CN110938134A - Preparation method and application of recombinant human erythropoietin - Google Patents

Abstract

The invention belongs to the field of biological medicine preparations, relates to the technical field of preparation of medicines for treating malaria, and particularly relates to a preparation method of recombinant human erythropoietin and application of the recombinant human erythropoietin as an oral medicine for treating mouse cerebral malaria. The recombinant human erythropoietin is obtained by expression preparation from tussah pupa or larva, the rhEPO freeze-dried powder is prepared from the tussah pupa, the requirement on equipment condition is low, the production process is simple, large-scale production can be carried out, the specific activity of the rhEPO in the freeze-dried powder is higher, and the recombinant human erythropoietin is suitable for developing auxiliary treatment medicines for oral administration of cerebral malaria and has practical value. Meanwhile, the utilization value of the tussah pupa is improved, and the method has important significance for future high-benefit and sustainable development of the tussah industry. Provides a new approach for the research of the auxiliary treatment medicine of the children cerebral malaria.

Description

Preparation method and application of recombinant human erythropoietin

Technical Field

The invention belongs to the field of biological medicine preparations, relates to the technical field of preparation of medicines for treating malaria, and particularly relates to a preparation method of recombinant human erythropoietin and application of the recombinant human erythropoietin as an oral medicine for treating experimental cerebral malaria of mice.

Background

Plasmodium falciparum (A) and (B)Plasmodium falciparum) Infect human body and parasitize in human red blood cells, cause red blood cell injury, and serious infection can cause complications, including injuries of tissues such as blood, liver, kidney and brain. Cerebral Malaria (CM) is one of the most serious complications, with a mortality rate of 15-20% occurring in african children under five years of age. Most children with cerebral malaria die within one to two days after central nervous system infection, and even surviving children are often accompanied by persistent neurological impairment, resulting in neurological sequelae such as cognitive, behavioral and motor dysfunction. The current research considers that the main pathological change of cerebral malaria is characterized by adhesion of red blood cells infected with plasmodium to cerebral vessels, resulting in local microcirculation disturbance of brain tissues and immunopathological damage, so that the blood brain barrier is damaged. Although artesunate is recommended as the first choice for treatment of cerebral malaria, this treatment does not address the clinical symptoms of cerebral malaria. The research and research on the auxiliary treatment medicine for the cerebral malaria has important significance for reducing blood brain barrier injury, improving the symptoms of the cerebral malaria and reducing the death rate.

Human Erythropoietin (EPO) is a multifunctional glycoprotein cytokine secreted mainly by the human kidney and mainly acts to stimulate the bone marrow to produce red blood cells. In addition, research shows that the medicine has various non-hematopoietic functions, such as antioxidation, anti-apoptosis, inflammation regulation, angiogenesis promotion and the like, and has protective effects on cells and organs such as nerves, brains, cardiovascular diseases, kidneys, stem cells and the like. Recombinant human erythropoietin (rhEPO) has been used clinically for individual therapy, and the primary structure of rhEPO includes 166 amino acid residues, which are identical to the native EPO protein. rhEPO is mainly used for treating anemia of patients caused by chronic renal failure, tumor chemotherapy, AIDS and the like by intravenous injection or subcutaneous injection, and no technology for applying rhEPO to adjuvant therapy of cerebral malaria exists so far.

Currently, commercially available rhEPO is a genetic engineering product for injection, which is produced by utilizing Chinese hamster ovary Cells (CHO) through large-scale amplification culture expression, and has high requirements on production equipment conditions, complex process and higher product price. It has also been reported that the rhEPO is expressed by silkworm chrysalis, the recombinant silkworm baculovirus, the expressed recombinant protein and the preparation and application thereof (application No. 201310636430.8), because the silkworm is diapaused in the non-pupal stage, the silkworm rapidly develops and changes into imago through the short pupal stage, which is not beneficial to the infection of the virus and the full expression of the gene. The tussah is a unique resource in China, and the annual output of the tussah cocoons is about 7 ten thousand tons. The tussah can be preserved for a long time due to the pupal stage diapause and overwintering, and the pupal body is large, so that the tussah can be considered to be developed into a high-efficiency bioreactor. The prior art does not report a method for preparing rhEPO samples by utilizing tussah pupae or larvae.

Disclosure of Invention

In view of the defects in the prior art, the invention aims to provide a preparation method of recombinant human erythropoietin and application thereof in preparing a medicament for treating mouse cerebral malaria, wherein the rhEPO is prepared by expression from tussah pupae or larvae. By establishing an application model of the rhEPO as the medicament for treating the mouse cerebral malaria, technical and theoretical supports are provided for the future use of the rhEPO as an oral medicament for the adjuvant treatment of the human cerebral malaria.

