CN104419724A - T lymphocyte model constructed by virtue of SV40LT gene inducing and cell tank thereof - Google Patents

Abstract

The invention relates to construction of an SV40LT gene induced T lymphocyte model and a cell tank of the SV40LT gene induced T lymphocyte model applied to the field of medical research. The construction method is mainly characterized by comprising the following steps: according to a conventional route, constructing an SV40LTag-pcDNA3.1(-) recombinant by using T4DNA ligase, BamHI, pcDNA3.1(-)DNA and SV40LTag DNA; purifying the recombinant by using competent escherichia coli, and transferring the recombinant to T lymphocytes cultured in vitro by virtue of a liposome transfection process to ensure that the recombinant is integrated with DNA of cells; performing subculture and extended culture on the cells which are screened by G418 and contain positive recombinants, screening the cells of which the cellular morphology, cell growth curves, chromosome karyotypes, carcinogenicity tests in nude mice, SV40 large T gene detection in the DNA of transfection cells, mRNA expression product determination and DNA sequence determination results can meet the biological characteristics of immortalized cells as a T lymphocyte model, and freezing and preserving the T lymphocyte model in liquid nitrogen to construct the SV40LT gene induced T lymphocyte model and the cell bank thereof so as to ensure that a method for performing subculture on the T lymphocytes in vitro for a long time can be established, and the method can be applied to in vitro studies on a T lymphocyte immune mechanism.

Description

SV40LT gene induced structure T lymphocyte model and cell bank thereof

Technical field

The present invention relates to SV40LT gene (simian virus 40 large T antigen gene) induction (mediation) and build T lymphocyte model and cell bank thereof, be mainly used in Medical Immunology research field, for carrying out T lymphocyte or the various researchs relevant to T lymphocyte provide cell model and preserve its Scientific Research Resource.

Background technology

Visible component in human blood is divided into red corpuscle, white corpuscle and thrombocyte.White corpuscle is wherein based on neutrophil leucocyte, lymphocyte and monocyte.Lymphocyte is divided into again T lymphocyte (T cell) and bone-marrow-derived lymphocyte (B cell) etc.

Known T cell is broken up by intrathymic lymphoid stem cell to form, and is that in lymphocyte, quantity is maximum, the class cell that function is the most complicated.Three subgroups can be divided into: helper T cell, suppressor T cell and cytotoxic T cell by its function.The cytolemma of T cell there are many different marks, mainly surface antigen and surface receptor.These surface markers are all the huge protein moleculars be combined on cytolemma.T cell is quite complicated uneven one, the again continuous subgroup upgrading in vivo, can there is different developmental phases or function at one time.Different by the function in immunne response, T cell can be divided into some subgroups again, consistent generally acknowledged having: helper T cell (Helper T cells, Th), has the function of assisting humoral immunization and cellular immunization; Suppressor T cell (Suppressor T cells, Ts), has the function of T suppression cell immunity and humoral immunization; Effector T cell (Effector T cells, Te), has the function of release lymphokine; Cytotoxic T cell (cytotoxic T cells, Tc), has the function of killing and wounding target cell; Tardive allergy T cell (Td), has the effect participating in IV allergic reaction type; Amplifier T cell (Ta), can act on Th and Ts, has the effect expanding immune effect; Virgin or nave T cell (Virgin or Natural T cells), be divided into effector T cell and memory T cell after they and antigen contact; Memory T cell (Tm), has the effect that memory specific antigens stimulates.

Wherein, Th cell is otherwise known as CD4+ cell, because it is at surface expression CD4 (cluster ofdifferentiation 4).Be activated by reacting with the polypeptide antigen of MHC II (major histocompatibility complex, majorhistocompatibility complex) submission.MHC II is at antigen presenting cell (antigen presenting cells, APCs) surface expression.Once activate, can secrete cytokines, adjustment or assistance immune response.Tc cell has another name called for CD8+ cell, and this kind of cell of its surface expression CD8. directly can be combined with antigen by MHCI.T lymphocyte also can be further divided into auxiliary/inducer T lymphocyte (CD3+CD4+), suppression/cytotoxic T cell (CD3+CD8+), the pure subgroup (CD4+CD45RA+/CD4+CD45RA+62L+) of CD4+T cell and memory subset (CD4+CD45RA-/CD4+CD45RO+), Functional subclass (CD28+), activate subgroup (CD38+, HLA-DR+), apoptosis subgroup (CD95+) etc.

