| In recent years,thrombotic diseases have attracted more and more attention.They are gradually increasing clinically and seriously threatening human life and health.Using thrombolytic drugs to eliminate thrombosis is a common clinical method.Although the existing thrombolytic drugs have a clear therapeutic effect,there are also problems such as narrow therapeutic window period,large dosage,low fibrin specificity,low reperfusion rate,high rate of systemic bleeding,short half-life,and large side effects.Desmodus rotundus salivary plasminogen activator alpha 1(DSPAal),also known as desmoteplase,which has high fibrin specificity,high thrombolysis efficiency,low risk of bleeding,long half-life and low side effects is expected to be a new and efficient thrombolytic drug.In this study,mammalian transgenic technology was used to obtain mammary gland-specific recombinant DSPA(recombinant DSPA,rDSPA)transgenic rabbits,and the expression products in rabbit milk were collected.Use self-made monoclonal antibodies for ELISA and Western-blot detection of rabbit milk.To establish a method for separating and purifying rDSPA from transgenic rabbit milk.The primary structure and bioactivity of rDSPA were analyzed,including the amino acid sequence of N-terminal and C-terminal,relative molecular weight and in vitro fibrinolytic activity.The thrombolytic effect and half-life of the purified DSPA in vivo were studied by rat tail thrombus model induced by carrageenan.1.Subculture of transgenic rabbits specifically expressing rDSPA in mammary glandsTwo primary rabbits were inbred and backcrossed by extended group breeding.A total of 134 transgenic rabbits with mammary gland-specific expression of rDSPA were obtained,among which 2 rabbits(F2 generation)were identified as homozygous by cross test.Rabbit milk was collected and centrifuged to separate whey.The rDSPA expression level was 1.19±0.26 mg/mL by ELISA Assay.The fibrinolytic activity of rDSPA in F0,F1 and F2 generation transgenic rabbit whey was detected by Fibrinolytic fibrinolytic Assay(FAPA).The dissolution diameter of rDSPA was basically the same(18.6-20.4 mm),and the results showed that the exogenous gene rDSPA could be stably transmitted to the offspring and could be stably expressed in the rabbit mammary gland.In addition,the homozygous rabbits can avoid the tedious workload of genotyping and provide a guarantee for the preservation of rDSPA transgenic rabbits.2.Construction of prokaryotic vector expressing desmoteplase(DSPA)geneUsing molecular biology technology,PCR was used to amplify the DNA sequence encoding the mature peptide of DSPAal on the pCL25/DSPA expression vector,which was used to connect with the prokaryotic expression vector to construct the pET-28a/DSPA prokaryotic expression vector.The pET-28a/DSPA prokaryotic expression vector was transferred into Rosetta competent cells to induce the expression of the target protein.The estimated size by SDS-PAGE electrophoresis is about 50 kDa.After purification,the purity of the target protein was 92%,which far met the purity requirement of the immunogen for the preparation of rDSPA monoclonal antibody.3.Preparation and screening of rDSPA monoclonal antibodyBALB/c mice were immunized with purified prokaryotic expression product plus Freund's adjuvant.B lymphocytes were isolated and fused with SP2/0 cells.Positive hybridoma cell lines were screened,and secrete PR-mAb hybridoma cells were injected into the abdominal cavity of the same strain of mice to prepare antibodies.A total of 36 positive hybridoma cells were obtained,and 12 cell lines(named M1-M12)with stable antibody secretion were screened out.After screening by ELISA-elution,the M3 and M4 strains are polyhydroxy-reactive monoclonal antibodies(PR-mAb),with a screening rate of 16.67%(2/12).The antibody titer of ascites in mice injected with M3 and M4 cell lines is 1:125000,and the ascites antibody can be used for ELSA