In order to achieve the purpose, the invention adopts the following technical scheme.

The recombinant human erythropoietin is obtained by expression preparation from tussah pupa or larva.

A method for preparing a recombinant human erythropoietin sample specifically comprises the following steps.

(1) Artificially synthesized human erythropoietin gene (hepo): 166 amino acids of mature human erythropoietin protein are coded by the gene, a restriction enzyme BamH I recognition sequence GGATCC and a protein initiation codon ATG are introduced at the 5 'end of the gene, and a restriction enzyme EcoR I recognition sequence GAATTC and a protein termination codon TGA are introduced at the 3' end of the gene;hepothe gene may be human tissue cDNAThe template is obtained by PCR amplification.

(2) Constructing a recombinant expression plasmid: transfer expression vector pApM748BE and tussah virushepoCarrying out double enzyme digestion treatment on the genes by using restriction enzymes BamH I and EcoR I respectively, recovering a vector and a gene fragment by agarose gel electrophoresis, connecting the vector and the gene fragment by using T4DNA ligase, and converting the vector and the gene fragment into escherichia coli; picking single colony for culturing, extracting plasmid DNA, performing enzyme digestion identification to obtain recombinant plasmid pApM748 BE-hepo。

(3) Preparing insect cells: the Tn-High Five cells are subcultured to logarithmic phase by using complete TNM-FH medium to prepare about 0.5-1 × 10⁶Transferring the cell suspension/mL into 6-well cell culture plate to make the cell density about 80% of the culture plate, culturing at 27 deg.C for 1-2 hr, and replacing 1-2mL serum-free cell culture medium (SF-900)^TMII SFM), cells can be used for transfection or recombinant virus screening.

(4) Cell transfection: pApM748BE `/5 `/4 `/S `/hepoRecombinant plasmid DNA and tussah ApNPV-Δph/egfp ⁺ The virus DNA is cotransfected to Tn-High Five cells by the following specific method: adding about 0.5 μ g of viral DNA and about 1 μ g of recombinant plasmid DNA to 100 μ L of serum-free cell culture medium SF-900^TMII; adding 8 muL transfection reagent Cellffectin into 100 muL serum-free cell culture medium SF-900^TMAnd II, uniformly mixing the DNA mixed solution and the transfection reagent mixed solution, transferring the DNA and transfection reagent mixed solution into a prepared cell at room temperature for 30-50 minutes, culturing for 4-5 hours at 27 ℃, replacing a complete TNM-FH culture medium, and continuously culturing for 4-5 days at 27 ℃. And (3) taking cell culture supernatant, injecting infected tussah pupae, incubating at the temperature of 18-22 ℃ for 10-15 days, wherein the pupae are about 50-100 mu L/pupae.

(5) Screening of recombinant viruses: screening the recombinant virus ApNPV by using Tn-High Five cells by adopting a terminal dilution method-Δph/Δegfp/hepo ⁺ . Will havehepoThe tussah pupa body fluid infected by the recombinant virus and the expression EGFP virus is cultured in the insect serum-free cell culture medium SF-900^TMII SFM according to 1: diluting with 100 vol%, filtering for sterilization, and performing 10-fold gradient on the sterile virus solutionDiluting to 10 deg.C^-1、10^-2、10^-3And 10^-4Virus solutions of different concentrations. The High Five cells are infected by the virus liquid which is diluted in a gradient way, fixed by 2 percent of low-melting point agarose, added with 1-2mL of complete TNM-FH culture medium and cultured for 4-5 days at 27 ℃. And taking out the upper culture solution, picking out cells without EGFP markers by using a glass pipette by adopting a reverse marker screening method, respectively suspending the cells in a small amount of serum-free cell culture medium, shaking and centrifuging, taking the supernatant, injecting the supernatant to infect tussah pupas, placing the tussah pupas at the temperature of 18-22 ℃ for 10-15 days, wherein each end of the supernatant is 50-100 uL. The purity of the screened virus is analyzed by microscopic observation or PCR detection method. This step can be repeated for one more round of virus screening if desired.

(6) Expression detection of rhEPO in tussah pupa: recombinant virus ApNPV-Δph/Δegfp /hepo ⁺ Injecting and inoculating into tussah pupa, and incubating at 18-22 deg.C; and (3) respectively taking tussah pupa body fluids infected for 7, 9, 11, 12, 13, 14 and 15 days to perform protein electrophoresis, performing Western-blot detection on an anti-EPO antibody, and performing quantitative analysis.