T cell is lymphocytic main ingredient, and it is extremely important that the normal function of T cell resists disease to the mankind.T cell does not produce antibody, and its immunne response is cellular immunization, as being combined with target cell specificity, destroys target cell membrane, direct killing target cell; Release lymphokine, finally makes immunological effect expand and strengthens; Auxiliary or suppression B cell produces antibody.Resisting, disease infects, tumour plays an important role in being formed.And B cell works by producing antibody.Antibody is present in body fluid, so the immunization of B cell is called humoral immunization.

Lymphocytic mean lifetime is generally about 7 days, can not the Secondary Culture of long period in vitro.Because be easy to dead at short notice when lymphocyte is cultivated in vitro, need the research of the lymphocyte biological function of long-term cultivation in vitro so be difficult to carry out.In order to address this problem, domestic and foreign literature report Epstein-Barr virus infection protocol makes bone-marrow-derived lymphocyte immortalization, sets up bone-marrow-derived lymphocyte system (strain), to meet the needs of above-mentioned scientific research.The principle setting up this immortalized B lymphocyte system has the acceptor of Epstein-Barr virus based on bone-marrow-derived lymphocyte surface.And the lymphocytic surface of T does not have the acceptor of Epstein-Barr virus, so just immortalization T lymphocyte series cannot be set up by the method.So far there are no about can the establishment method of T lymphocyte series cultivated of Long Term Passages in vitro.

Foreign literature is reported, simian virus 40 (SV40) can make some human cell that immortalization occurs.The research such as Poulin DL, Kung AL and Sullivan CS shows, the importing of SV40T antigen gene can accelerate the growth velocity of transformant, after immortalized cells repeatedly goes down to posterity in vitro, still there is metastable multiplication characteristic and functional status, also can retain many phenotypic differentiations of its initiating cell simultaneously, may be used for the foundation of the cell model of transgenic animal model and some kind of human and animal, the characteristic of initiating cell can be studied accordingly by means of research transgenosis immortalized cells and animal model in vitro, thus study its pathogenesis.

According to bibliographical information, vascular smooth muscle cell strain is set up in Reilly simian virus (SV40) large T antigen gene transformation, builds cell model to study the restraining effect mechanism of heparin for vascular unstriated muscle.Su etc. utilize the superficial cell strain transformed through SV40, build the regulating and controlling effect that cell model analyzes the synthesis of epithelial cell internal protein.The superficial cell strain that Miquel etc. transform with SV40, as the cell adhesion that cell model research ln 5 mediates.Webber etc. study physiological function and the secreting function of prostate epithelial cell as cell model with the prostate epithelial cell strain transformed through SV40.Racusen etc. transform with through Ad12-SV40 damage and the disease that proximal convoluted tubule studied by renal cells model.Hougton etc. transform with SV40 and set up Bone marrow Stromal cell as cell model to study under certain culture condition, and cell has the potential to adipocyte and osteoblastic two-way differentiation, further the osteoporotic mechanism of research.

Because of the needs of research work, almost often kind of disease all establishes respective cell model.As diabetes cell model, cancer cell model, transgenic cell model, climacteric syndrome cell model, endometrial cell model, epilepsy cell model, E-Cell models, alcoholic dementia cell model, cerebral edema cell model.Etc..

Summary of the invention

Cannot set up at present to solve can cultivate and can be used for the problem of the T lymphocyte model of in vitro study T lymphocyte immunity mechanism by Long Term Passages in vitro, present inventors have proposed the present invention.

The object of the invention is the construction process that will provide with the gene induced immortal T lymphocyte model of SV40LT and cell bank thereof, another object provides the gene induced immortal T lymphocyte model of SV40LT and cell bank thereof for studying the lymphocytic immunologic mechanism of T in vitro.

The object of the present invention is achieved like this: connect pcDNA3.1 (-) DNA and pcr amplification that cut through BamHI enzyme with T4DNA ligase enzyme simultaneously, the SV40LTag DNA that agarose gel electrophoresis is separated, build SV40LTag-pcDNA3.1 (-) recombinant plasmid, transform DH5a competent escherichia coli cell with amplification, purifying picking amp-R bacterium colony extracting plasmid, the T lymphocyte of vitro culture is imported with lipofection, the DNA of recon and cell is integrated, with the cell containing positive recombinant of G418 screening, go down to posterity, enlarged culturing, screening cellular form, cell growth curve, karyotype, nude mice tumorigenesis is tested, the large T gene test of SV40 in transfectional cell DNA, mrna expression product measures and determined dna sequence result meets immortalized cells characteristic and identical with primary cell or close person is frozen in liquid nitrogen as T lymphocyte model, SV40LT gene mediated T lymphocyte model and cell bank thereof is built with this.

