CN1891821B - Production method of animal cell with human lysozyme gene - Google Patents

Abstract

The invention relates to a manufacture method for cloning large size livestock by transgene of trans-human lysozyme gene. The operation process includes the following steps: constructing mammary gland special expression carrier containing human lysozyme gene; constructing cascade structure of human lysozyme gene mammary gland special expression carrier with double marked selecting carrier and single marked selecting carrier, inducting the cascade structure into livestock somatic nucleus to gain transgene cell; taking cell clone to transgene cell used as nucleus supplier to gain the transgene clone animal containing trans-human lysozyme gene. The invention has the same biological activity as natural human lysozyme. The milk containing reconstructed human lysozyme gene could be developed into heath milk and milk product. The purified reconstructed human lysozyme could be used to develop antiseptic, food additive, health products, and medicine.

Description

Change the production method of the zooblast of human lysozyme gene

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

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

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

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

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

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

N,O-Diacetylmuramidase (lysozyme) claims muramidase (muramidase) again, is natural non-specific immunity material, has the strong disinfecting effect.Human lysozyme (hLY) belongs to c type N,O-Diacetylmuramidase (Isabelle, 1990), has antibiotic, immunomodulatory and certain antitumor action, and the 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 iso-electric point is pH11.

N,O-Diacetylmuramidase has bacteriolysis with the cell walls of dissolving gram positive bacterium.Reason is (NAM between its energy hydrolytic bacteria cell walls mucopolysaccharide (polypeptidoglycan) N-acetylglucosamine (NAG) and the N-acetylglucosamine lactic acid (NAM) ₄-O-NAG ₅Glycosidic bond) β-1.4 glycosidic link.Along with the reduction of cell walls, bacterium can be dead because the effect of internal pressure is burst apart.People's such as Pellegrini result shows that the N,O-Diacetylmuramidase bacteriostatic action is not only relevant with its muramidase, but also relevant with its positively charged ion and hydrophobic characteristic.Experiment showed, when the suitableeest small molecules 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 N,O-Diacetylmuramidase: 1) antisepsis and anti-inflammation, N,O-Diacetylmuramidase is the alkaline hydrolysis enzyme of energy hydrolysis mucopolysaccharide, and mucopolysaccharide is one of main component of bacteria cell wall, its energy direct hydrolysis gram positive organism, 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 substance that bring out inflammation, makes its inactivation, and can strengthen the curative effect of microbiotic and other medicines, improves the mucopolysaccharide metabolism of periplast, thereby reaches the purpose of anti-inflammatory, repair tissue.2) antivirus action, N,O-Diacetylmuramidase can with electronegative viral protein effect, with DNA, RNA, apoprotein forms double salt, makes virally inactivated.Recently, some results show the source Ovum Gallus domesticus album, and the N,O-Diacetylmuramidase of human milk nuclear and human neutrophil has the activity that suppresses the HIV-1 growth.3) strengthening immunity, N,O-Diacetylmuramidase 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 vital role that keeps physiological equilibrium, can improve and strengthen engulfing and digestion ability of scavenger cell, activated leukocyte phagocytic function, and can improve the oligoleukocythemia that cytostatics causes, when being subjected to the infringement of pathogenic micro-organism, it brings into play anti-infectious function, is a kind of very important non-characteristic phylactic agent.4) it also has the local blood circulation obstacle 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 N,O-Diacetylmuramidase: 1) N,O-Diacetylmuramidase is as sanitas.Itself is a kind of natural protein, and nontoxicity is a kind of safe foodstuff additive.N,O-Diacetylmuramidase only can act on purpose microbial cell wall simultaneously, and can not act on other material.2) N,O-Diacetylmuramidase is medically and use.A. the application of department of eye: N,O-Diacetylmuramidase is applicable to the various inflammation of department of eye.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 acute pharyngitis, acute laryngitis, acute otitis medias especially.If in toothpaste, collutory, clean dose of mouth, chewing gum, manage to add a certain amount of N,O-Diacetylmuramidase, then can kill these germs, reach the purpose that prevents decayed tooth.Equally, available N,O-Diacetylmuramidase system collyrium, liquid wets one's whistle.B. the application of Dermatology Department: N,O-Diacetylmuramidase can be used for the treatment of various skin diseases such as verruca plana, molluscum contagiosum, ordinary property wart, pointed condyloma, zoster.For zoster, N,O-Diacetylmuramidase for oral administration gets final product.And, preferably take N,O-Diacetylmuramidase for oral administration and 5-fluor-uracil people solution to be coated with outward for the treatment of verruca plana and molluscum contagiosum etc., curative effect obviously improves.C. otherwise application: N,O-Diacetylmuramidase 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.N,O-Diacetylmuramidase can prevent and treat viral hepatitis, and especially the effect to post-transfusion hepatitis and acute hepatitis 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 infantile diarrhea and Sjogren ' s disease to sarcoma causes all have certain curative effect.D. the concentration of human body N,O-Diacetylmuramidase can also be as multiple medical diagnosis on disease index.3) application in food, the beverage.N,O-Diacetylmuramidase integrates pharmacology, health care and anticorrosion three functions.Without any side effects as a kind of natural protein.The adding of N,O-Diacetylmuramidase has replenished the intravital non-specific immunity factor of people, kills the corrupt coccus in the enteron aisle, keeps flora normalizing in the enteron aisle, promotes bifidus bacillus 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 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 N,O-Diacetylmuramidase.The adding of N,O-Diacetylmuramidase can be strengthened infant's anti-infection ability.4) preparation cell extractive substance.The maximum class medium component of consumption in the yeast plaster fermentation industry.Its preparation adopts yeast autolysis method or glycolysis, zymic way to make at present mostly.Prepare yeast extract paste if use N,O-Diacetylmuramidase instead, then not only can improve the yield of medicinal extract amount, can also shorten the preparation time of yeast extract paste greatly.Also can from yeast cell, prepare taste compound with N,O-Diacetylmuramidase, as in some N,O-Diacetylmuramidase except containing lysozyme activity, also contain the taste compound that the nucleic acid that can decompose in the yeast cell is the monokaryon acids of t-inosinic acid and guanylic acid.5) application in the scientific research.Because N,O-Diacetylmuramidase has the ability that can decompose to specificity cell walls, except the application in actual production, N,O-Diacetylmuramidase is applied in the scientific research widely.As characteristics, understand the structure of microorganism wall with N,O-Diacetylmuramidase specificity ground hydrolysis cell walls; Prepare protoplastis after decomposing cell walls, and be used for academic research and special agent such as Microbial Breeding and microorganism classification.

