CN104531762B - The slow virus carrier and its construction method and application of a kind of RAB7A and EGFP gene overexpression fusion green fluorescent protein - Google Patents
The slow virus carrier and its construction method and application of a kind of RAB7A and EGFP gene overexpression fusion green fluorescent protein Download PDFInfo
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Abstract
The invention provides a kind of RAB7A genes and EGFP gene overexpression fusion green fluorescent protein slow virus carrier and its construction method and application.Carrier of the present invention is with slow virus carrier LV11 as skeleton carrier, behind the CMV promoter of LV11 carriers EGFP gene and RAB7A genetic fragments are inserted by 5 ' to 3 ' directions, the fusion green fluorescent protein slow virus carrier of the EGFP and RAB7A gene overexpressions that the present invention builds, the problems such as solving in RAB7A gene studies over-express vector effectively and be directly perceived.The carrier ensure that eukaryotic gene high efficient expression with CMV as promoter；It is label to use EGFP, the effect of the degree and RAB7A genes of cell generation autophagy and endocytosis during cell hypoxia can be judged by the power of observation expression fluorescence directly perceived, for the function of studying RAB7A genes provides good instrument, for the correlative study of follow-up RAB7A provides basis.
Description
Technical field
The invention belongs to genetic engineering field, it is related to RAB7A genes and EGFP gene overexpression fusion green fluorescence egg
The structure of white slow virus carrier, and in particular to a kind of RAB7A genes and EGFP overexpression fusion green fluorescent protein slow virus carry
Body and its structure and the functional study in cell autophagy and endocytic processes.
Background technology
Rab families are the related EGTP associated proteins of a class RAS, and the regulation to Vesicle transport plays an important role.Rab albumen
Used as the molecular switch of vesicle transport, its function is interacted with the specific effector of upstream regulator and downstream, and and GTP
Combination be mutually coupled with hydrolytic process, played an important role during formation, transhipment, grappling, fusion in vesica etc..
Rab7 belongs to Rab protein families (Ras-lik protin in rat brain, Rab), with mediation late endosome
Merged with the film of lysosome, participate in the special biological that late protein is transported to lysosome process, Rab7 is molten by promoting
The transport of enzyme body can also remove intracellular virus, and Rab7 also take part in acceptor in addition to regulating and controlling the late period of endocytic processes and transporting
Transhipment, such as by angiotensinsAcceptor 1A, EGF-R ELISA is transported to lysosomal degradation, and the position where acceptor is determined
Determine the result of signal output.Thus it is speculated that Rab7 may play a significant role during signal transduction.
Rab albumen interacts by with the specific effector of upstream regulator and downstream, what they can be combined in GTP
Changed between the inactive form that activity form and GDP are combined, so as to regulate and control the links of Vesicle transport, such as shape of vesica
Into, move, anchor and fusion process.The mutation of Rab albumen or abnormal expression can cause vesicle transport exception, cause some diseases
The generation of disease.In recent years, increasing research shows that Rab albumen take part in vesicle transport and release and innate immunity,
The also wide participation process of signal transduction.Separately there are some researches show the letter that the regulation and control Vesicle transport that Rab albumen is participated in is participated in it
Number transduction has an inevitable contact, and the positioning of Rab albumen is closely related with their function.As be positioned at late endosomal or
The Rab7 of person's lysosome, its function is closely related with the late period transport of endocytosis body, although many experiments all support Rab7 at present
Late period endocytic processes are take part in, but detailed mechanism therein is not clear.At present, the comparing studied in the effector molecule of Rab7 is saturating
That thorough is Rab7 correlations lysosomal protein (Rab7-intracting lysosomal protin, RILP).It is primarily present in thin
Intracellular late endosomal/lysosome structure.Rab7 also turns in addition to being played a role in the Protein transport in endocytic processes late period with signal
Lead, the physiology course height correlation such as Apoptosis and antigen submission.Rab7 gene mutations in itself can cause genetic disease.
In addition, Rab7 escapes with pathogen, tumour occurs, lipid accumulation disease height correlation.Therefore, the structure and its work(of research Rab7
Energy.Its mechanism played a role in intracellular is illustrated, the announcement pathogenetic mechanism of disease is not only facilitated, and may be infectious disease
The treatment of the diseases such as disease, tumour provides scientific basis.
Slow virus is common to gene functional research instrument plasmid in a kind of molecular biology, can high-efficiency transfection protokaryon with
And eukaryotic, cell is synthesized and express genes of interest, whether expressed in the cell by comparative purpose gene or
Person's expression intensity, realizes that the effect to genes of interest in cell even body function is verified and analyzed.Therefore, slow virus
It is extremely wide in application of the research gene in cell and body function.Because Rab7 is present in autophagosome and late endosomal membrane
On, it is one of shared molecule necessary to autophagosome and endocytosis body maturation, intracellular goods can be guided and transported along micro-pipe, finally participate in
The fusion process of autophagosome and endocytosis body and lysosome, it is now recognized that the molecule is the key molecule for regulating and controlling autophagy and endocytosis.Cause
How intuitively, effectively the expression of observation RAB7A genes and its change in cell autophagy and endocytosis seem particularly important for this.
