CN101851638A - Cow fetus fibroblast cell containing Iprl macrophage metamerism expression vector - Google Patents

Abstract

The invention discloses a cow fetus fibroblast cell containing an Iprl macrophage metamerism expression vector. A cow fetus fibroblast cell line which contains an Iprl macrophage metamerism expression vector Psp-EGFP-Iprl, is based on the vector and contains the Iprl macrophage metamerism expression vector is established, and the cell line is used as a nucleus donor cell, which provides massy foundation for transgene cow with tuberculosis. In the invention, an exogenous expression vector Psp-EGFP-Iprl is transduced to the cow fetus fibroblast cell through an electrotransfection method, the expression condition of a marked gene EGFP is observed by a fluorescence microscope, positive cells are selected by G418, the target gene Iprl is confirmed to be integrated into the genome of the cow fetus fibroblast cell by PCR identification, and finally the cow fetus fibroblast cell subjected to the transfection of the Iprl gene is moved into cow denucleated oocyte as a donor to obtain a transgene clone embryo.

Description

A kind of bovine fetal fibroblast that comprises Ipr1 scavenger cell opposite sex expression vector

Technical field

The invention belongs to the transgenic and cloned animal technical field, relate to the structure of the nuclear donor cell of transgene clone embryo, particularly a kind of bovine fetal fibroblast that comprises Ipr1 scavenger cell specific expression vector.

Background technology

Mycobacterium tuberculosis is the pathogenic bacterium lungy of infecting both domestic animals and human, and that mycobacterium tuberculosis is divided into is human-like, three types on ox type and fowl type, and there is the cross infection phenomenon in the three.Mycobacterium bovis is mainly encroached on ox, bovine tuberculosis be by the caused a kind of infecting both domestic animals and human of Mycobacterium bovis chronic infectious disease, its characteristics of lesion is that the disease ox becomes thin gradually, forms nodositas granuloma and caseous necrosis in histoorgan.Bovine tuberculosis is because of its infectivity, and harm is serious, so OIE classifies it as category-B transmissible disease, and China classifies them as two class animal infections.It is in 1917 that bovine tuberculosis appears in the U.S. first, and countries such as Britain, France are in state of a control with bovine tuberculosis, and China relatively lags behind to the research of bovine tuberculosis, bovine tuberculosis took place in Guangdong in 2003 in China, once also cause citizen's fear, causing social extensive concern.

For diagnosis lungy bacteriodiagnosis and serodiagnosis (comprising some molecular biological methods) are arranged.For prevention lungy, people's tuberculosis can be used bacille Calmette-Guerin vaccine and tuberculosis dna vaccination etc. when prevention; But the vaccine for bovine tuberculosis does not also have commercialization to use, and therefore can not prevent by immunity.So the sickness rate height of bovine tuberculosis can only be strengthened the quarantine to ox, take the principle of slaughtering without exception in case find the positive ox of tuberculosis.At present can only adopt antibiotic therapy, as Streptomycin sulphate, kantlex etc. to the treatment of bovine tuberculosis.But the life-time service microbiotic not only can make bacterium develop immunity to drugs, and also has the residual problem of milk Chinese traditional medicine, so it is extremely urgent to seek a kind of new treatment method lungy.

Development and perfection along with molecular biology and molecular genetics and genetic engineering technique, for animal disease resistant breeding provides new thinking, adopt the eighties in 20th century procaryotic injection to produce transgenic animal, but this method exists the random integration of foreign gene and the uncertainty that gamete system transmits foreign gene, has limited further developing of this technology.The embryonic stem cell somatocyte can replace procaryotic injection, but embryonic stem cell is not also built and is on domestic animal, therefore can not be widely used.Along with the birth of Dolly sheep, the somatocyte transgenosis is cultivated the premium animal kind in conjunction with clone technology was once becoming engineered focus.

Somatic cell clone technique is produced transgenic animal and has been shown great vitality (GOO J, et al.An approach for producing transgenic cloned cows by nuclear transfer of cellstransfected with human alpha 1-antitrypsin gene[J] .Theriogenology, 2006,65 (9): 1800-1812.), the advantage of this technology is that transgenosis was carried out in the somatic cell culture stage, directly utilize the fixed somatocyte that is integrated with goal gene to carry out body-cell neucleus transplanting (SCNT) for nuclear donor, the natality that the selection of donorcells not only can improve transgenic animal before the body-cell neucleus transplanting can also reduce its production cost (WHEELER MB like this, WALTERS EM.Transgenictechnology and applications in swine[J] .Theriogenology, 2001,56:1345-69.).2006, U.S. scientist Maga etc. cultivate specifically expressing human lysozyme in mammary gland transgenic goat (Maga E A etal Consumption of milk from transgenic goat expressionhuman lysozyme in the mammary gland result in modulation of intestinalmicrofluro[J] .Transgenic Ras, 2006,15:515-519).2009, Biological Engineering Inst., Xibei Agriculture and Forest Science and Technolog cultivated the transgenosis milk cow of specifically expressing human alpha-defensin in mammary gland.Present comparative maturity for somatocyte transgenosis and clone technology grasp.

