TW200526781A - Transgenic animals producing biologically active human factor VIII in their milk driven by mammary-specific expression cassette - Google Patents

Abstract

Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with whole plasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy. One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. The present invention provides improved mammary expression cassettes useful for the expression of genes at high levels in transgenic milks. In particular, the present invention provides recombinant signal peptide sequences derived from α-lactalbumin and αS1-casein milk genes suitable for leading protein secretion in the mammary gland. These gene cassettes are capable of delivering different transgenic constructs which result in the production of full-length or B domain-deleted therapeutic levels of biologically active human FVIII in the transgenic animal in vivo. Within the scope of the invention are also method for producing the transgenic non-human mammal, such as mice, rabbits, goats, sheep, pigs and bovine species, capable of expressing human FVIII, and methods of making milk and methods of identifying protein from the transgenic milk.

Description

200526781 发明 Description of the invention: [Technical field to which the invention belongs] The present invention belongs to the field of biotechnology of genetically modified animals, and uses the development of a mammary gland-specific expression vector system to achieve the intermediate production of genetically modified animals and their offspring. Techniques and methods for preparing non-blood-derived human coagulation factor VIII biological agents. [Previous technology] Human eighth coagulation factor (human factOr; hFVHj) is an important coagulation factor that is essential to the intrinsic pathway of blood coagulation; once the human body lacks or reduces hFVIn, it will cause different degrees Problems with blood coagulation, leading to so-called hemophilia A (hemophUia A). Hemophilia A (hemophiUa A) is an X-link disease that is a genetic disease. It occurs in men with a probability of about 1 in 10,000. Human factor VIII (hFVIII) ) Is human ninth coagulation factor (hFIX) activated human tenth coagulation factor (hFX) cofactor [丨], and the human eighth coagulation jk factor precursor protein (precursorr pr0tein) contains "Η amino groups ^ ' Contains 19 amino acid signal peptides, and can distinguish six regions' in the order of A1-A2-B-A3-Cl-C2, etc. The single-chain precursor protein has a molecular weight of 256-kD. After hydrolysis, heterodimers are formed; 90 to 20 0-kD heavy chain (heaVy chain) is composed of A1-A2-B and 80-kD light chain (A3-C1-C2) Constitute P]. The pre-200526781 driver of human eighth coagulation factor is mainly synthesized in the liver, the FViii protein secreted into the plasma has a molecular weight of about 320kb and the coding region has i86kb on the X chromosome. Activated FVIII will remove the B domain amino acid sequence The month and month are targeted at patients with hemophilia A Treatment is usually performed by purifying hFVIII in normal human blood for intravenous injection; however, this treatment method is not only costly, but also may expose patients to a number of viral infections such as human immunodef iciency virus, HIV) or hepatitis virus (heptits virus), etc. Therefore, the possibility of using human transgenic methods to produce non-blood products of human eighth coagulation factor or initiating living gene therapy is still a priority. The demand for coagulation factor pharmaceutical protein is about USD 870 million, which has considerable market development value. Hemophilia is a hereditary abnormal blood coagulation disease. With current medical technology, this disease cannot be cured and must be cured. He has been treated for a lifetime. Hemophilia is divided according to the lack of coagulation factors: Hemophilia A: Those who lack the eighth coagulation factor; Hemophilia type 8: Those who lack the ninth coagulation factor; and Hemophilia C · · Those who lack the eleventh coagulation factor are divided according to the amount of coagulation factor deficiency: Severe: factor less than 1%; Moderate: factor between 1 % ~ 5%; 200526781 Mild: Factor is between 5% ~ 30%. There is one hemophilia patient per 10,000 people in the total population, of which A 蜇 accounts for about 80% ~ 85%, Type B accounts for about 15% to 20%, and one-third of the cases cannot be found without history. It is caused by genetic mutations, and there are also patients with acquired hemophilia. Hemophilia C is rare and the symptoms are mildly recessive in individuals. Symptoms occur in both men and women. Patients with hemophilia often have spontaneous bleeding or periodic bleeding throughout their lives; there are many reasons for this, such as bleeding in muscle joints, skin, mucous membranes, and tissues. Another congenital bleeding disorder has symptoms similar to or similar to hemophilia and is called hemophilia. Hemophilia in a broad sense is a congenital bleeding disorder caused by a lack of coagulation factor activity in addition to types A, B, and c. Hemophilia in the narrow sense is vonWUlebrand's disease, the most common congenital hemorrhagic disease, which is caused by a lack of vonWillebran's factor (V.WF.) amount or a qualitative abnormality caused by hemostatic disorders [3]. vOnWulebrand factor can protect the eighth debulking factor and is related to the platelet attachment effect, because the symptoms of Μη Willebrand's disease are mainly mild to moderate mucosal skin bleeding. 'Severe type, symptoms and severe type of blood Friendship is not easy to distinguish. The process of blood coagulation is through a series of enzyme reactions. The process can be divided into internal and external pathways. The eighth coagulation factor and the fifth coagulation factor play a very important cofactor role in the internal pathway; The amount is about 0.2ug / ml plasma to ensure the maintenance of blood coagulation function.-Once 200526781, the lack or reduction will cause abnormal bleeding. The eighth coagulation factor will combine with vWF to form a complex with non-covalent bonds in the blood circulation. It will protect "coagulation factor proteins from being decomposed by protein reading enzymes and increase their synthesis [4]. The eighth coagulation factor plays a role as a cofactor of the ninth coagulation factor (cofactor) and plays a role in calcium ions and scales. In the presence of lipid (phospholipid), it can catalyze the ninth coagulation factor activity: the tenth coagulation factor rate is more than 10,000 times' the absence of the eighth coagulation factor will cause hemophilia A (hemophilia A). The gene line is located at the tail of the long arm of Xq28 chromosome, close to the fragile X chromosome (fragile x chr__e). It is about 186kb in length and contains 26 exon and 25 Each lntron, cDNA is about 9-kb in length, the gene code region that can be translated into protein is 7.2_kb, and can make 235i amino acid [5]. Because the eighth coagulation factor is very low in blood, and The entire protein molecule is so huge that the structure of the eighth coagulation factor has not been fully understood so far. The following have been explored: ： Arg 372, Arg 74G, Arg 1689 have mutation cases, and use genetic engineering technology A study of the changes in Arg at three thrombin cleavage sites AU 372, Απ 740, and Arg 1 689 found that the 372 and Arg 1 689 cleavage sites are very important for the activation of the eighth coagulation factor, while Arg 740 is less important [6 Must be, because the eighth coagulation factor (2) B domain is not 200526781 after thrombin cleavage, the b domain is discarded to form an active eighth / spin factor, and the entire B d⑽ain molecule is quite large and is active for formation The eighth coagulation factor is also a hindrance [7]. (3) At the end of the eighth coagulation factor; the amino acid sequence is 21 73 2232 is the region where the eighth blood factor 4 binds to phosphoryl ipid Domain, and the C2 domain is the site responsible for binding to the platelet cell membrane [8]. (4) The junction of the A3 domain and the B domain is in the amino acid sequence of 1670169689; vWF binds to the eighth coagulation factor Position, and Tyr 168 sulfation (sulfation) is closely related to the eighth coagulation factor and vWF binding. (5) The structure of human eighth coagulation factor is very similar to that of the fifth coagulation factor. Among them, the active human eighth coagulation factor acts with eight 2 and A3 and the ninth coagulation factor, and the fifth coagulation factor A2 A A3 and the first Decoction acts and utilizes the cell membrane action of C2 domains and platelets. Patients with hemophilia are born with coagulation disorders for life and blood transfusion, but coagulation factor therapy can easily lead to the occurrence of viral infection, AIDS and chronic hepatitis, which are life-threatening. 基因 In recent years, genetically reconstituted coagulation factor abolition therapy has been used to solve blood transfusion. The problem of infection ', but treatment is expensive. At present, about 60% of hemophiliacs in the world cannot receive proper treatment due to insufficient medical resources, especially in developing countries. This medical problem is even more serious. Preventive treatment cannot be universally implemented. 10 200526781 During treatment, 30% of patients will develop anticoagulant antibodies. Patients still suffer from bleeding and joint loss of joint function. Therefore, the development of gene therapy can bring new hope to these patients. The human eighth coagulation factor gene is a rather large gene (l86 Kb long, no 26 exons). In theory, such a large gene is not easy to perform gene therapy. It requires an appropriate virus as a vector to implant a huge gene, but Fortunately, the following advantages of hemophilia led the National Health Organization to adopt a gene therapy program for hemophilia in 1997. 1. Although most of the coagulation factors are normally produced by hepatocytes, tests have shown that the use of human fibroblasts' muscle cells or vascular endothelial cells for gene therapy can successfully increase the eighth and ninth coagulation factor concentrations in the gold circulation. Coagulation genes can be implanted into multiple target cells, not limited to liver cells, and coagulation factors produced by fibroblasts and muscle cells can easily enter the gray fluid circulation [9]. 2. The range of effective physiological functions of coagulation factors in human blood is extremely loose, from 200%. The coagulation factors can fully perform physiological functions without the assistance of other organs, and there is no need to strictly control the effective range of gene therapy, so there will be no miss or invalidation. Treatment. 3. At present, animal experimental models of hemophilia rats and dogs can quite accurately reflect the normal physiological functions of human coagulation factor genes, making the test easy to proceed. [10] 〇200526781 4. Because only simple measurement of coagulopathy gene therapy is required Blood factor growth concentration can effectively evaluate hemophilia

