CN105039401A - Helper plasmid for efficiently establishing human stable expression cell strain and establishment method thereof - Google Patents

Abstract

The invention discloses a helper plasmid for efficiently establishing a human stable expression cell strain and an establishment method thereof. The helper plasmid and an exogenous gene cotransfect a human recipient cell, and the helper plasmid can manually form 167 nicks on a human genome specificity target set sequence to provide 167 integration hotspots for the exogenous gene, so that the efficiency of the integral entrance of the exogenous gene into a human genome is remarkably improved, and further the expression quantity of the exogenous gene is increased. The helper plasmid disclosed by the invention is simple in establishment process and cheap, can be independently established in an ordinary laboratory, can directly cotransfect the human recipient cell with different exogenous genes, is simple in operation method, can remarkably improve the efficiency of establishing the human stable expression cell strain, can realize the efficient establishment of various human cell models, and facilitates the study of human disease pathogenesis and the development of gene therapy.

Description

A kind of helper plasmid of efficient structure people stable expression cell strain and construction process thereof

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of helper plasmid and construction process thereof of efficient structure people stable expression cell strain.

Background technology

At present, the mechanism of people to a lot of human diseases is not clear, cannot in vivo study of disease occur whole process, simultaneously, carry out further investigated disease mechanism using people itself as experimental subjects, the development promoting medicine and pharmacology comes slow, and the experience of clinical accumulation not only all exists limitation over time and space, and many experiments morally with in method are also being restricted, needing to study to complete such by means of cell model and animal model for this reason.Present overwhelming majority's research is just based on rodent cells system level, and human body cell and rodent zooblast also exist significant difference, such as, normal human cell will through cellular immortalization in Carcinogenesis, the process such as prosoplasia and pernicious transfer, and human body cell and rodent zooblast all also exist different at immortalization and two stages of cell transformation, mammalian cell is easy to spontaneous transformation in vivo, and people's normal diploid cell middle spontaneous transformation frequency that infinitely goes down to posterity in vitro is low, the mammalian cell of vitro culture is generally easy to immortalization than people cell, in the vicious transformation stage, channel genes mammalian cell is easy to that cell is obtained and transforms, and the conversion of people's primary cell needs the participation of more polygene event, thus the difference of human body cell and animal cells in vitro conversion characteristic makes mammalian cell model can not simulate the mechanism of disease in human body completely, therefore pass through the generation of Research of Animal Model for Study human disease, the result that evolution process and mechanism thereof obtain is worth discussion, difference between kind has some limitations when making zooblast test-results be extrapolated to people (Pang Yaqin. the structure of transgenic cell model and the application [D] in carcinogenic activity detects thereof. Zhongshan University, 2015).Therefore, human cell's model, compared with laboratory animal cell model, has more substantivity and cogency.Meanwhile, using human cell's model as experimental study, have that condition is easily controlled, external environment is simple, be easy to the advantages such as screening acting factor.Therefore, set up human cell's model and play immeasurable great function in the research and gene therapy of human body diseases mechanism.

The importing carrier of effective goal gene and safety is the key efficiently building human cell line, particularly how foreign gene is transported to target cell by suitable carrier system.At present, foreign gene being transported to carrier conventional in target cell has plasmid, phage and virus etc., and the method for the structure stable expression cell strain of routine has eucaryon plasmid transfection, slow virus packaging.Slow virus wrapping cycle is longer, costly, requires high to laboratory hardware condition, significantly limit its application; Eucaryon plasmid transfection to have in host cell genetic stability, the feature such as easily separated, can be inserted in recipient cell genome by the mode of random integration, be widely applied to build people's stable expression cell strain process in the middle of.But the integration site of the foreign gene of random integration in acceptor gene group has uncertainty, blindness and randomness, it is not high that this makes eucaryon plasmid transfection shift destination gene expression amount, and the expression amount of foreign gene is subject to the impact of position effect and dosage effect, dosage effect is exactly the copy of foreign gene a certain position on chromosome when arriving some amount, then become tumor-necrosis factor glycoproteins and affect the expression of foreign gene.When Costantini found human beta-globin gene to proceed in β-thalassemia mouse in 1986, if genetically modified copy number is greater than 50, then thalassemia can be corrected, then invalid when copy number is 1, this illustrates that genetically modified copy number also likely affects the performance of genetically modified expression and function.

