Fusion protein SLIT2D2-HSA and its use in treatment of sepsis

Abstract

Disclosed is a fusion protein Slit2D2-HSA formed by fusion of a Slit2D2 polypeptide and a human serum albumin HSA, and use thereof for the manufacture of a medicament for prophylaxis and/or treatment of sepsis, and the said fusion protein Slit2D2-HSA retains the pharmacological activities of Slit2 in inhibition of neutrophil migration and treatment of sepsis, and has an improved stability, prolonged half-life and improved therapeutic effect of sepsis. Compared with protein of Slit2, the polypeptide of Slit2D2 has a smaller molecular weight which is much easier to be purified and separated in preparation and used in the development of drugs.

Claims

1. A fusion protein Slit2D2-HSA formed by the fusion of a D2 domain of Slit2 protein and a HSA protein; wherein said D2 domain of Slit2 comprises: a) an amino acid sequence shown by SEQ ID NO: 1, or b) an amino acid sequence with a same function obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the aforesaid sequence a); said HSA comprises: c) an amino acid sequence shown by SEQ ID NO: 2, or d) an amino acid sequence with a same function obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the aforesaid sequence c); wherein the C-terminal domain of said D2 domain of Slit2 is directly linked to the N-terminal domain of said HSA protein, or the N-terminal domain of said D2 domain of Slit2 is directly linked to the C-terminal domain of said HSA protein; and wherein up to 10 amino acid residues are changed for substitution and/or deletion and/or addition.

2. The fusion protein Slit2D2-HSA of claim 1, wherein the fusion protein contains an amino acid sequence shown by SEQ ID NO: 3, or, an amino sequence with a same function obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the aforesaid sequence shown by SEQ ID NO: 3.

3. A method of prophylaxis and/or treatment of sepsis comprising the steps of: preparing a pharmaceutically acceptable composition or a pharmaceutically acceptable adjuvant comprising the fusion protein Slit2D2-HSA formed by fusion of the D2 domain of Slit2 protein and HSA protein as in claim 1, and administering the composition or adjuvant to a subject for the prophylaxis or treatment thereof.

4. The method of claim 3, wherein the sepsis is severe sepsis; or septic shock.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a map of recombinant plasmid pZD-Slit2D2-HSA containing the fusion protein Slit2D2-HSA;

(2) FIG. 2 illustrates a graph of identification results of expression of the fusion protein Slit2D2-HSA; wherein the left graph is a 12% SDS-PAGE electrophoretogram of the fusion protein Slit2D2-HSA; the right graph is a SEC-HPLC spectra of the fusion protein Slit2D2-HSA using G3000swxl gel column with a flow rate of 0.5 ml/min, a period of 40 min and a detector of UV280;

(3) FIG. 3 illustrates an experiment result of inhibition of neutrophil migration in vitro with fusion protein Slit2D2-HSA in rats;

(4) FIG. 4 illustrates an experiment result of inhibition of LPS-induced cell migration with fusion protein Slit2D2-HSA in rats with acute lung injury; wherein the figure A illustrates a result of total cell number; the figure B illustrates a result of the numbers of eosinophils (EOS), macrophages (Mac), neutrophils (Neu) and lymphocytes (Lym), in figure B, the results of each group are normal control group, solvent group, dexamethasone group, fusion protein Slit2D2-HSA 1 mg/kg, fusion protein Slit2D2-HSA 5 mg/kg, from left to right, respectively.

(5) FIG. 5 illustrates an efficacy test result of CLP sepsis model of fusion protein Slit2D2-HSA, and ** indicates a p-value less than 0.01 compared with G2 group: CLP+ solvent (wilcoxon test);

(6) FIG. 6 illustrates a safety evaluation result of the fusion protein Slit2D2-HSA.

DETAILED DESCRIPTION OF THE INVENTION

(7) The following examples are provided to explain in greater detail the contents of the present invention. However, the content of the present invention is not limited to the following examples.

