CN101775400A - Rana chensinensis functional gene Rd-RNase2 sequence, construction method and amino acid sequence and application thereof - Google Patents
Rana chensinensis functional gene Rd-RNase2 sequence, construction method and amino acid sequence and application thereof Download PDFInfo
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Abstract
The invention discloses a rana chensinensis functional gene Rd-RNase2 sequence which is a nucleotide sequence shown in a sequence table SEQ ID No.1. The invention firstly extracts the genomic DNA of the rana chensinensis, designs auele specific primers and PCR amplified genes, and determines the gene sequence. After researching the database, the gene is determined as a novel gene, i.e. the rana chensinensis functional gene Rd-RNase2 sequence of the invention. Experiment proves that the protein of the rana chensinensis functional gene Rd-RNase2 sequence of the invention has application value on curing infectious diseases caused by antibiotic pathogenic bacteria, and the recombinant proteins of the invention has wide application value on preparing biomedicine for treating cancer cells.
Description
Technical field
The present invention relates to a kind of Rana temporaria chensinensis David function sequence and encoded protein sequence thereof, specifically a kind of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence and amino acid sequence coded thereof and its purposes.
Background technology
The cases of cancer of the annual new diagnosis in the whole world reaches 100,000,000, and per 100,000 philtrums, 250 people die from cancer, so beat cancer is one of the most great challenge of modern medicine.Although surgical operation can be cured resectable cancer, unresectable cancer can only adopt chemotherapy or radiotherapy.The subject matter that conventional medicine is used for the treatment of existence is, lacks selectivity, and toxicity reaches the immunogenicity of foreign protein strongly, causes side reaction through regular meeting, as temporary diarrhoea, feel sick, alopecia, easy infection.Sometimes the heart, lung, kidney, marrow are produced long-range effects.
Whether in close relations the existence of the apoptosis that medicine caused of most of degradation of dna and wild type p53 tumour containment thing is, and under the situation of p53 disappearance, the medicine of degradation of dna is invalid to tumour.Human at present most of tumour (especially parenchyma) can both make the p53 inactivation by variation, or produces p53 antibody.And, cause the medicine of dna damage often can make normal cell produce genovariation.
Therefore need medicine exploitation toxicity appropriateness, that the mankind have an immunotolerance to solve these problems.
RNases can cause necrocytosis by degradation of rna, is considered to a kind of toxin.Viable cell approximately contains 20 kinds of circumscribed and endoribonucleases, RNA is treated to mature form and rna regulation is transcribed.RNase participates in RNA metabolism, cell maturation, cell physiological death, promotes vasculogenesis, and the host defense function of RNA viruses is resisted in performance.
RNases enters cell when external source, and they may synthesize, cause apoptosis and cell killing by degradation of rna, weakening protein biology.
In fact, have been found that multiple rnase has cytotoxicity in experiment in vitro, but have no toxicity in the experiment in vivo.Typical case's representative of this family is ox pancreas rnase (RNase A), owing to be subjected to the effect of ubiquitous ribonuclease inhibitor (RI) in the mammalian body, does not possess antitumor action.And the rnase of the natural dimeric forms of from the ox seminal fluid, finding (BS-RNase), although with the amino acid sequence similarity of RNase A up to 80%, but be not subjected to the restraining effect of RI, all demonstrate powerful optionally antitumor action in the experiment in vivo and in vitro.BS-RNase not only acts on tumour cell, also has embryotoxicity, and spermatogenesis suppresses, immunosuppressive activity.EDN and ECP are the ribonuclease A superfamily members that derives from eosinophilic granulocyte, although sequence similarity has different functions.EDN has antiviral function, and ECP mainly shows powerful sterilization, parasiticidal function.Be separated to two kinds of rnase RNases A-1 and A-2 from tame chicken (Gallus gallus) white corpuscle, it is identical that the two has 81% aminoacid sequence, and the two all interacts with human plasma ribonuclease inhibitor (hRI).RNases A-2 has angiogenic activity and germicidal action simultaneously, and RNases A-1 does not have this two kinds of activity.Present discovers that the ribonuclease A superfamily member is the protein family of one group of tachytely, and the fine difference of rnase sequence causes the noticeable change of function in the different species.
The antineoplastic research of batrachia rnase of bibliographical information only is confined to the leopard frog, bullfrog and the Japanese rice field frog at present.The aminoacid sequence difference of different frog species rnases, the difference of structure causes the function difference.
In the Amphibians Rana, certified the earliest rnase is Onconase, be Ardelt in 1991 etc. from the R.pipiens ovum based on its antitumor action and isolating albumen.Illustrating of the complete amino-acid sequence of Onconase disclosed it and belonged to the ribonuclease A superfamily.Other three kinds of rnases are identified from ovum and the liver of the frog: bullfrog Rana catesbeiana (frog's egg rc-RNase, frog liver rc-L1) Japanese rice field frog Ranajaponica (frog's egg rj-lec).Huang in 1998 etc. at first isolate the cDNA of coding frog rnase, find mRNA only at the liver expression of the female frog, and final synthetic albumen is positioned in the frog's egg cell.Liao in 2000 etc. isolate cDNA sequence (the rc-01 rc-L1 that encodes of 5 coding bullfrog R.catesbeiana rnases from yellow particle extract of frog's egg and liver, rc-02, rc-03, rc-04, rc-06), Chen in 2000 etc. have found the cytotoxicity rnase (rp-rap) of sex-specific from R.pipiens.Calendar year 2001 Rosenberg etc. are template with bullfrog Rana catesbeiana genomic dna, adopt PCR obtained five new frog ribonuclease genes orders (rc-203, rc-204, rc-208, rc-212, rc-218).Show that the ribonuclease gene that derives from Rana selects the mode of differentiation fast to evolve by forward, hinted that this unique protein family may have new physiological function.
The most of pyrimidine bases specific ribonucleic acid enzymes that with the ribonuclease A are representative have four intramolecular disulfide linkage, and its position is very conservative.Frog albumen has kept three in four disulfide linkage of ribonuclease A, is bringing out existing new disulfide linkage near C.Although have similar three-dimensional structure to RNase A, the RNases that derives from Amphibians is not suppressed by people RI in experiment in vitro, and this sequence similarity owing to itself and RNase A is low.The lower fish in evolution status but are subjected to RI and suppress.
Isolated rnase Onconase enters cell by internalization to cause cytotoxicity from the leopard frog.Have been found that Onconase can reduce the size of animal in-vivo tumour, can strengthen the cell toxicant effect of several chemotherapeutics.But Onconase also has embryotoxicity, suppresses spermatogenesis, immunosuppressive activity and renal toxicity.Onconase enters clinical three phases experiment as the antitumour drug of the inoperable malignant mesothe of treatment.
From the ovum of the Japanese rice field frog, isolate a kind of rnase jSBL, the preferentially a large amount of various human and animals' of aggegation tumour cell, rather than normal red corpuscle, lymphocyte, or inoblast with agglutination activity.From the bullfrog ovum, also isolate rnase cSBL with the various tumour cell effects of aggegation.Rnase with agglutination activity can be discerned the concrete weave construction and the direction of the cell surface glycoprotein feature of a large amount of various tumour cells, and this provides an important basis diagnosis and a treatment for human cancer undoubtedly.
CSBL and jSBL albumen all show cytotoxicity to intravital multiple cancer cells, and wherein cSBL has been proved to be a kind of at the external chemotherapeutics that effectiveness is also arranged very much.In a relevant experimental study, the researchist injects the mouse peritoneum inner chamber with tumour cell, and beginning is treated part mouse wherein by doses every day after 24 hours.Result of study shows that the survival time of being treated mouse is significantly higher than not by the survival time of treatment mouse.
