CN101619315B - Functional gene Rdr-lec sequence of rana chensinensis lectin sample, construction method, amino acid sequence thereof and application thereof - Google Patents

Abstract

The invention discloses a functional gene Rdr-lec sequence of a rana chensinensis lectin sample, which is a nucleotide sequence shown by SEQ ID No.1 in a sequence table. Firstly, the genome DNA of rana chensinensis is extracted, a specific primer and a PCR amplification gene are designed, and the gene sequence is measured; and then the gene sequence is determined as new gene Rdr-lec, namely the functional gene Rdr-lec sequence of the rana chensinensis lectin sample through database retrieval. The experiment shows that the protein of the functional gene Rdr-lec sequence of the rana chensinensis lectin sample has application value in the aspect of treating infectious diseases caused by medicament-resistant pathogenic bacteria, and the recombinant protein has broad application value in preparing a biological medicament for treating tumor cells.

Description

Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence, construction process and aminoacid sequence and purposes

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 lectin sample anti-tumor function gene Rdr-lec sequence and amino acid sequence coded thereof and its purposes.

Background technology

Only study in the world at present and reported from the Japanese rice field frog (Rana Japonica), separate rnase jSBL ([1] FusaoSakakibara that obtains having activity of lectin in the bullfrog frogs such as (Ranacatesbeiana), Hiroaki Kawauchi, Giichi Takayanagi, Hitomilse.Egg lectinof Rana japonica and its receptor glycoprotein of ehrlich tumor cells.1979.Cancer research.39:1347-1352) with cSBL ([2] Kazuo Nitta, GiichiTakayanagi, Hiroaki Kawauchi, Sen-itiroh Hakomori.Isolation andCharacterization of Rana catesbeiana Lectin and Demonstration of theLectin-binding Glycoprotein of Rodent and Human Tumor Cell Membranes.1987.CANCER RESEARCH.47,4877-4883).

Sakakibara etc. ^[1]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.Nitta etc. ^[2]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 various cancer cells, and wherein cSBL has been proved to be a kind of at the external chemotherapeutics that effectiveness is 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, its enzymic activity is not subjected to the inhibition of human rna 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.

Rana temporaria chensinensis David is Chinese distinctive Amphibians resource, domestic research and development for Rana temporaria chensinensis David at present only are confined to wood frog oil and Wood frog antibiotic peptides etc., all belong to blank for the rnase and the Antitumor Effects thereof of its agglutinin international and domestic.

Summary of the invention

The objective of the invention is to remedy the defective of prior art and propose a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence.

Another purpose of the present invention is to propose a kind of construction process of above-mentioned Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence.

Another purpose of the present invention is to propose the aminoacid sequence of a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec.And be substituted, lack or add one or several amino acid whose and identical amino acid derived sequence with this aminoacid sequence function by this aminoacid sequence.

A further object of the present invention is to propose a kind of purposes of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence.

For achieving the above object, invention thinking of the present invention is:

Whether in close relations the existence of the apoptosis that medicine caused 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.And whether have nothing to do by the tumour cell aggegation of lectin sample rnase mediation with to the selective killing effect of tumour cell and the existence of wild type p53 tumour containment thing, and do not produce the variation of gene, therefore, the lectin sample rnase as degradation of rna has advantage at drug treatment.

Gene order of the present invention increases from the Rana temporaria chensinensis David genome and obtains, and is a new gene.The functional protein of its coding can be used as a kind of cytotoxin selectively, and it as novel, the application of anti-tumor function bio-pharmaceutical efficiently of preparation, has important pharmaceutical developments using value based on Rana temporaria chensinensis David.

The concrete technical scheme that the present invention adopts is:

A kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec 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

>Rdr-lec

cagaactggc?caacatttca?gcagaagcac?atttcaagca?atccaaccat?cgtctgtaac 60

agcatcatga?acaacatcat?atatatcgta?ggaggtcaat?gcaaggaacg?caacactttc 120

ataatttctt?ctgcaaccac?cgtaagggcc?atctgtagcg?gggcgtcaac?cgatagaaat 180

gtattaagta?ccacaagatt?ccagctcaac?acttgcattc?gtagtgccat?tgtaccccgc 240

ccttgtccat?atacctccag?accggaaact?aatgtgatat?gtgtaaaatg?tgagaataga 300

cttcccgtac?attttgttgg?aataggaagt?tgt 333

The aminoacid sequence of a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec, it is the aminoacid sequence shown in the SEQ ID No.2, and is specific as follows:

Obtain the mature protein coding region protein sequence by the gene order derivation

>Rdr-lec

QNWPTFQQKH?ISSNPTIVCN?SIMNNIIYIV?GGQCKERNTF?IISSATTVRA?ICSGASTDRN 60

VLSTTRFQLN?TCIRSAIVPR?PCPYTSRPET?NVICVKCENR?LPVHFVGIGS?C 111

The construction process of above-mentioned a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence, its concrete steps are: the genomic dna that extracts Rana temporaria chensinensis David, the design gene-specific primer carries out pcr amplification, the product electrophoresis, from running gel, reclaim the target dna fragment, be connected with plasmid, transfer to cell, extract plasmid, behind the PCR preliminary evaluation target gene, order-checking.

