CN103865920A - Method for cloning unique micro-molecule polypeptide ES61 encoding gene of embryonic stem cell - Google Patents
Method for cloning unique micro-molecule polypeptide ES61 encoding gene of embryonic stem cell Download PDFInfo
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- CN103865920A CN103865920A CN201310753802.5A CN201310753802A CN103865920A CN 103865920 A CN103865920 A CN 103865920A CN 201310753802 A CN201310753802 A CN 201310753802A CN 103865920 A CN103865920 A CN 103865920A
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Abstract
The invention provides a method for cloning unique micro-molecule polypeptide ES61 encoding gene of an embryonic stem cell. The method comprises the following steps: (1) extracting total RNA of human embryonic chorion tissue, and reversely transcribing the total RNA to be cDNA; (2) designing a PCR amplification primer to amplify a target gene; (3) recycling and purifying a PCR product; (4) performing enzyme digestion and connecting transformation on the gene. By adopting the method, the ES61 encoding gene can be effectively cloned, the quantitative determination of ES61 polypeptide gene is facilitated, and foundation is laid for researching the function of the ES61 polypeptide gene.
Description
Technical field
The invention belongs to genetically engineered field, be specifically related to the cloning process of the peculiar micromolecule polypeptide ES61 of a kind of embryonic stem cell encoding gene.
Background technology
Human embryo stem cell (Embryonic Stem cell, ES cell) is at the early stage class cell existing of fetal development, has totipotency, can be divided into each histoorgan into organism; The RP4-792G4.2(NCBI number of logging in HG492132.1) gene is ncRNA (the non-coding RNA that finds that at present a specific expressed class in the multipotential stem cells such as ES cell can not translated protein, ncRNA), research is found, RP4-792G4.2 gene a kind of polypeptide being formed by 61 amino acid of can encoding, called after ES61 polypeptide, the nucleotide sequence of coding ES61 polypeptide is by 183 based compositions, 61 amino acid of encoding, there is typical secretion signal peptide sequence through bioinformatic analysis in this amino acid.
Still nobody's report, for the cloning process of this polypeptide ES61 encoding gene, has therefore been difficult to the detection by quantitative to this ES61 polypeptide gene, further to study the function of this polypeptide at present.
Summary of the invention
The object of the invention is the technical problem that will solve for above, a kind of effective cloning process for the peculiar micromolecule polypeptide ES61 of embryonic stem cell encoding gene is provided.
In order to realize above technical purpose, the invention discloses the cloning process of the peculiar micromolecule polypeptide ES61 of a kind of embryonic stem cell encoding gene, step is as follows:
(1) extract the total RNA of human villi, then total RNA reverse transcription is become to cDNA;
(2) design pcr amplification primer increases out by target gene;
(3) recovery of PCR product and purifying;
(4) gene enzyme is cut to connect and is transformed.
The method according to this invention, the concrete operations of described step (2) are: after ES61, add HA sequence label, XhoI and HindIII restriction enzyme enzyme sequence are added respectively in upstream and downstream primer two ends, and primer amplification fragment total length is 234bp; Target fragment clone is connected in PLEGFP-N1 eukaryote expression vector, and primer sequence is as follows:
ES61-F:5'-CGG
CTCGAGACCATGAACTCGCAGATGCCGCTC-3'
Coordinating following reaction system: PCR with above-mentioned primer is the total system of 50 μ l, specifically 5 × Phusion HF Buffer10 μ l, 2.5mM dNTPmix5 μ l, the each 2 μ l of upstream and downstream primer of 10mM, the Phusion archaeal dna polymerase 0.8 μ l of 2U/ μ l, human villi cDNA template 2 μ l, supply 50 μ l systems with aqua sterilisa, and reaction conditions is " touchdown PCR ": 95 ℃ of 5min denaturations, interior 98 ℃ of 10s sex change circulate, from 1 ℃ of annealing of 68 ℃ of each cycle down, 72 ℃ are extended 20s, 10 circulations; 60 ℃ of annealing, carry out 25 circulations; After PCR reaction cycle, 72 ℃ are continued to extend 7min, then 16 ℃ of preservations.
The method according to this invention, in described step (4), above-mentioned PCR product and PLEGFP-N carrier are carried out to double digestion with XhoI and HindIII enzyme, endonuclease reaction system is as follows: 10X Buffer4 μ l, DNA approximately 2 μ g, (10U/ μ is each 1 μ l l), and sterilizing deionized water is supplied 40 μ l, 37 ℃ of reaction conditionss, 1hr for restriction endonuclease.