The N,O-Diacetylmuramidase of different sources, its bacteriolyze spectrum has nothing in common with each other.Hen's egg-white lysozyme only has bacteriolysis to gram-positive microorganism.In these N,O-Diacetylmuramidases, the lysozyme activity in the human body is the highest, secondly is the N,O-Diacetylmuramidase in the egg albumen, the N,O-Diacetylmuramidase 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 technology 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 N,O-Diacetylmuramidase.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 hen's egg-white lysozyme, and expression amount is 12mg/l.People such as Tsuchiya are the expressing human N,O-Diacetylmuramidase in aspergillus oryzae, and expression amount is 1.2mg/l, does not regrettably have biologic activity equally.This shows that the N,O-Diacetylmuramidase of expressing is biologically active not in yeast and aspergillus oryzae.In second stage, utilize transgenic mice expressing human N,O-Diacetylmuramidase.Maga has made extensive work in this respect, and people such as Maga had carried out the research of transgenic mouse milk expressing human N,O-Diacetylmuramidase in 1994, and they utilize alpha s1-casein promotor to induce the expression of N,O-Diacetylmuramidase.Existence at transgenic mouse milk tissue detection human lysozyme mRNA.The expression amount of N,O-Diacetylmuramidase is estimated as 0.2g/L to 0.7g/L, because the expression of N,O-Diacetylmuramidase, change has also taken place for the physics of milk and functional performance.1998, people's such as Maga result of study showed that further the milk sample of transgenic mice expressing human N,O-Diacetylmuramidase has with the identical activity of standard N,O-Diacetylmuramidase.These results are hinting that the transgenic research of N,O-Diacetylmuramidase has great application prospect in diary industry.2000, people such as Akinbi were in order to detect the effect of N,O-Diacetylmuramidase at respiratory system in vivo, and they have prepared the transgenic mice of expressing the rat N,O-Diacetylmuramidase 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 N,O-Diacetylmuramidase 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 N,O-Diacetylmuramidase, have obtained remarkable progress in this respect.1997, people's such as Nakajima result of study showed that low-level N,O-Diacetylmuramidase 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 Radix Dauci Sativae 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 N,O-Diacetylmuramidase and be discharged the thin film layer on root surface.The T4 N,O-Diacetylmuramidase 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 N,O-Diacetylmuramidase of reorganization can be expressed in rice suspension cell, and expression amount reaches 4% of about soluble proteins.Experimental result shows that the Ramy3D signal peptide can normally be sheared, and has the biological activity same with human lysozyme.Above result of study shows that it is feasible utilizing the N,O-Diacetylmuramidase 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