The SV40 large T antigen gene that the present invention purifies with pcr amplification, agarose gel electrophoresis imports T lymphocyte through genophore pcDNA3.1 and lipofection and builds its cell model, the defined medium containing 20ml/L foetal calf serum, 5 ~ 10nmol/L Regular Insulin has been selected during transfection, make cell can not grow too fast and affect the integration of SV40 large T antigen gene, also can not make cell with regard to premature death before not reaching requested number, not entering logarithmic phase because lacking nutrition or Growth of Cells stimulating factor, or logarithmic phase shortens; Clone recovery cultivation after frozen 1 month in-196 DEG C of liquid nitrogen of preparation, growth of all going down to posterity; When making clone Chromosome Identification, the consumption of colchicine and action time are conventional 5 ~ 10 times, and chromosome separation is increased mutually, are enough to counting and analyze; Build up T lymphocyte model and cell bank thereof thus, be based upon external Long Term Passages cultivate the lymphocytic method of T and in vitro study T lymphocyte immunity mechanism.

Embodiment

1, the extraction of SV40 large T antigen DNA: 1. SV40DNA enzyme is cut: the SV40 freeze dried powder or the SV40 plasmid that contain large T antigen gene from commercially available purchase, be dissolved in appropriate H ₂in O or TE damping fluid, add 2uL10 × enzyme cutting buffering liquid and 18uL H ₂o, adds restriction enzyme BamH I (1-5U/ugDNA), 37 DEG C of incubation 1h, 75 DEG C of heating 15min, and inactivator, adds 5uL electrophoresis sample loading buffer (also by adding 0.5mol/L EDTA) termination reaction in order to electrophoresis.2. SV40DNA electrophoresis: power taking swimming level agarose is made into 10% sepharose with electrophoretic buffer, pour on the gel casting platform sealed, plug sample comb, from glue platform removing envelope band after gelling is solid, extract comb, put into the electrophoresis chamber being added with enough electrophoretic buffers, damping fluid exceeds gel surface and is about 1mm, DNA sample is prepared with 10 appropriate × sample loading buffer, then with pipettor, sample is added in sample well, and do suitable DNA molecular amount standard control thing simultaneously, connect electrode, DNA anode is moved, when under the voltage of 1-10V/cm gel, electrophoresis is to the distance of enough isolation of DNA fragments, powered-down.3. from agarose, about 2600bp SV40 large T antigen DNA is separated: under 300-360nm long wave ultraviolet light source, (use long wave ultraviolet light source to prevent DNA damage) cut in loading dialysis tubing by the gel-tape containing target DNA fragments, 2ml electrophoretic buffer is added in dialysis tubing, make it submergence gel, and emptying steam bubble, dialysis tubing level is put into electrophoresis chamber (length direction is parallel with electrophoresis), add appropriate amount of buffer solution by dialysis tubing submergence (about 6-7mm), switch on power, 150 volts of electricity are washed, observe under ultraviolet lamp and treat that DNA all shifts out gel, change direction of an electric field and continue energising 1 minute, from dialysis tubing, sucking-off damping fluid is in 1-5ml Eppendorf pipe, add 1.5 times of volume propyl carbinols, EB is removed in mixing extracting, on desk centrifuge 2 minutes the most at a high speed, suck upper strata butanol solution, repetition secondary like this, equal-volume phenol chloroform (2) extracting 2 times is added in the solution of lower floor speech DNA, supernatant proceeds in another Eppendorf pipe and adds 1/10 times of volume 3M NaAc, 2 times of volume precooling dehydrated alcohols, spend the night in 20 DEG C, 12000g, at 4 DEG C centrifugal 10 minutes, obtain DNA precipitation, abandon supernatant, dry ethanol is abandoned after adding 70% washing with alcohol 2 times, add 50 μ l TE dissolving DNAs.In addition, also target DNA fragment is separated, is purified by available low melting-point agarose gel method, DNA filter membrane inserted sheet method etc. from gel.

2, the connection of SV40 large T antigen DNA and pcDNA3.1 genophore: get the above-mentioned DNA composition of 9 μ l (0.1-5 μ g), 10 μ l 2 × connect damping fluid, 1 μ l 10mmol/L ATP, T4 DNA ligase (20 ~ 500 sticky end unit) or e. coli dna ligase, pcDNA3.1 empty carrier mix, 15 DEG C of incubation 24h, are built into SV40T/pcDNA3.1 recon.