At present, hemophilia gene therapy tests are performed in vitro (e " and live selection (―) respectively, and the ninth coagulation factor test in vivo is more promising (because the human eighth coagulation factor gene is larger than the ninth coagulation factor, Moreover, the ninth coagulation factor is suitable for the selection of retrGvirus as a medium but must be implanted into the proliferating cells.-Generally the ninth coagulation factor is only suitable for human bone marrow cells and hepatocytes. When the human clotting gene vector virus is implanted from the portal vein, the human clotting gene can be used to nucleate the nuclear gene of the human target cell. Although there is a long gene expression effect, it may cause genetic disorder. In addition, the human eighth coagulation factor gene has a partial genetic code that can inhibit retroviruses. When the human eighth coagulation factor is generated, the vector virus retrQvirus will be gradually suppressed and disappeared, making the efficiency of the vector poor.

Although it has recently been developed to remove the B-d⑽ah portion from the human eighth coagulation factor cDNA and reduce the size to 4.5 kb, it is still difficult to use adenovirus as an implanted vector, even though implantability does not affect the production of human eighth Multi-target cells such as muscle cells, fibroblasts, etc. of the physiological functions of coagulation factors are still completely embedded in the genome of the target cells because of the adenovirus, so the risk of gene mutation caused by the adenovirus and the immune response of the human body can be taken into account Therefore, the adenoviral vector virus is eliminated from the host, causing clotting. 12 200526781 The gene expression period of factor 25 is shortened and the effect of gene therapy is greatly reduced. [11] Therefore, 'gene therapy with adenovirus is not the best strategy. . In addition, 'over-implantation of adenovirus will produce hepatotoxicity, and patients with hemophilia are mostly trapped by hepatitis virus infection during long-term use of coagulation factor therapy. Therefore, the number of implanted vectors is limited due to hepatotoxicity. The degree of coagulation factor production is insufficient. Therefore, the use of blood products produced by transgenic animals is currently a safe and secure method for hemophiliacs.