Example 1 Preparation of the Fusion Protein Slit2D2-HSA

(8) The fusion expression vector of the second domain Slit2D2 (Hohenester2008) in Slit2 and HSA was analyzed, designed and constructed based on the known sequence of Slit2 [GenBank: EAW92793.1].

(9) The gene fragment of slit2D2, as a template, was obtained by total gene synthesis, and then the gene sequence of slit2D2 with a length of 674 bp shown by SEQ ID NO: 4 was obtained by PCR amplification of primers T62F (with a sequence shown by SEQ ID NO: 7) and T62R (with a sequence shown by SEQ ID NO: 8).

(10) The gene fragment of HSA, as a template, was obtained by total gene synthesis, and then the gene sequence of HSA with a length of 1783 bp shown by SEQ ID NO: 5 was obtained by PCR amplification of primers T60F3 (with a sequence shown by SEQ ID NO: 9) and T59R (with a sequence shown by SEQ ID NO: 10).

(11) The gene sequence of slit2D2 shown in SEQ ID NO: 4 and gene sequence of HSA shown by SEQ ID NO: 5 obtained by PCR amplification were purified and recovered.

(12) The plasmid vector containing CMV promoter was double-enzyme digested by restriction endonuclease BssH II and EcoR I, and then a seamless connection reaction of the gene of slit2D2 obtained after purification and recovery with the gene of HSA was conducted by using one step directed cloning kit. The recombinant vector was transfected into E. coli Top10 followed by being transferred to a solid medium containing ampicillin (AMP) for breeding, the positive clones were screened, whether the construction of vector is successful was confirmed by sequencing, and then the strains were preserved.

(13) The recombinant plasmid in E. coli TOP10 was extracted by using endo-free plasmid extraction kit, and was then transfected into EXPI293 cells when the cell density of EXPI293 cells reached 2.510.sup.6 cell/ml. The supernatant of culture medium was collected and centrifuged at a high speed after being cultured for 5 days. The fusion protein Slit2D2-HSA was purified by a HiTrap Q HP anion column, and the molecular weight and purity of Slit2D2-HSA were detected by SDS-PAGE and SEC-HPLC, respectively.

(14) Wherein, the result of SDS-PAGE and the SEC-HPLC spectra are shown in FIG. 2. It is proved by FIG. 2 that a recombinant expression vector containing fusion protein Slit2D2-HSA was constructed successfully and the expression and purification of the fusion protein Slit2D2-HSA in the host cell was achieved.

Example 2 Affinity of Fusion Protein Slit2D2-HSA to Target Protein Robo1 Determined by SPR

(15) The affinity constant between the prepared protein and Robo1 was determined by SPR (Surface Plasmon resonance BIAcore200). The Robo1 (ORIGEN Company, number: TP327713) was bound on CM5 chip and used to analysis the interaction between fusion protein Slit2D2-HSA and receptor Robo1. Kinetic measurements were carried out according to the method referred by Canziani et al. (2004, Anal. Biochem. 325:301-307), simultaneously, the same method was applied to measure the affinity between Slit2N protein (Slit2N is a protein at N-terminal of slit2 with a molecular weight of about 120 KDa) and Robo1.

(16) TABLE-US-00001 TABLE 1 Result of affinity of fusion protein Slit2D2-HSA to receptor Robo1 measured by SPR No. Protein Ka (1/Ms) KD (M) Kd (nM) 1 Slit2N 7.436 10.sup.5 3.394 10.sup.3 4.5 2 Slit2D2-HSA 1.771 10.sup.5 5.099 10.sup.4 2.8

(17) The experiment results showed that the constructed fusion protein Slit2D2-HSA had a good affinity to Robo1, which was similar to the property of Slit2N.