Summary of the invention

First object of the present invention is to solve and builds the low problem of people's stable expression cell strain exogenous gene expression amount by eucaryon plasmid rotaring transfecting mode, provides a kind of helper plasmid of efficient structure people stable expression cell strain.Another object of the present invention is to the preparation method that above-mentioned helper plasmid is provided.

In previous research work, the present inventor finds in mouse genome, have a specific target sites sequence, this target site sequence has 32 copies in human genome, designed by this specific target sites and the helper plasmid built can for foreign gene provide 32 integrate focuses, the efficiency enabling exogenous origin gene integrator enter human genome reaches 21.23%.On the basis of above exploratory development, the present inventor finds in human genome, have a specific target sites sequence, this target site sequence has 167 copies in human genome, designed by this specific target sites and the helper plasmid built can for foreign gene provide 167 integrate focuses, the efficiency that exogenous origin gene integrator enters human genome can be significantly improved, and then improve the expression amount of foreign gene.On the basis of above exploratory development, present inventors have proposed helper plasmid of a kind of efficient structure people stable expression cell strain and preparation method thereof.

The helper plasmid of a kind of efficient structure people stable expression cell strain disclosed by the invention, it is characterized in that: this helper plasmid and foreign gene cotransfection people recipient cell, helper plasmid manually can form 167 breach in human genome specific target sites sequence, for foreign gene provides 167 to integrate focus.

The helper plasmid of above-mentioned a kind of efficient structure people stable expression cell strain, is characterized in that this helper plasmid has built according to following steps:

(1) by pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid enzyme restriction, enzyme cuts after product through agarose electrophoresis, reclaims the fragment containing pX330-U6-Chimeric_BB-CBh-hSpCas9;

(2) design is for the oligonucleotide of human genome specific target point sequence;

(3) oligonucleotide that step (2) designs is connected into that step (1) reclaims containing in the fragment of pX330-U6-Chimeric_BB-CBh-hSpCas9, product conversion bacillus coli DH 5 alpha competent cell will be connected, the well-grown mono-clonal of picking, 37 DEG C of shaking culture are spent the night, and extract plasmid.

The helper plasmid of above-mentioned a kind of efficient structure people stable expression cell strain, it is characterized in that on described human genome, specific target point sequence is as shown in SEQIDNO.1, corresponding oligonucleotide sequence is for shown in SEQIDNO.2 and SEQIDNO.3.

Beneficial effect of the present invention is: helper plasmid disclosed by the invention manually can form 167 breach on human genome specific target sites, focus is integrated for exogenous genetic fragment provides 167, the efficiency that exogenous origin gene integrator enters human genome can be significantly improved, and then improve the expression amount of foreign gene; The building process of helper plasmid disclosed by the invention is simple, cheap, and common laboratory also can complete structure voluntarily, its can directly from different foreign gene cotransfection people recipient cells, working method is simple; This helper plasmid effectively can improve the efficiency building all kinds of people's stable expression cell strain, can realize the efficient structure of various human cell's model, is conducive to the development of the pathogenetic research of human diseases and gene therapy.

Accompanying drawing explanation

In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:

Fig. 1 is selected by flow cytometry apoptosis transient transfection GFP positive-labeled cells result figure (A: blank; B:pSpCas9 (167T); C:pCMV-p53-2A-EGFP; D:pCMV-p53-2A-EGFP+pSpCas9 (167T)).

Fig. 2 is that selected by flow cytometry apoptosis passes 5 generation GFP-transfected positive-labeled cells result figure (A:pCMV-p53-2A-EGFP continuously; D:pCMV-p53-2A-EGFP+pSpCas9 (167T)).

Fig. 3 quantitative pcr amplification canonical plotting (A:P53 primer; B:IL-24 primer).

Embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.

Embodiment helper plasmid efficiently builds the strain of human colon carcinoma stably express p53 gene cell

(1) structure of helper plasmid

By pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (AddgeneplasmidID:42230, hereinafter referred to as pSpCas9 (BB)), cut with BbSI enzyme, 37 DEG C of water-baths are after 1 hour, the agarose electrophoresis of 1%, reclaims digestion products (TAKARA glue reclaims test kit).

It is as follows that enzyme cuts system:

Utilize online tool ZiFiTTargeterversion4.0 design for the oligonucleotide of human genome specific target sites sequence, be specially:

Specific target sites sequence: 5 '-GGAGGGGAACATCACATACC-3 ' (SEQIDNO.1).