Discoveries such as pan in 2005 are lungy to be relevant with intra-cellular pathogens resistant gene 1 (Ipr1), this studies have shown that the animal to the tubercule bacillus susceptible is because body does not have Ipr1 to express, and this gene is only expressed (Pan H in scavenger cell, et al.Ipr1 gene mediates innate immunity toTuberculosis[J] .Nature, 2005,434 (7034): 767-772).This provides new possibility for research prevents and treats tuberculosis: the mode of utilizing somatocyte transgenosis and somatic cell clone to combine makes the ox that lacks the Ipr1 expression realize the Ipr1 expression by the biotechnology means, to reach antituberculotic purpose.

Summary of the invention

The problem that the present invention solves is to provide a kind of Ipr1 of comprising scavenger cell opposite sex expression vector, and based on the bovine fetal fibroblast system that comprises Ipr1 scavenger cell opposite sex expression vector of this carrier, utilize this clone as the nuclear donor cell, for the transgenosis milk cow that solves bovine tuberculosis provides solid basis.

The present invention is achieved through the following technical solutions:

A kind of Ipr1 scavenger cell specific expression vector comprises goal gene Ipr1, at 5 of the Ipr1 gene ' sp promoter sequence of end insertion shown in SEQ.ID.NO.1.

Described Ipr1 scavenger cell specific expression vector also comprises antibiotic-screening gene and marker gene.

Described antibiotic-screening gene is the neo gene, and described marker gene is the EGFP gene.

Described Ipr1 scavenger cell specific expression vector is the Psp-EGFP-Ipr1 expression vector.

Described Psp-EGFP-Ipr1 expression vector, earlier by ECOR1 and Pst1 multiple clone site with the Ipr1 gene clone to the pSRA-EGFP carrier, and then by BglII and ECOR1I multiple clone site the sp promotor is cloned on the pSRA-EGFP carrier and obtains.

A kind of bovine fetal fibroblast that comprises goal gene Ipr1, its host cell are bovine fetal fibroblast, by transfection exogenous expression carrier Psp-EGFP-Ipr1, goal gene Ipr1 are incorporated into the genome of bovine fetal fibroblast.

Described host cell is the bovine fetal fibroblast in 3～10 generations.

Described bovine fetal fibroblast is by electrotransfection method transfection exogenous expression carrier Psp-EGFP-Ipr1.

The bovine fetal fibroblast that comprises goal gene Ipr1 is reorganization holstein cow fetal fibroblast-Psp-EGFP-Ipr1, and this cell is preserved in Chinese typical culture collection center, and preserving number is CCTCCC201005.

The described bovine fetal fibroblast that comprises goal gene Ipr1 makes up the application of the nuclear donor cell of transgene clone embryo as nuclear transplantation.

Compared with prior art, the present invention has following beneficial technical effects:

1, the present invention has made up a kind of Ipr1 scavenger cell specific expression vector, by in Ipr1 gene 5 ' insertion sp promotor, makes goal gene Ipr1 special efficiently expressing in the scavenger cell of ox.

2, the present invention is cloned into goal gene Ipr1 on the carrier is carrier Psp-EGFP, has made up Ipr1 scavenger cell specific expression vector Psp-EGFP-Ipr1, is transfected into bovine fetal fibroblast, makes up genetically modified bovine fetal fibroblast to be; Fluorescence microscopy is observed the expression of marker gene EGFP, and obtains positive cell through the G418 screening, and the performing PCR of going forward side by side is identified and confirmed that goal gene Ipr1 is incorporated into the genome of bovine fetal fibroblast.

3, make up the nuclear donor cell of transgene clone embryo as nuclear transplantation with the bovine fetal fibroblast that comprises goal gene Ipr1, obtain the transgene clone embryo by SCNT, the uterus that these embryos is moved into recipient cattle is expected to produce the ox that changes the Ipr1 gene, and this transgenic cattle can prevent generation lungy.

The preservation explanation

The preservation explanation

The present invention has carried out following preservation to described reorganization holstein cow fetal fibroblast-Psp-EGFP-Ipr1:

Depositary institution: Chinese typical culture collection center, address: wuchang, wuhan Luo Jiashan, preservation time: on March 28th, 2010; Preserving number is: CCTCC C201005, classification called after reorganization holstein cow fetal fibroblast-Psp-EGFP-Ipr1.

