CN1687425A - Bioreactor of animal mammary gland for producing recombined human milk ferritin-method of transgene cloning great cattle for human milk ferritin - Google Patents

Abstract

The production of animal mammary gland biology reactor for producing recombined human milk ferritin -- a method of transgenic cloning great domestic animal by human milk ferritin, it's operating steps: 1. We use hLFBAC DNA with complete gene of ferritin of human whey as the differential expressing carrier, 2. We mix hLFBAC DNA and double or single marked choosing carrier at a reasonable rate, and inject it to the core of animal cell, then obtain the transgenic cell which transmit offering gene in it, 3. The cell colons body cell by itself as the offering, then obtain the animal which has the gene of hLFBAC DNA. This recombined ferritin of animal mammary gland cloned transgenic can be developed to be health care product or drug, the milk containing recombined ferritin can also be developed to be heath milk of milk product with additional value.

Description

Produce animal mammary gland bioreactor---the method for human lactoferrin transgene cloning great cattle of restructuring lactoferrin

Technical field

The present invention relates to bioengineering field, particularly relate to transgenosis-clone technology field.

Background technology

Study on Transgenic Animal be human according to the wish of controlling oneself have purpose, planned, with good grounds, have pre-insight to change the genetic composition of animal, and the purpose that changes its genetic composition is diversified.Wish that such as the geneticist observing its phenotype by the genetic composition that changes animal changes, the physiologist wishes to study by the expression of specific gene the influence of this gene pairs organism physiology situation.Producing relevant transgenic research with animal then wishes to give animal new phenotypic character by transgenic technology.The research relies on molecular biology, animal embryo and gamete operative technique on experimental technique.

The making method of transgenic animal mainly contains the microinjection of pronuclear-stage embryos at present, retroviral infection is grown early stage animal embryo, the sperm vector method, the ES cell technology, the PGCs technology, somatic cell nuclear transfer technique, retroviral vector infects the ovocyte of MII phase, and sperm head and DNA merge injection ovocyte method.Improvement on these methods has promoted the process that Study on Transgenic Animal is transformed to production practice by the laboratory widely with raising.

Wherein traditional and the most the most frequently used method is the microinjection of pronuclear-stage embryos, and this method is one of method of using at present the making transgenic animal relatively more extensive, that effect is more stable by American Gordon invention.Promptly under the micrurgy instrument, foreign DNA is injected in the protokaryon of fertilised non-human eggs cell by a glass capillary, again this fertilized egg cell is transplanted into recipient cell intrauterine, when zygote is divided, foreign DNA may be integrated into the host chromosome group, treats that this development of fertilized ova maturation can obtain transgenic animal.But the efficient of the breeding transgenic livestock that microinjection obtains is extremely low, ox particularly, and sheep and pig etc. often are lower than 1%, and this will increase the cost of making breeding transgenic livestock greatly.

Along with the development of somatic cell clone technique, it is to become transgenic animal that gene manipulation techniques combines with clone technology, particularly the main mode of big Livestock Production.At present, the major progress that obtains is transgenic technology and the application of target position operative technique on cloned animal.At first, utilize the clone to carry out transgenosis and be meant before nuclear transplantation, earlier the fusion gene of goal gene and marker gene is imported the somatocyte of cultivating, screen genetically modified positive cell and clone thereof by the performance of marker gene again, and then transplant.1997, the scientist Schnieke of Britain PPL company and the Wilmut of Roslyn institute etc. were jointly by the somatic cell nuclear transfer technique Transgenic Sheep that takes the lead in having made in the world.Utilize fetal fibroblast system,, clone again through after the transfection.Transfected foreign gene contains the complete coding region and the β-BLG gene promoter of people's plasma thromboplastin component gene, and plasma thromboplastin component can be efficiently expressed, and comprises the plasma thromboplastin component albumen of 125 μ g in every milliliter of milk.Utilize the clone to carry out transgenosis, in case the nuclear transplantation success, theoretically, the transgenosis success ratio is 100%.The method of procaryotic injection can make a large amount of non-transgenic embryos by pregnancy, and this is a kind of wasting of resources, can not cause the surrogate mother to go conceived non-transgenic animal and utilize the clone to carry out transgenic technology.In addition, when utilizing the clone to carry out transgenosis, the sex of transgenic animal can be determined in advance.Like this, if expect can be in mammary gland marking protein, the transgenic animal that just can make the clone are jenny entirely.

Because the animal transgenic technology can change the production traits of animal according to people's wish, obtain the needed product of people, particularly some albumen medicine and healthcare products, scientists has begun the animal transgenic technology is applied in the middle of the practice, at present the animal transgenic technology mainly contains following application: (1), promote growth of animal, improve the output of live-stock product, improve product quality, (2), animal disease resistant breeding, (3) set up the animal model of diagnosing and treating human diseases, (4) produce pharmaceutical protein.

Animal mammary gland bioreactor is a kind of animal transgenic technology proteic technology such as express polypeptide medicine, industrial enzyme, vaccine and antibody in mammary gland cell of utilizing.The function of transforming mammary gland by engineered method helps the mechanism that we further study mammary cancer, improves the nutritive ingredient of milk, even can synthetic drugs.This technology has the low characteristics that drop into high production, and its efficient is 100 times that utilize with intestinal bacteria and animal cell culture technology, is a kind of very potential new and high technology.1987, people such as Simons successfully expressed sheep lactoglobulin gene first in transgenic mouse milk, and the protein content in the mouse milk sample approximately is that animal cell expression is proteic more than 400 times up to 23 grams per liters.This technology is once swift and violent development occurring just obtaining.At present, cow's milk albumin gene people organizes the profibr(in)olysin factor, and human growth hormone gene, human body antitrypsin gene, genes such as human urokinase gene and human interferon gene all obtain expressing in the mammary gland of mouse.Various famous scientists thinks that this will be the unprecedented revolution of livestock industry, may bring huge economic benefit to society.

The superiority that galactophore biological reactor is produced medicine allows pharmaceutical protein genetic expression at a certain privileged site, rather than expresses arbitrarily at health randomly.Scientists is consistent to be thought, with regard to medicine, it is mammary gland that ideal is expressed the place.Mainly contain following reason: 1, animal's mammary gland is the system of a sealing.The mammary tissue expressed protein overwhelming majority can not got back to blood circulation, the harm of so just avoiding the extrinsic protein confrontation animal health of great expression to cause.2, mammary tissue is a kind of effective protein proteins matter synthesizer.One cow head can be produced milk-protein 250～300kg in 1 year, a sheep and goat can be produced milk-protein 25～30kg, even a rabbit, annual galactopoiesis albumen also can reach 3～5kg, if centesimal milk-protein synthesis of medical albumen, its output is just very considerable.3, mammary tissue can be carried out correct modification and post-treatment to human body protein, and the biologic activity of product is near natural product.4, can heredity in the animal's mammary gland expressed exogenous gene.Produce certain valuable proteinic animal individual in case obtain one, can be with conventional livestock technology propagation production colony, though investigative technique is complicated, costly, its amplification process of researching and producing is easier to 5, the shortening new drug goes on the market the cycle.From drug manufacture construction cycle aspect, present a kind of new drug is examined from its development, medicine, up to listing, and whole process need 10～15 years, if utilize the galactophore of transgenic animal reactor, about 5 years of the cycle that new drug is produced.6, can obtain huge economic profit.Produce lactoferrin as Dutch golden hair horse company with transgenic cattle, estimate that annual to extract the Nutrious milk powder sales volume from milk be 5,000,000,000 dollars.

Utilize the method for galactophore biological reactor to transform milk cow, milk goat, make the milk composition of producing be similar to people's milk, both nutritious function, medicinal function is arranged again, this milk can make child look tall and big, promote brain and neurodevelopment, raise immunity, this new health care product necessarily has wide future.The nutrition or the physiological and biochemical property that improve milk also are that people wish one of target that realizes by the genetic composition that changes animal, promptly make so-called nutritional medicine (Nutraceuticals).Domestic animal milk and converted products thereof can provide human 30% nutrient protein, contain abundant indispensable amino acid, calcium and inorganic phosphate, and the very high casein of digestibility, are human high-quality source of nutrition; But breast is also not fully up to expectations on quality with domestic animal milk.Therefore, people are studying always and how to improve milk quality.Since Gordon foundes the animal transgenic technology, all try to be the first and carry out transgenic breeding and correlation technique research thereof in countries in the world.Studies show that foreign gene can not only obtain integrating and express in transgenic animal, and can obtain tissue specificity (mammary tissue, uterine tube) and development-specific expression, and can utilize molecular engineering to find all genes of regulating milk-content now.