Corresponding oligonucleotide is to being Oligo (+): 5 '-caccGGAGGGGAACATCACATACC-3 ' (SEQIDNO.2);

Oligo(-)：5’-aaacGGTATGTGATGTTCCCCTCC-3’(SEQIDNO.3)。

By two oligonucleotide annealing, form the short double-stranded DNA with sticky end, reaction system is as follows:

Above-mentioned reaction system is mixed in 200ulPCR pipe, then PCR pipe is processed 30min in 37 DEG C of water-baths, then put into 500ml boiling water, naturally cool to room temperature.

Linked system:

Double-strand short dna product with sticky end is connected into enzyme cut after pSpCas9 (BB) linear fragment, product conversion bacillus coli DH 5 alpha competent cell (TakaraCode:D9057A) will be connected, and coat overnight incubation on LB solid plate that Ampicillin concentration is 100 μ g/mL, the well-grown mono-clonal of picking, be in the LB liquid nutrient medium of 100 μ g/mL in 15mLAmpicillin concentration, 37 DEG C of shaking culture are spent the night, extract helper plasmid, called after pSpCas9 (167T).

(2) without the preparation of intracellular toxin plasmid DNA

A, get pSpCas9 (167T) helper plasmid 1 μ L add blow in 100 μ LDH5 α competent cells even, 20min is left standstill in ice, put into 42 DEG C of water-bath 90s again, be placed in rapidly ice bath 3min, add 500 μ LLB liquid nutrient mediums, place shaking table 180rpm37 DEG C of 1h, get bacterium liquid 100 μ L and be spread evenly across LB solid medium 37 DEG C of overnight incubation that Ampicillin concentration is 100 μ g/mL.

B, to get single bacterium colony be in the LB liquid nutrient medium of 100 μ g/mL in 3mLAmpicillin concentration, 250rpm, 37 DEG C of shaking culture 8 hours; Therefrom getting 300 μ L bacterium liquid, to be inoculated in 300mLAmpicillin concentration be in the LB liquid nutrient medium of 100 μ g/mL, and in 250rpm, 37 DEG C of shaking culture 12 ~ 16 hours;

C, collection bacterium liquid, then at 4 DEG C, centrifugal 15min under 4000rpm condition, abandon supernatant, collect thalline, then extract plasmid according to QIAGENEndoFreePlasmidMaxiKit test kit specification sheets operation steps, obtain without endotoxic pSpCas9 (167T) plasmid.

(3) exogenous gene expression carrier is built

The expression vector pCMV-p53-2A-EGFP of construction expression p53 gene, p53 gene (GenBank:AB082923.1) is synthesized by the raw work in Shanghai.

Carry greatly pCMV-p53-2A-EGFP plasmid, concrete with reference to step (2); Vector linearization method is with reference to Clontech company pEGFP-N1 specification sheets.

(4) cotransfection cell

First 3 days of transfection, recovery human colon cancer cell (CW-2 cell, Chinese Academy of Sciences's Shanghai cell bank), puts into the FBS+DMEM culturing bottle being added with 10%, in 37 DEG C, 5%CO by cell ₂incubator in cultivate, day before transfection, Secondary Culture recovery cell.Sucking-off nutrient solution, rinses 3 times with PBS, adds 0.25% appropriate trypsinase in bottle, add DMEM nutrient solution after 5min and stop digestion, piping and druming makes cell detachment, centrifugal (1000rpm gently, remove supernatant liquor 5min), make cell suspension, by 5 × 10 with DMEM nutrient solution ⁴the density Secondary Culture of individual/ml.

Utilize trysinization attached cell, centrifugal collecting cell, with PBS cleaning twice, careful suspension, counting, cell count is 7 × 10 ⁵individual/ml; Then, remove PBS, electroporation buffer is utilized to suspend, add linearizing pCMV-p53-2A-EGFP plasmid and each 10.5ug of pSpCas9 (167T) helper plasmid respectively, regulate and make final volume be 700ul, then mixed solution is put in the electric shock cup of 0.4cm, under selecting square wave condition, voltage 250V, 25ms, 1 subpulse parameter shocks by electricity.Cell suspending liquid after electric shock is transferred in Tissue Culture Flask, adds 5mlDMEM (containing 10%FBS) and continue to cultivate.

Separately distinguish transfection linearizing pCMV-p53-2A-EGFP plasmid, pSpCas9 (167T) plasmid, any plasmid of not transfection in contrast under the same conditions.