The method according to this invention, in described step (4), ligation system is as follows: object fragment DNA2 μ l, PLEGFP-N1 carrier 0.5 μ l, T4 ligase enzyme 1 μ l, 10X T4Buffer1 μ l sterilizing deionized water is supplied 10 μ l, and 22 ℃ connect 30min.
The method according to this invention, in described step (4), step of converting is as follows: get connection product and be added in 100 μ lDH5 α competent cells, mix ice bath 30 minutes; Above-mentioned conversion fluid is placed in to 42 ℃ of water-baths 90 seconds, after taking-up, is placed in immediately ice bath and places 2-3 minute; Add wherein the LB substratum of 900 μ l37 ℃ preheatings, 150rpm, 37 ℃ of shaking culture 45 minutes; The centrifugal 5min of 2500rpm, siphons away supernatant liquor, stays 100 μ l to mix bacterium liquid, is added to containing on Kana microbiotic LB solid nutrient agar, with aseptic elbow glass rod cell is evenly coated with and is opened gently; After planar surface is dry, be inverted flat board, cultivate 12-16 hour for 37 ℃.
The cloning process of the encoding gene of the peculiar micromolecule polypeptide ES61 of embryonic stem cell provided by the invention has added HA albumen label after ES61 peptide coding gene, can effectively clone this ES61 encoding gene, facilitate the detection by quantitative of later ES61 polypeptide gene, laid a good foundation for study ES61 polypeptide gene function later.
Accompanying drawing explanation
Fig. 1 is PLEGFP-N1 eukaryote expression vector structure collection of illustrative plates.
Fig. 2 is ES61 polypeptide gene pcr amplification figure, wherein P: target stripe M:Marker2000.
Fig. 3 is ES61 peptide coding gene nucleotide order-checking part peak figure result.
Fig. 4 is ES61 polypeptide signal peptide analysis result.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment are only not used in restriction range of application of the present invention for the present invention is described.
The total RNA of 1 human villi extracts and is transcribed into cDNA
The extraction of 1.1RNA
(1) process the early stage of sample: after getting about 30mg tissue and milling with liquid nitrogen, put into 1.5ml EP pipe, add 0.5ml BioPure, mix 5-10 minute, make to organize abundant cracking.Cell sample does not need to mill, and directly carries out next-step operation.
(2) phase-splitting: add 100 μ l volumes chloroform (for BioPure cumulative volume 1/5), fully put upside down and mix, under room temperature, leave standstill 5 minutes, 12000rpm4 ℃ centrifugal 15 minutes, solution layering, careful absorption containing the supernatant of RNA moves on in a new centrifuge tube.
(3) RNA precipitation: add 1 μ lRNA coprecipitator in supernatant in the supernatant of drawing, mix, adding the Virahol 250 μ l(of equivalent is generally Virahol: Trizol=0.5:1 again), put upside down and mix for several times, room temperature leaves standstill 10min, 12000rpm4 ℃ centrifugal 10 minutes, in the pipe visible RNA in end precipitation, abandon supernatant.
(4) RNA cleans: in centrifuge tube, add 1ml70% ethanol (the fresh preparation of DEPC water), put upside down and mix, 7500rpm4 ℃ centrifugal 5 minutes.Abandon carefully supernatant.
(5) RNA dissolves: open wide the centrifugal mouth of pipe, drying at room temperature makes residual liquid volatilization for 5～10 minutes, after RNA is slightly dry, adds 10 μ l DEPC water dissolution precipitations, the detection that takes a morsel, and all the other can be stored in-70 ℃ or liquid nitrogen.
1.2 reverse transcription reaction
| Total RNA | 1μg |
| Random?Hexamer?Primer（0.2μg/ul） | 1μl |
| Water,nuclease-free | To12μl |
65 ℃ of reaction 5min,, be placed on immediately 2-3min on ice.
Sample adds following reaction system after cooled on ice:
| 5×Reaction?Buffer | 4μl |
| RiboLockTMRNase?Inhibitor | 1μl |
| Transcriptase(200U/μl) | 1μl |
| 10mM?dNTP?Mix | 2μl |
25 ℃ of reaction 5min are then hatched 60min in 42 ℃, process 5min deactivation reversed transcriptive enzyme for last 70 ℃.
