CN104726493A - Construction method and application of overexpressed c2orf68 gene plasmid - Google Patents

Abstract

The invention discloses a construction method of an overexpressed recombinant plasmid pcDNA3.1 for c2orf68 genes and an application of the recombinant plasmid in gene function research. The invention relates to an mRNA of a c2orf68 gene. The nucleotide sequence of the mRNA is shown in SEQ ID NO.2 in a table. The invention also provides an overexpressed recombinant eukaryotic expression vector for c2orf68 genes. The sequence of the vector is shown in FIG 2. The invention also provides a construction method of the overexpressed vector, and an experiment method of c2orf68 gene research in human hepatocellular carcinoma and human colorectal carcinoma.

Description

A kind of process LAN c2orf68 gene plasmid construction process and application thereof

Technical field

The invention belongs to technical field of bioengineering, be specially a kind of energy process LAN c2orf68the eukaryon recombinant plasmid of genetic expression, and utilize this restructuring process LAN plasmid to study c2orf68function provides good tool, also in research people liver cancer, Human colorectal carcinoma and other malignant tumour c2orf68the function of gene provides effective experimental technique.

Background technology

Hepatocellular carcinoma (hepatoceLLuLar carcinoma, HCC), be called for short people's liver cancer, being worldwide the 5th modal cancer, is also one of cancer that mortality ratio is the highest.In China every year because the number of people's PLC mortality is more than 300,000, be only second to lung cancer, occupy the second of China's mortality of malignant tumors.Colon and rectum gland cancer is one of modal malignant tumour of the mankind, and in recent years, its sickness rate rises year by year, accounts for the second of all malignant tumours.In Europe, Colon and rectum gland cancer accounts for malignant tumour and to be correlated with the second of the lethal cause of disease.In some big cities of China, the sickness rate of colorectal cancer also reaches 2 to 5 of all Cancer Mortalities.Because most of malignant tumour has special biological characteristics, early diagnosis difficulty, is easy to shift, easily recurs after excision, so Long term therapy poor effect.The molecular mechanism of the early stage generation of malignant tumour is illustrated not yet completely, therefore understands the molecule mechanism that cancer occurs in depth, plays an important role for the treatment of research and development early diagnosis of cancer.

Genetically engineered, is also called gene recombination technology, be grow up nineteen seventies in vitro to a special kind of skill that DNA operates, be widely used in disease fundamental research, gene therapy, genetic immunization, gene vaccine etc.Its three elements are respectively: enzyme, goal gene, carrier.And carrier for expression of eukaryon is for building a kind of carrier of eukaryotic gene expression system in genetically engineered, in recent decades, scholars has carried out the correlative studys such as various disease, gene around carrier for expression of eukaryon, do a lot of work in genetically engineered, for medical scientific opens brand-new route. c2orf68in the PROTEIN C 2ORF68 of genes encoding, containing 126 amino acid whose UPF0561 structural domains, belong to UPF0561 protein family.Up to now, there is no any function about UPF0561 protein family report.

Therefore, the present invention uses gene recombination technology, for c2orf68gene, structure recombinant eukaryon expression vector pcDNA3.1 (+)- c2orf68, and by low for its transfection expression or do not express c2orf68the malignant cell such as liver cancer, colorectal cancer, build stable transfection c2orf68the cell strain of gene is research c2orf68function provides good tool, is also research further c2orf68the biological function of gene in the malignant tumour such as liver cancer, colorectal cancer lays the first stone, simultaneously also in research people liver cancer, Human colorectal carcinoma and other malignant tumour c2orf68the function of gene provides effective experimental technique.

Summary of the invention

The object of this invention is to provide a kind of novel eukaryon recombinant plasmid and preparation method thereof, and prove that described recombinant plasmid is c2orf68functional study provides good experimental tool, also in research people liver cancer, Human colorectal carcinoma etc. and other malignant tumour c2orf68the function of gene provides effective experimental technique.

People c2orf68gene is in GenBank registration, and number of registration NM_001013649.3, is positioned 2p11.2, total length 4283bp, 166 amino acid of encoding, and its nucleotides sequence to be classified as in sequence table described in SEQ ID NO:1.People c2orf68the tissue expression spectrum analysis display of gene: this gene all has expression in the gastrointestinal tumor such as Human colorectal carcinoma.Eukaryon recombinant plasmid of the present invention, by the CY-in the nucleotide sequence shown in sequence table SEQ ID NO:2 c2orf68fragment and eukaryotic vector build and form, described CY- c2orf68fragment is the nucleotide sequence of initiator codon downstream 73bp ~ 573bp.

Eukaryon recombinant plasmid of the present invention, its preparation method comprises the following steps: successively

(1) by the CY-of heminested PCR synthesis people c2orf68gene fragment;

(2) nucleotide fragments of above-mentioned acquisition is connected with eucaryon plasmid, the restriction enzyme site of this Insert Fragment is respectively Xho I and EcoR I, again described eukaryon recombinant plasmid is transformed in competence bacterial cell and cultivates, then filter out the clone of the correct eukaryon recombinant plasmid inserted;

(3) use alkaline lysis extracting eukaryon recombinant plasmid, utilize the eukaryon recombinant plasmid that restriction endonuclease map analysis is built, and carry out DNA sequence analysis.

