CN102286084B - Gingko biloba defensin, encoding gene and application thereof - Google Patents
Gingko biloba defensin, encoding gene and application thereof Download PDFInfo
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- CN102286084B CN102286084B CN2010102066861A CN201010206686A CN102286084B CN 102286084 B CN102286084 B CN 102286084B CN 2010102066861 A CN2010102066861 A CN 2010102066861A CN 201010206686 A CN201010206686 A CN 201010206686A CN 102286084 B CN102286084 B CN 102286084B
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Abstract
The invention discloses gingko biloba defensin, an encoding gene and an application thereof. The gingko biloba defensin disclosed by the invention is gingko biloba defensin GD01, or conservative variant polypeptide thereof, or active derivatives thereof, or polypeptide derivatives of antimicrobial gingko biloba defensin GD01 formed by substituting, lacking or adding one or more amino acid residues. Mature peptide of the gingko biloba defensin disclosed by the invention has the amino acid sequences described in SEQ ID NO:2 in a sequence list. The gingko biloba defensin disclosed by the invention is applied to preparing antimicrobial products, such as feed additives, veterinary medicines, antiseptics, medicinal products and the like.
Description
Technical field
The present invention relates to bioengineering field, be specifically related to a kind of Gingko biloba defensin, its encoding gene and application.
Background technology
American scientist Lehrer in 1985 etc. go out 3 kinds of little peptides of positively charged ion from people's neutrophil separation and purification, HNP-1, HNP-2 and HNP-3, by chromatogram, electrophoresis and functional analysis, with them and the former similar little peptide that separates from rabbit, cavy together called after alexin.Finding afterwards that the little peptide of this class was prevalent in the higher organism, pathogenic micro-organism is had the poisoning effect of wide spectrum, is the important defensive substance of higher organism defence pathogenic agent invasion.
At present, alexin (defensins) is molecule amount very little (general 15-30 residue), is rich in the cationic protein of halfcystine, belongs to a class of antimicrobial peptide.Be present in the biological groups such as vertebrates, invertebrates and plant.
Defence have the effect of very strong antibacterium, fungi and tool togavirus.Although at present alexinic antifungal mechanism be there is no clear and definite final conclusion, popular viewpoint is that alexin and the electronegative bacterial cell membrane of positively charged attracts each other, the alexin of dimerization or poly is worn the ionic channel that film forms cross-film, thereby membrane passage and cellular energy state have been upset, cause the cytolemma depolarize, respiration is suppressed and the cell ATP content decrease, finally makes target cell dead.Alexinic antivirus action then is to cause virus to lose biological activity by being combined with virus capsid protein.Just because of this special mechanism of action, make alexin possess two characteristics: the one, resistance spectrum is wide, and the 2nd, the purpose microorganism is difficult to alexin is produced resistant mutation.Scientists that Here it is is to the good important evidence of its application prospect.
Ginkgo (Ginkgo biloba) has another name called gingko, Gong Sunshu, is one of something lost plant the most ancient in the existing gymnosperm, person " living fossil ".Ginkgo wanted for two more than ten years from plantation to the result, and the ability large result can be lived more than 1,000 year old after 40 years, was the venerable old man or lady in the tree.Gingko seeds is nutritious, has the natural health effect, and long-term edible can delaying senility prolongs life, and is listed in imperial tribute in the Song dynasty.
The occurring in nature ginkgo can catch hardly, and investigators have carried out a large amount of research around this special plant.For example, Huang etc. (2000) from the ginkgo leaf separation and purification obtain a kind of chitin-binding protein, Shen etc. (2005) are cloned into the phylaxin gene that a kind of jasmonate relies on from ginkgo.Wang etc. (2000) isolate a kind of new antibacterial protein that suppresses the HIV-1 RT activity that has from food containing gingko almond.
The contriver has obtained a new Gingko biloba defensin gene order by making up the CDNA library, and alexin of the present invention and encoding gene thereof are searched in database, does not find any identical sequence.
Summary of the invention
First purpose of the present invention is to provide a kind of Gingko biloba defensin.
Second purpose of the present invention is to provide the encoding gene of Gingko biloba defensin of the present invention.
The 3rd purpose of the present invention is Gingko biloba defensin of the present invention is applied to prepare the Anti-bacterium series products.
The selected ginkgo of the present invention (Ginkgo biloba) is picked up from the Jiangsu Province, China Xinyi.
The present invention's method in construction cDNA library, the clone obtains the alexin family member from Chinese Xinyi food containing gingko almond.
The structure of food containing gingko almond cDNA library: at first separate food containing gingko almond, extract total RNA.Get total RNA and carry out synthetic cDNA the first chain product of reverse transcription.Get cDNA again and be used for ligation, conversion fluid is coated plate respectively, and all the other are used for shaking total storehouse bacterium liquid, picking mono-clonal conservation from the flat board.