Xianli's research showed that the gene of human eighth coagulation factor was placed in the small milk heavy metal thioprotein gene promoter (murine Jean i⑽ein J; mMT-1) for the development of transgenic animals, and its performance was only as good as ng / ml [12], which is 50% of the concentration of the eighth coagulation factor in normal human plasma

-'Therefore, it is not economically beneficial; In addition, the research group used a mouse Whey acidic prOtein; · Aρ] gene promoter to express human eighth coagulation factor on the basis of transgenic pigs. The good expression effect of 2 · 7 Ug / mi [13] 'cannot efficiently produce recombinant human eighth blood coagulants'. Obviously there is currently no suitable breast expression system for the production of human eight blood coagulation factors. The present invention focuses on this and selects a high-yield milk: lactalbumin promoter for laparotomy cows, and adopts mammary gland specificity: α-lactalbumin gene or alpha-casein gene release for cows Secrete the message sequence, and connect the dairy cow's auxin gene to the end of the gene, and develop a new high-performance mammary gland phenotype, a novel high-performance mammary phenotype, 13 200526781 as a technological platform for expressing human eighth coagulation factor recombinant protein . [Summary of the Invention] σ ° In view of the lack of a method for expressing human eighth coagulation factor in the milk of genetically modified animals, the present invention provides-a kind of specific and continuous performance in the genetically modified mammals' courtesy juice. A method for deriving a protein, the steps of which include: constructing a recombinant exogenous protein gene containing a complete or genetically engineered scissor in a vector having a mammalian mammary gland-specific phenotype; meal (b) combining the aforementioned exogenous protein Breast phenotype of f gene It is recommended to perform microinjection of embryo genes and embryo transfer into mammalian embryos, so that the aforementioned foreign protein is expressed in the milk of the transgenic mammal, and during lactation Sustainable performance Research on mass production of recombinant proteins or modification of milk composition by genetically modified animals has been regarded as an important research and development direction by all walks of life, especially when biotechnology has been listed as the key global technology of Mailu towards the 21st century If the animal's genetic background can be changed without seriously affecting the physiology of the animal, the animal's body tissue can be further upgraded to produce human beings. Plants that require pharmaceutical products to produce high-priced products. In addition, from nucleic acid to active protein f synthesis, in addition to nucleic acid replication, transcription, and translation, post-translational modification processes are also required, including: glycosylation , Phosphorylation, gamma acidification (γ-carboxylation), proteolysis, and covalent or non-covalent binding with cofactors 14 200526781, etc., can become a biologically active protein. In the production of humans Among the systems that require recombinant proteins, transgenic animal systems are the most advantageous, especially after translation, which is more suitable for human needs. In order to produce high-priced genetically engineered proteins, the mammary gland has become the main goal of genetically modified animal researchers. Tissues are relatively independent in physiology. When foreign genes are expressed in the mammary gland, the effect on the animal's hanging physiology can be minimized. In addition, gene products are also simpler when they are in milk. The aforementioned gene contains foreign protein genes. The construction of the expression vector includes four parts: the 5 gene promoter 5 and the regulatory sequence, which has breast epithelial cells. Performance-specificity and can regulate foreign protein genes for stable and stable performance during lactation in transgenic mammals; ⑺ Breast specificity-specific secretion message sequences can guide foreign proteins in transgenic mammals After the mammary epithelial cells are expressed, they are efficiently released into the milk; (3) the foreign protein gene is located after the 5 regulatory sequence and the secretory message and can be regulated to perform; (4) the gene 3, the regulatory sequence, It is located after the code sequence of the foreign protein gene, and contains the adenine polymerization message sequence, which is used to stabilize the integrity and post-transcriptional repair of mMA molecules transcribed by foreign genes. The etiquette method continues to display special biological functions in addition to the "self-property method. Its mammary gland expresses its gene promoter. The weekly control sequence 歹 j is a gene from a livestock pasture-a high-yield Dutch dairy cow gene using genetic breeding technology." In the body group, the obtained u, long-fat cow α-lactalbumin 15 200526781 promoter sequence was obtained. The aforementioned method for continuously expressing foreign proteins with special biological functions in the milk of transgenic mammals, wherein the "V sequence" of the protein release message is selected to the same high milk yield Dutch dairy cow genome There are two kinds of mammary gland high-performance j-lactalbumin gene or asb casein gene release message sequence. The aforementioned method for continuously expressing a foreign protein with special biological functions in the milk of a transgenic mammal, wherein the gene 3, the regulatory sequence, is located behind the code sequence of the foreign protein gene and contains a 0.5-kb Netherlands The cow's auxin gene polyadenylation signal sequence is used to stabilize the integrity and post-translational modification of mRNA molecules transcribed by foreign genes. The aforementioned transgenic exogenous protein gene line with special biological functions can be transmitted to offspring through sexual reproduction. Including the complete gene code region Human eighth coagulation factor group The aforementioned foreign protein gene can include (7.2-kb) or genetically engineered splicing (4.5-kb) reasons. The activity of the sibling occlusion factor can reach the milk performance of human blood transgenic milking animals. The agricultural degree can reach 50 g / L, and the purified coagulation plasma coagulation factor VIII is 14 times. The transgenic mammalian line prepared by the method of the present invention can continuously and stably express foreign proteins during lactation. Before ^ „& u The genetically modified mammalian animals returned by Pui Zei are cattle, sheep, pigs, rabbits or mice, preferably v 'is Ping, pigs and mice. 200526781 m described in the milk of mammalian transgenic mammals Continuous performance = method of exogenous protein, which can be advanced-add step after step (b): (C) collect the aforementioned transgenic milk of genes containing exogenous proteins with biological functions; and The exogenous protein is isolated from the juice, so as to obtain the aforementioned various exogenous proteins f from the milk of the transgenic mammal. Another object of the present invention is to provide a human transgenic mammal that continuously expresses human parasites in milk. The method of factor production includes the steps of: (i) constructing a plastid that contains the human eighth coagulation factor gene and can be expressed in the mammary glands of mammals; (b) gene-injecting the aforementioned plastid of the first human coagulation factor gene Humans and embryos are transplanted into mammals, so that the aforementioned human eighth coagulation factor is expressed in the milk of mammals with this gene, and during lactation: continuous performance; Transgenic milk of human eighth coagulation factor deleted by region or genetic engineering splicing B-d brewing; and ⑷ the aforementioned human coagulation factor was isolated from the aforementioned milk, so as to obtain purified Recombinant human eighth coagulation factor can be used as a medical protein for medical use in hemophilia patients. [Embodiment] The present invention provides the following five examples to further illustrate the expression of the recombinant human eighth coagulation factor in the breast according to the present invention. The novel and progressive method of animal production of genetically modified genes is provided. The present invention provides a method for producing exogenous proteins with the biological function of a transgenic mammal in the milk of a transgenic mammal. Carry the complete base of human clotting factor VIII (hFVIII) □ gain code region (7.2-kb) and B-domain deleted recombinant eighth clotting factor (4.5-kb) Construction of foreign genes. The milk secreted by the transgenic mammals (including suckling goats, pigs, and mice) of the present invention can be expressed in milk. The whole human eighth coagulation factor (rFVIn), and the recombinant eighth coagulation factor (Bx_hFVin) deleted by β-d 透过 ah, can be clearly measured in whey protein f through the preliminary purification and separation steps of milk protein. The coagulation activity test also verified that the genetically-transformed recombinant eighth coagulation factor has biological activity to reduce blood work. In addition, the breast phenotype vector of the present invention can strictly control exogenous human eighth coagulation factor protein only in lactation. It is expressed in the glandular tissues of the stage, and the transgenic exogenous protein line can be continuously and stably secreted during breastfeeding. Example 1 Production of a gene carrying exogenous intact human factor VIII (rFVIII) Transgenic dairy goats, pigs and mice The genetically transgenic mammalian breeding method of the present invention is to construct the complete human eighth tuff factor (rFVIII) gene (7.2-kb) in the bovine alpha milk albumin promoter ( bovine α-lactalbumin promoter, aLA; 2.0-kb)-, a lactalbumin release secretion message guide sequence (& ―), and then a message of adenine polymerization of a cow auxin gene The sequence (〇5 — kb) forms a mammalian vector with a glandular phenotype. The map of the transgenic gene construction is shown in Figure 1 18 200526781 A. The aforementioned constructed gene vector is purified and extracted from the bacterial body after a large amount of proliferation. Enzyme truncation and ultra-high-speed centrifugation are used to obtain high-purity transgenic genes. The transgenes are mixed in a suitable concentration in a suitable buffer solution, and the DNA is injected into mammalian embryos using microinjection technology, followed by embryo transfer. At the end of the pregnancy period, the individual animals were harvested to obtain tissue DNA for rapid PCR screening and southern blot analysis of the exogenous Γρνπ I transgene into the genomic genome. The results are shown in Figure 1. β and Figure 1c show in this way a total of two series of transgenic milk goats, two series of transgenic pigs, and 17 series of transgenic mice were obtained. The milk of the 111 transgenic ewes, sows, and female mice during lactation was collected and used for quantitative and qualitative analysis of foreign protein performance. The present invention further analyzes the number of embedded sets of exogenous rFVI 11 transgenic genes in the cells of the transgenic animals by Slot blot technology. The quantitative standard line is constructed and purified, and the transgenic genes are diluted at a concentration corresponding to the number of sets. In other words, the genetically modified animal DNA samples were taken with fixed 10ug DNA as the basis for quantification; after the hybridization of the radioactive probes, the X-ray film was used for automatic radiographic development. The results showed that the number of inserts of the transgenic genes was distributed in each Cells between 2-50 sets' are shown in Figure II. Example 2: Genes of recombinant human eighth coagulation factor (Bx-hFVm) gene carrying exogenous B-domain deletions are produced in transgenic dairy goats and mice. The human factor VIII factor A gene is a rather large gene ( 186 Kb long, 19 200526781 containing 26 = 〇nS), the CDNA part alone reaches 7.2 ~ kb, the precursor protein of human eighth coagulation factor contains 2351 amino acids, which can distinguish six regions, which