(5) mensuration of integration efficiency

Transfection, after 24 hours, utilizes trysinization attached cell, centrifugal collecting cell, with PBS cleaning twice, then, adopts selected by flow cytometry apoptosis transient transfection GFP positive-labeled cells.Result display only has in transfection pCMV-p53-2A-EGFP plasmid (Fig. 1 C) and pCMV-p53-2A-EGFP+pSpCas9 (167T) recombinant plasmid (Fig. 1 D) two groups can sub-elect GFP mark positive cell.And only do not sub-elect GFP in transfection pSpCas9 (167T) helper plasmid (Figure 1B) and any plasmid of not transfection (Figure 1A) two groups and mark positive cell.

Continued respectively to cultivate by sub-elect two groups of GFP mark positive cells, in 5 generations of biography, fully remove transient transfection interference continuously, and employing selected by flow cytometry apoptosis goes out the cell with green fluorescence.Result shows, and two groups of cells (pCMV-p53-2A-EGFP, pCMV-p53-2A-EGFP+pSpCas9 (167T)) are respectively 2.34% (Fig. 2 A) and 20.16% (Fig. 2 B) with green cells ratio.Illustrate that pSpCas9 (167T) helper plasmid built significantly can improve the efficiency that pCMV-p53-2A-EGFP plasmid integration enters human genome, and foreign gene p53 stably express can be made.

(6) mensuration of p53 cell strain gene copy number

Use SYBRGreen fluorescence dye real-time quantitative PCR method measure exogenous p 53 gene copy number (concrete grammar see: the strong .SYBRGreen real-time quantitative PCR in the village detects copy number of foreign gene [J]. Institutes Of Technology Of Zhejiang's journal, 2010,27 (1): 125-129).

The present invention is with human genome single copy gene interleukin-22 4 (IL-24) (LiuXY.Targetinggene-virotherapyofcanceranditsprosperity [J] .CellRes, 2006,16 (11): 879-886) copy number of foreign gene p53 in stable cell lines is detected as reference gene.For avoid SYBRGreen in quantitative fluorescent PCR reaction different amplified production because of the feature of its length and this body structure different, and cause the impact that the fluorescence intensity difference produced is brought, design construction goes out to contain list copy p53 gene and list copies the reference plasmid of IL-24 gene as standard substance, by drawing the typical curve of p53 primer and IL-24 primer respectively, thus determine the content of p53 gene and IL-24 gene in sample gene group, recycling proportionlity is between the two multiplied by the copy number of individual cells endogenous IL-24 gene to determine the integrate copy number of foreign gene p53.

Built the cell strain of stably express p53 gene by plasmid pCMV-p53-2A-EGFP, collect the cell concentration of 6 orifice plates, genome extraction step extracts test kit with reference to TakaraDNA.SYBRGreen real-time quantitative PCR is adopted to detect copy number of foreign gene.Primer amplification fragment for quantitative PCR controls between 50 ~ 150bp, and design primer sequence is as follows:

p53-F：5’-AGGAAATTTGCGTGTGGAGT-3’(SEQIDNO.4)

p53-R：5’-TACAGTCAGAGCCAACCTCA-3’(SEQIDNO.5)

IL-24-F：5’-AGATCTATGAATTTTCAACAGAGGC-3’(SEQIDNO.6)

IL-24-R：5’-ACGCGTTCAGAGCTTGTAGAATTTC-3’(SEQIDNO.7)

IL-24PCR reaction system is:

The response procedures parameter of PCR is 94 DEG C, 5min; 94 DEG C, 15s; 55 DEG C, 30s; 72 DEG C, 1min; 30 circulations.Collect pcr amplification product and purifying (see TAKARA, glue reclaims test kit), be cloned into (see TAKARA, pMD18-T test kit) in pDM18-T carrier.In like manner, pcr amplification is carried out to p53 gene.

The drafting of typical curve: by first diluting into about 10 with reference to plasmid pCMV-p53-2A-EGFP and PMD18T-IL-24 after purifying ⁴individual/ul, then continuous gradient dilutes 3 gradients, extension rate is 2.5 times, is designated as 10000,4000,1600,640/ul respectively.Using identical standard substance as template, draw the typical curve for p53 primer (Fig. 3 A) and IL-24 (Fig. 3 B) respectively.