3, the amplification of SV40T/pcDNA3.1 recon, Isolation and ldentification: the 1. preparation of E. coli competent: its basic skills is ice-cold CaCl ₂or the process such as multiple divalent positively charged ion bacterium, making it to enter competence is transformed, and uses CaCl ₂prepare fresh or freezing competent escherichia coli cell, be usually used in preparation in quantity competence bacterium, this law is applicable to most of coli strain, operating process is summarized as follows: cultivate picking single bacterium colony (as bacillus coli DH 5 2) the fresh plate of 16 ~ 20h from 37 DEG C, or 16 ~ 20h overnight culture that 1ml is fresh, forward one to containing in 1L or the 500ml flask of 100mlLB substratum, about 2 ~ 3h (rotary shaker 200 ~ 300r/min) is cultivated in 37 DEG C of violent joltings, OD600 value ≈ 0.4 is measured every 20 ~ 30min, aseptically bacterium is transferred to one, in ice-cold 50ml polypropylene centrifuge tube, 10 ~ 20min is placed on ice, in 4 DEG C with SorvallGS2 rotary head (or the rotary head matched with its centrifuge tube) with the centrifugal 10min of 4000r/min, to reclaim cell, nutrient solution reciprocal, pipe is inverted 1min to flow to end to make the trace nutrient solution of final residual, with the ice-cold 0.1mMCaCl of 10ml ₂resuspended every part of precipitation, be put on ice, in 4 DEG C with SorvallGS3 rotary head (or its corresponding rotary head) with the centrifugal 10min of 4000r/min, to reclaim cell, pour out nutrient solution, pipe is inverted 1min to flow to end to make the trace nutrient solution of final residual, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice precooling ₂resuspended every part is sunk calmly, now, rapidly cell can be distributed into aliquot, freezing in liquid nitrogen,-70 DEG C of storages are for subsequent use, from every kind of competent cell suspension, getting 200 μ l with the sterile pipette tip of cooling transfers in aseptic Eppendorf tube, DNA or ligation mixture (volume≤10 μ l should be added in every pipe, DNA≤50ng), rotate gently to mix content, 30min is placed in ice, centrifuge tube is put into pre-heating on the test-tube stand in the circulator bath of 40 DEG C, place 90s ~ 2min, do not shake test tube, fast pipe is transferred in ice bath, cell is made to cool 1 ~ 2min, every centrifuge tube adds 800 μ lSOC substratum, with water-bath, substratum is warmed to 37 DEG C, then pipe is transferred on 37 DEG C of shaking tables, incubation 45min makes bacteria resuscitation, and antibiotic-resistance marker's gene of expression plasmid coding, the competent cell that proper volume (each 90mm flat board can reach 200 μ l) has transformed is transferred to containing on 200mmol/LMgSO4 and corresponding antibiotic SOB substratum, flat board is placed in room temperature to be absorbed to liquid, be inverted plate, in 37 DEG C of cultivations, bacterium colony can be there is after 12 ~ 16h.2. the screening of recon, amplification and extraction: select single colony inoculation in the aseptic LB substratum of 5mL or rich medium (as super broth or TB super broth substratum) with sterile toothpick or disinfection inoculation pin, after overnight incubation, join 500mL again containing in the 2L flask of LB substratum (containing suitable microbiotic), then be cultured to state of saturation (OD in 37 DEG C ₆₀₀≈ 4, for improving output, the flask of the comparatively large and band traverse baffle of surface-area should be adopted to increase venting quality as far as possible, jolting speed should be greater than 400r/min), in 4 DEG C, the centrifugal 10min of 6000g, by the resuspended precipitation of 4mL GTL solution, and transfer to (bacterial precipitation can be preserved at-20 DEG C or-70 DEG C of indefinitely) in the high speed centrifugation pipe of a volume>=20mL, add the GTE solution containing 25mg/mL N,O-Diacetylmuramidase that 1mL newly joins, resuspended precipitation, 10min is placed in room temperature, add 10mL and newly join NaOH/SDS solution, and mixing is until liquid becomes homogeneous gently, limpid and thickness, in placing 10min on ice, add 7.5mL acetic acid solution, stir until viscosity declines and forms large precipitation gently with suction pipe, in placing 10min on ice, in 4 DEG C, the centrifugal 10min of 20000g, supernatant is poured into gently in another clean centrifuge tube, if there is visible drift can by several layers of filtered through gauze, add the Virahol of 0.6 times of volume, put upside down mixing, room temperature places 5 ~ 10min, in room temperature, the centrifugal 10min of 1500g, add the washing precipitation gently of 2mL70% ethanol, then of short duration centrifugal fast, suck ethanol, and vacuum-drying (precipitation can be preserved for a long time at 4 DEG C).3. the qualification of recon: the above-mentioned DNA (containing recon SV40T/pcDNA3.1) extracted from competence intestinal bacteria, the same method restriction enzyme BamH I carries out enzyme and cuts, 10g/L agarose gel electrophoresis is identified, obtain 2 bands that size is about 2600bp and 5600bp, the former meets the size of SV40T fragment in GenBank.