(4) by the Transfected Recombinant Plasmid SMMC7721 human liver cancer cell of above-mentioned acquisition and LoVo Human colorectal cancer cells, observing cell biology characteristic.

(5) experiment shows, described Transfected Recombinant Plasmid is entered people's liver cancer and Human colorectal cancer cells, and recombinant plasmid acts on people c2orf68gene can cause the propagation tying human liver cancer cell, and therefore, the present invention contributes to research c2orf68gene function, and be research c2orf68the effect that gene is fallen ill in people's liver cancer and Human colorectal carcinoma provides useful biology tool.

Accompanying drawing explanation

Fig. 1 is the structural representation of eucaryon recombinant vectors pcDNA3.1 (+) of the present invention.

Fig. 2 is eucaryon recombinant vectors pcDNA3.1 (+) of the present invention ^{+ CY- c2orf68} structural representation.

Fig. 3 is eucaryon recombinant vectors pcDNA3.1 (+) of the present invention ^{+ CY- c2orf68} enzyme cut qualification collection of illustrative plates.

Fig. 4 is eucaryon recombinant vectors pcDNA3.1 (+) of the present invention ^{+ CY- c2orf68} sequencing chromatogram.

Fig. 5 is eucaryon recombinant vectors pcDNA3.1 (+) of the present invention ^{+ CY-c2orf68}human liver cancer cell SMMC7721 photo.

Fig. 6 is eucaryon recombinant vectors pcDNA3.1 (+) of the present invention ^{+ CY-c2orf68}human colorectal cancer cells LoVo photo

Fig. 7 is the canonical plotting of people c2orf68 gene primer in fluorescent quantitative PCR experiment.

Fig. 8 is the canonical plotting of people's internal reference gapdh gene primer in fluorescent quantitative PCR experiment.

Fig. 9 is the SMMC-7721 human hepatocarcinoma cell of transfection eukaryon recombinant plasmid ^{+ CY-c2orf68}, the negative control group SMMC7721 of transfection empty carrier ^-NC, the histogram of the blank group SMMC7721 genetic expression of non-Pignus pignoris grain.

Figure 10 is the Human colorectal cancer cells strain LoVo of transfection eukaryon recombinant plasmid ^{+ CY-c2orf68}, the negative control group LoVo of transfection empty carrier ^-NC, the histogram of the blank group LoVo genetic expression of non-Pignus pignoris grain.

Figure 11 is that SDS-PAGE shows SMMC7721 ^{+ CY-c2orf68}, SMMC7721 ^-NC, SMMC7721 and LoVo ^{+ CY-c2orf68}, LoVo ^-NCand in LoVo cell strain C2ORF68 albumen at electrophoretic band figure.

Figure 12 is that C2ORF68 albumen is at SMMC7721 ^{+ CY-c2orf68}, SMMC7721 ^-NC, SMMC7721 and LoVo ^{+ CY-c2orf68}, LoVo ^-NCand expression level histogram in LoVo cell strain.

Figure 13 is the SMMC-7721 human hepatocarcinoma cell of transfection eukaryon recombinant plasmid ^{+ CY- c2orf68} , the negative control group SMMC7721 of transfection empty carrier ^-NC, the vegetative map of the blank group SMMC7721 cell of non-Pignus pignoris grain.

Figure 14 is the Human colorectal cancer cells strain LoVo of transfection eukaryon recombinant plasmid ^{+ CY- c2orf68} , the negative control group LoVo of transfection empty carrier ^-NC, the vegetative map of the blank group LoVo cell of non-Pignus pignoris grain.

Figure 15 is the SMMC-7721 human hepatocarcinoma cell of transfection eukaryon recombinant plasmid ^{+ CY- c2orf68} , the negative control group SMMC7721 of transfection empty carrier ^-NC, the streaming apoptosis of the blank group SMMC7721 cell of non-Pignus pignoris grain detects figure.

Figure 16 is the SMMC-7721 human hepatocarcinoma cell of transfection eukaryon recombinant plasmid ^{+ CY- c2orf68} , the negative control group SMMC7721 of transfection empty carrier ^-NC, the streaming apoptosis of the blank group SMMC7721 cell of non-Pignus pignoris grain detects figure histogram.

Figure 17 is the Human colorectal cancer cells strain LoVo of transfection eukaryon recombinant plasmid ^{+ CY- c2orf68} , the negative control group LoVo of transfection empty carrier ^-NC, the streaming apoptosis of the blank group LoVo cell of non-Pignus pignoris grain detects figure.

Figure 18 is the Human colorectal cancer cells strain LoVo of transfection eukaryon recombinant plasmid ^{+ CY- c2orf68} , the negative control group LoVo of transfection empty carrier ^-NC, the streaming apoptosis of the blank group LoVo cell of non-Pignus pignoris grain detects figure histogram.

Embodiment

Below in conjunction with embodiment, the invention will be further described.In following embodiment, all unreceipted specific experiment conditions, be according to the condition in normal condition well known to those skilled in the art, laboratory manual (New York:CoLd Spring Harbor Laboratory Press, 1989), the condition of advising according to manufacturer.

