1Fc fusion protein, and the fusion protein achieves the effect of inhibiting cerebral malaria by inhibiting excessive immune response of an organism, thereby providing a new method for clinically treating severe malaria such as cerebral malaria and the like.

1. Construction of PDL1-IgG1Fc fusion Gene

1.1 obtaining mouse PDL1 extracellular segment and IgG1Fc segment gene

1.1.1 design of primers

According to NCBI information, primers for amplifying PDL1 extracellular segment and IgG1Fc segment genes are designed, and meanwhile, a proper restriction enzyme cutting site is added for successfully inserting a pIRES2-EGFP vector, and in addition, a point mutation method is adopted for mutating cysteine into serine because 4 cysteine exist in a hinge region of an IgG1Fc molecule and a disulfide bond is formed.

PDL1F：CAGAGA TCTATG AGG ATA TTT GCT GGC ATT (Bgl II site with black underline)

PDL1R：CTCGAA TTCGTG AGT CCT GTT CTG TGG AGG (EcoR I site is black underline)

IgG1Fc F：GACGAA TTCGTG CCC AGG GAT AGT GGT AGT AAG CCT AGC ATA AGTACA GTC CCA GAA GTA TCA TCT (EcoR I site is black underline)

IgG1Fc R：ATTCCG CGGTCA TTT ACC AGG AGA GTG GGA (Black underline Sac II cleavage site).

1.1.2 extraction of Total RNA from mice and reverse transcription into cDNA

Mouse peripheral blood is obtained by performing eyeball bleeding on a mouse, mouse peripheral blood mononuclear lymphocytes (PBMC) are isolated by using mouse lymphocyte separation liquid, total RNA of the mouse PBMC is extracted, and the RNA is reversely transcribed into cDNA.

Total RNA extraction:

(1) every 5X 10⁶Adding 1ml of TRizol into the cells, uniformly blowing and mixing, and transferring to a 1ml of EP tube;

(2) adding 200 μ l chloroform into each tube, turning the EP tube upside down by hand for 15s, standing at room temperature for 10min, standing at 4 deg.C and 12000rpm, and centrifuging for 15 min;

(3) sucking the supernatant, transferring to a new 1.5ml EP tube, adding equal volume of precooled isopropanol, mixing uniformly, and precipitating at 4 ℃ for 10 min;

(4) centrifuging at 12000rpm at 4 deg.C for 10min, and removing supernatant;

(5) adding 1ml 75% ethanol (prepared fresh with DEPC treated water), washing the precipitate;

(6) centrifuging at 4 deg.C and 10000rpm for 5min, and discarding most of supernatant;

(7) centrifuging again at 4 deg.C and 10000rpm for 5min, and sucking off supernatant

(8) Drying at room temperature, volatilizing to remove volatile substances, adding 20 μ l RNase-free water, dissolving completely, ultraviolet analyzing to determine the concentration of extracted RNA, and performing reverse transcription reaction or freezing at-70 deg.C.

Reverse transcription of RNA to obtain cDNA

Taking 1 μ g of RNA as a template, and performing reverse transcription of the RNA into cDNA by using a TAKARA reverse transcription kit, wherein the reaction conditions are as follows: reacting at 37 ℃ for 1h, inactivating reverse transcriptase at 85 ℃ for 5s, and using the product as a next PCR template or freezing and storing at-20 ℃.

1.1.3 cloning of extracellular and IgG1Fc genes in mouse PDL1

Mouse cDNA is taken as a template, high-fidelity DNA polymerase of TAKARA company is adopted, PDL1 primer pair and IgG1Fc primer pair are respectively used for PCR amplification of PDL1 extracellular segment genes and IgG1Fc segment genes, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30 sec, annealing at 62 ℃ for 30 sec, extension at 72 ℃ for 1 min, 35 cycles in total, and final extension at 72 ℃ for 5 min. The results of 1.5% agarose gel electrophoresis are shown in FIG. 1, wherein (a) shows the results of PDL1PCR, lane 1 shows the results of PDL1 extracellular PCR amplification product, lane 2 shows marker, lane 1 shows the results of IgG1Fc PCR, lane 1 shows the results of IgG1Fc fragment amplification product, lane 2 shows marker, and PCR products were recovered.