and western blot detection.4.Isolation and purification of rDSPA from transgenic rabbit milkThe mouse asitoneal antibody was purified by rProtein A affinity chromatography and conjugated with CNBR-activated Bestarose 4 FF medium to produce an immunoaffinity chromatography column.The rDSPA transgenic rabbit milk was pretreated by ultracentrifugation and ammonium sulfate precipitation.The rDSPA was purified by self-made immunoaffinity chromatography,benzamidine affinity chromatography,cation-exchange chromatography and Cibacron blue affinity chromatography.The results showed that 98%purity rDSPA was obtained from rDSPA transgenic rabbit milk by ultracentrifugation,55%ammonium sulfate precipitation,benzamidine affinity chromatography,cation-exchange chromatography and Cibacron blue affinity chromatography.Anion exchange chromatography was used to remove the endotoxin in the purified product,and the result of endotoxin detection kit showed that the endotoxin content of purified rDSPA was less than 0.015 EU/mL,which met with the standard of injection.5.Structure and function detection of the purified rDSPA expressed specifically in mammary glandsThe N-terminal amino acid sequence of rDSPA was determined by Edman degradation method,and the results showed that the N-terminal amino acid sequence of rDSPA was Val Ala Cys Lys Asp Glu IIe Thr Gln Met Thr Tyr Arg Arg Gln,which was completely consistent with the theoretical amino acid sequence at the 4-18 position at the N-terminal of the designed rDSPA coding sequence,indicating that the signal peptide of the goat?-lactoglobulin(?-lactoglobulin)at the N-terminus of the rDSPA coding sequence has been completely removed.The C-terminal amino acid sequence and other parts of rDSPA were determined by liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS).The results showed that the C-terminal amino acid sequence of rDSPA was Asp Val Pro Gly Val Tyr Thr Lys Tyr Leu Gly Trp ?e Arg Asp Asn Met His Leu,which was completely consistent with the C-terminal of the designed rDSPA coding sequence,with a total of 47%(208 amino acids)of the sequences were completely homologous to the DSPA coding sequence.The relative molecular weight of rDSPA was determined to be 53126.75 Da by the high resolution mass spectrometer,which was higher than the theoretical 49535.94 Da,which was speculated that it was caused by posttranslational glycosylation modification.The fibrinolytic activity of rDSPA(773333 IU/mg)was slightly higher than that of Alteplase(580000 IU/mg)in vitro by fibrinolytic Assay Fibrin Agarose Plate(FAPA).6.Preliminary evaluation of thrombolytic effect of rDSPA in vivo and in vitroBlood was collected from abdominal aorta of SD rats.The blood clot was cut into 0.1 cm3 squares and weighed,and divided into 7 groups(0.5 ?g/mL,l?g/mL,2?g/mL DSPA;0.5 ?g/mL,1 ?g/mL,2 ?g/mL Alteplase;normal saline),incubated at 37? for 16 h.Weighed the remaining blood clots and calculated the dissolution rate of blood clots.The results showed that the blood clot dissolution rate of rDSPA group(48.34±2.25%)was significantly higher than that of Alteplase group(40.91±1.36%)(p<0.05),indicating that the thrombolysis activity of rDSPA was stronger than that of Alteplase in vitro.8-week-old male SD rats were randomly divided into 4 groups with 6 rats in each group,divided into low,medium and high dose rDSPA groups(2 mg/kg,8 mg/kg,32 mg/kg)and Alteplase group(8 mg/kg),placebo group and blank control group.Blood samples were collected from the heart of rats in each group to detect the activation of partial thrombin time(APTT),prothrombin time(PT),thrombin time(TT),fibrinogen(FIB)and D-dimer levels,and simultaneously measuring rat tail thrombus length.The results show that rDSPA has a significant thrombolytic effect.The APTT and TT of rDSPA low-(APTT:18.80± 1.26 s;TT:35.75±0.41 s),medium-(APTT:27.78± 1.88 s;TT:39.18±0.82 s)and high-dose groups(APTT:36.51 ± 1.29 s;TT:37.56± 