CN1669405A - Method for producing human lysozyme transgene cloning large-scale domestic animal as animal mammary gland bioreactor to recombine human lysozyme - Google Patents

Abstract

The invention relates to the animal galactophore biological reactor to reconstruct human antalzyme, a method to clone large livestock's by transudation the gene of the human antalzyme. It comprises the following steps:(1)construct an galactophore specifically expression carrier which includes human antalzyme genes; (2)construct double signs and single sign choice carrier, and get the gene cell through cell transudation by electronic; (3) Clone the body cell to get the transudation gene creature considering the cell as a nucleus. The cattle, created by transudation gene of human antalzyme which is an technique of clone with body cell, will produce a great deal of milk containing active human antalzyme. The human antalzyme produced by this invention, has the biological activity as the nature ones, and it could be developed into health milk with great appended value or purified the human antalzyme into medicine and health food.

Description

Produce animal mammary gland bioreactor---the method for human lysozyme transgene cloning great cattle of recombinant human lysozyme

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 own wish have purpose, planned, with good grounds, the genetic constitution of pre-insight change animal is arranged, and the purpose that changes its genetic constitution is diversified.Wish that such as the geneticist observing its phenotype by the genetic constitution 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 operating technology on experimental technique.

The preparation 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 egg mother cell of MII phase, and sperm head and DNA merge injection egg mother cell method.Improvement on these methods has promoted the process that Study on Transgenic Animal is transformed to production practices 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 micromanipulation 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 fertilized egg 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 operating technology on cloned animal.At first, utilize the clone to carry out transgenosis and be meant before nucleus transplantation, earlier the fusion of genes of interest and marker gene is imported the somatic cell 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 research 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.Utilize the clone to carry out transgenosis, in case the nucleus transplantation success, theoretically, the transgenosis success rate 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 health 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 technology of utilizing animal transgenic technology albumen such as express polypeptide medicine, industrial enzyme, vaccine and antibody in mammary glandular cell.The function of transforming mammary gland by engineered method helps the mechanism that we further study breast cancer, improves the nutrient component of milk, even can synthetic drug.This technology has the low characteristics that drop into high production, and its efficient is 100 times that utilize with Escherichia coli and animal cell culture technology, is a kind of very potential new and high technology.1987, people such as Simons successfully expressed sheep milk globulin gene first in transgenic mouse milk, and the protein content in the mouse milk sample approximately is more than 400 times of animal cell expression albumen 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 plasminogen 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 breeding, may bring huge economic benefit to society.

The superiority that galactophore biological reactor is produced medicine allows pharmaceutical protein gene expression at a certain privileged site, rather than expresses arbitrarily at health randomly.Scientists is consistent to be thought, with regard to medicine, desirable expression place is a mammary gland.Mainly contain following reason: 1, animal's mammary gland is the system of a sealing.The breast 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, breast 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, breast tissue can be carried out correct modification and back processing to human body protein, and the biologic activity of product is near natural prodcuts.4, can heredity in the animal's mammary gland expressed exogenous gene.In case obtain an animal individual of producing certain valuable protein, 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 cow, 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, milch 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 constitution 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 essential amino acid, calcium and inorganic phosphate, and the very high casein of digestibility, are human high-quality source of nutrition: but domestic animal milk is on quality or not fully up to expectations.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 (breast tissue, oviduct) and development-specific expression, and can utilize molecular engineering to find all genes of regulating milk composition 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 lysozyme (4%) content height, and lack beta lactoglobulin and κ-casein.

Lysozyme (lysozyme) claims muramidase (muramidase) again, is natural nospecific immunity material, has the strong disinfecting effect.Nineteen twenty-two, Britain bacterium scientist Fleming has the enzyme of dissolution of bacteria cell wall first in finder's saliva and the tears, because of it has bacteriolysis, so the called after lysozyme.After this at the multiple tissue of humans and animals, secretion, also found the existence of lysozyme in certain plants, the microorganism.Lysozyme can be divided into three types: the c type (hen egg-white lysozyme, Chicken white lysozyme, cly), the g type (the goose hen egg white lysozyme, googse egg-white lysozyme, gly), phage lysozyme (Canfield and Mcmurry, 1967).Most of lysozyme all belongs to the c type, and the lysozyme that extracts from human milk, urine and placenta also belongs to the c type.Human lysozyme (hLY) belongs to c type lysozyme (Isabelle, 1990), has antibiotic, immunological regulation and certain antitumor action, and potential clinical value is arranged.

Human lysozyme is that a kind of molecular weight is 14.7KD by 18 kinds of 130 basic polypeptides that amino acid is formed, and isoelectric point is pH11.Its precursor comprises 148 amino acid, and wherein preceding 18 amino acid are signal peptide (NP_000230).The structure of human lysozyme is simple relatively, comprises 7 α-spirals and 1 beta-sheet and four pairs of disulfide bond of being made up of 3 strands of peptide chains.All structures all are made up of a peptide chain, and do not have other subunits.From the surface texture of enzyme three-dimensional structure, the structure of enzyme is elliposoidal more closely, and most of polar groups are distributed on the surface of enzyme, be convenient to combine with solvent, and non-polar group are hidden in the inside of enzyme, and a long and narrow depression is arranged in the whole enzyme molecule.

Lysozyme has bacteriolysis with the cell wall of dissolving Gram-positive bacteria.Reason is β-1.4 glycosidic bond of (NAM4-O-NAG5glycosidic bond) between its energy hydrolytic bacteria cell wall mucopolysaccharides (polypeptidoglycan) N-acetylglucosamine (NAG) and the N-acetylglucosamine lactic acid (NAM).Along with the reduction of cell wall, bacterium can be dead because the effect of internal pressure is burst apart.People's such as Pellegrini result shows that the lysozyme bacteriostasis is not only relevant with its muramidase, but also relevant with its cation and hydrophobic characteristic.Experiment showed, when the suitableeest little molecule substrate combines with enzyme, just in time with the enzyme molecule in microscler depression chimeric, enzyme active center is made up of at a distance of nearer Asp53 and Glu35 amino acid residue tertiary structure.

The effect of lysozyme: 1) anti-inflammation, lysozyme is the basic hydrolysis enzyme of energy hydrolysis mucopolysaccharides, and mucopolysaccharides is one of main component of bacteria cell wall, its energy direct hydrolysis gram positive bacteria, in the presence of immune protein A, complement, can also the hydrolysis gram-negative bacteria.In addition, it also can combine with the various acidic materials that bring out inflammation, makes its inactivation, and can strengthen the curative effect of antibiotic and other medicines, improves the mucopolysaccharides metabolism of periplast, thereby reaches the purpose of anti-inflammatory, repair tissue.2) antivirus action, lysozyme can with electronegative virus protein effect, with DNA, RNA, apoprotein forms double salt, makes virally inactivated.Recently, some results show that the source egg is clear, and the lysozyme of human milk nuclear and human neutrophil has the activity that suppresses the HIV-1 growth.3) enhancing immunity, lysozyme is as one of body nonspecific immunity factor, participate in the reaction of body panimmunity, in normal defense function of body and nonspecific immunity, have the important function that keeps physiological equilibrium, can improve and strengthen engulfing and digestion power of macrophage, activated leukocyte phagocytic function, and can improve the leukopenia that cytostatics causes, when being subjected to the infringement of pathogenic microorganism, it brings into play anti-infectious function, is a kind of very important non-characteristic immune substance.4) it also has the local blood dyshaemia that the hematoblastic function of activation can be improved tissue, decomposes fester, strengthens local defense function, thereby embody effects such as hemostasis, detumescence.Can also tissue local be shielded as a kind of resistance factor.

