CN103045647A - Method for establishing and identifying core fucosyltransferase gene silencing cell model - Google Patents
Method for establishing and identifying core fucosyltransferase gene silencing cell model Download PDFInfo
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- CN103045647A CN103045647A CN2012104965546A CN201210496554A CN103045647A CN 103045647 A CN103045647 A CN 103045647A CN 2012104965546 A CN2012104965546 A CN 2012104965546A CN 201210496554 A CN201210496554 A CN 201210496554A CN 103045647 A CN103045647 A CN 103045647A
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Abstract
The invention discloses a method for establishing a core fucosyltransferase gene silencing cell model, which comprises the following steps of: selecting the most effective RNA (ribonucleic acid) interference target sequence; synthesizing two sections of complementary oligonucleotides in vitro; and recombining the siRNA fragment of Fut8 gene into retrovirus plasmid pSINsi-hU6 by use of the enzyme digestion sites BamH I and Cla I on a carrier. Through 293T cell packing, the titer of the recombinant retrovirus can reach 2.1*10<5>CFU/ml, and the B cell strain is subjected to 400-1,000mu g/ml G418 screening to obtain a stable expression strain before infection of the generated recombinant retrovirus. The detection result of Real time-PCR and high performance liquid chromatography (HPLC) indicates that the activity of Fut8mRNA and enzyme is obviously reduced in the Fut8siRNA replication-defective recombinant retrovirus-infected 70Z/3 cell. The method disclosed by the invention has the advantages of easiness in operation, high gene silencing efficiency, good repeatability and the like. The establishment of a Fut8 gene silencing cell model lays a foundation for the biological function study of Fut8 gene.
Description
Technical field
The present invention relates to a kind of biological function research field, more particularly, relate to the reticent cell model establishment method of a kind of core fucosyl transferase gene.
Background technology
The core fucosylation is general protein post-translational modification process.Fut8 is unique glycosyltransferase (as shown in Figure 8) of modifying the core fucosylation, can regulate space structure and the biological function of protein, such as the correct location of molecular interaction, cell and intercellular mutual identification, protein, cell signaling etc.Fut8
-/-Mouse has the pulmonary emphysema phenotype, and this is because the damaged binding ability that then reduces TGF 1 and acceptor of the core fucosido on the TGF-1 acceptor activates the expression of matrix metalloproteinase (MMP), causes the pulmonary emphysema pathology; The Fut8 gene knockout causes the vascular endothelial growth factor receptor expression amount of cell surface to descend.In addition, the discovery core fucosylation degree such as Stubbs can affect molecular conformation and the flexibility of the N-sugar chain on the glycoprotein molecule.
Method commonly used is to utilize RNA to disturb (RNA interference, RNAi) or gene knockout method at present, suppresses this genetic expression, observes losing of corresponding function.The gene knockout method is not only time-consuming but also need most advanced and sophisticated experimental installation.RNA i is found to be the New Policy that people provide specific inhibition genetic expression.RNA i can make the target gene degraded, and have high specificity, suppression efficiency high by the double-stranded RNA of the 21bp of transfection in cell and target gene identical sequence.At present siRNA introduction method commonly used is the carrier that utilizes liposome to import double-stranded siRNA or contain siRNA sequence, but aforesaid method is degraded after existing siRNA to enter cell easily, in problems such as intracellular duration of effect are short, siRNA expression vector transfection efficiency is low.For this situation, using at present more is the carriers such as retrovirus, adenovirus, liposome.
At present, article and the patent of the reticent cell model establishment method of relevant a kind of core fucosyl transferase gene are as follows:
(1) is used for the method and composition (patent publication No.: CN101883850A) of inactivating alpha-1,6 fucosyltransferase (FUT8) genetic expression
(2) Methods and compositions for inactivating alpha 1,6fucosyltransferase (FUT8) gene expression (patent publication No.: US2009042250 (A1))
(3) Compositon for suppressing the expression of fucosyltransferase (patent publication No.: US2009221065 (A1))
(4)Wang?X,Inoue?S,Gu?J,et?al.Dysregulation?of?TGF-beta1receptor?activation?leads?to?abnormal?lung?development?and?emphysema-like?phenotype?in?core?fucose-deficient?mice.Proc?Natl?Acad?Sci?USA.2005,102(44):15791-6.