Example 3 Experiment of Inhibition of Neutrophil Migration In Vitro with Fusion Protein Slit2D2-HSA in Rats

(18) Extraction of Rat Neutrophils

(19) 16 ml of fresh anticoagulation was taken from rat and then mixed with 16 ml of tissue dilution. Every 2 ml of the mixture obtained was added into 10 ml EP tube containing 2 ml of liquid A of neutrophils extraction kit, followed by being centrifuged at 500 g for 25 min for stratify. Then cells in the first and second layer were discarded, meanwhile cells in the third and forth layer were collected and placed into a test tube containing 10 ml of cell washing liquid, followed by being centrifuged at 500 g for 30 min to remove the supernatant. Then 3-5 times the cell volume of red blood cell lysate was added into the precipitation and the mixture was mixed, and cell lysis was performed for 1-2 min. The mixture obtained was centrifuged at 500 g for 5 min and then the supernatant was discarded. The cell lysis was repeated once. Washing was performed 1-2 times as follows: proper amount of PBS solution was added to make the precipitate resuspended and then the mixture obtained was centrifuged at 500 g for 3 min to remove the supernatant. The precipitate was resuspended by 1640 complete medium and transferred to a 6 cm plate, rat neutrophils were extracted successfully and incubated in an incubator.

(20) The neutrophils were resuspended after centrifugation, and then 110.sup.6 cells were added into the upper transwell chamber followed by adding 100 l of BSA (10 mg/ml) without serum and corresponding drugs as shown in FIG. 3, while 600 l medium containing serum and 10 mg/ml of BSA was added into the sublayer, except the PBS group, other groups were added by fMLP with a final concentration of 100 M. 3 hours later, the chamber was taken out and cells in the lower culture medium were centrifuged, collected and concentrated 6 times by being suspended in 100 l of PBS, and then added into a blood cell counting chamber for counting, the total number of cells in four large squares, upper left square, upper right square, lower left square and lower right square, were statistic calculated.

(21) Experimental results showed that, as shown in FIG. 3, the fusion protein Slit2D2-HSA could significantly inhibit rat neutrophil migration (P<0.01) in vitro, and had a better activity of inhibiting neutrophil migration.

Example 4 Experiment of Inhibition of LPS-Induced Neutrophil Migration with Fusion Protein Slit2D2-HSA in Rats with Acute Lung Injury

(22) Acute lung injury (ALI) is an uncontrolled systemic inflammatory response caused by varieties of direct or indirect injury factors, accompanied by damage of alveolar epithelial cells and capillary endothelial cells. The pathogenesis of ALI is not clear, and currently, there is a lack of effective treatment means for ALI. In the present invention, ALI is induced by intratracheal lipopolysaccharide (LPS) infusion in rats which is a commonly used animal model. Animals were injected compounds via tail vein and 4 hours later, the fluid of lung lavage and bronchioalveolar lavage were collected and used for measurement by COMP-U-DIFF, and the measurement results represented the efficacy of testing compounds in prevention of LPS-induced ALI. The experiment design of animal grouping is shown in Table 2.

(23) In addition to the first group, other groups of animals were treated with lipopolysaccharide lung perfusion. Concretely, using a micro-cannula sprayer, 100 l of LPS solution (1 mg/ml) was imported into the tracheal cannula of rats which were anesthetized with 3-5% isoflurane in advance, and 4 hours later the animals were sacrificed using chloral hydrate (750 mg/kg). Lung lavage were carried out three times in situ using 4 ml of PBS+1% albumin. After lavage, bronchioalveolar lavage fluid (BALF) was stored in ice and then the amount of eosinophils (EOS), macrophages (Mac), neutrophils (Neu), lymphocytes (Lym) and total cells were counted. The results of total cell number of 5 groups are illustrated in FIG. 4 and the number of EOS, Mac, Neu and Lym are shown in FIG. 5.

(24) In this study, the total cell number of the second group, as LPS model group, had an increase, while the number of neutrophils and the total cells decreased significantly with dexamethasone at a dosage of 10 mg/kg as a positive control. Compared with the model group, fusion protein Slit2D2-HSA at a dosage of 1 mg/kg and 5 mg/kg could significantly inhibit the increase of cell number and control the number of neutrophils.