The application of lysozyme: 1) lysozyme is as preservative.Itself is a kind of native protein, and avirulence is a kind of safe food additives.Lysozyme only can act on purpose cells of microorganisms wall simultaneously, and can not act on other material.2) lysozyme is medically and use.A. the application of ENT dept.: lysozyme is applicable to the various inflammation of ENT dept..Studies show that it all has certain curative effect to the acute and chronic inflammation in Eye Ear Nose And Throat and oral cavity etc., obvious to effects such as acute inflammation such as acpuei pharyngitis, acute laryngitis, otitis media acutas especially.If in toothpaste, mouthwash, clean dose of mouth, chewing gum, manage to add a certain amount of lysozyme, then can kill these germs, reach the purpose that prevents decayed tooth.Equally, available lysozyme system eyedrops, liquid wets one's whistle.B. the application of dept. of dermatology: lysozyme can be used for the treatment of various skin diseases such as flat wart, molluscum contagiosum, ordinary property wart, condyloma acuminatum, herpes zoster.For herpes zoster, lysozyme for oral administration gets final product.And, preferably take lysozyme for oral administration and 5-fluor-uracil people solution to be coated with outward for the treatment of flat wart and molluscum contagiosum etc., curative effect obviously improves.C. otherwise application: lysozyme can be used for children's and nursing infant's asthmatic bronchitis, the swelling of respiratory tract inner membrance etc., and it can make the very fast elimination of mucous membrane swelling, makes sputum thinning, respiratory passage unblocked.Lysozyme can prevent and treat virus hepatitis, and especially the effect to post-transfusion hepatitis and oxyhepatitis is comparatively remarkable.In body, it also has the growth of resisiting influenza virus and adenovirus, and can prevent herpesvirus infection.In addition, pain, cureless baby diarrhea and Sjogren ' s disease to sarcoma causes all have certain curative effect.D. the concentration of human body lysozyme can also be as multiple disease diagnosis index.3) application in food, the beverage.Lysozyme integrates pharmacology, health care and anticorrosion three functions.Without any side effects as a kind of native protein.The adding of lysozyme has replenished the nospecific immunity factor in the human body, kills the corrupt coccus in the enteron aisle, keeps flora normalization in the enteron aisle, promotes Bifidobacterium propagation, strengthens white clear sterilization albumen, and the defense factor of Y-ball egg strengthens resistance infection.Add in the beverage, in calcium milk, lactic acid drink, also can play anti-decayed tooth, tasty and refreshing effect.Particularly humanized milk powder is very important to infant's growth.Lysozyme content in the milk is few, and the lysozyme content in the breast milk is very abundant.For remedying the deficiency of milk powder, can make female emulsifying power milk powder to wherein adding an amount of lysozyme.The adding of lysozyme can be strengthened infant's anti-infection ability.4) preparation cell extract.The maximum class medium component of consumption in the yeast plaster fermentation industry.Its preparation is to adopt the way of yeast autolysis method or glycolysis, yeast to make at present mostly.Prepare yeast extract if use lysozyme instead, then not only can improve the yield of medicinal extract amount, can also shorten the preparation time of yeast extract greatly.Also can from yeast cells, prepare taste compound with lysozyme, as in some lysozyme except containing lysozyme activity, also contain the taste compound that the nucleic acid that can decompose in the yeast cells is the monokaryon acids of inosinicacid and guanylic acid.5) application in the scientific research.Because lysozyme has the ability that can decompose to selectivity cell wall, except the application in actual production, lysozyme is applied in the scientific research widely.As characteristics, understand the structure of microorganism wall with lysozyme selectivity ground hydrolysis cell wall; Prepare protoplast after decomposing cell wall, and be used for academic research and special agent such as Microbial Breeding and microorganism classification.

The lysozyme of separate sources, its bacteriolyze spectrum has nothing in common with each other.HEL only has bacteriolysis to gram-positive bacteria.In these lysozymes, the lysozyme activity in the human body is the highest, secondly is the lysozyme of egg in white, the lysozyme that minimum is in the cow's milk.Human lysozyme has good biocompatibility, non-stimulated to organizing, avirulent advantage.Business-like human lysozyme can extract from milk, neutrophil leucocyte and urine, yet this resource is very limited after all, utilizes recombinant technique to produce human lysozyme so people begin one's study.This process has mainly experienced three phases.First stage, initial, utilize yeast and aspergillus oryzae to test and express lysozyme.1986, people such as Jigami utilized yeast to carry out human lysozyme and express, but this recombinant protein does not have bacteriostatic activity.Next, Castanon has also done similar work with Yoshimura, does not also obtain important breakthrough.Nineteen ninety, human aspergillus oryzaes such as Archer are expressed HEL, and expression is 12mg/l.People such as Tsuchiya are the expressing human lysozyme in aspergillus oryzae, and expression is 1.2mg/l, does not regrettably have biologic activity equally.This shows that the lysozyme of expressing is biologically active not in yeast and aspergillus oryzae.In second stage, utilize transgenic mice expressing human lysozyme.Maga has made extensive work in this respect, and people such as Maga had carried out the research of transgenic mouse milk expressing human lysozyme in 1994, and they utilize alpha sl-casein promotor to induce the expression of lysozyme.Existence at transgenic mouse milk tissue detection human lysozyme mRNA.The expression of lysozyme is estimated as 0.2g/L to 0.7g/L, because the expression of lysozyme, change has also taken place for the physics of milk and functional characteristic.1998, people's such as Maga result of study showed that further the milk sample of transgenic mice expressing human lysozyme has with the identical activity of standard lysozyme.These results are hinting that the transgenic research of lysozyme has great application prospect in dairy industry.2000, people such as Akinbi were in order to detect the effect of lysozyme at respiratory system in vivo, and they have prepared the transgenic mice of expressing the rat lysozyme at airway epithelial cell.This transgenic mice can improve in lung to the kill capability 5-30 of bacterium doubly.This shows that the method for utilizing transgenic mice to express the lysozyme of biologically active is feasible, and huge potential application foreground is arranged.Three phases, in recent years, people begin to utilize the plant experimental system to express lysozyme, have obtained remarkable progress in this respect.1997, people's such as Nakajima result of study showed that low-level lysozyme also can express at tobacco leaf, and can improve the activity of the antimycotic and bacterium of plant.2000, Takaichi and Oeda utilized transgenic technology also successfully to express human lysozyme in the carrot root.People such as Ahreholta transfer to the T4 lysozyme gene in the potato.The root epithelial cell of discovery potato can be expressed the T4 lysozyme and be discharged the thin layer on root surface.The T4 lysozyme has activity, can killing bacteria.And this sterilizing ability does not rely on age and the growing environment of plant.2002, the lysozyme gene of a codon optimized synthetic forwarded the rice callus tissue to by particle gun, and the lysozyme of reorganization can be expressed in rice suspension cell, and expression reaches 4% of about soluble protein.Experimental result shows that the Ramy3D signal peptide can normally be sheared, and has the biologically active same with human lysozyme.Above result of study shows that it is feasible utilizing the lysozyme of expression of plants biologically active.Can imagine, contain be bound to people's the life of the fruit of human lysozyme and food and bring glad tidings.

Studies show that human lysozyme is genetically modified from above-mentioned, also do not see the transgene clone report of human lysozyme aspect large-scale livestock.

Summary of the invention

Blank at above-mentioned technical field, transgenosis, cell transfecting and clone technology are combined, provide a kind of animal mammary gland bioreactor of producing recombinant human lysozyme---the method for human lysozyme transgene cloning great cattle, to realize " milk people emulsification ".

Produce animal mammary gland bioreactor---the method for human lysozyme transgene cloning great cattle of recombinant human lysozyme, its operating procedure is as follows: (1) makes up the mammary gland-specific expression vector that contains human lysozyme gene, (2 make up double alternative carrier and single mark selection carrier, carry out cell transfecting by electric-shocking method, obtain transgenic cell, (3) transgenic cell carries out somatic cell clone as nuclear donor, obtains transgenic and cloned animal.

Described human lysozyme gene contains the human lysozyme gene signal peptide sequence, 1-4 exon, 1-3 intron.

The specific expression carrier of described human lysozyme gene is pBC1-HLY or pBC2-HLY.

Described human lysozyme gene also contains ox beta-casein signal peptide sequence, 1-4 exon, 1-3 intron.

The specific expression carrier of described human lysozyme gene is pBC1-sigHLY or pBC2-sigHLY, pBC2 is at the constructed carrier of the disappearance 31bp of the 6407-6438bp place of pBC1, the human lysozyme of expressing in the transgene mouse model of recombinant human lysozyme is consistent with natural human lysozyme size, has and the identical immunocompetence of natural human Ruzhong lysozyme.

Described double alternative carrier is pBC2-sigHLY-EGFP-NEO, and it is pBC2-sigHLY-NEO that single mark is selected carrier.

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.

Produce recombinant human lysozyme in order to prepare the cow mammary gland bio-reactor that efficiently expresses human lysozyme, the mouse model that mammary gland efficiently expresses human lysozyme has at first been set up in this laboratory, has made up pBC1-hLY, pBC2-hLY, four kinds of different lysozyme gene expression vectors of pBC1-sighLY and pBC2-sighLY.Utilize micro-injection method injection protokaryon phase mice embryonic to prepare transgenic mice.

PBC-hLY lysozyme expression vector comprises coded sequence (lysozyme signal peptide), goat beta-casein promotor, the terminator of lysozyme gene and the exon and the intron of partly not encoding thereof.Obtain 6 altogether through microinjection and be the transgenic positive mouse, detect, in the milk of 3 female mouse of survival transgenic positive, can detect the expression of recombinant human lysozyme through Western Blotting.Wherein the lysozyme expression of No. 6 transgenic mices reaches 0.2mg/ml, and No. 34 expression is 0.12mg/ml, does not detect the expression of reorganization lysozyme for No. 41.Through bacteriolyze experiment detection and to the calculating of reorganization lysozyme than vigor, the reorganization lysozyme has with the similar ratio vigor of human lysozyme.