To studies show that cSBL carries out, the enzymic activity of cSBL is not subjected to the inhibition of human RNase enzyme inhibition factor, and in a single day they enter in the cell, and the interaction energy of its degraded tumour cell RNA, kill cancer cell is brought into play well, helps using as cancer therapy drug.
The frog has unique specific rnase of pyrimidine bases, and structural similitude is in the rnase of tortoise, lizard, chicken.But the rnase that derives from lizard is not found cytotoxicity.Although carried out a large amount of research, yet, be not that all experimental result is all consistent.Antitumor subject matter: immunogenicity, Cytotoxic selectivity, stability.
Summary of the invention
The objective of the invention is to additional the deficiencies in the prior art and propose a kind of rana chensinensis functional gene Rd-RNase2 sequence.
Another purpose of the present invention is to propose a kind of construction process of above-mentioned rana chensinensis functional gene Rd-RNase2 sequence.
Another purpose of the present invention is to propose the aminoacid sequence of above-mentioned Rana temporaria chensinensis David functional gene Rd-RNase2 sequence and is substituted, lacks or added one or several amino acid whose and identical with this aminoacid sequence function amino acid derived sequence by this aminoacid sequence.
A further object of the present invention is to propose a kind of purposes of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence.
For achieving the above object, invention thinking of the present invention is:
The Anticancer Effect and Mechanism of Onconase is based on its internalization and enters tumour cell, degradation of rna, arrestin matter synthetic, inducing apoptosis of tumour cell.The cell that is in logarithmic phase is behind Onconase drug effect 24h, and cell proliferation stops, and the not effect of cell to being in lag phase.This may be relevant with cell recognition, and under drug effect dosage, Onconase only acts on tumour cell, and to the not influence of normal grown cell.Except the action target spot different with chemicals, the susceptibility that improve tumour cell oxygen pressure, resistant cell is recovered to medicine is another factor that Onconase reduces solid tumor, also is and the synergistic reason of other medicines.In solid tumor, the increase of iuntercellular hydrodynamicpressure often causes the conveying obstacle of antitumor drug: Onconase induced tumor capillary blood vessel pressure raises, and blood flow is accelerated, and the tumor vessel oxidative pressure is significantly increased.Therefore it may become new radiopotentiator, secures permission about research in the body of its radioreaction.The gene of the present invention's clones coding Onconase sample rnase maturation protein from the genome of Rana temporaria chensinensis David finds the rnase with Onconase sample anti-tumor activity, as the application of preparation treatment knubble biological medicine.The present invention is with a kind of new Onconase sample gene order Rd-RNase2 of preparation among the Rana temporaria chensinensis David DNA, serves as basic as novel, the application of anti-tumor function bio-pharmaceutical efficiently of preparation with it, has important pharmaceutical developments using value.
The concrete technical scheme that the present invention adopts is:
A kind of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence, it is the nucleotide sequence shown in the SEQ ID No.1 in the sequence table, and is specific as follows:
The mature protein coding region gene order
caagactgga?aaacatttca?gaagaagcac?ctgacaaaag?cccgggatat?taaatgtgat 60
aatatcatgt?caaaaacctt?gttcaactgc?aaagacacaa?acacttttat?cttttcactt 120
cctgggccag?ttaaggccct?ctgtagagga?attaaagtct?ccaaaaatgt?gttaagtcgt 180
tcagagtttg?atctctctga?gtgcaatgta?aaatccaagc?cctgcaagta?taaattaaag 240
aaaaaaagtg?atggaatttg?tataacatgt?agggatgaag?ctccggtaca?ttttgtcggt 300
gtcggaagtt?gc 312
A kind of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence amino acid sequence, it is the aminoacid sequence shown in the SEQ IDNo.2 in the sequence table, and is specific as follows:
Obtain the mature protein coding region protein sequence by the gene order derivation
Gln?Asp?Trp?Lys?Thr?Phe?Gln?Lys?Lys?His?Leu?Thr?Lys?Ala?Arg?Asp
1 5 10 15
Ile?Lys?Cys?Asp?Asn?Ile?Met?Ser?Lys?Thr?Leu?Phe?Asn?Cys?Lys?Asp
20 25 30
Thr?Asn?Thr?Phe?Ile?Phe?Ser?Leu?Pro?Gly?Pro?Val?Lys?Ala?Leu?Cys
35 40 45
Arg?Gly?Ile?Lys?Val?Ser?Lys?Asn?Val?Leu?Ser?Arg?Ser?Glu?Phe?Asp
50 55 60
Leu?Ser?Glu?Cys?Asn?Val?Lys?Ser?Lys?Pro?Cys?Lys?Tyr?Lys?Leu?Lys
65 70 75 80
Lys?Lys?Ser?Asp?Gly?Ile?Cys?Ile?Thr?Cys?Arg?Asp?Glu?Ala?Pro?Val
85 90 95
His?Phe?Val?Gly?Val?Gly?Ser?Cys
100
The derived sequence of above-mentioned a kind of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence amino acid sequence, its be by the aminoacid sequence shown in the SEQ ID No.2 in the sequence table be substituted, lack or add one or several amino acid whose and with sequence table in the identical aminoacid sequence of aminoacid sequence function shown in the SEQ ID No.2.
The construction process of above-mentioned a kind of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence, its concrete steps are: the genomic dna that extracts Rana temporaria chensinensis David, the design gene-specific primer, carry out pcr amplification, the product electrophoresis, described gene-specific primer is the nucleotide sequence shown in SEQ ID No.3 and the SEQ ID No.4 in the sequence table, for positive anti-primer right:
Forward primer N275:TAT TTG CAG TTG TTT TGA GTC TCA C
Reverse primer C715:CTG CTT ATC ACA TCC CTG TTG TC
From running gel, reclaim the target dna fragment, be connected, transfer to cell, extract plasmid with plasmid, behind the PCR preliminary evaluation target gene, order-checking.
The construction process of above-mentioned a kind of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence amino acid sequence, wherein, described PCR reaction system and parameter:
Genomic templates 100ng, dNTPs (10mM) 1 μ L, 10 * Taq Buffer, 5 μ l, Taq DNAPolymerase 3U and primer (N275, C715) each 30pmol, benefit aseptic ultrapure water to cumulative volume are 50 μ l, mix;
Amplification condition is: 94 ℃ of 30s, and 48 ℃ of 30s, 72 ℃ of 50s, 35 circulations, 72 ℃ are extended 7min and finish.
The construction process of above-mentioned a kind of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence amino acid sequence, wherein, the described concrete steps that are connected with plasmid are:
(1) target dna segment (20ng), pGM-T carrier (50ng), T4 DNA Ligase 3U, 10 * T4DNA Ligase Buffer, 1 μ l mend aseptic ultrapure water to cumulative volume 10 μ l;
(2) 16 ℃ connect 10～12 hours and are prepared into the gene that connects carrier;
(3) sample that step (2) is connected carries out the PCR detection; (T7, SP6) each 10pmol mend aseptic ultrapure water to cumulative volume 50 μ l, mix to connect product 1 μ l, dNTPs (10mM) 1 μ L, 10 * Taq Buffer, 5 μ l, Taq DNA Polymerase 3U and plasmid universal primer; The pcr amplification condition: 94 ℃ of 30s, 48 ℃ of 30s, 72 ℃ of 50s, 35 circulations, 72 ℃ are extended 7min and finish;
(4) go up the sample electrophoresis detection in the 2.5%TAE sepharose, in the uv analyzer, observe the DNA band.