(25) TABLE-US-00002 TABLE 2 Experiment of inhibition of neutrophil migration with fusion protein Slit2D2-HSA Number Administration Concentrate Dosage Dosage Group Test agent of rats route mg/mL mL/kg mg/kg regimen 1 Normal control 3 N/A 0 0 0 N/A group 2 solvent 6 intravenous TBD 10 TBD TBD drip 3 dexamethasone 6 oral 1 10 10 3 hours before LPS infusion, oral 4 fusion protein 6 intravenous 0.5 2 1 3 hours before Slit2D2-HSA drip LPS infusion, intravenous drip 5 fusion protein 6 intravenous 2.5 2 5 3 hours before Slit2D2-HSA drip LPS infusion, intravenous drip

Example 5 Detection of Therapeutic Effect on CLP Sepsis Model

(26) The cecalligation-peferation (CLP) test of rodent was widely adopted to build an experimental sepsis model. This experiment evaluated the efficacy of testing drugs on CLP sepsis induced in C57BL/6 mice.

(27) The CLP surgery was conducted on six to eight-week-old male C57BL/6SDF mice weighed 18-22 g as experiment animals. Mice were anesthetized by intraperitoneal injection of ketamine (50 mg/kg body weight), and an incision with a length of 1 cm was made in the ventral midline of the mice, mesenteric and cecum were dissociated, and the end of cecum was ligated with a 4-silk suture at 1.2 cm from the tip of cecum. Subsequently, the end of the cecum was pierced for two times with a sterile 21-needle, and a little excreta was squeezed into the abdominal cavity, and then intestinal segment was returned and the peritoneal and skin were stitched in turn. Animal grouping and drug administration are described as the following table 3, and the death status of each group were observed and recorded.

(28) TABLE-US-00003 TABLE 3 Detection experiment of therapeutic effect of Slit2D2-HSA on CLP sepsis model Number Administration Concentration Dosage Dosage Group Testing agent of rats route mg/mL mL/kg mg/kg regimen 1 sham 10 N/A 1 10 0 N/A operating group 2 CLP + 10 intraperitoneal 1 10 0 0-6 day, solvent injection Once a day an hour before CLP 3 CLP + 10 oral 1 10 10 surgeon and dexamethasone postoperative 18 hours each time 4 CLP + 10 intraperitoneal 0.1 10 1 0-6 days, once a Slit2D2-HSA injection day 5 CLP + 10 intraperitoneal 0.5 10 5 0-6 days, once a Slit2D2-HSA injection day

(29) The experimental results are shown in FIG. 5, and FIG. 5 shows that the survive rate of CLP model was significantly increased by tail intravenous administration of fusion protein Slit2D2-HSA. The survive rate of CLP model animal reached 90% at a dosage of 5 mg/kg, while the control group only reached 20%, which indicated that the fusion protein Slit2D2-HSA had a good therapeutic effect on the sepsis and could be an effective drug for prophylaxis and treatment of sepsis.

Example 6 Safety Evaluation of the Fusion Protein Slit2D2-HSA

(30) In order to evaluate the safety of the fusion protein Slit2D2-HSA in animals sufficiently, mice were administered continuous, and the changes of body weight and the survival status of the mice were observed to evaluate the effect of drugs on mice.

(31) TABLE-US-00004 TABLE 4 Safety evaluation experiment of the fusion protein Slit2D2-HSA Dosage Administration Number Group Treatment Dose regimen route of mice 1 solvent(PBS) 0 daily/ intraperitoneal 10 30 days injection 2 Slit2D2-HSA 10 mg/kg daily/ intraperitoneal 10 30 days injection 3 Slit2D2-HSA 5 mg/kg daily/ intraperitoneal 10 30 days injection 4 Slit2D2-HSA 1 mg/kg daily/ intraperitoneal 10 30 days injection

(32) The results of body weight changes over time of four group mice are shown in FIG. 6. The figure shows that there was no significant change in body weight in the three dose groups and animals were in good condition after 30 days of intravenous administration of tail vein in mice, which indicated a good drug safety of fusion protein Slit2D2-HSA.