(7) Preparing rhEPO tussah pupa freeze-dried powder: recombinant virus ApNPV-Δph /Δegfp /hepo ⁺ Infecting tussah pupae in batches; collecting infected tussah pupas, homogenizing, filtering with gauze to remove residues, and freeze-drying the homogenate of the tussah pupas; collecting lyophilized powder, packaging and storing in-80 deg.C refrigerator.

Application of recombinant human erythropoietin in preparing oral medicine for treating mouse cerebral malaria.

Compared with the prior art, the invention has the following beneficial effects.

The invention provides a method for preparing rhEPO samples by utilizing tussah pupas or larvae. The rhEPO freeze-dried powder is prepared by utilizing the tussah pupa for the first time, the requirement on equipment conditions is low, the production process is simple, the large-scale production can be carried out, the specific activity of the rhEPO in the freeze-dried powder is higher, the rhEPO freeze-dried powder is suitable for developing auxiliary treatment medicines for oral administration of cerebral malaria, and the rhEPO freeze-dried powder has practical value. Meanwhile, the utilization value of the tussah pupa is improved, and the method has important significance for future high-benefit and sustainable development of the tussah industry.

The invention provides the prepared rhEPO which is administrated in an oral (intragastric) way, and establishes a model for treating mouse cerebral malaria by the rhEPO. The rhEPO orally taken is proved for the first time to obviously protect the integrity of a blood brain barrier and prevent the occurrence of cerebral malaria of mice. Provides a new approach for the research of the auxiliary treatment medicine of the children cerebral malaria.

Drawings

FIG. 1 ishepoAnd (3) the PCR detection result of the recombinant virus, wherein 1, 2 and 3 are the PCR detection of the recombinant virus, and 4 is the recombinant plasmid pApM748 be/hepo.

FIG. 2 shows the results of detecting the expression of rhEPO in tussah pupae, wherein 1 is the non-infected tussah pupae, 2-8 are the infected tussah pupae with recombinant virus, and the body fluid of the tussah pupae is obtained at days 7, 9, 11, 12, 13, 14 and 15.

FIG. 3 is the in vitro activity test of tussah pupa expressing rhEPO protein.

FIG. 4 is the in vivo activity assay of tussah pupae expressing rhEPO protein. Wherein A is the reticulocyte percentage; b is the absolute number of reticulocytes. Compared with wild tussah freeze-dried powder, the EPO group and the rhEPO group,P<0.05；^#compared with rhEPO tussah freeze-dried powder,P<0.05。

FIG. 5 shows that tussah pupae express rhEPO to improve the brain pathological injury of experimental cerebral malaria of mice, which is an Evans blue staining experiment and the staining condition of the brains of various groups of mice after perfusion.

FIG. 6 shows that tussah pupae express rhEPO to improve the brain pathological injury of experimental cerebral malaria of mice, and the severe condition of blood brain barrier disorder is suggested for quantifying the content of Evans blue in each group.

FIG. 7 shows that tussah pupae expressing rhEPO improves the brain pathological lesions of experimental cerebral malaria in mice, stained for Hematoxylin and Eosin (HE) and immunohistochemical analysis of anti-ICAM-1 and-VCAM-1 antibodies.

FIG. 8 shows that the tussah pupae expressing rhEPO improves the brain pathological damage of experimental cerebral malaria of mice, which is the number of ICAM-1 and VCAM-1 positive blood vessels, wherein^*Differences in comparison of each infected group to the uninfected group,P<0.05；^#the differences compared between the various infection groups were,P<0.05。

FIG. 9 shows that the tussah pupae expressing rhEPO reduces the pro-inflammatory response of PbA infected mice, ELISA measures the expression of cytokines in spleen cell culture supernatant at different time points, wherein A is IFN-gamma, B is TNF- α, C is IL-10, and D is TGF- β.

Fig. 10 is the absolute numbers of CD11c + and CD11c + MHCII + cells at different time points in spleen tissue from tussah pupae expressing rhEPO inhibiting the differentiation and maturation of dendritic cells.

FIG. 11 shows that the tussah pupae expressing rhEPO reduces the proliferation and activity of Th1 cells in the spleen of PbA infected mice, which is the absolute log number of CD4+ T-beta + IFN-gamma + T cells at different time points in the spleen tissue.