(5)Stubbs?HJ?et?al.Influence?of?core?fucosylation?on?the?flexibility?of?a?biantennary?N-linked?oligosaccharide.Biochemistry?1996,35:937-947.
Patent 1-3 invention comes the method and composition of inactivation FUT8 gene with the fusion rotein that comprises zinc finger protein and cutting structure territory or cut half structural domain.The polynucleotide of encoding said fusion protein also are provided, and the cell that comprises described polynucleotide and fusion rotein.
The damaged binding ability that then reduces TGF1 and acceptor of core fucosido on the TGF-1 acceptor introduced in article 4, activates the expression of matrix metalloproteinase (MMP), causes the pulmonary emphysema pathology.
Molecular conformation and the flexibility that core fucosylation degree can affect the N-sugar chain on the glycoprotein molecule introduced in article 5.
At present, the research of Fut8siRNA replication defect type recombinant retrovirus structure Fut8 gene silencing cell model there is no report.
Summary of the invention
The present invention utilizes recombinant retroviral vector to make up the pSINsi-hU6-Fut8siRNA carrier, many drawbacks of conventional carriers have been overcome, with stably express Fut8siRNA, mode Rapid Establishment Fut8 gene silencing cell model by cell infection is intended to lay the foundation for the functional study of Fut8 biology of gene.
In order to achieve the above object, the establishment method of the reticent cell model of a kind of core fucosyl transferase gene of the present invention comprises the steps:
Step1, according to the Fut8 gene order, design 1 21 nucleotide double Fut8siRNA sequences:
CUGAUCACU?CCAGCAGAGA（759-778）；
And the double-stranded bob clamping structure of design Fut8siRNA:
siRNA-sense:
5′-GATCCA
CTGATCACTCCAGCAGAGATTCAAGAGA
TCTCTGCTGGAGTGA TCAGTTTTTTAT-3′;
siRNA-antisense:
5′-CGATAAAAAAA
CTGATCACTCCAGCAGAGATCTCTTGAA
CTCTGCTGGA GTGATCAGTG-3′
The foundation of Step 3, Fut8 gene silencing cell model comprises following substep:
Step 31, with the liposome transfection method with pSINsi-hU6-Fut8siRNA, the pE-ampho carrier, the common transfection 293T of pGP carrier cell, packing carries the retrovirus of pSINsi-hU6-Fut8siRNA;
Step 32, with 3-5 * 10
4Target cell carries out 9-12h to be cultivated, utilize 7-10ug/mL polybrane to hatch after, by the virus infection mode with the Fut8siRNA transfection to target cell;
Step 33, screen with 400-1000ug/ml G418, and the picking individual cells, carry out enlarged culturing, obtain stable Fut8 gene silencing cell model.
Under the optimal way, among the Step 31 liposome and the volumetric molar concentration ratio of pSINsi-hU6-Fut8siRNA, pE-ampho carrier, pGP carrier be 4-5:1.5-2:1-1.5:1-1.5; Ratio among the Step 32 between virus liquid and the cell culture fluid is 1-2:10.
The present invention also provides the detection method of the reticent cell model of above-mentioned core fucosyl transferase gene, selects real-time quantitative PCR to identify that wherein, the primer sequence of described real-time quantitative PCR is:
sense:AACAGCTTGTTAAGGCCAAAG，
antisense:GCATGTCTTTGGAGTTCATTTC。
The present invention also provides the detection method of the reticent cell model enzymic activity of above-mentioned core fucosyl transferase gene, utilizes the HPLC method to measure the Fut8 enzymic activity, and the cell pyrolysis liquid protein content is 0.2-0.5mg, and the reaction times is 2-5 hour.