4, the lymphocytic collection of T: 1. with aseptic technique be extracted in do other experiments after unnecessary, discarded containing the lymphocytic sample of T (as anticoagulant heparin blood specimen).2., after sample and equivalent RPMI-1640 being mixed, in the centrifuge tube that tube wall joins containing 4ml lymphocyte separation medium gradually (mixing blood: lymphocyte separation medium=6:4), 3000 leave the heart 10 minutes.3. draw leukocytic cream, move in another centrifuge tube, add not containing the RPMI-1640 6ml of serum, mix gently, carry out first time washing.1500 leave the heart 15 minutes.4. abandon supernatant liquor, then add the full nutrient solution of 6ml1640 carry out second time washing, centrifugal 1500 turns 15 minutes, abandon supernatant liquor.

5, T lymphocyte preculture: above-mentioned cell is inoculated in the RPMI1640 liquid containing 5 ~ 10nmol/L Regular Insulin, 20% foetal calf serum, or in the low sugar DMEM cell culture medium being inoculated in containing 20% foetal calf serum, 5 ~ 10nmol/L Regular Insulin, be generally inoculated in the freshly prepared substratum of 3ml (1.6%1M HEPES damping fluid; 15% foetal calf serum (FBS); 1% penicillin and Streptomycin sulphate; PRMI1640 supplies 100%), be placed in 37 DEG C, in volume fraction 5%CO2 incubator, cultivate 1-2 days, centrifugal, remove supernatant liquor, for subsequent use.

6, the importing of SV40T/pcDNA3.1 and enlarged culturing: prepare following solutions in 1.5ml Eppendorf tube: pipe A, SV40T/pcDNA3.1 is dissolved in 100 μ l serum-free mediums (foetal calf serum concentration is 20ml/L); 20 μ lLipofectamine are dissolved in 80 μ l serum-free mediums, are mixed by pipe A and pipe B, the underlying 45min of room temperature by pipe B.Above-mentioned T lymphocyte is washed 2 times with serum-free medium.In Lipofectamine-SV40T/pcDNA3.1 mixture, add 1ml serum-free medium, mix gently, then drop in above-mentioned T lymphocyte, then add 1ml serum-free medium (foetal calf serum concentration is 20ml/L), at CO ₂10h cultivated by incubator, and sucking-off transfection liquid, adds 4ml complete culture solution (foetal calf serum concentration is 20%), continues to cultivate 20h, and discard nutrient solution, replacing concentration is 400mgL ^-1g418 nutrient solution continue to cultivate, within 8 days, select after viable cell makes enlarged culturing afterwards, then strengthen G418 concentration to 800mgL ^-1, in the G418 environment of high density, amplification cultivation can be proceeded by the cell of stable growth.Cultivate about 9 days Microscopic observations, visible lymphocyte obviously increases, and occurs clustering phenomena.If cell enlargement is slow, or cell density is low, or medium pH value is in acid, and sucking-off partly measures nutrient solution, carries out equivalent oil changing.Transfer in 75ml culturing bottle when total amount reaches 14ml, every 2-3 week adds 5-10ml fresh culture.Cell cultures is about 6-8 week (the about the 55th generation), and be still in logarithmic growth, namely incubation time and cell are accelerated in multiplication relation, and dead cell is less than 10% and (is judged the increase situation of cell quantity by the scale reading culture vessel; Dead cell and viable cell is differentiated by trypan blue staining.Because normal viable cell, after birth structural integrity, can repel, and trypan blue can not be entered in born of the same parents; And the cell of loss of activity, the permeability of after birth increases, and can be dyed blueness, can be judged as that cell is dead by trypan blue.Method draws weekly a certain amount of suspension culture, rearmounted room temperature 5 ~ 10 minutes are mixed with Trypan Blue agent, then make cell sheet, count 1000 total cellular score under the microscope, calculate the per-cent of painted dead cell and non-staining viable cell).After this along with the cultivation increase of algebraically and the prolongation of incubation time, the increase of cell quantity is slack-off, dead cell gets more and more, until cell no longer increases, even dissolves, reduces, all death.When total amount reaches 45ml, put in 50ml centrifuge tube, centrifugal 1500 turns, 10 minutes, abandon supernatant, add 3ml freezing media (5% DMSO (dimethylsufoxide), 95%FBS) mix, (cell concn is about 10 to become cell suspending liquid ⁵/ ml).Cryopreservation tube packing, 1ml/ manages, and puts-20 DEG C of 2h, then puts-70 DEG C of 2h, then frozen in-196 DEG C of liquid nitrogen (or under zero setting immediately 80 degree, move in liquid nitrogen container after 1-2 hour).Immortalization T lymphocyte model is built with this.