The protein content of cow's milk is than the height of human milk, and cow's milk is 33g/L, and human milk only is 10g/L.Wherein the most important thing is protein content of whey in the human milk (68%) than casein (32%) ratio height, have immunocompetent lactoferrin (15%) and N,O-Diacetylmuramidase (4%) content height, and lack beta-lactoglobulin and κ-casein.Yet, containing higher various caseins in the cow's milk, corresponding protein content of whey is less.Beta-lactoglobulin in the albumen cow's milk and ALA are main anaphylactogens, newborn infant about 7.5% has stomach, skin and respiratory hypersensitivity reaction, this is expected to by transgenic technology sealing, disappearance beta-lactoglobulin gene or inserts people lactoprotein's gene and regulate its expression level in bovine mammary cell, thereby make cow's milk more near human milk, satisfy consumer demand.Animal bioreactor is one of focus of transgenic research in the present world wide, not only national governments' investment, and some private financial groups do not stint yet and drop into substantial contribution and researched and developed.

Human lactoferrin (Human lactoferrin is a kind of glycosylated protein that belongs to iron-binding protein family HLF), finds first by Sorensen, and by Blanc and Isliker name.Lactoferrin can reversibility ground in conjunction with two iron ions, its ionic bond intensity is 300 times of transferrin (in vivo iron being played the major protein of transportation effect).Human lactoferrin is made up of 692 amino acid, and molecular weight is about 80,000Da.Two foliation structures with iron ion binding ability are arranged in lactoferrin, and one is amino petiolarea, i.e. N-leaf (N-lobe), and another is the carboxyl petiolarea, i.e. C-leaf (C-lobe).There is 40% homologous amino acid sequence in two intervals.Each district can be in conjunction with an iron ion, simultaneously in conjunction with a dicovalent carbon acid radical anion.Human lactoferrin is modified by the glycosyl that the N end connects.Through enzymatic reaction, oligomerization glycosidic linkage with covalent bonds on tactic l-asparagine acid residue with Asn-Xaa-Thr/Ser.Human lactoferrin have three may be by glycosylated site, lay respectively on the 479th, the 624 l-asparagines acid residue of the 138th l-asparagine acid residue, N leaf of C leaf.In three sites, be positioned at 138,479 l-asparagine acid residue preferentially by glycosylation, the glycosylation of the 624th l-asparagine acid residue is subjected to the glycosylated restriction of the 479th l-asparagine acid residue.Lactoferrin extensively is present in the secondary granule of mammiferous body fluid and neutrophilic granulocyte, and the physiological function that lactoferrin has mainly comprises: the antibacterial of wide spectrum, anti-inflammatory function and immune physiological regulation function.These functions are directly obtained iron ion by lactoferrin or are combined in cell surface and make the somatic cells permeability increase realization.Lactoferrin can be in conjunction with the iron ion in zone that is inflamed, thereby stops the free iron ion to participate in the deleterious oxidizing reaction of catalysis.Lactoferrin also can influence the amplified reaction of T cell simultaneously.Some studies show that lactoferrin anti-microbial effect in vivo is more than external strong.This may be owing to exist antibacterial and potential immunoloregulation function district on the lactoferrin.In vivo, lactoferrin is brought into play antibiotic and immunoregulation effect by combining with intravital cell.The performance of many physiological functions of human lactoferrin depends on and the combining of specific cell acceptor.Have now found that the human lactoferrin acceptor is present in various kinds of cell, as intestinal cells, thrombocyte, Mammals epithelial cell, lymphocyte etc.Other human lactoferrin potential function also comprises the adjusting that medullary cell is generated, the generation of aerobic metabolism, somatomedin effect, DNA combined function, and potential RNA enzyme function.But some result of study about the lactoferrin function is controversial.Its some biological function is also fuzzy, still awaits further research work and illustrates.

Lactoferrin has widely biologic activity, and it mainly shows: (1) .. is to the effect of baby's iron metabolism.Some description of test lactoferrins can promote baby's iron to absorb, and find then that in rhesus monkey and people's experiment absorption does not have obvious facilitation to lactoferrin to iron.(2). anti-microbial effect.Lactoferrin is the intravital a kind of wide spectrum antimicrobial proteins of people, to pathogenic bacterium, and fungi, protozoon, virus all has lethal effect.(3). the effect in inflammatory reaction.Lactoferrin can be regulated inflammatory reaction by multiple different approach.A. regulate the complement activation approach.Lactoferrin can be blocked the C3 conversion enzyme in the classical pathway, makes complement factor C3 deposition, thereby stops the erythrocyte splitting of complement-mediated.B. the deferrization lactoferrin can be consumingly in conjunction with free iron, thereby limited the generation of active oxygen radical, and further suppressed the infringement of active oxygen radical pair cell, stops the peroxidation of cytolemma lipid.C. lactoferrin can mobilize the polymorphic nucleus monocyte to migrate to inflammation part fast.D. lactoferrin can influence the generation of the various kinds of cell factor, and these cytokines are being brought into play epochmaking effect in inflammatory reaction.Experiment in vitro shows that lactoferrin can suppress lipopolysaccharide-induced monocyte and produce IL-1, IL-6 and TNF-α, and the minimizing of IL-1 has caused the minimizing of GM-CSF, has further suppressed the generation of medullary cell.In addition, lactoferrin can strengthen the secretion of human polymorphonuclear leukocyte to IL-8.E. lactoferrin can with combined with lipopolysaccharide.Its combining site is positioned at the contiguous zone of N end 1-5 and two locus of 28-34.Lactoferrin combines lipopolysaccharides with the lipopolysaccharide binding protein competition, has therefore reduced the lipopolysaccharides of lipopolysaccharide binding protein mediation and combining of CD14, plays the effect of regulating inflammatory reaction.(4). other effect.In the brain of suffering from the Parkinsonism mouse, the enhancing that detects lactoferrin is expressed, and lactoferrin certain effect of performance in body defence Parkinsonism is described.This external many autoimmune diseases patient has detected the antibody of lactoferrin on one's body, as rheumatoid arthritis, vasculitis, systemic lupus, elementary sclerosis vasculitis, illustrates that lactoferrin tool in these treatment of diseases and diagnosis has certain effect.Recently, there is the people to find that also lactoferrin can suppress the growth and the transfer of tumour cell.

The multi-functional attribute of human lactoferrin has caused the research of people to its encoding gene, so that on this basis this albumen is carried out scale operation.People such as people such as M.J.Powell and M.W.Rey have delivered the cDNA sequence and the portion gene group sequence of human lactoferrin first respectively in nineteen ninety.Studies show that afterwards, human lactoferrin are positioned at the position of people's No. three karyomit(e) q21-q23, and the about 35kb of total length has 17 exons and forms, and the size of intron is between 300bp-3.3kb.Based on the research of human lactoferrin gene, many investigators begin to be devoted to the genetically modified research of human lactoferrin, to understand by the possibility of genetically modified mode mass production restructuring lactoferrin and the character of restructuring lactoferrin.The final purpose of human lactoferrin transgenic research is to produce restructuring lactoferrin identical or close with human lactoferrin on a large amount of functions by transgenic technology, makes it to benefit for the mankind.

The past people were the host with fungi, yeast, plant, animal cell line, animal once, had carried out the human lactoferrin transgenic research.Following table has been summarized the situation of this aspect research.

The overview of table 1 human lactoferrin transgenic research

Table?1???????The?outline?of?the?HLF?transgenic?study

Transgenic???????????hLF??????????????Promoter?????????????rhLF?Expressing????????????????Published?year?&

host?????????????????sequence??????????????????????????????level??????????????????????????author

Fungi????????????????2.3kb????????????300bp????????????????5μg/ml(total)?????????????????1992，Pauline?P.

(Aspergillus?????????cDNA?????????????A.nidulans?alcA??????1.5μg/ml(secreted)????????????Ward，et?al

nidulans)?????????????????????????????promoter?????????????(ELISA?analysis)1

Fungi????????????????2.1kb????????????1.1kb?????????????????2,000μg/ml(secreted)?????????1995，Pauline?P.