PBC2-sigHLY lysozyme expression vector comprises the coded sequence (containing ox beta-casein signal peptide) of lysozyme gene, and pBC2 is at the constructed carrier of the disappearance 31bp (GAATTCATTTCCTAATCATGCAGATTTCTAG) of the 6407-6438bp place of pBC1.The transgenic mice of human lysozyme gene is changeed in the microinjection that the pBC2-sigHLY carrier is carried out mouse protokaryon embryo, has two can only detect efficiently expressing of recombinant human lysozyme in three positive mouse milk, is respectively 2mg/ml and 0.5mg/ml; Detect through bacteriolyze experiment, show that recombinant human lysozyme has the bacteriolyze activity, wherein the enzymic activity of No. 61 mouse is 2160U/ml, No. 63: 948U/ml, the enzyme concentration of negative mouse is: 25.98U/ml, the people is: 1224U/ml.Compare with negative mouse, the lysozyme activity of No. 61 transgenic mice wheys has improved 83 times, has improved 36 times No. 63.Compare with people's milk, the enzymic activity of No. 61 mouse milk has improved nearly one times, and the lysozyme activity of also relatively suckling near the people for No. 63.Show that pBC2-sigHLY expression vector that we make up can efficiently express the recombinant human lysozyme of bacteriolyze activity in animal's mammary gland.And the expression of pBC2-sigHLY expression vector human lysozyme in transgenic mice milk and the active human lysozyme expression vector pBC1-HLY (＜0.2mg/ml and 480U/ml) that contains self signal peptide sequence that makes up apparently higher than us.

The present invention explores and perfect transgenic technology, cell transfecting technology and somatic cell clone technique combine produces and carries the external source functional gene and (comprise goat beta-casein 5 ' and 3 ' controlling element, the code area of genes of interest) approach of transgenic animal, and transgenic animal have greatly been improved, the make efficiency of the big domestic animal of transgenosis particularly, technology path used in the present invention is applicable to the underlying group production of transgenic cow, goat, sheep, pig and rabbit and expands numerous.

Present business-like human lysozyme mainly extracts from human milk, neutrophil leucocyte and urine etc., yet this resource is very limited after all, so we utilize the transgenic cow that somatic cell clone technique is produced changes human lysozyme gene, thereby has active human lysozyme by a large amount of production of the mammary gland of high yield cow, both can develop health-care milk, and also can be purified into human lysozyme and be developed to medicine and health products with high added value.The recombinant human lysozyme that the present invention studied has the biologically active identical with the natural human lysozyme; The milk that contains recombinant human lysozyme can be developed to health-care milk and dairy produce; The recombinant human lysozyme of purifying can be developed to preservative, food additives, health products and medicine.

Description of drawings

Fig. 1: the physical map of lysozyme gene expression vector pBC1-hLY

Wherein Pcasein is the goat beta-casein promotor on the pBC1, Casein E1-E2 is goat beta-casein the noncoding the 1st, 2 exons, Casein E7-E8-E9 is goat beta-casein the noncoding the 7th, 8 and 9 exons, Casein 3`genome DNA are goat beta-casein 3 ' ending regulating sequence.

Fig. 2: the PCR of pBC1-hLY transgenic positive mouse detects

0: non-transgenic mouse genome ,+: people's genome; 6,30,34,41,48,50: be respectively the positive transgenic mice that PCR detects

Fig. 3: the Southern blot of pBC1-hLY transgenic positive mouse detects

0: non-transgenic mouse genome, 6,30,34,41,48,50: be respectively the positive transgenic mice that PCR detects, positive control comprises 3 bands (10,5,1), is equivalent to genomic 10,5,1 copy respectively.

Fig. 4: it be the female mouse of the transgenosis sample of suckling that the Western Blot of the female mouse milk of pBC1-hLY transgenosis detects 41,34,6, and human is people's contrast of suckling, the positive contrast of CK.

Fig. 5: the pcr amplification of human lysozyme gene code area.

M is 1kb DNA marker, and 1 is the PCR product of hly

Fig. 6: utilize XhoI and KpnI that p7zf-sighLY11 is carried out enzyme and cut evaluation

M is 1kb DNA marker, and 11 is the p7zf-sighLY11 plasmid DNA, and KpnI is that p7zf-sighLY11 is cut the result by the KpnI enzyme, and XhoI is that p7zf-sighLY11 is cut the result by the KpnI enzyme.

Fig. 7: utilize the XhoI enzyme to cut pBC2-sighLY13 is identified

M is 1kb DNA marker, and 13 is the pBC-sighLY13 plasmid DNA, and BC1 is the pBC1 plasmid DNA, and XhoI cuts pBC-sighLY13 result for the XhoI enzyme.

Fig. 8: the pBC2-sighLY13 direction is identified

M is 1kb DNA marker, and 13 is the pBC-sighLY13 plasmid DNA, and CalI cuts pBC2-sighLY13 result for the CalI enzyme, and KpnI cuts pBC2-sighLY13 result for the KpnI enzyme.

Fig. 9: the physical map of lysozyme expression vector pBC2-sighLY

Wherein Pcasein is the goat beta-casein promotor on the pBC2, Casein E1-E2 is goat beta-casein the noncoding the 1st, 2 exons, Casein E7-E8-E9 is goat beta-casein the noncoding the 7th, 8 and 9 exons, Casein 3`genome DNA are goat beta-casein 3 ' ending regulating sequence.

Figure 10: the PCR of pBC2-sighLY transgenic positive mouse detects

M is 1kb DNA marker, 0 negative contrast, the positive contrast of h, 6,12,43,61 and 63 positive transgenic mices.

Figure 11: the Southern blot of pBC2-sighLY transgenic positive mouse detects

M is 1kb DNA marker, 0 negative contrast, and the positive contrast of h, 6,12,43,61 and 63 positive transgenic mices, 1,3,5 are respectively 1,3 and 5 copy of positive control.

Figure 12: the Western Blot of the female mouse milk of pBC2-sighLY transgenic positive detects

The negative mouse milk of CK sample, 12,61 and 63 are transgenic mice milk sample, human is the human milk contrast.

Figure 13: the structure chart 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 enhancer, and pEF321 is the EF321 promotor

Figure 14: single mark is selected the structure chart 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

Figure 15: the linear collection of illustrative plates after the pBC2-sighLY-EGFP-NEO enzyme is cut

B-casein promoter is the goat beta-casein promotor on the pBC1, and human lysozyme gene is human lysozyme gene (containing ox b-casein sequence), and GFP is for strengthening green fluorescence protein gene, and Neomycin is a neomycin resistance gene

Figure 16: the linear collection of illustrative plates after the pBC2-sighLY-NEO enzyme is cut

B-casein promoter is the goat beta-casein promotor on the pBC2, and human lysozyme gene is human lysozyme gene (containing ox b-casein sequence), and GFP is for strengthening green fluorescence protein gene, and Neomycin is a neomycin resistance gene

Figure 17: the amplification of hLY primer.

M:1kb ladder; 1: expect the baby; 2: Jin Wa; 3: positive control; 4: negative control; 5:blank

Figure 18: the amplification of hLY primer.

M:1kb ladder; The 1:329 ox; The 2:0365 ox; 3 is 020613,4: positive control; 5: negative control; 6:blank

Figure 19: the amplification of NEO primer.

M:1kb ladder; 1: expect the baby; 2: Jin Wa; 3: positive control; 4: negative control; 5:blank

Figure 20: the amplification of NEO primer

M:1kb ladder; 1: negative control; The 2:329 ox; The 3:0365 ox; 4 is 020613,5: positive control

Figure 21: the amplification of EGFP primer.

M:1kb ladder; The 1:329 ox; The 2:0365 ox; 3 is 020613,4: positive control; 5: negative control; 6:blank

Figure 22: pBC2 primer amplification result:

Swimming lane 1; 4 is common clened cows genomic DNA; swimming lane 2; 3 are respectively commentaries on classics pBC2-sighLY gene clone cow genome group DNA; swimming lane 5 is pBC2-sighLY-NEO (disappearance is arranged) plasmid DNA on pBC, swimming lane 6 is pBC2-sighLY transgenic mice DNA, and swimming lane 7 is complete pBC1; swimming lane 8 is non-clened cows DNA, and swimming lane 9 is a blank.

Figure 23: the Southern result of hLY transgene clone ox

Swimming lane 1 is for expecting the baby, and swimming lane 2 is golden baby, the negative contrast of swimming lane 3-5, and swimming lane 6-8 is respectively the positive control of 1,2 and 5 copy.

Figure 24 p7zf-sighLY11 plasmid records the comparison result of sequence

The signal peptide sequence of beta-casein is indicated with underscore and black matrix; The sequence of exons 1 marks with underscore; XhoI and Csp45I restriction enzyme site are indicated with grey

Figure 25: the sequencing result of carrier exon 2 and with the comparison of lysozyme gene sequence

The sequence of exon 2 is marked by underscore and black matrix, the sequencing sequence of last behavior exon 2, the gene order of following behavior lysozyme.

Figure 26: the sequencing result of carrier exon 3 and with the comparison of lysozyme gene sequence

The sequence that shows son 3 marks with underscore and black matrix.The gene order of last behavior lysozyme, following behavior exon 3 and flanking sequence thereof.