Detailed Description

The present invention is further described below in conjunction with the appended drawings and specific examples, which enable one of ordinary skill in the art to more fully understand the present invention, but do not limit it in any way. The test methods referred to in the examples are, unless otherwise specified, conventional methods or methods recommended by the manufacturer; the reagents, materials, and the like used are commercially available unless otherwise specified. The gene encoding the amino acid sequence of the mature human erythropoietin protein was synthesized by Beijing Baisheng Gene technologies, Inc. The transfer expression vector pApM748BE was provided by the institute of biotechnology, Dai Liaoning institute of agricultural sciences.

Example 1 preparation of recombinant human erythropoietin samples.

1. Construction of recombinant plasmid of transfer vector of human erythropoietin gene tussah silkworm virus.

(1) Mature human erythropoietin protein consists of 166 amino acids (GenBank Acc. NM-000799.2). Artificially synthesizing the gene thereof based on this sequence (hepo) Introducing a recognition sequence GGATCC of a restriction enzyme BamH I and an ATG of a protein initiation codon at the 5' end of the gene; the 3' end of the gene is introduced with a recognition sequence GAATTC of the restriction enzyme EcoR I and a protein stop codon TGA. The synthesized gene is further verified by cloning and sequencing.

（2）hepoThe gene is double cut by restriction enzyme BamH I and EcoR I, and is connected with a transfer expression vector pApM748BE treated by the same restriction enzyme to obtain a recombinant plasmid pApM748 BE-hepoWhereinhepoThe expression of (a) is regulated by ApNPV polyhedrin gene promoter, and the expression product is not fused with any protein tag. Recombinant plasmid DNA was extracted and quantified by UV spectrophotometer for transfection, as shown in FIG. 1.

2. Recombinant plasmid pApM748 BE-hepoTransfecting the cell.

(1) Subculturing High Five cells to logarithmic phase with complete TNM-FH culture medium, and gently blowing and beating with a pipette to obtain about 0.5-1 × 10⁶Transferring the cell suspension to a 6-well cell culture plate to ensure that the cell density is about 80% of the cell suspension, culturing at 27 ℃ for 1-2 hours, and replacing 1-2mL of serum-free cell culture medium (SF-900)^TMII SFM)。

(2) The transfection reagent Cellffectin was used for cell transfection. About 0.5 mug of ApNPV-Δph/egfp ⁺ Virus DNA and about 1 μ g of pApM748BEhepoAdding the recombinant plasmid DNA into 100 mu L serum-free cell culture medium SF-900^TMII; adding 8 muL transfection reagent Cellffectin into 100 muL serum-free cell culture medium SF-900^TMAnd II, uniformly mixing the DNA mixed solution and the transfection reagent mixed solution, transferring the DNA and transfection reagent mixed solution to the cells prepared in the step (1) at room temperature for 30-50 minutes, and culturing for 4-5 hours at 27 ℃. Complete TNM-FH medium was changed and cultured at 27 ℃ for 4-5 days. And (3) taking the cell culture solution, injecting the cell culture solution into the tussah pupae with the diapause removed, incubating for 10-15 days at 18-22 ℃ at about 50-100 mu L/pupae, and observing the infection condition of the tussah pupae under a fluorescence inverted microscope.

3.hepoRecombinant virus ApNPV-Δph /Δegfp /hepo ⁺ Screening and identifying.

(1) Will havehepoThe tussah pupa body fluid infected by the recombinant virus and the expression EGFP virus is cultured in the insect serum-free cell culture medium SF-900^TMII SFM is diluted according to the volume ratio of 1:100, filtered and sterilized, and then the sterile virus solution is diluted by 10 times in a gradient way to prepare 10^-1、10^-2、10^-3And 10^-4Virus solutions of different concentrations. Infecting High Five cells with the virus solution diluted in gradient, discarding the virus solution after 1 hour, and using 1-2mL of serum-free cell culture medium SF-900^TMII SFM cleaning infected cells, discarding cell sap, adding 1mL of 2% low melting point agarose, after the agarose is solidified, adding 1-2mL of complete TNM-FH culture medium, culturing at 27 ℃ for 4-5 days, and observing virus occurrence conditions under a microscope.

(2) And taking out the upper culture solution, selecting a cell hole infected by a lower virus concentration, randomly picking out cells without EGFP markers by a glass pipette by adopting a reverse marker screening method, respectively suspending the cells in a small amount of serum-free cell culture medium, taking the supernatant after shaking and centrifuging, injecting the supernatant into infected tussah pupas, wherein each head of each cell is 50-100 uL, and placing the cell culture medium at 18-22 ℃ for 10-15 days. The recombinant virus is propagated and amplified, and the purity of the virus is analyzed and screened by a microscope observation or PCR detection method. This step can be repeated for one more round of virus screening if desired. ApNPV-Δph/Δegfp/hepo ⁺ The tussah pupae infected by the recombinant virus has no polyhedron formation and no EGFP protein expression.