1.07 s)and Alteplase group(APTT:27.00± 1.76 s;TT:34.05±0.99 s)were prolonged compared with placebo group(APTT:12.53±0.54 s;TT:26.87± 1.31 s),and the difference was extremely significant(p<0.01).The PT and D-dimer of rDSPA medium-(PT:14.28±0.79 s;D-dimer:2.49±0.07 mg/L)and high-dose groups(PT:17.07±1.19 s;D-dimer:3.12±0.55 mg/L)and Alteplase group(PT:14.26±0.93 s;D-dimer:2.53±0.12 mg/L)were significantly higher than those in the placebo group(PT:12.29±0.76 s;D-dimer:2.30±0.07 mg/L,p<0.05),indicated that both rDSPA and Alteplase can effectively activate fibrinolytic system in rats.The FIB level of the Alteplase group(200.22±6.09 mg/dL)was lower than that of the rDSPA low-(222.40±7.89 mg/dL),medium-(228.30±1.72 mg/dL),and high-dose groups(228.19± 1.89 mg/dL,p<0.05).There was no significant difference in the FIB level between the rDSPA low-,medium-,and high-dose groups,indicating that the FIB consumption of rDSPA in the process of thrombolysis was less than that of Alteplase,which was less likely to cause intracranial hemorrhage and has more safety compared to Alteplase.The relative length percentage of rat tail thrombosis in the low-(48.96±9.42%),medium-(24.71±6.26%),and high-dose groups(0.00±0.00%)of rDSPA and the Alteplase group(49.19±6.21%)were significantly smaller than that of the placebo group(67.76±6.06%),that means,the relative length of tail thrombosis elimination in the low-,medium-and high-dose groups of rDSPA was significantly longer than that in the placebo group,and was proportional to the dose,and no thrombosis formed in the tail of the rats in the high dose rDSPA group.The relative length percentage of rat tail plugs in the same dose of rDSPA medium-dose group(24.71±6.26%)and Alteplase group(49.19±6.21%)were significantly different(p<0.01),indicating that the thrombolytic ability of the purified product rDSPA was significantly better than that of Alteplase.8-week-old SD male rats were randomly divided into two groups with 4 rats in each group.One was rDSPA group and another was Alteplase group.rDSPA group and Alteplase group were injected with 8mg/kg of rDSPA or Alteplase through tail vein.Plasma was collected for the determination of plasminase-anti-plasminase complex(PAP),and the half-life of rDSPA was calculated.The plasma PAP concentrations of rats in the rDSPA group(133.06±63.90 ng/mL)and Alteplase group(219.44±1.18 ng/mL)reached their peaks at 6 min and 3 min.The plasma half-life of rDSPA and Alteplase in SD rats were 52.41 min and 10.41 min respectively.The half-life of rDSPA was calculated to be 5 times longer than that of Alteputrase,indicating that rDSPA can be used for the preparation of single injection thrombolytic drugs.In conclusion,transgenic rabbits with mammary gland-specific expression of rDSPA were successfully bred,and rDSPA transgenic rabbits had high expression level.An effective laboratory process for rDSPA isolation and purification was established,and the original fibrinolytic and thrombolytic activity were maintained,indicating that the research has taken the first step in clinical application.The primary structure analysis of rDSPA showed that the rDSPA coding sequence could be expressed in transgenic rabbit mammary epithelial cells,and the heterologous signal peptide sequence could be cut and removed,and then processed into mature protein with thrombolytic activity after translation.This result has not been reported at home and abroad.In vivo and in vitro thrombolytic tests in rats,rDSPA showed better thrombolytic performance than Alteplase,and the half-life of rDSPA in rats was longer than that of Alteplase,and in vivo thrombolytic tests showed safer and more continuous thrombolytic effect.The results have not been reported at home and abroad.The results of this study lay the experimental foundation for the development and production of new efficient thrombolytic drugs. |
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