The present invention selects the most effective RNA to interfere target sequence in the Fut8 of 4 kinds of decision designs gene-specific RNA interference target sequence.According to the oligonucleotide of this sequence in external synthetic two sections complementations, utilize BamH I and Cla I restriction enzyme site on the carrier, Fut8 gene siRNA fragment is recombinated among the retroviral plasmid pSINsi-hU6.After 293T cell packing, the recombinant retrovirus titre can reach 2.1 * 10
5CFU/ml, the recombinant retrovirus of generation infects pre B cell strain (70Z/3 cell), obtains stably express strain, called after Fut8-KD-70Z/3 by 400-1000ug/ml G418 screening.Show that with Real time-PCR and high performance liquid chromatography (HPLC) detected result Fut8siRNA replication defect type recombinant retrovirus infects in the 70Z/3 cell, Fut8mRNA and enzymic activity significantly descend.This invention has simple to operate, the advantages such as gene silencing efficient height, good reproducibility.The Fut8 biology of gene functional study that is established as of Fut8 gene silencing cell model is laid a good foundation.
Description of drawings
Fig. 1 is the double-stranded sequences of 4 Fut8siRNA;
Fig. 2 is the optimal proportions of TransIT-TKO transfection reagent and double-stranded siRNA;
Fig. 3 is the ordered sequence of screening Fut8siRNA;
Fig. 4 is that the PCR of retroviral plasmid pSINsi-hU6-Fut8siRNA identifies;
Fig. 5 is the Fut8 gene silencing 70Z/3 cell through the G418 screening;
Fig. 6 is that the Fut8 gene expression amount obviously descends in the Fut8 gene silencing 70Z/3 cell;
Fig. 7 is that the Fut8 enzymic activity obviously descends in the Fut8 gene silencing 70Z/3 cell;
Fig. 8 is Fut8 catalyzed reaction figure.
Embodiment
Technical scheme of the present invention is: at first design 4 kinds of Fut8 gene-specific RNA interference target sequences, select the most effective RNA to interfere target sequence.The Fut8siRNA fragment is recombinated among the retroviral plasmid pSINsi-hU6.The recombinant retrovirus that produces after 293T cell packing infects pre B cell strain (70Z/3 cell), obtains stably express strain, called after Fut8-KD-70Z/3 by the G418 screening.This invention has simple to operate, the advantages such as gene silencing efficient height, good reproducibility.The Fut8 biology of gene functional study that is established as of Fut8 gene silencing cell model is laid a good foundation.
Embodiment 1:
(1) the online http://rnaidesigner.invitrogen.com/sirna of the selection utilization software program of Fut8siRNA fragment design 4 21 nucleotide double siRNA sequences (19 Nucleotide+overhang TT).From Fut8 gene (GenBank Accession NM_016893), select respectively 4 kinds of siRNA sequences (Fig. 1).Utilize TransIT-TKO transfection reagent (Mirus Co.Madison, USA) that 4 double-stranded siRNA are transfected in the target cell, after 24 hours, the screening specificity suppresses the most effective siRNA sequence that Fut8 expresses.The ratio of TransIT-TKO transfection reagent and double-stranded siRNA is 8:3(Fig. 2).Through enzyme assay, determine that finally Fut8siRNA (759-778:CUG AUC ACU CCA GCA GAGATT) is optimal sequence (Fig. 3).