7, the qualification of immortalization T lymphocyte model biological characteristics:: after using SV40 to set up immortalization T lymphocyte model, the key issue of qualification has: one is that this cell of requirement has lasting multiplication capacity, i.e. T antigen stably express in clone; Two is that requirement its form, Basic Biological Character etc. remain unchanged.1. observation of cell form: visible lymphocyte obviously increases, and occurs clustering phenomena, has lymphocyte blastogenesis feature.2. observation of cell growth curve: get the good transfectional cell of growth, make cell suspension, through counting, get 1.4 × 10 respectively ⁴cell is inoculated in 30 containing 15FBS low sugar DMEM culture medium culturing bottle.Get 2 bottles of cells every day to count, computation of mean values, Continuous Observation, until cell quantity obviously declines, is cultivated after 3 days and was changed liquid every 2 days to the cell of no count, adopts same method to observe the growing state of transfectional cell in hepato ZYME-SFM serum free medium.Result take incubation time as transverse axis, and cell quantity is the longitudinal axis (logarithm), be depicted in semi-logarithmic scale to make after curve the growth curve of this cell, immortalized cell line is formed in typical " S " feature or " arched roof "; 3. karyomit(e) is checked: by analyzing karyotype, if karyotype is diploid " 46; XX " or " 46; XY ", then illustrate that this clone vicious transformation does not occur and (whether occurs abnormal DNA colony in available flow cytometry analysis clone simultaneously, as not having, illustrate that knurl feature does not appear in clone).Chromosome karyotype analysis method is: by the 250ug/ml colchicine 100ul adding preheating in 5mL nutrient solution, mixes rearmounted 37 DEG C of incubators 4 hours, through centrifugal, remove supernatant liquor, hypotonic, fixing, film-making, G aobvious band post analysis karyotype; 4. nude mice tumorigenesis test: by the cell of SV40LT gene immortalization with 3 × 10 ⁷inoculation nude mice dorsal sc, after 2 months, 4 nude mices are showed no tumour and are formed, and prove that this cell is non-malign cells; 5. the large T gene test of SV40 in transfectional cell DNA: as with Immunohistochemical detection, in the nucleus of SV40 transfection, the visible a large amount of brown particle of dyeing, shows that SV40T antigen has been integrated in cell; Also the expression of T antigen in cell can be detected by RT-PCR method, the wherein primer of T antigen: upstream primer (A4239) 5 '-GTT ATG ATT ATA ACT GTT ATG-3 ', downstream primer (S4496) 5 '-GAAATG CCA TCT AGT GAT-3 '; Amplified production length is 268bp, and amplification condition is 94 DEG C, 5min, that is: and (94 DEG C, 1min; 55 DEG C, 1min ,-0.5 DEG C/circulation; 72 DEG C, 1min) × 30, (94 DEG C, 30S; 40 DEG C, 30S, 72 DEG C, 30S) × 15, amplification system is 50 μ l:[Mg ²⁺] 2mmol/L, dNTPs200 μm ol/L, primer concentration 0.4 μm of ol/L, Taq1U, template 5 μ l; Experimental group with the 19th generation cell cDNA for template (with reference to commercially available cDNA first chain synthetic agent box carry out cDNA first chain synthesis, product-20 DEG C preservation); Negative control establishes two, template is done respectively with the cDNA of sterilized water, primary cell, positive control is that template (extracts SV40DNA with reference to SDS-proteinase-K pathway with SV40DNA, because SV40 virus is without coating, do not use SDS rupture of membranes, get 5 μ l and carry out 1.5% agarose gel electrophoresis detection, all the other-20 DEG C save backup); 6. mrna expression product measures: T antigen mRNA RT-PCR product checks order: the amplified production getting 100 μ l systems, test kit (Takara is reclaimed with gel, Japan) reclaim product, get 2 μ l DNA solutions and dilute 100 times, survey concentration, remaining DNA and each 10 μ l of upstream and downstream primer checks order.7. Flow cytometry: the cell proportion detecting synthesis in the 19th continuous cell line, division, if its multiplication capacity obviously strengthens than not building the normal cell being, explanation is the result that SV40 large T antigen is integrated, expressed.8. determined dna sequence: sequenator detects routinely, display SV40 large T antigen DNA sequence dna.

