CN106047927A - Overexpression TMEM88 gene plasmid, construction method and application thereof - Google Patents

Overexpression TMEM88 gene plasmid, construction method and application thereof Download PDF

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CN106047927A
CN106047927A CN201610425684.9A CN201610425684A CN106047927A CN 106047927 A CN106047927 A CN 106047927A CN 201610425684 A CN201610425684 A CN 201610425684A CN 106047927 A CN106047927 A CN 106047927A
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tmem88
gene
reaction system
process lan
cell
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徐涛
潘林鑫
李小枫
倪明明
杨扬
孟晓明
黄成�
张磊
李俊
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Abstract

The invention discloses an overexpression TMEM88 gene plasmid which is constructed by recombination of a TMEM88 gene CDS sequence and a pEGFP-C2 eukaryotic expression vector. A nucleotide of the overexpression TMEM88 gene plasmid is as shown in a sequence table SEQ ID NO:3 and is located on the 10th-489th bit of a TMEM88 gene; and an amino acid sequence of the overexpression TMEM88 gene plasmid is as shown in a sequence table SEQ ID NO:4. The invention further discloses a method for constructing the overexpression TMEM88 gene and application thereof. The overexpression TMEM88 gene plasmid has the advantages of being capable of remarkably inhibiting proliferation of human hepatoma cells and remarkably promoting apoptosis of the human hepatoma cells.

Description

A kind of process LAN TMEM88 gene plasmid, construction method and application thereof
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of process LAN TMEM88 gene plasmid, construction method And application.
Technical background
Hepatocarcinoma is a kind of common alimentary system malignant tumour, mainly includes constitutional and the big class of Secondary cases two, Commonly primary hepatocarcinoma (PHC), it falls ill in intrahepatic biliary epithelium cell or hepatic parenchymal cells, have grade malignancy high, M & M high, epidemiological survey data show, PHC occupies the 5th of tumor incidence, and mortality rate is especially Up to the 3rd, it is only second to gastric cancer and pulmonary carcinoma, brings huge threat to the health of the mankind and development.And the morbidity of PHC is deposited In certain region, being apt to occur in Asia and Africa, in China southeastern coastal areas, the patient being diagnosed as PHC every year is the highest Reaching more than 30 ten thousand person-times, account for 55% in world's new cases, and also have the trend of cumulative year after year in recent years, therefore China faces The severeest PHC situation, means and the medicine of finding effective treatment PHC are particularly important.
Genetic engineering, is also called gene recombination technology, is entering DNA in vitro of growing up nineteen seventies A special kind of skill of row operation, is widely used in disease basic research, gene therapy, genetic immunization, gene vaccine etc..Its three elements divide It is not: enzyme, genes of interest, carrier.And carrier for expression of eukaryon is for building the one of eukaryotic gene expression system in genetic engineering Planting carrier, in recent decades, scholars has carried out the correlational studyes such as various disease, gene around carrier for expression of eukaryon, at base Because engineering aspect is done a lot of work, open brand-new route for medical scientific.
TMEM88 is the member of TMEM88 protein family subfamily, and its gene mapping is in chromosome 17p13.1, Lee et al. Confirming and belong to and being positioned in embryonic cell on cell membrane grabbing toad, be twice transmembrane protein, TMEM88 is by 159 aminoacid Residue forms, and its molecular weight is about 17KD, and in 159 aminoacid, 43-63 amino acid residue constitutes first cross-film knot Structure territory, and 88-108 amino acid residue constitutes second membrane spaning domain.About 100 amino of the PDZ domain of DVL Acid, comprises 6 beta-strands and 2 a-helices composition, and and the PDZ of DVL that combines of the V-W-V sequence of TMEM88 The sequence of domain is positioned between beta-strands and a-helices, and TMEM88 can be by the V-W-V sequence of its C end Be combined with the PDZ domain of DVL1, suppress classical Wnt/b-by cutting off the combination of DVL Yu Frrizle and LRP5/6 Catenin path, thus play its biological action.
Summary of the invention
It is an object of the invention to provide a kind of process LAN TMEM88 gene plasmid, construction method and application thereof.And prove institute Stating recombiant plasmid is that TMEM88 functional study provides good experimental tool, also for the merit of TMEM88 gene in research people's hepatocarcinoma It is provided that effective experimental technique.
