CN103421766B - Method for extracting anti-oxidant anti-tumor Latcripin-3 gene segment from mushroom C91-3 strain - Google Patents

Abstract

The invention discloses a method for extracting an anti-oxidant anti-tumor Latcripin-3 gene segment from a mushroom C91-3 strain. The method is implemented sequentially according to the following steps: extracting mycelium total RNA from the mushroom C91-3 strain; inversely transcripting the mycelium total RNA to synthesize cDNA; taking the obtained cDNA as a template and performing polymerase chain reaction (PCR) on PCR reaction primers which are inserted into two limited incision enzyme sites BamHI and XhoI, wherein the upstream primer BamHI is (5'-3')GGCTGATATCGGATCCCTTGCCCAAGATCTTACCG and the downstream primer XhoI is (5'-3')GGTGGTGGTGCTCGAGGTCGGGGCAGTGAGGAATT; and performing 1% agarose gel electrophoresis on the obtained PCR product, purifying a remarkable stripe near 750 bpMarker, cutting colloid and recovering the DNA segment.

Description

The method of anti-oxidant, antitumor Latcripin-3 gene fragment is extracted from mushroom C91-3 bacterial strain

Technical field

The present invention relates to a kind of preparation method of anti-oxidant, anti-tumor protein Structure and function domain, especially a kind of extract from mushroom C91-3 bacterial strain anti-oxidant, antitumor latcripinthe method of-3 gene fragments.

Background technology

Fungi is rich in multiple bioactive molecules, comprises ribosome inactivating protein, antifungal protein, lectin, ubiquitin-like protein, polysaccharide and kinases.The advantages such as antineoplastic component is wherein few with untoward reaction, evident in efficacy have distinctive feature in oncotherapy.It is Basidiomycotina Basidiomycetes Pleurotaceae that Lentinus Edodes fungus C91-3 belongs to fungi, and this bacterial classification was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in 2013, and preserving number is CGMCC No.7354.Lentinus Edodes fungus C91-3 contains invigorating the spleen and benefiting QI, strengthening vital QI to eliminate pathogenic factors, enhancing body immunizing power, the effects such as anti-curing cancers.[Zhao C，Sun H，Tong X，et a1．An Antitumor leetin from edible Mushroon Agroebe aegerita[J]．Bioehem J，2003，374：321—327]。Containing multiple protein composition in Lentinus Edodes fungus C91-3 fermented liquid, confirm that some protein ingredient has good antitumor action [Huang Min by animal experiment in vivo, Ning'an is red, Zhang Zhuoran etc. the research [J] of mushroom C91-3 hypha fermentation liquid mouse Anticancer effect in vivo. Chinese microecology magazine, 1996,8 (3): 38-40].Experiment in vitro also confirms, some protein ingredients in its fermented liquid have inducing apoptosis of tumour cell ability [wear soldier, Huang Min, Ning'an is red. mushroom C91-3 hypha fermentation liquid eggs suppresses mouse cervix JEG-3 U14 to grow and apoptosis-induced experimental study in vain. Zhejiang Medical, 2004,26(9); 656-658].But, up to now also not about extract from mushroom C91-3 bacterium expressed albumen have antioxidase to antioxidant radical, can the gene fragment relevant report of inducing apoptosis of tumour cell.

Summary of the invention

The present invention is the above-mentioned technical problem in order to solve existing for prior art, provides one to extract from mushroom C91-3 bacterial strain anti-oxidant, antitumor latcripinthe method of-3 gene fragments.

Technical solution of the present invention is that one is extracted anti-oxidant, antitumor from mushroom C91-3 bacterial strain latcripinthe method of-3 gene fragments, is characterized in that carrying out as follows successively:

A. the mycelium total serum IgE of mushroom C91-3 bacterial strain is extracted;

B. by mycelium total serum IgE reverse transcription synthesis cDNA;

C. pcr amplification target gene:

With obtained cDNA for template, react primer with the PCR inserting BamH I and Xho I two restriction endonuclease sites and carry out PCR reaction; It is as follows that described PCR reacts primer sequence:

Upstream primer BamH I:

( 5′-3′)GGCTGATATCG GATCCCTTGCCCAAGATCTTACCG

Downstream primer Xho I:

( 5′-3′)GGTGGTGGTGC TCGAGGTCGGGGCAGTGAGGAATT；

D. purifying DNA fragment is reclaimed:

Obtained PCR primer is carried out 1% agarose gel electrophoresis, bright near purifying 750 bpMarker

Aobvious band, cuts glue and reclaims DNA fragmentation.