Description of drawings

Fig. 1 is the figure as a result that ECOR1 and Pst1 double digestion are identified the pTAIpr1 carrier;

Fig. 2 is the plasmid map of scavenger cell specific expression vector Psp-EGFP-Ipr1;

Fig. 3 is the electrophoresis qualification result figure of the sp promoter sequence of pcr amplification;

Fig. 4 is the figure as a result that BglII and BamH1 double digestion are identified positive recombinant plasmid Psp-EGFP-Ipr1;

Fig. 5 is the figure as a result of the recombinant bovine fetal fibroblast of fluorescence microscope Psp-EGFP-Ipr1 carrier positive expression;

Fig. 6 is to the figure as a result of the agarose gel electrophoresis after the genomic Ipr1 gene amplification of the bovine fetal fibroblast of Psp-EGFP-Ipr1 carrier positive expression;

Fig. 7 is the fate map that comprises the reconstituted embryo of Psp-EGFP-Ipr1 expression vector.

Embodiment

The present invention at first makes up the scavenger cell specific expression vector Psp-EGFP-Ipr1 that contains Ipr1 and green fluorescent protein (EGFP) gene, the electricity consumption infection protocol imports bovine fetal fibroblast with exogenous expression's carrier Psp-EGFP-Ipr1 then, the expression of fluorescence microscope marker gene EGFP, and through G418 screening acquisition positive cell, identify through PCR, confirm that goal gene Ipr1 is incorporated into the genome of bovine fetal fibroblast; At last, the bovine fetal fibroblast with transfection Ipr1 gene moves into the ox enucleation oocyte as nuclear donor, acquisition transgene clone embryo.

Concrete related reagent and material are as follows: G418, DMEM substratum are available from U.S. GIBICO (Invitrogen) company, EDTA and Trypsin are available from U.S. Sigma company, foetal calf serum is available from Chinese Hangzhou folium ilicis chinensis company, Tissue Culture Plate and culture dish are Denmark Nunclon company product, plasmid extraction kit and cellular genome are extracted test kit all available from sky root company, Hot start archaeal dna polymerase and T4DNA ligase enzyme are available from Takara company, electrotransfection instrument (ECM2001) is available from U.S. BTX company, and restriction enzyme is available from MBI company.The C57BL/6J mouse is available from Shanghai Communications University, Xi'an medical college, and the total RNA extraction reagent box is available from promega company.Opti-

I Reduced Serum Media is available from Invitrogen company.

The present invention is described in further detail below in conjunction with accompanying drawing and experiment, described be the present invention is explained rather than limit.

1, the structure of scavenger cell specific expression vector Psp-EGFP-Ipr1

1.1 the clone of goal gene Ipr1

Ipr1 gene order design primer P1 and P2 according to GeneID:AY845948 announced extract the total RNA of lung tissue of C57 mouse, obtain cDNA through reverse transcription, are that template is carried out pcr amplification with primer P1, P2 with cDNA, and described primer is as follows to sequence:

Forward primer P1:aggaacccct taactaatcc aggca 25;

Reverse primer P2:gctgggacac tcagaggctc aaag 24;

The PCR reaction system of 25 μ L is: 10 * PCR Buffer:5 μ L, dNTP (2.5mmol/L): 2 μ L, P1 (10 μ mol/L): 1 μ L, P2 (10 μ mol/L): 1 μ L, the LA enzyme: 0.3 μ L, template 1 μ L adds ultrapure water to 25 μ L.

The PCR reaction conditions is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s, 64.4 ℃ of annealing 45s, 72 ℃ are extended 2min, 35 PCR circulations; 72 ℃ are extended 10min again;

PCR product behind the purifying spent the night at 4 ℃ with pMD-18T to be connected, and the back transformed competence colibacillus cell E.coli DH5 α that spends the night selects recon by the blue hickie screening of α-Hu Bu, shakes bacterium, upgrading grain, has made up recombinant plasmid pTAIpr1.Identify positive recombinant plasmid pTAIpr1 and deliver the order-checking of Nanjing Jin Site company through ECOR1 and Pst1 double digestion.

The result that enzyme is cut evaluation as shown in Figure 1, the positive recombinant plasmid pTAIpr1 of swimming lane A wherein, swimming lane M is Maker, can see that the pTAIpr1 carrier can be seen the purpose band of 1614bp after the double digestion.Carrier pTAIpr1 sequencing result shows that the order-checking of Ipr1 and GeneID: the CDS district homology of the Ipr1 gene order (AY845948) of announcement is 100%, so far, and Ipr1 gene clone success.

1.2Psp-EGFP-Ipr1 carrier structure and building process thereof

Comprise goal gene Ipr1, as the sp promoter sequence of controlling element, and the scavenger cell idiosyncratic carrier Psp-EGFP-Ipr1 of resistance screening gene (neo) and marker gene EGFP, its element arrangements as shown in Figure 2:

Wherein, sp promotor downstream is goal gene Ipr1, the downstream of Ipr1 gene is SV40PolyA and resistance screening gene neo, and marker gene EGFP is positioned at the downstream of CMV promotor, and screening-gene neo and marker gene EGFP are applied to screen the reconstitution cell that Psp-EGFP-Ipr1 is integrated; The sp promotor is the giant cells specificity promoter, and the sp promotor just can instruct goal gene Ipr1 specific expression in the ox scavenger cell like this.