Above-mentioned a kind of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence construct method, wherein, the described concrete steps of transferring to cell are:
(1) product (recombinant plasmid pGM-RNase2) that 5 μ l (50ng) target genes are connected with plasmid joins among the 100 μ l competent cell DH5 α, mixing, ice bath 30min;
Heat shock 90s in (2) 42 ℃ of water-baths; Place ice bath 2min after the taking-up immediately;
(3) add 500 μ l, 37 ℃ of good not containing in the antibiotic LB substratum of preheating in advance, 37 ℃, the 150rpm shaking table is cultivated 45min;
(4) the centrifugal 2min of 4000rpm abandons the part supernatant liquor, gets 100 μ l after concentrating and is coated with the conversion flat board, smoothens, and drying is inverted flat board, and 37 ℃ of thermostat containers are cultivated 12～16h;
(5) good, the medium sized white colony of picking separation is inoculated into and contains in the antibiotic LB liquid nutrient medium, and 37 ℃ of shaking tables, 180rpm cultivate 12～16h.
In the construction process of above-mentioned a kind of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence, PCR reaction system and parameter that described extraction plasmid PCR identifies are:
Recombinant plasmid (pGM-RNase2) 1 μ l, dNTPs (10mM) the 0.5 μ L, 10 * Taq Buffer2 μ l, Taq DNA Polymerase 1.5U and the plasmid universal primer (T7 that extract, SP6) each 5pmol, mend aseptic ultrapure water to cumulative volume 20 μ l, mix;
The pcr amplification condition: 94 ℃ of 30s, 48 ℃ of 30s, 72 ℃ of 50s, 35 circulations, 72 ℃ are extended 7min and finish.
A kind of amplimer of described Rana temporaria chensinensis David functional gene Rd-RNase2 sequence is the nucleotide sequence shown in SEQ IDNo.3 in the sequence table and the SEQ ID No.4, for just anti-primer is right.
The application of a kind of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence in preparation treatment antitumor drug that above-mentioned Rana temporaria chensinensis David is above-mentioned.
Advantage of the present invention and benefit:
The present invention at first extracts the genomic dna of Rana temporaria chensinensis David, the design Auele Specific Primer, and the pcr amplification gene is measured gene order.Through database retrieval, be defined as the new gene of Rana temporaria chensinensis David rnase.
Whether wood frog functional gene Rd-RNase2 sequence of the present invention has nothing to do, and the stability of this gene is better compared to prior art through the tumour cell aggegation of rnase mediation with to the selective killing effect of tumour cell and the existence of wild type p53 tumour containment thing.This gene expression research shows that the protein of this genes encoding has cytotoxicity, can suppress the growth of tumour cell, meets the condition for preparing antitumor drug.In pressing down the knurl experiment as can be seen, Rd-RNase2 gene recombinant fusion protein of the present invention has very powerful lethal effect to tumour cell under extremely low concentration, therefore but normal cell is not had lethal, have very big advantage than existing genomic medicine aspect the preparation antitumor drug.And recombinant protein Rd-RNase2 has good anti-oxidant function, and this is its important factor in order to tumour cell antiproliferative, cytotoxic activity.Because the redox state of cell influences all respects of cell function.The cytotoxic effect of many medicines is regulated by active oxygen.Oxidative stress can disturb the cancer chemotherapy, comprises that chemotherapeutic agents such as Zorubicin cause that Burkitt lymphoma cell apoptosis can be suppressed by catalase.Recombinant protein Rd-RNase2 has good effect aspect oncotherapy, and it is in the pathological conditions relevant with oxidative stress, has the potential of application as treatment of diseases aspects such as inflammation, autoimmune disease, atherosclerosis, septic shock, amyotrophic lateral sclerosis (spinal cord) lateral sclerosis.
The structure of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence of the present invention has very big instructive effect to further research RNases.The significant difference of aminoacid sequence and existing sequence, functional stronger, can be used as a kind of cytotoxin selectively, have important pharmaceutical developments using value.
The present invention will be further described below in conjunction with accompanying drawing and preferred forms, so that the public has whole to summary of the invention and understand fully, and is not qualification to protection domain of the present invention.Aforementioned part fully discloses the protection domain that the present invention can implement, and therefore allly any well known in the artly is equal to replacement according to what the disclosure of invention was carried out, all belongs to infringement of the present invention.
Description of drawings
Fig. 1 is target gene PCR amplification figure;
Fig. 2 is carrier joint detection figure as a result;
Fig. 3 is for transferring to transformation figure as a result;
Fig. 4 is plasmid pcr amplification detected result figure;
Fig. 5 does not have the figure of influence for the empty carrier abduction delivering to host's bacteria growing;
Fig. 6 has restraining effect figure to host's bacteria growing for Rd-RNase2 behind the IPIG abduction delivering;
Fig. 7 is the SDS-PAGE figure of intestinal bacteria lysate;
Fig. 8 detects the purity figure of recombinant protein behind the Affi-Gel purifying for SDS-PAGE;
Fig. 9 is that the recombinant protein Western blotting of intestinal bacteria lysate total protein and purifying detects figure;
Figure 10 is a recombination fusion protein to C6 spongiocyte oncocyte with to the growth-inhibiting action diagram of CHO-K1 cell;
Figure 11 recombinant protein Rd-RNase2 is to the influence of murine liver tissue mda content.
Embodiment
Specific embodiment of the invention material therefor and equipment source:
1, laboratory animal: Rana temporaria chensinensis David (Rana dybowskii) source: Huadian City, Jilin Province; Choice criteria: recognition feature: color of the leather mostly is Vandyke brown, the tripe yellow, and the slightly blunt circle of mouth end, the ear-drum limit has goat's horn to deceive scar, and there is " ∧ " shape blackspot at the nape place, and four limbs have belt blackspot.
2, used bacterial classification and carrier: competent escherichia coli cell DH5 α, pGM-T carrier (all purchasing Bioisystech Co., Ltd) in TIANGEN.
3, toolenzyme and biochemical reagents:
Sepharose DNA reclaims test kit, silica gel membrane type PCR product (dna segment) purification kit, the little extraction reagent kit of common plasmid, Tryptone, Yeast Extract, Agarose, penbritin, X-Gal, IPTG etc. (all purchasing Bioisystech Co., Ltd) in TIANGEN, dNTPs, T4DNA Ligase, λ DNA/HindIII, Taq DNA Polymerase, EcoR I, HindIII, 1kb DNA Ladder, DNA Marker I (all purchasing company) in TaKaRa, genome DNA extracting reagent kit, nucleic acid dye GoldView, 50bp DNALadder (purchasing) in match Parkson, Beijing Bioisystech Co., Ltd, other reagent is commercially available analytical pure.
4, equipment and equipment
TGL-16C type desk centrifuge, Anting Scientific Instrument Factory, Shanghai;
HQ-60 type vortex mixer, the north is with positive biotech development company;
Pcr amplification instrument 9700 types, Gene Amp company;
The desk-top constant temperature oscillator of THZ-D type, Taicang experimental installation factory;
UV-IV type uv analyzer, Beijing's new science and technology Applied Research Laboratory;
SPN202F type electronic balance, plum Teller-Tuo benefit Weight Equipment System Co., Ltd;
DYY-6C type electrophoresis apparatus Beijing Liuyi Instrument Factory;
DYCP-31DN type electrophoresis chamber, Liuyi Instruments Plant, Beijing;
The biochemical incubator of LRH-250 type, Shanghai one permanent Science and Technology Ltd.;
LS-835L type vertical pressure steam sterilizing pot Jiangyin Jiang Bin Medical Equipment Plant;
SWCJ-B type clean work station, ring experimental electric furnace company limited in the Tianjin;
DZKW type electronic thermostatic water-bath, Bright brand.