1.2 construction of the PDL1-IgG1Fc fusion Gene

A PDL1-IgG1Fc-pIRES2 plasmid is constructed by taking a pIRES2-EGFP vector as a framework, a pIRES2-EGFP plasmid map is shown in figure 2, and a multiple cloning site of the pIRES2-EGFP plasmid map contains Bgl II, EcoR I and Sac II enzyme cutting sites.

1.2.1 construction of IgG1Fc-pIRES2 plasmid

Carrying out double digestion on pIRES2-EGFP plasmid and IgG1Fc PCR fragment by using EcoR I and Sac II respectively, carrying out electrophoresis on 1.5% agarose gel, recovering digestion products, taking pIRES2 as a vector, connecting the IgG1Fc fragment to the vector by using T4DNA ligase, transforming the ligation products into XL-10 competent cells, coating the cells on a kanamycin-resistant solid LB culture medium, carrying out inverted culture overnight, picking a monoclonal colony, extracting the plasmid, carrying out double digestion identification on the EcoR I and Sac II, and carrying out sequencing identification on correct digestion products by using an organism company under the condition of enzyme digestion identification as shown in FIG. 3, wherein a lane 2 is an EcoRI and Sac II double digestion product, a small fragment is IgG1Fc, and a lane 1 is Maker.

1.2.2 construction of the PDL1-IgG1Fc-pIRES2 plasmid

Identifying correct IgG1Fc-pIRES2 plasmid and PDL1PCR fragment, performing double enzyme digestion by Bgl II and EcoR I respectively, performing 1.5% agarose gel electrophoresis, recovering enzyme digestion products, using IgG1Fc-pIRES2 as a carrier, using T4DNA ligase to connect PDL1 fragment to the carrier, transforming the ligation products to XL-10 competent cells, coating the cells on a kanamycin-resistant solid LB medium, performing inverted culture overnight, picking up a monoclonal colony, extracting the plasmid, performing double enzyme digestion identification on Bgl II and EcoR II, and referring to FIG. 4, (a), lane 1 is Maker, lane 2 is a double enzyme digestion product of Bgl II and EcoR I, a small fragment is PDL1, and performing sequencing identification on the product with correct enzyme digestion identification by a department of biotechnology company.

To this end, a complete PDL1-IgG1Fc fusion gene was constructed and integrated into the pIRES2-EGFP plasmid, see FIG. 4(b), and the expression products were PDL1 extracellular and IgG1Fc fusion proteins.

1.3 PDL1-IgG1Fc-pIRES2 plasmid transfection experiment

The PDL1-IgG1Fc-pIRES2 plasmid is verified to be constructed correctly, and is transfected into 293A cells to observe the expression of green fluorescent protein.

Recovering 293A cells, starting the experiment after the growth condition of the cells is good, and starting the transfection step when the cell fusion degree is 80-90% (taking a 24-well plate as an example);

(1) diluting 0.8. mu.g plasmid DNA with 50. mu.L serum-free medium, gently blowing and sucking for 3-5 times, mixing, and standing at room temperature for 5 min.

(2) The transfection reagent was mixed by gentle inversion, 2.0. mu.L Lipofectamine TM2000 was diluted with 50. mu.L of serum-free medium, mixed by gentle pipetting 3-5 times, and allowed to stand at room temperature for 5 min.

(3) Mixing the transfection reagent and the plasmid DNA diluent, gently blowing and sucking for 3-5 times, mixing uniformly, and standing for 20min at room temperature.

(4) The transfection complex was added to a 24-well cell plate at 100. mu.L/well and mixed well by gently shaking the cell plate back and forth.

(5) The cell plates were incubated at 37 ℃ with 5% CO₂Culturing in incubator for about 6 hr, changing culture medium to 10% serum, and culturing at 37 deg.C under 5% CO₂The incubator continues the cultivation.