The present invention specifically is connected the goal gene of being cloned into and controlling element respectively with different carrier, be building up to together by multiple clone site then, specifically is to be that skeleton carrier makes up with psp-EGFP carrier and pSRA-EGFP-Ipr1 carrier.

1.2.1 psp-EGFP carrier (Macrophage-specific overexpression of antimicrobialpeptide PR-39 inhibits intracellular growth of Salmonella enterica serovarTyphimuriumin macrophage cells) is a kind of expression vector that comprises the scavenger cell specificity promoter, comprising the sp promotor; The present invention is with the clone template of this carrier as the sp promotor, and concrete clone is as follows:

With psp-EGFP is that template is carried out pcr amplification with primer, and wherein, primer is to being:

Forward primer P3:ga AgatctTa ataaaagcga cttcctcttt ccagcagaaa agga 44;

Reverse primer P4:cg GaattcGc tagcgactgg gtggcctcca gtgctccc 38;

Line in the middle of primer P3, the P4 partly is respectively BglII and ECORI restriction enzyme site;

The PCR reaction conditions is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s, 71.1 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 PCR circulations; 72 ℃ are extended 7min again.

The result that pcr amplification product is carried out gel electrophoresis as shown in Figure 3, wherein, swimming lane M is Maker, and swimming lane A is the sp promoter sequence of pcr amplification, the sp promoter sequence of 346bp of can having seen pcr amplification, its concrete nucleotide sequence is shown in SEQ.ID.NO.1.Behind pcr amplification product glue recovery purifying, behind the BglII/ECORI double digestion, glue reclaims the fragment that purifying has cohesive terminus.

1.2.2pEGFP-C1 carrier comprises G418 resistance screening gene neo and has the marker gene EGFP of CMV promotor, this carrier just is purchased and can obtains; In the pEGFP-C1 carrier transfection mammalian cell, marker gene EGFP is expressed the back and is produced green fluorescence.

The present invention is inserted into goal gene Ipr1 on the pEGFP-C1 carrier by multiple clone site ECOR1 and Pst1; And the control region of goal gene also comprises G/T bunch of polyA tailing signal and control Transcription Termination, these sequential structures are specifically provided by the pSRA-EGFP-Ipr1 carrier to the transcribing of mRNA, in intracytoplasmic stability and mediate its translational control and play important effect.

The pSRA-EGFP-Ipr1 vector construction is as follows:

With clone to Ipr1 sequence and pEGFP-C1 use EcoRI and PstI double digestion respectively, reclaim small segment and the big fragment of carrier, the T4DNA ligase enzyme spends the night, the transformed competence colibacillus bacillus coli DH 5 alpha, next day the picking positive colony, extract plasmid and cut evaluation with the EcoRI/Pst enzyme, correct called after: pEGFP-C1-Ipr1.

With the human blood genome is template, according to the SR-A promotor main transcription initiation site upstream-245～+ 46 between, amplified production 291bp, design of amplification primers is right, described amplimer is to being:

Forward primer P5:ga AgatctTa ataaccactg cactccaccc tggtgagac 39;

Reverse primer P6:g GaattcCtc atagtatttc agcatctggt ac 32;

5 ' end at forward primer P5 adds BglII restriction enzyme site and TAATAA, adds the EcoRI restriction enzyme site at reverse primer P65 ' end, and underscore is labeled as restriction enzyme site.

The PCR condition is 94 ℃ of sex change 30s; 59 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 30 circulations.After pcr amplification product separated with 2% agarose gel electrophoresis, glue reclaimed the band that meets the goal gene size.

PEGFP-C1-Ipr1 and SRA are used BglII and EcoRI double digestion respectively, reclaim the SR-A enzyme and cut product and the big fragment of pEGFP-C1-Ipr1, the T4DNA ligase enzyme spends the night, the transformed competence colibacillus bacillus coli DH 5 alpha, next day the picking positive colony, extract plasmid and identify correct name pSRA-EGFP-Ipr1 with the BglII/PstI double digestion.

1.2.3 after the skeleton carrier structure was finished, the structure of Psp-EGFP-Ipr1 carrier was as follows:

With pSRA-EGFP-Ipr1 BglII and ECORI double digestion, the double digestion system is: pSRA-EGFP-Ipr1:20 μ L, and BglII:2 μ L, ECORI:2 μ L adds ultrapure water to 40 μ L.

The big fragment of pSRA-EGFP-Ipr1 carrier with BglII and ECORI double digestion, with the sp promoter sequence fragment behind BglII and ECORI double digestion, spend the night with 4 ℃ of connections of T4 dna ligase, connect and finish transformed competence colibacillus cell E.coli DH5 α afterwards, recon is selected in blue hickie screening by α-Hu Bu, identifies positive recombinant plasmid with BglII and BamH1 double digestion behind the extraction plasmid; Qualification result as shown in Figure 4, wherein swimming lane M is Marker; Swimming lane A is that BglII and BamH1 double digestion are identified positive recombinant plasmid, can see two bands of about 4700bp and 1960bp, and the 4700bp band is the big fragment of pEGFP-C1 carrier that enzyme is cut, and the band of 1960bp is sp promotor and Ipr1 gene order fragment; With the recombinant plasmid called after Psp-EGFP-Ipr1 expression vector that obtains.