At the blank of above-mentioned technical field, transgenosis, cell transfecting and clone technology are combined, provide a kind of production method of changeing the transgene cloning great cattle of human lysozyme gene, to realize " milk people emulsification ".

The invention provides a kind of production method of changeing the zooblast of human lysozyme gene, its operation steps is as follows: (1) makes up the mammary gland-specific expression vector that contains human lysozyme gene, (2) make up the cascaded structure that human lysozyme mammary gland-specific expression vector and double alternative carrier or single mark are selected carrier, cascaded structure DNA is imported in the animal somatic cell nuclear, obtain transgenic cell, described mammary gland-specific expression vector is pBC2-sigHLY, and described pBC2 is at the constructed skeleton carrier of the disappearance 31bp of the 6407-6438bp place of pBC1; Described sigHLY is by ox beta-casein signal peptide sequence, the 1-4 exon of human lysozyme and 1-3 intron are formed, described mammary gland-specific expression vector and double alternative carrier cascaded structure DNA are pBC2-sigHLY-EGFP-NEO, it is pBC2-sigHLY-NEO that described mammary gland-specific expression vector and single mark are selected carrier cascaded structure DNA, and described animal is ox, goat, sheep, pig or rabbit.

Described introduction method is the electroporation transfection method.

Described animal is an ox.

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

PBC1-HLY N,O-Diacetylmuramidase expression vector comprises encoding 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 human lysozyme through Western Blotting.Wherein the N,O-Diacetylmuramidase expression amount of No. 6 transgenic mices reaches 0.2mg/ml, and No. 34 expression amount is 0.12mg/ml, does not detect the expression of reorganization N,O-Diacetylmuramidase for No. 41.Through bacteriolyze experiment detection and to the calculating of reorganization N,O-Diacetylmuramidase than vigor, the reorganization N,O-Diacetylmuramidase has with the similar ratio vigor of human lysozyme.

PBC2-sigHLY N,O-Diacetylmuramidase expression vector comprises the encoding 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 human lysozyme in three positive mouse milk, is respectively 2mg/ml and 0.5mg/ml; Detect through bacteriolyze experiment, show that human lysozyme has the bacteriolyze activity, wherein the enzymic activity of No. 61 mouse is 2160U/ml, No. 63: 948U/ml, the enzyme concn 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 the pBC2-sigHLY expression vector that we make up can efficiently express the active human lysozyme of bacteriolyze in animal's mammary gland.And the expression amount 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 coding region of goal gene) approach of transgenic animal, and transgenic animal have greatly been improved, the make efficiency of the big domestic animal of transgenosis particularly, technological line used in the present invention is applicable to the underlying group production of transgenic cattle, goat, sheep, pig and rabbit and expands numerous.

N,O-Diacetylmuramidase integrates pharmacology, health care and anticorrosion three functions, and is without any side effects as a kind of natural protein.And N,O-Diacetylmuramidase destroys and the ratio vigor of dissolution of bacteria is about 300 times of cow's milk in the human milk, be the important antimicrobial factors in the human milk, so human lysozyme is considered to a kind of health care albumen and pharmaceutical protein with high exploitation value.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 cattle that somatic cell clone technique is produced changes human lysozyme gene, thereby the mammary gland mass production by high yield cow has active human lysozyme, both can develop nourishing milk, and also can be purified into human lysozyme and be developed to medicine and healthcare products with high added value.The human lysozyme that the present invention studied has the biological activity identical with the natural human N,O-Diacetylmuramidase; The milk that contains human lysozyme can be developed to nourishing milk and milk preparation; The human lysozyme of purifying can be developed to sanitas, foodstuff additive, healthcare 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, Casein3`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: the Western Blot of the female mouse milk of pBC1-HLY transgenosis detects

41,34,6 is the female mouse milk of transgenosis sample, and human is people's contrast of suckling, the positive contrast of CK.