(2) structure that the recombinant retroviral vector that contains the Fut8 gene makes up pSINsi-hU6-Fut8 becomes double-stranded hairpin structure with siRNA hairpin Oligonucleotide Sequence Designer software design, siRNA-sense:5 '-
CTGATCACTCCAGCAGAGATTCAAGAGA
TCTCTGC TGGAGTGATCAGTTTTTTAT-3 '; SiRNA-antisense:5 '-
CTGATCAC TCCAGCAGAGATCTCTTGAA
CTCTGCTGGAGTGATCAGTG-3 '.5 ' end of the most effective siRNA sequence two strands is introduced Bam H I site (italic represents), 3 ' end is Cla I site (italic represents), 2 restriction enzyme sites all are sticky end, middle 19 Nucleotide are applicable to form hairpin ring structure (expression of rolling off the production line), near the 3 ' termination signal of holding the U6 promotor of TTTTTT.The siRNA sequence two strands is connected with pSINsi-hU6's, and 16 ℃ of connections are spent the night, and connects product pSINsi-hU6-Fut8 and transforms e. coli jm109, and coated plate is selected to cultivate, and selects white colony.Carry out polymerase chain reaction (PCR) amplification with Auele Specific Primer, specific primer sequence is as follows: Sense:5 '-GATCCACTGATCACTCCA GCAGAGATTCAAGAGATCTCTGCTGGAGTGATCAGTTTTTTAT-3 ' (containing BamH I restriction enzyme site); Antisense:5 '-TAATTGAGATGCATGCTTTG-3 ' (Cla I restriction enzyme site downstream).Select positive colony, extract in a small amount plasmid and order-checking.BamH I and Cla I double digestion identify that 1.5% agarose gel electrophoresis inspection enzyme is cut the result, determines the Insert Fragment size.The Fut8siRNA fragment is recombinated among the retroviral plasmid pSINsi-hU6, carry out PCR and identify (170bp band), the result shows the Fut8siRNA carrier, and pSINsi-hU6-Fut8siRNA successfully constructs (Fig. 4).
(3) retrovirus packing and virus titer are measured and are pressed Wizard Plus SV Minipreps DNA purification kit operation instructions extraction pSINsi-hU6-Fut8siRNA, with the liposome transfection method with pSINsi-hU6-Fut8siRNA, the pE-ampho carrier, the common transfection packing cell of pGP carrier 293T, form the pseudotyped retroviral virion with single infection of restructuring, obtained to carry the retrovirus of the siRNA of Fut8 target.The ratio of liposome and plasmid is 4:1.5:1:1.48h after the transfection, trysinization, diluted passage behind the cultivation 24h, screens with 800ug/ml G418.After two weeks, select monoclonal cell, change 24 well culture plates over to and cultivate, after amplification, change the 6cm culture dish over to and cultivate.With every 6cm plating 5 * 10
4The NIH3T cell is inoculated rear 24h, after hatching with polybrene 9ug/mL, inoculation 1:10,1: 100,1: 1000,1:10000, the virus liquid of dilution in 1: 100000 is after continuing to cultivate 24h, trysinization, cultivate 24h, add again after 800ug/ml G418 screened for 2 weeks, fix through formaldehyde, Giemsa dyeing, meter resistant cell clone number calculates virus titer with this, represents (CFU/ml) with clonogenic unit.
(4) the Fut8 gene silencing 70Z/3 cystic cancer cell line preparatory stage: first day gets 3 * 10
4In 70Z/3 cell six orifice plates, add certain nutrient solution, be put into CO
2Cultivate in the incubator.Infective stage: change liquid, add polybrene 7ug/mL and recombinant retrovirus liquid, at CO
2Hatch 4-6h in the incubator, after append again the 1ml nutrient solution in six orifice plates, CO
2The incubator incubated overnight.Ratio between virus liquid and the cell culture fluid is 1:10.G418 screening: add G418(400ug/ml) in nutrient solution, then changed the nutrient solution that contains G418 every 3 days.After two weeks, get single 70Z/3 cell at microscopically and carry out the cultivation of 96 orifice plates, obtain to stablize Fut8 gene silencing cell strain (Fut8-KD-70Z/3 cell).Import the 70Z/3 cell of empty carrier as the Mock cell.Utilize the 293T cell to carry out the retrovirus packing, and obtain to contain the retroviral particle of Fut8siRNA.By the virus infection experiment, infect the 70Z/3 cell, screen with 400ug/mlG418 again.The picking individual cells is inoculated into and carries out enlarged culturing in 96 orifice plates, obtains stable Fut8 gene silencing cell model (Fig. 5).