People source TMEM88 gene is registered at GenBank, and number of registration NM_203411.1 is positioned 17p13.1, total length 887bp, wherein CDS sequence 480bp, encode 159 aminoacid.The tissue expression analysis of spectrum of people source TMEM88 gene shows: should Gene all has expression in many tumors of people.
The present invention solves above-mentioned technical problem by following technical proposal: a kind of process LAN TMEM88 gene plasmid, bag The recombination to construct including TMEM88 gene C DS sequence and pEGFP-C2 carrier for expression of eukaryon forms；Its nucleotide such as sequence table SEQ ID NO:3, is positioned at the 10-489 position of TMEM88 gene；Aminoacid sequence is as shown in sequence table SEQ ID NO:4.
A kind of method building above-mentioned process LAN TMEM88 gene, comprises the steps:
(1) collection, cell lysis, extraction RNA carries out reverse transcription and obtains TMEM88cDNA；
(2) use round pcr, amplify the CDS sequence fragment of artificial T MEM88 gene；
(3) purification of the CDS sequence fragment of TMEM88 gene；
(4) genetic fragment obtained and pEGFP-C2 carrier for expression of eukaryon are carried out KpnI and EcoRI double digestion, go forward side by side Row connects；
(5) product will be connected convert in e. coli tg1, and choose positive colony and cultivate, then extract plasmid.
Preferably, after extracting RNA, degeneration is carried out；Concrete processing step: after RNA is placed 5-10min in 65 DEG C, It is transferred in frozen water place.
Preferably, the reaction system of reverse transcription includes: 5 × RT Master Mix, RNA, Nuclease-free Water； Concrete processing step includes:
(1) preparation reaction system: 5 × RT Master Mix 2 μ L+RNA 2 μ L+Nuclease-free Water 6 μ L；
(2) above-mentioned reaction system is placed 37 DEG C, and act on 15min；
(3) above-mentioned reaction system is transferred to 50 DEG C, and acts on 5min；
(4) above-mentioned reaction system is transferred to 98 DEG C, and acts on 5min；
(5) above-mentioned reaction system is transferred to 4 DEG C, frozen after cooling, i.e. obtain cDNA.
Preferably, according to the CDS sequence of TMEM88, design upstream and downstream primer；Upper and lower primer sequence is as follows:
TMEM88-F:5’-CCGGAATTCCGGATGGCGGATGTCCCCG-3’
TMEM88-R:5’-GGGGTACCCCTTAGACCCAGACCTTTCCT-3’。
Preferably, the reaction system of PCR is as follows: 2 × PrimeSTAR Max, cDNA sample, upstream and downstream primer, high purity water；
Concrete processing step includes:
(1) preparation reaction system: each 1 μ L+ high purity water 22 μ L of 2 × PrimeSTAR Max 25 μ L+cDNA 1 μ L+ primer；
(2) put into PCR instrument device after above-mentioned reaction system being mixed, following program be set and run:
(3) above-mentioned PCR primer is carried out 1-1.2% agarose gel electrophoresis；
(4) target DNA fragment is reclaimed.
Preferably, the PCR primer of TMEM88 and pEGFP-C2 carrier are carried out the double digestion of EcoR I and KpnI；Enzyme action body It is as follows: restricted enzyme (EcoRI and KpnI), the PCR primer of 10 × Buffer Tango, TMEM88, pEGFP-C2 carry Body；Concrete processing step includes:
(1) preparation reaction system: the PCR of each 0.5 μ L+TMEM88 of 10 × Buffer Tango 2 μ L+EcoRI and KpnI produces Thing or and pEGFP-C2 carrier 7 μ L；
(2) above-mentioned reaction system is placed in 37 DEG C of water-baths, and act on 4～6h；
(3) 1-1.2% agarose gel electrophoresis is carried out；
(4) observed result reclaim target DNA fragment.
Preferably, before proceeding, carrier is carried out dephosphorylation process；
Dephosphorylation system (10 μ L): CIAP1 μ l, 10 × CIAP buffer 1 μ l, carrier 8 μ l
Linked system (10 μ L): carrier 0.5 μ l after CIAP dephosphorylation, 10 × T4DNA ligase buffer 1 μ l, T4DNA ligase 1 μ l, PCR enzyme action purified product 7.5 μ l；
Method of attachment: above-mentioned reaction system is placed in the centrifuge tube of 0.5ml mixing, and 16 DEG C of water-baths are overnight.