Fig. 5: the pcr amplification of human lysozyme gene coding region.

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 N,O-Diacetylmuramidase 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, Casein3`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 iron of double alternative carrier pEGFP-NEO

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

Figure 14: single mark is selected the structure iron of carrier pNEO

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

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: hLY transgene clone cow's milk sample PAGE result, swimming lane 1 protein standard, swimming lane 2 is the human lysozyme standard substance, and swimming lane 3 is a human milk, and swimming lane 4-7 is respectively Jin Wa, expects the baby, 329 and 0365.

Figure 25: HLY transgene clone cow's milk sample Southern results of hybridization, swimming lane 1 is the human lysozyme standard substance, and swimming lane 2 is a human milk, and swimming lane 3-6 is respectively Jin Wa, expects the baby, 329 and 0365.

Embodiment

Below in conjunction with embodiment the present invention being made progressive describes in detail.

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

1.4 culture medium preparation

SOB substratum: dispose every liter of substratum, should add at the 950ml deionized water: peptone: 20g yeast extract: 5g NaCl0.5g. shakes the vessel solute and dissolves fully, add 250mmol/L KCl solution 10ml then, be adjusted to pH7.0 with 5mol/L NaOH.Sterilization

The LB liquid nutrient medium: peptone 10g, yeast powder 5g, NaCL10g is dissolved in the 800ml water, regulates pH value to 7.5, constant volume system 1000ml, autoclaving.

The LB solid medium: peptone 10g, yeast powder 5g, 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 nutrient solution: DMEM powder 13.4g, NaHCO33.7g, penicillin 66mg, Streptomycin sulphate 100mg, Mill-Q ultrapure water constant volume to 1 liter, PH7.0-7.4, osmotic pressure are 280-320mOsm/kg, with the sterilization of 0.2 μ m membrane filtration, 4 ℃ of preservations.Add 10% serum during use.

0.1%Trypsin/EDTA enzymic digestion liquid: Trypsin0.1g, KCl 0.04g, NaHCO ₃, NaCl 0.8g, EDTA 0.02g, the Mill-Q ultrapure water dissolving with sterilization is settled to 100ml, with the sterilization of 0.2 μ m membrane filtration ,-20 ℃ of preservations.DPBS liquid: DPBS powder 9.7g, Mill-Q ultrapure water constant volume to 1 liter, 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 ultrapure water constant volume to 1 liter, PH 7.0-7.4, osmotic pressure are 280-320mOsm/kg, with the sterilization of 0.2 μ m membrane filtration, 4 ℃ of preservations.The Gimsa mother liquor: Gimsa powder 0.5g adds a small amount of glycerine the Gimsa powder is fully ground in mortar.Adding glycerine to total amount again is 22ml, 56 ℃ of insulation 2h, and then add 33ml methyl alcohol, and be kept in the brown reagent bottle, standby.Cell pyrolysis liquid: 10mM Tris.Cl (PH 8.0), 0.1M EDTA (PH8.0), 0.5%SDS.The Proteinase K stock solution: Proteinase K 100mg, be dissolved in the 5mL aqua sterilisa, packing ,-20 degree are preserved.TBS liquid: NaCl 8.0g, KCl 0.2g, Tris.Cl 3.0g is dissolved in the 1L redistilled water autoclaving.G418 stores liquid: 1g G418 is dissolved in the 1mL 1mol/mL HEPES solution (PH7.0-7.2), adds Mill-Q10mL, filtration sterilization, and-20 degree are preserved.

Twice electric shock damping fluid (2 * HeBS): 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 damping fluid: 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, 100ml 0.5Mol/L EDTA (pH8.0) is settled to 1000ml after the dissolving.Penbritin (Amp) stock solution: sodium ampicillin is dissolved in behind the aqua sterilisa with the filter filtration sterilization of 0.22 μ m, is mixed with 100mg/ml, be stored in-20 ℃.

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

1mol/L 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/LNaCl, 1%SDS.

