| Since the last century, thrombotic disease has been the primary risk factor to human health and safety, and thrombolytic therapy was one of the most wide and effective method of treatment in currently clinical. Thrombolytic drugs have passed through three generations of development. Human tissue-type plasminogen activator (t-PA) is a serine protease synthesized and secreted from vascular endothelial cell, which is a favorable second-generation thrombolytic drugs due to it’s effective and specific thrombolysis. Recombinant human plasminogen activator (rhPA) was a recombination mutant of natural tPA, and it is known as the novel third-generation thrombolytic drugs with more superior thrombolysis function than natural t-PA. There are many advantages of using mammary gland bioreactor to produce t-PA and recombinant human plasminogen activator than other expression systems, including higher yield, lower cost, post-translational modification and higher bioactivity. One of the most commonly purified strategies is to use various purification (chromatography) technology combined with each other, which can easily, quickly and efficiently isolate and purify the target proteins from milk of animals. Currently, there have been many reports at home and abroad.In this study, we aimed to obtain a novel high-efficient thrombolytic drug, ultimately. In order to created transgenic rabbits of highly expressed rhPA with thrombolytic biological activity, we constructed reasonable and effective rhPA mammary-specific expression vectors (F, E, K1 domains deletion mutant of t-PA), and also studied the effect factors of the expression levels and activity functions. We introduced a strategy of combining various purification techniques to purify high-purity and high-activity rhPA from milk of transgenic rabbits. The thrombolytic effect of the purified product (rhPA) was also evaluated on mouse tail model of thrombosis induced by carrageenan. Meanwhile, in order to obtain polyol-responsive monoclonal antibody (PR-mAb) for affinity chromatography, we also studied on the methods of preparing and screening monoclonal antibody against rhPA. The main methods and results were as follows:1. We used conventional molecular biology techniques to construct mammary gland-specific expression vectors PCL25/rhPA, BLC14/rhPA and AP/rhPA, including goat p-casein gene, goat β-lactoglobulin gene and bovine as 1-casein gene as regulatory elements and natural t-PA deletant in F, E, K1 domain as the encoded protein sequence, respectively. By restriction enzyme digestion mapping and sequence blasting, the results revealed that the vectors were completely consistent with theoretical sequence.2. We established goat mammary gland epithelial cells (GMECs) isolation and culture system by collagenase digestion, and electrobloted the three vectors. After two weeks of 600ug/mL G418 screening, the monoclonal cell colonies with vigorous growth and better performance were selected for expanding subculture. PCR was used to screen integration, and the three vectors (PCL25/rhPA, BLC14/rhPA, AP/rhPA) achieved 79,66,63 integrate positive monoclonal cell strains, respectively. These colonies were induced with 5μM prolactin, and then the media were gleaned respectively at 72h. It was found that there were 25 PCL25/rhPA and 3 BLC14/rhPA colonies expressing recombinant human plasminogen activator (rhPA) by ELISA, while AP/rhPA colonies had not expressed it. All expressing cell strains have thrombolytic biological activity in vitro by fibrin agarose plate assay (FAPA). It was suggested that PCL25/rhPA and BLC14/rhPA vector could effectively promote specific expression of rhPA gene at the cellular level, better in PCL25/rhPA vector. Moreover, in this study, our creatively designed rhPA have high efficiency thrombolytic biological activity.3. Fertilized eggs were microinjected with the three expression vectors (PCL25/rhPA, BLC14/rhPA, AP/rhPA) to generate transgenic rabbits. In this study, a total of 94 receptors were transplanted, among which 71 were pregnanted and 319 pups were born. A total of 85 rabbits were detected as transgenic integrated positive by PCR, and 57 survived (36$21$) with a pregnancy rate of 75.5%(71/94)and an integration rate of 26.6%(85/319). We obtained a total of 12 mammary gland-specific expressed rhPA transgenic rabbits with the expression levels of 5.2-630μg/mL (PCL25/rhPA 11, BLC14/rhPA 1) by ELISA. The SDS-PAGE electrophoresis and Western Blot showed a 39KDa target-specific band in the milk from 12 expressed transgenic rabbits. Compared with alteplase, the maximum thrombolytic specific activity was up to 360-fold in vitro by FAPA and ELISA. The descendants of the high expressed transgenic rabbits A29 mated, and all descendants of one F2 generation transgenic rabbit A29F2-09 were integrated positive (100%,13/13) by testcross. The relative copy number of this rabbit was about twice more than other homologous series of transgenic rabbits by real-time PCR, which proved that the rabbit A29F2-09 was homozygous transgenic rabbit. The rhPA expressing level of this homozygous transgenic rabbit was about 1000μg/mL and more significantly