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

Human coagulation factor V?(hFV?) is an important clotting factor,widely used for prevention or the treatment of hemophilia A in clinic.Presently,hFV? 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 hFV? using mammary gland bioreactor,we did research for the expression system of hV? on the level of cell and transgenic mice respectively.In the first part of this thesis,hFV? cassettes were constructed,respectively driven by cytomegalovirus(CMV),goat ?-casein regulation sequences(P1A3)or murine serum albumin gene(Palb)promoters with hFV? cDNA full length(hFV?)or B domain deleted(hFV?BDD).Cell experiments show that the six vectors all can express hFV? or hFV?BDD in cells at transcriptive level.ELISA results demonstrated that hFV? protein can be expressed in the milk after injection of hFV? vectors into mammary gland of lactating mice.The highest h FV? concentration was 2.01±0.23?g/mL(CMV-hFV?)vector and 1.20±0.21?g/mL(P1A3-hFV?)vector,no detection for Palb-hFV? and Palb-hFV?BDD.The results of hFV?BDD vectors were consistent with hFV?,with concentration 2.81±0.31?g/m L(CMV-hFV?) and 1.82±0.25?g/m L(P1A3-hFV?BDD).The hFV? concentration of expressed hFV?BDD target gene was higher than hFV? 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 hFV? cassettes directed by CMV were translated in the whole body of transgenic mice,while the hFV? cassettes driven by Palb or P1A3 were expressed specifically in liver or mammary gland.ELISA indicated that the expression level of hFV? was varied among different transgenic mouse lines;the average h FV? concentration of in the milk of P1A3-hFV?BDD transgenic line was 2.45±0.80?g/mL while hFV? of P1A3-hFV? transgenic line was 1.16±0.80?g/mL,about 6-25 fold higher than that in normal human plasma.Furthermore,the average coagulation activity was about662±114 mU/mL for P1A3-hFV?BDD line while 337±118 mU/mL for P1A3-hFV? 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 hFV? in the mammary gland of transgenic mice.P1A3-hFV? and P1A3-hFV?BDD 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 hFV?,thus in this study we constructed P1A3-hFV?BDD-IRES-VWF co-expression vector and prepared the P1A3-hFV?BDD-IRES-VWF co-expression transgenic mice.The bioactivity and concentration of hFV? were detected at various time points under different storage conditions.The results showed that the half-life of hFV? bioactivity degration in vitro(Td_1/2v) in P1A3-h FV?BDD-IRES-vWF co-expression mouse was significantly longer than P1A3-hFV?BDD 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-hFV?BDD/CMV-v WF double heterozygous mice mating with P1A3-h FV?BDD transgenic mice for synchronously expressing hFV? and vWF.Detection for the h FV? in the milk of double heterozygous mice showed that the Td_1/2v of hFV? bioactivity in milk of P1A3-hFV?BDD/CMV-vWF double heterozygous mouse was similar to P1A3-h FV?BDD-IRES-vWF co-expression mouse,apparently demonstrating that the vWF transgene expression in the hFV? transgenic mice can efficiently improve the stabilization of hFV? 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-hFV?BDD-IRES-vWF co-expression would be more valuable and low cost for the production of hFV? transgenic mammary gland bioreactor.This study results show that P1A3 promoter can direct hFV? or h FV?BDD gene to specifically express bioactive hFV? in the mammary gland of transgenic mice safely.Meanwhile in the milk of the transgenic mice,the hFV? expression level of B domain deleted hFV? vectors is higher than that of full-length hFV? vectors.Furthermore,vWF trasgene can significantly increase the stability of hFV? protein in the transgenic mouse milk.