Embodiment 1: the concrete construction process of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence of the present invention:
1, design of primers
Gene-specific primer:
Forward primer N275:TAT TTG CAG TTG TTT TGA GTC TCA C
Reverse primer C715:CTG CTT ATC ACA TCC CTG TTG TC
2, the pcr amplification of target gene
(1) uses the poba gene group to extract test kit, from the wood frog fresh blood, extract genomic dna, 1% agarose electrophoresis detection molecules amount and concentration.
(2) in aseptic PCR pipe, add genomic templates 100ng, dNTPs (10mM) 1 μ L, 10 * Taq Buffer5 μ l, Taq DNA Polymerase 3U and primer (N275, C715) each 30pmol, benefit aseptic ultrapure water to cumulative volume are 50 μ l, mix;
(3) the PCR pipe that will add sample is put into preheating good PCR instrument is carried out the PCR reaction.The pcr amplification condition: 94 ℃ of 30s, 48 ℃ of 30s, 72 ℃ of 50s, 35 circulations, 72 ℃ are extended 7min and finish.
(4) after PCR finishes, in the 2.5%TAE sepharose on sample electrophoresis detection and cut the glue purification target gene.In the uv analyzer, observe the DNA band.
Through pcr amplification and agarose gel electrophoresis as shown in Figure 1, with Marker I is reference standard, in uv analyzer, can see about the 400bp position and (comprise the part signal sequence, exact value is 387bp) the bright fluorescence band of appearance, the proof goal gene increases successfully, can be used for ensuing carrier and connects.
3, the pGM-T carrier connects
(1) in aseptic PCR pipe, adds target gene segment (20ng), pGM-T carrier (50ng), T4DNALigase 3U, 10 * T4DNA Ligase Buffer, 1 μ l, mend aseptic ultrapure water to cumulative volume 10 μ l;
(2) 16 ℃ connect 10～12 hours and are prepared into the carrier that connects target gene;
(3) sample that connects being carried out PCR detects.(T7, SP6) each 10pmol mend aseptic ultrapure water to cumulative volume 50 μ l, mix to add connection product 1 μ l, dNTPs (10mM) 1 μ L, 10 * Taq Buffer, 5 μ l, Taq DNA Polymerase3U and plasmid universal primer in aseptic PCR pipe.
(4) the PCR pipe that will add sample is put into preheating good PCR instrument is carried out the PCR reaction.The pcr amplification condition: 94 ℃ of 30s, 48 ℃ of 30s, 72 ℃ of 50s, 35 circulations, 72 ℃ are extended 7min and finish.
(5) after PCR finishes, in the 2.5%TAE sepharose on the sample electrophoresis detection.In the uv analyzer, observe the DNA band.
The result that 16 ℃ of connections are spent the night carries out PCR and agarose electrophoresis detects as shown in Figure 2, in uv analyzer, is reference standard with Marker I, can see at the bright fluorescence band of 600bp position appearance, with the expected results basically identical, prove the carrier successful connection, can carry out subsequent experimental.
4, preparation transforms dull and stereotyped
(1) preparation LB and LB/Agar substratum
1. LB liquid nutrient medium preparation (500ml): Tryptone 5g, Yeast Extract 2.5g, NaCl 5g, add 400ml water, abundant stirring and dissolving, Dropwise 5 N NaOH (about 0.2ml), adjust pH to 7.0, add water again and be settled to 500ml (take out 200ml and be used to prepare solid medium, two bottles of 300ml packing, each 150ml).121 ℃, 0.1MPa, autoclaving 20min.Deposit for 4 ℃.
2. LB/Agar/Amp solid medium preparation: be ready to by the LB liquid nutrient medium, add Agar 3g/200ml, autoclaving before the autoclaving, be cooled to 50～60 ℃, on super clean bench to wherein adding microbiotic (penbritin) 0.2ml, mixing, fall dull and stereotyped (20ml/ plate), deposit for 4 ℃.
(2) on super clean bench, contain corresponding antibiotic LB/Agar/Amp solid culture primary surface and add 16 μ l IPTG (50mg/ml), 40 μ l X-Gal (20mg/ml) to completing, use the aseptic rod that is coated with to smoothen, place in 37 ℃ of thermostat containers of lucifuge after 1～3 hour and use.
5, transformed competence colibacillus cell DH5 α
(1) 5 μ l (50ng) connects product and joins in the 100 μ l competent cells mixing, ice bath 30min.
Heat shock 90s in (2) 42 ℃ of water-baths.Place ice bath 2min after the taking-up immediately.
(3) add 500 μ l, 37 ℃ of LB substratum (not containing microbiotic) that preheating is good in advance, 37 ℃, the 150rpm shaking table is cultivated 45min.
(4) the centrifugal 2min of 4000rpm abandons the part supernatant liquor, gets 100 μ l after concentrating and is coated with the conversion flat board, smoothens, and drying is inverted flat board, and 37 ℃ of thermostat containers are cultivated 16h.
Conversion results after 16h cultivates, grows blueness and two kinds of bacterium colonies of white through the goal gene that connects, transform as shown in Figure 3 on substratum, wherein white colony is required purpose bacterium, after detecting,, then can be used for liquid culture and plasmid and extract if the result is correct through bacterium colony.
6, extract plasmid, amplification and order-checking
(1) good, the medium sized white colony of picking separation is inoculated into and contains in the antibiotic LB liquid nutrient medium, and 37 ℃ of shaking tables, 180rpm cultivate 12h.
(2) extract plasmid with plasmid extraction kit.Detect plasmid with 0.8% agarose gel electrophoresis.
(3) PCR detects the insertion dna fragmentation in the recombinant plasmid.In aseptic PCR pipe, add recombinant plasmid (pGM-RNase2) 1 μ l, dNTPs (10mM) 0.5 μ L, 10 * Taq Buffer, 2 μ l, Taq DNAPolymerase 1.5U and the plasmid universal primer (T7 that extracts, SP6) each 5pmol, mend aseptic ultrapure water to cumulative volume 20 μ l, mix.The pcr amplification condition: 94 ℃ of 30s, 48 ℃ of 30s, 72 ℃ of 50s, 35 circulations, 72 ℃ are extended 7min and finish.2.5% sepharose detects.To cumulative volume 20 μ l, mix.
The target gene fragment is inserted as shown in Figure 4 on the pcr amplification detection plasmid, cultivate gained bacterium liquid through 12h and carry out the plasmid extraction, the gained plasmid is carried out PCR reaction and agarose electrophoresis detection, with Marker I is reference standard, can see at the bright fluorescence band of 600bp position appearance, with the expected results basically identical, it is correct to prove that plasmid extracts the result.
(4) with the plasmid order-checking of extracting.
