CN100439505C

CN100439505C - Carrier structure and use for transfection cell separation containing CD34 labelled gene

Info

Publication number: CN100439505C
Application number: CNB2004100499995A
Authority: CN
Inventors: 裴雪涛; 岳�文; 曲笑霞; 秦立篷; 高艳红; 闫舫
Original assignee: Institute of Field Blood Transfusion Chinese Academy of Military Medical Sciences
Current assignee: Institute of Field Blood Transfusion Chinese Academy of Military Medical Sciences
Priority date: 2004-06-25
Filing date: 2004-06-25
Publication date: 2008-12-03
Anticipated expiration: 2024-06-25
Also published as: CN1712535A

Abstract

The present invention relates to a method for constructing a carrier containing a CD34 marker gene for cell sorting infection and an application thereof, which belongs to the field of biomedicine. The present invention constructs a carrier containing a nucleotide sequence for encoding a cell surface molecule CD34 and uses a construction form of a multigene coexpression carrier for inserting a target nucleotide sequence into the polycloning site of the carrier for infecting cells, and then the CD34 gene in the carrier and the target nucleotide sequence can realize coexpression. A new cell surface marker obtained by the infected cells uses the principle of immune combination for realizing the purpose of sorting positive cells. The present invention provides the new method for sorting infected cells so as to not only simplify traditional complex and time-consuming processes for sorting infected cells, but also enhance the sorting efficiency of the infected cells, and moreover, the present invention has admirable application prospect in the field of biomedicine.

Description

Contain structure and application thereof that the CD34 marker gene is used for the carrier of transfectional cell sorting

Technical field

The present invention relates to biomedical sector, a kind of specifically CD34 of containing cell surface marker gene is used for the construction of carrier and the application thereof of transfectional cell sorting.

Background technology

In genetically engineered, foreign gene is carried into host cell by carrier, and the technology that foreign gene is efficiently expressed that increases in host cell has application very widely.But after with the carrier transfectional cell, how from the cell mass of enormous amount, to detect and sub-elect, just become a key setting up the eukaryotic cell expression system to the few transformant of number.

Used method is generally and utilizes the marker gene of carrying in the carrier at present, and by adding corresponding resistance medicine or screen by other modes in substratum, the most frequently used is G418 (geneticin) screening.G418 has toxicity to eukaryotic cell, but when the neomycin resistance gene that carries in the carrier (neo) is expressed in eukaryotic cell, can produce a kind of phosphotransferase, make the G418 inactivation, thereby the cell that transfection is advanced carrier and expressed neomycin resistance gene can be avoided being killed.Also have methods such as thymidine kinase gene (tk) selective system, dihydrofolate reductase gene (dhfr) selective system, chloramphenicol acetyl transferasegene (cat) detection system in addition, but because marker gene and goal gene might not be expressed simultaneously in the transfectional cell, when therefore transfectional cell being screened by these class methods, efficient not only consuming time but also low will sub-elect the time that the cell strain of having expressed goal gene need expend one even some months usually.

Some characteristic that can also utilize transfectional cell to obtain is in some cases carried out sorting by flow cytometer, but the required expense of airflow classification is higher, it is applied be restricted.

In recent years, by immunologic method, some marker molecule that promptly utilizes cell surface has a wide range of applications in biomedical scientific research and clinical study by the method for the cell of Ag-Ab association reaction sorting type, for example utilizes immunomagnetic beads method (MACS) separation of C D34 ⁺Hematopoietic stem or the like.We combine immunologic cell sorting method and are applied to the sorting of transfectional cell with the construction strategy of polygene co-expression carrier, not only simplified the loaded down with trivial details time-consuming procedure again of traditional transfectional cell sorting, and improved the efficiency of separation of transfectional cell.

In the selection of cell surface marker gene, the CD34 gene has received our concern because of its significant advantage.At present, for CD34 ⁺The sorting of cell has very sophisticated method, and many commercial reagent and equipment are arranged, and can make things convenient for, sorting CD34 efficiently ⁺Cell.We by change over to goal gene the time, change marker gene CD34 over to cell together, also obtain the CD34 surface marker when making transfectional cell express goal gene, thereby utilize the CD34 surface marker, easily the sorting transfectional cell.

Summary of the invention

The purpose of this invention is to provide a kind of carrier that is used for the transfectional cell sorting.It is characterized in that, the nucleotide sequence that comprises Codocyte surface molecular CD34 in the carrier, the purpose nucleotide sequence is inserted this carrier multiple clone site after transfectional cell, both can realize co expression, by transfectional cell the new CD34 surface marker molecule that obtains utilize immune bonded principle to realize the purpose of sorting positive cell.