So the cell model of the present invention SV40LT gene that has been 1. T lymphocyte transfection becomes can the Long Term Passages immortalization T lymphocyte model of cultivating; 2. the karyotype of T lymphocyte model is diploid " 46, XX " or " 46, XY "; 3. the growth curve of cell is as taken incubation time as transverse axis, and cell quantity is the longitudinal axis, is that " S " feature or " arched roof " are formed in hepato ZYME-SFM serum free medium; 4. cell can not grow (forming clone) in soft agar; 5. cell is non-malign cells, nude mice tumorigenesis negative; Incorporate SV40 large T antigen gene in the DNA of 6. cell, express SV40 large T antigen mRNA product; 7. in-vitro cultivation 6-8 week (the 55th generation) is still in logarithmic phase; 8. can repeatedly cultivate by frozen, Long Term Passages; 9. for studying the lymphocytic function of T and immunologic mechanism; 10. the relevant genetic resources of T lymphocyte is preserved on recyclability ground.

8, SV40LT gene mediated T lymphocyte model and cell bank thereof: screen and continue to go down to posterity, enlarged culturing meets immortalized cells characteristic and the cell identical or close with primary cell after above-mentioned qualification, get the cell that growth conditions is good, be in the different generations of logarithmic phase, through centrifugation (1200r/min, 6min), with the frozen storing liquid 0.5 ~ 1ml re-suspended cell containing methyl-sulphoxide, cell density is 5 × 10 ⁵individual/ml, adds cryopreservation tube, through 4 DEG C, and 0.5h;-20 DEG C, 2h;-70 DEG C, spend the night, enter-196 DEG C of liquid nitrogen cryopreservations, build the stable immortalization T lymphocyte model of biological characteristics and cell bank thereof in this way.

9, the application of T lymphocyte model and cell bank thereof: for concentrated preservation genetic resources, for other researchs provide scientific research material; Directly as the cell model of T lymphocyte relative disease pathogenesis in vitro study, to accelerate the new way expanding the research of T lymphocyte relative disease, study from cell levels the mechanism such as transgenation, genetic expression, changing function, physiological property, biology conduction that T lymphocyte relative disease cell affects by physics, chemistry, biology, heredity etc. in vitro.Such as:

1) T lymphocyte model is made to be in the artificial nuisance with different content or concentration manufactured as physics (as X-ray), chemistry is (as formaldehyde, gasoline, plumbous, mercury), biological (as rubella virus, cytomegalovirus, simplexvirus) condition under cultivate, then the viable cell of the different cycles of external Long Term Passages is got, the apoptotic cell of succeeding generations, nutrient solution containing the meta-bolites produced in Secondary Culture, application ordinary method is as gene chip, miRNA chip, comparative genomic hybridization hybrid chip (CGH), differential methylation hybridization hybrid chip (DMH) and SNPs etc. screen gene and the polymorphism thereof of difference, methylation level, the gene rate of rotation in the technology for detection gene place such as in situ hybridization (FISH), Northern blot, Real-time PCR, CHIP, EMSA research cell model is used to express, locate and regulation and control, utilize the meta-bolites of protein metabolism process and key in enzyme reaction, metabonomic technology identification of cell model Long Term Passages, application two-dimensional electrophoresis, MALDI-TOF Mass Spectrometric Identification, yeast two-hybrid and co-immunoprecipitation etc. the protein function of technical study cell model in Long Term Passages and the interaction between protein, from viable cell culturing process dynamically, the T lymphocyte model that studies for a long period of time tolerance in all cases and immunologic mechanism.

2) make cell model and compared with control cells respectively containing 0.1,1.0,5.0,10.0, cultivate in the nutrient solution of 20.0pmol/L benzopyrene, detect in the apoptosis that can be used as cytotoxicity index of cultivation 2 weeks, the different incubation time such as 4 weeks, 6 weeks, 8 weeks, 16 weeks, necrosis, dikaryocyte rate and the micronuclear rates, caryoplasm bridge rate, the core bud rate that can be used as genetoxic index, micronucleus number, caryoplasm bridge number and core bud number namely under an optical microscope in counting 10000 dikaryocytes; Necrosis and apoptosis cell count in 500 viable cell, dikaryocyte number, and detection cell survival rate, namely tetrazolium-based colorimetric assay (mtt assay) is applied, after sucking original fluid, 96 orifice plates add the serum-free medium of the 5mg/mlMTT of 20%, continue to cultivate 4h, supernatant liquor in hole is abandoned in careful suction, every hole adds 150 μ L methyl-sulphoxides, vibration 10min, purple crystal thing is fully dissolved, and this microplate reader measures the absorbance in each hole with 490nm, calculates cell survival rate.Can also other normal experiment methods detect different incubation time containing toxic substance with containing the transgenation, proteomics, emiocytosis function, chromosome aberration, cell survival rate (life-span) etc. of respectively organize cell in toxic substance, T lymphocyte model and normal cell, with dynamic from cell levels from viable cell culturing process, the bad environmental factor that studies for a long period of time on T lymphocyte function, machine-processed impact.