Preferably,
(1) taking-up is stored in the TG1 competent cell of 80 DEG C, and ice bath adds 10 μ l and connects products, mixing, ice after thawing Upper standing 30min；
(2) 42 DEG C of heat shocks 90s, take out and put 3min on ice；
(3) 500 μ l LB culture fluid, 37 DEG C, 250rpm shaken cultivation 45min are added；
(4) take out 100 μ l conversional solution to be applied on the LB solid medium with corresponding resistant, 37 DEG C be inverted cultivate 8～ 12h。
A kind of use above-mentioned process LAN TMEM88 gene plasmid suppression human liver cancer cell propagation, promote human liver cancer cell The application of apoptosis.
Described Transfected Recombinant Plasmid is entered in Bel7402, it is possible to significantly inhibit the propagation of human liver cancer cell, and show Write the apoptosis promoting human liver cancer cell.Therefore, the present invention contributes to studying TMEM88 gene function, and for studying it people liver In cancer, the effect of morbidity provides useful biology tool.The present invention uses gene recombination technology, for TMEM88 gene, Build recombinant eukaryon expression vector pEGFP-C2-TMEM88, and transfected to hepatoma cell strain, study TMEM88 process LAN On hepatoma cell proliferation and the impact of apoptosis, for studying the function of TMEM88 further, and the gene therapy of hepatocarcinoma is established Basis.
Accompanying drawing explanation
Fig. 1 is the structural representation of carrier for expression of eukaryon pEGFP-C2 of the present invention.
Fig. 2 is the structural representation of eucaryon recombinant vector pEGFP-C2-TMEM88 of the present invention.
Fig. 3 is that the enzyme action of eucaryon recombinant vector pEGFP-C2-TMEM88 of the present invention identifies collection of illustrative plates.
Fig. 4 is the Sequencing chromatogram of eucaryon recombinant vector pEGFP-C2-TMEM88 of the present invention.
Fig. 5 is the eucaryon recombinant vector pEGFP-C2-TMEM88 of the present invention expression figure in HEK 293T cell
Fig. 6 be the apoptotic cell numerical value of untransfected group of the present invention affect situation map
Fig. 7 is that eucaryon recombinant vector pEGFP-C2-TMEM88 of the present invention affects situation map to hepatoma cell line apoptosis
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.In following embodiment, all unreceipted specific experiment conditions , it is according to the condition in normal condition well known to those skilled in the art, laboratory manual, according to proposed by manufacturer Condition.Embodiment 1
Build process LAN TMEM88 gene plasmid
1, the extraction of TMEM88cDNA
(1) take out Tissue Culture Dish and abandon clean culture medium, clean cell face (2-3 time) with the PBS of 1ml.
(2) add the Trizol cell lysis 10min of 1ml, after mixing cell gently with pipettor, cell pyrolysis liquid is turned Moving in centrifuge tube, rear chamber is gentle and quiet puts 5min in reverse mixing.
(3) adding the chloroform (4 DEG C of pre-coolings) of 1/5 amount of Tri201, acutely the mixing gentle and quiet 5min that puts of rear chamber, high speed is low The centrifugal 15min (4 DEG C, 12000r/min) of temperature.
(4) 0.4ml supernatant liquid is transferred in new centrifuge tube, adds isopyknic isopropanol, quiet after reverse mixing Putting 60min (-20 DEG C), high-speed low temperature abandons supernatant after being centrifuged 15min (4 DEG C, 12000r/min).
(5) vibration mixing after the ethanol solution of 1ml75% is added, after high-speed low temperature is centrifuged 5min (4 DEG C, 12000r/min) Abandon supernatant, and dry 5min.
(6) add 20 μ L without enzyme water dissolution precipitate after, for reverse transcription, it is possible to frozen in-80 degree.
(7) degeneration of RNA
The RNA extracted is carried out degeneration, and denaturing step is as follows: after RNA is placed 5min in 65 DEG C, be transferred quickly to Frozen water is placed 2min.
Reverse transcription reaction system (10 μ L) is as follows:
Processing step includes:
(1) above-mentioned reaction system is placed 37 DEG C, and act on 15min；
(2) above-mentioned reaction system is transferred to 50 DEG C, and acts on 5min；
(3) above-mentioned reaction system is transferred to 98 DEG C, and acts on 5min；
(4) above-mentioned reaction system is transferred to 4 DEG C, frozen after cooling, i.e. obtain cDNA.