Because plasmid is bred in intestinal bacteria, easily bacterial endotoxin is taken in the end product with common plasmid extracting method, the existence of bacterial endotoxin can influence plasmid transfection efficient and cell growth, so having used, the present invention removes endotoxic plasmid extraction kit, to reduce the negative impact of intracellular toxin to test-results.The endotoxic plasmid purification test kit of going with promega company extracts concentration and the purity of measuring plasmid behind the plasmid with the nucleic acid-protein determinator, after measured, the concentration of plasmid is 508.3ng/ μ l, and OD260/280 is 2.12, illustrate that plasmid is purer, can be used for follow-up cell transfecting.

2, Psp-EGFP-Ipr1 expression vector transfection bovine fetal fibroblast makes up nuclear donor clone

2.1 the cultivation of bovine fetal fibroblast

The bovine fetal fibroblast of getting a pipe third generation holstein cow from liquid nitrogen thaws in 38 ℃, and is centrifugal.Discard waste liquid, it is resuspended to add 3ml cell suspending liquid (DMEM that contains 10%FBS), is inoculated in the culture dish of 60mm (DMEM that contains 10%FBS), places CO ₂Cultivate under 37 ℃ of conditions in the incubator.

Treat that bovine fetal fibroblast reaches 80% when converging, inhale and abandon nutrient solution, with no Ca ²⁺, Mg ²⁺PBS flushing cell, add pancreatin and EDTA mixture slaking liquid, observation of cell under inverted microscope.When treating that most cells becomes circle, intercellular substance expansion, the DMEM cell culture fluid that contains 10% foetal calf serum with equal-volume stops digestion, and behind pipettor piping and druming mixing, centrifugal collection suspends, and is inoculated in the 60mm culture dish in 1: 3 ratio, puts into CO ₂Cultivate in the incubator, choose the cell that 3～10 generations went down to posterity and reach 90% bovine fetal fibroblast that converges, as the host cell that is used for transfection.

The present invention with G418 as screening of medicaments, owing on the skeleton of expression vector Psp-EGFP-Ipr1 G418 resistant gene neo is arranged, under the bovine fetal fibroblast that foreign gene Psp-EGFP-Ipr1 obtains expressing can be survived in the nutrient solution that contains finite concentration G418, and the normal cell of untransfected can be dead under this concentration, and the minimum lethal concentration that therefore needs to determine normal cell G418 is used as screening concentration.

The mensuration of G418 minimum lethal concentration: the G418 that adds concentration gradient in bovine fetal fibroblast nutrient solution (DMEM cell culture fluid) respectively screened for 1 week, measured Normocellular G418 minimum lethal concentration.When the concentration of G418 in the cell culture fluid during more than or equal to 600 μ g/ml, all dead at the microscopically visible cell, so the minimum lethal concentration of G418 is 600 μ g/ml.

2.2Psp-EGFP-Ipr1 expression vector transfection bovine fetal fibroblast

Transgenic method has a variety of, comprises electroporation, microinjection, particle bombardment, virus vector method, receptor-mediated method etc.The present invention specifically adopts the electrotransfection method, with expression vector Psp-EGFP-Ipr1 transfection bovine fetal fibroblast.

Cell reaches 90% when converging, and centrifugal behind the peptic cell (1000r/min, 4min) collecting cell are used Opti-then

I Reduced Serum Media washed cell 2 times, will be gently in each process of cleaning slowly piping and druming cell, prevent that cell from suffering damage, with electrotransfection liquid (cell salt: opti=3: 1V/V; Cell salt: Kcl:120mm; Cacl2:0.15mm; K2HPO4:10mm; Mgcl2:50mm; Transfer PH to 7.6; Opti is Opti-

I Reduced Serum Media) and the mixed solution (800 μ l) of expression vector Psp-EGFP-Ipr1 (20 μ g) be added in the centrifugal centrifuge tube that discards nutrient solution (adding opti to 800 μ L after adding plasmid), blow and beat cell gently, it is dispelled fully, mixing.Above-mentioned cell suspension is transferred in the BTX electric shock cup (4mm gap, yellow lid, 800 μ L volumes), left standstill 10min; Be provided with the electrotransfection parameter as follows: voltage: 500V; Burst length: 1ms; Number of shocks 3 times leaves standstill to finish and carries out electrotransfection afterwards.

After transfection was finished, cell left standstill 10min, then cell suspension was all transferred in the 60mm culture dish; The DMEM complete culture solution 3mL that contain 10% foetal calf serum that add 37 ° of pre-temperature place 37 °, 5% CO with nutrient solution ₂In the incubator, treat cell attachment after, discard nutrient solution (contain in the nutrient solution electrotransfection liquid influence cell growth), change into through the DMEM of 37 ° of incubations complete culture solution 4mL, add behind the 24h G418 to final concentration be 600 μ g/ml.