The plasmid sample is by the order-checking of Beijing three rich polygala root biotechnology limited liability companys.Obtaining the mature protein coding region gene order is shown in the SEQ ID No.1, is specially:
caagactgga?aaacatttca?gaagaagcac?ctgacaaaag?cccgggatat?taaatgtgat 60
aatatcatgt?caaaaacctt?gttcaactgc?aaagacacaa?acacttttat?cttttcactt 120
cctgggccag?ttaaggccct?ctgtagagga?attaaagtct?ccaaaaatgt?gttaagtcgt 180
tcagagtttg?atctctctga?gtgcaatgta?aaatccaagc?cctgcaagta?taaattaaag 240
aaaaaaagtg?atggaatttg?tataacatgt?agggatgaag?ctccggtaca?ttttgtcggt 300
gtcggaagtt?gc 312
Obtain the mature protein coding region protein sequence shown in SEQ ID No.2 by the gene order derivation, be specially:
Gln?Asp?Trp?Lys?Thr?Phe?Gln?Lys?Lys?His?Leu?Thr?Lys?Ala?Arg?Asp
1 5 10 15
Ile?Lys?Cys?Asp?Asn?Ile?Met?Ser?Lys?Thr?Leu?Phe?Asn?Cys?Lys?Asp
20 25 30
Thr?Asn?Thr?Phe?Ile?Phe?Ser?Leu?Pro?Gly?Pro?Val?Lys?Ala?Leu?Cys
35 40 45
Arg?Gly?Ile?Lys?Val?Ser?Lys?Asn?Val?Leu?Ser?Arg?Ser?Glu?Phe?Asp
50 55 60
Leu?Ser?Glu?Cys?Asn?Val?Lys?Ser?Lys?Pro?Cys?Lys?Tyr?Lys?Leu?Lys
65 70 75 80
Lys?Lys?Ser?Asp?Gly?Ile?Cys?Ile?Thr?Cys?Arg?Asp?Glu?Ala?Pro?Val
85 90 95
His?Phe?Val?Gly?Val?Gly?Ser?Cys
100
Above-mentioned sequencing result and Genbank database are carried out sequence alignment, and the result shows that Rana temporaria chensinensis David functional gene Rd-RNase2 sequence of the present invention is new gene.
Embodiment 2: the Chinese woods functional gene Rd-RNase2 sequence that the present invention makes up and the biological experiment of aminoacid sequence
One, material and method
1, material
(1) bacterial classification and carrier
E. coli bl21 (DE3) competent cell is purchased the Bioisystech Co., Ltd in TIANGEN, and the pFLAG-CTS carrier is purchased in sigma-aldrich.
(2) toolenzyme and biochemical reagents:
Tryptone, Yeast Extract, Agarose, penbritin, X-Gal, IPTG etc. (all purchasing Bioisystech Co., Ltd) in TIANGEN, other reagent is commercially available analytical pure.
(3) equipment and equipment
Equipment
The desk-top constant temperature oscillator of THZ-D type, Taicang experimental installation factory;
SPN202F type electronic balance, plum Teller-Tuo benefit Weight Equipment System Co., Ltd;
The biochemical incubator of LRH-250 type, Shanghai one permanent Science and Technology Ltd.;
LS-835L type vertical pressure steam sterilizing pot Jiangyin Jiang Bin Medical Equipment Plant;
SWCJ-B type clean work station, ring experimental electric furnace company limited in the Tianjin;
2, experimental technique
The e. coli bl21 (DE3) that carries empty plasmid pFLAG-CTS or carry the recombinant plasmid pFLAG-RNase2 of Rana temporaria chensinensis David Rd-RNase2 gene coated on the LB/agar/Amp flat board activate, single bacterium colony of overnight growth on the picking LB/agar/Amp flat board, be inoculated in the 20ml LB/Amp substratum, 37 ℃, 180rpm shakes bacterium and spends the night.Add fresh LB/Amp substratum 20ml next day in 20ml bacterium liquid again, 37 ℃, 180rpm shakes bacterium to logarithmic growth (OD in mid-term
600=0.8), tell 20ml bacterium liquid in sterilizing and being preheated in 37 ℃ the triangular flask, adding final concentration is the IPTG abduction delivering of 20uM, does not add IPTG in the remaining 20ml bacterium liquid, induces contrast as non-, regularly detects OD
600, record IPTG induces in the expression of recombinant proteins process inhibition situation to host's bacteria growing.
3, experimental result
(1) the empty carrier abduction delivering does not have influence to host's bacteria growing, as shown in Figure 5, induce the Escherichia Coli BL21 (DE3) that carries empty plasmid pFLAG-CTS to express with 20uM IPTG after, growing state of host bacterium and induction phase ratio not do not have difference.As seen the abduction delivering of empty carrier does not influence the growth of host bacterium.
(2) in the recombinant plasmid pFLAG-RNase2 abduction delivering process host's bacteria growing had obvious restraining effect, as shown in Figure 6, after inducing EscherichiaColi BL21 (DE3) the express recombinant protein 30min that carries recombinant plasmid pFLAG-RNase2 with 20uM IPTG, the growth of obviously visible host bacterium is suppressed, inhibiting rate reaches 19% behind the 60min, induce 120min after inhibiting rate can reach 64%.
4, conclusion
Carry the e. coli bl21 (DE3) of the recombinant plasmid pFLAG-RNase2 of anti-tumor function gene Rd-RNase3 of the present invention, transcriptional start and expression Rd-RNase2 gene after adding inductor IPTG, the recombinant protein secretion stores to colibacillary pericentral siphon chamber.
Experimental result shows that inductor IPTG does not have restraining effect to the intestinal bacteria that have empty carrier, illustrates that the abduction delivering of empty carrier itself does not have cytotoxicity to intestinal bacteria.
Inductor IPTG has significant inhibitory effect to the intestinal bacteria that have Rd-RNase2 recombinant plasmid pFLAG-RNase2, illustrates that the abduction delivering of recombinant protein has significant cytotoxicity to intestinal bacteria.Especially it should be noted that the host bacterium that carries the pFLAG-CTS plasmid all is the antibiotic resistant organism of tolerance Amp, the cytotoxicity that recombinant protein has been described is stronger, these recombinant proteins might have using value aspect the infectious diseases that causes of treatment pathogenic bacteria of drug-resistant, have illustrated that equally also these recombinant proteins have using value in the treatment tumour cell.
Embodiment 3: the preparation of the recombination fusion protein of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence of the present invention
One, material and equipment
1, bacterial classification and carrier
E. coli bl21 (DE3) competent cell is purchased the Bioisystech Co., Ltd in TIANGEN, and the pFLAG-CTS carrier is purchased in sigma-aldrich.
2, toolenzyme and biochemical reagents
Tryptone, Yeast Extract, Agarose, penbritin, X-Gal, IPTG etc. (all purchasing Bioisystech Co., Ltd) in TIANGEN, mouse anti FLAG monoclonal antibody (purchasing company) in Sigma, the goat-anti mouse two that is connected with alkaline phosphatase is anti-, BCIP/NBT colouring reagents box (purchasing biotech firm of China fir Golden Bridge in Beijing), and other reagent is commercially available analytical pure.