Described a step gets the mycelium of the cultivation mushroom C91-3 bacterial strain of 18 days, adds liquid nitrogen and fully grind in mortar, and room temperature places 5min, then adds chloroform, after gentle concussion, and 4 C, 12000r/min is centrifugal, 15min; Draw upper strata aqueous phase solution and be transferred in another centrifuge tube, add equal-volume Virahol, the static 15min of room temperature after mixing, 4 C again, 12000r/min is centrifugal, 10min, abandon supernatant, then add 75% washing with alcohol and drying, finally by DEPC process water dissolution, obtain the mycelium total serum IgE of mushroom C91-3 bacterial strain.

The PCR reaction system 50 ul:Premix Taq 25 μ l of described step c, cDNA template 2 μ l, upstream primer 1 μ l, downstream primer 1 μ l, sterile purified water 21 μ l; Reaction conditions: 95 DEG C of denaturation 6min; 94 DEG C of sex change 30s, 55 DEG C of renaturation 45s, 72 DEG C extend 45s, 30 circulations; 72 DEG C extend 10min; 4 DEG C of cooling 10min.

The present invention is from mushroom C91-3 mycelium transcript profile, clones one section of specific gene fragment, called after latcripin-3 gene fragments, the albumen expressed by this gene fragment has the antioxidase to antioxidant radical, the various biological functions such as inducing apoptosis of tumour cell.This gene fragment can be carried out gene recombination, and efficient solubility expression is carried out in pET32-a prokaryotic expression system, expression product is carried out isolation andpurification by affinity chromatography, obtain the protein of this genes encoding, this albumen can be used for the mechanism in apoptosis of studying and the effect in cell-signaling pathways thereof, also can be used for the medicine preparing prevention or treatment tumour.

Accompanying drawing explanation

Fig. 1 is the mycelium total serum IgE 1.0% agarose gel electrophoresis figure of the mushroom C91-3 bacterial strain that the embodiment of the present invention is extracted.

Fig. 2 is that embodiment of the present invention PCR primer is through 1% agarose gel electrophoresis figure.

Fig. 3 is that the expressive host bacterium of embodiment of the present invention recombinant expression plasmid is through 1% agarose gel electrophoresis figure.

Fig. 4 is the solubility expression 1% agarose gel electrophoresis figure of embodiment of the present invention target protein.

Fig. 5 is that embodiment of the present invention target protein Western-blotting identifies colouring reagents box colour developing schematic diagram.

Fig. 6 is the target protein 12%SDS-PAGE electrophorogram after embodiment of the present invention purifying.

Fig. 7 is the SRB experimental result schematic diagram of the target protein target protein after embodiment of the present invention purifying.

Mushroom C91-3 bacterial strain preservation date: on March 15th, 2013;

Classification And Nomenclature: mushroom ( lentinula edodes);

Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC);

Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City

Preserving number: CGMCC No.7354.

Embodiment

A. the mycelium total serum IgE of mushroom C91-3 bacterial strain is extracted:

Take the mycelium of the culture medium culturing mushroom C91-3 bacterial strain of 18 days, add liquid nitrogen and fully grind in mortar, room temperature places 5min, then adds 0.2ml chloroform, after gentle concussion, and 4 C, 12000r/min is centrifugal, 15min; Draw upper strata aqueous phase solution 0.5ml and be transferred to another clean 1.5ml centrifuge tube, add equal-volume Virahol, put upside down the rear static 15min of room temperature of mixing, 4 C again, 12000r/min is centrifugal, 10min, abandon supernatant, then add 75% ethanol 1ml to wash and drying, finally by DEPC process water dissolution, the mycelium total serum IgE of mushroom C91-3 bacterial strain;

Ultraviolet spectrophotometer checking purity, OD260/280 ratio is between 1.8 ~ 2.0.

Carry out 1.0% agarose gel electrophoresis checking, result as shown in Figure 1, shows that RNA DNA purity is higher.