2.3Psp-EGFP-Ipr1 the cell screening of carrier positive expression

Behind the Psp-EGFP-Ipr1 transfection bovine fetal fibroblast 24h, in containing the DMEM cell culture fluid of serum, add the G418 screening of the minimum lethal concentration of 600 μ g/ml; With the negative contrast of the bovine fetal fibroblast of untransfected Psp-EGFP-Ipr1, its cell culture fluid is added with the G418 (600 μ g/ml) of same concentration.

Behind the Psp-EGFP-Ipr1 transfectional cell 24h, expression vector can be seen green fluorescence after being expressed under fluorescent microscope.When genetically modified bovine fetal fibroblast sends as shown in Figure 5 green fluorescence, illustrate that Psp-EGFP-Ipr1 has entered cell and positive expression; Behind the Psp-EGFP-Ipr1 transfectional cell 7d, the cell of control group is all dead, the G418 concentration that will comprise Psp-EGFP-Ipr1 transfection positive cells group reduces by half and continues screening at the bottom of cell covers with ware, can see positive cell and form island shape cell mass, under fluorescent microscope, still can see green fluorescence; Peptic cell usefulness does not add the normal nutrient solution enlarged culturing of G418 then.

The positive cell of the present invention's screening all is the cell of stable transfection Psp-EGFP-Ipr1, and goal gene Ipr1 is incorporated on the genome of cell, rather than is free on outside the genome; In the process of stable transfection, the gene that carries on the plasmid Psp-EGFP-Ipr1 can be incorporated on the genome of host cell; Screen 7 days lasting purposes that reach stable transfection of screening of half-value dose again by G418, show as reporter gene continuous expression in transfected positive cell, therefore the positive cell of screening continues to send green fluorescence and presents the G418 resistance.

What more than make up passes through integration Psp-EGFP-Ipr1 carrier to cellular genome, obtains comprising the holstein cow fetal fibroblast of goal gene Ipr1.

2.4Psp-EGFP-Ipr1 the PCR of carrier positive expression cell identifies

Get the bovine fetal fibroblast of the Psp-EGFP-Ipr1 positive expression after the enlarged culturing, extract cell genomic dna, with the genomic dna is template, PCR identifies whether the Ipr1 gene is incorporated in the cellular genome, negative control is the normal bovine fetal fibroblast of untransfected, and PCR identifies that the primer is to being P1 and P2;

The PCR reaction conditions is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s, 64 ℃ of annealing 40s, 72 ℃ are extended 2min, 35 PCR circulations; 72 ℃ are extended 10min again; Reclaim the PCR product and carry out the detection of 0.8% agarose gel electrophoresis, detected result as shown in Figure 6, wherein, swimming lane 1 is DNAMarker, swimming lane 2 is the bovine fetal fibroblast of Psp-EGFP-Ipr1 transfection, swimming lane 3 is the bovine fetal fibroblast of untransfected, swimming lane 2 is compared with swimming lane 3, obviously can see a band (goal gene Ipr1 is 1614bp) about 1600bp, and see not that as the swimming lane 3 of negative control illustration purpose gene Ipr1 has been incorporated into the genome of host cell bovine fetal fibroblast.

When the Ipr1 gene integration has arrived the genome of host cell bovine fetal fibroblast, sp promotor, EGFP also can be incorporated into the genome of bovine fetal fibroblast with goal gene Ipr1.

The present invention is to the positive reconstitution cell of expression vector Psp-EGFP-Ipr1 of having verified correct transfection of the PCR through reorganization screening and reconstitution cell, deliver Chinese typical culture collection center (CCTCC) and carry out preservation, classification called after reorganization holstein cow fetal fibroblast-Psp-EGFP-Ipr1, preserving number is: CCTCC C201005.

3, with above-mentioned genome conformity the bovine fetal fibroblast of goal gene Ipr1 be the nuclear donor cell, make up the transgene clone embryo

3.1 the maturation of ovocyte is cultivated

Ovary picks up from the slaughterhouse, Xi'an, the aseptic holstein cow ovary of taking, in 37 ℃ of stroke-physiological saline solution, transport the laboratory back in 4～6 hours, extract the ovarian follicle of 3～8mm diameter, collect ovarian cumulus ovocyte complex body, select under the stereomicroscope and have complete cumulus cell more than three layers, the uniform ovocyte of kytoplasm is used for ripe the cultivation.The maturation culture solution of ovocyte is: TCM199 (Gibico) adds 10% foetal calf serum, the Urogastron of 10ng/ml, and culture condition is: 38.5 ℃, 5%CO ₂, 95% air atmosphere surrounding, saturated humidity; Remove cumulus cell with 0.2% Unidasa behind the ripe 20h of cultivation,, select mature oocyte and be used for nuclear transplantation and test with the discharging of first polar body determination flag as oocyte maturation.