Al-A2-B-A3-Cl-C2 ^^^^^^ (sirigle_cha; n precursor protein) has a molecular weight of 256-kD and is hydrolyzed by proteolytic enzymes to form heterodimers; respectively, 90 to 200-heavy chain * A1-A2 and 80-kD light chain (ught chain) are composed of A3-Π-C2, so B-domain does not No, experimental analysis shows that the B-domain region of the human eighth coagulation factor gene has endogenous negative regulatory sequences, thereby suppressing the content of human eighth coagulation factor in the blood. In view of this, the present invention further removes the human eighth coagulation factor B-domain region by genetic engineering methods, and reserves two d⑽ain regions of biological functions A1, A2, and A3-Cl-C2, and constructs 4.5. -kb Bx-hFVIII transgenic gene binds it to the bovine lactalbumin promoter (2.0-kb) and the aSl-saccharin secretion message guide sequence (48-bp), and then connects to a cow auxin The message sequence (0.5-kb) of adenine polymerization of genes forms a mammalian vector of mammary gland phenotype. The map of Bx-hF VIII transgenic gene construction is shown in Figure 3a. Nucleic acid sequencing analysis of the linkage of the recombined gene code region. Figure 3B shows that after the 16 amino acid aSl-casein secretion message guide sequences, the A-d⑽ain sequence of the human eighth coagulation factor gene was correctly connected; Fig. 3C shows that after the Al-A2 doma i η sequence, the human 20 200526781 苐 eight clotting factor gene A3-C1 -C2 doma i η sequence was correctly connected, and the attachment points were Ser amino acids at positions 741, respectively. With Leu amino acid at position 1,643 B doma i η of the amino acid sequence is completely removed. The constructed Bx-hFVIII gene vector was purified and extracted from the bacterial body after a large amount of proliferation, and a high-purity transgenic gene was obtained through the preparation process of special enzyme truncation and ultra-high speed centrifugation, and the transfection was performed in the same manner as in Example 1. Microinjection of mammalian embryo genes into genes, and embryo transfer. At the end of pregnancy, the individual animals were harvested to obtain tissue DNA for rapid Korean detection and Southern blot analysis of exogenous Bx-FVIII transgenic genes. The results of the genomic genome insertion pattern are shown in Figure 4B and Figure 4c. In this way, two series of genetically transgenic milking goats and 14 series of genetically transgenic stings were obtained. The B-bomain contained in the gene transgenic dairy goat of the present invention deletes the recombinant human eighth coagulation factor gene, and is mated to produce the f 1 progeny. After PCR amplification, it is determined that the progeny have a foreign gene It shows that the gene-transplanted dairy goat produced by the present invention has the ability of the gonad to inherit foreign genes to the offspring. It is further verified by automatic nucleic acid sequencing technology that the first offspring of the gene-transplanted dairy goat still carries the same form as the parent. Foreign genes, as shown in Figure 5. Example three, the eighth human breast phenotype; suspected blood cause? Gene transgenic animals have stage-specific and tissue-specific performance regulation characteristics. The mammary gland-specific expression vector system of the present invention is used in gene transfer animals. 21 200526781