Adopt the building mode of polygene co-expression carrier, realize the co expression of CD34 gene and purpose nucleotide sequence.Every vector construction strategy of two or more different genes co expression of can realizing all can be applied to this, commonly used at present is to make up the bicistronic mRNA carrier, connect with certain nucleotide sequence between two genes, realize its co expression, for example the construction process that two genes is connected with sequences such as IRES (internal ribosome entry site), 2A.We utilize the IRES sequence, make up the bicistronic mRNA carrier.

Behind the carrier transfectional cell described in the present invention, when expressing goal gene, transfectional cell can obtain a kind of new cell surface marker CD34.Utilize this kind cell surface marker, adopt immunologic method, certain mark (for example immunomagnetic beads) is incorporated on the cell surface, can realize the sorting of transfectional cell then by corresponding cell sorting technology (for example immune magnetic sorting).

This carrier that we make up provides a kind of thinking and method of new sorting transfectional cell, has simplified loaded down with trivial details in the past transfectional cell assorting room, has improved the efficiency of separation of transfectional cell, has splendid application prospect at biomedical sector.

Below with the example that is configured to of plasmid vector pcDNA3.1 (+)-IRES-CD34, and in conjunction with the accompanying drawings the present invention is described in detail.

Description of drawings

Fig. 1 is the structure schema of plasmid vector pcDNA3.1 (+)-IRES-CD34.

Fig. 2 is that the PCR of plasmid vector pcDNA3.1 (+)-IRES-CD34 identifies.

The M:DL2000 molecular weight marker;

1:pcDNA3.1 (+)-IRES-CD34 is through the partial sequence of pcr amplification IRES;

2:pcDNA3.1 (+)-IRES-CD34 is through pcr amplification CD34 sequence;

Fig. 3 is that the enzyme of plasmid vector pcDNA3.1 (+)-IRES-CD34 is cut evaluation.

The M1:DL2000 molecular weight marker;

The M2:DL15000 molecular weight marker;

1:pcDNA3.1 (+)-IRES-CD34 is through the single enzymic digestion of Hind III.

2:pcDNA3.1 (+)-IRES-CD34 is through the two enzymic digestions of EcoR I, Xba I.

Fig. 4 is the collection of illustrative plates of plasmid vector pcDNA3.1 (+)-IRES-CD34.

Embodiment

The structure of embodiment 1, pcDNA3.1 (+)-IRES-CD34 carrier

One, CD34-cDNA inserts the T-easy carrier.The CD34 gene isolation is from CD34 ⁺Cell is also introduced Nco I and Xba I, Sph I restriction enzyme site synthetic primer respectively according to the sequence of CD34-cDNA coding region, and the condition of primer sequence and polymerase chain reaction thereof (PCR) is as follows:

P1：5′-CGC CCATGGTGCTGGTCCGCAGGGGC-3′

P2：5′-CGC GCATGCTCTAGATTAGCGGCGATTCATCAGGAA-3′

50 μ l reaction systems comprise: 2ul cDNA, primer1 and 2 each 2 μ l (10 μ M), 4 μ l dNTP (2.5mM), 0.5 μ l high-fidelity Taq archaeal dna polymerase (BioDev company), 5 μ l, 10 * PCR Buffer, 34.5 μ l aqua sterilisas.Reaction conditions is: 94 ℃ of sex change 5min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 80sec, 33 circulations; 72 ℃ of 10min.Reaction product is cloned into pGEM-T easy carrier (Promega company, the U.S.), obtains cloned plasmids T-CD34.

Two, IRES (EMCV) inserts T-CD34.Plasmid pIRES2-EGFP digests with restriction enzyme Nco I, Sal I, reclaim on Nco I that purpose fragment IRES is inserted into the T-CD34 carrier, the Sal I site, the carrier T-IRES-CD34 of IRES and marker gene CD34 is carried in acquisition, after the two enzymic digestions of Sal I, Xba I, the TAE agarose gel electrophoresis obtains the band of 1.6kb, meets with expected results.

Three, the structure of pcDNA3.1 (+)-IRES-CD34 carrier.With the two restriction enzyme digestion of Sal I, Xba I T-IRES-CD34 carrier, obtain the IRES-CD34 fragment, insert multiple clone site through pcDNA3.1 (+) carrier of the two restriction enzyme digestion of Xho I, Xba I.After the two enzymic digestions of EcoR I, Xba I, obtain the 1.6kb band, meet with expected results; After the single enzymic digestion of Hind III, obtain the 300bp band, meet with expected results.Identify that through PCR CD34 is positive.The design primer identifies that IRES is as follows:

P1：5′-CGGTGGGAGGTCTATATAAGC-3′

P2：5′-TTATTCCAAGCGGCTTCG-3′。

20 μ l reaction systems comprise: 1 μ l plasmid, primer1 and 2 each 1 μ l (10 μ M), 2 μ l dNTP (2.5mM), 0.2 μ l rTaq enzyme (TakaRa company), 2 μ l, 10 * PCR Buffer, 12.8 μ l aqua sterilisas.Reaction conditions is: 94 ℃ of sex change 5min; 94 ℃ of 30s, 48 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 7min.The IRES qualification result is positive.It is checked order and analysis revealed, and carrier pcDNA3.1 (+)-IRES-CD34 successfully constructs.