Embodiment 1: structure recombinant eukaryon expression vector pcDNA3.1 (+)- c2orf68

1, c2orf68the clone of gene

According to the encoding sequence of Gene Bank HLA-G, with primer premier 5 software design primer c2orf68 S1, c2orf68 S2, c2orf68 A, as follows respectively:

c2orf68 S1：5’-gtgggcgggtggctgttgtt-3’

c2orf68 S2: 5 '-agtgtg gaatcatggaggcggggccgcatcc-3 ' (containing EcoRI restriction enzyme site)

c2orf68 A: 5 '-tctaga ctc gagtcagtgttggctctggcgctttg-3 ' (containing Xho I restriction enzyme site)

PCR reaction is heminested PCR (Takara company, PrimeSTAR HS fidelity enzyme)

PCR use for the first time, c2orf68 S1with c2orf68 Atwo primers, second time is used c2orf68 S2, c2orf68 Atwo primers

Reaction conditions: first time PCR 94 DEG C of 40 sec

65℃ 40 sec

72℃ 40 sec

30 cycLe

Reaction system: 20uL

2 x primStar HS mix 10 uL

primStar HS enzyme 0.25 uL

Primer c2orf68 S10.25 uL

Primer c2orf68 A0.25 uL

Human cDNA 0.25 uL

ddH2O 13.75uL

The template of second time PCR is directly used as the first time after PCR reaction product

Reaction conditions: second time PCR 94 DEG C of 40 sec

62℃ 40 sec

72℃ 40 sec

30 CycLe

Reaction system: 50uL

2 x primStar HS mix 25 uL

primStar HS enzyme 0.5 uL

Primer c2orf68 S20.5 uL

Primer c2orf68 A0.5 uL

First time PCR primer 0. 5 uL

ddH ₂O 23 uL

Heminested PCR Successful amplification goes out people c2orf68gene coded sequence (CDS), obtains amplified production band at 501bp place after PCR primer electrophoresis, basically identical with goal gene clip size.

2, PCR primer purified after electrophoresis reclaims, and test kit is that the PCR purifying of Promega company reclaims test kit, operation by specification.Experimental procedure:

1) cut PCR primer position gel, weigh, c2orf68gene is respectively 0.18 g;

2) add 180uL binding buffer, 55 DEG C of water-bath temperature are all melted to gel;

3) 2 step gained liquid are all moved into adsorption column, centrifugal 1 minute of 12000rpm, abandons filtered solution;

4) add 500 uL washing buffer in adsorption column, centrifugal 1 minute of 12000rpm, abandons filtered solution;

5) add 750 uL washing buffer in adsorption column, centrifugal 1 minute of 12000rpm, abandons filtered solution;

6) adsorption column recentrifuge, centrifugal 3 minutes of 12000rpm, abandons filtered solution;

7) adsorption column is moved into clean 1.5mL EP to manage, add 45 uL nuclease free waters eluted dna in adsorption column;

8) centrifugal 1 minute of 12000rpm, collects filtered solution, is the DNA solution of purifying.

3, carrier for expression of eukaryon linearizing with c2orf68gene connects

Reaction conditions: 37 DEG C of water-bath incubation 60min.

Digestion products reclaims (test kit reclaims with PCR primer), enzyme cut after carrier and PCR primer for next step ligation, ligation system is as follows:

Reaction system 10 uL

Reaction conditions :25 DEG C of water-bath incubation 60min

4, eukaryon recombinant plasmid pcDNA3.1 (+)-C2ORF68 transforms DH5a competent cell, and experimental procedure is as follows:

1) DH5a competence is melted on ice, packing, often pipe 50 uL;

2) add 5uL and connect product, flick mixing fast, leave standstill ice bath 30min;

3) 42 DEG C of water-bath incubation 90 sec;

4) ice bath 3 min is left standstill;

5) antibiotic-free LB substratum 200 uL is added, 37 DEG C of water-bath incubation 40 sec;

6) half culture coating LB agar plate (containing Amp 100ug/mL) is got, 37 DEG C of overnight incubation;

7) the single colonies of picking identifies positive bacterium colony for PCR.

c2orf68picking 3 clone, numbering is respectively 57,58,59.Each positive bacterium colony mixes in 10uL sterilized water, gets 0.5uL and reacts for pcr template

Reaction conditions: 94 DEG C of 5 min

94 ℃ 40 sec

62℃ 40 sec

72℃ 90 sec

30 cycLe

PCR product gets 3 uL for 1% agarose electrophoresis qualification positive colony, and electrophoresis result is as figure

4, positive bacterium colony overnight incubation is extracted plasmid (plasmid extraction kit: Promega company, Cat:A1330)

, enzyme cuts qualification.Reaction system 20 uL, restriction enzyme digestion and electrophoresis result is as figure.

Reaction conditions :37 DEG C of water-bath incubation 60min

5, positive bacterium colony overnight incubation is extracted plasmid, select two cloning and sequencing qualifications (the raw biological company limited of upper Hypon), sequencing result is shown in Fig. 6, and sequencing result shows, Sequencing chromatogram is made up of red, black, green and blue order-checking peak, represent different base sequences, the sequencing result of this experiment shows the signal of four look fluorescence comparatively by force, and comparatively single, by sequencing result by BLAST comparison, find consistent with the sequence announced in GencBank, goal gene fragment length is real is 501bp.