(6) After 48-72h, the expression of green fluorescent protein of the transfected cells was observed using a fluorescence microscope.

After transfection with the PDL1-IgG1Fc-pIRES2 plasmid, the cells exhibited bright yellow-green fluorescence when observed under a fluorescent microscope, see FIG. 5.

2. Recombinant adenovirus construction for expressing PDL1-IgG1Fc/IgG1Fc protein

An AdMax adenovirus packaging system is adopted, the working principle is that an adenovirus shuttle plasmid carrying an exogenous gene and an auxiliary packaging plasmid carrying most of the genome of an adenovirus are cotransfected with HEK293 cells, and recombination is realized by the action of a Cre/loxP recombinase system to generate recombinant adenovirus, as shown in figure 6. The system is simple and convenient to operate, high in recombination efficiency, high in yield of the obtained virus and high in expression level of the target gene.

2.1 shuttle vector construction

This procedure linked the PDL1-IgG1Fc gene to the shuttle plasmid vector PMT85, while the IgG1Fc fragment was linked to the shuttle plasmid vector PMT85 as the former control.

2.1.1 IgG1 Fc' -pIRES2 vector construction

Since IgG1Fc is the 5' end of IgG1 gene, there is no promoter and signal peptide, and primer redesign and promoter and signal peptide introduction are required, the same promoter and signal peptide as PDL1 was used in this experiment.

IgG1Fc’Primer F：AGCAGA TCTATG AGG ATA TTT GCT GGC ATT ATA TTC ACAGCC TGC TGT CAC TTG CTA CGG GCG GTG CCC AGG GAT AGT GGT AGT (Red sequence PDL1 promoter and signal peptide sequence)

IgG1Fc’Primer R：ATTCCG CGGTCA TTT ACC AGG AGA GTG GGA

IgG1Fc-pIRES2 plasmid is used as a template, high fidelity DNA polymerase of TAKARA company is adopted, and the IgG1Fc fragment is amplified by PCR by the primer pair, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30 sec, annealing at 62 ℃ for 30 sec, extension at 72 ℃ for 1 min, 35 cycles in total, and final extension at 72 ℃ for 5 min. 1.5% agarose gel electrophoresis was performed to recover the PCR product, IgG1 Fc'.

Carrying out double digestion on pIRES2-EGFP plasmid and IgG1Fc 'PCR fragment by Bgl II and EcoR I respectively, carrying out 1.5% agarose gel electrophoresis, recovering digestion products, taking pIRES2 as a carrier, connecting the IgG1 Fc' fragment to the carrier by using T4DNA ligase, transforming the ligation products into XL-10 competent cells, coating the cells on a kanamycin-resistant solid LB culture medium, carrying out inverted culture overnight, picking a single colony, extracting the plasmid, carrying out double digestion identification on the Bgl II and the EcoR I, and sending the products with correct digestion identification to an engineering biological company for sequencing and identification.

2.1.2 PDL1-IgG1Fc-/IgG1 Fc' -PMT85 shuttle vector construction

For convenient connection with a PMT85 shuttle vector, XbaI restriction enzyme cutting sites and Cla I restriction enzyme cutting sites are respectively introduced at two ends of a PDL1-IgG1Fc/IgG1 Fc' gene.

Primer 1：G AAC CGT CAG ATCTCTAGAGCC ACC ATG AGG ATA TTT GCT GGC ATTATA (Xbal I restriction site with black underline)

Primer 2：G AGG TTG ATTATCGATTCA TTT ACC AGG AGA GTG GGA GAG (Black underline Cla I cleavage site)

PDL1-IgG1Fc/IgG1Fc 'fragment is amplified by PCR by using PDL1-IgG1Fc-pIRES2/IgG1 Fc' -pIRES2 plasmid as a template, a high fidelity DNA polymerase of TAKARA company and Primer 1 and Primer 2 as primers, wherein the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1 min, 35 cycles in total, and final extension at 72 ℃ for 5 min. 1.5% agarose gel electrophoresis was performed to recover the PCR product.