(Aspergillus?????????cDNA?????????????glucoamylase?gene????(ELISA?analysis)???????????????Ward，et?al

awamori)??????????????????????????????promoter

Yeast????????????????Full?length??????Yeast?????????????????20-30μg/ml(total)????????????1993，Qianwa

(Saccharomyces???????cDNA?????????????chelatin?promoter?????1.5-2.0μg/ml?????????????????Liang，et?al

cerevisiae)?????????????????????????????????????????????????(secreted)2

Baby-hamster?????????2.3kb?full???????MT-1??????????????????20μg/ml(secreted)????????????1991，Stowell

kidney?cells?????????length?cDNA??????promoter??????????????(Determined?by?the????????????K.M.，et?al

A280?of?the?pooled?column

fractions)

Tobacco?cells????????Full?length??????CaMV?35s??????????????0.6-2.5％?of?the?total????????1994，Amitava

(Nicotiana?tabacum???cDNA?????????????promoter??????????????cellular?protein??????????????Mitra，et?al

L.)

Tobacco??????????????2.1kb?full???????DP35ScaMV????????????(1)0.1％?of?total??????????????1998，Valérie

Plant????????????????length?cDNA??????promoter?????????????extracted?leaf?proteins????????Salmon，et?al.

(2)0.3％?of?total

extracted?leaf?proteins3

(ELISA)

Potato?plant?????????2.3kb?full???????1.Mas?P2?????????????(1)0.01％of?total??????????????2000，Daniel?K.X.

length?cDNA??????promoter?????????????soluble?protein????????????????et?al

2.CaMV?35s???????????(2)0.1％of?total

promoter?????????????soluble?protein4

(ELISA?analysis)

Mice?????????????????Full?length??????7.7kb????????????????0.1-36μg/ml???????????????????1994，Gerard

cDNA????????????asl-casein???????????????(RIA?analysis)???????????J.Platenburg，et?al

promoter

Mice???????????Genomic?????????6.2kb????????????????????＞1,000μg/ml????????????1993，De?Wit，

sequence????????asl-casein?gene???????????????????????????????????I.C.M，et?al

promoter

Mice???????????1.hLF???????????asl-casein???????????????400-1,300μg/ml??????????1997，Jan?H.

cDNA????????????gene?promoter????????????300-3,800μg/ml??????????Nuijens，et?al.

2.hLF???????????????????????????????????(RIA?analysis)6

genomic

sequence

Mice???????????2.5kb?hLF???????2kb?bovine???????????????1-200μg/ml&7????????????1994，1997，Sun

cDNA????????????β-casein?gene??????????(ELISA?analysis)??????????Jung?Kim?et?al

promoter

Mice???????????27kb????????????10kb?bovine??????????????60-6,600μg/ml8??????????1999，Sun?Jung

genomic?????????β-casein?gene??????????(ELISA?analysis)??????????Kim，et?al

sequence????????promoter

Cattle?????????Genomic?????????6.2kb????????????????????300-2,800μg/ml9?????????2002，Patrick?H.C.

sequence????????asl-casein?gene?????????(ELISA?analysis)??????????Van?Berkel

promoter

In the research in the past, human milk iron egg cDNA once was widely used in all kinds of transgenic researches.This mainly is because human lactoferrin cDNA than the easier acquisition of genom sequence gene, because the cDNA sequence is shorter, also is easier to be built in the expression vector go.Simultaneously, cDNA can be used for procaryotic expression, and it is used in the transgenic research earlier.Reported first in 1993 such as Qianwa Ling the research in yeast, expressed of restructuring lactoferrin.In this research, human lactoferrin and yeast sucrase signal sequence district are used to guide the secretion of restructuring lactoferrin respectively.The yeast that contains yeast sucrase signal sequence has higher restructuring lactoferrin expression amount, and the yeast that the contains the human lactoferrin signal sequence then expression amount of restructuring lactoferrin is very low.In fungi, the Aspergillus bacterial classification is secreted glycosylated protein under field conditions (factors), and this attribute makes it become a kind of host of ideal express recombinant glycosylated protein, and it has been used for commercially producing of glycosylated protein now.Useful human lactoferrin gene transforms the report of Aspergillus nidulans and Aspergillus awamori.In these two researchs, the promotor of the gene of high expression level is used to guide the expression of restructuring lactoferrin in the host.In Aspergillusawamori, human lactoferrin cDNA and link together at the glucoamylase gene that Aspergillus awamori efficiently expresses middlely separates with the KEX-2 cleavage site.Through translation, fusion rotein cuts with KEX-2, forms independent restructuring lactoferrin.In this research, in the transformant of express recombinant human lactoferrin, introduce sudden change, can obtain the expression amount (2,000 μ g/ml) of higher restructuring lactoferrin.This expression amount be up to now in the human lactoferrin transgenic research of yeast, fungi, clone the restructuring lactoferrin expression amount the highest.This may be because adopted the method that merges to carry out transfection also the cause of Aspergillus awamori as transformed host.According to the result of former studies, the expression level of reorganization human lactoferrin in yeast, fungi, clone in the used host of transgenosis, promotor and the remarkably influenced of secretion signal region sequence.Owing to the effect of human lactoferrin antimicrobial, influenced the normal growth of some yeast and fungi, after cultivation, do not reach higher concentration, their growth is suppressed by the excretory restructuring lactoferrin.Like this, select the insensitive bacterium of restructuring lactoferrin bacteriostatic action is just seemed very important.When the promotor of transgenosis host's autogene and signaling zone sequence are used for transgenic research, tend to obtain the expression level of higher external secretion restructuring lactoferrin.Up to the present, the full gene of lactoferrin of also having no talent carries out the report of transgenic research in fungi and clone.Also has the further leeway of research in this respect.

1993, reported first such as Gerard J.Platenburg the human lactoferrin gene transgenic mice obtained success.The research in early stage this field only is confined to human lactoferrin cDNA.Discovering afterwards, the amount of transgenic mice excretory restructuring lactoferrin that carries human lactoferrin gene group complete sequence is far above the amount that carries human lactoferrin cDNA.The lactoferrin cDNA that choose respectively such as Jan H.Nuijens in 1997 are connected with the ALPHAsl-casein gene promoter with human lactoferrin whole genome sequence fragment and are built into the mammary tissue specific expression vector.The result shows that the expression scope of cDNA transgenic mice restructuring lactoferrin is 400-1,300 μ g/ml, and be 300-3 in the transgenic mice of the full gene fragment of genome, 800 μ g/ml.In the transgenic experiments that Sun Jung Kim in 1999 etc. carry out, show, use at the same time under the situation of Beta-casein as promotor, the lactation amount that contains the segmental transgenic mice of human lactoferrin gene complete sequence reaches as high as 6,600 μ g/ml, and the expression amount of the restructuring lactoferrin of the transgenic mice that obtains with cDNA is no more than 30 μ g/ml.In some cases, can obtain higher Recombinant Protein Expression amount as transgenic line with cDNA.CDNA[51 as human body protein C], people IGF-1cDNA and people EPO cDNA etc.But under the other situation, can not get the high expression level amount with the cDNA transgenosis.For this reason, have research adopted the pattern attached component (matrix attachment elements, MARs), locuscontrol region territory (locus control regions, LCRs), or yac clone etc. overcomes position effect, to obtain the recombinant exogenous protein of high expression level amount.Work in the past shows, has in the intron of some genes to exist controlling element forward or negative sense, regulating and control expression of gene.Human lactoferrin is genetically modified to be studies show that, exists important controlling element in human lactoferrin gene inside, the expression of human lactoferrin is being brought into play the regulating and controlling effect of forward.Still need now and further study with controlling element in investigator's lactoferrin intron having expressed the mechanism of regulating and controlling effect.In the past, genetically modified research mainly is confined on the mouse to mammiferous human lactoferrin, relates to big domestic animal is rare.The transgenic research that big domestic animal such as ox, sheep is carried out human lactoferrin will be next step developing direction.

Summary of the invention

The present invention is directed to the blank of above-mentioned prior art, plan genetic engineering technique, the cell transfecting technology combines with somatic cell clone technique, employing contains the BAC sequence that is about 150kb of whole person's bovine lactoferrin gene as transgenic structure, provide a kind of method of changeing the transgene cloning great cattle that human lactoferrin gene is arranged of producing, in order to the restructuring lactoferrin of scale operation biologically active.