Embodiment 2: recombinant eukaryotic expression plasmid pcDNA3.1 (+)- c2orf68determination of activity

1, cell cultures

With the volume ratio of foetal calf serum be 10% DMEM high glucose medium (from HyCLone company buy) in 37 DEG C, 5% CO ₂cell culture incubator in cultivator SMMC-7721 cell line and people's Human colorectal carcinoma LoVo cell strain (SMMC-7721 and LoVo buys from ATCC company of the U.S.) to confluent culture bottle.

2, eukaryon expression plasmid pcDNA3.1 (+) ^{- c2orf68} transfected with human SMMC-7721 Cell Line

2.1 get 1/24 human hepatocarcinoma BEL-7402 in culturing bottle is respectively inoculated in each hole of 24 porocyte culture plates;

After 2.2 24h, Tissue Culture Plate fraction of coverage about reaches 70% ~ 80%, abandons former substratum.Cell in 24 orifice plates 500 μ LDMEM high glucose mediums (serum-free, antibiotic-free) are cultivated 4h;

2.3 A liquid configurations: 1 μ g+50 μ LDMEM substratum (serum-free, antibiotic-free), mixes;

B liquid configures: 2.5 μ L Lipofectamine 2000 (invitrogen company)+50 μ LDMEM substratum (serum-free, antibiotic-free), mix; Room temperature leaves standstill 5min;

C liquid configures: A liquid+B liquid, and mix, room temperature leaves standstill 25min;

2.4 abandon original substratum in Tissue Culture Plate, and every hole adds 400 fresh μ L DMEM high glucose mediums (serum-free, antibiotic-free);

2.5 by C liquid dropwise join in culture plate, each 100 μ L in every hole;

Transfected with human SMMC-7721 Cell Line cell to be put into 37 DEG C by 2.6,5% CO2 cell culture incubator hatches; Setup Experiments 3 groups, experimental group SMMC7721 ^{+ CY- c2orf68} , the negative control group SMMC7721 of transfection empty carrier ^-NC, blank group SMMC7721;

After 2.7 6h, abandon original substratum in Tissue Culture Plate, every hole adds the DMEM high glucose medium of 2mL containing 10% foetal calf serum; Put into 37 DEG C, 5% CO ₂cell culture incubator is hatched.With the DMEM high glucose medium containing 600 μ g/mLG418 after 48h, screening 14d, changes a subculture every 3d.Select monoclonal cell group and maintain G418 resistance culture, the maintenance concentration of G418 is 300 μ g/mL, obtains the SMMC-7721 cell strain (see figure 5) of stable transfection.

Structure and the screening process of Human colorectal carcinoma LoVo stable cell line are consistent with said process.

3, the extraction of total serum IgE

3.1 human hepatocarcinoma BEL-7402 getting experimental group (transfection pcDNA3.1 (+)-C2ORF68) after stable transfection, blank group, negative control group (transfection pcDNA3.1 (+)), add TRIzol reagent (invitrogen company) 1mL, then transfer in the Eppendorf pipe of 1.5mL, room temperature leaves standstill 5min;

3. 2 chloroforms adding 0.2mL, cover tightly lid, shake up about 15 seconds, room temperature leaves standstill 2-3min, in 4 DEG C of whizzers centrifugal (12000r/min, 15min);

Supernatant is transferred in new pipe (the Eppendorf pipe of 1.5mL) by 3.3, adds the Virahol of 0.5mL, fully mixes, room temperature leaves standstill 10min, in 4 DEG C of whizzers centrifugal (12000g/min, 15min), abandon supernatant, bottom pipe, the little throw out of visible oyster white, is RNA;

3.4 ethanol adding 1mL mass concentration 75%, mixing concussion, in 4 DEG C of whizzers, centrifugal (7500g/min) 5min, abandons supernatant, and ventilate, dry 5min in room temperature, allows ethanol volatilize; Then the DEPC process water dissolution RNA of 50 μ L mass concentrations 0.1% is added;

3.5 gel electrophoresises and spectrophotometer detectable level and purity, ensure that the value of the OD260/OD280 of each sample is between 1.8 ~ 2.0, and the gray-scale value being detected 28S and the 18S band of each sample by agarose electrophoresis carrys out strict control RNA quality further, and the RNA of each sample all uses DNA enzymatic to carry out processing to remove genome pollution.

4, the synthesis of cDNA first chain

Get total serum IgE sample 1 μ g, OLigo dT 1 μ L enters PCR reaction tubes, add the DEPC process water of mass concentration 0.1% again to cumulative volume 12 μ L, 65 DEG C, react 5 minutes, reaction terminates rear taking-up and is placed in and hatches 2min on ice, add damping fluid (Buffer) 4 μ L again, the dNTP 2 μ L of RNA inhibitor 1 μ L, concentration 10mmol/L, reversed transcriptive enzyme 1 μ L reaches 20 μ L to cumulative volume, mixed solution is placed in 37 DEG C of reactions 60 minutes, again in 70 DEG C of reactions 5 minutes, cooled on ice termination reaction afterwards ,-20 DEG C of preservations.

5, fluorescent quantitative PCR experiment

5.1 fluorescence quantification PCR primer designs

According to people ( homo sapiens) c2orf68opening code-reading frame (Open Reading Frame, the ORF) sequence of full length gene cDNA, primer needed for design quantitative fluorescent PCR.Software used is Primer Premier 5.0 and OLig6.Ensure that PCR primer length is at 100bp ~ 250bp.After design of primers is good, whether the specificity further by BLAST comparison primer on NCBI is good.Employing people ( homo sapiens) gapdh(deoxynucleotide sequence is shown in ncbi database), as internal reference, primer sequence sees the following form.