The content of the invention
Goal of the invention：Under prior art conditions, first purpose of the present invention is to solve needed for RAB7A gene studies
The over-express vector wanted, and the slow virus carrier of EGFP gene and RAB7A gene overexpressions fusion green fluorescent protein is provided；This
Second purpose of invention is to provide EGFP and RAB7A gene overexpression slow virus carrier construction methods, can pass through EGFP's
Green fluorescent protein carrys out positioning and the expression of spike RAB7A albumen；Third object of the present invention is to provide this overexpression
The exemplary applications of carrier.The fusion green fluorescence slow virus carrier of the EGFP and RAB7A gene overexpressions that the present invention builds, solves
In RAB7A gene studies over-express vector efficiently and it is directly perceived the problems such as, the carrier ensure that eucaryon with CMV as promoter
Gene efficient expression, it is label to use EGFP, can judge cell by the power of observation expression fluorescence directly perceived and occur certainly
The effect during cell hypoxia with the degree and RAB7A genes of endocytosis is bitten, for the function of studying RAB7A genes is provided
Good instrument, for the correlative study of follow-up RAB7A provides basis.In the present invention, RAB7A genes and EGFP gene cross table
HK-2 cells (renal cells) and 293T cells are transferred into up to fusion green fluorescence slow virus carrier.By RAB7A and
The position of EGFP and fluorescence degree are reflected in the degree of HK-2 cell autophagies and endocytosis.
Technical scheme：To realize the purpose of foregoing invention, the technical solution adopted by the present invention is：A kind of RAB7A and EGFP
Gene overexpression merges the slow virus carrier of green fluorescent protein, the side that EGFP gene and RAB7A genes are passed through bridging PCR
Method, is connected with one section of nucleotide sequence between EGFP gene and RAB7A genes and obtains+one section of nucleotides sequence of EGFP gene fragment
Row+RAB7A genetic fragments, then with slow virus carrier LV11 as skeleton carrier, behind CMV promoter in the skeleton carrier of LV11
Obtained final product according to 5 ' to 3 ' directions insertion EGFP gene+one section of nucleotide sequence+RAB7A genetic fragment of fragment.RAB7A genes are thin
Born of the same parents occur to be played a significant role in autophagy and endocytic processes, observe its expression and can determine whether that cell occurs the journey of autophagy and endocytosis
Degree.
Above-mentioned one section of nucleotide sequence such as SEQ ID NO：Shown in 1.SEQ ID NO：1：TCCGGACTCAGATCT.On
A kind of construction method of the slow virus carrier of RAB7A and EGFP gene overexpression the fusion green fluorescent protein stated, including it is as follows
Step：In 5 ' primers of the end design containing restriction enzyme digestion sites of EGFP gene, limit is introduced at the end of RAB7A genes 3 '
Property endonuclease digestion site processed primer, expands EGFP gene and RAB7A genes, then set with 5 ' ends of EGFP gene with two pairs of primers
Count the primer containing restriction enzyme digestion sites and introduce restriction enzyme digestion sites at the end of RAB7A genes 3 ' and draw
Thing ,+one section of nucleotide sequence+RAB7A genetic fragment of EGFP gene fragment is obtained with bridging PCR method, after the completion of PCR reactions,
Using agarose gel electrophoresis and+one section of nucleotide sequence+RAB7A genetic fragment of gel extraction EGFP gene fragment, finally
+ one section of nucleotide sequence+RAB7A gene fragment clone of EGFP gene fragment is to obtaining final product in carrier LV11.
Specific construction step is as follows：
1) renal cells (HK-2) of people total RNA, the cDNA with reverse transcription are extracted as template, PCR amplifications
RAB7A genetic fragments, the primer：RAB7F:ATGACCTCTAGGAAGAAAG；RAB7R:TCAGCAACTGCAGCTTTCT, will
1.5% Ago-Gel is reclaimed and obtains PCR primer importing PMD18-T carriers, the nucleotide sequence of sequence verification RAB7A genes；
The RAB7A gene orders of the people on sequencing result and NCBI are compared.