2, the clone of TMEM88 gene
CDS sequence according to TMEM88, designs upstream and downstream primer, and primer sequence is as follows:
TMEM88-F:5 '-CCGGAATTCCGGATGGCGGATGTCCCCG-3 ' (containing EcoRI restriction enzyme site)
TMEM88-R:5 '-GGGGTACCCCTTAGACCCAGACCTTTCCT-3 ' (containing KpnI restriction enzyme site)
Enzyme used in the process of PCR is preferably the PrimeSTAR Max high-fidelity enzyme of Takara company, its reaction system and Response procedures is as follows:
Reaction system (50 μ L):
Put into PCR instrument device after above-mentioned reaction system being mixed, following program be set and run:
(3) above-mentioned PCR primer is carried out 1-1.2% agarose gel electrophoresis；
(4) observed result reclaim target DNA fragment.
PCR primer is carried out agarose gel electrophoresis, by the contrast with DNA Marker, found that go out at 500bp Obtaining a band, size is basically identical with the size of the fragment of TMEM88 mesh (including restriction enzyme site), shows that Successful amplification goes out people The CDS sequence of TMEM88 gene.
3, the purification of the fragment of TMEM88 mesh
Test kit used by purification is preferably likes that the glue of the company that pursues progress reclaims test kit, and purification step is as follows:
(1) under uviol lamp, cut the agarose gel containing target DNA, exhaust gel surface liquid with filter paper and cut Broken.Calculated for gel weight, every 100mg is equal to 100 μ l volumes.
(2) the Buffer DE-A of 3 gel volumes is added, in 75 DEG C of heating (low melting-point agarose gels after mix homogeneously In 40 DEG C of heating), it is interrupted mixing (every 2～3min), until gel piece is completely melt.
(3) the Buffer DE-B of 0.5 Buffer DE-A volume, mix homogeneously are added.When the DNA fragmentation separated is less than During 400bp, the isopropanol of 1 gel volume need to be added.
(4) mixed liquor in aspiration step 3, transfers in DNA preparation pipe (being placed in 2ml centrifuge tube), and 12,000rpm are centrifuged 1min.Abandon filtrate.
(5) putting back into 2ml centrifuge tube by preparing pipe, add 500 μ l Buffer W1,12,000rpm are centrifuged 30s, abandon filtrate.
(6) putting back into 2ml centrifuge tube by preparing pipe, add 700 μ l Buffer W2,12,000rpm are centrifuged 30s, abandon filtrate.With Same method washed once 12,000rpm with 700 μ l Buffer W2 again and is centrifuged 1min.
(7) putting back in 2ml centrifuge tube by preparing pipe, 12,000rpm are centrifuged 1min.
(8) it is placed in preparing pipe in the 1.5ml centrifuge tube of cleaning, adds 25～30 μ l Eluent or go preparing film central authorities Ionized water, room temperature stands 1min.12000rpm is centrifuged 1min eluted dna.
4, PCR primer and the double digestion of carrier for expression of eukaryon
The PCR primer of TMEM88 and pEGFP-C2 carrier are carried out the double digestion of EcoR I and KpnI, enzyme action system and side Method is as follows:
Enzyme action system (10 μ L):
Enzymatic cleavage methods: above reaction system is placed in the centrifuge tube of 0.5ml mixing, 37 DEG C of water-baths 4～6h (temperature and time Between selection gist restriction endonuclease description depending on).Rapid observed result reclaim purpose after 1.2% agarose gel electrophoresis DNA fragmentation (the same 2.2.3.3 of recovery method), it is to avoid ultraviolet long-term irradiation.
5, the connection of digestion products
Being attached the double digestion product of TMEM88 and pEGFP-C2, the enzyme used by connection is preferably T4 ligase.? Before connection, for avoiding the recirculation of carrier, therefore carrier is carried out dephosphorylation process.
Dephosphorylation system (10 μ L): CIAP 1 μ l, 10 × CIAP buffer 1 μ l, carrier 8 μ l
Linked system (10 μ L): carrier 0.5 μ l after CIAP dephosphorylation, 10 × T4DNA ligase buffer 1 μ l, T4DNA ligase 1 μ l, PCR enzyme action purified product 7.5 μ l；
Method of attachment: above-mentioned reaction system is placed in the centrifuge tube of 0.5ml mixing, and 16 DEG C of water-baths are overnight.