Table 1 quantitative PCR primer

5.2 fluorescent quantitative PCR

SYBR GREEN dye method is adopted to carry out fluorescent quantitative PCR, and according to PCR kit for fluorescence quantitative process specifications (TakaRa company) Control release standardization.Get successively 2 μ L step 4 synthesize cDNA template, 25 μ L SYBR MIX(quantitative PCR kit), the stoning sour water of 21 μ L, the upstream primer of 1 μ L, the downstream primer of 1 μ L, reaction cumulative volume 50 μ L, then put into eppendorf quantitative real time PCR Instrument to increase, internal reference gapdhamplification slope is-3.329, and Y-axis intercept is 25.38, and amplification efficiency is 1.00, and relation conefficient is 0.999.Goal gene c2orf68amplification slope is-3.397, and Y-axis intercept is 26.17, and amplification efficiency is 0.97, and relation conefficient is 0.999.Test experience group, blank group, negative control group respectively c2orf68the expression level of the mRNA of gene.After completion of the reaction experimental result is analyzed, c2orf68gene primer canonical plotting see Fig. 6, gapahgene primer canonical plotting is shown in Fig. 7.Experimental result shows, after human hepatocarcinoma BEL-7402 transfection, experimental group, negative control group and blank group c2orf68gene relative expression levels be respectively 2.675,0.438 and 0.493(P<0.005) (see figure 9).After Human colorectal carcinoma LoVo cell transfecting, experimental group, negative control group and blank group c2orf68gene relative expression levels be respectively 2.58,0.892 and 0.849(P<0.005) (P<0.005) (see figure 10).

Embodiment 3:Western bolt detects c2orf68testing goal genetic expression

1 total protein extraction (operating on ice)

Discard nutrient solution, every bottle of cell adds 200uL protein lysate and 20ulPMSF; In cracking 10min on ice, period rocks Tissue Culture Flask 2-3 time gently; Lysate is collected in EP pipe, centrifugal (12000 rpm, 15min, 4 DEG C) with cell scraper; In-80 DEG C of preservations in transfer supernatant to new EP pipe.

2 BCA albuminimetries measure protein content

Ox typical curve is prepared according to BCA determination of protein concentration test kit (Beyotime); Quantity per sample, adds 1 volume BCA reagent B (50:1) by 50 volume BCA reagent A and prepares appropriate BCA working fluid, fully mix.The little indoor of BCA working fluid room temperature 24 are stablized; Get 2ul protein sample aerial to the sample of 96 orifice plates, add standard dilutions and supply 20uL; Control tube adds 50ul deionized water; Each hole adds 200uLBCA working fluid, places 30min for 37 DEG C; Measure A570nm place and read A value; The protein concentration of sample is calculated according to typical curve.

3 SDS-polyacrylamide gel (SDS-PAGE) electrophoresis

Sample preparation: the protein sample after quantitatively by volume adds 5 × SDS sample-loading buffer, and in 100 DEG C of boiling water, 10min makes the abundant sex change of albumen; According to the molecular weight preparation separation gel of 12% and the concentrated glue of 4% of albumen to be detected.Every hole adds 40ug albumen, and with starting voltage 80V electrophoresis, treat that sample electrophoresis is to separation gel, Voltage Cortrol is 120V, and continue electrophoresis and be about 1.5h, bromjophenol blue has just been run out of gel and namely stopped electrophoresis.Transferring film is carried out after suitable cutting after taking off gel.

4 transferring films

Prepare 6 filter paper and 1 pvdf membrane of suitable size, pvdf membrane be placed in methyl alcohol 15s, become translucent after be transferred in transferring film damping fluid and soak 5min.Transferring film instrument installs according to the order of sponge-tri-metafiltration paper one gel one pvdf membrane one or three metafiltration paper-sponge, gets rid of bubble, 60V constant voltage transferring film in transferring film damping fluid.The size of transferring film basis of time molecular weight of albumen and different.

5 immune responses

After transferring film, film is put into confining liquid and (containing in 1 × TBST (1 × TBS, the 0.1% Tween20) solution of the skim-milk of 5%, under room temperature, shaking table is closed.After 1h, on shaking table, wash film 3 times with TBST liquid, each 15min.The primary antibodie 4 DEG C of 5% skim-milk dilution is hatched.After spending the night, on shaking table, wash film 3 times with 1 × TBST, each 15min.Two anti-methods of hatching are the same, after incubated at room temperature 1-2h, wash film 3 times, each 15min with TBST liquid on shaking table.

6 detect

Manage A liquid and the B liquid of medium volume mixture ECL liquid at EP, be added to the front of pvdf membrane.Be placed in gel imaging system, exposure is 10min altogether, and every 30s collects one, photo.

7 gel images analyses

Target protein and internal reference β-actin protein band Quantity one analysis software (Bio-Rad, the U.S.) analyze, survey its absorbance (optical density, OD), the relative content of ratio as target protein of the absorbancy of target protein and internal reference β-actin albumen is got.