Double digestion of PMT85 plasmid and PDL1-IgG1Fc/IgG1Fc 'fragment with Xbal I and Cla I, respectively, 1.5% agarose gel electrophoresis, recovery of the digestion products, ligation of PDL1-IgG1Fc/IgG1 Fc' fragment to the vector with PMT85 as the vector using T4DNA ligase, transformation of the ligation products into XL-10 competent cells, plating on kanamycin-resistant solid LB medium, overnight inverted culture, picking of single colony, plasmid extraction, validation by PCR:

verification primer

Primer ID(+)CGCAAATGGGCGGTAGGCGTG

Primer ID(－)GAAATTTGTGATGCTATTGC

Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1 min, 35 cycles in total, final extension at 72 ℃ for 5min, and detection by 1.5% agarose gel electrophoresis. The results are shown in FIG. 7, in which (a) PDL1-IgG1Fc was confirmed by PCR, lane 1 was Maker, lane 2 was PDL1-IgG1Fc PCR product, and (b) IgG1Fc was confirmed by PCR, lane 1 was Maker, lane 2 was IgG1Fc PCR product. The construction of the two shuttle plasmid vectors was completed, and the map is shown in FIG. 8.

2.2 recombinant adenovirus packaging

(1) Recovering HEK293 cells, and using after the growth condition of the cells is good;

(2) the day before transfection, cells are inoculated into a 10cm cell culture dish, and the density of the cells during transfection is controlled to be 70-90%;

(3) taking out the cell culture vessel one hour before transfection, removing the original cell culture medium, adding 10ml of Opti-MEM culture medium, and returning the cells to the incubator;

(4) preparation of complexes of transfection reagents and plasmids:

taking one 1.5ml Ep tube, adding a virus vector plasmid to be transfected, supplementing the virus vector plasmid to 500 mu l by using an Opti-MEM culture medium, and gently mixing the mixture;

adding a 1.5ml Ep tube into the Trans-EZ solution, filling the mixture to 500 mu l by using an Opti-MEM culture medium, and gently mixing the mixture;

dripping the Trans-EZ diluent into the plasmid diluent, gently mixing while adding, and then placing at room temperature for 20 minutes to ensure that the DNA and the Trans-EZ are fully combined to form a stable transfection complex;

(5) taking out the cell culture plate, adding the DNA-Trans-EZ complex obtained above into a cell culture vessel, marking, and putting back into an incubator;

(6) absorbing the culture medium after 6h, washing with PBS once, adding 10ml of fresh growth culture medium for culturing, if a large amount of cells float up, not removing supernatant, adding 6ml of complete culture medium, culturing overnight at 37 ℃, and changing the culture solution the next day;

(7) the change was made once every three days, approximately 7-15 days or so, with viral plaques appearing.

After most of the cells had typical lesions and 50% of the cells had detached (as shown in FIG. 9, the cells had floated up and expressed a large amount of green fluorescent protein), the cells were collected and frozen and thawed repeatedly at-70 ℃ and 37 ℃ for 3 times, and the virus supernatants were collected and stored at-70 ℃.

2.3 Small amplification of recombinant adenovirus

(1) When the cultured HEK293 cells reach 80-90% confluence, removing the culture solution, and leaving a little to cover the cell surface;

(2) adding 10 μ l of the virus solution into a 10cm cell culture dish by using a pipette, uniformly mixing, and placing into a 5% CO2 incubator at 37 ℃;

(3) after 2h, 10ml of culture solution is supplemented, and the mixture is placed into an incubator for culture;

(4) after 4-5 days, HEK293 to be cultured becomes round and is detached from the wall, some cells float, and the cells are changed from orange to yellow, so that the cells are diseased, and the cells can be collected by a pipette gun and stored at-70 ℃ for later use.

2.4 Mass amplification and gradient centrifugation purification of recombinant adenovirus

This recombinant adenovirus was used in animal experiments and was purified by the classical cesium chloride gradient centrifugation method of Hitt et al (1998).