(5) real-time quantitative PCR gets 1 * 10
6Individual 70Z/3 cell and Fut8-KD-70Z/3 cell are pressed the operation instructions extraction cell total rna that TrizolRNA extracts test kit.Take Oligo (dT) as initial primers, add total RNA 1 μ g, behind 70 ℃ of 10min, add successively the water without RNase, 10 * RT buffer, 250 μ mol/LdNTPs, RNase-Inhibitor, RT Reverse Transcriptase, reaction system is 20 μ l.In the PCR instrument, setting program, 42 ° of C 90min, 85 ° of C 5min, 4 ° of C 1min utilize SYBR Green to detect GAPDH by real-time quantitative PCR simultaneously, the amount of cDNA among 70Z/3 and the Fut8-KD-70Z/3.With SYBR Green Real-time PCR test kit, add a certain amount of reagent, in the PCR instrument, setting program: sex change: 93 ° of C 10s, 52 ° of C 20s, 35 circulations of 72 ° of C 1min are extended in 94 ° of C 3min annealing.Reverse transcription product is carried out Real-time PCR, measure the expression amount of cDNA.Use house-keeping gene GAPDH that the RNA that joins in the reverse transcription reaction is carried out the homogenization processing.Extract mRNA in 70Z/3 and the Fut8-KD-70Z/3 cell, carry out real-time quantitative PCR and detect.Compare with the 70Z/3 cell, the Fut8mRNA of Fut8-KD-70Z/3 cell expresses descend (Fig. 6).
(6) the Fut8 enzyme assay gets 1 * 10
6Individual 70Z/3 cell and Fut8-KD-70Z/3 cell wash twice with PBS, and rear usefulness contains 0.05% trypsinase and 0.02%EDTA is hatched 45min at 37 ° of C.Utilize 1%Triton-100 to carry out cracking, the centrifugal 5min of 10000rpm gets supernatant, surveys protein content, reacts with substrate.Substrate is the S:GnGn-bi-Asn-PABA of PABA:4-(2-pyridylamino) butylamine mark.By HPLC its product (P:GnGnFuc-bi-Asn-PABA) is analyzed.Fut8 enzymic activity (pmol/hr/mg) is by calculating according to formula [P (pmol)/reaction times (hr)/protein content (mg)].Protein content is 0.200mg, and the reaction times is 2 hours.Extract protein in 70Z/3 and the Fut8-KD-70Z/3 cell and carry out the HPLC detection.Compare the enzymic activity of Fut8-KD-70Z/3 cell obviously descend (Fig. 7) with the 70Z/3 cell.
Embodiment 2:
(1) the online http://rnaidesigner.invitrogen.com/sirna of the selection utilization software program of Fut8siRNA fragment design 4 21 nucleotide double siRNA sequences (19 Nucleotide+overhang TT).From Fut8 gene (GenBankAccession NM_016893), select respectively 4 kinds of siRNA sequences (Fig. 1).Utilize TransIT-TKO transfection reagent (Mirus Co.Madison, USA) that 4 double-stranded siRNA are transfected in the target cell, after 24 hours, the screening specificity suppresses the most effective siRNA sequence that Fut8 expresses.The ratio of TransIT-TKO transfection reagent and double-stranded siRNA is 8:5(Fig. 2).Through enzyme assay, determine that finally Fut8siRNA (759-778:CUG AUC ACU CCA GCA GAGATT) is optimal sequence (Fig. 3).