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

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

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

TCM199 nutrient solution: claim TCM199 powder 9.9g, NaHCO ₃2.2g, Sodium.alpha.-ketopropionate 0.1375g, EDTA 0.372g is with 1L ultrapure water constant volume, PH7.2-7.4, malleation filtration sterilization, 4 ℃ of preservations.Cultivate with adding 10%FCS in the working fluid.Operation liquid H199: in the TCM199 that contains 25mM Hepes, add 10%FCS.

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

Unidasa: Unidasa 0.2g, being dissolved in 10ml does not have in the calcium DPBS solution, filtration sterilization ,-20 ℃ of storages.Working fluid: use towards 10 times of ovum liquid dilutions.

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

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

The CRlaa nutrient solution:

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

1.6 key instrument equipment

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

2) inverted microscope and fluorescent microscope: Japanese Nikon company

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

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

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

6) Bechtop: Beijing treating plant factory

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

8) ice-making machine: Japanese SANYO company

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

10) refrigerated centrifuge: Eppendorf company

11) high speed freezing centrifuge: BECKMAN company

12) gel imaging system: ALPHA INNOTECH company

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

14) autosterilization pot: Japanese SANYO company

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

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

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

18) ABI 377DNA sequenator: 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

2.1.1pBC1-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 coding region of lysozyme gene, it is cloned on the PCR-XL-TOPOvector, 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 N,O-Diacetylmuramidase at last.

2) pcr amplification of human lysozyme gene coding region

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), initiator codon is positioned at 547 sites, TATA box is positioned at the 491-496 position.In order to obtain N,O-Diacetylmuramidase coding region 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 TGACCT AGC AGT CAA C 3`, and downstream 5`CCG CTC GAG TCT GTT 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 coding region dna fragmentation

For the PCR product being checked order and verifying, we at first are cloned into it and obtain the pTOPO-HLY recombinant vectors 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 non-coding 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 coding region 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 mono-clonal 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 pBC1-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 coding region 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 segmental direction.We identify by selectional restriction restriction endonuclease Drd direction of travel 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 segmental direction of insertion of external source.

7) sequence verification

In order to guarantee the exactness 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 expression vector 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 test kit, be dissolved in the special-purpose TE of transgenosis, adjusting DNA concentration is 2 μ g/ml.

(2) microinjection

Inject 1200 pieces in zygote 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: 5TTA TAC ACA CGG CTT TAC 3` downstream: 5`CAG CAT GAG CGA TGT TAT CT3`) 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 pBC1-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 pBC1-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 detected 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 N,O-Diacetylmuramidase of the resulting estimate transgenic mice by hybridization: the content of human milk's N,O-Diacetylmuramidase 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 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 human lysozyme detects

A. N,O-Diacetylmuramidase is set up the typical curve of micrococcus lysodeikticus bacteriolysis

50,75,100,125,150,175,200U/mL the configuration standard N,O-Diacetylmuramidase, 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 zou, come the production standard curve as X-coordinate with the mean value of the difference of 0 to 1 minute OD450 with the concentration of standard enzyme.According to typical 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 calculation formula of whey is: x=2000 (y+0.002) * 3.

B. the activity of human lysozyme detects

According to aforesaid 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 N,O-Diacetylmuramidase 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 N,O-Diacetylmuramidase 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 N,O-Diacetylmuramidase 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 human lysozyme can significantly improve the vigor of N,O-Diacetylmuramidase in the mouse milk, show that the human lysozyme of expressing by mammary gland has the biologic activity of bacteriolyze.

2.1.2pBC2-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 N,O-Diacetylmuramidase 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 Csp451 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 N,O-Diacetylmuramidase coding region 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 TGGCCCTTGCA AAGGTCTT 3`, oppositely 5`C GAAGACCTT T GCAAGGGCCA GAGCCACCAGGCAGGCAAGG ATGAGGACCTTCAT C 3`.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.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 Kpn I 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 coding region (not containing signal peptide sequence)

In order to obtain the coding region of human lysozyme gene, the BAC that we utilize probe to transfer from the BAC storehouse and contain lysozyme gene clones (BAC RP11-1143G9).In containing the LB liquid nutrient medium of paraxin, 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 N,O-Diacetylmuramidase coding region (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 GGCCAG AAC TCT G 3`, the 5` end contains Csp45I restriction enzyme site, downstream 5`T respectively AA GCT TCT CGA GACCAT CCT GGC TAA CAC G 3`, the 5` end comprises Hind III 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 vectors on the pPCR-XL-TOPO vector.

