The Research Of The CDNA Clone, Expression & Purification Of Human Antithrombin Ⅲ And The Therapeutic Effect Of Its Recombinant Protein On DIC Rat Model

Compared with the traditional medicines, recombinant protein drugs have several advantages, such as high activity, specificity, low toxicity, clear biological function, facilitate clinical application and can be mass production. Therefore, at the present time, the production of recombinant drug protein is hotspot in the field of drug research in domestic and abroad, and the focus of the recombinant drug protein research is economical and efficient production and application of this kind of proteins. There are several exogenous protein expression systems for recombinant protein production, such as in the bacteria, fungi, insect cells, animal cells, genetically modified plants and genetically modified animals, but they have some irreparable defects. To explore the feasibility of the use of mammary gland as expression system to production of medical proteins, in this study, we selected human antithrombin (hAT) - one of the important anticoagulant protein in human plasma proteins as the research object, and expressed bioactive recombinant human antithrombin (rhAT) by construction of recombinant adenovirus vector containing human antithrombin cDNA sequence and direct infusion of recombinant adenovirus vectors into the goat mammary gland. The rhAT was purified from the goat milk and used in the treatment of disseminated intravascular coagulation (DIC) rats. Through the above experiments, we want to provide the theory basis and the technical support for rhAT production in the mammary gland by adenovirus vectors infection and its application in the treatment of human DIC diseases.This research mainly includes the following content: (1) The extraction of total RNA from human embryonic liver tissue, obtaining the sequence of human antithrombin cDNA by RT-PCR techniques, and sequencing the cDNA sequences to verify the cDNA sequence we obtained was the human antithrombin cDNA. (2) Construction the recombinant expression plasmid of p3AT, and transfection of p3AT into cultured goat mammary epithelial cells to verify fact that the cDNA sequence we obtained can be used to express rhAT. (3) Construction the recombinant shuttle vector plasmid of pShAT, and obtained the plasmid of recombinant adenovirus vector encoding human antithrombin cDNA (pAd-hAT) by recombination between the plasmid of pShAT and Adeasy-1 in the bacteria of BJ5183. (4) Transfection of pAd-hAT into HEK293 cells, and obtained the recombinant adenovirus vector of Ad-hAT by the virus packaging and amplification in HEK293 cells. (5) Transfection of Ad-hAT into cultured goat mammary epithelial cells to verify the fact that Ad-hAT can expressed the rhAT in goat mammary epithelial cells efficiently. (6) Direct infusion of Ad-hAT into goat mammary gland and expressed rhAT in goat mammary gland epithelial cells efficiently, detection the recombinant protein in goat milk, purification of rhAT from the goat milk by heparin agarose gel 6FF affinity chromatography, and obtaining the freeze-dried powder a freeze dryer. (7) Construction of the rat DIC model, and treatment of the rat DIC model with human plasma antithrombin (hpAT) and rhAT, decision the therapeutic effect of two ATs on DIC rats by analysis the DIC indexes of the treated DIC rats. (8) Comparison the therapeutic effect of two ATs on DIC rats, exploration the replacement possibility of the hpAT by rhAT in DIC patient treatment.Through above study, we obtained the following results:(1) The antithrombin cDNA gene was successfully obtained from a human embryo liver tissue with RT-PCR techniques. The cDNA sequence of hAT was in accordance with the sequence in Genbank (NM₀00488) by DNA sequence analysis.(2) Two recombinant expression plasmid containing human antithrombin cDNA gene, p3AT and pShAT, were constructed on base of the plasmid of pcDNA3.1+, pIRES and pShuttle-CMV.The neomycin resistance gene (Neor), which permits selection of transformed cells and the green fluorescence protein (GFP) gene, which served as a live marker for tracking infected cells or tissues in animal study, were constructed in p3AT. The GFP gene was also constructed into pShAT served as a live marker. Thus construction facilitated checking the express of target gene and screening out the transformed cells.(3) The identified pShAT vector was linearized by Pme I, then the linearized vectors was directly transformed the competent E. Coli. BJ5183 contained with Adeasy-1 plasmid for homologous recombination to obtain the recombinant adenoviral vector of Ad-hAT. The result of directly transforming of linearized pShAT into the competent E. Coli. BJ5183 which contained with Adeasy-1 plasmid validated the fact that the"two step transforming"greatly enhanced the efficiency of homologous recombination.(4) The recombinant adenoviruses of Ad-hAT was successfully packaged and amplified in HEK293 cells.(5) The rhAT was obtained in cultured goat mammary gland epithelial cells mediums either transfection with p3AT or infection with Ad-hAT. The expression level of rhAT which obtained by transfection with p3AT was up to 700±90 mg/L, and the activity of rhAT for 110%～120%. This result confirmed the fact that the cDNA sequence we obtained by RT-PCR techniques could express rhAT properly. The expression level of rhAT which obtained by infection with Ad-hAT was up to 900±85 mg/L, and the activity of rhAT for 120%～130%. This result confirmed the fact that Ad-hAT could infect goat mammary gland epithelial cells and express rhAT with high efficiency. It also provided the foundation for the next step to directly infuse the Ad-hAT into the goat mammary for the rhAT high expression.(6) Directed infusion of Ad-hAT into the goat mammary gland. The expression level of rhAT in goat milk was up to 1.58±0.21 g/L (detected 11 times in 21 days) averagely, the highest expression level of rhAT was 3.1 g/L among the lactation days, the activity of rhAT was up to 102.5±1.44 %;after purification with heparin agarose gel affinity chromatography, the recovery rate and the purity of rhAT were up to 54.7±1.0 % and 92.5±0.5 % separately.(7) The rat DIC model was constructed successfully.(8) Treatment the DIC rats with hpAT and rhAT, the results showed that both two ATs alleviated the condition of DIC rats (p>0.05) and had good curative effect on DIC rats. In blood of the DIC rats, the consumption of fibrinogen and platelet was reduced significantly, meanwhile the levels of antithrombin and thrombin-antithrombin (TAT) were augmented significantly; and the increase of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentration was restrained efficiently. The rhAT produced with this method has potential as a substitute for hpAT in the therapy of DIC patients.In present study, the rhAT was expressed in goat mammary gland with high efficiency by recombinant adenovirus vector infection. After the recombinant protein was purified with heparin agarose gel affinity chromatography, high recovery rate and high purity of rhAT (freeze-dried powder) were obtained. For the curative effect on DIC rats, hpAT and rhAT all alleviated the condition of DIC rats (p>0.05) and had good curative effect on DIC rats. All the above results showed that the method we explored for rhAT production was feasible, and the rhAT produced with this method has potential as a substitute for hpAT in the therapy of DIC patients. These will greatly promote the hAT production mode from traditional to low-cost, high-efficiency, mass production, and the availability of rhAT with high quality will expand the application fields and create more economic and social benefit in future.