(1) Preparing cells: using a 30X 150mm petri dish, when all infected cells were rounded and partially floated, the dish was scraped and the cells and supernatant were transferred to a 50ml centrifuge tube, 750g, and centrifuged for 10 minutes. Resuspend with 15ml of 0.1M Tris-HCl (pH 8.0) and store at-80 ℃.

(2) The sample was lysed and 1.5ml of 5% sodium deoxycholate was added per 15ml of cell lysate, mixed well and incubated for 30 minutes at room temperature. A relatively clear, high viscosity suspension was obtained.

(3) Add 150. mu.l magnesium chloride and 75. mu.l DNase I solution to each 15ml of cell lysate, mix and incubate at 37 ℃ for 30-60 minutes, mix every 10 minutes, at which time the viscosity should be reduced and only slightly more viscous than water.

(4) A high speed bench centrifuge was used, and high speed centrifugation was carried out at 4 ℃ for 15 minutes.

(5) Simultaneously, a cesium chloride gradient (ultrapure tubes with SW41 rotor for 5ml samples) was prepared: 0.5ml of a 1.5g/cc solution of cesium chloride was added to each tube, and 3ml of a 1.35g/cc solution of cesium chloride was gently layered thereon, followed by gently layering a 3ml layer of a 1.25g/cc solution of cesium chloride. The gradient may not be disturbed after addition.

(6) 5ml of the supernatant obtained in step 4 was added to each tube of the gradient.

(7) Centrifugation with SW41 rotor at 4 deg.C and 35000rpm for 1 hour (acceleration and deceleration set-up 1)

(8) Viral bands were collected (should be between 1.25g/cc and 1.35 g/cc).

(9) The resulting viral bands were transferred to sterile tubes with SW50.1 rotors, the tubes were topped up with 1.35g/cc of solution) and mixed well. Centrifugation was carried out for 16-20 hours using SW50.1 rotor at 4 ℃ and 35000rpm (or for 16-24 hours using SW41 rotor at 10 ℃ and 35000 rpm).

(10) The virus (usually 0.5-1ml) is collected in as small a volume as possible and transferred to a dialysis bag and dialyzed against 500 volumes (or more) of 10mM Tris-HCl, pH 8.0 at 4 ℃ for at least 24 hours with 2-3 changes of solution.

(11) After dialysis, the purified virus was aliquoted into small portions-80 for storage.

2.5 recombinant adenovirus Titer assay

(1) HEK293 cells in good condition were selected and suspended to 5.0X 10 cells by using complete culture medium⁵Cell suspension of one/ml, 1ml of cells were seeded into each well of 24-well plate at 37 ℃ with 5% CO₂And (5) culturing.

(2) Preparing virus samples with 10-fold gradient dilution (preparing 7 sterile Ep tubes, adding 990 μ l of complete culture medium to the first Ep tube, adding 900 μ l of complete culture medium to each of the remaining 6 tubes; diluting the virus solution to be tested, adding 10 μ l of adenovirus stock solution to 990 μ l of Ep tubes, and performing 1:100 dilution (10: 10)^-2) (ii) a Starting from this, 100. mu.l of the dilution were then added to 900. mu.l of Ep tube for a 1:10 dilution (10)^-3) Until it is diluted to 10^-8) Then sequentially mixing 10^-5To 10^-8The diluted virus solution was added to 24 well plates at 100. mu.l per well, one well for each dilution.

(3) Infection with 5% CO2 was carried out at 37 ℃ for 48 hours.

(4) The medium was gently removed and 0.5ml of pre-cooled methanol was slowly added along the side wall of the 24-well plate and fixed at-20 ℃ for 20min (the tip did not touch the cells).

(5) Cells were gently washed 3 times with PBS for 5min each (rather than washing the cells up).

(6) 0.2ml of 1% BSA was added and blocked at 37 ℃ for 1 hour.

(7) 0.2ml of primary antibody solution was added to each well and incubated at 37 ℃ for 1 hour.

(8) Cells were gently washed 3 times 5min each with PBS.

(9) 0.2ml of secondary antibody was added to each well and incubated at 37 ℃ for 1 hour.

(10) Cells were gently washed 3 times 5min each with PBS.