The present invention passes through the extensive sequencing of above recombinant clone, obtain 6 est sequence coding Gingko biloba defensin family members, applicant general's called after Gingko biloba defensin GD01, the Gingko biloba defensin mature peptide is comprised of 48 amino acid, iso-electric point is 9.05, and molecular weight is 5.43 kilodaltons, and Gingko biloba defensin has the feature of typical alexin primary structure, its aminoacid sequence is shown in SEQ ID NO:2 in the sequence table, and is specific as follows:
Arg Thr Cys Lys Ser Gln Ser His Lys Phe Lys Gly Tyr Cys Leu Ser Asp Thr
1 10
Asn Cys Arg Asn Val Cys Arg Thr Glu Gly Phe Gly Thr Gly Ser Cys Asp Phe
20 30
Ala Ser Arg Lys Cys Tyr Cys Tyr Lys Pro Cys Val
40
This mature peptide can obtain by separation and purification, and HPLC identifies that its purity reaches 96.5%, MALDI-TOF and identifies that its molecular weight is correct.
Gingko biloba defensin of the present invention is following (a) or (b) described polypeptide:
(a) have the described aminoacid sequence of SEQ ID NO:2 in the sequence table, i.e. Gingko biloba defensin GD01;
(b) polypeptide of being derived by (a) with identical function that the aminoacid sequence in (a) is formed through replacement, disappearance or the interpolation of indivedual amino-acid residues.
Above-mentioned (b) described Gingko biloba defensin, the C-terminal amidation modifier of preferred Gingko biloba defensin GD01.
Gingko biloba defensin of the present invention can be the product that obtains by chemosynthesis, dna recombinant expression.
The polynucleotide sequence of coding Gingko biloba defensin H is shown in SEQ ID NO:1 in the sequence table, and is specific as follows:
1 cggacgtgca aaagtcaaag ccacaagttc aaagggtatt gcttgagcga caccaattgc
61 agaaatgtgt gcagaacaga gggatttggg actgggagct gtgatttcgc cagccgaaag
121 tgctactgct ataaaccctg cgtt
Polynucleotide of the present invention are following (a) or (b) described nucleotide sequence:
(a) polynucleotide of the precursor peptide of coding Gingko biloba defensin H;
(b) polynucleotide complementary with polynucleotide (a).
Polynucleotide of the present invention preferably have the polynucleotide of the described nucleotide sequence of SEQ ID NO:1 in the sequence table.
The Gingko biloba defensin GD01 that the present invention obtains has biological activity, has anti-microbial effect.The alexin GD01 of separation and purification can suppress part malignant bacteria and fungi when 20nM.Therefore, Gingko biloba defensin of the present invention can be applicable to prepare the Anti-bacterium series products, and the Anti-bacterium series products comprises fodder additives, veterinary drug, sanitas, pharmaceutical prod such as antibacterials etc.
Description of drawings
Fig. 1 is the MALDI-TOF evaluation figure behind the Gingko biloba defensin GD01 purifying;
Fig. 2 is the experimental result that Gingko biloba defensin GD01 suppresses Escherichia coli Growth.
Embodiment
Embodiment one: the structure of food containing gingko almond cDNA library and est sequence analysis
The extraction of total RNA is synthetic with cDNA: separates food containing gingko almond, carry out poison with reference to the TRIZOL LS reagent specification sheets of Gibco BRL company and manage total RNA extraction.Library construction adopts the Clontech Smart cDNA Library Construction Kit of company test kit, and utilizes the subsidiary primer of test kit itself, carrier, restriction enzyme etc. to test.Get the total RNA of 1ug food containing gingko almond with SMART III oligonucleotide segment (5 '-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3 ') and CDSIII/3 ' PCR primer (5 '-ATTCTAGAGGCCGAGGCGGCCGACATG-d (T)
30N
-1N-3 ') carries out synthetic the first chain of reverse transcription, obtain 10 μ l cDNA the first chain product.
The structure of food containing gingko almond cDNA library and evaluation: get cDNA 1.5 μ l and be used for ligation, reaction system 5 μ l transform wherein 1 μ l, get respectively coated plate of 5ul conversion fluid, get the 300ul conversion fluid and are used for shaking total storehouse bacterium colony.The picking mono-clonal carries out the PCR evaluation at random, and the result shows that it is 850bp that recombination fraction surpasses 95% average Insert Fragment length.Picking six plate mono-clonals shake bacterium, conservation, extraction plasmid, the unidirectional order-checking of T7 primer at random from the flat board, manual BLASTN and the online homology comparison of BLASTX, SEQTOOL, the ClustalX 1.83 programanalysis sequencing results of using.