4. The expression of rhEPO in tussah pupa is detected.

For detecting recombinant viruseshepoWhether the gene can be correctly expressed in tussah pupa, and the recombinant virus ApNPV- Δph/Δegfp/hepo ⁺ Injecting and inoculating into tussah pupa, and incubating at 18-22 deg.C. And (3) respectively taking tussah pupa body fluid infected for 7 days, 9 days, 11 days, 12 days, 13 days, 14 days and 15 days to carry out protein electrophoresis, and carrying out Western-blot detection on an anti-EPO antibody. The detection results show that the expression of the rhEPO protein can be detected in the tussah pupa body fluid inoculated with the recombinant virus, but the expression of the rhEPO protein can not be detected in the tussah pupa body fluid infected with the wild type virus, as shown in figure 2. An Erythropoietin (EPO) detection kit is used for drawing an EPO content standard curve, the expression quantity of the rhEPO in different periods is detected and calculated, the expression of the rhEPO reaches a peak at the 11 th day of the tussah pupa infected recombinant virus, and the expression level is 429.12ng/ml pupa body fluid, as shown in figure 2.

5. And (3) preparing rhEPO tussah pupa freeze-dried powder.

Recombinant virus ApNPV-Δph /Δegfp /hepo ⁺ Infecting tussah pupae in batches, and incubating at 18-22 ℃. After infection for 10-12 days, collecting tussah pupae, homogenizing, and filtering with gauze to remove residue such as pupa skin. Oak (Oak, Pernyi Japonica Makino, Onychoma Onyi, Onychoma GmbH)And (4) placing the silkworm pupa homogenate liquid into a freeze dryer for freeze-drying treatment. Collecting lyophilized powder, packaging and storing in-80 deg.C refrigerator. Detecting and analyzing the activity unit of rhEPO in tussah pupa freeze-dried powder to be not less than 200 IU/mg.

Example 2 in vitro and in vivo activity assays of tussah pupae expressing rhEPO protein.

1. And (3) detecting the in vitro activity of the tussah pupa expression rhEPO protein.

The in vitro biological activity of the tussah pupa rhEPO is tested by utilizing an MTT method to detect the growth and proliferation promoting effect of the tussah pupa expression rhEPO protein on human blood leukemia cells (TF-1). EPO protein standard and wild type tussah pupa lyophilized powder (Wildsilkworm, WS) are respectively used as positive and negative controls. The experimental result shows that the tussah pupa rhEPO has obvious in vitro biological activity, can promote the proliferation of TF-1 cells like the EPO standard product, and the in vitro biological activity of the tussah pupa rhEPO and the in vitro biological activity of the TF-1 cells are increased along with the increase of the concentration of the EPO. When the concentration is more than 1.25IU/mL, the cell growth begins to slow down, as shown in FIG. 3.

2. And (3) detecting the in vivo activity of the tussah pupa expression rhEPO protein.

(1) And (5) constructing a renal anemia mouse model. 42 female B/c mice were selected, aged 5 weeks, weighed 17. + -.1G, and randomly divided into 7 groups (groups A-G), 3 groups A, 9 groups B, and 6 groups each of the other 5 groups. Group A was used as a blank control group and was normally bred, and the other 6 groups were used for gastric lavage to induce renal anemia model. After continuous gavage for 2 weeks at an adenine dose of 100mg/kg, eyeball blood sampling was performed on 3 mice in A, B groups, and blood routine analysis and renal function detection were performed to determine whether the mouse renal anemia model was successfully constructed.

(2) The tussah pupa-expressed rhEPO has the biological activity in an animal model. The in vivo detection of the tussah pupa rhEPO efficacy is carried out on each group of model mice, and an EPO protein standard substance and wild tussah pupa freeze-dried powder are respectively used as positive and negative controls. The experimental result shows that the tussah pupa rhEPO and the EPO standard substance have obvious drug effect compared with a negative control group. Statistical analysis showed that the experimental results were significant (P < 0.05), as shown in fig. 4.

Example 3 evaluation of the effect of tussah pupae expressing rhEPO on the treatment of experimental cerebral malaria mice.