2) it is template amplification EGFP gene with the carrier of PEGFP-C1, the digestion position of BamHI is introduced at the end of B1F primers 5 '
Point, expands EGFP gene.1.5% Ago-Gel is reclaimed, the primer：
B1F：GATAGGAATTCGCCACCATGGTGAGCAAGGGCGAGGAG
B1R：AGAGGTCATAGATCTGAGTCCGGACTTGTAC；
3) with RAB7A genes as template, amplification RAB7 genes introduce restriction enzyme site EcoRI at the end of primer 3 ' of B2R simultaneously,
The primer：
B2F：CTCAGATCTATGACCTCTAGGAAGAAAGTGT；
B2R:GTATGGGATCCTCAGCAACTGCAGCTTTCTGCCGAGGC；
4) EGFP+TCCGGACTCAGATCT+RAB7A sequences are expanded with the method for bridging PCR, with B1F and B2R primers, with
Step 2) and step 3) obtained by product for template amplification obtain EGFP+TCCGGACTCAGATCT+RAB7A two genes
The sequence for linking together, the primer：
B1F：GATAGGAATTCGCCACCATGGTGAGCAAGGGCGAGGAG
B2R:GTATGGGATCCTCAGCAACTGCAGCTTTCTGCCGAGGC；
5) it is restriction enzyme site by BamHI and EcoRI, the sequence of EGFP+TCCGGACTCAGATCT+RAB7A genes is connected
The recombinant plasmid of the green fluorescent protein for obtaining RAB7A and EGFP fusion overexpression is connected on LV11 carriers (Fig. 6).First use
BamHI and EcoRI carries out digestion to EGFP+RAB7A genetic fragments, 37 DEG C of digestions 2 hours；Again with BamHI and EcoRI to carrier
LV11 carries out digestion, 37 DEG C of digestions 2 hours.Electrophoresis, EGFP+RAB7A genetic fragments and load are reclaimed with DNA gel QIAquick Gel Extraction Kit
Body LV11.The EGFP+RAB7A genetic fragments that double digestion is obtained and the carrier for linearizing, 22 DEG C of companies are connected with T4DNA ligase
Connect 2 hours；
6) by connection product transformed competence colibacillus cell, choose bacterium and be placed in being cultivated in bacterium shaking table, 37 DEG C, 200 revs/min,
Culture 16 hours.The plasmid for having extracted carries out digestion identification.
7) by checking, the recombinant plasmid of the fusion green fluorescent protein of RAB7A and EGFP overexpression is obtained.Obtain
RAB7A and EGFP gene overexpression merge the slow virus carrier of green fluorescent protein.The fusion green of RAB7A and EGFP overexpression
Function of the slow virus carrier of fluorescin in research RAB7A genes and the effect in cell autophagy and endocytic processes.
The slow virus carrier of a kind of above-mentioned RAB7A and EGFP gene overexpression fusion green fluorescent protein is in research cell
Application in autophagy and endocytosis and RAB7A gene functions.
The present invention constructs the slow virus carrier of a kind of RAB7A genes and EGFP gene overexpression fusion protein, not only solves
Over-express vector in RAB7A gene studies of having determined is efficient, moreover it is possible to which observation RAB7A genes directly perceived occur autophagy and endocytosis mistake in cell
Change in location in journey.The slow virus carrier with CMV as promoter, by one section of nucleotide sequence EGFP gene and RAB7A
Gene connect together insertion LV11 carriers in the middle of, not only obtain high efficient expression, moreover it is possible to by EGFP be label, can intuitively see
Examine the height of position occurs after the expression of RAB7A genes change and expression quantity.By the height of RAB7A gene expression amounts
Low, there is the degree of autophagy and endocytosis in observation of cell directly perceived, understand effect of the RAB7A genes in cell, finally influence cell
Function, for the function of studying RAB7A genes provides good instrument.RAB7A and EGFP merges the green fluorescent protein of overexpression
Application of the slow virus carrier in HK-2 cells and 293T cells.HK-2 cells and 293T cells are transferred to, by scanning copolymerization
Focusing microscope shows that the RAB7A albumen and EGFP in HK-2 cells form fusion protein, and RAB7A albumen is expressed in endochylema, lead to
Cross the change in location of EGFP albumen energy spike RAB7A albumen and the concentration of expression.
Beneficial effect：There is advantages below compared with prior art：
(1) RAB7A genes are connected with EGFP gene by 1 short nucleotide chain, and LV11 carriers, construction expression are transferred to jointly
The slow virus carrier of the fusion green fluorescent protein of RAB7A and EGFP, method is easy.
(2) by observe the position of green fluorescent protein not only traceable RAB7A albumen being transferred to intracellular position, and
And can in real time, the accurate expression for understanding RAB7A genes under varying environment it is strong and weak, and know that cell occurs the strong of autophagy and endocytosis
Degree.
(3) because the RAB7A and EGFP gene expressed fusion protein fluorescence intensity in cell that are transferred to are high, therefore under being
One step furthers investigate RAB7A genes and the function of albumen provides reliable means.