The construction process of above-mentioned a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence, wherein, described gene-specific primer is:

Forward primer NP1:ATG TGT GCA AAA TCT CTT CTT CTG G

Reverse primer CP0:GTA AAG CGT TGC TTG ACC AAG TAA TT

The construction process of above-mentioned a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec 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 (NP1, CP0) 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 lectin sample functional gene Rdr-lec sequence, wherein, the described concrete steps that are connected with plasmid are:

(1) target dna segment (20ng), pGM-T carrier (50ng), T4DNA 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 Polymerase3U 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.

The construction process of above-mentioned a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence, wherein, the described concrete steps of transferring to cell are:

(1) product (recombinant plasmid pGM-lec) 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 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.

The construction process of above-mentioned a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence, wherein, PCR reaction system and parameter that described extraction plasmid PCR identifies are:

Recombinant plasmid (pGM-lec) 1 μ l, dNTPs (10mM) the 0.5 μ L, 10 * Taq Buffer, 2 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 Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence of the present invention can be used in preparation treatment antitumor drug.

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 new gene Rdr-lec, Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence promptly of the present invention.Our experiments show that, the albumen of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence of the present invention has and has using value aspect the infectious diseases that causes of treatment pathogenic bacteria of drug-resistant, and recombinant protein of the present invention is with a wide range of applications in the bio-pharmaceutical of preparation treatment tumour cell.

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 for the recombinant protein abduction delivering to host's bacteria growing;

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 the growth-inhibiting action diagram of recombination fusion protein to the HeLa tumour cell.

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, 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, T4 DNA 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.

The concrete construction process of a kind of Rana temporaria chensinensis David lectin of the present invention sample anti-tumor function gene Rdr-lec sequence:

1, design of primers

Gene-specific primer

Forward primer NP1:ATG TGT GCA AAA TCT CTT CTT CTG G

Reverse primer CP0:GTA AAG CGT TGC TTG ACC AAG TAA TT

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 (NP1, CP0) 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.

Target gene pcr amplification result as shown in Figure 1, through pcr amplification and agarose gel electrophoresis, with 50bpDNALadder is reference standard, in uv analyzer, can see the bright fluorescence band of (complete open reading frame comprises signal sequence, and exact value is 495bp) appearance about the 500bp position, the proof goal gene increase successfully, cuts to can be used for ensuing carrier connection behind the glue purification.

3, the pGM-T carrier connects

(1) in aseptic PCR pipe, adds target gene segment (20ng), pGM-T carrier (50ng), T4 DNALigase 3U, 10 * T4 DNA 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.

PGM-T carrier joint detection result as shown in Figure 2, the result that 16 ℃ of connections are spent the night carries out PCR and agarose electrophoresis detection, in uv analyzer, with Marker I is reference standard, 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 55 ℃, 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 lIPTG (50mg/ml), 40 μ lX-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.

(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.

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) extracts plasmid with plasmid extraction kit.Detect plasmid with 0.8% agarose gel electrophoresis.

(2) PCR detects the insertion dna fragmentation in the recombinant plasmid.In aseptic PCR pipe, add recombinant plasmid (pGM-lec) 1 μ l, dNTPs (10mM) 0.5 μ L, 10 * Taq Buffer, 2 μ l, Taq DNA Polymerase1.5U 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.

(3) 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:

>Rdr-lec

cagaactggc?caacatttca?gcagaagcac?atttcaagca?atccaaccat?cgtctgtaac 60

agcatcatga?acaacatcat?atatatcgta?ggaggtcaat?gcaaggaacg?caacactttc 120

ataatttctt?ctgcaaccac?cgtaagggcc?atctgtagcg?gggcgtcaac?cgatagaaat 180

gtattaagta?ccacaagatt?ccagctcaac?acttgcattc?gtagtgccat?tgtaccccgc 240

ccttgtccat?atacctccag?accggaaact?aatgtgatat?gtgtaaaatg?tgagaataga 300

cttcccgtac?attttgttgg?aataggaagt?tgt 333

Obtain the mature protein coding region protein sequence shown in SEQ ID No.2 by the gene order derivation, be specially:

>Rdr-lec

QNWPTFQQKH?ISSNPTIVCN?SIMNNIIYIV?GGQCKERNTF?IISSATTVRA?ICSGASTDRN 60

VLSTTRFQLN?TCIRSAIVPR?PCPYTSRPET?NVICVKCENR?LPVHFVGIGS?C 111

Above-mentioned sequencing result and Genbank database are carried out sequence alignment, and the result shows that Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence of the present invention is new gene.