6) acquisition of .p7zf-sighLY recombinant plasmid

In order to obtain containing the lysozyme gene coding region of ox beta-casein signal peptide DNA, we utilize Hind III and Csp45I that the lysozyme gene coding region on the pTOPO-HLY recombinant vectors (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 N,O-Diacetylmuramidase coding region 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 N,O-Diacetylmuramidase coding region 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 N,O-Diacetylmuramidase coding region, 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 .pBC2-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 (pBC1L1:AATTCATTTCCTAATCATGCAGATTTCTAGG, with pBC1 L2:AATTCCTAGAAATCTGCATGATTA GGAAATG, 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 mono-clonal 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 segmental direction.Our selectional restriction restriction endonuclease KpnI and Csp45I and ClaI and Csp45I double digestion direction of travel are identified, Csp45I has 1 restriction enzyme site at the segmental 5 ' end of external source, 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 segmental direction of insertion of external source.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, coding region 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 pBC2-sigHLY expression vector.This has guaranteed that advantageously the pBC2-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 N,O-Diacetylmuramidase, 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.

9) 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 test 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 zygote 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: 5TTA 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 detected result these mouse all change pBC2-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 detected 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 Mendel's regulavity 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 pitocin and the massage breast of abdominal 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 N,O-Diacetylmuramidase.Contain N,O-Diacetylmuramidase 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 N,O-Diacetylmuramidases of not recombinating, as Figure 12.

(7), the activity of human lysozyme detects

A. N,O-Diacetylmuramidase is set up the typical curve of micrococcus lysodeikticus bacteriolysis

Method as previously mentioned

B. the activity of human lysozyme detects

According to aforesaid 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 N,O-Diacetylmuramidase 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, can both in transgenic mouse milk, have the active human lysozyme of bacteriolyze by specifically expressing, and have the pBC2-sigHLY expression vector expression amount 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.

2.1.3pBC2-sigHLY-EGFP-NEO and pBC2-sigHLY-NEO expression vector establishment

1) construction strategy of .pBC2-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 selective marker carriers (pEGFP-NEO) of neomycin gene and neomycin gene list selective marker carrier (pNEO), respectively two labeled vectors are connected with the pBC2-sigHLY carrier that makes up before this then to be built into pBC2-sigHLY-EGFP-NEO and pBC2-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) pBC2-sigHLY-EGFP-NEO expression vector establishment

Cut pBC2-sigHLY and pEGFP-NEO with NotI and SalI enzyme, both connections promptly constituted pBC2-sigHLY-EGFP-NEO, cut and sequencing analysis, determine the success of pBC2-sigHLY-EGFP-NEO vector construction, carrier figure following (Figure 15) by enzyme:

5) pBC2-sigHLY-NEO expression vector establishment

Cut pBC-sigHLY and pNEO with NotI and SalI enzyme, both connections promptly constituted pBC2-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 cultures, gene transfection and positive cell screening

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

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

2.2.2.2 it is freezing

2.2.2.3 recovery

2.2.3 the transfection preparation of DNA

Big upgrading grain pBC2-sigHLY-EGFP-NEO and pBC2-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

DdH2O 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 dehydrated 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 cultures to the is used 0.25% tryptic digestion 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 cultured continuously, changed liquid once, and added G418 simultaneously in per 3～4 days.Every day, observation of cell was grown or death condition.

2.2.5 electric shock transfection

2.2.6 the mono-clonal of positive cell is cultivated

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

2.3 transgenosis somatic cell clone

2.3.1 the maturation of ovocyte is cultivated

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

2.3.2 the stoning of ovocyte

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

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

2.3.4 the activation of reconstructed embryo and cultivation

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

2.3.5 embryo transfer detects with gestation

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

2.4 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 CAGCGA TGT TAT CT 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 CTG3` downstream: 5`AAGAAC TCG TCAAGAAGG CGATAGAAG 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`CTCAGG TAG TGG TTG 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 ' AGAGGAGAAGAAGTGAAG 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 homology higher (pBCF1 and 2 primers and ox casein promoter region homology 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 pBC2-sigHLY-NEO, and has a commentaries on classics that pBC2-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 pBC2-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 cattle can efficiently express human lysozyme in mammary gland.