Figure 27: the sequencing result of exon 4 and with the comparison of lysozyme gene sequence

The sequence of exon 4 marks with underscore, and coded sequence wherein shows with black matrix again.

Embodiment

Below in conjunction with embodiment the present invention is done progressive detailed description.

Embodiment

1 experiment material

1.1 plasmid and bacterial strain

Host bacterium bacillus coli DH 5 alpha and DH10B, pBC1 milk expression vector kit and PCR-XL-TOPO vector are available from Invitrogen company, pCMV-EGFP-IRES-NEO and pIRES-NEO are available from Clontech company, pGEM-7zf is available from Promega company, and BAC RP11-1143G9 purchases the genome company in the U.S.

1.2 laboratory animal

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

1.3 main agents

DMEM/F ₁₂Powder, DMEM powder culture medium, trypan blue (trypan blue), TCM199 powder, glutamine and G418 are Gibco company product; Hyclone 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, ampicillin (Amp), kanamycin (Kan), keyhole limpet hemocyanin (KLH) are all available from magnificent biotech firm; Saturated phenol is ancient cooking vessel state bio-engineering corporation product; Trypsase, penicillin streptomycin, EDTA, Hepes, FSH, LH, 17 β-E2, EGF, hyaluronidase, HEPES, FAF BSA, cytochalasin B, Hoechest33342, cycloheximide, A23187,6-DMAP, D-mannitol, Sodium Pyruvate, liquaemin, mineral oil and other mineral salt are Sigma company product.

1.4 culture medium preparation

SOB medium: dispose every liter of medium, 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/L NaOH.Sterilization LB liquid nutrient medium: peptone 10g, dusty yeast 5g, NaCL10g is dissolved in the 800ml water, regulates pH value to 7.5, constant volume system 1000ml, autoclaving.

The LB solid culture medium: peptone 10g, dusty yeast 5g, NaCL10g 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 culture fluid: DMEM powder 13.4g, NaHCO33.7g, penicillin 66mg, streptomycin 100mg, Mill-Q ultra-pure 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: Trypsin 0.1g, KCl 0.04g, NaHCO ₃, NaCl 0.8g, EDTA 0.02g, the Mill-Q ultra-pure 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 ultra-pure water constant volume to 1 liter, PH 7.0-7.2 is with the sterilization of 0.2 μ m membrane filtration, 4 ℃ of preservations.No calcium magnesium PBS solution: KCl 0.2g, KH ₂PO ₄0.2g, NaCl 8.0g, Na ₂HPO ₄12H ₂O 2.88g, Mill-Q ultra-pure 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 buffer solution (2 * HeBS): 280mM Nacl, 10mM Kcl, 1.5mM Na2HPO4,12mM Glucose, 50mM Hepes, 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 buffer solution: 10mMol/L NaCL, 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, 100ml0.5Mol/L EDTA (pH8.0) is settled to 1000ml after the dissolving.Ampicillin (Amp) stock solution: ampicillin sodium 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 CaCl ₂: in 200ml water, dissolve 54gCaCl ₂.6H ₂O, 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 culture fluid: claim TCM199 powder 9.9g, NaHCO ₃2.2g, Sodium Pyruvate 0.1375g, EDTA 0.372g is with 1L ultra-pure water constant volume, PH7.2-7.4, malleation filtration sterilization, 4 ℃ of preservations.Cultivate with adding 10%FCS in the working solution.

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

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

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

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

Merge liquid: 0.3M 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 culture fluid, 10u/ml FSH, 100U/ml LH, 1ug/ml estradiol, 100U/ml penicillin, 100ug/ml streptomycin.

The CRlaa culture fluid:

Composition final concentration (mM) addition 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) CO ₂Incubator: U.S. Forma Scientific Inc.

2) inverted microscope and fluorescence microscope: Japanese Nikon company

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

4) electric shock instrument: U.S. BTX company (ECM2001)

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

6) superclean bench: Beijing cleaning equipment factory

7) ℃ ultra low temperature freezer-80: Japanese SANYO company

8) ice machine: Japanese SANYO company

9) ultra-pure 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: Oligo4.0

Dna sequence analysis software: Chromas

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

2 experimental techniques

2.1 vector construction

2.1.1 pBC1-hLY expression vector establishment and transgene mouse model are set up

1) construction strategy of pBC1-hLY expression vector

At first utilize the method for long segment PCR to obtain the code area of lysozyme gene, it is cloned on the PCR-XL-TOPO vector, and carry out that enzyme is cut and sequence verification.And then it is connected on the mammary gland-specific expression vector pBC1, identify through direction of insertion and order-checking, obtain the expression vector of lysozyme at last.

2) pcr amplification of human lysozyme gene code area

Lysozyme gene total length 6807bp contains 4 exons.MRNA (519-682,2246-2410,4349-4427,5281-6374) and CDS (601-682,2246-2410,4349-4427,5281-5347), initiation codon is positioned at 547 sites, TATAbox is positioned at the 491-496 position.In order to obtain lysozyme code area fragment, we carry out DNA cloning with lysozyme gene group dna sequence dna for masterplate designs primer.The primer of design is: upstream 5`CCC TCG AGA CTC TGA CCT AGC AGT CAA C 3`, and downstream 5`CCG CTC GAG TCTGTT TCT ATC ATT TGG 3`, the 5` end contains the XhoI restriction enzyme site respectively.Amplified production total length 5652bp comprises lysozyme gene signal peptide dna fragmentation partly.PCR condition: 94 ℃ of 3min, 94 ℃ of 1min, 60 ℃ of 1min, 68 ℃ of 5min, 68 ℃ of 10min, 4 ℃ of 1h; 30cycles.PCR amplify the specific fragment of 1 treaty 5.5kb,, be cloned on the pPCR-XL-TOPO carrier so we reclaim this PCR product return.

3) clone of human lysozyme gene code area dna fragmentation

For the PCR product being checked order and verifying, we at first are cloned into it and obtain the pTOPO-hLY recombinant vector on the PCR-XL-TOPO vector.After transforming, cultivate, extract plasmid at last.The positive recombinant plasmid that obtains is carried out the enzyme evaluation of cutting and check order.

4) structure of pBC1-hLY expression vector

The present invention adopts pBC1 as the skeleton carrier that makes up mammary gland expression vector, pBC1 is the commercialization mammary gland expression vector of Invotrogen company, total length is 21628bp, comprise goat beta-casein promotor, exons 1,2 and 7,8,9 noncoding region, beta-casein 3` ends the beta-globin separaant of subarea and 2 copies, and the protokaryon sequence.Wherein between the exon 2 and 7 of goat beta-casein, there is an XhoI restriction endonuclease sites, is used for the code area of foreign gene is connected up.

When making up the lysozyme gene expression vector, we carry out the XhoI enzyme to pBC1 and pTOPO-hLY at first respectively and cut and reclaim, again by 1: 10 ratio mixing in 16 ℃ of connections of spending the night, carry out the electricity conversion again.Choose the monoclonal bacterium colony and shake bacterium extraction plasmid, screening recombinant plasmid pBC-hLY positive colony.

5) enzyme is cut and is identified and the PCR detection

The XhoI enzyme is cut the pBC-hLY positive plasmid, and the result shows the skeleton fragment of the pBC1 that can cut out a 5kb external source fragment and 21kb, shows that the XhoI site of recombinant plasmid has been connected into 5kb external source fragment.

Be further checking, we are template design primer with the lysozyme gene sequence: the upstream: 5`TTA TAC ACA CGG CTT TAC 3` downstream: 5`CAG CAT CAG CGA TGT TAT CT 3`PCR condition: 94 ℃ of 5min, 94 ℃ of 40sec, 53 ℃ of 40sec, 72 ℃ of 40sec, 72 ℃ of 7min, 4 ℃ of 1 ∞; 30cycles.The result show with No. 4 be that template can increase and be the identical dna fragmentation of template size to contain human lysozyme gene BAC with No. 12 plasmids, show that the human lysozyme gene code area has been connected on the pBC1 carrier

6) direction is identified

Because above-mentioned connection belongs to the single endonuclease digestion site and connects, identify so must insert the direction of fragment.We identify by selectional restriction restriction endonuclease Drd travel direction have a Drd site to be positioned at lysozyme gene 5` end (538), also have corresponding restriction enzyme site (2442,12109,12219,15035,17540,17953) simultaneously on pBC1.According to the dna fragmentation size that enzyme is cut product, can judge the direction of insertion of external source fragment.

7) sequence verification

In order to guarantee the correctness of this expression vector sequence, we check order to the part of the exons 1-4 of pBC1-hLY positive plasmid again, the result shows in the PCR process, do not cause the sudden change of lysozyme gene exon partial sequence, what be connected into pBC1 is intact human lysozyme gene genomic DNA.