(1) Histopathological and immunohistochemical assessment of blood brain barrier integrity in mice. When PbA + ddH₂When mice in group O developed neurological symptoms of Experimental Cerebral Malaria (ECM), 3 mice per group were injected caudally with 200. mu.L of phosphate buffer plus 2% Evans blue (Sigma, St Louis, USA). After 1 hour of injection, the mice were anesthetized with ether and sacrificed by cardiac perfusion with 20 mL PBS to lavage without Evans blue. The rat brain is quickly taken out and soaked in formamide (2 ml) for 48 hours at the temperature of 37 ℃, then the supernatant is taken and analyzed by a spectrophotometer at the wavelength of 620 nm, and standard Evans Blue is used for standard curve analysis, and the content of the Evans Blue in the soaking solution represents the damage degree of the blood brain barrier of the rat. The results show that the brain evans blue content of the rhEPO-treated mice was significantly reduced compared to the PbA-only infected group and the wild-type insect vector-treated group, indicating that rhEPO treatment improved microcirculation impairment in the mice brain, as shown in fig. 5 and 6.

Meanwhile, 3 mice in each group were anesthetized with ether, brains were fixed with 4% paraformaldehyde, and paraffin embedding was performed after 24 hours, including gradient dehydration and multistage paraffin immersion. Continuously taking 5-micron thick slices of the embedded brain, baking the slices at 60 ℃ for 12 hours, and performing gradient dewaxing and debenzolization. After deparaffinization and debenzolization, the sections were repaired with 3% sodium citrate antigen at 90 ℃ for 15 minutes. Respectively taking brain tissue sections of each group of mice to carry out Hematoxylin Eosin (HE) staining to detect microvascular obstruction and endothelial cell injury, and taking the blood vessel with leukocyte infiltration in the blood vessel as positive. And taking brain tissue sections of each group of infected mice, performing immunohistochemical staining, after dewaxing and antigen retrieval, incubating 3% BSA at room temperature for 30 minutes to block nonspecific antigens, and incubating specific rabbit polyclonal antibodies against ICAM-1 (Abcam, Cambridge, UK) and VCAM-1 (Abcam, Cambridge, UK) respectively after blocking to detect the expression conditions of the cell adhesion factors of the cerebrovascular endothelial cells ICAM-1 and VCAM-1. And the number of blood vessels of VCAM-1+ and ICAM-1+ was counted under a microscope. The results show that hematoxylin and eosin stained brain sections show that rhEPO-treated mice have reduced leukocyte infiltration vessels compared to the control group; the immunohistochemically stained brain sections showed that the number of blood vessels staining positive for VCAM-1 and ICAM-1 in the rhEPO treatment group and the staining intensity were also significantly lower than those in the control group, indicating that rhEPO has the effect of protecting the integrity of the blood brain barrier, and oral administration of rhEPO reduced the injury of the blood brain barrier related to mouse ECM, as shown in FIGS. 7 and 8.

Example 4 study of the mechanism of tussah pupae expression rhEPO to protect the integrity of the blood brain barrier of mice.

(1) Assessment of the pro-inflammatory response in mice.

3 mice in each group were taken 3 days and 5 days after infection, and spleen tissues of the mice were taken for detection of related inflammation and anti-inflammatory factors. Spleen tissue was ground, lysed with 0.17M ammonium chloride, washed 3 times with RPMI1640 medium, counted on cell counting plates, and splenocytes collected from each group were 1X 10⁶Culturing at 37 deg.C under 5% CO₂. Culturing for 48h, collecting upper layer culture solution for cytokine detection, and adopting commercial ELISA kit according to the instruction (R)&D Systems, Minneapolis, USA) including levels of TNF- α, IFN- γ, IL-10 and TGF- β the method is briefly described as follows, first, diluting each recombinant cytokine standard with Calibrator Diluent RD5-16, diluting the standard for 7 gradients, using undiluted standard as high standard, Diluent as zero standard, according to the instructions, adding 50ul AssayDiluent RD1N to each well, then adding 50ul standard, control or sample per well, incubating at room temperature for 2 hours, washing the wells 4 times with 200ul wash buffer per well, adding 100ul mouse cytokine conjugates per well, incubating at room temperature for 2 hours, then repeating the washing step, adding 100ul substrate solution per well, keeping out of light for 30 minutes at room temperature, adding 100ul stop buffer per well, measuring Optical Density (OD) values at 450nm with a microplate reader, finally, calculating the concentration of recombinant cytokine in each well using a standard curve generated by using IFN-gamma-derived from the samples, calculating significantly lower IFN-5 concentrations in the IFN- α, IFN-gamma-10 and EPO-595 concentrations after infection of the control and EPO-EPO, showing significantly lower levels after TNF-10 and EPO contents, significantly lower in the control or EPO-7 ml treated groups, and EPO contents after addition of the untreated mice, and EPO, respectively, andDW and PbA + NS groups. The results indicate that rhEPO treatment can reduce the expression of inflammatory factors and increase the expression of anti-inflammatory factors in the early stage of PbA infection, which indicates that rhEPO has the effect of inhibiting the pro-inflammatory response of PbA infected mice, as shown in fig. 9.