The Rana temporaria chensinensis David lectin sample anti-tumor function gene Rdr-lec sequence that embodiment 2 the present invention make up and the biological experiment of aminoacid sequence:

In the recombination fusion protein abduction delivering process of Rana temporaria chensinensis David Rdr-lec gene to the Study of cytotoxicity of host bacterium

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

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-lec of Rana temporaria chensinensis David Rdr-lec 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 phase (OD 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.

Two, 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 ColiBL21 (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-lec abduction delivering process host's bacteria growing had obvious restraining effect.

The recombinant protein abduction delivering has restraining effect to host's bacteria growing as shown in Figure 6, after inducing Escherichia Coli BL21 (DE3) the express recombinant protein 30min that carries recombinant plasmid pFLAG-lec with 20uM, 40uM and 100uM IPTG respectively, the growth of obviously visible host bacterium is suppressed, inhibiting rate reaches 11.9% respectively behind the 60min, 9.4%, 8.7%, induce 210min after inhibiting rate can reach 36.4%, 31.8%, 33.5%.

Three, conclusion

Carry the e. coli bl21 (DE3) of the recombinant plasmid pFLAG-lec of Rana temporaria chensinensis David Rdr-lec gene, transcriptional start and expression Rdr-lec 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 Rdr-lec recombinant plasmid pFLAG-lec, 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, and these recombinant proteins have using value aspect the infectious diseases that causes of treatment pathogenic bacteria of drug-resistant.Same these recombinant proteins of explanation have in the treatment tumour cell and have good using value.

The preparation of the recombination fusion protein of embodiment 3 Rana temporaria chensinensis David Rdr-lec genes

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 (all 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 blotting 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-lec of Rdr-lec gene are cultured to logarithmic phase (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 in the lysate recombination fusion protein is arranged in expection molecular weight position band as shown in Figure 7, Fig. 7 be that the SDS-PAGE of intestinal bacteria lysate schemes.Among the figure, the Far Left swimming lane is a ribonuclease A, and middle swimming lane is the low molecular weight protein (LMWP) standard, and the rightmost side is the intestinal bacteria lysate.As seen from the figure, there is target protein to express at the about 14.2kDa of molecular weight place.

2, Affi-Gel purification of Recombinant fusion rotein Rdr-lec

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 Rdr-lec is resolved from Affi-Gel.Rdr-lec albumen behind the wash-out is neutralized to pH neutral with neutralizer immediately.The purity of electrophoresis detection recombinant protein Rdr-lec.The result is shown in Figure 8, and Fig. 8 detects the purity figure of recombinant protein behind the Affi-Gel purifying for SDS-PAGE; As seen from the figure, being about 14.2kDa 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, shows as Fig. 9.Fig. 9 is that the recombinant protein Western blotting of intestinal bacteria lysate total protein and purifying detects figure.Among the figure, the left side is the pure product of Rdr-lec, and the right is the intestinal bacteria lysate.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.

Four, conclusion

This experiment will import the intestinal bacteria of recombinant plasmid and cultivate, preparation rnase Rdr-lec 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 rnase Rdr-lec recombination fusion protein, the SDS-PAGE electrophoresis detection is to the homogeneous band of molecular weight about about 14.2kDa.Western blotting method has been identified the Rdr-lec recombination fusion protein.Set up the preparation method of effective Rdr-lec recombination fusion protein.

The extracorporeal anti-tumor function research of the recombination fusion protein of embodiment 4 Rana temporaria chensinensis David Rdr-lec genes

One, material and equipment

1, cell strain, substratum and biochemical reagents

Human cervical carcinoma cell HeLa preserves for this laboratory.RPMI 1640 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

The HeLa cell grows in RPMI RPMI-1640 (Gibco company), and including volume fraction is 10% calf serum (56 ℃ of deactivation 30min), 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, is 5%CO in 37 ℃, volume fraction ₂Incubator in 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 Rdr-lec 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 * 10 ⁴Cell/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 Rdr-lec, 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 570nm wavelength detects each hole absorbance on microplate reader, draws the cell survival rate curve.