(2) structure that the recombinant retroviral vector that contains the Fut8 gene makes up pSINsi-hU6-Fut8 becomes double-stranded hairpin structure with siRNA hairpin Oligonucleotide Sequence Designer software design, siRNA-sense:5 '-
CTGATCACTCCAGCAGAGATTCAAGAGA
TCTCTGC TGGAGTGATCAGTTTTTTAT-3 '; SiRNA-antisense:5 '-
CTGATCAC TCCAGCAGAGATCTCTTGAA
CTCTGCTGGAGTGATCAGTG-3 '.5 ' end of the most effective siRNA sequence two strands is introduced Bam H I site (italic represents), 3 ' end is Cla I site (italic represents), 2 restriction enzyme sites all are sticky end, middle 19 Nucleotide are applicable to form hairpin ring structure (expression of rolling off the production line), near the 3 ' termination signal of holding the U6 promotor of TTTTTT.The siRNA sequence two strands is connected with pSINsi-hU6's, and 16 ℃ of connections are spent the night, and connects product pSINsi-hU6-Fut8 and transforms e. coli jm109, and coated plate is selected to cultivate, and selects white colony.Carry out polymerase chain reaction (PCR) amplification with Auele Specific Primer, specific primer sequence is as follows: Sense:5 '-GATCCACTGATCACTCCA GCAGAGATTCAAGAGATCTCTGCTGGAGTGATCAGTTTTTTAT-3 ' (containing BamH I restriction enzyme site); Antisense:5 '-TAATTGAGATGCATGCTTTG-3 ' (Cla I restriction enzyme site downstream).Select positive colony, extract in a small amount plasmid and order-checking.BamH I and Cla I double digestion identify that 1.5% agarose gel electrophoresis inspection enzyme is cut the result, determines the Insert Fragment size.The Fut8siRNA fragment is recombinated among the retroviral plasmid pSINsi-hU6, carry out PCR and identify (170bp band), the result shows the Fut8siRNA carrier, and pSINsi-hU6-Fut8siRNA successfully constructs (Fig. 4).
(3) retrovirus packing and virus titer are measured and are pressed Wizard Plus SV Minipreps DNA purification kit operation instructions extraction pSINsi-hU6-Fut8siRNA, with the liposome transfection method with pSINsi-hU6-Fut8siRNA, the pE-ampho carrier, the common transfection packing cell of pGP carrier 293T, form the pseudotyped retroviral virion with single infection of restructuring, obtained to carry the retrovirus of the siRNA of Fut8 target.The ratio of liposome and plasmid is 5:2:1.5:1.5.48h after the transfection, trysinization, diluted passage behind the cultivation 24h, screens with 800mg/L G418.After two weeks, select monoclonal cell, change 24 well culture plates over to and cultivate, after amplification, change the 6cm culture dish over to and cultivate.With every 6cm plating 5 * 10
4The NIH3T cell is inoculated rear 24h, after hatching with polybrene 9ug/mL, inoculate 1: 10,1: 100,1:1000,1:10000, the virus liquid of dilution in 1: 100000 is after continuing to cultivate 24h, trysinization, cultivate 24h, add again after 800ug/ml G418 screened for 2 weeks, fix through formaldehyde, Giemsa dyeing, meter resistant cell clone number calculates virus titer with this, represents (CFU/ml) with clonogenic unit.
(4) the Fut8 gene silencing 70Z/3 cystic cancer cell line preparatory stage: first day gets 5 * 10
4In 70Z/3 cell six orifice plates, add certain nutrient solution, be put into CO
2Cultivate in the incubator.Infective stage: change liquid, add polybrene 10ug/mL and recombinant retrovirus liquid, at CO
2Hatch 4-6h in the incubator, after append again the 1ml nutrient solution in six orifice plates, CO
2The incubator incubated overnight.Ratio between virus liquid and the cell culture fluid is 2:10.G418 screening: add G418(1000ug/ml) in nutrient solution, then changed the nutrient solution that contains G418 every 3 days.After two weeks, get single 70Z/3 cell at microscopically and carry out the cultivation of 96 orifice plates, obtain to stablize Fut8 gene silencing cell strain (Fut8-KD-70Z/3 cell).Import the 70Z/3 cell of empty carrier as the Mock cell.Utilize the 293T cell to carry out the retrovirus packing, and obtain to contain the retroviral particle of Fut8siRNA.By the virus infection experiment, infect the 70Z/3 cell, screen with 1000ug/mlG418 again.The picking individual cells is inoculated into and carries out enlarged culturing in 96 orifice plates, obtains stable Fut8 gene silencing cell model (Fig. 5).