Brief description of the drawings
Fig. 1 is the length one that RAB7A genes, amplification and gene are expanded from renal cells (HK-2 cells)
Cause；1 and 2 is RAB7A genes 624bp, and M is DL 1000bp maker；
Fig. 2 is that RAB7A genes are transferred to PMD-18T, the comparison result of the RAB7A genes in sequencing and NCBI；
Fig. 3 is the restriction enzyme site for introducing BamHI, expands EGFP gene；1 is the EGFP bases of the restriction enzyme site with BamHI
Because of 717bp, M is DL 1000bp maker；
Fig. 4 is the restriction enzyme site for introducing EcoRI, expands RAB7A genes；1 is the RAB7A of the restriction enzyme site with EcoRI
Gene 624bp, M are DL 1000bp maker；
Fig. 5 is the restriction enzyme site for introducing BamHI and EcoRI, and EGFP passes through mono- section of nucleotide sequence of TCCGGACTCAGATCT
Connect the fragment of RAB7A genes；1 is that EGFP+RAB7A the genes 1356bp, M of the restriction enzyme site with BamHI and EcoRI is
DL1000bp maker；
Fig. 6 is the collection of illustrative plates of LV11-CMV carriers；
Fig. 7 is the BamHI/EcoRI digestions identification picture of LV11-CMV-EGFP-RAB7A carriers；1 is LV11-EGFP+
RAB7A recombinant plasmids (V6101-1) BamHI and EcoRI double digestions；2 is markerFermentas SM0331；3 is marker
Fermentas SM0331；
Fig. 8 is RAB7A genes and EGFP gene overexpression fusion green fluorescent protein slow virus carrier infection HK-2 cells
Situation.1 is the HK-2 cells of untransfected slow virus LV11-CMV-EGFP-RAB7A；2 is transfection slow virus LV11-CMV-
The HK-2 cells (10 ×) of EGFP-RAB7A；3 is the HK-2 cells (20 ×) of transfection slow virus LV11-CMV-EGFP-RAB7A；4
It is the HK-2 cells (40 ×) of transfection slow virus LV11-CMV-EGFP-RAB7A；
Fig. 9 is RAB7A genes and EGFP gene overexpression fusion green fluorescent protein slow virus carrier LV11-CMV-
The expression of the dye core and RAB7A genes and EGFP gene fusion protein of EGFP-RAB7A transfection HK-2 cells is under scanning copolymerization Jiao
Photo.1 is transfection RAB7A genes and EGFP gene overexpression fusion green fluorescent protein slow virus carrier LV11-CMV-
EGFP-RAB7A transfects the dye core (DAPI) of HK-2 cells, and 2 is the HK-2 cells of transfection slow virus LV11-CMV-EGFP-RAB7A
(20 ×), 3 situations about merging for the HK-2 cells and dye core (DAPI) of transfection slow virus LV11-CMV-EGFP-RAB7A；
Figure 10 is RAB7A genes and EGFP gene overexpression fusion green fluorescent protein slow virus carrier LV11-CMV-
EGFP-RAB7A transfects the situation of 293T cells；1 is that transfection RAB7A genes and EGFP gene fusion overexpression green fluorescence are sick slowly
The 293T cells (10 ×) of poisonous carrier LV11-CMV-EGFP-RAB7A；2 is that transfection RAB7A genes and EGFP gene overexpression melt
Close the 293T cells (40 ×) of green fluorescent protein slow virus carrier LV11-CMV-EGFP-RAB7A；
Figure 11 is real-time fluorescence quantitative PCR and Western blot analysis RAB7A genes and the fusion of EGFP gene overexpression
The mRNA and egg of the RAB7A genes of green fluorescent protein slow virus carrier LV11-CMV-EGFP-RAB7A transfection HK-2 cells
The result figure of white expression contents；1 is the testing result of real-time fluorescence quantitative PCR；2 is the result of Western blot；
Figure 12 is RAB7A genes and EGFP gene overexpression fusion green fluorescent protein slow virus carrier LV11-CMV-
Under anaerobic conditions, cell after autophagy and endocytosis EGFP-RAB7A transfection HK-2 cells occurs, and the expression of RAB7A genes occurs
Change；The expression and change in location of RAB7A genes are shown by scanning confocal microscope；1 is normal oxygen condition
Lower HK-2 cells contaminate core (DAPI), and 2 is the expression of the RAB7A genes of HK-2 cells under normal oxygen condition, and 3 under normal oxygen condition
The dye core (DAPI) and the expression combination situation of RAB7A genes of HK-2 cells.4 is HK-2 cells dye core under anaerobic condition
(DAPI), 5 be anaerobic condition under HK-2 cell RAB7A genes expression, 6 be anaerobic condition under HK-2 cells contaminate core (DAPI) and
The expression combination situation of RAB7A genes；
Figure 13 is RAB7A genes and EGFP gene overexpression fusion green fluorescent protein slow virus carrier LV11-CMV-
EGFP-RAB7A transfects HK-2 cells under normal oxygen and anaerobic condition, and real-time fluorescence quantitative PCR and Western blot are analyzed
RAB7A genes and EGFP gene overexpression fusion green fluorescent protein slow virus carrier LV11-CMV-EGFP-RAB7A transfections HK-
The result figure of the expression contents of the mRNA and albumen of the RAB7A genes of 2 cells；1 is the detection knot of real-time fluorescence quantitative PCR
Really；2 is the result of Western blot.
Specific embodiment
Hereinafter implement to be advantageously used for the explanation present invention, but be not limited to protection scope of the present invention.The present invention relates to
The LV11 carriers for arriving are purchased from Shanghai Ji Ma companies.If not specializing, the technological means used in embodiment is this area skill
Conventional meanses known to art personnel.Enzyme used is Dalian treasured biotech firm product without special instruction.