Table 1Realtime-PCR result

Realtime-PCR detects p53 gene and the IL-24 gene of the cell strain that transfection PCMV-p53-2A-EGFP plasmid obtains, the mean CT-number (table 1) obtained.According to the typical curve that p53 primer and IL-24 primer are drawn, and the mean CT-number that Realtime-PCR obtains, the IL-24 gene copy number that can obtain the cell strain built by plasmid PCMV-p53-2A-EGFP is the copy number of 5011 ± 12.21/ul, p53 gene is 3715 ± 24.11/ul.The integrate copy number of p53 equals the copy number of IL-24 on genome and is multiplied by both quantitative ratio, i.e. 1 × (5011 ÷ 3715)=1.34.

Calculate by identical method, in the cell strain built by plasmid pCMV-p53-2A-EGFP+pSpCas9 (167T), p53 gene copy number is 3.89.

(7) mensuration of p53 cell strain expressing quantity

Adopt piping and druming method, collect in the centrifuge tube of 1.5ml by cultivation strain cell 6 orifice plate cells and cell culture fluid, multigelation passes through multigelation, to make cytoclasis and to release composition in cell.Centrifugal 20 minutes (3000 revs/min), carefully collect supernatant.

The dilution of standard substance and application of sample: at enzyme mark bag by the accurate sample wells 10 of bidding on plate hole, add standard substance 100 μ l respectively in first, second hole, in first, second hole, then add standard dilutions 50 μ l, mixing; Then from the first hole, the second hole, respectively get 100 μ l be added to the 3rd hole and the 4th hole respectively, then add standard dilutions 50 μ l respectively in the 3rd, the 4th hole, mixing; Then in the 3rd hole and the 4th hole, first respectively get 50 μ l to discard, respectively get 50 μ l and be added to respectively in the 5th, the 6th hole, then in the 5th, the 6th hole, add standard dilutions 50ul respectively, mixing; From the 5th, the 6th hole, respectively get after mixing that 50 μ l are added to the 7th respectively, in octal, again the 7th, add standard dilutions 50 μ l respectively in octal, after mixing from the 7th, get 50 μ l respectively octal and be added in the 9th, the tenth hole, standard dilutions 50 μ l is added respectively again in the 9th, the tenth hole, from the 9th, the tenth hole, respectively get 50 μ l after mixing discard (after dilution, each hole application of sample amount is all 50 μ l, and concentration is respectively 30 μ g/mL, 20 μ g/mL, 10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL).

Establish blank well (blank control wells does not add sample and enzyme marking reagent, and respectively step operation is identical for all the other), testing sample hole respectively.In enzyme mark bag is by testing sample hole on plate, first adds sample diluting liquid 40 μ l, and then adds testing sample 10 μ l (the final extent of dilution of sample is 5 times).

Elisa detects cell strain p53 expressing quantity, and concrete operation method step is with reference to (Yan Jin bio tech ltd, Shanghai, ELISA kit specification sheets).Result shows, by the stably express p53 gene cell strain of transfection CMV-p53-2A-EGFP plasmid and pCMV-p53-2A-EGFP+pSpCas9 (167T) plasmid construction, OD value be respectively 0.17 and 0.41, p53 protein concentration be respectively 3.5ug/ml and 10.29ug/ml.

Can be found out by above data, helper plasmid pSpCas9 (167T) participates in copy number and the equal showed increased of p53 expressing quantity of the p53 cell strain p53 gene built.

What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.

Claims (3)

1. one kind efficiently builds the helper plasmid of people's stable expression cell strain, it is characterized in that: this helper plasmid and foreign gene cotransfection people recipient cell, helper plasmid manually can form 167 breach in human genome specific target sites sequence, for foreign gene provides 167 to integrate focus.

2. the helper plasmid of a kind of efficient structure people stable expression cell strain according to claim 1, is characterized in that this helper plasmid has built according to following steps:

(1) by pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid enzyme restriction, enzyme cuts after product through agarose electrophoresis, reclaims the fragment containing pX330-U6-Chimeric_BB-CBh-hSpCas9;

(2) design is for the oligonucleotide of human genome specific target point sequence;

(3) oligonucleotide that step (2) designs is connected into that step (1) reclaims containing in the fragment of pX330-U6-Chimeric_BB-CBh-hSpCas9, product conversion bacillus coli DH 5 alpha competent cell will be connected, the well-grown mono-clonal of picking, 37 DEG C of shaking culture are spent the night, and extract plasmid.

3. the helper plasmid of a kind of efficient structure people stable expression cell strain according to claim 1 and 2, it is characterized in that on described human genome, specific target point sequence is as shown in SEQIDNO.1, corresponding oligonucleotide sequence is as shown in SEQIDNO.2 and SEQIDNO.3.