Claims (7)

1. the structure of the gene induced T lymphocyte model of the SV40LT for medical research field and cell bank thereof, its principal character is routinely with T4DNA ligase enzyme, BamHI, pcDNA3.1 (-) DNA and SV40LTag DNA builds SV40LTag-pcDNA3.1 (-) recon, with competence intestinal bacteria purification of Recombinant, the T lymphocyte of vitro culture is imported with lipofection, the DNA of recon and cell is integrated, with the cell containing positive recombinant of G418 screening, go down to posterity, enlarged culturing, screening cellular form, cell growth curve, karyotype, nude mice tumorigenesis is tested, the large T gene test of SV40 in transfectional cell DNA, it is frozen in liquid nitrogen as T lymphocyte model that mrna expression product mensuration and determined dna sequence result meet immortalized cells biological nature person, SV40LT gene mediated T lymphocyte model and cell bank thereof is built with this, cultivate the lymphocytic method of T to be based upon external Long Term Passages and to study T lymphocyte immunity mechanism for external (substituting human body or animal straightway testing).

2. the structure of the gene induced T lymphocyte model of SV40LT according to claim 1 and cell bank thereof, is characterized in that the indication cell model SV40LT gene that has been the transfection of (1) T lymphocyte and become can the Long Term Passages immortalization T lymphocyte model of cultivating; (2) karyotype of T lymphocyte model is diploid " 46, XX " or " 46, XY "; (3) growth curve of cell is as taken incubation time as transverse axis, and cell quantity is the longitudinal axis, is that " S " feature or " arched roof " are formed in hepato ZYME-SFM serum free medium; (4) cell can not grow (forming clone) in soft agar; (5) cell is non-malign cells, nude mice tumorigenesis negative; (6) because incorporating SV40 large T antigen gene in the DNA of cell, so express SV40 large T antigen mRNA product; (7) in-vitro cultivation 6-8 week (the 55th generation) is still in logarithmic phase; (8) can repeatedly cultivate by frozen, Long Term Passages; (9) for studying the lymphocytic function of T and immunologic mechanism; 10. the relevant genetic resources of T lymphocyte is preserved on recyclability ground.

3. the structure of the gene induced T lymphocyte model of SV40LT according to claim 1 and cell bank thereof, it is characterized in that primary T lymphocyte take from gathers because other experiments are required and unnecessary, discardedly after other experiments contain the lymphocytic sample of T.

4. the structure of the gene induced T lymphocyte model of SV40LT according to claim 1 and cell bank thereof, it is characterized in that nutrient solution used is the RPMI1640 containing 20% foetal calf serum, 5 ~ 10nmol/L Regular Insulin or the low sugar DMEM nutrient solution containing 20% foetal calf serum, 5 ~ 10nmol/L Regular Insulin, during transfection, foetal calf serum concentration is 20ml/L.

5. the structure of the gene induced T lymphocyte model of SV40LT according to claim 1 and cell bank thereof, is characterized in that being used as lipofection imports the T lymphocyte of recon, is in 1-2 days after preculture.

6. the structure of the gene induced T lymphocyte model of SV40LT according to claim 1 and cell bank thereof, when it is characterized in that making chromosome karyotype analysis, adds 250ug/ml colchicine 100ul in every 5mL nutrient solution, mixes rearmounted 37 DEG C of incubators 4 hours.

7. the structure of the gene induced T lymphocyte model of SV40LT according to claim 1 and cell bank thereof, is characterized in that the structure of cell bank comprises (1) screening and after qualification, meets immortalized cells characteristic and the T lymphocyte that mediates of the SV40LT identical or close with primary cell; (2) go down to posterity, enlarged culturing, get the attached cell that growth conditions is good, be in the different generations of logarithmic phase; (3) through digestion, termination, centrifugal (1200r/min, 6min) step harvested cell; (4) preparing density with the frozen storing liquid containing methyl-sulphoxide is 5 × 10 ⁵the cell suspension of individual/ml; (5) according to 4 DEG C, 0.5h;-20 DEG C, 2h;-70 DEG C, spend the night; Enter the program freeze-stored cell of-196 DEG C of liquid nitrogen; (6) the long-term SV40LT of preservation in recyclability ground mediates T lymphocyte model, for making genetic resources and scientific research material.