Embodiment two: mensuration and the analysis of ginkgo antifungal genes GD01 sequence
The sequence of ginkgo antifungal genes gd01 is from the est sequence of 6 height homologies of being cloned in the embodiment one food containing gingko almond cDNA library.Utilize tool software SEQTOOL that its base sequence is analyzed, and obtain its maximum opening code-reading frame, length 237bp (not comprising terminator codon), the precursor peptide of 79 amino-acid residues of coding.We by high performance liquid chromatography from the kernel extract in addition separation and purification the mature peptide sequence corresponding with this gene order, it is described that its mature peptide nucleotide sequence corresponds in the sequence table SEQ ID NO:1, and the aminoacid sequence of mature peptide is as described in the SEQ ID NO:2 in the sequence table.
Embodiment three: the separation and purification of Gingko biloba defensin GD01
The 50g ginkgo kernel is at room temperature ground to form pasty state, add the 100mL Extraction buffer, by 40% and 80% (NH4)
2The SO4 salt fractionation, CM Sephorose F.F. ion exchange chromatography and Superdex 75 gel permeation chromatographies, with the extraction buffer solution elution, 215nm and 280nm detect online, as seen three ultraviolet absorption peaks, collect on the 1st peak C18 reversed-phase column and separate, the acetonitrile gradient wash-out separablely is purified to a simple spike.Through flying time mass spectrum analysis, molecular weight is 5428 dalton (Fig. 1), and this theoretical molecular (considering four pairs of disulfide linkage) with the expressed sequence of inferring according to the gene of cloning is consistent.Adopt the order-checking of N-end to confirm that further this sequence is the antibiotic GD01 of ginkgo.
Embodiment four: the bacteriostatic activity experiment of Gingko biloba defensin GD01
Add blank meat soup 100 μ l (except the first hole) in each hole of 96 orifice plates.Experimental concentration according to design adds 200 μ l testing sample 5ug/ml alexin GD01 at first row, draws 100 μ l to the second holes, and piping and druming mixes rear absorption 100 μ l to the three holes up and down, and last 100 μ l discard.Volume fraction corresponding to every like this hole is 1,0.5,0.25.Do three parallel.The last 100 μ l that add in every hole dilute good bacterium liquid, put into the constant incubator incubated overnight.
Take in aperture fully the lowest concentration of drug of bacteria growing inhibiting as MIC.The obvious growth test of bacterium is just meaningful in positive control hole (namely not containing microbiotic).The result shows that a large amount of bacterial growths are arranged in the control tube, adds in the hole of alexin GD01, does not have bacterial growth (Fig. 2) in 1: 1 the hole fully.
Gingko biloba defensin GD01 of the present invention can suppress part malignant bacteria and fungi.Therefore, Gingko biloba defensin of the present invention can be applicable to prepare the Anti-bacterium series products, as: fodder additives, veterinary drug, sanitas, antibacterials etc.
Sequence table
＜110〉Guangzhou and Shi Kang Bioisystech Co., Ltd
＜120〉Gingko biloba defensin, encoding gene and application
<160>3
<170>Patent In version 3.1
<210>1
<211>144
<212>DNA
＜213〉ginkgo (Ginkgo biloba)
<400>1
1 cggacgtgca aaagtcaaag ccacaagttc aaagggtatt gcttgagcga caccaattgc
61 agaaatgtgt gcagaacaga gggatttggg actgggagct gtgatttcgc cagccgaaag
121 tgctactgct ataaaccctg cgtt
<210>2
<211>48
<212>PRT
＜213〉ginkgo (Ginkgo biloba)
<400>2
Arg Thr Cys Lys Ser Gln Ser His Lys Phe Lys Gly Tyr Cys Leu Ser Asp Thr
1 10
Asn Cys Arg Asn Val Cys Arg Thr Glu Gly Phe Gly Thr Gly Ser Cys Asp Phe
20 30
Ala Ser Arg Lys Cys Tyr Cys Tyr Lys Pro Cys Val
40
Claims (2)
1. a Gingko biloba defensin is characterized in that its aminoacid sequence is shown in SEQ ID NO:2.
2. polynucleotide, its aminoacid sequence claimed in claim 1 of encoding.
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| CN1062017C (en) * | 1995-09-01 | 2001-02-14 | 中国科学院遗传研究所 | Plant expression carrier plasmid of phylaxin gene |
| CN1772896A (en) * | 2004-11-10 | 2006-05-17 | 中国科学院海洋研究所 | Bay scallop phylaxin gene and its coded protein and cloning method |
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| Title |
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| Plant defensins;Thomma等;《Planta》;20021231;第216卷(第2期);197 * |
| Thomma等.Plant defensins.《Planta》.2002,第216卷(第2期),193-202. |
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