Produce animal mammary gland bioreactor---the method for human lactoferrin transgene cloning great cattle of restructuring lactoferrin, its operation steps is as follows: (1) utilizes and contains the hLF BAC DNA of whole person's bovine lactoferrin gene as mammary gland-specific expression vector, (2) select carrier to be mixed in proportion hLF BACDNA and double alternative carrier or single mark, import in the animal somatic cell nuclear, carry out cell transfecting, acquisition changes the transgenic cell of hLF BAC DNA over to, (3) cell carries out somatic cell clone as nuclear donor, obtains to change the transgenic and cloned animal that hLF BAC DNA is arranged.

Described specific expression vector is the hLF BAC DNA that is about 150kb, and the human lactoferrin of expressing in the transgene mouse model of recombinant human milk-protein is consistent with natural human lactoferrin size, has and the identical immunocompetence of natural human Ruzhong lactoferrin.

The human lactoferrin gene expression amount of described transgenic mice is 0.5-7.8g/l.

Described double alternative carrier is pEGFP-NEO, and it is pNEO that described single mark is selected carrier.

It is 1: 3 that described hLF BAC DNA and double alternative carrier or single mark are selected carrier blended mol ratio.

Described introduction method is the micro-injection altogether of somatocyte.

Described transgenic cell is the positive cell that screens acquisition by G418.

Described heavy livestock is ox, goat, sheep, pig or rabbit.

Described heavy livestock is an ox.

Utilization of the present invention contains the HLF BAC that the is about 150kb (5 ' control region that comprises about 90kb of human lactoferrin gene, 3 ' the control region of the coding region of about 30kb and 30kb) as transgenic structure, obtain to change the transgenic mice that total length HLF BAC DNA is arranged by zygote protokaryon embryo microinjection, restructuring lactoferrin is at every heavy stably express that all detects of the female mouse mammary gland of the positive, expression amount is 0.5-7.8g/l, show and utilize complete human lactoferrin gene expression system can in other mammal galactophore, obtain efficient special expression, also show with the disconnected DNA of lengthy motion picture and can reduce the influence of position effect transgene expression.

Cell transfecting for large fragment DNA (more than the 100kb) rarely has bibliographical information both at home and abroad, and the present invention has also attempted a lot of methods, but because goal gene is oversize, adopts electric-shocking method and other cell transfecting method to carry out cell transfecting and be difficult to obtain positive cell.The HLF BAC DNA that the present invention will obtain efficiently expressing in mouse mammary gland and double alternative carrier pEGFP-NEO or single mark select carrier pNEO in molar ratio example (1: 3) mix, carry out cell transfecting, import in the animal somatic cell genome by micro-injection altogether, screen the acquisition positive cell by G418.Positive cell mono-clonal is again cultivated and amplification, and the transgenic cell that detects the HLF BAC NDA that 150kb is arranged through PCR just can carry out somatic cell clone, obtains the transgene clone ox.Detect through PCR and Southern, obtain the transgene clone ox that change at two the HLF BAC that contains the total length human lactoferrin gene altogether.According to restructuring lactoferrin expression in the transgenic mice, can infer that these transgenic cattles also will efficiently express the human lactoferrin of biologically active.The restructuring lactoferrin of these transgenosis cow mammary gland specifically expressings can be developed to healthcare products and medicine, and the milk that contains restructuring lactoferrin also can directly be developed to the nourishing milk and the milk preparation of high added value.

Utilize the gene regulating element and the goal gene of mammary gland specifically expressing to be built into mammary gland-specific expression vector, this carrier is imported the animal gene group, thereby acquisition can be an animal mammary gland bioreactor the proteic transgenic animal of mammary gland specific high-efficiency expression exogenous medicinal.Mammary tissue can be carried out correct modification and post-treatment to human body protein, the biologic activity of product is near natural product, so animal mammary gland bioreactor is a kind of animal transgenic technology proteic technology such as express polypeptide medicine, industrial enzyme, vaccine and antibody in mammary gland cell of utilizing.Like this, transgenic and cloned animal just becomes " pharmaceutical factory ", for wide application prospect has been opened up in the exploitation and the production of medical protein and albumen healthcare products.

The present invention explores and perfect cell transfecting technology and somatic cell clone technique combine produces the approach of the transgenic animal of carrying the pulsating external source functional gene of complete length, and transgenic animal have greatly been improved, the make efficiency of the big domestic animal of transgenosis particularly, technological line used in the present invention are suitable for utilizing the disconnected DNA of this lengthy motion picture to carry out the underlying group production of transgenic cattle, goat, sheep, pig and rabbit and expand numerous as transgenic structure.

Description of drawings

Fig. 1: hLF transgenic mice Southern results of hybridization

Digitized representation mouse number, P represents positive control, and N represents negative control.

Fig. 2: hLF transgenic mice whey sample SDS/PAGE gel electrophoresis after the sex change of subtracting property damping fluid

Swimming lane 5,6 is the whey sample of hLF transgenic positive mouse, and swimming lane 7 is people's whey sample, and swimming lane 9 is the human lactoferrin standard, and swimming lane 10 is the molecular weight of albumen standard substance, and other swimming lane is a non-transgenic mouse whey sample.

Fig. 3: hLF transgenic mice breast sample western results of hybridization after the sex change of subtracting property damping fluid

L to 7 swimming lane is the whey sample of hLF transgenic positive mouse: 8 swimming lane behaviour whey samples, as positive control; 9 swimming lanes are non-transgenic mouse whey sample, as negative control; The 10th swimming lane is the molecular weight of albumen standard substance.

Fig. 4: the structure iron of double alternative carrier pEGFP-NEO

EGFP is for strengthening green fluorescence protein gene, and NEO is a neomycin resistance gene, and IRES is a ribosome bind site, and SalI, NotI, AscI and BamHI are restriction enzyme site, and CMV-IE Enhancer is the CMV-IE enhanser, and pEF321 is the EF321 promotor.

Fig. 5: single mark is selected the structure iron of carrier pNEO

NEO is a neomycin resistance gene, SalI, and NotI, AscI and BamHI are restriction enzyme site, and pPGK is the PGK promotor, and Amp is an ammonia benzyl resistant gene.

Fig. 6: the PCR of hLF transgene clone ox detects

M:100bp marker, A: be hLF 5 ' primer, B: be the hLF primer, C:hLF 3 ' primer .1 is the iron baby, and 2 is auspicious baby, 3 positive contrasts, 4 negative contrasts, 5 is blank.

Fig. 7: the Southern result of hLF transgene clone ox.1: the iron baby; 2: auspicious baby; 3: negative control; 4-6: the positive control that is respectively 1,5 and 10 copy.

Embodiment:

The present invention is described in further detail below in conjunction with embodiment.

Embodiment

1 experiment material

1.1 plasmid and bacterial strain

Host bacterium bacillus coli DH 5 alpha and DH10B, pIRES-NEO and pCMV-EGFP-IRES-NEO are available from Clontech company.

1.2 laboratory animal

The fetus at about 3 monthly ages of Holstein milk cow is taken from the slaughterhouse, and the ox ovary is from slaughterhouse, surrounding area, Beijing.

1.3 main agents

DMEM/F12 powder, DMEM powder culture medium, trypan blue (trypan blue), TCM199 powder, glutamine and G418 are Gibco company product: foetal calf serum is a Hyclone company product; DNTP and PCR primer are purchased in Shanghai bio-engineering corporation; The Taq enzyme is purchased in China Agricultural University Agricultural biotechnologies laboratory; Restriction endonuclease SspI and BamHI are Huamei Bio-Engrg Co.,'s product; IPTG, X-gal, penbritin (Amp), kantlex (Kan), keyhole limpet hemocyanin (KLH) are all available from magnificent biotech firm: saturated phenol is ancient cooking vessel state bio-engineering corporation product; Trypsinase, penicillin streptomycin, EDTA, Hepes, FSH, LH, 17 β-E2, EGF, Unidasa, HEPES, FAF BSA, cytochalasin B, Hoechest33342, cycloheximide, A23187,6-DMAP, D-N.F,USP MANNITOL, Sodium.alpha.-ketopropionate, heparin sodium, mineral oil and other inorganic salt etc. are Sigma company product.

1.4 culture medium preparation

SOB substratum: dispose every liter of substratum, should add at the 950ml deionized water: peptone: 20g yeast extract: 5g NaCl 0.5g. shakes the vessel solute and dissolves fully, add 250mmol/L KCl solution 10ml then, be adjusted to pH7.0 with 5mol/LNaOH.Sterilization.