During the lactation period, the 5-regulatory sequence of α-lactalbumin gene promoter (20-place) and 19 amino acids at the front end of the alpha-lactalbumin gene code sequence were extracted from the genome of high-yield Dutch dairy cows. Or the 16 amino acids of the asi-casein gene are composed of special sequences of mammary gland-specific secretion information peptides, which have the ability to regulate the lactation stage and mammary gland specificity and regulate foreign protein genes to Transgenic mammals continue to be stable during lactation; in order to verify the regulatory effect of this mammary specific carrier, individuals of transgenic animals were selected to extract various tissues during lactation, including the heart (H), Liver (L), lung (Lu), muscle (M), breast (Ma), brain (β), pancreas (P), and adipose tissue of mammary gland from non-gene transgenic individuals -PCR analysis of the specificity of the expression of exogenous hFVIn mRNA; as shown in Figure 6A, the transcription of the recombinant human factor VIII clotting factor is strictly controlled in breast tissue during lactation, and is expressed in other tissues such as the heart Liver, lung, muscle, brain, pancreas and mammary adipose tissue of non-transgenic individuals will not be shown. Figure 6B shows the analysis of the staged performance of transgenic ㈣…, which were delivered in the transgenic females. The first three days (D-3), the first day of lactation (D1), the eighth day of lactation (D8), the 15th day of lactation (D15), the 22nd day of lactation (D22), and the first week after weaning (D29) After the second week (D36) after weaning, the mRNA of the breast tissue was analyzed by RT-pCR, and the results showed that the continuity of the hFV 丨 I recombinant gene could not be measured in the pre-lactation, middle and late stages; and before delivery Table 22 200526781 shows that the exogenous gene is turned off after weaning. The mammary gland phenotype vector of the present invention exhibits the ability to regulate the recombinant human eighth coagulation factor gene specific to breast tissue and specific to physiological stages. &Quot; Implementation Example 4: Analysis of the expression of recombinant human first human factor TF protein in the mammary gland tissue of transgenic ewes and female mice and the release analysis in milk

The collected gene transgenic milk was centrifuged to remove the upper courtesy fat and Shen Dianzhi protein aggregates, and the whey clear solution was taken at undiluted ratios (UGX and 2GX), and the purified milk was subjected to polyacrylamide colloid electrophoresis Isolation (SDS-PAGE) and Coomassie Mue staining, and the protein on the electrophoretic film was transferred to the fiber membrane for Western blot experiments. The specific antibody (rabbitantihFviH

Polyclonal antibody) detection of human eighth coagulation factor. As shown in Figure 7 (A), H — (^ “11 and Lchain heterogeneous recombinant protein can be measured at the same time, and its molecular weight is between 8 and 5.”; Figure Seven