Present embodiment is to have made up the bicistronic mRNA carrier that contains cell surface marker gene C D34, adopt internal ribosome entry site (the internal ribosome entry site of encephalomyocarditis virus EMCV, IRES) sequence connects, to realize the co expression of goal gene and cell surface marker gene.

The structure of embodiment 2, recombinant vectors pcDNA3.1 (+)-IRES-CD34-luc

Design primer from carrier pAP1-luc amplification luc gene, and introduce NheI, EcoRI restriction enzyme site, (PCR) is as follows for primer sequence and polymerase chain reaction thereof:

P1：5′- GCTAGCATGGAAGACGCCAAAAACA-3′

P2：5′- GAATTCAATTACACGGCGATCTTTCC-3′。

50 μ l reaction systems comprise: 2 μ l plasmids, primer1 and 2 each 2 μ l (10 μ M), 4 μ l dNTP (2.5mM), 0.3 μ l Pyrobest Taq enzyme (TakaRa company), 5 μ l, 10 * Pyrobest Buffer, 34.7 μ l aqua sterilisas.Reaction conditions is: 94 ℃ of sex change 5min; 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 90s, 33 circulations; 72 ℃ of 10min.Reaction product is cloned into pGEM-T easy carrier (Promega company, the U.S.), obtains cloned plasmids T-luc.

With the two restriction enzyme digested plasmid T-luc of NheI, EcoRI, the goal gene luc fragment (about 1650bp) that obtains is inserted the multiple clone site through the 3.1-IRES-CD34 carrier of the two restriction enzyme digestion of NheI, EcoRI.After PCR evaluation, enzyme are cut evaluation correctly, it is carried out sequencing.Obtain recombinant vectors pcDNA3.1 (+)-IRES-CD34-luc.

Embodiment 3, recombinant vectors pcDNA3.1 (+)-IRES-CD34-luc transfectional cell

The Chinese hamster ovary celI routine that growth conditions is good is cultured to 50%～75% to be converged, and uses the trysinization collecting cell, and the electroporation damping fluid flushing twice with precooling is suspended in cell in this damping fluid then again, makes the final concentration of cell be (0.5-1.0) * 10 ⁷Individual cell/ml.Get the 0.8ml cell suspension and put into electric revolving cup, add an amount of recombinant vectors pcDNA3.1 (+)-IRES-CD34-luc and thorough mixing, place 10min in the ice bath, electric shock once.After the electric shock electric revolving cup is put into ice bath 10min.With suitable perfect medium diluting cells, place 37 ℃, 5%CO ₂Cultivate in the incubator.

Embodiment 4, utilization MACS system sorting transfectional cell

Transform the back cell routine and cultivated 24～48 hours, the foreign gene of conversion is expressed.Discard old substratum, add the digestion of trysinization liquid, sucking-off Digestive system gently, add 10ml PBS (containing 2mM EDTA) pressure-vaccum gently, be transferred to centrifugal (800g in the centrifuge tube, 10min) collecting cell is washed once with 10ml PBS (containing 2mM EDTA), adopts Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec) to utilize the method for immunological magnetic bead sorting (MACS) that it is carried out sorting afterwards.The cell that sorting obtains continues to cultivate after can directly carrying out next step analysis or adding substratum.

Adopt the carrier among the present invention to compare with conventional carrier, can greatly improve the transfectional cell efficiency of separation, it is thin to shorten transfection The cycle of born of the same parents' sorting.

Claims

1, a kind of CD34 of containing marker gene is used for the carrier of transfectional cell sorting, it is characterized in that, is the plasmid-type bicistronic mRNA carrier for expression of eukaryon that utilizes IRES and CD34 to make up.

2, the described construction of carrier of claim 1, it is characterized in that, adopt the bicistronic mRNA construction of carrier, the multiple clone site that the purpose nucleotide sequence is inserted this carrier obtains the co expression of purpose nucleotide sequence and CD34 gene after transfectional cell.

3, the application chosen at the transfectional cell branch of the described carrier of claim 1 promptly utilizes the CD34 cell surface marker of transfectional cell new acquisition of institute when expressing purpose Nucleotide and the purpose cell is carried out sorting.

4, application according to claim 3 is characterized in that, when described transfectional cell carries out sorting to the purpose cell, utilizes immune bonded principle to adopt immunomagnetic beads to separate or the immunoabsorption separation means.