The LB liquid nutrient medium: peptone 10g, yeast powder 5g, NaCl 10g is dissolved in the 800ml water, regulates pH value to 7.5, constant volume system 1000ml, autoclaving.

The LB solid medium: peptone 10g, yeast powder 5g, NaCl 10g is dissolved in the 800ml water, adds cosmetics 15g, regulates pH value to 7.5, is settled to 1000ml, autoclaving.

1.5 the preparation of reagent

The DMEM nutrient solution: DMEM powder 13.4g, NaHCO33.7g, penicillin 66mg, Streptomycin sulphate 100mg, Mill-Q ultrapure water constant volume to 1 liter, PH 7.0-7.4, osmotic pressure are 280-320mOsm/kg, with the sterilization of 0.2 μ m membrane filtration, 4 ℃ of preservations.Add 10% serum during use.

0.1%Trypsin/EDTA enzymic digestion liquid: Trypsin0.1g, KCl 0.04g, NaHCO3, NaCl 0.8g, EDTA 0.02g, the Mill-Q ultrapure water dissolving with sterilization is settled to 100ml, with the sterilization of 0.2 μ m membrane filtration ,-20 ℃ of preservations.

DPBS liquid: DPBS powder 9.7g, Mill-Q ultrapure water constant volume to 1 liter, PH7.0-7.2 is with the sterilization of 0.2 μ m membrane filtration, 4 ℃ of preservations.

No calcium magnesium PBS solution: KCl0.2g, KH2PO4 0.2g, NaCl 8.0g, Na2HPO4 12H2O 2.88g, Mill-Q ultrapure water constant volume to 1 liter, PH7.0-7.4, osmotic pressure are 280-320mOsm/kg, with the sterilization of 0.2 μ m membrane filtration, 4 ℃ of preservations.

The Gimsa mother liquor: Gimsa powder 0.5g adds a small amount of glycerine the Gimsa powder is fully ground in mortar.Adding glycerine to total amount again is 22ml, 56 ℃ of insulation 2h, and then add 33ml methyl alcohol, and be kept in the brown reagent bottle, standby.

Cell pyrolysis liquid: 10mM Tris.Cl (PH 8.0), 0.1M EDTA (PH8.0), 0.5%SDS.

The Proteinase K stock solution: Proteinase K 100mg, be dissolved in the 5mL aqua sterilisa, packing ,-20 degree are preserved.

TBS liquid: NaCl 8.0g, KCl 0.2g, Tris.Cl 3.0g is dissolved in the 1L redistilled water autoclaving.

G418 stores liquid: 1g G418 is dissolved in the 1mL 1mol/mL HEPES solution (PH7.0-7.2), adds Mill-Q10mL, filtration sterilization, and-20 degree are preserved.

Twice electric shock damping fluid (2 * HeBS): 280mMNacl, 10mMKcl, 1.5mMNa2HPO4,12mMGlucose, 50mMHepes, PH7.0-7.4, filtration sterilization, the 1mL packing ,-20 degree are preserved.

1Mol/L Tris.Cl (pH8.0): 121.1gTris alkali is dissolved in the 800ml water, transfers pH value to 8.0 with HCl, is settled to 1000ml, autoclaving.

0.5Mol/L EDTA (pH8.0): the 126.1g disodium ethylene diamine tetraacetate joins in the 800ml water, transfers pH value to 8.0 with NaOH, is settled to 1000ml, autoclaving.

RNase solution: RNaseA is dissolved in 10mMol/L Tris.Cl (pH7.5), among the 5mMol/L NaCL, is made into the concentration of 10mg/ml,, slowly cool to room temperature, be distributed into aliquot and be stored in-20 ℃ in 100 ℃ of heating 15min.

TE damping fluid: 10mMol/LNaCL, 10mMol/L Tris.Cl (pH8.0), 1mMol/L EDTA (pH8.0), autoclaving.

After 5mol/L LiCl:30.2g lithium chloride two water (LiCl.2H2O) are dissolved in 80ml water fully, be settled to the 100ml autoclaving.

50 * TAE:242gTris alkali, the 57.1ml glacial acetic acid, 100m10.5Mol/L EDTA (pH8.0) is settled to 1000ml after the dissolving.

Penbritin (Amp) stock solution: sodium ampicillin is dissolved in behind the aqua sterilisa with the filter filtration sterilization of 0.22 μ m, is mixed with 100mg/ml, be stored in-20 ℃.

3mol/L NaAc (pH5.2): the 24.604g anhydrous sodium acetate is dissolved in the 80ml water, regulates pH value to 5.2 with glacial acetic acid, is settled to 100ml, autoclaving.

1mol/L CaCl2: dissolve 54gCaCl2.6H2O in 200ml water, filtration sterilization is distributed into every part of 10ml, is stored in-20 ℃, takes out portion during the preparation competence and is diluted to 100ml, filtration sterilization, pre-cold standby.

The ethidium bromide stock solution: add the 1g ethidium bromide in 100ml water, preserve in 4 ℃ of brown bottles the dissolving back.

DNA extraction extraction buffer: 50mMol/L Tris.Cl (pH8.0), 100mMol/L EDTA (pH8.0), 100mMol/L NaCl, 1%SDS.

2 * Cracking buffer:0.2N NaOH, 0.5%SDS, 20% sucrose.

Phenol: imitative (1: 1): equal-volume phenol, chloroform mix, and are stored in 4 ℃ of brown bottles.

Proteinase K solution: water is made into 20mg/ml ,-20 ℃ of preservations.

TCM199 nutrient solution: claim TCM199 powder 9.9g, NaHCO ₃2.2g, Sodium.alpha.-ketopropionate 0.1375g, EDTA 0.372g is with 1L ultrapure water constant volume, PH7.2-7.4, malleation filtration sterilization, 4 ℃ of preservations.Cultivate with adding 10%FCS in the working fluid.

Operation liquid H199: in the TCM199 that contains 25mM Hepes, add 10%FCS.

Towards ovum liquid: add 10%FCS in the DPBS solution.

Unidasa: Unidasa 0.2g, being dissolved in 10ml does not have in the calcium DPBS solution, filtration sterilization ,-20 ℃ of storages.

Working fluid: use towards 10 times of ovum liquid dilutions.

Merge liquid: 0.3M N.F,USP MANNITOL, 0.1mM MgSO ₄, 0.05mM CaCl ₂, 0.5mMHEPES, 0.05%BSA transfers PH to 7.2-7.4, filtration sterilization.

Maturation culture solution: add 10%FBS in the M199 nutrient solution, 10u/ml FSH, 100U/ml LH, 1ug/ml estradiol, 100U/ml penicillin, 100ug/ml Streptomycin sulphate.

The CR1aa nutrient solution:

Composition final concentration (mM) add-on g/100ml

NaCl??????????114.7??????????????0.67

KCl???????????3.1????????????????0.023

NaHCO ₃???????26.2???????????????0.22

Na?Pyruvate???20.4???????????????0.002

Phenol?Red????1μl/ml????????????100μl

Above-mentioned composition is added 90ml ddH ₂Among the O, treat that all reagent thoroughly after the dissolving, add 0.055g Hemicalcium L-lactate (5mM).Adjusting pH is 7.4, uses ddH ₂O is added to 100ml, and osmotic pressure is between the 265-285mOsm.Filtration sterilization.

1.6 key instrument equipment

1) CO2 incubator: U.S. Forma Scientific Inc.