In one step, the protein expression of the human eighth coagulation factor recombinant gene was analyzed using a single antibody of 1-1 ^ 乂 111, and the results showed that a chain recombinant protein molecule of 80 kDa can be completely measured. It has been shown that the recombinant eighth coagulation factor expressed in breast tissue can be efficiently released into milk, and its release effect can maintain its stable performance during the lactation period due to transgenic animals. In order to confirm that the genetically transgenic dairy goat produced in the present invention has the ability to express and release recombinant human human coagulation factor 8 protein in the ritual juice during lactation, sexually mature genetically transgenic ewes are bred Milk samples were collected at different lactation stages (D3, D11, and D22) after childbirth 23 200526781 and centrifuged to remove the upper layer of milk fat and the casein agglomerates from Shendian, and the clarified liquid of whey was taken at different dilution rates. The milk was separated by polypropylene ammonium colloid electrophoresis separation (SDS-PAGE) and Coomassie blue staining, and the proteins on the electrophoresis film were transferred to pVDF fiber membranes for western transfection experiments, using specific antibodies (rabbit anti -hFVIII pOlyclonal antibody) was used to detect the recombinant human eighth coagulation factor in goat milk. A standard sample of 100 ng of human eighth coagulation factor was used as the control group, and the negative control group was extracted from As a comparison, the milk of non-transgenic goat milk is compared. The experimental results are shown in Figure 8. The difference between Η-chain and L-Chain can be measured at the same time. A qualitative recombinant protein with a molecular weight between 80-250 kDa; non-transgenic goat milk has no antibodies. Example 5: Eighth in the milk of transgenic goats and transgenic female mice The coagulation factor coagulation activity test is to confirm the biological activity of human eighth coagulation factor white matter produced in transgenic milk.

The steps of the embodiment of the Western Rupture Method and EUSA

Juice, using the same quantitative analysis as before, and then at a dilution factor of 1 Q 24 200526781 to 120 times. The following table shows the release and coagulation activity of recombinant human eighth coagulation factor protein in the transgenic milk of individuals with different sets of genes and different offspring.

No. of transgenic mice Lactations and days (L / D) Coagulation activity assay (U / ml) a rFVIII concentration (pg / ml) Low copy 2roup (l-5 copies) aLAF15 (Fo) L1 / D7 4.82 ± 0.43 22.84 ± 1.65 (n-1) L1 / D12 7.11 ± 0.76 30.58 soil 1.37 L1 / D19 3.60 ± 0.51 19.87 soil 0.96 aLAF36 (FI) L1 / D7 6.38 ± 1.85 29.26 ± 3.58 (n-4) L1 / D17 5.64 ± 2.66 24, 92 persons 3.04 L2 / D7 13.41 persons 2.87 50.21 ± 5.73 L2 / D17 9.46 persons 2.04 40.84 ± 4.91 Middle copy sroupn〇-20 copies) aLAF18 (Fl) L1 / D7 0 · 98 persons 0 · 39 7.03 persons 2.56 (n-3) L1 / D12 6.14 ± 0.98 32.07 ± 4.30 L1 / D17 8.91 ± 2.15 41.37 i 7.78 aLAF28 (F2) L1 / D7 3.01 ± 0.95 9.23 ± 3.74 (n = 3) L1 / D12 6.55 persons 2.07 35.56 persons 6.08 L2 / D17 7.81 persons 2.53 38.11 ± 5.72 High copy sroup (40-50copies >) aLAF25 (FI) L1 / D8 11.28 ± 2.33 46.33 soil 4.57 (n-3) L1 / D15 8.72 People 1.79 32.92 i 3.97 L1 / D21 1 · 56 Soil 0.24 15.08 Soil 4.84 aLAF25 (F2) L1 / D8 7.92 Soil 2.01 39.29 i 6.89 (n = 3) L1 / D15 8.58 Soil 2.78 43.48 Soil 5.21 L1 / D21 1.22 i 0 .61 13.32 士 5.05 a One unit of rFVIII was defined as equivalent to the amount of human FVIII normally present in 1ml of plasma, approximately 200 ng. Results presented are the average of two independent assays