(11) Add 0.2ml of company's working solution to each well and incubate at room temperature for 5-10min (incubation time does not exceed 10 min).

(12) Discard the working solution, wash 2 times with PBS, and add 1ml PBS per well.

(13) The number of positive cells was counted using an optical microscope at 10 × objective lens with 5 fields randomly selected per well.

(14) The average number of positive cells per well and the virus titer were calculated.

And (3) calculating the result:

the average number of positive cells in the experiment calculated in 5 visual fields under the microscope is 28.2, and the virus in the hole is diluted by 10⁷Times, according to the above formula:

PDL1-IgG1Fc recombinant adenovirus titer: 2.23X 10¹¹pfu/ml

IgG1Fc recombinant adenovirus titers: 2.29X 10¹¹pfu/ml。

2.6 recombinant expression detection qPCR

2.6.1 Real time PCR detection of target Gene expression

qPCR primers:

PDL1-IgG1Fc F:AATCAACCAGAGAATTTCC

PDL1-IgG1Fc R:TTGTCCAGATTACCTCAG

IgG1Fc F:CCATTACTCTGACTCCTAA

IgG1Fc R:CCTCCACATCATCTACAA

PDL1-IgG1Fc recombinant adenovirus and IgG1Fc recombinant adenovirus transfect 293T cells, after 5 days, cell RNA is extracted and is reversely transcribed into cDNA, the transcription condition of target gene mRNA is detected by using the primer qPCR,

reaction conditions (two-step process): pre-denaturation at 95 ℃ for 30 seconds, 95 DEG C5 seconds of denaturation, 30 seconds of annealing and extension at 60 ℃, 40 cycles, and 2-delta. Ct analysis for gene transcription, the target genes (PDL1-IgG1Fc, IgG1Fc) were over-expressed, as shown in tables 1 and 2, PDL1-IgG1Fc and IgG1Fc were over-expressed by 1 × 10 respectively, compared to the control cells (transfected empty adenovirus)⁸And 3.3X 10⁷And (4) doubling.

Table 1 qPCR validation of expression efficiency after transfection of recombinant adenovirus into cells (PDL1-IgG1Fc)

Sample (I)	Actin(CT)	PDL1-IgG1Fc(CT)	-ΔCt	-ΔΔCt	2-ΔΔCt
						PMT85	18.22405	40	-21.7759	0	1
PSB2030	18.73667	13.93551	4.801158	26.57711	1×108
						BLANK	19.00078	Undetermined			Is free of

Table 2 qPCR validation of expression efficiency after transfection of recombinant adenovirus into cells (IgG1Fc)

Sample (I)	Actin(CT)	IgG1Fc(CT)	-ΔCt	-ΔΔCt	2-ΔΔCt
						PMT85	18.22405	40	-21.7759	0	1
PSB2030	18.73667	15.13091	4.801158	26.57711	32666477
						BLANK	19.00078	Undetermined			Is free of

Wherein BLANK is 293T cells; PMT85 was a control adenovirus plasmid transfected 293T cells; psb2030 is a 293T cell group transfected by PDL1-IgG1Fc gene overexpression adenovirus plasmid; psb2032 is a 293T cell group transfected by an IgG1Fc gene overexpression adenovirus plasmid.

2.6.2Western blot detection of expression of target proteins

(1) Collecting samples, extracting and quantifying total protein:

receiving cells of a cell platform, discarding a cell culture solution, adding a proper amount of 1 × Lysis Buffer according to the amount of the cells, cracking the cells for 10-15 min at 4 ℃, and marking an empty 1.5ml EP tube;

scraping or blowing down the cells by using a cell scraping or liquid transfer gun, transferring the cells into a correspondingly marked 1.5ml EP tube, and carrying out ice bath for 10-15 min;

centrifuging at 12000 g for 5min at 4 deg.C, and slowly sucking the supernatant into a new 1.5ml EP tube;

protein quantification by the BCA method (the specific steps refer to the kit instruction), and protein concentration is calculated;

treating the protein sample: adding 5 Xloading Buffer, heating at 95-100 deg.C for 5min, and storing at-20 deg.C.