3, equipment
The desk-top constant temperature oscillator of THZ-D type, Taicang experimental installation factory;
The biochemical incubator of LRH-250 type, Shanghai one permanent Science and Technology Ltd.;
LS-835L type vertical pressure steam sterilizing pot Jiangyin Jiang Bin Medical Equipment Plant;
Bechtop, Shanghai new talent medicine equipment Manufacturing Co., Ltd;
YC-1 chromatography cabinet, Beijing rich doctor health laboratory apparatus company limited;
High speed freezing centrifuge (SORVALL RC6PLUS), THERMO;
Freeze drier (FD-1B-50), inferior safe Cologne, Beijing experiment Science and Technology Development Center;
The supersonic cell crusher, NingBo XinZhi Biology Science Co., Ltd;
Ten thousand/electronic balance (AR2140), Ao Haosi company;
PH instrument (PHSJ-3F), Shanghai thunder magnetic
As seen-and ultraviolet spectrophotometer (UV-1700), SHIMADZU;
Vertical electrophoresis groove (DYC2-24D), Liuyi Instruments Plant, Beijing;
Electrophoresis apparatus (DYY-8C), Liuyi Instruments Plant, Beijing
Two, experimental technique
1, the cultivation of thalline and ultrasonic degradation
With carrying the thalline of recombinant plasmid, on the flat board of LB/Agar/Amp substratum, rule overnight incubation in 37 ℃ of thermostat containers.Inferior daily inoculating needle picking separates good single bacterium colony, inserts in the LB/Amp liquid nutrient medium, places shaking table, and in 37 ℃, 180rpm cultivated 12 hours.Be inoculated in the good LB/Amp substratum of preheating according to 5% inoculum size, cultivate 6L bacterium liquid altogether.The IPTG that adds final concentration when making bacteria growing to logarithmic phase (OD600=0.6～0.8) and be 20uM induces 4h.
Centrifugal collection thalline, PBS washing three times.Add broken damping fluid, ice-bath ultrasonic, cracking thalline.Centrifugal collection supernatant liquor.Resolving gel concentration is that 12% SDS-PAGE and western blot detect the target protein in the lysate.
2, Affi-Gel purification of Recombinant fusion rotein
Have FLAG label (its aminoacid sequence is EFPGTRSVDYKDDDDK) at the C of recombinant protein end, FLAG can not influence target protein and correctly be folded into three-dimensional structure, can not influence the activity of target protein yet.Can utilize mouse anti FLAG monoclonal antibody link coupled agarose Affi-Gel (purchasing company) to carry out purifying easily in sigma.Concrete grammar is: with cell pyrolysis liquid high speed frozen centrifugation (4 ℃, 20000rpm, 30min) after, get supernatant, hatch with Affi-Gel, 4 ℃, spend the night.It is centrifugal that (4 ℃, 1500rpm 2min), collects Affi-Gel.Wash glue with TBS, five times.In Affi-Gel, add 1ml glycine elutriant, jog 1min, centrifugal (4 ℃, 1500rpm 2min), gets supernatant, puts into a centrifuge tube that the 10ul neutralizer is arranged, and the co-elute Affi-Gel is 5 times in batches, collects 5 pipe eluted proteins.
3, test sample purity
The eluted protein that obtains is merged freeze-drying after water is fully dialysed.After lyophilized powder usefulness 100ul TBS dissolving, measure protein concn with the Bradford method, carry out SDS-PAGE, testing product purity, westernblotting identifies recombinant protein.
Three, experimental result
1, the detection of recombination fusion protein in the intestinal bacteria lysate
The intestinal bacteria that carry the recombinant plasmid pFLAG-RNase2 of Rd-RNase2 gene are cultured to the logarithm middle and later periods (OD600=0.6～0.8) in the LB/Amp substratum, the adding final concentration is after the IPTG of 20uM induces 4h, centrifugal collection thalline, ice-bath ultrasonic lysing cell behind the washing thalline, the lysate that obtains is carried out SDS-PAGE (resolving gel concentration is 12%), detect the band that recombination fusion protein is arranged in expection molecular weight position in the lysate.As shown in Figure 7, be the SDS-PAGE figure of intestinal bacteria lysate, among the figure: the Far Left swimming lane is the intestinal bacteria lysate, and middle swimming lane is a ribonuclease A, and the rightmost side is the low molecular weight protein (LMWP) standard.As seen from the figure, there is target protein to express at the about 13.7kDa of molecular weight place.
2, Affi-Gel purification of Recombinant fusion rotein Rd-RNase2
With cell pyrolysis liquid high speed frozen centrifugation (4 ℃, 20000rpm, 30min) after, get supernatant, the Affi-Gel overnight incubation with being connected with anti-FLAG antibody is adsorbed on the Affi-Gel target protein.After repeatedly washing Affi-Gel removal foreign protein, adopt branch's pickling to take off, target protein Rd-RNase2 is resolved from Affi-Gel.Rd-RNase2 recombinant protein behind the wash-out is neutralized to pH neutral with neutralizer immediately.The purity of electrophoresis detection recombinant protein Rd-RNase2.Result such as Fig. 8 show, are the purity figure of recombinant protein behind the SDS-PAGE detection Affi-Gel purifying, and as seen from the figure, being about 13.7kDa place recombinant protein at molecular weight is the homogeneous band.Adopt the Affi-Gel purifying to obtain the one-component of recombinant protein.
Respectively with the recombinant protein of intestinal bacteria lysate and purifying after the SDS-PAGE of 12% resolving gel concentration separates, be transferred on the pvdf membrane, use anti-FLAG mouse monoclonal antibody (Sigma respectively, 300 times of dilutions), two is anti----goat anti-mouse antibody that alkaline phosphatase connects (biotech firm of middle China fir Golden Bridge, 20 times of dilutions) and antigen-reactive, recombinant protein is identified in the BCIP/NBT colour developing, as shown in Figure 9, be the recombinant protein Western blotting detection figure of intestinal bacteria lysate total protein and purifying, swimming lane 1: intestinal bacteria lysate total protein; Swimming lane 2: the recombinant protein of purifying.Among the figure, the left side is the intestinal bacteria lysate, and the right is the pure product of Rd-RNase2.But in the identification of escherichia coli lysate total protein expression of recombinant proteins is arranged thus, through having obtained the pure product of recombinant protein behind the Affi-Gel purifying.
4, conclusion
This experiment will import the intestinal bacteria of recombinant plasmid and cultivate, preparation rnase Rd-RNase2 recombination fusion protein.Because rnase is an intracellular enzyme, at first carries out cytoclasis, supernatant liquor is crude enzyme liquid.Adopt the Affi-Gel separation and purification, obtain wood frog Rd-RNase2 recombination fusion protein, the SDS-PAGE electrophoresis detection is to the homogeneous band of molecular weight about about 13.7kDa.Western blotting method has been identified the Rd-RNase2 recombination fusion protein.Set up the preparation method of effective Rd-RNase2 recombination fusion protein.
Embodiment 4: the extracorporeal anti-tumor function research of the recombination fusion protein of Rana temporaria chensinensis David Rd-RNase2 gene
One, material and equipment
1, cell strain, substratum and biochemical reagents
Rat C6 glioma cell line is available from the neural department of microbiology of Harbin Medical University, and Chinese hamster ovary CHO-K1 cell is available from Wuhan University China typical culture collection center.DMEM high glycoform nutrient solution, RPMI1640 substratum are purchased in Gibco, and calf serum, penicillin, Streptomycin sulphate, MTT, trypan blue, DMSO purchase in ancient cooking vessel state Bioisystech Co., Ltd, and other reagent is commercially available analytical pure.
2, equipment
CO2gas incubator (Japanese Forma);
Microplate reader (Lab systems Dragon well scan MK3);
Inverted microscope (Nikon 808434);
Vertical pressure steam sterilizing pot (LS-835L type) Jiangyin Jiang Bin Medical Equipment Plant;
Clean work station (SWCJ-B type), Shanghai new talent medicine equipment Manufacturing Co., Ltd.