6, the conversion of product is connected
E. coli competent used by conversion is: TG1, and step of converting is as follows:
(1) taking-up is stored in the TG1 competent cell of 80 DEG C, and ice bath adds 10 μ l and connects products after thawing, mix gently Even, stand 30min on ice.
(2) 42 DEG C of heat shocks 90s (not rocking), take out rapidly and put 3min on ice.
(3) 500 μ l LB culture fluid, 37 DEG C, 250rpm shaken cultivation 45min are added.
(4) take out about 100 μ l conversional solution be applied on the LB solid medium with corresponding resistant, 37 DEG C be inverted cultivate 8～ 12h。
7, a small amount of extracting of plasmid
Choose in the centrifuge tube that 8 positive colonies add containing 2mlLB culture medium (ammonia benzyl resistance), 37 DEG C, 250rpm shakes Swinging cultivation 8～12h, then bacterium solution carries out plasmid extraction, test kit used is the plasmid extracting examination in a small amount liking the company that pursues progress Agent box, extraction steps is as follows:
(1) with the monoclonal of sterilizing toothpick picking antibacterial, put into and contain the 2～3ml LB culture fluid containing corresponding antibiotic In 15ml centrifuge tube, 37 DEG C, 250rpm shaken cultivation 8～12h.
(2) proceeding in 1.5ml centrifuge tube by about 1ml bacterium solution, 4 DEG C, 12,000rpm is centrifuged 1min, supernatant.
(3) add 250 μ l Buffer S1 suspended bacterial precipitations, suspend and need uniformly, not leave little truffle.
(4) 250 μ l Buffer S2 are added, gentle and spin upside down 4～6 times fully and make thalline fully crack, until shape Become bright solution.
(5) adding 350 μ l Buffer S3, gentleness also spins upside down mixing 6～8 times fully, and 12,000rpm are centrifuged 10min。
(6) the centrifugal supernatant in aspiration step 5 and transfer to preparation pipe (being placed in 2ml centrifuge tube), 12000rpm be centrifuged 1min, abandons filtrate.
(7) putting back into centrifuge tube by preparing pipe, add 500 μ l Buffer W1,12,000rpm are centrifuged 1min, abandon filtrate.
(8) putting back into centrifuge tube by preparing pipe, add 700 μ l Buffer W2,12,000rpm are centrifuged 1min, abandon filtrate；With same The method of sample washed once with 700 μ l Buffer W2 again.Abandon filtrate.
(9) putting back in 2ml centrifuge tube by preparing pipe, 12,000rpm are centrifuged 1min.
(10) move into preparing pipe in new 1.5ml centrifuge tube, add 60～80 μ l sterilized water, room temperature preparing periosteum central authorities Standing 1min, 12,000rpm are centrifuged 1min, abandon preparation pipe.
(11) sample put 20 DEG C standby.Fig. 2 is the structural representation of eucaryon recombinant vector pEGFP-C2-TMEM88.
8, plasmid is carried out enzyme action qualification and the double digestion mirror sending order-checking that the plasmid extracted carries out EcoR I and KpnI Fixed, enzyme action system and method are as follows:
Enzyme action system (10 μ L):
Enzymatic cleavage methods: above-mentioned reaction system is placed in the centrifuge tube of 0.5ml mixing, after 37 DEG C of enzyme action 4-6h, agarose Gel electrophoresis, ultraviolet spectrometer are observed, preservation taken pictures by gel imaging instrument.And enzyme action is identified, and correct plasmid send raw work biology public Department's order-checking, is shown in Fig. 3.Wherein 1:pEGFP-C2 double digestion；2:pEGFP-C2-TMEM88 double digestion, the PCR primer of 3:TMEM88.
Sequencing result is shown in that Fig. 4, sequencing result show, Sequencing chromatogram is made up of red, black, green and blue order-checking peak, represents not Same base sequence, the sequencing result of this experiment shows that the signal of four color fluorescence is relatively strong and the most single, is passed through by sequencing result BLAST comparison, finds consistent with the sequence announced in GencBank, genes of interest fragment length actually 480bp.
Embodiment 2: the expression of process LAN TMEM88 gene plasmid
1, cell is cultivated
The cell of the required cultivation of the present invention is: HEK 293T, HepG2, SMMC-7402, SMMC-7721, SGC-7901 are thin Born of the same parents, first recovery cell, and with containing the DMEM high glucose medium that volume ratio is 10% hyclone in 37 DEG C, 5%CO2 thin Born of the same parents' incubator is cultivated, and changes liquid in time and pass on.