2) inverted microscope and fluorescent microscope: Japanese Nikon company

3) culture dish, culturing bottle, centrifuge tube and cell cryopreservation pipe: Nunc company

4) electric shock instrument: U.S. BTX company (ECM 2001)

5) electrophoresis apparatus: DYY-III2 voltage stabilizing electrophoresis apparatus, Liuyi Instruments Plant, Beijing

6) Bechtop: Beijing treating plant factory

7) ℃ Ultralow Temperature Freezer-80: Japanese SANYO company

8) ice-making machine: Japanese SANYO company

9) ultrapure water instrument: U.S. MILLIPORE company

10) refrigerated centrifuge: Eppendorf company

11) high speed freezing centrifuge: BECKMAN company

12) gel imaging system: ALPHA INNOTECH company

13) PHS-3C acidometer: go up marine rainbow benefit instrument plant

14) autosterilization pot: Japanese SANYO company

15) sequenator: ABI 377 type DNA sequencer, U.S. PERKIN ELMER company

16) water bath with thermostatic control instrument: U.S. LIFESCIENCE company

17) 9700 type PCR instrument: U.S. PERKIN ELMER company

18) ABI 377 dna sequencing instrument: U.S. PERKIN ELMER company

1.7 analysis tool software and network address:

Homology analysis software: DNAMAN

PCR primer-design software: Oligo6.0

Dna sequence analysis software: Chromas

DNA and protein sequence database: NCBI/GenBank/Nucleotides, Protein

2 experimental techniques

2.1 the preparation of BAC DNA and the foundation of transgene mouse model

2.1.1 hLF gene BAC positive strain

From the people BAC library of Genome Systems Inc, screen acquisition hLF gene BAC positive strain, used PCR primer such as table 2 by PCR.The skeleton carrier of BAC is pBeloBAC11, and its length is 7.35kb.Two NotI sites are arranged on it, be used to connect the external source fragment.HLF gene fragment length used in this experiment is about 150kb.Wherein 5 ' the distolateral wing sequence length is about 90kb, and 3 ' the distolateral wing sequence length is about 35kb.Show that through restriction analysis and order-checking this BAC fragment contains 17 exons at interior complete hLF encoding gene.

2.1.2 a large amount of extractions and the purifying of BAC

A large amount of extractions of BAC are described referring to " molecular cloning experiment guide " a large amount of extraction bacteriums from bacterium.BAC extracts after the NotI enzyme is cut, and electroelution reclaims behind the hLF gene fragment pulse electrophoresis after enzyme is cut.

Design a pair of primer, amplification hLF gene 3 ' end is to identify gene.Primer sequence is as follows:

HLF5????5’CCTGGGCAACAAAGCGAGAC?3’

HLF6????5’GCTATGGCTCCTTCTCTACTG?3’

The annealing temperature of PCR is respectively: 68 ℃.The length of PCR product is 1313bp.

2.1.3 microinjection prepares transgenic mice

The complete hLF fragment that reclaims prepares transgenic mice through microinjection

2.1.4 transgenic mice PCR and Southern identify

Identify that for the PCR that changes the human lactoferrin mouse we have designed a pair of primer, primer sequence and reaction conditions such as table 2 respectively at hLF gene coding region 5 ' end and 3 ' end;

Table 2: the PCR primers designed and the reaction conditions that change the human lactoferrin gene mouse

Primer name primer sequence product annealing temperature

Length

BAC70FSL????5’-TTGACTGGATCCCGTAGAGG-3’?????310bp??????60℃

BAC70FSR????5’-TCAGTCCAGGGTAAGGAGGA-3’

BAC70RSL????5’-CACTAAACTCATCGCCACCA-3’?????303bp??????60℃

BAC70RSR????5’-CACCTCTCCCTTTTCTTCCTG-3’

The detection of PCR shows, in 152 that obtain after microinjection former generation mouse, has 11 PCR tests to be positive, and positive rate is about 7%, and wherein 4 is public mouse.

Southern detects: the about 10 μ g of transgene clone ox DNA that get the PCR test positive digest with EcoRI, low pressure is electrophoresis at a slow speed, after changeing film, carry out Southern hybridization, hybridizing used probe is the hLF gene coding region fragment of the isotope-labeled 2.5kb of α-P32dCTP.

Result such as Fig. 1:

2.1.5 the Western of transgenic mice milk sample analyzes

Newborn sample to transgenic mice dilutes three times with heavily steaming aqua sterilisa, removes butterfat, goes caseic processing, obtains whey portion at last.Lactoferrin promptly is present in the whey.Fig. 2 is 2 μ l for applied sample amount, with the PAGE glue figure after the last sample Buffer of subtracting property (reducing) the SDS sex change.As can be seen from FIG., exist evident difference between the transgenic mice of the 5th, 6 swimming lane breast sample and the non-transgenic mouse breast sample.

Behind the PAGE gel electrophoresis of hLF transgenic mice breast sample with the last sample Buffer of subtracting property (reducing) SDS sex change, change film, carry out Western hybridization then and handle, obtain result as shown in Figure 3.Can get from Fig. 3, the rhLF in transgenic mice Ruzhong has consistent mobility with lactoferrin in the human milk of contrast.Detect through Western hybridization, F0 generation 14,19,20,44, No. 74 mouse, F1 generation 15, No. 55 mouse, totally seven female mouse are positive.

The result of rhLF125I radioimmunoassay reaction shows, the expression amount of the transgenic mice rhLF in 7 F0 generations and F1 generation is between 490mg/l to 7.8g/l.In F0 generation,, daughter's (No. 55) of a public mouse had the highest expression of recombinant proteins amount.

2.2 pEGFP-NEO and pNEO select vector construction

For the ease of the screening positive cell, we have at first made up and have contained green fluorescence protein gene (EGFP) and two selective marker carriers (pEGFP-NEO) of neomycin gene and neomycin gene list selective marker carrier (pNEO).

1) structure of pEGFP-NEO

Cut the segment that pBC1 reclaims about 6kb with NotI and SalI enzyme, connect one and contain SalI successively, the Linker of BamHI and NotI, be built into pXM, cut pCMV-EGFP-IRES-NEO and reclaim 4kb segment EGFP-IRES-NEO with BamHI and NotI enzyme, cut pXM with BamHI and NotI enzyme simultaneously, and be connected with EGFP-IRES-NEO and promptly constitute pEGFP-NEO, pEGFP-NEO such as Fig. 4.

2) structure of pNEO

Cut the segment that pBC1 reclaims about 6kb with NotI and SalI enzyme, connect one and contain SalI successively, the Linker of BamHI and NotI, be built into pXM, cut pIRES-NEO and reclaim 2kb segment IRES-NEO with BamHI and NotI enzyme, cut pXM with BamHI and NotI enzyme simultaneously, and be connected with IRES-NEO and promptly constitute pNEO, pNEO such as Fig. 5.

2.3 cell cultures, gene transfection and positive cell screening

2.3.1 it is foster that fetal fibroblast former is commissioned to train

In Bechtop, fetus is immersed in 30sec in 70% alcohol,, takes off the several piece face tissue of ear and ridge in the DMEM+10%FCS nutrient solution with the instruments of sterilizing with PBS rinsing several times.

↓

Fetal tissue's chopping is become the fragment of organizing of 1mm3 size in diameter 60mm plate, will organize fragment to be transferred in the 15ml centrifuge tube again, centrifuge washing for several times under the 1000rpm.

↓

With sterilization glass elbow suction pipe the small org fragment is drawn in the 25cm2 culturing bottle, tissue block is evenly distributed on bottle wall by the glass pipette elbow.

↓

Careful upset culturing bottle (preventing that tissue block from falling) allows tissue block attach on bottle wall, places 37 degree, places 2～4h in the 5%CO2 incubator, treat tissue block adherent after, just putting culturing bottle again, add an amount of nutrient solution and continue to cultivate.

2.3.2 the going down to posterity of fetal fibroblast, freezing, and recovery

Primary cell to be planted grows to 80% when converging, and a cell part is freezing, and a part goes down to posterity and continues to cultivate.

2.3.2.1 go down to posterity

Carefully absorb nutrient solution in the culturing bottle with sterilization glass elbow suction pipe, inject no calcium magnesium PBS (37 degree incubation) along the opposite bottle wall of cell growth wall and wash cell twice, to dispel remaining serum and cell metabolite.

↓

With add 0.05% trypsinase/0.02%EDTA Digestive system with quadrat method, add-on gets final product for covering cell monolayer that (diameter 100mm culture dish generally adds the 1ml Digestive system, add 200u L Digestive system in the 35mm culture dish), put into incubator and digest 1-2min.Examine under a microscope, treat that cell begins to become bowlder, beat the culture dish wall, make cell thoroughly take off wall, add nutrient solution then and stop digestion with finger.

↓

Blow and beat repeatedly with sterilization glass elbow suction pipe, cell makes its dispersion become individual cells, centrifugal 5min collecting cell under the 1000rpm.With nutrient solution by 1: 2 or 1: 3 dilution proportion and again inoculating cell to culture dish, cultivate.

2.3.2.2 it is freezing

After the attached cell digestion, collecting cell is in centrifuge tube, and centrifugal 5min is to remove most supernatant under 1000rpm.