The blood coagulation activity unit was measured, and the results are shown in the table above. It was found that recombinant human eighth coagulation factor protein of varying concentrations can be measured in the breast milk of individuals with different sets of transgenic genes, as well as the mother, first, and second-generation transgenic mother mice, and their performance Will increase with lactation parity. The collected base 25 200526781 is the eighth in humans due to the right χ @ 3 in transgenic milk; the spinal factor Chunsheng _1, which is a factor contained in normal human plasma. ~ 50 · 2 ^ delete times; using the _ method to analyze the whole milk ^ chen activity twelve _… — higher than human small coagulation II = milk of human transgenic milk goats during lactation group human human eighth coagulation factor & And # I, release of heavy ^ ^ 卞 The blood spinning activity is also analyzed by a single-step blood government test (aPTT test), and normal human plasma with different dilution rates as standard samples () Household = Qu Jun, the standard for blood activity test. Zhi Zhi Zai Ru _ 9 is 4 transgenic milk goat milk = :, =, coefficient; Figure 9 (c) is the value of the first-generation gene. The method of measuring blood coagulation activity and blood suspicion of the blood clotting factor and the blood clot of the human sibling factor was successfully transfected two different forms of the human occlusive factor of foreign genes into mammalian goats, pigs, and young Mouse body = Genes transfected between feeding animals are sustained and stable. A large number of eggs are expressed. ^ ^ Has coagulation activity. 'Exogenous genes are only expressed in breast tissues. The normal physiological functions of transgenic tadpoles. Even the transgenic 2 of the mammary gland phenotype does not contain any additional anti-tolerance screening genes, the present invention has considerable progress, and furthermore, the mammals transgenic by the technology of the present invention produce The progeny also carry a transgenic exogenous gene, and their performance is no less than that of the parent who received the transgenic technology, further highlighting the advantages of the present invention. The above detailed description is a specific description of a feasible embodiment of the present invention, but this embodiment is not intended to limit the scope of the patent of the present invention, and any equivalent implementation or change that does not depart from the technical spirit of the present invention should be It is included in the patent scope of this case. As mentioned above, this case is not only technically innovative, but also can be used to enhance the above-mentioned multiple effects compared with Xi 26 200526781. It should have fully complied with the novel and progressive statutory invention patent requirements, and filed an application in accordance with the law. I urge your office to approve this patent application for invention, to encourage invention, and to have a sense of virtue. [Brief description of the figure] FIG. 1 shows the embedding of the human eighth coagulation factor transgenic gene on the genome of the genetically transgenic mice and the goats and the F1 progeny of the mammary gland phenotype vector of the present invention. Insertion pattern analysis: Figure 1A; Construction map of the mammary-specific phenotype vector and the complete gene code sequence of human eighth coagulation factor (7.2-kb); Figure 1B; Rapid detection of parental gene transgenic mice, NC represents the control group of normal mice; Figure 1C: Rapid PCR detection of parental and F1 offspring gene transgenic dairy goats, NC represents the control group of normal dairy goats; Figure 1D: Human eighth coagulation factor gene in gene transfer Southern blot analysis of the genomic insertion pattern of hereditary animals. Figure 2 shows the analysis of the number of sets of 17 parental gene transgenic animals of the present invention containing exogenous human eighth coagulation factor gene in the genome: Figure 2A: Gene transfer animals. Samples and standards relative Location map; the standard is diluted with the corresponding number of transgenic genes to construct and purify the quantification. The DNA samples of the transgenic animals are fixed with 10ug of DNA as the basis for quantification. Figure 2B: Parent gene Quantitative analysis of sl0t_bl0t of transgenic animals. After hybridization with radioactive probes, X-ray films were used for automatic radiographic development. The results showed that the number of inserts of transgenic genes was distributed between 2-50 sets per cell; 27 200526781 Figure 3 shows the construction of the recombinant human eighth coagulation factor gene (4.5-kb) deleted from the B-domain of the present invention in the breast phenotype vector and the sequence analysis of the nucleic acid sequence: Figure 2A. The breast phenotype vector The construction elements 'including the alpha lactalbumin gene promoter' secretory message peptide guide sequence, the B-domain deleted recombinant human eighth coagulation factor gene, and the adenine polymerization message sequence are 70.0-kb in length; three B: 16-aa leader sequence of the secretory message peptide of the oCS 1 glutenin gene in the breast phenotype vector; Figure 3C: Nucleic acid sequencing analysis showing the construction of a recombinant human eighth coagulation factor gene deleted by B-domain In this case, the gene's endogenous negative regulatory sequence B-domain is deleted, and the biologically-functional A- and C-domain are concatenated with a Ser-Leu amino acid sequence; Figure 4 shows the gene conversion of the present invention. Detection of B-doma i η deleted recombinant human eighth coagulation factor gene contained in genomic milk of goats and mice: Figure 4A: Map of transgenic genes; Figure 4B: Map of transgenic mice pcr rapid detection and analysis, using the promoter and hFVIII front-end junction region, hFVIII AC domain junction region as primer amplified fragments to verify; Figure 4C: Southern genome of transgenic dairy goats and non-transgenic sheep genomes Blot analysis showed that the transgenic dairy goats contained 1-2 additional heterozygous fragments of genes, respectively. Figure 5 shows the Bboma i η deletion and recombination contained in the transgenic milk goat of the present invention 28 200526781 Human eighth coagulation factor has the ability to pass the gonads to F1 progeny; after PCR amplification, it is then sequenced by automatic nucleic acid Analysis technology verified that the first offspring of the transgenic dairy sheep still carried the same form of foreign genes as their parents. Figure 6 shows the breast-specific expression vector system of the present invention in gene transgenic animals, milk, / month, showing the specificity of breast tissue specificity and physiological stage specificity, and the regulation of human 苐 eight clotting factor genes: Figure A uses rt-PCR to analyze the human heart coagulation factor recombinant gene's difficult expression. It is strictly controlled in the lactation of breast tissue for transcription, but in other tissues such as heart, liver, lung, muscle, brain, Pancreas and others will not show; Figure 6B: Analysis of the staged performance of transgenic mRNA, showing the continuous performance of the #hFVIII recombinant gene can be measured in the pre-lactation, middle and late stages. ® is a recombinant human human "clotting 0 · protein" that expresses secretion in the milk of transgenic animals during lactation when the breast-specific expression vector system of this test X Ming shows: Figure 7A: Analysis of human antibodies using anti-hFVIII polyclonal antibodies The protein expression of the eight clotting factor recombinant gene has been analyzed by Western analysis to show that the heterogeneous recombinant protein of H-chain and L-chain can be detected at the same time. Figure 7B: Antl-hFVIII monoclonal antibody is used to analyze human eight clotting factor recombination. The results of gene protein expression showed that the 80-His L-chain recombinant protein molecule can be completely eliminated. 29 200526781 ® The eight-line specific breast-specific expression vector system of the present invention is expressed in the milk of genetically transgenic milk goats during lactation. Releasing secreted recombinant human human coagulation factor VIII protein. Using anti-hFVIII antibody to analyze the protein expression of human recombinant coagulation factor VIII gene, the results show that Η-chain and L-chain heterogeneous recombinant proteins can also be measured at the same time. Releasing in goat milk. Figure 9 shows the performance of the transgenic dairy goat of the present invention in milk during lactation and the release of recombinant human human Analysis of coagulation activity possessed by eight clotting factors. Figure IX. A single-step blood agglutination test (apTT 忭, normal plasma with different dilution rates as standard samples); Figure IX is the standard curve and correlation coefficient of coagulation activity test; Nine C: The value of the coagulation activity of human eighth coagulation factor secreted in the milk of the first-generation transgenic milk goat. [References]

[1] Fujio et al., 1996 [2] Evgueni et al., 1998 [3] Alexander et al., 1993 [4] Leon et al., 1994 [5] William et al., 1984 [6] Phililp et al., 1991 30 200526781 [7] Rekha et al., 1997 [8] Evgueni et al., 1998 [9] Palmer TD et al., 1995 [10] High KA et al., 1996 [11] Kaleko M et al., 1998 [12] Niemann et al., 1999