(5) the Fut8 enzyme assay gets 1 * 10
6Individual 70Z/3 cell and Fut8-KD-70Z/3 cell wash twice with PBS, and rear usefulness contains 0.05% trypsinase and 0.02%EDTA is hatched 5min at 37 ° of C.Utilize 1%Triton-100 to carry out cracking, the centrifugal 5min of 10000rpm gets supernatant, surveys protein content, reacts with substrate.Substrate is the S:GnGn-bi-Asn-PABA of PABA:4-(2-pyridylamino) butylamine mark.By HPLC its product (P:GnGnFuc-bi-Asn-PABA) is analyzed.Fut8 enzymic activity (pmol/hr/mg) is by calculating according to formula [P (pmol)/reaction times (hr)/protein content (mg)].Protein content is 0.500mg, and the reaction times is 5 hours.Extract protein in 70Z/3 and the Fut8-KD-70Z/3 cell and carry out the HPLC detection.Compare the enzymic activity of Fut8-KD-70Z/3 cell obviously descend (Fig. 7) with the 70Z/3 cell.
According to above-mentioned two embodiment, the inventive method is summarized as follows, and step is:
The selection of S1, best Fut8siRNA fragment, concrete substep is as follows:
S11, according to the Fut8 gene order, design 4 21 nucleotide double siRNA sequences; CUGAUCACU CCAGCAGAGA(759-778); CAGCUUGUUAAGGCCAAAG(918-936); UCUCAGAAUUGGCGCUAUG(1386-1404); CAGGCUUAUAUCCCUCCUA(2302-2320).
S12, utilize TransIT-TKO transfection reagent to be transfected into each double-stranded siRNA in the target cell, measure its Fut8mRNA with real-time quantitative PCR, screen at last specificity and suppress the most effective Fut8siRNA sequence that Fut8mRNA expresses;
The structure of S2, replication defect type Fut8siRNA recombinant retroviral vector (pSINsi-hU6-Fut8siRNA), concrete substep is as follows:
S21, the double-stranded bob clamping structure of the most effective Fut8siRNA sequences Design Fut8siRNA of basis.
S22, the Fut8siRNA fragment is recombinated among the retroviral plasmid pSINsi-hU6, make up the pSINsi-hU6-Fut8siRNA carrier.
The foundation of S3, Fut8 gene silencing cell model
S31, with liposome (lipofectamine) infection protocol with pSINsi-hU6-Fut8siRNA, the pE-ampho carrier, the common transfection 293T of pGP carrier cell, packing carries the retrovirus of pSINsi-hU6-Fut8siRNA.
S32, with 3-5 * 10
4Target cell carries out 9-12h to be cultivated, utilize 7-10ug/mL polybrane to hatch after, by the virus infection mode with the Fut8siRNA transfection to target cell.
S33, screen with 400-1000ug/ml G418, and the picking individual cells, carry out enlarged culturing, obtain stable Fut8 gene silencing cell model.
The most effective Fut8siRNA sequence of step S12 is (759-778:CUGAUCACUCCAGCAGAGA).Step S12 concrete steps are: utilize the ratio of Trans IT-TKO transfection reagent and Fut8siRNA to be 8:3-8:5.
Be siRNA-sense:5 '-GATCCA in the double-stranded bob clamping structure of the Fut8siRNA of step S21 sequence
CTGATCACTCCAGCAGAGATTCAAGAGA
TCTCTGCTGGAGTGA TCAGTTTTTTAT-3 ';
siRNA-antisense:5′-CGATAAAAAAA
CTGATCACTCCAGCAGAGATCTCTTGAA
CTCTGCTGGAGTGATCAGTG-3′。
In step S31 liposome (lipofectamine) and pSINsi-hU6-Fut8siRNA, the pE-ampho carrier, the volumetric molar concentration ratio of pGP carrier is 4-5:1.5-2:1-1.5:1-1.5.Virus liquid and the ratio between the cell culture fluid at step S32 are 1-2:10.