higher than non-homozygous rabbits. The copy number of different transgenic rabbit lines were not correlated with expression levels. Hence, it seemed that the PCL25/rhPA vector could high-effectively promote specific expression of rhPA with activity in the mammary gland of transgenic rabbits. Moreover, the expressed levels were mainly correlated with the integration site but not with the copy number. However, when the integration site was consistent, the expressed level would correspondingly increase with the increasing copy number.4. The mice were immunized by the traditional Freund’s and QuickAntibody adjuvant, and the results showed that the QuickAntibody adjuvant were greatly superior to Freund’s adjuvant in serum titer, immunizing dose and immune cycle (1010 vs.109.20 μg vs.1300 μg and 35d vs.70d. respectively). The splenic lymphocytes was fused with SP2/0 myeloma cells by using electrofusion and PEG-fusion, and the results showed that the electrofusion rate was significantly higher than PEG-fusion (166% vs.88.4%). After HAT-HT screening and limiting dilution assay. 12 hybridoma cell lines (labeled C1-C12) were achieved from subclone and could stably secrete high levels of antibodies. Three of them were polyol-responsive monoclonal antibodies (PR-mAbs) by ELISA-elution, accounted for 25%(3/12). The PR-mAb was prepared by using induced ascites and purified by rProteinA affinity chromatography. The ascitic titers of C1, C4 and C8 were 108,1010 and 108. respectively, and no non-specific-cross reaction occurred. Therefore, they can be used for affinity chromatography.5. The rabbit milk was treated with centrifugation to defat,35% ammonium sulfate to precipitate, ultrafiltration and dialysis. Immunoaffinity chromatography column was prepared by coupling the C4 PR-mAb with CNBr activated Bestarose 4FF, which coupling efficiency was up to 96.5%. In order to obtain the best elution effect, five different kinds of eluents (A:0.1 mol/L Glycine, pH 2.5; B:0.1 mol/L Glycine, pH 3.5; C:0.1 mol/L Glycine, pH 5.0; D:TE buffer with 0.75mol/L ammonium sulfate and 40% propanediol, pH 7.9; E:0.2M NaH2PO4 citric acid, pH 2.2) were applied. The results showed that the optimal eluent was D, and the eluents A, B and E could also elute the target protein. However, the eluent C with higher pH eluted more other unrelated proteins and the coefficient of recovery was very low. Further purification of the target protein (rhPA) was performed by using Chromdex75 prep grade gel filtration chromatography column, and the results showed that the target protein could be completely separated at the sample loading volume of 1% and 2.5%, with two independent peaks; and showed incompletely separated the target protein and two peaks overlap with each other at the volume of 5%. Immunodetection and Time of Flight Mass Spectrometry (TOF) identified that the purified product was rhPA. Compared with BSA (pl=4.7, Mr=66KDa, purity of 96-99%), two-dimensional electrophoresis showed that the molecular weight and isoelectric point of purefied product was about 39KDa (35-40KDa) and 7.1, respectively, and the purity of rhPA was also up to about 96-99% or more. Compared with alteplase, circular dichroism spectra analysis of rhPA showed that the secondary structure was similar to alteplase; and the thrombolytic specific activity of our purfied product (rhPA) was up to 214-fold by FAPA and much higher than alteplase.6. The endotoxin was removed by the HitrapTM CaptoRMQ-1mL ion exchange column and it was detected lower than 0.015 EU/mL by endotoxin-detected kit. In order to establish mice thrombus model,0.3mL of 1%,0.5%,0.1%,0.05%,0.01% carrageenan were used to induce thrombosis in mice tail via intraperitoneal injection and 0.05% was shown the optimal concentration. The "thrombus mice" were treated with the rhPA at different "administration" concentrations including low-dose group (0.1 mg/mice), middle-dose group (0.4 mg/mice) and high-dose group (1.6mg/mice), with alteplase and normal saline as the control groups. The results showed that the rhPA was significantly superior to alteplase in efficacy and dose; moreover, the "thrombotic disease" were completely cured 32 h after three administration of 1.6 mg/mice.In conclusion, these results suggested that the constructed mammary-specific expression PCL25/rhPA vector (F, E, K1 domains deletion mutant of t-PA) was reasonable and effective, which could be effectively expressed in mammary glands of transgenic rabbits. The expression product rhPA was relatively individually presented in rabbit milk and could be purified. The purified product was of higher recovery rate, higher purity and higher thrombolytic activity, and rhPA had a more favorable efficacy in treating mice model of thrombotic disease compared with alteplase. Furthermore, we created the series of technical system was relatively consummate and feasible. As far as we know, these have been rarely reported at home and abroad, and it provides a new idea for future research and development of novel recombinant thrombolytic drugs, and also establishes a strong foundation for further clinical trials. |
PDF Full Text Request