Three, experimental result

1, MTT experiment detect the recombination fusion protein pair cell the growth-inhibiting effect as shown in figure 10, Figure 10 is the growth-inhibiting action diagram of recombination fusion protein to the HeLa tumour cell.As seen from the figure, recombination fusion protein Rdr-lec has remarkable restraining effect to the growth of HeLa cell, its IC ₅₀Be about 12.3ug/ml.

Four, conclusion

Recombination fusion protein Rdr-lec has powerful vitro cytotoxicity for the HeLa tumour cell that is tried under extremely low concentration, its IC ₅₀Value be about 12.3ug/ml, and present tangible dose-dependent effect.As seen, recombination fusion protein Rdr-lec has the cytotoxicity of killing tumor cell.Clinical treatment for tumour has important development and application values.

Sequence table

＜110〉Biochemical Engineering College of Beijing Union University

＜120〉Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence, construction process and aminoacid sequence and purposes

<130>

<140>

<141>

＜213〉artificial sequence

<400>

cagaactggc?caacatttca?gcagaagcac?atttcaagca?atccaaccat?cgtctgtaac 60

agcatcatga?acaacatcat?atatatcgta?ggaggtcaat?gcaaggaacg?caacactttc 120

ataatttctt?ctgcaaccac?cgtaagggcc?atctgtagcg?gggcgtcaac?cgatagaaat 180

gtattaagta?ccacaagatt?ccagctcaac?acttgcattc?gtagtgccat?tgtaccccgc 240

ccttgtccat?atacctccag?accggaaact?aatgtgatat?gtgtaaaatg?tgagaataga 300

cttcccgtac?attttgttgg?aataggaagt?tgt 333

<212>DNA

<211>333

Rana temporaria chensinensis David lectin sample functional gene Rdr-lec sequence

＜213〉artificial sequence

<400>

QNWPTFQQKH?ISSNPTIVCN?SIMNNIIYIV?GGQCKERNTF?IISSATTVRA?ICSGASTDRN 60

VLSTTRFQLN?TCIRSAIVPR?PCPYTSRPET?NVICVKCENR?LPVHFVGIGS?C 111

<212>PRT

<211>111

The aminoacid sequence of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec

Claims (8)

1. a Rana temporaria chensinensis David lectin sample functional gene Rdr-lec is characterized in that its nucleotide sequence is shown in SEQID No.1.

2. a Rana temporaria chensinensis David lectin sample functional gene Rdr-lec is characterized in that its aminoacid sequence is shown in SEQID No.2.

3. the preparation method of a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec according to claim 1, it is characterized in that its concrete steps are: extract the genomic dna of Rana temporaria chensinensis David, the design gene-specific primer, carry out pcr amplification, the product electrophoresis reclaims the target dna fragment from running gel, be connected with plasmid, transfer to cell, extract plasmid, behind the PCR preliminary evaluation target gene, order-checking;

Described gene-specific primer is:

Forward primer NP1:ATG TGT GCA AAA TCT CTT CTT CTG G

Reverse primer CP0:GTA AAG CGT TGC TTG ACC AAG TAA TT.

4. the preparation method of a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec according to claim 3 is characterized in that the reaction system of described pcr amplification and parameter are:

Genomic templates, dNTPs, 10 * Taq Buffer, Taq DNA Polymerase and gene-specific primer are mixed, and mending aseptic ultrapure water to cumulative volume is 50 μ l, mixes;

Amplification condition is: 94 ℃ of 30s, and 48 ℃ of 30s, 72 ℃ of 50s, 35 circulations, 72 ℃ are extended 7min and finish.

5. the preparation method of a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec according to claim 3 is characterized in that the described concrete steps that are connected with plasmid are:

(1) target dna segment, carrier, T4DNA Ligase 3U, 10 * T4DNA Ligase Buffer are mixed, 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 step (2) is connected carries out the PCR detection; To connect product, dNTPs, 10 * TaqBuffer, Taq DNA Polymerase and plasmid universal primer and mix, 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.

6. the preparation method of a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec according to claim 3 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.

7. the preparation method of a kind of Rana temporaria chensinensis David lectin sample functional gene Rdr-lec according to claim 3 is characterized in that, the PCR reaction system and the parameter of described PCR preliminary evaluation target gene are:

The recombinant plasmid, dNTPs, 10 * Taq Buffer, Taq DNA Polymerase and the plasmid universal primer that extract are mixed, mend aseptic ultrapure water, 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.

8. the application of the described Rana temporaria chensinensis David lectin of claim 1 sample functional gene Rdr-lec in preparation treatment human cervical carcinoma medicine.

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