2.5 transgenic cattle restructuring lactoferrin expression analysis

2.5.1 medicine lactagogue

Press of the transgene clone Niu Jinhang medicine lactagogue of 1/4 dosage with the lactagogue pin that Cao Tan pharmaceutical factory, Xi'an produces to the 6-8 monthly age, simultaneously with double-tagging carrier transgene clone ox, common clened cows, carry out lactagogue in contrast with common Fresian of monthly age, it also is contrast with the ordinary milk, collect the milk sample every day 3 times, collected for two weeks altogether.

2.5.2 PAGE electrophoresis, Western and the ria-determination of transgenosis milk

Present embodiment is analyzed four-head transgene cow milk sample.Newborn sample to transgenic cattle dilutes three times with heavily steaming aqua sterilisa, removes butterfat, goes caseic processing, obtains whey portion at last.Human lysozyme promptly is present in the whey.After newborn sample carried out degreasing and remove casein to transgenosis milk sample and contrast, the PAGE glue of the last sample Buffer of usefulness subtracting property (reducing) SDS sex change carried out electrophoresis, result such as Figure 24.From then on scheming as can be seen, transgenosis milk sample has one and human lysozyme standard substance specific band of the same size.Carry out Western hybridization, result such as Figure 25 with the anti-human lysozyme antibody of rabbit.From Western result as can be seen transgene cow milk sample and people milk have one with human lysozyme standard substance specific band of the same size, show in the transgenic cattle mammary gland can The expressed human lysozyme.

Utilize the radioimmunoassay method to detect the expression amount of human lysozyme in the transgenosis milk sample.The human lysozyme standard substance are used ¹²⁵I chloramine-t method mark, the newborn sample of getting 6 time points detects.The expression amount mean value gold baby who records human lysozyme in the transgene cow milk sample is 3.49g/l, expect that the baby is 0.55g/l, 329 is 0.52g/l, and 0365 N be 2.48g/l, shows that we can efficiently express complete human lysozyme at employed human lysozyme gene structure in transgenic cattle mammary gland.

2.5.3 the activity of human lysozyme detects

Typical curve is set up (transgenic mice) as previously mentioned, after testing, transgenic cattle gold baby's human lysozyme enzymic activity is 3065U/ml, expect baby: 922U/ml, No. 329: 986U/ml, No. 0365: 2740U/ml, the enzymic activity of negative mouse (ck) is: 25.98U/ml, the people is: 1224U/ml.As seen the human lysozyme that the present invention produced has the biological activity suitable with the natural human N,O-Diacetylmuramidase, even surpasses natural human N,O-Diacetylmuramidase biological activity.

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

1 MKALIVLGLV?LLSVTVQGKV?FERCELARTL?KRLGMDGYRGISLANWMCLA

51 KWESGYNTRA?TNYNAGDRST?DYGIFQINSR?YWCNDGKTPG?AVNACHLSCS

101?ALLQDNIADA?VACAKRVVRD?PQGIRAWVAW?RNRCQNRDVR?QYVQGCGV

Claims (3)

1. the production method of the zooblast of a transgene cloning great cattle that changes human lysozyme gene, its operation steps is as follows: (1) makes up the mammary gland-specific expression vector that contains human lysozyme gene, (2) make up the cascaded structure that human lysozyme mammary gland-specific expression vector and double alternative carrier or single mark are selected carrier, cascaded structure DNA is imported in the somatic cell nuclear of heavy livestock, obtain transgenic cell, described mammary gland-specific expression vector is pBC2-sigHLY, and described pBC2 is at the constructed skeleton carrier of the disappearance 31bp of the 6407-6438bp place of pBC1; Described sigHLY is by ox beta-casein signal peptide sequence, the 1-4 exon of human lysozyme and 1-3 intron are formed, described mammary gland-specific expression vector and double alternative carrier cascaded structure DNA are pBC2-sigHLY-EGFP-NEO, it is pBC2-sigHLY-NEO that described mammary gland-specific expression vector and single mark are selected carrier cascaded structure DNA, and described heavy livestock is ox, goat, sheep, pig or rabbit.

2. production method according to claim 1, described introduction method are the electroporation transfection method.

3. production method according to claim 1, described heavy livestock are ox.

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