The connection of embodiment 1EGFP genes and RAB7A genes
1st, extract the total RNA of renal cells (HK-2) of people, reverse transcription into cDNA be template, then be with cDNA
Template PCR amplifications RAB7 genetic fragments, the primer is：RAB7F:ATGACCTCTAGGAAGAAAG, RAB7R:
TCAGCAACTGCAGCTTTCT, expands RAB7A genes.PCR reaction systems：
PCR amplification conditions：95 DEG C of 5min, 1cycle, 94 DEG C of 40sc, 56 DEG C of 45s, 72 DEG C of 45s, 35cycles, 72 DEG C
10min, 1cycle.1.5% Ago-Gel is reclaimed, and imports PMD-18T carriers, the RAB7A of the people on sequencing result and NCBI
Gene order is compared (Fig. 1 and 2).
2nd, by primer B1F：GATAGGAATTCGCCACCATGGTGAGCAAGGGCGAGGAG and B1R：
AGAGGTCATAGATCTGAGTCCGGACTTGTAC with the carrier of PEGFP-C1 be template amplification EGFP gene.In B1F primers 5 '
End introduces the restriction enzyme site of BamHI, expands EGFP gene.
PCR reaction systems：
PCR amplification conditions：95 DEG C of 5min, 1cycle, 94 DEG C of 40s, 56 DEG C of 45s, 72 DEG C of 45s, 35cycles, 72 DEG C
10min, 1cycle.1.5% Ago-Gel reclaims (Fig. 3).
3rd, by primer B2F：CTCAGATCTATGACCTCTAGGAAGAAAGTGT and B2R:
, with RAB7A genes as template, amplification RAB7 genes are simultaneously in B2R for GTATGGGATCCTCAGCAACTGCAGCTTTCTGCCGAGGC
The end of primer 3 ' introduce restriction enzyme site EcoRI.
PCR reaction systems：
PCR amplification conditions：95 DEG C of 5min, 1cycle, 94 DEG C of 40s, 56 DEG C of 45s, 72 DEG C of 45s, 35cycles, 72 DEG C
10min, 1cycle.1.5% Ago-Gel reclaims (Fig. 4).
4th, EGFP+SEQ ID NO are expanded with the method for bridging PCR：1+RAB7A sequences, with B1F and B2R primers, with step
2) and step 3) obtained by product for template amplification condition, first 5 circulations are not added with primer, add B1F and B2R after 5 circulations
Primer, 30 cyclic amplifications.Obtain EGFP+SEQ ID NO：The sequence that two genes of 1+RAB7A link together, EGFP bases
Cause and RAB7A genes are by SEQ ID NO：What 1 sequence was coupled together.
Reaction system：
PCR amplification conditions：95 DEG C of 5min, 1cycle, 94 DEG C of 40s, 56 DEG C of 45s, 72 DEG C of 45s, 5cycles；94 DEG C of 40sc,
60 DEG C of 45s, 72 DEG C of 45s, 30cycles, 72 DEG C of 10min, 1cycle.1.5% Ago-Gel reclaims (Fig. 5).Import PMD-
18T carriers, sequence verification.
Embodiment 2：EGFP gene+SEQ ID NO：The sequence of 1+RAB7A genes is connected on LV11 carriers
It is restriction enzyme site by BamHI and EcoRI, the sequence of EGFP+RAB7A genes is connected on LV11 carriers and is obtained
The recombinant plasmid of the green fluorescent protein of RAB7A and EGFP fusion overexpression.First with BamHI and EcoRI to EGFP+RAB7A bases
Because fragment carries out digestion, 37 DEG C of digestions 2 hours；Carry out digestion to carrier LV11 with BamHI and EcoRI again, 37 DEG C of digestions 2 are small
When.Electrophoresis, EGFP gene+SEQ ID NO are reclaimed with DNA gel QIAquick Gel Extraction Kit：1+RAB7A genetic fragments and carrier LV11.
The EGFP+SEQ ID NO that double digestion is obtained are connected with T4DNAligase：1+RAB7A genetic fragments and the carrier of linearisation, 22
DEG C connection 2 hours, linked system is as follows：
By connection product transformed competence colibacillus cell, choose bacterium and be placed in being cultivated in bacterium shaking table, 37 DEG C, 200 revs/min, training
Support 16 hours.The plasmid for having extracted carries out digestion identification (Fig. 7).Endonuclease reaction system is as follows：
By checking, the recombinant plasmid of the green fluorescent protein of RAB7A and EGFP fusion overexpression is obtained.Obtain RAB7A
With the slow virus carrier that EGFP gene overexpression merges green fluorescent protein.RAB7A and EGFP merges the green fluorescence of overexpression
Function of the slow virus carrier of albumen in research RAB7A genes and the effect (Fig. 8) in cell autophagy and endocytic processes.