B. by mycelium total serum IgE reverse transcription synthesis CDNA;

Use TaKaRa 3 '-Full RACE Core Set Ver.2.0 test kit reverse transcription synthesis cDNA, this test kit is purchased from precious biotechnology (Dalian) company limited

Reaction system: shiitake mushroom hypha total serum IgE (1 ug/ul) 1 ul, 3 ' RACE Adaptor (5 uM) 1 ul, dNTP Mixture (10 mM each) 1 ul, RNase Free dH2O 4.5 ul;

Reaction conditions: 70 DEG C, 10 min place 2 minutes immediately on ice;

Then following component is added: 5 × M-MLV Buffer 2 ul, RNase Inhibitor (40 U/ul) 0.25 ul, Reverse Transcriptase M-MLV (RNase H-) (200 U/ ul) 0.25 ul, system cumulative volume reaches 10 ul.

Reaction conditions: 42 DEG C, 60 min 70 DEG C, 15 min obtain inverse transcription reaction liquid (cDNA).

C. pcr amplification target gene;

With obtained cDNA for template, react primer with the PCR inserting BamH I and Xho I two restriction endonuclease sites and carry out PCR reaction; It is as follows that described PCR reacts primer sequence:

Upstream primer BamH I:

( 5′-3′)GGCTGATATCG GATCCCTTGCCCAAGATCTTACCG

Downstream primer Xho I:

( 5′-3′)GGTGGTGGTGC TCGAGGTCGGGGCAGTGAGGAATT；

Primer entrusts the synthesis of precious biological (Dalian) company.

PCR reaction system 50 ul:Premix Taq 25 ul, cDNA template 2 μ l, upstream primer 1 ul, downstream primer 1 ul, sterile purified water 21 ul.Reaction conditions: 95 DEG C of denaturation 6min; 94 DEG C of sex change 30 s, 55 DEG C of renaturation 45 s, 72 DEG C extend 45 s, 30 circulations; 72 DEG C extend 10min; 4 DEG C of cooling 10min.PCR product through 1% agarose gel electrophoresis as shown in Figure 2, proves this gene fragment of successful clone.

D. purifying DNA fragment is reclaimed;

Obtained PCR primer is carried out 1% agarose gel electrophoresis, bright near purifying 750 bpMarker

Aobvious band, cuts glue and reclaims DNA fragmentation, and name life is latcripin-3 gene fragments.

Experiment:

1. Latcripin-3 gene fragment order measures: the Latcripin-3 gene fragment that embodiment reclaims be connected with pMD19-T cloning vector, construction recombination plasmid pMD19T-Latcripin-3, transform competent E. coli JM109.Recombinant vectors order-checking is measured by the precious biotech firm in Dalian, and mushroom C91-3 Latcripin3 nucleotide sequencing is as SEQ ID NO.3.

2. the clonal expression of Latcripin-3 gene fragment

The structure of 2.1 Latcripin-3 gene fragment recombinant expression plasmids: with the double digested pET32a (+) of BamH I and Xho I, 1.0% agarose gel electrophoresis is selected to be separated, purified linear pET32a (+) carrier segments is reclaimed with DNA gel extraction, connect the Latcripin3 gene fragment of pET32a (+) carrier segments and embodiment of the present invention acquisition with T4DNA ligase enzyme, then obtain recombinant expression plasmid pET32a (+)-Latcripin3 gene fragment.Recombinant expression plasmid transformation of E. coli JM109, is spread evenly across on the LB flat board of Pyocianil by converted product, cultivate 14 hours in 37 DEG C.The positive bacterium colony of picking mono-clonal, be inoculated in 3ml respectively containing in the LB liquid nutrient medium of Pyocianil, at 37 DEG C, 190 rpm/min shaking culture are spent the night; With Plasmid Miniprep Kit V3.l extracting plasmid, and carry out nucleic acid sequencing qualification.Nucleic acid sequencing qualification result shows that sequence is consistent with Latcripin-3 gene fragment order.