8) foundation of pBC1-hLY transgene mouse model and detection

(1) recovery and the detection of the NotI of pBC1-hLY expression vector and SalI endonuclease bamhi

The physical map of pBC1-hLY expressionvector as shown in Figure 1, this experiment is removed the redundance of plasmid with NotI and SalI double digestion, reclaims the fragment of about 26kb size with the Qigen kit, be dissolved in the special-purpose TE of transgenosis, adjusting DNA concentration is 2 μ g/ml.

(2) microinjection

Inject 1200 pieces in fertilized egg altogether, 70 of transplant recipient mouse, 32 of the mouse of becoming pregnant, 83 of commensal mouses

(3) PCR of transgenic mice detects

Get the tail of 2-3 transgenic mice in age in week and organize sample, extract DNA.With the used a pair of primer in front (upstream: 5`TTA TACACA CGG CTT TAC 3` downstream: 5`CAG CAT CAG CGA TGT TAT CT 3`) respectively to mouse DNA sample pcr amplification, with common mouse genome as negative control, with the people's gene group as positive control, as shown in Figure 2, found that having 6 mouse is the transgenic positive mouse, is respectively 6,30,34,41,48 and No. 50.The PCR product that mouse amplifies is consistent with positive control, and our these mouse are the transgenic mice that carries pBC-hLY

(4) the Southern blot of transgenic mice detects

6 mousetails of PCR test positive are organized sample DNA, get about 10 μ g and digest with the PstI restriction endonuclease, low pressure is electrophoresis at a slow speed, behind the commentaries on classics film, carries out Southern hybridization, and hybridizing used probe is α-P ³²The isotope-labeled PCR product of dCTP.With the positive contrast of pBC-hLY plasmid, with the negative contrast of negative mouse genome, hybridization can obtain the 5kb fragment.The result as shown in Figure 3, the result shows all positive mouse of transgenic mice 6,30,34,41,48 and No. 50, and is consistent with the PCR testing result, and the genome of negative mouse (0) does not have hybridization signal.According to Southern hybridization signal intensity, the copy number of the exogenous origin gene integrator of estimation transgenic mice.6, No. 48,3 copies; No. 30,1 copy; 34, No. 41,2 copies; No. 50,8 copies.

(5) the Western blot of transgenic mice detects

Milk is gathered in behind transgenic mice cub mouse the 5th day.The milk of gathering is carried out the centrifugal 10min of 4500rpm, remove butterfat.Add 2 times of volume aqua sterilisas, remove casein about adjust pH to 4.0, at last the pH value is transferred to 8.0, whey is stored in-70 ℃ of refrigerators.The expression contents of lysozyme of the resulting estimate transgenic mice by hybridization: the content of human milk's lysozyme is: 0.5mg/ml, can tentatively estimate thus, and No. 6 whey reorganization lysozyme content is: 0.2mg/ml; No. 34 whey reorganization lysozyme content is: 0.12mg/ml; Do not express for No. 41.(Fig. 4)

34, No. 6 mouse milk sample can detect the expression of recombinant human lysozyme as can be seen from Figure, preliminary estimation, and No. 6 whey reorganization lysozyme content is: 0.2mg/ml; No. 34 whey reorganization lysozyme content is: 0.12mg/ml; Do not express for No. 41

(6), the activity of recombinant human lysozyme detects

A. lysozyme is set up the calibration curve of micrococcus lysodeikticus bacteriolysis

50,75,100,125,150,175,200U/mL the configuration standard lysozyme, concentration is respectively:.Get the 90ul bacterial suspension and place cuvette, add enzyme liquid 10ul, blow evenly rapidly with pipettor, measure the absorbance value (OD450) under the 450nm.From adding the timing of enzyme liquid, write down 1 time every 1min, every group of data will repeat more than 3 times.As ordinate, come the production standard curve as abscissa with the mean value of the difference of 0 to 1 minute OD450 with the concentration of standard enzyme.According to calibration curve, we can obtain regression equation: y=0.0005x-0.002, and y represents the difference of OD450, and x represents enzyme activity (U/ml).Formula can be derived thus: x=2000 (y+0.002).In the preparation process of whey, diluted 3 times, so the enzyme activity computing formula of whey is: x=2000 (y+0.002) * 3.

B. the activity of recombinant human lysozyme detects

According to said method, measure transgenic mice, people, negative mouse whey OD respectively to the micrococcus lysodeikticus effect ₄₅₀Value changes, and measures three times, gets 0 to 1 minute OD450 difference.Can calculate the concentration of lysozyme in the whey according to x=2000 (y+0.002) * 3 formula.The enzyme activity that No. 6, transgenic mice is 480U/ml, No. 34: 301U/ml, No. 41: 37.98U/ml, the enzyme activity of negative mouse is: 25.98U/ml, the people is: 1224U/ml.This shows, compare with negative mouse that the lysozyme enzyme activity of transgenic mice whey has very obviously and to improve, wherein No. 6 mouse have been improved 18 times, have improved 11 times No. 34, but No. 41 are not almost improved.Compare with people's whey, the lysozyme enzyme activity degree in the transgenic mice whey is relatively low, wherein accounts for for No. 6 and accounts for 1/4 2/5, No. 34.This shows that the expression of recombinant human lysozyme can significantly improve the vigor of lysozyme in the mouse milk, show that the recombinant human lysozyme of expressing by mammary gland has the biologic activity of bacteriolyze.

2.1.2 pBC2-sigHly expression vector establishment and transgene mouse model are set up

1) construction strategy of .pBC2-sighLY expression vector

In order to make up the lysozyme expression vector that contains beta-casein signal peptide dna sequence dna, we have designed the strategy of following construction of expression vector.At first synthetic ox beta-casein signal peptide dna sequence dna, 5 ' and 3 ' end contains XhoI and Csp45I respectively, utilizes these two restriction enzyme sites to link and obtains the p7zf-sig plasmid on the pGEM-7zf plasmid.Utilize the coding region sequence of long segment PCR method amplification human lysozyme gene again, 5 ' end has the Csp45I restriction enzyme site, and 3 ' end has XhoI and two restriction enzyme sites of HindIII successively.Utilize Csp45I and HindIII restriction enzyme site to link and obtain the p7zf-sighLY plasmid on the p7zf-sig.Make up the pBC2 carrier, ox signal peptide and lysozyme code area fusion sequence are scaled off, link the XhoI site of pBC2 with XhoI.

2). the design of ox beta-casein signal peptide is with synthetic

In order to obtain ox beta-casein signal peptide, we have synthesized two single stranded DNA sequences respectively.Sequence is respectively: forward 5`TCGAG ATGA AGGTCCTCAT CCTTGCCTGC CTGGTGGCTC TGGCCCTTGC A AAGGTCTT 3`, oppositely 5`C GAAGACCTT T GCAAGGGCCA GAGCCACCAG GCAGGCAAGG ATGAGGACCT TCATC3`.Black matrix line partial sequence is an ox beta-casein signal peptide dna sequence dna.After two single stranded DNAs were annealed into two strands, the 5` end can form XhoI restriction enzyme site part, and the 3` end forms Csp45I restriction enzyme site part.With synthetic signal peptide DNA dissolving, final concentration reaches 825ng/ μ l.Respectively get 10 μ l mixings then, boiling water boils and carried out sex change in 5 minutes, and the room temperature cooling is annealed then, so just can form the ox beta-casein signal peptide DNA that 5` end and 3` end comprise XhoI and the partially digested site of Csp45I respectively

3). the clone of ox beta-casein signal peptide DNA

We utilize XhoI on the ox beta-casein signal peptide DNA and the partially digested site of Csp45I to be connected to and obtain the p7zf-sig recombinant plasmid on the pGEM-7zf carrier.In order to identify the recombinant plasmid of attach signal peptide, we utilize KpnI to carry out enzyme and cut evaluation.Because attach signal peptide dna fragmentation between 7zf plasmid XhoI and Csp45I, original KpnI restriction enzyme site is removed.Therefore, p7zf-sig can not be cut by KpnI, and pGEM-7zf can be cut open.Simultaneously, it is checked order, reconfirm that signal peptide sequence links on the pGEM-7zf, and entirely true.

4). the acquisition of lysozyme gene code area (not containing signal peptide sequence)

In order to obtain the code area of human lysozyme gene, the BAC that we utilize probe to transfer from the BAC storehouse and contain lysozyme gene clones (BACRP11-1143G9).In containing the LB liquid nutrient medium of chloramphenicol, shake bacterium.Extract BAC then as stated above,, concentration is about 100ng/L.Next, we carry out the coding region sequence fragment that pcr amplification obtains lysozyme gene with BAC RP11-1143G9 as template.