[13] Paleyanda et al., 1997

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Claims (1)

200526781 Scope of patent application: A method for continuously expressing foreign proteins with special biological functions in the milk of transgenic mammals, including the following steps: a. Constructing genes containing special biological functional proteins and capable of Manifested in mammalian mammary phenotype vectors; and b. Prokaryotic embryo gene microinjection of mammary phenotype vectors with special biological function protein genes and transfer of embryos to a mammal to make foreign proteins with special biological functions Expressed in the milk of the transgenic mammal «milk animals, and can continue to perform during lactation. Bu Ru, the scope of patent application! The method for the continuous expression of a foreign protein with special biological functions in the milk of a genetically modified 嗔 milk animal according to the above item, wherein v step a · contains a gene of a special biological function protein and can be expressed in a mammalian mammary gland phenotype vector, The lines include: c. 5 promoter sequences of gene promoters, which have the ability to regulate the lactation stage and the exclusive expression of the mammary gland, and can regulate the foreign protein genes to maintain stable performance during the lactation period of transgenic mammals ; And d. Protein shellfish secretion guide sequence, which has the ability to guide mammary epithelial cells to express foreign proteins in the lactation stage of transgenic mammals, which can be efficiently released into the vesicles and collecting ducts, and e. a foreign protein gene, which is located after the gene promoter's secretory message sequence and can be regulated and expressed; and 32 200526781 f. a gene 3 'regulatory sequence, which is located after the foreign protein gene code sequence and contains a A message sequence for adenine polymerization. The method for continuously expressing a foreign protein f with special biological functions in the milk of a transgenic mammal, as described in item (i) of the scope of the patent application, in step b. Exogenous protein gene lines in beans can be transformed with the gene Crohn's milk animals reproduce sexually and are passed on to their offspring. 4. The method for continuously expressing foreign proteins with special biological functions in gene transfection and animal milk as described in item i of the patent application scope, wherein step b. The foreign protein gene line includes human eighth coagulation factor is complete Genotype and B-dQmai_ divided by recombinant human eighth coagulation factor gene. The method for continuously expressing foreign proteins with special biological functions in the milk of transgenic mammals as described in item 4 of the scope of the patent application, in which the expression vector construction of the recombinant human first and second hematopoietic factors with B-domain deletion is performed 'Departs from the negative negative dragon sequence material to remove and biologically-A- and C-domain reservations 忭 W with Ser-Leu amino acid sequence in series. For example, the method of patent application No. 2 includes the method of continuously expressing foreign proteins with special biological functions in the milk of transgenic mammals, in which the gene promoter 5 of c. Regulates you _ ^ J is the alpha-lactalbumin promoter of high-yield Dutch dairy cows. 200526781, as described in item 2 of the scope of the patent application, the method of continuous expression of exogenous proteins with special biological functions in the milk of transgenic mammals, the guidance of the protein release information of the mammary gland of casein gene. The sequence is the α-lactalbumin gene or α S1-sex release message sequence of high-yield Dutch dairy cows. 'As described in item 2 of the scope of the patent application, a method for continuously expressing a foreign protein with a special biological function in the milk of a U gene transgenic mammal, wherein the e · foreign protein gene line includes the human VIII clotting factor intact 7.2kb gene Type and 4.5-kb recombinant human eighth coagulation factor gene in B-d⑽ai. 9 10 The method for the continuous expression of exogenous proteins with special biological properties in the milk of transgenic milked animals as described in item 2 of U Li Gao Wai, gene 3 of f., The regulatory sequence, is located in the exogenous After the dense sequence of the protein gene, a nitroglycerin polymerization message sequence containing a rutin-1 gene was used to stabilize the integrity and modification of the niRNA molecule transcribed from the foreign gene. 'As described in the scope of the patent application 箆 1 jg &, +, and 1 described in the method of the continuous expression of a biologically modified At + 4 foreign protein in the milk of transgenic mammals, wherein step b · The transgenic feeding of human genes is as follows: The concentration of human eighth coagulation factor in the milk of a private body can reach 20-50 g / L and its hemolytic activity can reach 13 times the coagulation efficiency of normal human plasma. . 34 200526781 η, as described in item j of the scope of the application for patent, continued to perform a special bio-technical plant due to transgenic taste milk animal milk human step b. The gene transgenic feeding -r and release exogenous protein. It is continuous performance during lactation. 12 13 Second, the scope of patent application for the transgenic mammalian milk as described in item 11: Medium continuous performance with special M function. Exogenous eggs: during lactation, including pupa period, Chinese food. The method for expressing foreign proteins with special biological functions in the milk of transgenic mammals as described in item U of the scope of the patent application, wherein the transgenic mammals are suckling goats and mice . 14, ^ please patent scope! The method for continuously expressing a foreign protein with a special biological function in the milk of a genetically modified mammal according to the item, wherein the genetically transformed mammal of step b · is a cow, a sheep, a pig, a rabbit, a rat, or a mouse. 15 As described in item i of the patent application, the method for continuously expressing a foreign protein f with special biological functions in the milk of a genetically modified mammal can further include the following steps after step b: Collect the genetically transgenic mammal The milk of animals containing foreign proteins with special biological functions; and the purification and isolation of foreign proteins with special biological functions from milk to obtain foreign proteins. 35 200526781 1 6. A method for producing a recombinant protein that continuously expresses human eighth coagulation factor in the milk of a transgenic mammal, which purifies and isolates the recombinant human eighth coagulation factor pharmaceutical protein from milk and can be applied to hemophiliacs For medical use. 36