The expressive host bacterium of 2.2 recombinant expression plasmids transforms selects freeze-thaw method, recombinant expression plasmid pET32a (+)-Latcripin3 gene fragment is proceeded in expressive host bacterium intestinal bacteria Rosetta-gami (DE3) competent cell, coat on the LB flat board containing four kinds of microbiotic (Pyocianil, tsiklomitsin, paraxin, kantlex), 37 DEG C of incubated overnight.The positive bacterium colony of mono-clonal choosing grow on plates is inoculated in containing four kinds of microbiotic (Pyocianils, tsiklomitsin, paraxin, kantlex) LB liquid culture based on 37 DEG C cultivate 14 hours, preserve bacterial strain, adopt precious biotech firm Plasmid Miniprep Kit V3.l extracting plasmid simultaneously, after BamH I and Xho I is double digested, carry out 1.0% agarose gel electrophoresis detection validation, 1.0% agarose gel electrophoresis result as shown in Figure 3, in Fig. 3,1 is empty pET32a (+) double digestion, 2 is pET32a (+)-Latcripin3 double digestion, show object pET32a (+)-Latcripin3 gene fragment successful conversion in the bacterial strain of Rosetta-gami (DE3).

The solubility expression of 2.3Latcripin3 albumen

The TB that the bacterial strain that 2.2 steps are preserved is inoculated in containing four kinds of microbiotic (Pyocianil, tsiklomitsin, paraxin, kantlex are high density) in 1:100 ratio is supported in base, wherein contain the glucose of 1%, at 37 DEG C, 190 rpm/min shaking culture 5 hours, treat bacterial concentration OD ₆₀₀when=0.8, add IPTG and make its solution final concentration be 1mmol/L, 16 DEG C of inductions of spending the night, morning 4 DEG C next day, 8000r/min, within 10 minutes, reclaim bacterium.With PBS, bacterium is washed 3 times, resuspended with appropriate PBS, add PMSF trash ice and hatch ultrasonic degradation, low temperature (4 DEG C) centrifugal 10000rpm/min, 5min, does 12%SDS-PAGE electrophoresis detection (as Fig. 4) by supernatant protein, in Fig. 4,1 is Latcripin-3 target protein, 2 is pET-32a empty carrier expressing protein, tentatively shows that Latcripin3 target protein has expression.

2.4 target protein Western-blotting identification and analysis

2.3 step gained soluble proteins samples are carried out Western blot qualification, be transferred on NC film by the mode of wet transferring film, after 5% skimmed milk room temperature confining liquid closes 120 minutes, first anti-Mouse Anti-His Tag Monoclonal Antibody, 4 DEG C of overnight incubation, add the second anti-horseradish enzyme labelling goat anti-mouse IgG (H+L) incubated at room again 90 minutes, colouring reagents box colour developing (as Fig. 5), shows Latcripin3 target protein successful expression.

The purifying of 2.5 target proteins

Above-mentioned 2.4 step gained soluble proteins samples are mixed with the binding buffer equal-volume containing 10mM imidazoles, by nickel post, utilizes the affinity chromatography of HIS label to carry out the purifying of albumen, dense by difference

The gradient elution (concentration is 200mM, 300mM, 500mM) of degree imidazoles, obtains single target protein 12%SDS-PAGE electrophoresis checking (as Fig. 6), 1 whole bacterial protein, and 2 spread liquid, 3 elutriants; Collect protein concentrate.Electrophoresis the result shows: can successfully obtain Latcripin3 target protein after ni-sepharose purification.

2.6 protein-active analyses

A. adopt the target protein of existing method to 2.5 step purified concentration to check order, its aminoacid sequence, as shown in SEQIDNO:4, names Latcripin-3 albumen.

Aminoacid sequence and functional structure domain analysis are analyzed: analyzed by the coded product of DNASTAR software package to embodiment of the present invention nucleic acid.

Adopt Protean module, analyze the general aspects of polypeptide;

Similarity system design completes with BLAST on NCBI website;

Adopt Pfam database, known protein sequence is carried out retrieval analysis, its functional domain of prediction peroxidase albumen, has anti-oxidant protease activity, have scavenging free radicals and anti-tumor activity.Analysis shows, Latcripin3 protein structure domain and peroxidase albumen have certain sequence similarity, illustrates that Latcripin-3 albumen has anti-oxidant protease activity, have scavenging free radicals and anti-tumor activity.