Experimental result shows: C2ORF68 protein expression level is at hepatoma cell strain SMMC7721 ^{+ CY- c2orf68} 51.41% is raised in cell strain than in SMMC7721 cell.C2ORF68 protein expression level is at colorectal cancer cell lines LoVo ^{+ CY- c2orf68} 44.50% is raised in cell strain than in LoVo cell.Its difference all has statistical significance (P<0.005; P<0.005).

Embodiment 4: transfection c2orf68gene after impact on human hepatoma cell strain SMMC-7721 and Human colorectal carcinoma LoVo cell proliferative conditions

MTT experiment is adopted to detect the proliferative conditions of human hepatoma cell strain SMMC-7721.To be in the SMMC-7721 cell strain of growth index phase, and cultivate in 96 orifice plates, adjustment cell concn is 1 × 10 ⁴individual/hole, after 24 h, Tissue Culture Plate fraction of coverage about reaches 70% ~ 80%, then carries out transfection, and step is with the step 1 in embodiment 2.Often kind of cell strain arranges three group Setup Experiments, 3 groups, experimental group SMMC7721 respectively ^{+ CY- c2orf68} , the negative control group SMMC7721 of transfection empty carrier ^-NC, blank group SMMC7721, often group establishes 6 holes.Add 15uLMTT (5mg/mL prepares with PBS, filtration sterilization) respectively at 0h, 12h, 24h, 36h and 48h after transfection at each reacting hole, continue to hatch 4h.Stoste is abandoned in suction, adds 150uL DMSO.37 DEG C hatch 15min after to detect the light absorption value (OD) at 490nm wavelength place, each hole by microplate reader, represent the proliferative conditions of cell by the average of each group of light absorption value.Detected result is shown in Figure 13 and Figure 14.The processing mode of Human colorectal carcinoma LoVo cell is identical with people liver cancer SMMC7721 cell.

MTT experiment shows: after cytotostatic is expressed, compared with blank group and negative control group, experimental group SMMC7721 cell and Human colorectal carcinoma LoVo cell occur significantly breeding phenomenon (P<0.005, P<0.005), process LAN is described c2orf68the propagation that obviously can promote human liver cancer cell SMMC7721 of gene.

Embodiment 5: transfection c2orf68gene after impact on human hepatoma cell strain SMMC-7721 apoptosis situation

Flow cytometry is adopted to detect the apoptosis situation of SMMC-7721 cell.To be in the SMMC-7721 cell strain of growth index phase, cultivate in 6 orifice plates, after 24 h, Tissue Culture Plate fraction of coverage about reaches 70% ~ 80%, then carries out transfection, and step is with the step 1 in embodiment 2.Often kind of cell strain arranges three group: experimental group SMMC7721 respectively ^{+ CY- c2orf68} , the negative control group SMMC7721 of transfection empty carrier ^-NC.After transfection 24h, collecting cell, on whizzer with 2000r/min centrifugal 5 minutes, abandon substratum, adjustment cell concn was 3 × 105/pipe.PBS washed cell twice, with 400 μ L binding buffer liquid (triumphant base is biological) re-suspended cell, adds the triumphant base biology of 5 μ L Annexin V-FITC(), mixing, room temperature reaction 10 min, then adds 5 μ L Propidium iodide(PI) (triumphant base is biological), 4 DEG C of lucifuges hatch 30min.Detect with flow cytometer (BD FACSAria II Cell Sorter, the U.S.) and analyze, each sample collection 10000 fluorescent signals.The processing mode of people's Human colorectal carcinoma LoVo cell is identical with people liver cancer SMMC7721 cell.

Detected result is shown in Figure 16 and Figure 18, transfection c2orf68gene after to SMMC-7721 human hepatocarcinoma cell ^{+ CY- c2orf68} , negative control cell strain SMMC7721 ^-NC, the apoptosis rate of blank group SMMC7721 is respectively 7.5 ± 0.43,12.9 ± 0.19 and 11.3 ± 0.64%, transfection c2orf68gene after to LoVo ^{+ CY- c2orf68} , negative control cell strain LoVo ^-NCthe apoptosis rate of blank group LoVo is respectively 6.3 ± 0.42,13.1 ± 0.22 and 12.7 ± 0.32%, find that the apoptosis rate of experimental group is significantly higher than negative control group and blank group (P<0.01 by T inspection, P=0.01), the apoptosis rate no significant difference (P>0.05) of negative control group and blank group.Experimental result shows, c2orf68the expression of gene obviously can reduce the apoptosis of people liver cancer SMMC7721 cell and Human colorectal cancer cells LoVo.

