Efficient And Stable Expression Of Human Coagulation Factor VⅢ In Transgenic Animal Milk

Human coagulation factor VⅢ（hFVⅢ） is an important clotting factor,widely used for prevention or the treatment of hemophilia A in clinic.Presently,hFVⅢ is generally produced by plasma and mammalian cell lines,with deficiencies such as risk of blood-borne virus infection,high cost and low yield.Recent developments indicate that more attention was focused on transgenic animal mammary gland as a bioreactor to produce pharmaceuticals by manipulating the milk with the advantages of high biological activity,low cost and high yield.In order to facilitate the production of hFVⅢ using mammary gland bioreactor,we did research for the expression system of hVⅢ on the level of cell and transgenic mice respectively.In the first part of this thesis,hFVⅢ cassettes were constructed,respectively driven by cytomegalovirus（CMV）,goat β-casein regulation sequences（P1A3）or murine serum albumin gene（Palb）promoters with hFVⅢ cDNA full length（hFVⅢ）or B domain deleted（hFVⅢBDD）.Cell experiments show that the six vectors all can express hFVⅢ or hFVⅢBDD in cells at transcriptive level.ELISA results demonstrated that hFVⅢ protein can be expressed in the milk after injection of hFVⅢ vectors into mammary gland of lactating mice.The highest h FVⅢ concentration was 2.01±0.23μg/mL（CMV-hFVⅢ）vector and 1.20±0.21μg/mL（P1A3-hFVⅢ）vector,no detection for Palb-hFVⅢ and Palb-hFVⅢBDD.The results of hFVⅢBDD vectors were consistent with hFVⅢ,with concentration 2.81±0.31μg/m L（CMV-hFVⅢ） and 1.82±0.25μg/m L（P1A3-hFVⅢBDD）.The hFVⅢ concentration of expressed hFVⅢBDD target gene was higher than hFVⅢ in milk after transfection into mammary gland of lactating mice.In the second part of this thesis,transgenic mice were generated by using pronuclear microinjection technique and stably germ-line transmitted in different lines.According to real-time PCR,Immunohistochemistry and Western blot analysis,the hFVⅢ cassettes directed by CMV were translated in the whole body of transgenic mice,while the hFVⅢ cassettes driven by Palb or P1A3 were expressed specifically in liver or mammary gland.ELISA indicated that the expression level of hFVⅢ was varied among different transgenic mouse lines;the average h FVⅢ concentration of in the milk of P1A3-hFVⅢBDD transgenic line was 2.45±0.80μg/mL while hFVⅢ of P1A3-hFVⅢ transgenic line was 1.16±0.80μg/mL,about 6-25 fold higher than that in normal human plasma.Furthermore,the average coagulation activity was about662±114 mU/mL for P1A3-hFVⅢBDD line while 337±118 mU/mL for P1A3-hFVⅢ line assayed by thrombin instrument.Compared with non-transgenic mice,all transgenic mice had no significant differences in physiological characteristics,lactation or breeding.Thus,we conclude P1A3 promoter can safely direct specifically expression of high level bioactive hFVⅢ in the mammary gland of transgenic mice.P1A3-hFVⅢ and P1A3-hFVⅢBDD vectors were more suitable for the generation of mammary gland bioreactor.In the third part of this thesis,Von willebrand factor（vWF） was believed conducive to stabilize the hFVⅢ,thus in this study we constructed P1A3-hFVⅢBDD-IRES-VWF co-expression vector and prepared the P1A3-hFVⅢBDD-IRES-VWF co-expression transgenic mice.The bioactivity and concentration of hFVⅢ were detected at various time points under different storage conditions.The results showed that the half-life of hFVⅢ bioactivity degration in vitro(Td_1/2v) in P1A3-h FVⅢBDD-IRES-vWF co-expression mouse was significantly longer than P1A3-hFVⅢBDD mouse（77.21 h vs.43.82 h at 4oC,32.45h vs.19.67 h at room temperature and 7.40 h vs.3.44 h at 37oC,respectively.P<0.05）.At the same time,CMV-vWF transgenic mice were obtained to produce P1A3-hFVⅢBDD/CMV-v WF double heterozygous mice mating with P1A3-h FVⅢBDD transgenic mice for synchronously expressing hFVⅢ and vWF.Detection for the h FVⅢ in the milk of double heterozygous mice showed that the Td_1/2v of hFVⅢ bioactivity in milk of P1A3-hFVⅢBDD/CMV-vWF double heterozygous mouse was similar to P1A3-h FVⅢBDD-IRES-vWF co-expression mouse,apparently demonstrating that the vWF transgene expression in the hFVⅢ transgenic mice can efficiently improve the stabilization of hFVⅢ bioactivity in the milk.However,the preparation of double heterozygous animals was time-consuming and laborious.In addition,the genetic characters of the double heterozygote may be segregated with the generations.Thus,P1A3-hFVⅢBDD-IRES-vWF co-expression would be more valuable and low cost for the production of hFVⅢ transgenic mammary gland bioreactor.This study results show that P1A3 promoter can direct hFVⅢ or h FVⅢBDD gene to specifically express bioactive hFVⅢ in the mammary gland of transgenic mice safely.Meanwhile in the milk of the transgenic mice,the hFVⅢ expression level of B domain deleted hFVⅢ vectors is higher than that of full-length hFVⅢ vectors.Furthermore,vWF trasgene can significantly increase the stability of hFVⅢ protein in the transgenic mouse milk.