Two, experimental technique
1, cell cultures
C6 spongiocyte oncocyte and Chinese hamster ovary CHO-K1 cell grow in DMEM high glycoform nutrient solution and RPMI RPMI-1640 (Gibco company) respectively, including volume fraction is 10% calf serum (56 ℃ of deactivation 30min), 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, in being the incubator of 5%CO2,37 ℃, volume fraction cultivate, per 2～3d goes down to posterity once, take the logarithm vegetative period cell be used for the experiment.
2, the MTT experiment detects the restraining effect of recombination fusion protein Rd-RNase2 cell growth
Collect the logarithmic phase cell, use 0.25% trysinization, be mixed with cell suspension, count with blood counting chamber in microscopically, adjusting the cell starting point concentration is 1 * 104 cell/ml.100 μ l (every hole contains 1000 cells) are inoculated in every hole in 96 well culture plates.After cultivating 24h, the sucking-off nutrient solution, add the nutrient solution that contains different pharmaceutical concentration respectively by experimental design, with substratum preparation recombination fusion protein Rd-RNase2, if 5 concentration: 50 μ g/l, 25 μ g/l, 12.5 μ g/l, 6.25 μ g/l, 3.12 μ g/l, the blank group adds the equal-volume nutrient solution that does not contain medicine, establishes 3 parallel holes for every group, every hole application of sample 100 μ l.Putting 37 ℃, volume fraction is to discard nutrient solution after cultivating 48h in the 5%CO2 incubator, washes 2 times with nutrient solution, and to remove soup, every hole adds 0.5%MTT solution (RPMI 1640 preparations) 20 μ l.37 ℃ of insulation 4h abandon supernatant liquor, and every hole adds DMSO 200 μ l dissolving Formazan particle, dull and stereotyped shaker vibration 10min, and the 490nm wavelength detects each hole absorbance on microplate reader, draws the cell survival rate curve.
Three, experimental result
1, the MTT experiment detects the growth-inhibiting effect of recombination fusion protein pair cell, experimental result as shown in figure 10, be the growth-inhibiting action diagram of recombination fusion protein to C6 spongiocyte oncocyte and Chinese hamster ovary CHO-K1 cell, as seen from the figure, recombination fusion protein has remarkable restraining effect to the growth of C6 spongiocyte oncocyte, to CHO-K1 cell unrestraint effect.
Four, conclusion
Glioma is listed in the first place of lethality tumour in the central nervous system, from the mean time of diagnosing death only be 1 year, existing methods of treatment is difficult to receive satisfied curative effect, the medicine of therefore seeking the antagonism glioma is extremely urgent.
Recombination fusion protein has powerful vitro cytotoxicity for the C6 spongiocyte oncocyte that is tried under extremely low concentration, and presents tangible dose-dependent effect.To CHO-K1 cell unrestraint effect.As seen, recombination fusion protein has the cytotoxicity of selective killing tumour cell.Clinical treatment for tumour has important development and application values.
Embodiment 5: the recombination fusion protein antioxygenation of Rana temporaria chensinensis David Rd-RNase2 gene
One, material and equipment
1, laboratory animal and reagent
The ICR mouse, body weight 18～22g, male and female dual-purpose.Beijing Vital River Experimental Animals Technology Co., Ltd. provides.
Reagent such as thiobarbituricacid, trichoroacetic acid(TCA), ferrous sulfate, hydrogen peroxide are commercially available analytical pure.
2, equipment
Ten thousand/electronic balance (AR2140), Ao Haosi company;
High speed freezing centrifuge (SORVALL RC6PLUS), THERMO;
TGL-16C type desk centrifuge, Anting Scientific Instrument Factory, Shanghai;
DZKW type electronic thermostatic water-bath, Bright brand.
As seen-and ultraviolet spectrophotometer (UV-1700), SHIMADZU;
Two, experimental technique
Detect the effect of recombinant protein to Mouse Liver foundation of microsomal Lipid Peroxidation product mda.
1, the preparation of liver homogenate liquid: get mouse liver, weigh after cleaning with cold saline, 0.5% suspension is made in homogenate under the ice bath, centrifugal, and (4 ℃, 5000rpm gets supernatant liquor after 20min) and is used for oxidation-resistance and measures.
The thiobarbituricacid method: the final product mda of peroxidatic reaction of lipid is total to (100 ℃ of heat with thiobarbituricacid under acidic conditions, 20min), generate pink material, absorption peak is arranged in 532nm, can calculate the amount of mda, thereby draw the situation of lipid peroxidation.
2, the mensuration of murine liver tissue mda content: respectively get liver homogenate liquid 1ml, the recombinant protein 1ml that adds different concns, the FeSO4100 μ l that adds 6mmol/L, add 60mmol/L H20240 μ l at last, after 1h is hatched in 37 ℃ of water-baths, add 1ml 15% trichoroacetic acid(TCA) and finish reaction, add 1ml 6.7% thiobarbituricacid again, the 20min that develops the color in boiling water bath, the cooling back is centrifugal, gets supernatant in 532nm place mensuration absorbance.
Data processing: the result checks through t.
Three, experimental result
As shown in Figure 11, because the OH that the Fe2++H2O2 system generates has extremely strong damageability, cause Mouse Liver foundation of microsomal Lipid Peroxidation product mda content to increase, after adding recombinant protein Rd-RNase2, the amount that mda produces reduces, illustrate that recombinant protein Rd-RNase2 can suppress the OH that the Fe2++H2O2 system generates, thereby suppress the carrying out of hepatomicrosome peroxidatic reaction of lipid.
Four, conclusion
The redox state of cell influences all respects of cell function.The cytotoxic effect of many medicines is regulated by active oxygen.Oxidative stress can disturb the cancer chemotherapy, comprises that chemotherapeutic agents such as Zorubicin cause that Burkitt lymphoma cell apoptosis can be suppressed by catalase.Recombinant protein Rd-RNase2 has anti-oxidant function, and this may be its important factor to tumour cell antiproliferative, cytotoxic activity.Recombinant protein Rd-RNase2 may have the potential of application in oncotherapy and the pathological conditions relevant with oxidative stress as treatment of diseases aspects such as inflammation, autoimmune disease, atherosclerosis, septic shock, amyotrophic lateral sclerosis (spinal cord) lateral sclerosis.