3.2 the structure of transgene clone embryo

Adopting body-cell neucleus transplanting (SCNT) technology that donorcells is transferred to removes in the nuclear mature oocyte, wherein, the donorcells that is integrated with foreign gene is crucial, the present invention controls to donorcells in 10 generations, and this mainly considers to reduce the accumulation of sudden change in culturing process that vitro culture causes.

Nuclear transplantation is specially: micrurgy liquid is for containing 10%FBS, the PBS of 5 μ g/ml cytochalasin Bs, stoning pipe sucking-off first polar body and part kytoplasm on the micrurgy instrument with internal diameter 20 μ m, with 10 μ g/mlHoechst, 33342 dyeing 10min, under fluorescent microscope, choose complete non-nucleus egg mother cell; Remove first polar body fully and chromosomal ovocyte is used to nuclear transplantation.

The notes nuclear and the fusion of ovocyte, detailed process is as follows: the He Sitan bovine fetal fibroblast of the transfection Psp-EGFP-Ipr1 in 6～10 generations of 0.25% tryptic digestion contact inhibition 3d is used as donor cell.With the stoning pipe donorcells is injected under the ovocyte zona pellucida of stoning success.The caryoplasm complex body merges balance 3min in the liquid at electricity, merge with the microelectrode method, use the microelectrode tip that is connected with the micrurgy instrument to arrange recombinant chou, make the film contact surface vertical with the line of two electrodes, push down recombinant chou gently with the microelectrode point, give electricimpulse and carry out the electricity fusion.Fusion voltage is 28V, and time of fusion is 10 μ m.Recombinant chou after the fusion is put into the M199 of 10%FBS, 38.5 ℃, 5%CO ₂, full closing under the humidity cultivate, and observes the fusion situation behind the 2h.

3.3 the activation of transgene clone embryo and vitro culture

The reconstituted embryo that merges is in containing the M199 of 10%FBS behind the balance 2h, with containing 5 μ mol/L ionomycin (Ionomycin, available from SIGMA company) mSOFaa nutrient solution (available from SIGMA company) handle 5min, in the mSOFaa nutrient solution that contains 2mmol/L dimethylaminopurine (6-DMAP), cultivate 4h then, clean change after 3 times that mineral oil covers over to and in advance in the CO2 incubator balance cultivate among the mSOFaa of 2h at least, culture density is 5 each reconstituted embryo of μ L, at 38.5 ℃, 5%CO ₂, cultivate under the saturated humidity, checked the blastaea developmental state on the 7th～9 day, as shown in Figure 7, the normal development of clone's embryo is to blastaea, black part is divided into inner cell mass among the figure, the periphery is the trophocyte.And integrated the successfully constructing of transgene clone embryo of scavenger cell specific expression vector Psp-EGFP-Ipr1, for the prevention bovine tuberculosis provides solid basis.

The nucleotides sequence tabulation

＜110〉Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology, clone limited-liability company of unit of Yang Ling section

＜120〉a kind of bovine fetal fibroblast that comprises Ipr1 scavenger cell opposite sex expression vector