The 2PCR amplimer target gene that designs and increase
Use primer premier5.0 software design PCR primer; after ES61, adding HA sequence label draws two-wire and marks; upstream and downstream primer two ends are added respectively XhoI and HindIII restriction enzyme enzyme sequence and are drawn single line and mark, and primer amplification fragment total length is 234bp; Target fragment clone is connected in PLEGFP-N1 eukaryote expression vector, and as shown in Figure 1, primer detailed sequence is as follows for carrier structure collection of illustrative plates:
ES61-F:5'-CGG
CTCGAGACCATGAACTCGCAGATGCCGCTC-3'
After ES61, add HA sequence label (drawing two-wire marks), XhoI and HindIII restriction enzyme enzyme sequence (drawing single line marks) are added respectively in upstream and downstream primer two ends.
Pcr amplification reaction system and condition
Coordinating following reaction system: PCR with above-mentioned primer is the total system of 50 μ l, specifically 5 × Phusion HF Buffer10 μ l, 2.5mM dNTPmix5 μ l, the each 2 μ l of upstream and downstream primer of 10mM, the Phusion archaeal dna polymerase 0.8 μ l of 2U/ μ l, human villi cDNA template 2 μ l, supply 50 μ l systems with aqua sterilisa, and reaction conditions is " touchdown PCR ": 95 ℃ of 5min denaturations, interior 98 ℃ of 10s sex change circulate, from 1 ℃ of annealing of 68 ℃ of each cycle down, 72 ℃ are extended 20s, 10 circulations; 60 ℃ of annealing, carry out 25 circulations; After PCR reaction cycle, 72 ℃ are continued to extend 7min, then 16 ℃ of preservations.
PCR has reacted and has got 2 μ l PCR products and carry out 1.5% agarose electrophoretic analysis, and result as shown in Figure 2.
The purifying of 3PCR product and recovery
Carry out purified product according to gel-purified test kit specification sheets, reclaim fragment.
4 enzymes are cut, are connected and transform
Above-mentioned PCR product and PLEGFP-N carrier are carried out to double digestion with XhoI and HindIII enzyme, endonuclease reaction system is as follows: 10X Buffer4 μ l, and DNA approximately 2 μ g, (10U/ μ is each 1 μ l l), and sterilizing deionized water is supplied 40 μ l for restriction endonuclease.37 ℃ of reaction conditionss, 1hr.
Enzyme cuts back to close purifying
Enzyme is cut rear recovery purifying enzyme and is cut product.Carry out purified product according to purification kit specification sheets.
Connect
Ligation system is as follows:
The about 150ng of object fragment DNA2 μ l(), the about 50ng of PLEGFP-N1 carrier 0.5 μ l(), T4 ligase enzyme 1 μ l, 10X T4Buffer1 μ l sterilizing deionized water is supplied 10 μ l, and 22 ℃ connect 30min.
Transform
(1) get connection product and be added in 100 μ l DH5 α competent cells, mix ice bath 30 minutes.
(2) above-mentioned conversion fluid is placed in to 42 ℃ of water-baths 90 seconds, after taking-up, is placed in immediately ice bath and places 2-3 minute.
(3) add wherein LB (containing the microbiotic) substratum of 900 μ l37 ℃ preheatings, 150rpm, 37 ℃ of shaking culture 45 minutes.
(4) the centrifugal 5min of 2500rpm, siphons away supernatant liquor, stays 100 μ l to mix bacterium liquid, is added to containing (antibiotic concentration 100 μ g/ml) on Amp microbiotic LB solid nutrient agar, with aseptic elbow glass rod cell is evenly coated with and is opened gently.After planar surface is dry, be inverted flat board, cultivate 12-16 hour for 37 ℃.
Clone is identified in 5DNA order-checking
Single bacterium colony on the random above-mentioned flat board of picking, carries out whether success of DNA sequencing identified gene clone, and line sequence is to be inserted into target sequence ES61 in PLEGFP-N1 carrier and HA sequence label wherein to draw two-wire be HA sequence label; Line sequence two ends are the sequence on PLEGFP-N1 carrier, and sequencing sequence and peak figure result are as shown in Figure 3.
Sequencing result confirms ES61 peptide coding gene clone success.
GCTAGCGCTACCGGACTCAGATCTCGAGACC
ATGAACTCGCAGATGCCGCTCAGGGTCAGCTTCTTCTGCGGGCTC TGCAGGATGGCCATGGTGATGAGCGCGATGTACGAGTGTGGACAAATCCTCCAAGATTTAACTTCCAAGAAAACCGGCGG
6ES61 polypeptid acid sequence information and signal peptide analysis
The amino acid composition sequence that ES61 is detailed is as follows:
MNSQMPLRVSFFCGLCRMAMVMSAMYECGQILQDLTSKKTGGGAGGGGEDVWPMHAECKAA
ES61 polypeptide signal peptide analysis
Whether there is secretion property signal peptide through SignalP4.1 software analysis ES61 polypeptide.Result shows that ES61 polypeptide exists typical secretion property signal peptide, and 1-23 amino acid is the signal peptide sequence of ES61 polypeptide, and analytical results as shown in Figure 4.