The final method that detects the reticent cell of described core fucosyl transferase gene is real-time quantitative PCR.The primer sequence of real-time quantitative PCR is sense:AACAGCTTGTTAAGGCCAAAG, antisense:GCATGTCTTTGGAGTTCATTTC.
When utilizing the HPLC method to measure the Fut8 enzymic activity, the cell pyrolysis liquid protein content is 0.2-0.5mg, and the reaction times is 2-5 hour.
The above; only be the better embodiment of the present invention; but protection scope of the present invention is not limited to this; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, all should be encompassed within protection scope of the present invention.
Claims (5)
1. the establishment method of the reticent cell model of core fucosyl transferase gene is characterized in that, comprises the steps:
S1, according to the Fut8 gene order, design 1 21 nucleotide double Fut8siRNA sequences:
CUGAUCACU?CCAGCAGAGA（759-778）；
And the double-stranded bob clamping structure of design Fut8siRNA:
siRNA-sense:
5′-GATCCA
CTGATCACTCCAGCAGAGATTCAAGAGA
TCTCTGCTGGAGTGA TCAGTTTTTTAT-3′;
siRNA-antisense:
5′-CGATAAAAAAA
CTGATCACTCCAGCAGAGATCTCTTGAA
CTCTGCTGGA GTGATCAGTG-3′
S2, this Fut8siRNA fragment is recombinated among the retroviral plasmid pSINsi-hU6, make up the pSINsi-hU6-Fut8siRNA carrier;
The foundation of S3, Fut8 gene silencing cell model comprises following substep:
S31, with the liposome transfection method with pSINsi-hU6-Fut8siRNA, the pE-ampho carrier, the common transfection 293T of pGP carrier cell, packing carries the retrovirus of pSINsi-hU6-Fut8siRNA;
S32, with 3-5 * 10
4Target cell carries out 9-12h to be cultivated, utilize 7-10ug/mL polybrane to hatch after, by the virus infection mode with the Fut8siRNA transfection to target cell;
S33, screen with 400-1000ug/ml G418, and the picking individual cells, carry out enlarged culturing, obtain stable Fut8 gene silencing cell model.
2. the establishment method of the reticent cell model of described core fucosyl transferase gene according to claim 1, it is characterized in that, among the step S31 liposome and the volumetric molar concentration ratio of pSINsi-hU6-Fut8siRNA, pE-ampho carrier, pGP carrier be 4-5:1.5-2:1-1.5:1-1.5.
3. the establishment method of the reticent cell model of described core fucosyl transferase gene according to claim 2 is characterized in that the ratio among the step S32 between virus liquid and the cell culture fluid is 1-2:10.
4. the detection method for the reticent cell model of above-mentioned core fucosyl transferase gene is characterized in that, real-time quantitative PCR identifies, wherein, the primer sequence of described real-time quantitative PCR is,
sense:AACAGCTTGTTAAGGCCAAAG，
antisense:GCATGTCTTTGGAGTTCATTTC。
5. the detection method for the reticent cell model enzymic activity of above-mentioned core fucosyl transferase gene is characterized in that, utilizes the HPLC method to measure the Fut8 enzymic activity, and the cell pyrolysis liquid protein content is 0.2-0.5mg, and the reaction times is 2-5 hour.
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| CN105969747A (en) * | 2016-06-24 | 2016-09-28 | 大连大学 | Method for secreting and producing human-derived core fucose-base transferases by aid of pichia pastoris expression systems |
| CN106661562A (en) * | 2014-05-27 | 2017-05-10 | 中央研究院 | Fucosidase from bacteroides and methods using the same |
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| CN106661562A (en) * | 2014-05-27 | 2017-05-10 | 中央研究院 | Fucosidase from bacteroides and methods using the same |
| CN105969747A (en) * | 2016-06-24 | 2016-09-28 | 大连大学 | Method for secreting and producing human-derived core fucose-base transferases by aid of pichia pastoris expression systems |
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