<160>2

<210>1

<211>352

<212>DNA

＜213〉the sp promotor of synthetic

<400>1

ttcgctgaag?gagaaaggtc?gtcttttcct?cttcatcctc?ggttctaaag?gtttgagaca 60

ccaacggaac?ttcgctgaag?gagaaaggtc?ttcgctgaag?gagaaaggtc?gtcttttcct 120

cttcatcctc?gaggatgaag?aggaaaagac?ggttctaaag?gtttgagaca?ccaacggaac 180

gaggatgaag?aggaaaagac?gtcttttcct?cttcatcctc?gacctttctc?cttcagcgaa 240

atgggaggga?cgggggacag?ggctggcgct?gttttcgctg?aaggagaaag?gtcacgtaaa 300

ttccgcgtcg?gaccttcacg?gtccctcgtg?acctccggtg?ggtcagcgat?cg 352

<210>2

<211>1614

<212>DNA

＜213〉the Ipr1 gene of synthetic

<400>2

aggaacccct?taactaatcc?aggcagtgac?ctgggagaac?tcgggagaac?ccgtggcagc 60

cctcagcatc?caggatgttc?actctgacca?aagccttgga?aaaggctctt?ctccagcatt 120

tcatatacat?gaaggtgaac?atcgcctatg?ccatcaacaa?gccattcccc?ttcttcgaag 180

cgctccggga?caattccttc?atcactgaga?gaatgtacaa?ggaatctctg?gaagcctgtc 240

aaaatctggt?ccctctgtcc?aaagtggtgc?acaatattct?caccagtctg?gagcagactt 300

tccacccgtc?agtgctgctg?acgttgttca?gcaaggtcaa?cctccgggaa?taccccagcc 360

tggtggcaat?tttcagaagc?ttcagaaacg?ttggttatac?ctacgaagag?aaaaacagac 420

ccccactgac?cctgcttgaa?gacctggcca?acccagcaga?agggtgctcc?cttcagacac 480

tgctgccacc?accccgaccc?cagatatcgc?tgccaagtca?tctgtcctca?gcaccgagag 540

tctgtgaccc?cagagcaacc?gcacagccaa?tcattgagat?cctggatgag?cagcccagtc 600

cttctccccg?agctgtgcct?ctccttggct?gcattcagga?aggaaaaacc?actccagtgt 660

cctccagaga?tcaccagaga?aaagataagg?aagactctcg?agagatgccc?cacagtccct 720

caggacccga?gtcagtggta?aaagatgact?ctccagcagc?aaatgacctg?gaaatggcca 780

gggaagtacc?ctgcacacct?gcaaacaaga?aagcaagaag?aaaaaaacgt?ccgaactggt 840

caaattccaa?aagaagacgg?cagaaaaaaa?agccccgtca?agatgagatg?atgggagtgg 900

cctcacctgg?acatggagtt?caagagaagc?tcaaggcagt?gagcaggagg?actttgtgga 960

aagatgactc?atctacgaac?gtgaaggagg?tgaccaagac?acagagaaca?aggatgaggc 1020

gtgcccagac?atccaattca?caagagatca?gcaaagaggc?atcaaaaaca?agtggtagaa 1080

agaggcccag?cacagcacga?agaaccacac?aagttccaga?gaagaccaag?aatgacgctg 1140

tggatttctc?tcccacactc?cctgtgacct?gtggtaaggc?caaagggact?ttgttccaag 1200

agaaactgaa?gcaaggagcc?tcaaaaaagt?gcattcagaa?tgaggcagga?gattggctca 1260

ctgtaaagga?atttttaaat?gaagggggaa?gggccacatc?aaaagactgg?aagggcgtta 1320

tacgttgtaa?cggggagaca?ttaagacatc?tggagcagaa?aggacttttg?ttctttacct 1380

ccaagagtaa?acctcaaaag?aagggtgcct?agcagatgaa?ctcctgacct?gatatgtgct 1440

cagctcctgt?gctcgctcgc?actctgtctc?tgtctctctg?tctctctctg?tctctctcta 1500

tctctgtctc?tctctctctc?tctctctctc?tctctctctc?tctctctgtg?tgtgtgtgtg 1560

tgtgtgtgtg?tgtgtgtgtc?tgaactttgc?ctttgagcct?ctgagtgtcc?cagc 1614

Claims (10)

1. an Ipr1 scavenger cell specific expression vector is characterized in that, comprises goal gene Ipr1, at 5 of the Ipr1 gene ' sp promoter sequence of end insertion shown in SEQ.ID.NO.1.

2. Ipr1 scavenger cell specific expression vector as claimed in claim 1 is characterized in that Ipr1 scavenger cell specific expression vector also comprises antibiotic-screening gene and marker gene.

3. Ipr1 scavenger cell specific expression vector as claimed in claim 2 is characterized in that described antibiotic-screening gene is the neo gene, and described marker gene is the EGFP gene.

4. Ipr1 scavenger cell specific expression vector as claimed in claim 1 is characterized in that, described Ipr1 scavenger cell specific expression vector is the Psp-EGFP-Ipr1 expression vector.

5. Ipr1 scavenger cell specific expression vector as claimed in claim 4, it is characterized in that, described Psp-EGFP-Ipr1 expression vector, earlier by ECOR1 and Pst1 multiple clone site with the Ipr1 gene clone to the pSRA-EGFP carrier, and then by Bg1II and ECORI multiple clone site the sp promotor is cloned on the pSRA-EGFP carrier and obtains.

6. bovine fetal fibroblast that comprises goal gene Ipr1, it is characterized in that, its host cell is a bovine fetal fibroblast, by transfection exogenous expression carrier Psp-EGFP-Ipr1, goal gene Ipr1 is incorporated into the genome of bovine fetal fibroblast.

7. the bovine fetal fibroblast that comprises goal gene Ipr1 as claimed in claim 6 is characterized in that, described host cell is the bovine fetal fibroblast in 3～10 generations.

8. the bovine fetal fibroblast that comprises goal gene Ipr1 as claimed in claim 6 is characterized in that, described bovine fetal fibroblast is by electrotransfection method transfection exogenous expression carrier Psp-EGFP-Ipr1.

9. the bovine fetal fibroblast that comprises goal gene Ipr1 as claimed in claim 6, it is characterized in that, the bovine fetal fibroblast that comprises goal gene Ipr1 is reorganization holstein cow fetal fibroblast-Psp-EGFP-Ipr1, this cell is preserved in Chinese typical culture collection center, and preserving number is CCTCC C201005.

10. the described bovine fetal fibroblast that comprises goal gene Ipr1 makes up the application of the nuclear donor cell of transgene clone embryo as nuclear transplantation.

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