Claims (5)
1. a cloning process for the peculiar micromolecule polypeptide ES61 of embryonic stem cell encoding gene, step is as follows:
(1) extract the total RNA of human villi, then total RNA reverse transcription is become to cDNA;
(2) design pcr amplification primer increases out by target gene;
(3) recovery of PCR product and purifying;
(4) gene enzyme is cut, connects, is transformed.
2. method according to claim 1, the concrete operations that it is characterized in that described step (2) are: after ES61, add HA sequence label, XhoI and HindIII restriction enzyme enzyme sequence are added respectively in upstream and downstream primer two ends, and primer amplification fragment total length is 234bp; Target fragment clone is connected in PLEGFP-N1 eukaryote expression vector, and primer sequence is as follows:
ES61-F:5'-CGG
CTCGAGACCATGAACTCGCAGATGCCGCTC-3'
Coordinating following reaction system: PCR with above-mentioned primer is the total system of 50 μ l, specifically 5 × Phusion HF Buffer10 μ l, 2.5mM dNTPmix5 μ l, the each 2 μ l of upstream and downstream primer of 10mM, the Phusion archaeal dna polymerase 0.8 μ l of 2U/ μ l, human villi cDNA template 2 μ l, supply 50 μ l systems with aqua sterilisa, and reaction conditions is " touchdown PCR ": 95 ℃ of 5min denaturations, interior 98 ℃ of 10s sex change circulate, from 1 ℃ of annealing of 68 ℃ of each cycle down, 72 ℃ are extended 20s, 10 circulations; 60 ℃ of annealing, carry out 25 circulations; After PCR reaction cycle, 72 ℃ are continued to extend 7min, then 16 ℃ of preservations.
3. method according to claim 1, it is characterized in that: in described step (4), above-mentioned PCR product and PLEGFP-N carrier are carried out to double digestion with XhoI and HindIII enzyme, endonuclease reaction system is as follows: 10X Buffer4 μ l, DNA approximately 2 μ g, (10U/ μ is each 1 μ l l), and sterilizing deionized water is supplied 40 μ l, 37 ℃ of reaction conditionss, 1hr for restriction endonuclease.
4. method according to claim 1, is characterized in that: in described step (4), ligation system is as follows: object fragment DNA2 μ l, PLEGFP-N1 carrier 0.5 μ l, T4 ligase enzyme 1 μ l, 10X T4Buffer1 μ l sterilizing deionized water is supplied 10 μ l, and 22 ℃ connect 30min.
5. method according to claim 1, is characterized in that in described step (4), step of converting is as follows: get connection product and be added in 100 μ lDH5 α competent cells, mix ice bath 30 minutes; Above-mentioned conversion fluid is placed in to 42 ℃ of water-baths 90 seconds, after taking-up, is placed in immediately ice bath and places 2-3 minute; Add wherein the LB substratum of 900 μ l37 ℃ preheatings, 150rpm, 37 ℃ of shaking culture 45 minutes; The centrifugal 5min of 2500rpm, siphons away supernatant liquor, stays 100 μ l to mix bacterium liquid, is added to containing on Kana microbiotic LB solid nutrient agar, with aseptic elbow glass rod cell is evenly coated with and is opened gently; After planar surface is dry, be inverted flat board, cultivate 12-16 hour for 37 ℃.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994002602A1 (en) * | 1992-07-24 | 1994-02-03 | Cell Genesys, Inc. | Generation of xenogeneic antibodies |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994002602A1 (en) * | 1992-07-24 | 1994-02-03 | Cell Genesys, Inc. | Generation of xenogeneic antibodies |
Non-Patent Citations (4)
| Title |
|---|
| HARROW ET AL.: "HG492132.1", 《GENBANK》 * |
| HARROW ET AL.: "HG492132.1", 《GENBANK》, 23 October 2013 (2013-10-23), pages 1 - 2 * |
| 周军等: "CDH22基因克隆及其真核表达载体的构建与表达", 《中国热带医学》 * |
| 谢向阳等: "雌激素受体β表达载体的构建及其转录活性测定", 《生物技术通讯》 * |
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