In order to obtain lysozyme code area (not containing signal peptide sequence) fragment, we carry out DNA cloning with lysozyme gene group dna sequence dna for masterplate designs primer.The primer of design is: upstream 5`A TT CGA AAG GTG TGA GTT GGC CAG AAC TCT G 3`, the 5` end contains Csp45I restriction enzyme site, downstream 5`T respectively AA GCT TCT CGA GAC CAT CCT GGC TAA CAC G 3`, the 5` end comprises HindIII and two restriction enzyme sites of XhoI.Amplified production total length 5255bp does not comprise the dna fragmentation that lysozyme gene is secreted peptide moiety.PCR condition: 94 ℃ of 3min, 94 ℃ of 1min, 60 ℃ of 1min, 68 ℃ of 5min, 68 ℃ of 14min, 4 ℃ of 1 ∞; 30cycles.Extracting the BAC (RP11-1143G9) that contains lysozyme gene by above-mentioned method again, is that template is carried out pcr amplification with BAC, PCR result following (Fig. 5).

5) clone of .PCR product

For the PCR product being checked order and verifying, we at first are cloned into it and obtain the pTOPO-sighLY recombinant vector on the pPCR-XL-TOPO vector.

6) acquisition of .p7zf-sighLY recombinant plasmid

In order to obtain containing the lysozyme gene code area of ox beta-casein signal peptide DNA, we utilize HindIII and Csp45I that the lysozyme gene code area on the pTOPO-hLY recombinant vector (the not signal peptide of lysozyme own) scaled off earlier, be connected on the p7zf-sig plasmid, thereby obtain the recombinant plasmid p7zf-sighLY that ox beta-casein signal peptide DNA and lysozyme gene connect together.In order to identify the positive recombinant plasmid of p7zf-sighLY11, utilize KpnI and XhoI to carry out enzyme respectively and cut, because in the process that connects signal peptide sequence, the KpnI restriction enzyme site is removed, so KpnI can not cut recombinant plasmid.And since 5 ' end of signal peptide and lysozyme code area 3 ' hold and contain the XhoI restriction enzyme site respectively, so enzyme is cut the 7zf skeleton fragment that can obtain 3kb and signal peptide and the lysozyme code area fragment of 5kb.Enzyme is cut the result as shown in Figure 6, confirms the certain positive recombinant plasmid of plasmid No. 11.

P7zf-sighLY11 is checked order, and the result shows the signal peptide sequence that comprises ox beta-casein on this carrier and the partial sequence of lysozyme code area, and any sudden change does not take place these sequences.The p7zf-sighLY11 plasmid records comparison result such as Figure 24 of sequence, and this sequencing result shows that the signal peptide sequence of beta-casein successfully is connected with the hly sequence

7) structure of .pBC-sighLY exprssion vector

The present invention adopts pBC2 as the skeleton carrier that makes up mammary gland expression vector, and pBC2 is that pBC1 is through revising the carrier that is obtained, promptly at the constructed carrier of the disappearance 31bp (GAATTCATTTCCTAATCATGCAGATTTCTAG) of the 6407-6438bp place of pBC1.This section sequence is arranged in pBC1 beta-casein promoter region, and the purpose that makes up pBC2 promptly is will detect this section sequence whether to influence the expression of foreign gene in mammal galactophore.

The pBC2 building process is as follows: cut the pBC1 plasmid DNA with ClaI and XhoI enzyme, reclaim the fragment of 4kb, be connected acquisition pGEM-CX1 by ClaI with pGEM-7zf with the XhoI restriction enzyme site, by the EcoRI site with deletion sequence (pBC1 L1:AATTCATTTCCTAATCATGCAGATTTCTAGG, with pBC1 L2:AATTCCTAGAAATCTGCATGATTAGGAAATG, two sequences annealing forms double-stranded) be connected with pGEM-CX1 and obtain pGEM-CX2, cut pGEM-CX2 and reclaim the fragment of 4kb with ClaI and XhoI enzyme, be connected with pBC1 with the XhoI restriction enzyme site by ClaI and can obtain pBC2.

When making up the lysozyme gene expression vector, we carry out the XhoI enzyme to pBC2 and p7zf-sighLY at first respectively and cut and reclaim, again by 1: 10 ratio mixing in 16 ℃ of connections of spending the night, carry out the electricity conversion again.Choose the monoclonal bacterium colony and shake bacterium extraction plasmid, the XhoI site of screening group plasmid has been connected into about 5kb external source fragment (as Fig. 7).Because above-mentioned connection belongs to the single endonuclease digestion site and connects, identify so must insert the direction of fragment.Our selectional restriction restriction endonuclease KpnI and Csp45I and ClaI and Csp45I double digestion travel direction are identified, Csp45I has 1 restriction enzyme site at 5 ' end of external source fragment, on pBC2, the ClaI restriction enzyme site is positioned at 4529bp, KpnI is positioned at 13772bp, has a Csp45I restriction enzyme site in the position of 19kb.According to the dna fragmentation size that enzyme is cut product, can judge the direction of insertion of external source fragment.The enzyme of No. 13 recombinant plasmids is cut result such as Fig. 8, shows that No. 13 recombinant plasmids are forward type recombinant plasmid.

8). the order-checking at carrier exon position

In order to guarantee the accuracy of constructed lysozyme gene expression vector, we will check order to the position, code area of carrier.Because order-checking has been finished to the position of exons 1 in the front, so mainly exon 2,3,4 is checked order here.Result as Figure 25,26,27. comprehensive order-checkings shows that any sudden change and mispairing do not take place the exon sequence of pBC-sighLY expression vector.This has guaranteed that advantageously the pBC-sighLY expression vector can correctly transcribe and translate.

The sequencing result of carrier exon 2 and with the comparison of lysozyme gene sequence: as Figure 25, through comparison, the own sequence of the sequence of exon 2 and lysozyme gene is in full accord.The sequence that yet shows intron 2 is not undergone mutation.

The sequencing result of carrier exon 3 and with the comparison of lysozyme gene sequence: as Figure 26, through comparison, any sudden change does not take place in the sequence of exon 3 yet.The gene order of last behavior lysozyme, following behavior exon 3 and flanking sequence thereof.

The sequencing result of exon 4 and with the comparison of lysozyme gene sequence: as Figure 27, through and the lysozyme gene sequence alignment, CDS4 and exon 4 are not all undergone mutation.

10) foundation of pBC2-sighLY transgene mouse model and detection

(1) recovery and the detection of the NotI of .pBC2-sighLY expression vector and SalI endonuclease bamhi

Produce transgenic mice with microinjection, therefore linear DNA must be cut into people pBC2-sighLY linear back (Fig. 9) than annular DNA integration efficiency height, can carry out microinjection; In addition, unnecessary plasmid fragment also influences the DNA integration efficiency.So the redundance of plasmid, is removed in this experiment with NotI and SalI double digestion, reclaim the fragment of about 26kb size with the Qigen kit, the OD260/OD280 value is 1.75, and its concentration is transferred to 2ng/ μ l, sets up transgenic mice by microinjection.

(2). microinjection

Inject 1500 pieces in fertilized egg altogether, 73 of transplant recipient mouse, 34 of the mouse of becoming pregnant, 63 of commensal mouses

(3). the PCR of transgenic mice detects

Get the tail of 2-3 transgenic mice in age in week and organize sample, extract DNA.With the used a pair of primer in front (upstream: 5`TTA TAC ACA CGG CTT TAC 3` downstream: 5`CAG CAT CAG CGA TGT TAT CT 3`) respectively to mouse DNA sample pcr amplification, with common mouse genome as negative control, with the people's gene group as positive control, found that having 5 mouse is the transgenic positive mouse, positive rate is 7.9%, as Figure 10. show that from testing result these mouse all change pBC-sighLY is arranged.

(4). the Southern blot of transgenic mice detects

5 mousetails of PCR test positive are organized sample DNA, get about 10 μ g and digest with the PstI restriction endonuclease, low pressure is electrophoresis at a slow speed, behind the commentaries on classics film, carries out Southern hybridization, and hybridizing used probe is α-P ³²The isotope-labeled PCR product of dCTP.Hybridization can obtain the 5kb fragment.The result as shown in figure 11, the result shows all positive mouse of transgenic mice 6,12,43,61 and No. 63, and is consistent with the PCR testing result, and the genome of negative mouse does not have hybridization signal (0).The calculating of transgenic mice copy number: positive control comprises 3 bands (5,3,1), is equivalent to genomic 5,3,1 copy respectively.According to Southern hybridization signal intensity, the copy number of the exogenous origin gene integrator of estimation transgenic mice.No. 6,1 copy; No. 12,2 copies; 43 and No. 61, be higher than 30 copies, No. 63,8 copies.

(5). the detection of transgenosis heredity

The Founder transgenic mice carries out mating with the non-transgenic mouse at once after identifying.Have 6 positive Founder mouse, F1 adopts the program same with identifying first ancestor's transgenic mice to extract genomic DNA and PCR detection for totally 44.Wherein 6,12, No. 61 mouse offsprings' positive rate near or less than 50%; No. 63 offspring is 76.9%; No. 43 offspring is 100%.In theory, transgenic mice offspring's positive rate is at least 50%, and meets broad-mouthed receptacle for holding liquid Dare law of segregation.