Rab7 is present on autophagosome and late endosomal membrane, is one of shared molecule necessary to autophagosome and endocytosis body maturation, can lead
Draw intracellular goods to be transported along micro-pipe, the fusion process of autophagosome and endocytosis body and lysosome is finally participated in, it is now recognized that the molecule
It is the key molecule for regulating and controlling autophagy and endocytosis.
The slow virus carrier transfectional cell of the fusion green fluorescent protein of embodiment 3RAB7A and EGFP overexpression and grinding
Study carefully functional study and the function of RAB7A genes in cell autophagy and endocytosis.
HK-2 and 293T cellular invasions are tested, 1) target cell bed board：24 orifice plates add 0.5 × 105Cells/ holes are (according to thin
Born of the same parents' species is adjusted), 0.5mlDMEM culture mediums are containing 10% hyclone, 37 DEG C, 5%CO2Overnight；2) virus is diluted：Dilution
(DMEM is containing 2% hyclone) μ g/ml of 400 μ l+ final concentrations 5 transfection enhancer Polybrene, by slow virus stoste (20-
100 μ l) it is added in dilution；3) original (DMEM culture mediums are containing 10% hyclone) cell culture fluid is removed, step is added
Virus liquid after rapid 2) dilution, while setting up control (blank, feminine gender), 37 DEG C, 5%CO2Overnight；4) 12-24h removes cell and invades
Virus liquid after dye, adds 0.5mlDMEM culture mediums containing 10% hyclone, 37 DEG C, 5%CO2Overnight；5) according to cellular
State and type, if necessary separate 1/3-1/5, add 0.5mlDMEM culture mediums containing 10% hyclone, continue to cultivate 24-
48h；6) using experimental techniques such as RT-PCR and Western blot, the expression or regulation and control of transgene are analyzed.
Specific experiment method：
First, RAB7A genes and EGFP gene overexpression fusion green fluorescent protein slow virus carrier LV11-CMV-EGFP-
The packaging of RAB7A slow virus and the renal cells (HK-2) of transfected with human and 293T cells.
1.293T passages
1) culture medium is discarded, 5ml 1 × PBS solutions of sterilizing are added, is gently rocked, then washing 293T cell growths face is abandoned
Go 1 × PBS solution；2) 2ml pancreatin digestive juices, digestion 1-2min is added to digest completely until cell；3) add and contain 10%
Hyclone and the dual anti-DMEM culture medium 5ml of 100U/ml, are blown and beaten for several times, by the 293T cells punching in bottle wall with measuring pipette
Wash；4) it is divided into two new blake bottles after mixing 293T cells, continues to cultivate.
2. virus is packed
1) 293T cells cultivated in 10cm culture dishes to 80-90% merge when, be inoculated with 15cm culture dishes；2) incline culture
Liquid, with 1mlD-Hank ' s solution washed cells twice；3) 1ml Trypsin-EDTAsolution are added, after mixing,
37 DEG C are placed 2-3 minutes；4) trypsin solution is carefully sucked, DMEM nutrient solutions of the 2ml containing 10%FBS is added, piping and druming makes cell shape
Into single cell suspension；5) single cell suspension is inoculated with 15cm culture dishes, adds DMEM nutrient solutions of the 18ml containing 10%FBS, mixed
37 DEG C of 5%CO afterwards2Overnight incubation；6) 1.5ml serum-free DMEM are added in an aseptic 5ml centrifuge tube, by 2:2:3 ratios
Viral shuttle plasmid (psPAX2), packaging plasmid (Pmd2.G) and LV11-CMV-EGFP-RAB7A are added, is mixed, take another
Aseptic 5ml centrifuge tubes, add 1.5ml serum-frees DMEM to mix, and room temperature mixes two pipes after placing 5 minutes, and room temperature places 20-
25 minutes；7) nutrient solution in 15cm culture dishes is removed, the DMEM nutrient solutions of 8ml serum-frees are added；8) by transfection mixture by
Be added dropwise in 15cm culture dishes, the culture dish that lightly rocks back and forth to mix compound, in 37 DEG C of 5%CO2Incubated in incubator
4-6 hours；9) inhale and abandon transfection liquid, add DMEM nutrient solutions of the 18ml containing 10%FBS.37 DEG C of 5%CO2Continue to cultivate 72 hours.