(2) Formulation of SDS-PAGE

(3) Loading and carrying out electrophoresis at 100V for 1.5 hours;

(4) transferring the membrane, and performing electrotransfer for 120min under the condition of constant current of 400 mA;

(5) sealing the PVDF membrane by TBST solution containing 5% of skimmed milk;

(6) primary antibody incubation: covering the sealed PVDF membrane by using a PE glove, adding an antibody diluted by a sealing solution, and incubating overnight at 4 ℃;

(7) washing the membrane with 1 × TBST for 3 times, 10min each time;

(8) and (3) secondary antibody incubation: diluting the corresponding secondary antibody with the blocking solution, and incubating the PVDF membrane for 2h at room temperature

(9) Washing the membrane: the membrane was washed 3 times with 1 × TBST for 10min each time.

(10) ECL development, X-ray development, film to obtain display strips was performed in a dark room.

As shown in FIG. 10, lane 2 shows control cells (untransfected adenovirus), lane 3 shows PDL1-IgG1Fc adenovirus transfected cells, and lane 4 shows IgG1Fc adenovirus transfected cells, and the target protein is expressed in a larger amount compared to the control.

3. PDL1-IgG1Fc protein Activity assay (cellular level)

The over-activated CD8+ T cells play an important and key role in the pathogenesis of severe malaria, for example, the over-activation of CD8+ T cells can be inhibited by PDL1-IgG1Fc fusion protein, so that the PDL1-IgG1Fc fusion protein is expected to be used for inhibiting the occurrence of severe malaria, and the experiment aims to detect the capability of PDL1-IgG1Fc in vitro for inhibiting the activation of T cells.

The results of Western blot detection of PDL1-IgG1Fc and IgG1Fc (as a control) proteins expressed and purified in vitro in cooperation with the biochemical laboratory of this department are shown in FIG. 11, wherein lane 1 is PDL1-IgG1Fc fusion protein, and lane 2 is IgG1Fc protein.

3.1 PDL1-IgG1Fc inhibits spleen cell proliferation

The T lymphocyte can be proliferated under a certain stimulation condition, so the detection of the proliferation efficiency of the PDL1-IgG1Fc for inhibiting the T cell can evaluate the activity of PDL1-IgG1 Fc.

3.1.1 determination of conditions for ConA stimulation of spleen cell proliferation

The mice were sacrificed by cervical dislocation and the spleens were removed under aseptic conditionsGrinding dirty cells, sieving with a 500-mesh sieve, collecting single cell suspension, lysing erythrocytes with erythrocyte lysate, collecting spleen cells, and counting; using 96-well plates, a drug concentration gradient (ConA concentration gradient: 0, 05, 1, 2, 4, 6, 8, 10. mu.g/ml), a cell number gradient (number of splenocytes 1X 10, respectively) were used⁵、2×10⁵、5×10⁵、1×10⁶Individual cells/well) and stimulation time gradient (stimulation time 48h, 72h), cell proliferation rate was determined using CCK8 kit to determine 1 × 10 per well⁶Cells/well, ConA concentration 2. mu.g/ml, and stimulation for 72 hours was the optimal stimulation condition for T cell proliferation (as shown in FIG. 12 (a)).

3.1.2 PDL1-IgG1Fc inhibits spleen cell proliferation

Under optimal ConA stimulation conditions (number of splenocytes 1X 10)⁶Cells/well, ConA concentration 2 μ g/ml, stimulated for 72 hours), using a PDL1-IgG1Fc concentration gradient (0, 0.3, 1, 3, 10 μ g/ml), 10 μ g/ml PDL1-IgG1Fc was able to significantly inhibit splenocyte proliferation compared to the control (IgG1Fc protein) (cell proliferation was significantly inhibited as shown in fig. 12 (b).