(6). the Western blot of transgenic mice detects

Behind transgenic mice cub mouse the 5th day gathered milk, and gathered the 5th day milk of a negative mouse in contrast.Before gathering milk, need female mouse and newborn mouse were isolated more than 3 hours, and could collect milk smoothly by the oxytocins and the massage breast of lumbar injection doses., carry out Western with negative mouse milk sample as negative control and detect as positive control with people's milk.Negative mouse do not have a hybridization signal, the people has strong hybridization signal as positive control.For transgenic mice, the milk sample of 61 and No. 63 mouse has strong hybridization signal, detects the expression of reorganization lysozyme.Contain lysozyme 0.5mg/ml in the human milk, estimate according to the intensity of signal, the content of 61 extra source human lysozymes is about: 2mg/ml; Be about for No. 63: 0.5mg/ml; And the expression of No. 12 lysozymes of not recombinating, as Figure 12.

(7), the activity of recombinant human lysozyme detects

A. lysozyme is set up the calibration curve of micrococcus lysodeikticus bacteriolysis

Method as previously mentioned

B. the activity of recombinant human lysozyme detects

According to said method, measure transgenic mice, people, negative mouse whey respectively the OD450 value of micrococcus lysodeikticus effect is changed, measure three times, get 0 to 1 minute OD450 difference.Can calculate the concentration of lysozyme in the whey according to x=2000 (y+0.002) * 3 formula.The enzymic activity that No. 61, transgenic mice is 2160U/ml, No. 63: 948U/ml, No. 12: 30U/ml, the enzymic activity of negative mouse (ck) is: 25.98U/ml, the people is: 1224U/ml.This shows that compare with negative mouse, the lysozyme concentration of transgenic mice whey is significantly increased, wherein No. 61 mouse have been improved 83 times, have improved 36 times No. 63, but No. 12 are not almost improved.Compare with people's whey, No. 61 lysozyme concentration has improved nearly 1 times, No. 63 also near the lysozyme concentration of people's whey, be about 0.8 times.

As can be seen from the above results, pBC1-Hly and pBC2-sigHly expression vector that we make up, recombinant human lysozyme that can both specifically expressing has the bacteriolyze activity in transgenic mouse milk, and have the pBC2-sigHly expression vector expression of ox beta-casein signal peptide sequence and relatively the bacteriolyze activity all will be apparently higher than the pBC1-Hly expression vector, so the pBC2-sigHly expression vector is that we carry out next step experiment and promptly carry out the transgene clone ox and make the main carrier that is adopted.And as can be seen from the expression of transgenic mice, consistent at the pBC2 of goat beta-casein promoter region disappearance 31bp sequence with the expression effect of complete pBC1, prove that the pBC2 that we make up also can be used for the research of galactophore of transgenic animal specifically expressing.

2.1.3pBC-sigHly-EGFP-NEO and pBC-sigHly-NEO expression vector establishment

1) construction strategy of .pBC-sighLY marker expression carrier

For the ease of the screening positive cell, we have at first made up and have contained green fluorescence protein gene (EGFP) and two selected marker carriers (pEGFP-NEO) of neomycin gene and neomycin gene list selected marker carrier (pNEO), respectively two labeled vectors are connected with the pBC-sighLY carrier that makes up before this then to be built into pBC-sigHly-EGFP-NEO and pBC-sigHly-NEO expression vector

2) 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, as Figure 13.

3) 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, as Figure 14.

4) pBC-sigHly-EGFP-NEO expression vector establishment

Cut pBC1-sigHLY and pEGFP-NEO with NotI and SalI enzyme, both connections promptly constituted pBC-sigHIy-EGFP-NEO, cut and sequencing analysis, determine the success of pBC-sigHly-EGFP-NEO vector construction, carrier figure following (Figure 15) by enzyme:

5) pBC-sigHly-NEO expression vector establishment

Cut pBC-sigHLY and pNEO with NotI and SalI enzyme, both connections promptly constituted pBC-sigHly-NEO, cut and sequencing analysis, determine the success of pBC-sigHly-NEO vector construction, carrier figure following (Figure 16) by enzyme:

2.2 cell culture, gene transfection and positive cell screening

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

In superclean bench, fetus is immersed in 30sec in 70% alcohol,, takes off the several piece epidermal tissue of ear and ridge in the DMEM+10%FCS culture fluid with the operating theater instruments of sterilizing with PBS rinsing several times.

↓

In diameter 60mm plate, fetal tissue's chopping is become 1mm ³Size organize fragment, will organize fragment to be transferred in the 15mlmlml centrifuge tube again, centrifuge washing for several times under the 1000rpm.

↓

With sterilization glass elbow suction pipe the cell fragment is drawn to 25cm ²In the blake bottle, piece of tissue is evenly distributed on bottle wall by the glass pipette elbow.

↓

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

2.2.2 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.

2.2.2.1 go down to posterity

Carefully absorb culture fluid in the blake 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% trypsase/0.02%EDTA digestive juice with quadrat method, addition gets final product for covering cell monolayer that (diameter 100mm culture dish generally adds the 1ml digestive juice, add 200 μ L digestive juices 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 culture fluid 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 culture fluid by 1: 2 or 1: 3 dilution proportion and again inoculating cell to culture dish, cultivate.

2.2.2.2 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 freezing liquid (DMEM+20%FCS+10%DMSO) the re-suspended cell precipitation of 4 degree precoolings, make cell concentration be about 10 ⁷Individual cell/ml, transitional cell is to frozen pipe.

↓

Put into 4 degree refrigerator precooling 30min, frozen pipe is inserted in be placed on the stifling 2h of liquid nitrogen liquid level on the thick foam again, drop into liquid nitrogen then rapidly and preserve.

2.2.2.3 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 freezing liquid thoroughly thaws, be transferred in the centrifuge tube with 10 times of fresh medium dilutions and with lysate, centrifugal 5min removes DMSO to extenuate freezing liquid toxicity under 1000rpm.

↓

With fresh culture fluid suspension cell precipitation, cell dilution to desired concn, is continued to cultivate.

2.2.3 the transfection preparation of DNA

Big upgrading grain pBC-sigHly-EGFP-NEO and pBC-sigHly-NEO, with the SalI digested plasmid, endonuclease reaction is as follows:

The about 10-20 μ of plasmid DNA g

10 * reaction buffer, 20 μ l

Restriction enzyme 10-20U

Dd H2O supplies system to 200 μ l

After mentioned reagent adds well, mixing, 37 degree water-bath digestion 2h, whether get 5 μ L enzymes cuts product and detects enzyme with 0.7% agarose gel electrophoresis and cut complete, if enzyme cuts entirely, add phenol, each extracting of phenol/chloroform once, add 2 times of volume absolute ethyl alcohols and 0.1 times of volumes of acetic acid sodium (PH5.2), mixing postposition-20 is spent night; The centrifugal 15min of 12000rpm reclaims precipitation, 70% washing with alcohol once, vacuum drying adds the TE dissolving.

2.2.4 bovine fetal fibroblast is to G418 toxicity sensitivity Detection

Cell culture to the is used 0.25% trypsinization after 3 generations, by every hole 0.5～1 * 10 ⁵The dispersion so that cell is tried one's best in individual cell inoculation to the 13 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 continuous culture, changed liquid once, and added G418 simultaneously in per 3～4 days.Every day, observation of cell was grown or death condition.

2.2.5 electric shock transfection

The DNA that before the transfection 10～50 μ g 1.2.1 purifying is reclaimed incorporates among 500 μ L, 2 * HeBS, replenishes the sterilization ultra-pure water to 1m1, the ice bath precooling.

↓

Before transfection 2～3 days, with 0.25% trypsin solution digestion cultured cell.With 1 * 10 ⁶Individual cell is transferred in 75cm ²In the blake bottle, add complete culture solution, put 37 degree, 5%CO ₂Cultivate in the incubator.

↓

Before the transfection, under inverted microscope, observe cultured cells, determine that cell is in the logarithmic growth later stage.With 0.25% trypsin solution vitellophag, the centrifugal 5min sedimentation cell of 1000rpm.

↓

Cell precipitation with 1 * HeBS centrifuge washing of ice bath precooling 1 time, is carried out cell counting simultaneously.

↓

The cell of some is suspended in the buffer electrode that is added with DNA again, changes in the electric shock cup ice bath 10min before electric shock after mixing over to.

↓

When cell is ready for electric shock, electric-field intensity (E) and burst length (t) are set, try earlier, guaranteed to produce required pulse.The cup that will shock by electricity inserts in the electric shock tank, according to selected electric-field intensity (E) and the burst length (t), and the electric shock cell.

↓

To behind the cell ice bath 10min after the electric shock, change 75cm over to ²Blake bottle in, add in the DMEM culture fluid contain 20%FCS in 37 degree, 5%CO ₂Cultivate in the incubator.

↓

Treat the cell growth state recovery after 1-2 days, cell is enlarged 10 times of cultivations, add an amount of G418 and screen.

↓

Changed liquid once, and added G418 simultaneously in per 3～4 days.Every day, observation of cell was grown or death condition.After 6-10 days, select the positive colony cell.