(2) Flow cytometry assessed changes in dendritic cells.

Dendritic Cells (DCs) and their subsets were labeled with CD11c + and CD11c + MHCII + at 1X 10⁶Splenocytes were stained on the cell surface with the corresponding antibody, and after staining, cells were disrupted with a fixed membrane breaker at 4 ℃ for 20min, followed by FITC-anti-CD11c and PE-anti-MHCII staining as described above. After washing, PBS was resuspended at 4 ℃ and stored for detection by flow cytometry. The results showed that mature DCs (CD 11c + and CD11c + MHCII +) in spleen tissue of the control and rhEPO-treated groups were not different between the three groups of D0, PbA + ddH₂Both O and PbA + WS controls were significantly higher than D0 at D3 and D5; the number of DCs cells matured in the rhEPO-treated group at D3 was significantly lower than that of PbA + ddH₂O control group; the number of DCs matured in the rhEPO-treated group at D5 was significantly lower than that of PbA + ddH₂O and PbA + WS control. The results suggest that DCs in mouse spleen tissue all start to be activated in the early stages of PbA infection, expressing MHCII molecules, compared to PbA + ddH₂In the O and PbA + WS control group, there was no significant increase in DCs after treatment with rhEPO, indicating that rhEPO reduced the maturation of DCs, inhibited the proliferation of DCs and the expression of MHCII molecules, and effectively inhibited the activation effect of DCs, as shown in fig. 10.

(3) Changes in Th1 cells were assessed by flow cytometry.

To determine the in vivo effect of rhEPO on Th 1-type cell differentiation, a Th1 cell subtype was quantified. 3 mice from each group were sacrificed at 3d and 5d after PbA infection under ether anesthesia and spleen tissues were taken for relevant flow cytometric assays. Uninfected mice were used as a flow cytometric assay for quality control at day 0. All flow antibodies were purchased from BioLegend (San Diego, USA). All stained cells were detected and analyzed by FACSCELESTIR flow cytometer (BD Bioscience), each group was provided with antibody single markers and negative controls, and the number of cell detection was 1X 10⁵. Data analysis was performed with FlowJo software (TreeStar).

Selecting a Th1 cell marker from CD4+ T-beta + IFN-gamma + markers, and taking splenocytes to obtain 2 x 10 cells⁶Put into 24-well plates containing 50 ng/ml PMA and 1mM ionomycin (Sigma, Stlouis, USA) in 5% CO₂Stimulation was performed at 37 ℃ for 6 hours and Brefeldin A (2. mu.M) was added to block cytokine secretion. After cell culture, all splenocytes were first stained for the cell membrane surface of PerCP-anti-CD 4. After the surface staining is finished, the membrane is broken for 20min at 4 ℃ by using a fixed membrane breaking agent (BD Bioscience), and after the membrane is fixed and penetrated, intracellular staining is carried out by using FITC-anti-T-beta and APC-anti-IFN-gamma. After the dyeing is finished, the PBS is resuspended at 4 ℃ after washing by matching membrane-penetrating washing liquid, and is stored until being detected by a flow cytometer. The results showed that Th1 (CD 4+ T-beta + IFN-. gamma. +) cells were not different between the D0 groups, PbA + ddH₂The O control group is obviously higher than D0 at D3 and D5 days, and the PbA + WS control group is obviously higher than D0 at D5 days; the number of Th1 cells of the rhEPO treated group at D5 is obviously lower than that of PbA + ddH₂O and PbA + WS control. The results suggest that PbA infection can effectively promote proliferation of Th1 cells, while the proliferation of Th1 cell number in the rhEPO-treated group is not obvious compared with the control group, which indicates that rhEPO inhibits the proliferation effect of Th1 cells, as shown in fig. 11.