(1) cell recovery: taken out by cell frozen in liquid nitrogen, immerses rapidly in the warm water of 37 DEG C and jiggles to complete Complete melting (rewarming time should control within two minutes), transfer cell suspension is in centrifuge tube and add 5ml culture medium rapidly, Abandon most supernatant after low-speed centrifugal (1000r/min, 2min), be seeded in culture dish after adding 5ml culture medium suspension cell gently Start to cultivate.
(2) cell changes liquid: discard old cell culture medium gently, and PBS gentleness adds fresh complete medium after cleaning (containing the DMEM in high glucose of 10% hyclone) continues to cultivate (note: PBS and fresh culture medium need 37 DEG C of preheatings).
(3) passage: add the pancreatin of 1ml in cell and be placed in incubator and digest, see under the microscope Examining cellular morphology change, add complete medium in time and terminate digestion, transfer cell suspension is to low-speed centrifugal in centrifuge tube Abandon supernatant after (1000r/min, 2min), add 0.2ml culture medium suspension cell after washed once with PBS and be again seeded to training Support and ware continues cultivate.
(4) cell cryopreservation: abandon supernatant after the cell suspension low-speed centrifugal (1000r/min, 2min) in succeeding generations, add After entering 1ml cells frozen storing liquid (formula is shown in) suspension cell gently, subpackage is frozen to carrying out classification in cell cryopreservation tube, i.e. 4 DEG C 0.5- 1h ,-20 DEG C of 1-2h ,-80 DEG C of about 24h, liquid nitrogen cryopreservations.
2, plasmid transient transfection (liposome method)
(1) diluting the liposome of 5 μ l with the Opti-MEM of 250 μ L and total amount is about the DNA of 2 μ g, gentle mixing, room temperature is quiet Put 5min.
(2) by two kinds of solution gentleness mixings in (1), room temperature stands 20min, to form DNA-liposome complex.
(3) discard the culture fluid in culture dish, add 1ml Opti-MEM and transfect liquid, the DNA-liposome in (2) is multiple Compound is slowly dropped in culture dish, jiggles culture dish mixing.
(4) 37 DEG C, 5%CO2After cultivating 6h, with the PBS of pre-temperature once, training is continued after renewing fresh complete culture solution Support about 24h.
3, immunoblotting analysis
(1) total protein extraction: centrifugal collecting cell, after PBS 2-3 time, adds 0.5ml cell pyrolysis liquid, on ice Cracking 10min, then high speed frozen centrifugation 10min, collect 0.4ml supernatant, adds sample-loading buffer, boiling water bath 5min, puts Put and treat electrophoresis on ice.
(2) installing glue plate and hunt leak, (formula is shown in, the selection of resolving gel concentration is according to observed first to prepare separation gel The size of protein content), with 1ml isopropanol line ball, after fully solidifying (about 1h), preparation concentrates glue, inserts required comb, waits to fill Use after fractional condensation is solid.
(3) electrophoresis system is installed, electrophoresis tank adds 500ml electrophoretic buffer, after 10 μ L sample are added loading hole Start electrophoresis (80V electrophoresis 0.5h+100V electrophoresis 1.5h).
(4), after electrophoresis terminates, cut SDS-PAGE glue, application sandwich mode (be the most successively filter paper+glue+ Pvdf membrane+filter paper) carry out wet turn
(5) installing electricity and transfer from one department to another system, the electricity adding 500ml pre-cooling in electricity turn trough turns buffer (formula is shown in), and by whole Electricity turn trough is put in frozen water, starts electricity and turns (100V electricity turns 1h or 200mA electricity and turns 2h)
(6), after electric turn of end, take off pvdf membrane gently, identify that transferring film is the most successful with Ponceaux dyeing, after transferring film is successful, Washing away Ponceaux with TBST, room temperature closes at least 1h.
(7) after having closed, after washing film (3 times, 5min) with TBST, addition one anti-solution (one resists: GFP antibody, and rabbit resists, Western mono-anti-diluent 1:500 dilutes) 4 DEG C of overnight incubation.
(8) next day, after washing film (3 times, 10min) with TBST, (two resist: the goat of Radix Cochleariae officinalis enzyme labelling resists to add two anti-solution Rabbit two anti-TBST solution 1:5000 dilutes) incubated at room 1-2h.Then film (3 times, 20min) is washed with TBST.
(9) with the band on ECL luminous agent detection PVDF, and X-ray film scotography is used.