2.2.6 the monoclonal of positive cell is cultivated

Under fluorescence microscope, observe the luminous situation of said 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 culture fluid, 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 culture fluids; The clone who chooses continues to cultivate, and reduces G418 concentration to 300 μ g/ml and keeps screening; After treating that clone cell quantity enlarges the back or converges, a part of cell transfer is continued enlarged culture 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 transgenosis somatic cell clone

2.3.1 the maturation of egg mother cell 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, the ovarian cumulus-egg mother cell-complex (COCs) of compact structure, 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-egg mother cell-complex is put into four orifice plates that contain ripe liquid with 50-60 piece/hole, at 38.5 ℃, 5%CO ₂After maturation is cultivated 18～20h in the incubator, the pipe that the egg mother cell of maturation is put into 0.1% hyaluronidase vibrates behind 2～3min, blow and beat gently with glass tube again, cumulus cell and egg mother cell are broken away from fully, select complete form, cytoplasm evenly and the egg mother cell of discharging first polar body be the somatic cell nuclear acceptor.

2.3.2 the stoning of egg mother cell

The egg mother cell 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, oolemma cut an osculum with a glass needle, again with internal diameter be 20 μ m glass tube with first polar body with and the egg mother cell of below in chromosome 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.

2.3.3 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 somatic cell of 10～12 μ m has gone its immigration in the egg mother cell oolemma of nuclear with 20 μ m diameter glass tubes, put it into then in the Zimmerman liquid [15] and put into integration slot behind balance 3～5min, rotating egg cell makes donorcells vertical with electric field with the egg mother cell contact-making surface, field intensity at DC pulse is 2.5kV/cm simultaneously, burst length is 10 μ s, pulse number is 2 times, pulse spacing 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.

2.3.4 the activation of reconstructed embryo and cultivation

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

2.3.5 embryo transplantation detects with gestation

Clone's blastaea of the 7d that form is good moves in the uterine horn 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.60d after transplanting carries out rectum and detects, to determine pregnancy rate.

2.4 the PCR of transgene clone and Southern identify

The present invention obtains the transgene clone ox that four-head changes pBC-sighLY-NEO altogether, and a commentaries on classics has the transgene clone ox of pBC-sighLY-EGFP-NEO.Gather transgenosis, non-transgenic clened cows and common holstein cow ear organizes sample, extract DNA, carry out then that PCR identifies and the Southern evaluation.

The primer sequence that PCR detects is: upstream: 5`TTA TAC ACA CGG CTT TAC 3` downstream: 5`CAG CAT CAG CGA TGT TATCT 3`.PCR condition: 94 ℃ of 5min; 94 ℃ of 40sec, 53 ℃ of 40sec, 72 ℃ of 40sec, 30cycles; 72 ℃ of 7min, 4 ℃ of 1 ∞, product length is 700bp.Result such as Figure 17 and 18.

For the NEO gene, primer sequence is: the upstream: 5`AGG ATC TCC TGT CAT CTC ACC TTG CTC CTG 3` downstream: 5`AAG AAC TCG TCA AGA AGG CGA TAG AAG GCG 3`.PCR condition: 94 ℃ of 5min; 94 ℃ of 40sec, 60 ℃ of 40sec, 72 ℃ of 40sec, 30cycles; 72 ℃ of 7min, 4 ℃ of 1 ∞, product length is 490bp.Result such as Figure 19 and 20.

For the EGFP gene, primer sequence is: the upstream: 5`TGC AGT GCT TCA GCC GCT AC 3` downstream: 5`CTC AGG TAG TGGTTG TCG GG 3`.PCR condition: 94 ℃ of 5min; 94 ℃ of 40sec, 62 ℃ of 40sec, 72 ℃ of 40sec, 30cycles; 72 ℃ of 7min, 4 ℃ of 1 ∞, product length is 380bp.Result such as Figure 21.

Our employed pBC2 carrier promotor is the goat casein promoter, and at promoter region the 31bp disappearance is arranged.We have designed a pair of primer (pBCF1:5 ' GGG GAC AGA AAA ATA GGA AG 3 ', pBCF1:5 ' AGA GGA GAA GAA GTGAAG GA 3 '), PCR condition: 94 ℃ of 5min at the disappearance place; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 30sec, 30cycles; 72 ℃ of 7min, 4 ℃ of 1 ∞, the amplified production of complete pBC1 is 174bp, and pBC2 is 143bp.Amplification such as Figure 22, swimming lane 3 and 6 amplified productions are about 140bp, and this is because pBC2-sighLY-NEO plasmid DNA and transgenic mice DNA all contain the pBC2 of deletion sequence; And 1,2,3, No. 4 oxen, the amplified production of contrast ox and complete pBC all contains one and is about the 170bp band, this may be that 2 and No. 3 clened cows then also contains a band that is about 140bp owing to the cause of the casein promoter district autoploidy higher (pBCF1 and 2 primers and ox casein promoter region autoploidy are 100% and 95%) of Niu Yuyang, proves that further 2 and No. 3 clened cows are for changeing the transgene clone ox that pBC2-sighLY-NEO is arranged.

From above PCR result as can be seen, the four-head clened cows that obtains at us all changes pBC-sighLY-NEO, and has a commentaries on classics that pBC-sighLY-EGFP-NEO is arranged in addition.

The about 10 μ g of transgene clone ox DNA that get the PCR test positive digest with the PstI restriction endonuclease, and low pressure is electrophoresis at a slow speed, behind the commentaries on classics film, carry out Southern hybridization, and hybridizing used probe is α-P ³²The hLY gene of the isotope-labeled 5kb of dCTP.With the positive contrast of pBC-sigHLY-NEO plasmid; with the negative contrast of non-transgenic clened cows genome; hybridization can obtain the 5kb fragment; further determined golden baby as shown in figure 23 and expected that the baby changes lysozyme gene is arranged; can estimate golden baby and expect the human lysozyme gene that the baby changes 20 copies; according to the transgenic mice result, can infer that this two transgenic cow can efficiently express human lysozyme in mammary gland.

Attached: SEQ ID NO 1: this sequence is an amino acid sequence for the human lysozyme primary structure, totally 148 amino acid, and wherein preceding 18 amino acid are signal peptide.

1 MKALIVLGLV?LLSVTVQGKV?FERCELARTL?KRLGMDGYRG?ISLANWMCLA

51 KWESGYNTRA?TNYNAGDRST?DYGIFQINSR?YWCNDGKTPG?AVNACHLSCS

101 ALLQDNIADA?VACAKRVVRD?PQGIRAWVAW?RNRCQNRDVR?QYVQGCGV

Claims (9)

1. produce animal mammary gland bioreactor---the method for human lysozyme transgene cloning great cattle of recombinant human lysozyme, its operating procedure is as follows: (1) makes up the mammary gland-specific expression vector that contains human lysozyme gene, (2) make up double alternative carrier and single mark and select carrier, carry out cell transfecting by electric-shocking method, obtain transgenic cell, (3) transgenic cell carries out somatic cell clone as nuclear donor, obtains transgenic and cloned animal.

2. the animal mammary gland bioreactor of production recombinant human lysozyme according to claim 1---the method for human lysozyme transgene cloning great cattle, described human lysozyme gene contains the human lysozyme gene signal peptide sequence, 1-4 exon, 1-3 intron.

3. the animal mammary gland bioreactor of production recombinant human lysozyme according to claim 1---the method for human lysozyme transgene cloning great cattle, the specific expression carrier of described human lysozyme gene are pBC1-HLY or pBC2-HLY.

4. the animal mammary gland bioreactor of production recombinant human lysozyme according to claim 1---the method for human lysozyme transgene cloning great cattle, described human lysozyme gene also contains ox beta-casein signal peptide sequence, the 1-4 exon, the 1-3 intron.

5. the animal mammary gland bioreactor of production recombinant human lysozyme according to claim 4---the method for human lysozyme transgene cloning great cattle, the specific expression carrier of described human lysozyme gene is pBC1-sigHLY or pBC2-sigHLY, the human lysozyme of expressing in the transgene mouse model of recombinant human lysozyme is consistent with natural human lysozyme size, has and the identical immunocompetence of natural human Ruzhong lysozyme.

6. the animal mammary gland bioreactor of production recombinant human lysozyme according to claim 4---the method for human lysozyme transgene cloning great cattle, described double alternative carrier is pBC2-sigHLY-EGFP-NEO, and it is pBC2-sigHLY-NEO that single mark is selected carrier.

7. the animal mammary gland bioreactor of production recombinant human lysozyme according to claim 4---the method for human lysozyme 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 recombinant human lysozyme according to claim 1---the method for human lysozyme transgene cloning great cattle, described heavy livestock are ox, goat, sheep, pig or rabbit.

9. the animal mammary gland bioreactor of production recombinant human lysozyme according to claim 8---the method for human lysozyme transgene cloning great cattle, described heavy livestock are ox.

Images