↓

Add refrigerating fulid (DMEM+20%FCS+10%DMSO) the re-suspended cell precipitation of 4 degree precoolings, make cell concn be about 107 cell/ml, transitional cell is to frozen pipe.

↓

Put into 4 degree refrigerator precooling 30min, more frozen pipe is inserted on the thick foam, place the liquid nitrogen liquid level to fumigate 2h then, drop into liquid nitrogen then rapidly and preserve.

2.3.2.3 recovery

From liquid nitrogen, take out frozen pipe, drop into fast in the 37 degree water-baths, and in water-bath, constantly shake frozen pipe fast to thaw rapidly.

↓

After treating that refrigerating fulid thoroughly thaws, be transferred in the centrifuge tube with 10 times of fresh medium dilutions and with lysate, centrifugal 5min removes DMSO to extenuate refrigerating fulid toxicity under 1000rpm.

↓

With fresh nutrient solution suspension cell precipitation, cell dilution to desired concn, is continued to cultivate.

2.3.3 the transfection preparation of DNA

A large amount of extractions of hLf BAC are described referring to " molecular cloning experiment guide " a large amount of extraction bacteriums from bacterium.BAC extracts after the NotI enzyme is cut, and electroelution reclaims behind the hLF gene fragment pulse electrophoresis after enzyme is cut, and uses injection TE the hLF gene fragment dissolved dilution of purifying to 5ng/ μ l.Extract two marks simultaneously and select carrier pEGFP-NEO and single mark to select carrier pNEO, electroelution recovery through the NotI enzyme is cut after is used injection TE the marker gene fragment dissolved dilution of purifying to 1ng/ μ l.

2.3.4 bovine fetal fibroblast is to G418 toxicity sensitivity Detection

Cell cultures to the is used 0.25% tryptic digestion after 3 generations, by the dispersion so that cell is tried one's best in 0.5～1 * 105 cell inoculations to 13 in every hole diameter 35mm culture dish.Next day therein in 12 holes gradient with 100 μ g/ml add the G418 of final concentration successively from 100 μ g/ml to 1200 μ g/ml, also have in the hole not add G418 in contrast.In three weeks of cultured continuously, changed liquid once, and added G418 simultaneously in per 3～4 days.Every day, observation of cell was grown or death condition.

2.3.5 the micro-injection altogether of goal gene and marker gene

Cover glass after cleaning, sterilizing is placed in six orifice plates,, treat to take out when cell grows to 60-80% for the cytogene injection to reach the 3-6 fetal fibroblast inoculation culture in generation.

↓

Dilute hLF BAC and double alternative carrier plasmid pEGFP-NEO DNA or single mark respectively with injection TE and select vector plasmid pNEO DNA to 5ng/ μ l and 1ng/ μ l, and then the equal-volume mixing, make the injection concentration of hLF BAC and double alternative carrier be respectively 2.5ng/ μ l and 0.5ng/ μ l.

↓

Growth is had the cover glass of fetal fibroblast to move to fill in the diameter 100mm culture dish of operation liquid H199, with suction have mixed base because of entry needle gene is injected nucleus, extract entry needle rapidly after waiting nucleus to expand.

↓

Cell cultures behind the gene injection is after 2 days, and pair cell carries out the G418 screening, the positive cell mono-clonal is cultivated and the mono-clonal of amplification 2.3.6 positive cell is cultivated

Under fluorescent microscope, observe the luminous situation of aforesaid method gained cell clone, live good dispersion (away from other clone) and the many fluorescence clones of cell quantity with the marking pen circle; Absorb nutrient solution, with no calcium magnesium PBS rinsing cell secondary, under the high power anatomical lens, 30-50ul 0.25% tryptic digestive juice is dripped on the good cell clone of mark, after most cells to be cloned is shunk and is taken off wall, do not wait cell suspension, draw cell clone fast, but near other clone's cell noting not sucking; Transitional cell is blown and beaten the cell dispersion agglomerate in four orifice plates that are added with 500 μ l nutrient solutions; The clone who chooses continues to cultivate, reduce G418 concentration to 300 μ g/ml and keep screening: after treating that clone cell quantity enlarges the back or converges, a part of cell transfer is continued enlarged culturing in the 35mm culture dish, another part is used for transgenosis and detects, and the cell after other enlarges is freezing as early as possible.

2.3.7 the PCR of transfectional cell detects

Because this experiment is adopted is the HLF BAC DNA of 150kb of 150kb and the micro-common injecting method of pEGFP-NEO or pNEO, therefore can not guarantee that with the G418 screening positive colony point is the transgenic cell that changes the HLF BAC DNA that 150kb is arranged, identify so before carrying out somatic cell clone, must carry out PCR to transgenic cell.In order to ensure the HLF BAC DNA that changes complete 150kb over to, we hold at the 5 ' end and 3 ' of the HLF of 150kb BAC DNA, and the HLF coding region has been designed a pair of primer respectively and carried out pcr amplification.

2.3.7.1 the extraction of cell genomic dna

With trypsinase/EDTA liquid digestion collecting cell, and use the PBS washed twice.

↓

Add 200 μ l cell pyrolysis liquids, final concentration is the Proteinase K of 100 μ g/ml, and 55 degree water-bath digestion are spent the night.

↓

With phenol, phenol/chloroform, each extracting of chloroform once.

↓

Add equal-volume dehydrated alcohol and 0.1 times of volumes of acetic acid sodium deposit D NA.

↓

70% washing with alcohol once after, be dissolved among the TE ,-20 degree are preserved.

2.3.7.2 PCR reaction

Primer sequence is as follows:

Table 3: the PCR primers designed and the reaction conditions that change the human lactoferrin clened cows

The primer name	Primer sequence	Product length (bp)	Annealing temperature (℃)
				????hLF?5’PF	?TGCTTTGTITGTATTGAGGGTC	????906	????60
????hLF?5’PR	?CCAGGAACAAACTTACGGAG
		????hLF?PF	?AGGACACAGACAGCAGACAC	????503	????60
????hLF?PF	?CAGCCCTCTTCTTCCTCTTC
		????hLF?3’PF	?TTCCTTCCACCACTGTTGAG			????633	????60
????hLF?3’PR	?CAAATACCTCTGCCGCTGTT

Reaction system: template DNA, 3 μ l; Primer 1 μ l; 2.5mMdNTP, 3 μ l; 10 * Tag enzyme Buffer, 5 μ l; The Tag enzyme, 10U: add ddH2O at last and supply system to 50 μ l.

Reaction parameter: 94 ℃ 5 '; 94 ℃ 40 " → 60 ℃ 40 " → 72 ℃ 1 '; 72 ℃ 10 of 25 cycles '; 4 ℃ of preservations.

2.4 transgenosis somatic cell clone

2.4.1 the maturation of ovocyte is cultivated

Collect the ovary of the ox that grows up from the slaughterhouse, place 30 ℃ physiological saline in 4h, to deliver to the laboratory, after cleaning three times in 37 ℃ the PBS liquid, is the ovarian follicle of 2～8mm with the diameter for the 0.7mm syringe needle extracts diameter, the recovery form is even, ovarian cumulus-the ovocyte of compact structure-complex body (COCs), with twice of ripe liquid (M199+10%FBS+0.01U/ml bFSH+0.01U/ml bLH+1 μ g/ml estradiol) washing, then ovarian cumulus-ovocyte-complex body is put into four orifice plates that contain ripe liquid with 50-60 piece/hole, at 38.5 ℃, after maturation is cultivated 18～20h in the 5%CO2 incubator, the pipe that sophisticated ovocyte is put into 0.1% Unidasa vibrates behind 2～3min, blow and beat gently with Glass tubing again, cumulus cell and ovocyte are broken away from fully, select complete form, tenuigenin evenly and the ovocyte of discharging first polar body be the somatic cell nuclear acceptor.

2.4.2 the stoning of ovocyte

The ovocyte that will have first polar body moves into and contains in the operation liquid of M199+10%FBS+7.5 μ g/ml cytochalasin B, under 200 power microscopes, above polar body, zona pellucida cut an osculum with a glass needle, again with internal diameter be 20 μ m Glass tubing with first polar body with and the ovocyte of below in karyomit(e) absorb in the lump, the solution of putting into M199+20%FBS again give a baby a bath on the third day after its birth all over after, place incubator standby.