3. virus titer detection
1) 293T cells cultivated in 10cm cell bottles to 80-90% merge when, incline nutrient solution, with 3mlD-Hank ' s
Solution washed cells are twice；2) add 1ml Trypsin-EDTA solution, after mixing, carefully suck trypsin solution, 37
DEG C place 3-5 minutes；3) DMEM nutrient solutions of the 2ml containing 10%FBS is being added, piping and druming makes cell form single cell suspension；4) by 3
×104The concentration of cells/well is inoculated with 96 orifice plates, mixes after 37 DEG C of 5%CO2Culture 24h；5) by the μ l of slow virus stoste 10, use
The DMEM trainings 3-5 gradient of ten times of dilutions of liquid of 10%FBS (according to cell state, can add final concentration of 5 μ g/ml if necessary
Transfection enhancer Polybrene)；6) nutrient solution in 96 orifice plates is sucked, the virus liquid of 100 μ l dilutions is added per hole, together
When set up blank control group, in 37 DEG C of 5%CO2Culture 24h；7) the dilution virus liquid abandoned in 96 orifice plates is inhaled, 100 μ is added per hole
The DMEM training liquid of l10%FBS, (according to cell state, 1/3-1/5 can be separated if necessary) is in 37 DEG C of 5%CO2Continue to cultivate
72h；8) fluorecyte is counted by fluorescence microscope or FACS, it is 10 to calculate virus titer with reference to extension rate9。
4.HK-2 and 293T cell infections are tested
1. target cell bed board：24 orifice plates, add 0.5 × 105Cells/ holes (adjust) according to cell category, 0.5mlDMEM
Culture medium is containing 10% hyclone, 37 DEG C, 5%CO2Overnight；2. virus is diluted：Dilution (DMEM is containing 2% hyclone)
The μ g/ml of 400 μ l+ final concentrations 5 transfect enhancer Polybrene, and slow virus stoste (20-100 μ l) is added in dilution；
3. original cell culture fluid (DMEM culture mediums are containing 10% hyclone) is removed, the virus liquid after step 2 dilution is added, together
Shi Jianli compares (blank, feminine gender), 37 DEG C, 5%CO2Overnight；4.12-24h removes the virus liquid after cellular invasion, adds
0.5mlDMEM culture mediums containing 10% hyclone, 37 DEG C, 5%CO2 overnight；5. according to cell state and type, if necessary
1/3-1/5 is separated, 0.5mlDMEM culture mediums is added containing 10% hyclone, continues to cultivate 24-48h；6. using RT-PCR and
The experimental techniques such as Western blot, analyze the expression or regulation and control of transgene.
2nd, RAB7A genes and EGFP gene overexpression fusion green fluorescence slow virus carrier LV11-CMV-EGFP-RAB7A
The renal cells (HK-2) of transfected with human and the checking of 293T cells.
1. HK-2 cell-like cells (control group) and viral metainfective HK-2 cells (overexpression group) are collected, is used
Trizol extracts total serum IgE, is cDNA by RNA reverse transcriptions, as template according to Reverse Transcriptase kit (TaKaRa) operating procedure
Carry out real-time fluorescence quantitative PCR.With housekeeping gene glyceraldehyde -3- phosphate dehydrogenases (GAPDH) as internal reference, contaminated using SYBR fluorescence
Material kit (Nanjing Vazyme Biotechnology Co., Ltd.) determines genes of interest RAB7A relative expression's contents.RAB7A
(156bp) primer：F:CATCCTGGGAGATTCTGGAGTC；R:TGTGTCCCATATCTGCATTGTG
GAPDH (90bp) primer：F:GGAAGGTGAAGGTCGGAGTCA；R:
GCAACAATATCCACTTTACCAGAGTTAA.Gene specific upstream and downstream primer amplification system is：95 DEG C of predegenerations 4 minutes,
Then reacted 10 seconds according to 95 DEG C, 60 DEG C are reacted 40 seconds, and continuing 40 circulations carries out genes of interest amplification.RAB7A genes are thin
Relative expression quantity uses 2 in born of the same parents RNA-△△CTCalculate, relative expression's fold differences use fold between overexpression group and control group
Chang=2-△△CTCalculate.Same relative expression's fold differences between anoxic group and the expression quantity of normal oxygen concentratio group use fold
Chang=2-△△CTCalculate.Using RIPA lysate cell lysis, and cell pyrolysis liquid is collected, tried using BCA protein determinations
Agent box is determined, and extract solution protein concentration carries out Western blot analyses.Using the anti-human RAB7A monoclonal antibodies (1 of mouse:
2000, ABCAM companies) and GAPDH (1:3000, PROTECH) as primary antibody, the goat-anti marked with horseradish peroxidase HRP
Mouse antibody (PROTECH) is used as secondary antibody, ECL colour developings.
2. the green fluorescence expression after observation of cell infection under fluorescence microscope, collects metainfective cell and uses
Real-time PCR and Western blot technologies observe transfection.(Fig. 8, Fig. 9, Figure 10 and Figure 11)
3. by 2 groups of cells according to 1 × 104/ hole density is inoculated into the burnt special ware of copolymerization, is inhaled after cell is attached completely and abandoned
Culture medium, is cleaned 3 times with culture medium.Respectively in normal oxygen concentratio (20%O2, 5%CO2) and anoxic concentration (1%O2, 5%CO2)
37 DEG C of incubated 24h.Cell is rinsed using 1 × PBS 3 times, the 4% paraformaldehyde normal temperature of 1ml is then added per ware
Fixed 30min, 4% paraformaldehyde is rinsed using 1 × PBS.Add the dye nuclear material (DAPI) of 100 μ l, 37 DEG C of shaking tables
5min.Rinsed using 1 × PBS.Cell is observed under being placed in scanning confocal fluorescent microscope.Intracellular RAB7A
The observation of gene, RAB7A genes and EGFP gene overexpression fusion green fluorescent protein slow virus carrier LV11-CMV-EGFP-
Under anaerobic conditions, cell after autophagy and endocytosis RAB7A transfection HK-2 cells occurs, the change of the expression generation of RAB7A genes
Change.The expression and change in location of RAB7A genes are shown by scanning confocal microscope.(Figure 12 and Figure 13)
3rd, experimental result
1.LV11-CMV-EGFP-RAB7A virus infection HK-2 cells after 48 hours, the HK-2 cells under fluorescence microscope
Green fluorescence (Fig. 8 and 9) is rendered as, shows that LV11-CMV-EGFP-RAB7A builds and transfects successfully.By infecting 293T cells
(Figure 10), further verifies that LV11-CMV-EGFP-RAB7A builds and transfects successfully.