B., after the Latcripin-3 target protein of purified concentration being carried out micro-BCA protein quantification, typeⅡ pneumocyte is selected to carry out cell toxicant SRB experiment.SRB(Sulforhodamine B Sulforhodamine B) be a kind of water-soluble protein dyestuff that can be combined with the basic aminoacids of biomacromolecule, its be incorporated into measure in cell number can reflect total protein concentration so that reflection cell count number.OD value at 515 nm wavelength places and viable count are good linear relationship.The SRB experimental result of target protein as shown in Figure 7, illustrates that the albumen of genetic expression of the present invention has restraining effect significantly to typeⅡ pneumocyte, when protein concentration is 15ug/ml, tumour inhibiting rate close to 30%, and is concentration dependent.

Sequence table

 

<110> Dalian Medical Univ

 

<120> extracts anti-oxidant, antitumor from mushroom C91-3 bacterial strain latcripinthe method of-3 gene fragments

 

 

<160> 4

 

<210> 1

<211> 35

<212> DNA

<213> artificial sequence

 

<220>

<223> primer

 

<400>1

GGCTGATATCGGATCCCTTGCCCAAGATCTTACCG 35

 

<210> 2

<211> 35

<212> DNA

<213> artificial sequence

 

<220>

<223> primer

 

<400>2

GGTGGTGGTGCTCGAGGTCGGGGCAGTGAGGAATT 35

 

<210> 3

<211> 732

<212> DNA

<213> mushroom C91-3 bacterial strain ( lentinula edodes)

<400> 3

1 CTTGCCCAAG ATCTTACCGA GACACTATTC GAGAACCAGT GTGGTGAAAC

51 GGCCCATGAA GTGCTTCGTT TGAGCTTCCA TGATGCTATT GCTATCTCTC

101 AATCCCTCGG TCCGTCGGCT GGAGGAGGCG CTGACGGTTC TATGTTGATC

151 TTCCCGGACG TCGAGCCCAA CTTCGCCGCC AACCTCGGAA TCTCCGACAG

201 CGTGAACGAC CTCGCTCCAT TCTTGGCTTC AGGAAAATTC CCGACTATTA

251 CTGCGGGAGA TATGATCCAG TTCGGTGCCG CTGTCGCTGT CGGCCTTTGT

301 CCTGGTGCAC CTCAATTGGA GTTCCGCGCC GGACGACCCA ACGCTACCGC

351 TCCTGCTGTT GACGGTCTGA TCCCTGAACC GCAAAATACT GTCGATGAAA

401 TTTTGGCACG TTTCCAGGAT GCTGCAAACA TGAATGCTGA GGACATTGTT

451 TCTCTGCTTG TATCACACAC AGTGGCCCGA GCCGATCACG TCGATCCCAC

501 TTTGGACGCT GCACCTTTCG ACTCGACTCC TTTCACCTTT GACTCACAGT

551 TCTTCCTCGA AACCCTCTTG ACTGGTGTCG GATTCCCTGG AACTACCAAC

601 AACACTGGTG AAGTTTCCTC CCCTCTTCCC CTCACTGTTG GCGACAACGT

651 TGGAGAACTC CGACTCCAAT CGGACTTCGA GCTGGCTCGT GACTCCCGAA

701 CTGCCTGTTT CTGGCAATCA ATGATCAACC AG 732

   

<210> 4

<211> 244

<212> PRT

<213> mushroom C91-3 bacterial strain ( lentinula edodes) expression product

 

<400> 4

1 LAQDLTETLF ENQCGETAHE VLRLSFHDAI AISQSLGPSA GGGADGSMLI

51 FPDVEPNFAA NLGISDSVND LAPFLASGKF PTITAGDMIQ FGAAVAVGLC

101 PGAPQLEFRA GRPNATAPAV DGLIPEPQNT VDEILARFQD AANMNAEDIV

151 SLLVSHTVAR ADHVDPTLDA APFDSTPFTF DSQFFLETLL TGVGFPGTTN

201 NTGEVSSPLP LTVGDNVGEL RLQSDFELAR DSRTACFWQS MINQ 244

Claims (1)

1. a mushroom C91-3 bacterial strain latcripinthe application of-3 genetic expression albumen in preparation treatment lung-cancer medicament, described mushroom C91-3 bacterial strain latcripinthe aminoacid sequence of-3 genetic expression albumen is as shown in SEQ ID NO:4.