Experimental result shows, recombinant eukaryotic expression plasmid pEGFP-C2-TMEM88 in eukaryotic cell can normal expression, Lay a good foundation for follow-up research, see Fig. 5.Wherein 1: the 293T cell pyrolysis liquid of untransfected；2: represent transfection pEGFP-C2- The 293T cell pyrolysis liquid of TMEM88.
Embodiment 3: to human liver cancer cell HepG2, SMMC-7402 after process LAN TMEM88 gene,
The impact of SMMC-7721, SGC-7901 proliferative conditions
The cell of exponential phase is passed on, obtains cell suspension, and with suitable density (about 1 × 108·L-1) will Cell is, in the amount renewed vaccination of every hole 100 μ L to 96 orifice plates, to cultivate about 24h, until cell density is about 80%, to carry out The transient transfection of pEGFP-C2-TMEM88, adds 20 μ L debita spissitudo (5mg mL after transfection a period of time in every hole-1) MTT, continues to cultivate 4h.Discard the culture medium in every hole, and add the DMSO of 150 μ L, after shaken at room temperature dissolves 10min, Survey absorbance (A value) at 492nm wavelength, represent the proliferative conditions of cell by the average of each group of light absorption value.
MTT experiment shows: compared with untransfected group, transfected pEGFP-C2-TMEM88 hepatoma cell line (HepG2, SMMC-7402, SMMC-7721, SGC-7901) occur in that and significantly breed phenomenon (P < 0.05), process LAN TMEM88 base is described Because can substantially suppress the propagation of human liver cancer cell, it is shown in Table 1.
Impact on human hepatoma cell proliferation situation after table 1 process LAN TMEM88 gene
Embodiment 4: the impact of process LAN TMEM88 gene pairs human hepatoma cell strain SMMC-7721 apoptosis
The cell of exponential phase is passed on, obtains cell suspension, and with suitable density (about 1 × 108·L-1) will Cell is inoculated in 6 orifice plates, cultivates about 24h, until cell density is about 80%, carries out the instantaneous of pEGFP-C2-TMEM88 Transfection, collects cell suspension after transfection a period of time.Low-speed centrifugal 5min, collects cell, discards culture medium, and buffer with PBS Liquid cleans cell 2-3 time.With 400ul Binding Buffer Eddy diffusion cell, add 5ul Annexin V dye liquor, gently 15min is hatched under the conditions of placing 4 DEG C of lucifuges after mixing.Add 10ul PI, hatch under the conditions of placing 4 DEG C of lucifuges gently after mixing 5min, flow cytometer detection apoptosis, experiment is repeated 3 times.
Flow cytometry results shows: compared with the apoptotic cell numerical value of untransfected group, transfected pEGFP-C2-TMEM88 The apoptotic cell numerical value of group is all increased significantly, and difference exists statistical significance (P ＜ 0.05).Process LAN TMEM88 base is described Because Fig. 6-7 can be seen substantially from the apoptosis of promotion human liver cancer cell.
The above, only present pre-ferred embodiments, therefore can not limit, with this, the scope that the present invention implements, i.e. depend on Equivalence change and the modification that scope of the present invention patent and description are made, all should still belong to the model that patent of the present invention contains In enclosing.

Claims (10)

1. a process LAN TMEM88 gene plasmid, it is characterised in that: include TMEM88 gene C DS sequence and pEGFP-C2 eucaryon The recombination to construct of expression vector forms；Its nucleotide such as sequence table SEQ ID NO:3, is positioned at the 10-489 of TMEM88 gene Position；Aminoacid sequence is as shown in sequence table SEQ ID NO:4.
2. the method building process LAN TMEM88 gene as claimed in claim 1, it is characterised in that include walking as follows Rapid:
(1) collection, cell lysis, extraction RNA carries out reverse transcription and obtains TMEM88cDNA；
(2) use round pcr, amplify the CDS sequence fragment of artificial T MEM88 gene；
(3) purification of the CDS sequence fragment of TMEM88 gene；
(4) genetic fragment obtained and pEGFP-C2 carrier for expression of eukaryon are carried out KpnI and EcoRI double digestion, and carry out even Connect；
(5) product will be connected convert in e. coli tg1, and choose positive colony and cultivate, then extract plasmid.
The construction method of a kind of process LAN TMEM88 gene the most according to claim 2, it is characterised in that after extracting RNA, Carry out degeneration；Concrete processing step: after RNA is placed 5-10min in 65 DEG C, is transferred in frozen water place.