RAB7A mRNA and protein expression content are significantly high after 2.LV11-CMV-EGFP-RAB7A virus infection HK-2 cells
The HK-2 cells (Figure 11) of untransfected.
3rd, anaerobic condition HK-2 cells occur autophagy and endocytosis, in the HK-2 cells of transfection the expression of RAB7A genes with
The increase of autophagy and endocytosis, the expression of RAB7A increases, and fluorescence intensity is directly proportional to intracellular RAB7A contents.1 in Figure 12,
2nd, 3 is normal oxygen concentratio overexpression group control group, and 4,5,6 is overexpression group of the RAB7A genes in anoxic, and HK-2 cells occur certainly
Bite with after endocytosis, the expression of RAB7A genes increases, and green fluorescence is strengthened, fluorescent places aggregation, the overexpression HK-2 of anoxic is thin
The fluorescence of intracellular compares by force concentration, and the HK-2 of normal oxygen concentratio intracellular fluorescence compares disperse, and fluorescence intensity is compared with anoxic group
Fluorescence it is weak (Figure 12).
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (1)
1. a kind of RAB7A and EGFP gene overexpression merge the construction method of the slow virus carrier of green fluorescent protein, its feature
It is to comprise the following steps：In 5 ' primers of the end design containing restriction enzyme digestion sites of EGFP gene, in RAB7A
The end of gene 3 ' introduces restriction enzyme digestion sites primer, expands EGFP gene and RAB7A genes with two pairs of primers, then use
Design the primers containing restriction enzyme digestion sites and introduced in restricted at the end of RAB7A genes 3 ' in 5 ' ends of EGFP gene
Enzyme cutting restriction enzyme site primer ,+one section of nucleotide sequence+RAB7A genetic fragment of EGFP gene fragment is obtained with bridging PCR method,
After the completion of PCR reactions, using agarose gel electrophoresis and+one section of nucleotide sequence+RAB7A base of gel extraction EGFP gene fragment
Because of fragment, will finally be obtained final product in+one section of nucleotide sequence+RAB7A gene fragment clone of EGFP gene fragment to carrier LV11, had
Body step is as follows：
1）The renal cells of people total RNA, the cDNA with reverse transcription are extracted as template, PCR amplification RAB7A gene pieces
Section, the primer：RAB7F: ATGACCTCTAGGAAGAAAG；RAB7R:TCAGCAACTGCAGCTTTCT, by PCR primer
Import PMD18-T carriers, the nucleotide sequence of sequence verification RAB7A genes；
2）It is template amplification EGFP gene with the carrier of PEGFP-C1, the restriction enzyme site of BamHI is introduced at the end of B1F primers 5 ', expands
Increase EGFP gene；
The primer：
B1F：GATAGGAATTCGCCACCATGGTGAGCAAGGGCGAGGAG
B1R：AGAGGTCATAGATCTGAGTCCGGACTTGTAC；
3）With RAB7A genes as template, amplification RAB7 genes introduce restriction enzyme site EcoRI at the end of primer 3 ' of B2R simultaneously, used
Primer：
B2F：CTCAGATCTATGACCTCTAGGAAGAAAGTGT ；
B2R:GTATGGGATCCTCAGCAACTGCAGCTTTCTGCCGAGGC；
4）EGFP+ TCCGGACTCAGATCT+RAB7A sequences are expanded with the method for bridging PCR, with B1F and B2R primers, with step
Rapid 2）With step 3）Resulting product is two genes company that template amplification obtains EGFP+ TCCGGACTCAGATCT+RAB7A
The sequence being connected together, the primer：
B1F：GATAGGAATTCGCCACCATGGTGAGCAAGGGCGAGGAG
B2R:GTATGGGATCCTCAGCAACTGCAGCTTTCTGCCGAGGC；
5）It is restriction enzyme site by BamHI and EcoRI, the sequence of EGFP+ TCCGGACTCAGATCT+RAB7A genes is connected
Onto LV11 carriers, and the recombinant plasmid of the fusion green fluorescent protein of RAB7A and EGFP overexpression is obtained by checking.
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