Claims (3)

1. The recombinant human erythropoietin is prepared by expressing from tussah pupa or larva.

2. A preparation method of a recombinant human erythropoietin sample is characterized by comprising the following steps:

(1) artificially synthesizing human erythropoietin gene: 166 amino acids of mature human erythropoietin protein are coded by the gene, a restriction enzyme BamH I recognition sequence GGATCC and a protein initiation codon ATG are introduced at the 5 'end of the gene, and a restriction enzyme EcoR I recognition sequence GAATTC and a protein termination codon TGA are introduced at the 3' end of the gene;hepothe gene can also be obtained by PCR amplification by using human tissue cDNA as a template;

(2) constructing a recombinant expression plasmid: transferring tussah virus to expression vector pApM748BE andhepocarrying out double enzyme digestion treatment on the genes by using restriction enzymes BamH I and EcoR I respectively, recovering a vector and a gene fragment by agarose gel electrophoresis, connecting the vector and the gene fragment by using T4DNA ligase, and converting the vector and the gene fragment into escherichia coli; picking single colony for culturing, extracting plasmid DNA, performing enzyme digestion identification to obtain recombinant plasmid pApM748 BE-hepo；

(3) Preparing insect cells: the Tn-High Five cells are subcultured to logarithmic phase by using complete TNM-FH medium to prepare about 0.5-1 × 10⁶Transferring the cell suspension to a 6-hole cell culture plate to ensure that the cell density accounts for about 80 percent of the cell culture plate, culturing for 1-2 hours at 27 ℃, and replacing 1-2mL of serum-free cell culture medium SF-900^TMII SFM, cells can be used for transfection or recombinant virus screening;

(4) cell transfection: pApM748BE `/5 `/4 `/S `/hepoRecombinant plasmid DNA and tussah ApNPV-Δph/egfp ⁺ Viral DNA was co-transfected into Tn-High Five cells by the following procedure: adding about 0.5 μ g of viral DNA and about 1 μ g of recombinant plasmid DNA to 100 μ L of serum-free cell culture medium SF-900^TMII; adding 8 muL transfection reagent Cellffectin into 100 muL serum-free cell culture medium SF-900^TMII, uniformly mixing the DNA mixed solution and the transfection reagent mixed solution, transferring the DNA and transfection reagent mixed solution into a prepared cell at room temperature for 30-50 minutes, culturing for 4-5 hours at 27 ℃, replacing a complete TNM-FH culture medium, and continuously culturing for 4-5 days at 27 ℃; taking cell culture supernatant, injecting infected tussah pupae with the volume of 50-100 mu L/pupae, and incubating for 10-15 days at 18-22 ℃;

(5) screening of recombinant viruses: screening the recombinant virus ApNPV by using Tn-High Five cells by adopting a terminal dilution method-Δ ph/Δegfp/hepo ⁺ Will havehepoThe tussah pupa body fluid infected by the recombinant virus and the expression EGFP virus is cultured in the insect serum-free cell culture medium SF-900^TMII SFM according to 1: diluting with 100 vol%, filtering for sterilization, and diluting the sterile virus solution 10 times in gradient to obtain 10-fold sterile virus solution^-1、10^-2、10^-3And 10^-4Virus solutions of different concentrations; diluting the virus solution in gradientInfecting HighFive cells, fixing with 2% low-melting-point agarose, adding 1-2mL of complete TNM-FH culture medium, and culturing at 27 ℃ for 4-5 days; taking out the upper culture solution, picking out cells without EGFP markers by a glass pipette by adopting a reverse marker screening method, respectively suspending the cells in a small amount of serum-free cell culture medium, shaking and centrifuging, taking the supernatant, injecting the supernatant to infect tussah pupas, placing each end of the supernatant at 50-100 uL, and standing at 18-22 ℃ for 10-15 days; analyzing the purity of the screened virus by using a microscope observation or PCR detection method;

(6) expression detection of rhEPO in tussah pupa: recombinant virus ApNPV-Δph/Δegfp /hepo ⁺ Injecting and inoculating into tussah pupa, and incubating at 18-22 deg.C; respectively taking tussah pupa body fluid infected for 7, 9, 11, 12, 13, 14 and 15 days to carry out protein electrophoresis, carrying out Western-blot detection on an anti-EPO antibody, and carrying out quantitative analysis;

(7) preparing rhEPO tussah pupa freeze-dried powder: recombinant virus ApNPV-Δph /Δegfp /hepo ⁺ Infecting tussah pupae in batches; collecting infected tussah pupas, homogenizing, filtering with gauze to remove residues, and freeze-drying the homogenate of the tussah pupas; collecting lyophilized powder, packaging and storing in-80 deg.C refrigerator.

3. Application of recombinant human erythropoietin in preparing oral medicaments for treating mouse cerebral malaria.

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