1

4283

DNA

People

 

1

gcggagctct gtgggcgggt ggctgttgtt ggggccgtcg aggcggcggc gactctgcgt 60

ccccggctcc tg atggaggc ggggccgcat ccccggccgg ggcactgctg caagcctggg 120

gggcggctgg acatgaacca cggcttcgtg caccatatcc gacggaacca gatcgctcgg 180

gacgactatg acaagaaggt gaagcaggcg gccaaggaga aggtgaggag gcggcacacg 240

cccgcgccga cgcggccccg caagccagac ctgcaggtgt acctgccgcg acaccgagat 300

gtctctgccc acccacgcaa cccagactat gaagagtccg gtgaaagcag cagtagtgga 360

ggctctgagc tggagccttc tggccatcag ctcttctgct tagaatacga ggcagacagt 420

ggagaggtca catcagttat cgtctatcag ggtgatgacc caggaaaggt gagtgagaag 480

gtgtcggcac acacgcctct ggatccaccc atgcgagaag ccctcaagtt gcgtatccag 540

gaggagattg caaagcgcca gagccaacac tgaccatgtt gaaggcgttc tctccaggct 600

ggattcactg cactcggaag aattctgccc agggaattta gtgtgggggt accaggacca 660

gtttgtctga tcttgagacc cccagagctg ctgcatccat agggtgttgc aggactacac 720

ctggcctgcc ttgcagtcat tctttcttat atgttgaccc atttgcccag cctgatattc 780

tggaattctt tttttttttt tttttttttt ttttttgagg attttgtgtg gctaatgtgt 840

tctagaagca gagactccag ggagaaccag aatttatgaa gcctcgtgca acatggtgct 900

ttctcaccga ggtcatatgc ctggctgctg ctgttccact cagctccatg agccacgttt 960

gttattttat gtttcttctg tgcttttgct catttccacc catgtgttta tagacctttt 1020

ttcagccctt tttctttgtt cctttccctc atctttttgc ctcaggtaga atccatcagt 1080

tttcctcccc ctccaaatga ctgtgtacct ccagctgctc aggactttgg aggtgggggc 1140

ggggcctggg gatctaccct tctaaaaaac tctccaagta attctgatac caataaagtg 1200

ggagacctcc atttttgagg cctatttgca agcatgtctc cctccctttc catggctttt 1260

tcgtttctcg catgttccac tgctttgcac tgtctagaca ggaaacctag gaagatgttg 1320

gtgctaaact gaaaagattt cttacagcaa aacctgcctc ggccagggct acagaattcc 1380

aactttcaac ttgattccta gccgtgaaca gactgtcctc tgctcagact aaaagggaat 1440

tataaacaag tgggaactta ctctccagtt gtgacttaac agttctgaac atacctacaa 1500

agggaaagtc aatattagca ataattgcct gattgacagc acgccagagg gaatgaaatg 1560

aggccttctc tcctacctcc caggagtatg taactcagat ccttagcata aagatcccca 1620

ggaataccac tgctctaacc cggtgaacat tttttctaat gcagggttag gcttacgtct 1680

gaattccaca agacatcctc cccctccagt acggaagttc caaggcactt gttttccagc 1740

atatcagcct aacctcagtg ccttgaaata tggctttaag cctttgagaa ctgagatttc 1800

ctgaaaccat aggcccttgc cccaggggtt tctccacatc cgggtgttaa gacacctgat 1860

ggcactgttg gtttgtcccc tataccccag aaaatctatc ctgcaaggta gctacttcaa 1920

tcttgtcatt aaaatgtgtc aagtcacagc tcgcaatgcc aaaggaatgc tggggcagta 1980

agtgaggtgg ataagtgaga agggcctggt ggtgaaagcg gccagggacc agaatgctcc 2040

agacctacag agctggtcaa ggttaagtgc cttaaactta ccaatcctgg gctcagttct 2100

cctttgaaag gagaaagtct ttgtcctcta cttaggcagc tgggctagaa gtgccttttg 2160

acttctaatg ttaactaccc tccaaagcct cctgggtcaa gaaggctctc ccaaactcca 2220

cccctgttct tcctggtcag agaaccagtc agtcattcta gtcttctagt ccttaaactg 2280

atctgatgac ttggaacata ggatttcact gcaagtctgg ctttttagtc tggaaataca 2340

tttgaggtct gttttcgcac agctggatca cctgtttcct ggttttatta cacaattcag 2400

aaggctagaa gaggagtttg ggggcttggc acctgaaaat ttagaccgca ttcttattaa 2460

gtaaaggaag aggtagggag gccgaggcgg gcggatcatg aaatcaggag atccagaccg 2520

tcctggctaa cacagaaacc ccgtctctac taaaaataca aaaaattagc tgggcatggt 2580

gacatgtgcc tgtagtccat cccagctact cgggaggccg aggcaagaga atcacctaaa 2640

cctggaaggc ggaggttgca gtgagccgag atcacaccac tgcacttcag gctgggcagc 2700

agagcgagac tccatctcaa aaaaaaaaaa aaaaggaaga ggtaaaaggc aaggcagcat 2760

ttaataagta cctgttgtat ccttttaagt gtttgttgtg gtaatcctca caaagaccgg 2820

gactgatgga aactccttgc tattaaactt tttttcttga ggaattttgc ttttcaagtg 2880

catatacact attaatattt tttacccaag agagcattct aagctaattt atgcagtgtg 2940

actgtattaa gcattaagct tccttcagag ctggcctatc ggagatgcta ctgccctctc 3000

tacagatgtg tctgaaatgc ctgcccaagg atggccctta gccagttaac agctttatag 3060

cccatcctca ttgcttactg ccacccctca gctggggtcc aaggcagtac tattcagttt 3120

attcaccaga cctgcctcca gacatctact tctttcaaaa attagtgttt tccatcaagg 3180

agcatgttcc agagcatttc ccagagatgt cccaaagaac actgtccggt gctgtggcgt 3240

acagtggcaa cagcattaga ctaagtggaa catcccagca ggctgcttta gaatccgctc 3300

atttgactag atacgatgta attggctgtc tttaaaaaac gcgcacacac acacaatctg 3360