Sequence table
＜110〉Biochemical Engineering College of Beijing Union University
＜120〉Rana temporaria chensinensis David functional gene Rd-RNase2 sequence, construction process and aminoacid sequence and purposes
<130>
<160>4
<170>PatentIn?version?3.5
<210>1
<211>312
<212>DNA
＜213〉Rana temporaria chensinensis David functional gene Rd-RNase2 sequence
<400>1
caagactgga?aaacatttca?gaagaagcac?ctgacaaaag?cccgggatat?taaatgtgat 60
aatatcatgt?caaaaacctt?gttcaactgc?aaagacacaa?acacttttat?cttttcactt 120
cctgggccag?ttaaggccct?ctgtagagga?attaaagtct?ccaaaaatgt?gttaagtcgt 180
tcagagtttg?atctctctga?gtgcaatgta?aaatccaagc?cctgcaagta?taaattaaag 240
aaaaaaagtg?atggaatttg?tataacatgt?agggatgaag?ctccggtaca?ttttgtcggt 300
gtcggaagtt?gc 312
<210>2
<211>104
<212>PRT
＜213〉Rana temporaria chensinensis David functional gene Rd-RNase2 aminoacid sequence
<400>2
Gln?Asp?Trp?Lys?Thr?Phe?Gln?Lys?Lys?His?Leu?Thr?Lys?Ala?Arg?Asp
1 5 10 15
Ile?Lys?Cys?Asp?Asn?Ile?Met?Ser?Lys?Thr?Leu?Phe?Asn?Cys?Lys?Asp
20 25 30
Thr?Asn?Thr?Phe?Ile?Phe?Ser?Leu?Pro?Gly?Pro?Val?Lys?Ala?Leu?Cys
35 40 45
Arg?Gly?Ile?Lys?Val?Ser?Lys?Asn?Val?Leu?Ser?Arg?Ser?Glu?Phe?Asp
50 55 60
Leu?Ser?Glu?Cys?Asn?Val?Lys?Ser?Lys?Pro?Cys?Lys?Tyr?Lys?Leu?Lys
65 70 75 80
Lys?Lys?Ser?Asp?Gly?Ile?Cys?Ile?Thr?Cys?Arg?Asp?Glu?Ala?Pro?Val
85 90 95
His?Phe?Val?Gly?Val?Gly?Ser?Cys
100
<210>3
<211>25
<212>DNA
＜213〉forward primer N275
<400>3
tatttgcagt?tgttttgagt?ctcac 25
<210>4
<211>23
<212>DNA
＜213〉reverse primer C715
<400>4
ctgcttatca?catccctgtt?gtc 23
Claims (10)
1. a Rana temporaria chensinensis David functional gene Rd-RNase2 sequence is characterized in that, is the nucleotide sequence shown in the SEQ IDNo.1 in the sequence table.
2. a Rana temporaria chensinensis David functional gene Rd-RNase2 aminoacid sequence is characterized in that, is the aminoacid sequence shown in the SEQ ID No.2 in the sequence table.
3. the derived sequence of the described Rana temporaria chensinensis David functional gene of claim 2 a Rd-RNase2 aminoacid sequence, it is characterized in that, its be by the aminoacid sequence shown in the SEQ ID No.2 in the sequence table be substituted, lack or add one or several amino acid whose and with sequence table in the identical aminoacid sequence of aminoacid sequence function shown in the SEQ ID No.2.
4. the construction process of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence according to claim 1 is characterized in that its concrete steps are: extract the genomic dna of Rana temporaria chensinensis David, the design gene-specific primer carries out pcr amplification, the product electrophoresis; Described gene-specific primer is the nucleotide sequence shown in SEQ ID No.3 and the SEQ ID No.4 in the sequence table, for positive anti-primer right:
Forward primer N275:TAT TTG CAG TTG TTT TGA GTC TCA C
Reverse primer C715:CTG CTT ATC ACA TCC CTG TTG TC
From running gel, reclaim the target dna fragment, be connected, transfer to cell, extract plasmid with plasmid, behind the PCR preliminary evaluation target gene, order-checking.
5. the construction process of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence according to claim 4 is characterized in that, described PCR reaction system and parameter:
Genomic dna, dNTPs, H
2O, 10 * Taq Buffer, Taq DNA Polymerase and gene-specific primer mix to cumulative volume 50 μ l;
Amplification condition is: 94 ℃ of 30s, and 48 ℃ of 30s, 72 ℃ of 50s, 35 circulations, 72 ℃ are extended 7min and finish.
6. the construction process of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence according to claim 4 is characterized in that, it is characterized in that, the described concrete steps that are connected with plasmid are:
(1) target dna segment, carrier, T4DNA Ligase, 10 * T4 DNA Ligase Buffer are to cumulative volume 10 μ l;
(2) 16 ℃ connect 10～12 hours and are prepared into the carrier that connects target gene;
(3) sample that step (2) is connected carries out the PCR detection; Connect product 1 μ l, dNTPs, 10 * TaqBuffer, 5 μ l, Taq DNA Polymerase3U and each 10pmol of plasmid universal primer, mend aseptic ultrapure water, mix to cumulative volume 50 μ l; The pcr amplification condition: 94 ℃ of 30s, 48 ℃ of 30s, 72 ℃ of 50s, 35 circulations, 72 ℃ are extended 7min and finish;
(4) go up the sample electrophoresis detection in the 2.5%TAE sepharose, in the uv analyzer, observe the DNA band.
7. the construction process of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence according to claim 4 is characterized in that, it is characterized in that, the described concrete steps of transferring to cell are:
(1) product that 5 μ l target genes are connected with plasmid joins in the 100 μ l competent cells, mixing, ice bath 30min;
Heat shock 90s in (2) 42 ℃ of water-baths; Place ice bath 2min after the taking-up immediately;
(3) add 500 μ l, 37 ℃ of preheatings in advance good do not contain antibiotic LB substratum, 37 ℃, the 150rpm shaking table is cultivated 45min;
(4) the centrifugal 2min of 4000rpm abandons the part supernatant liquor, gets 100 μ l after concentrating and is coated with the conversion flat board, smoothens, and drying is inverted flat board, and 37 ℃ of thermostat containers are cultivated 16h;
(5) good, the medium sized white colony of picking separation is inoculated into and contains in the antibiotic LB liquid nutrient medium, and 37 ℃ of shaking tables, 180rpm cultivate 12h.
8. the construction process of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence according to claim 4 is characterized in that, it is characterized in that, PCR reaction system and parameter that described extraction plasmid PCR identifies are:
The recombinant plasmid, dNTPs, 10 * Taq Buffer, the H that extract
2O, Taq DNA Polymerase and primer mix to cumulative volume 20 μ l;
The pcr amplification condition: 94 ℃ of 30s, 48 ℃ of 30s, 72 ℃ of 50s, 35 circulations, 72 ℃ are extended 7min and finish.
9. the amplimer of the described Rana temporaria chensinensis David functional gene of claim 1 a Rd-RNase2 sequence is characterized in that, is the nucleotide sequence shown in SEQ ID No.3 in the sequence table and the SEQ ID No.4, for just anti-primer is right.
10. the application of Rana temporaria chensinensis David functional gene Rd-RNase2 sequence in preparation treatment antitumor drug.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103285381A (en) * | 2012-02-22 | 2013-09-11 | 贵州神奇集团 | A combination of ribonucleases and cantharidin |
| CN105907774A (en) * | 2016-05-13 | 2016-08-31 | 山西大学 | Application of migratory locust intestinal tract RNase gene 2 to pest prevention and control |
| CN106636399A (en) * | 2016-12-21 | 2017-05-10 | 中国中医科学院中药研究所 | Method for performing true and false identification on oviductus ranae and special primer |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103285381A (en) * | 2012-02-22 | 2013-09-11 | 贵州神奇集团 | A combination of ribonucleases and cantharidin |
| CN103285381B (en) * | 2012-02-22 | 2015-06-24 | 贵州神奇集团 | A combination of ribonucleases and cantharidin |
| CN105907774A (en) * | 2016-05-13 | 2016-08-31 | 山西大学 | Application of migratory locust intestinal tract RNase gene 2 to pest prevention and control |
| CN105907774B (en) * | 2016-05-13 | 2019-06-25 | 山西大学 | A kind of application of migratory locusts enteron aisle nucleic acid hydrolysis enzyme gene in control of insect |
| CN106636399A (en) * | 2016-12-21 | 2017-05-10 | 中国中医科学院中药研究所 | Method for performing true and false identification on oviductus ranae and special primer |
| CN106636399B (en) * | 2016-12-21 | 2020-03-03 | 中国中医科学院中药研究所 | Method for identifying authenticity of oviductus ranae and special primer |
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