The construction method of a kind of process LAN TMEM88 gene the most according to claim 2, it is characterised in that reverse transcription anti- System is answered to include: 5 × RT Master Mix, RNA, Nuclease-free Water；Concrete processing step includes:
(1) preparation reaction system: 5 × RT Master Mix 2 μ L+RNA 2 μ L+Nuclease-free Water 6 μ L；
(2) above-mentioned reaction system is placed 37 DEG C, and act on 15min；
(3) above-mentioned reaction system is transferred to 50 DEG C, and acts on 5min；
(4) above-mentioned reaction system is transferred to 98 DEG C, and acts on 5min；
(5) above-mentioned reaction system is transferred to 4 DEG C, frozen after cooling, i.e. obtain cDNA.
The construction method of a kind of process LAN TMEM88 gene the most according to claim 2, it is characterised in that according to TMEM88 CDS sequence, design upstream and downstream primer；Upper and lower primer sequence is as follows:
TMEM88-F:5’-CCGGAATTCCGGATGGCGGATGTCCCCG-3’
TMEM88-R:5’-GGGGTACCCCTTAGACCCAGACCTTTCCT-3’。
The construction method of a kind of process LAN TMEM88 gene the most according to claim 2, it is characterised in that the reaction of PCR System is as follows: 2 × PrimeSTAR Max, cDNA sample, upstream and downstream primer, high purity water；Concrete processing step includes:
(1) preparation reaction system: each 1 μ L+ high purity water 22 μ L of 2 × PrimeSTAR Max 25 μ L+cDNA 1 μ L+ primer；
(2) put into PCR instrument device after above-mentioned reaction system being mixed gently, following program be set and run:
(3) above-mentioned PCR primer is carried out 1-1.2% agarose gel electrophoresis；
(4) target DNA fragment is reclaimed.
The construction method of a kind of process LAN TMEM88 gene the most according to claim 2, it is characterised in that to TMEM88's PCR primer and pEGFP-C2 carrier carry out the double digestion of EcoR I and KpnI；Enzyme action system is as follows: restricted enzyme (EcoRI And KpnI), the PCR primer of 10 × Buffer Tango, TMEM88 or pEGFP-C2 carrier；Concrete processing step includes:
(1) preparation reaction system: the PCR primer of each 0.5 μ L+TMEM88 of 10 × Buffer Tango 2 μ L+EcoRI and KpnI or With pEGFP-C2 carrier 7 μ L；
(2) above-mentioned reaction system is placed in 37 DEG C of water-baths, and act on 4～6h；
(3) above-mentioned digestion products is carried out 1-1.2% agarose gel electrophoresis；
(4) observed result reclaim target DNA fragment.
The construction method of a kind of process LAN TMEM88 gene the most according to claim 2, it is characterised in that in connection Before, carrier is carried out dephosphorylation process；
Dephosphorylation system (10 μ L): CIAP1 μ l, 10 × CIAP buffer 1 μ l, carrier 8 μ l
Linked system (10 μ L): after CIAP dephosphorylation, carrier 0.5 μ l, 10 × T4DNA ligase buffer 1 μ l, T4DNA connect Enzyme 1 μ l, PCR enzyme action purified product 7.5 μ l；
Method of attachment: above-mentioned reaction system is placed in the centrifuge tube of 0.5ml mixing, and 16 DEG C of water-baths are overnight.
The construction method of a kind of process LAN TMEM88 gene the most according to claim 8, it is characterised in that
(1) taking-up is stored in the TG1 competent cell of 80 DEG C, and ice bath adds 10 μ l and connects products after thawing, mix, the most quiet Put 30min；
(2) 42 DEG C of heat shocks 90s, take out and put 3min on ice；
(3) 500 μ l LB culture fluid, 37 DEG C, 250rpm shaken cultivation 45min are added；
(4) take out 100 μ l conversional solution to be applied on the LB solid medium with corresponding resistant, be inverted for 37 DEG C and cultivate 8～12h.
10. one kind uses process LAN TMEM88 gene plasmid as claimed in claim 1 in the suppression propagation of human liver cancer cell, rush Enter the application of the apoptosis of human liver cancer cell.
CN201610425684.9A 2016-06-14 2016-06-14 Overexpression TMEM88 gene plasmid, construction method and application thereof Pending CN106047927A (en)

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