2.4.3 move nuclear and merge

The donorcells of serum starvation 2～4d is digested 2～4min with 0.25%trypsin, the selection diameter is that the somatocyte of 10～12 μ m has gone its immigration in the ovocyte zona pellucida of nuclear with 20 μ m diameter Glass tubings, put it into then in the Zimmerman liquid [15] and put into integration slot behind balance 3～5min, rotating ovum makes donorcells vertical with electric field with the ovocyte contact surface, field intensity in DC pulse is 2.5kV/cm simultaneously, burst length is 10 μ s, pulse number is 2 times, recurrent interval is after merging (fusion instrument is the ECM-2001 of BTX company) under the condition of 1s, reconstructed embryo is moved in the M199+10%FBS liquid rapidly, observe fusion rate after placing 0.5h, select the fusion embryo and carry out next step activation processing.

2.4.4 the activation of reconstructed embryo and cultivation

Reconstructed embryo is put into 5 μ mol/L Ionomycin (Sigma) liquid, change to behind the 4min in the 1.9mmol/L 6-DMAP liquid, move into again behind the 4h in the CR1aa+5%FBS liquid, observe division rate after in 38.5 ℃, 5%CO2 incubator, cultivating 2d, observe blastocyst rate behind the 7d

2.4.5 embryo transfer detects with gestation

Clone's blastaea of the 7d that form is good moves in the horn of uterus of the receptor cow of the same period.What receptor cow was selected all is the multiparity cow, and wherein red is that the clone embryos of southern Hebei ox is moved into the Holstein milk cow, and the clone embryos of Holstein milk cow is moved into Luxi Yellow cattle, and the 60d after transplanting carries out rectum and detects, to determine pregnancy rate.

2.5 the PCR of transgene clone and Southern identify

2.5.1 the PCR of transgene clone ox identifies

Identify for the PCR that changes the human lactoferrin clened cows, because the transgenosis that we adopt is the hLF BAC of 150kb, in order to detect genetically modified integrity, we have designed a pair of primer at hLF upstream region of gene control region 5 ' end, designed a pair of primer in its coding region, a pair of primer, primer sequence and reaction conditions such as table 4 have also been designed at its downstream control region 3 ' end;

Table 4: the PCR primers designed and the reaction conditions that change the human lactoferrin clened cows

The primer name	Primer sequence	Product length (bp)	Annealing temperature (℃)
				????hLF?5’PF	????TGCTTTGTTTGTATTGAGGG ?TC	????906	????60
????hLF?5’PR	????CCAGGAACAAACTTACGGA ?G
		????hLF?PF	????AGGACACAGACAGCAGACA ?C	????503	????60
????hLF?PF	????CAGCCCTCTTCTTCCTCTTC
		????hLF?3’PF	????TTCCTTCCACCACTGTTGAG			????633	????60
????hLF?3’PR	????CAAATACCTCTGCCGCTGTT

PCR result such as Fig. 6, as can be seen from Figure 6, we amplify corresponding special product by three pairs of primers of design in the clened cows genome of two, show that two transgene clone ox all integrates complete hLF BAC DNA.

2.5.2 the Southern of transgene clone ox identifies

Southern detects the about 10 μ g of transgene clone ox DNA that then get the PCR test positive and digests with EcoRI, low pressure is electrophoresis at a slow speed, after changeing film, carry out Southern hybridization, hybridizing used probe is the hLF gene coding region fragment of the isotope-labeled 2.5kb of α-P32dCTP.

Fragment with a 2.5kb size of bovine lactoferrin gene coding region is identified this two transgene clone Niu Jinhang Southern.As can be seen from Figure 7, change the bovine lactoferrin gene that the people is arranged in this two clened cows genome really, and iron baby's transgenosis copy number is 1, auspicious baby's transgenosis copy number is 2.

Attached: SEQ ID NO 1: the primary structure of human lactoferrin is an aminoacid sequence, totally 711 amino acid

1?????MKLVFLVLLF?LGALGLCLAG?RRRRSVQWCA?VSQPEATKCF?QWQRNMRRVR

51????GPPVSCIKRD?SPIQCIQAIA?ENRADAVTLD?GGFIYEAGLA?PYKLRPVAAE

101???VYGTERQPRT?HYYAVAVVKK?GGSFQLNELQ?GLKSCHTGLR?RNAGWNVPIG

151???TLRPFLNWTG?PPEPIEAAVA?RFFSASCVPG?ADKGQFPNLC?RLCAGTGENK

201???CAFSSQEPYF?SYSGAFKCLR?DGAGDVAFIR?ESTVFEDLSD?EAERDEYELL

251???CPDNTRKPVD?KFKDCHLARV?PSHAVVARSV?NGKEDAIWNL?LRQAQEKFGK

301???DKSPKFQLFG?SPSGQKDLLF?KDSAIGFSRV?PPRIDSGLYL?GSGYFTAIQN

351???LRKSEEEVAA?RRARVVWCAV?GEQELRKCNQ?WSGLSEGSVT?CSSASTTEDC

401???IALVLKGEAD?AMSLDGGYVY?TAGKCGLVPV?LAENYKSQQS?SDPDPNCVDR

451???PVEGYLAVAV?VRRSDTSLTW?NSVKGKKSCH?TAVDRTAGWN?IPMGLLFNQT

501???GSCKFDEYFS?QSCAPGSDPR?SNLCALCIGD?EQGENKCVPN?SNERYYGYTG

551???AFRCLAEDAG?DVAFVKGVTV?LQNTDGNNNE?AWAKDLKLAD?FALLCLDGKR

601???KPVTEARSCH?LAMAPNHAVV?SRMDKVERLK?QVLLHQQAKF?GRNGSDCPDK

651???FCLFQSETKN?LLFNDNTECL?ARLHGKTTYE?KYLGPQYVAG?ITNLKKCSTS

701???PLLEACEFLR?K

Claims (9)

1. produce animal mammary gland bioreactor---the method for human lactoferrin transgene cloning great cattle of restructuring lactoferrin, its operation steps is as follows: (1) utilizes and contains the hLF BAC DNA of whole person's bovine lactoferrin gene as mammary gland-specific expression vector, (2) select carrier to be mixed in proportion hLF BACDNA and double alternative carrier or single mark, import in the animal somatic cell nuclear, carry out cell transfecting, acquisition changes the transgenic cell of hLF BAC DNA over to, (3) cell carries out somatic cell clone as nuclear donor, obtains to change the transgenic and cloned animal that hLF BAC DNA is arranged.

2. the animal mammary gland bioreactor of production restructuring lactoferrin according to claim 1---the method for human lactoferrin transgene cloning great cattle, described specific expression carrier is the hLF BAC DNA that is about 150kb, the human lactoferrin of expressing in the transgene mouse model of recombinant human milk-protein is consistent with natural human lactoferrin size, has and the identical immunocompetence of natural human Ruzhong lactoferrin.

3. the animal mammary gland bioreactor of production restructuring lactoferrin according to claim 2---the method for human lactoferrin transgene cloning great cattle, the human lactoferrin gene expression amount of described transgenic mice is 0.5-7.8g/l.

4. the animal mammary gland bioreactor of production restructuring lactoferrin according to claim 1---the method for human lactoferrin transgene cloning great cattle, described double alternative carrier is pEGFP-NEO, it is pNEO that described single mark is selected carrier.

5. it is 1: 3 that the animal mammary gland bioreactor of production restructuring lactoferrin according to claim 1---the method for human lactoferrin transgene cloning great cattle, described hLF BAC DNA and double alternative carrier or single mark are selected carrier blended mol ratio.

6. the animal mammary gland bioreactor of production restructuring lactoferrin according to claim 1---the method for human lactoferrin transgene cloning great cattle, described introduction method are the micro-injection altogether of somatocyte.

7. the animal mammary gland bioreactor of production restructuring lactoferrin according to claim 1---the method for human lactoferrin transgene cloning great cattle, described transgenic cell are to screen the positive cell of acquisition by G418.

8. the animal mammary gland bioreactor of production restructuring lactoferrin according to claim 1---the method for human lactoferrin transgene cloning great cattle, described heavy livestock are ox, goat, sheep, pig or rabbit.

9. the animal mammary gland bioreactor of production restructuring lactoferrin according to claim 6---the method for human lactoferrin transgene cloning great cattle, described heavy livestock are ox.

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