3.2 PDL1-IgG1Fc inhibits CD8+ T cell proliferation

3.2.1 magnetic bead method for isolation of CD8+ T cells

Killing the mouse by breaking the neck, taking out the spleen of the mouse under the aseptic condition, grinding, sieving by a 500-mesh sieve, collecting single cell suspension, cracking red blood cells by using a red blood cell lysate, collecting spleen cells, and counting;

gently beating spleen cells, counting the collected cells, and collecting 1 × 10⁷Spleen cells are separated and counted by using a kit for separating CD8+ T cells (negative selection) and CD8+ T cells, and the separation efficiency is over 90% by using CD3 and CD8 antibody flow cytometry.

3.2.2 ConA stimulation of CD8+ T cell proliferation

From experiments on splenocyte proliferation, conditions were determined under which ConA stimulated CD8+ T cell proliferation: number of splenocytes 1X 10⁵Cells/well (CD8+ T cells account for about 10% of total splenocytes), ConA concentration of 2. mu.g/ml, and stimulation for 72 hours, the results of which are shown in FIG. 13 (a).

3.2.3 PDL1-IgG1Fc inhibits CD8+ T cell proliferation

Under optimal ConA stimulation conditions (CD8+ T cell count 1X 10)⁵Cells/well, ConA concentration 2 μ g/ml, stimulation 72 hours), using a PDL1-IgG1Fc concentration gradient (0, 0.3, 1, 3, 10 μ g/ml), 10 μ g/ml PDL1-IgG1Fc was able to significantly inhibit CD8+ T cell proliferation compared to the control (IgG1Fc protein), as shown in fig. 13 (b).

And (4) conclusion: PDL1-IgG1Fc has obvious capacity of inhibiting CD8+ T cell proliferation, CD8+ T cell activation is inhibited, and the medicine is expected to be used for relieving severe malaria.

4 PDL1-IgG1Fc recombinant adenovirus in vivo experiment

Establishing an experimental cerebral malaria model: healthy C57BL6 mice aged 6-8 weeks were intraperitoneally inoculated 1X 10⁶Red blood cells (irbcs) infected with an ampa strain of p.burgii, mice developed brain-type malaria and died 7-14 days later.

Dividing healthy C57 mice into three groups, each group comprises 10 mice, ① PDL1-IgG1Fc recombinant adenovirus, ② IgG1Fc recombinant adenovirus and ③ null adenovirus, and each mouse is inoculated with 1 × 10⁶Individual irbcs, establishing cerebral malaria; each mouse was injected via tail vein with 0.2ml of titer 1X 10 the day before, the first day, the third day, and the fifth day after the inoculation of iRBC¹⁰Each set of pfu/ml corresponds to recombinant adenovirus (total 2X 10)⁹pfu/mouse), the level of protozoa blood disease of the mice is detected every day from the third day after infection, and the survival condition of the mice is observed at the same time to draw a survival curve. Compared with a control group, the PDL1-IgG1Fc recombinant adenovirus can obviously prolong the survival time of a mouse, and as shown in figure 14, a green curve is the injection of PDL1-IgG1Fc recombinant adenovirus, so that the PDL1-IgG1Fc protein can obviously reduce the occurrence of experimental cerebral malaria of the mouse and prolong the survival time of the infected mouse, thereby providing precious time for clinically treating severe malaria.

Claims (4)

1. The application of PDL1-IgGFc fusion protein in preparing a medicine for treating severe malaria is characterized in that the medicine is a medicine for inhibiting cerebral malaria;

   the action target of the medicine is PD-1 molecules expressed on the surface of an activated T cell, and CD8+ T cell over-activation is inhibited through a PD-1/PD-L pathway;

   the nucleotide sequence of the PDL1-IgGFc fusion protein is shown in SEQ ID No. 1.
2. 2. The use of claim 1, wherein said PDL1-IgGFc fusion protein is a fusion protein consisting of an extracellular domain of PDL1 molecule and an Fc fragment of IgG molecule.
3. 3. The use according to claim 1, wherein PDL1-IgGFc fusion protein is administered in a cell in an amount of 1 μ g/ml to 100 μ g/ml.
4. 4. The use according to claim 1, wherein the PDL1-IgGFc fusion protein is administered to the animal in an amount of 1 to 100 mg/kg.

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