ataggcatat ctcatgccca ttcaatatgg aatgttcttc gcttgctgaa tttaagcctg 3420

tattttaagg ttttgtggtt cctcggccac aatgggtgat gtcactgata gaacgaagct 3480

gagtttccaa gggtttgggg ctgtgcaaga gtaaacacta gagcttgagt tgttatccag 3540

ctggcaagca cggaagtctt tgaagaatgt aatgtaaaaa gggaaaagaa tgtaaagctt 3600

tttgtaccaa atgagagttg gagcccagcc aacaaatgct tttccctgtg taaaagtctc 3660

tctggaaggg acattccatc tccatggtgc actctgaggg gcactgtcaa ctagagattg 3720

gccccatcca ggtgggagga acccctttgg gatggtgagt atccaatctg ctgtgcattt 3780

gacaggatct ctgaatggct aggtaatgga tcccaagcag gctcacaaat ttaaatgagg 3840

gctttgtgtg cagaaagagg aataagtaca gattattttc ctaccactag atttttgggg 3900

agagtcacca tggaatgttg acaattactt aaaatatttt aagctccctt gctgaattcc 3960

tgtcctgtcc ctgaggaatc agatggtcat acagccatag gcacccaccc gaaatttccc 4020

taggagttgg agtaatgcta gaattgaaga ccttctgagt aaagggcttc tctgccttct 4080

cagaggcagg agaatttgca ctggttgtgt taaatgtata aaaagctata tgttcaccag 4140

tttactcatt tccaatgtgt agatgaataa aatgtagtgt acaaattatt tgaaaatccc 4200

agaaggaagg tacttttcaa atacagtatt ttttttaaca aataaactta cgatttttac 4260

agcacaaaaa aaaaaaaaaa aaa 4283

2

501

DNA

People

 

atggaggc ggggccgcat ccccggccgg ggcactgctg caagcctggg gggcggctgg 130

acatgaacca cggcttcgtg caccatatcc gacggaacca gatcgctcgg gacgactatg 190

acaagaaggt gaagcaggcg gccaaggaga aggtgaggag gcggcacacg cccgcgccga 250

cgcggccccg caagccagac ctgcaggtgt acctgccgcg acaccgagat gtctctgccc 310

acccacgcaa cccagactat gaagagtccg gtgaaagcag cagtagtgga ggctctgagc 370

tggagccttc tggccatcag ctcttctgct tagaatacga ggcagacagt ggagaggtca 430

catcagttat cgtctatcag ggtgatgacc caggaaaggt gagtgagaag gtgtcggcac 490

acacgcctct ggatccaccc atgcgagaag ccctcaagtt gcgtatccag gaggagattg 550

caaagcgcca gagccaacac tga 573

Claims (7)

1. an eukaryotic expression recombination plasmid, is characterized in that: its c2orf68gene C Y-c2orf68 fragment and eukaryon expression plasmid recombination to construct.

2. the deoxynucleotide sequence of its aim sequence is as shown in SEQ ID NO.2, is positioned at c2orf68gene 73rd ~ 573.

3. eukaryon expression plasmid according to claim 1, is characterized in that its carrier for expression of eukaryon is following one wherein: pcDNA3.1(+), pcDNA3.1(-), GPU6, pGPU6/GFP/Neo, Pgpu6/Hygro, Pgph1/GFP/Neo, CMV-RFP-MCS (MIR30)-SV40-Neomycin, CMV-EGFP-MCS (MIR30)-SV40-Neomycin.

4. a preparation method for eukaryon recombinant plasmid, as follows in preparation process of its feature:

(1) by the CY-of heminested PCR synthesis people c2orf68gene fragment;

(2) nucleotide fragments of above-mentioned acquisition is connected with eucaryon plasmid, the restriction enzyme site of this Insert Fragment is respectively Xho I and EcoR I, again described eukaryon recombinant plasmid is transformed in competence bacterial cell and cultivates, then filter out the clone of the correct eukaryon recombinant plasmid inserted;

(3) use alkaline lysis extracting eukaryon recombinant plasmid, utilize the eukaryon recombinant plasmid that restriction endonuclease map analysis is built, and carry out DNA sequence analysis.

5. recombinant plasmid according to claim 1, builds after stablizing human hepatoma cell strain and stable Human colorectal cancer cells strain, it is characterized in that at people liver cancer SMMC7721 ^{+ CY-c2orf68}in cell, c2orf68 gene expression dose experimental group is than blank group rising 45.4%(P<0.001), experimental group to rise 46.4%(P<0.001 than negative control group), process LAN c2orf68gene obviously can promote the propagation of human liver cancer cell SMMC7721, inhibited apoptosis.

After 6.LoVo cell transfecting, be characterised in that at Human colorectal carcinoma LoVo ^{+ CY-c2orf68}experimental group, negative control group and blank group in cell c2orf68gene relative expression levels be respectively 2.58,0.892 and 0.849(P<0.005).

7. a recombinant plasmid as claimed in claim 1, can be used for research people's liver cancer and Human colorectal carcinoma and other malignant tumour c2orf68gene function.