CN107043758A - A kind of bufo gargarizans Cantor lysozyme - Google Patents
A kind of bufo gargarizans Cantor lysozyme Download PDFInfo
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- CN107043758A CN107043758A CN201710062396.6A CN201710062396A CN107043758A CN 107043758 A CN107043758 A CN 107043758A CN 201710062396 A CN201710062396 A CN 201710062396A CN 107043758 A CN107043758 A CN 107043758A
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- lysozyme
- bufo gargarizans
- gargarizans cantor
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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Abstract
The invention discloses a kind of bufo gargarizans Cantor lysozyme, it has SEQ ID NO：Nucleotide sequence shown in 1, the lysozyme recombination expression product is inhibited to two kinds of gram-positive bacteriums, two kinds of gramnegative bacteriums and two kinds of fungi growths.
Description
Technical field
The present invention relates to biomedical sector, and in particular to a kind of bufo gargarizans Cantor lysozyme.
Background technology
Ecological environment disaster and the influence caused by them to human health that abuse of antibiotics triggers in the world today
The concern in the world is increasingly triggered.Find activity more preferably and the higher Substitutes For Antibiotic of specificity turns into the task of top priority
Thing, therefore, polypeptides matter-antibacterial peptide (AMP) with antibacterial activity turn into new study hotspot, wherein, with Maganin
It is especially noticeable for the amphibian source AMP of representative.The research of amphibian animal skin secretion focuses mostly in polypeptide
The identification of matter and functionally.Such as Africa xenopus Xenopys laevis antibacterial polypeptide Magainin and Chinese Bombina maxima
Bombina maxima antibacterial polypeptide Maximin just shows lethal effect in extremely low concentration to tumour cell, and they can
It is selectively applied to tumour cell and on body normal tissue cell without influence.In addition with the secretion of more amphibian animal skins
Polypeptide just shows the powerful and antibacterial activity of wide spectrum in very low concentrations level, and the antibacterial peptide Maximin3 of such as Chinese Bombina maxima exists
Can just be completely inhibited during 1.5 μ g/mL contents level Escherichia coli, bacillus megaterium Bacillus magaterium with
And shigella dysenteriae Shigella dysenteriae growth.Also there are some peptides that there is certain side effect to human body, as in
The Maximin H antibacterial peptides of state's Bombina maxima, the Brevinin-1 antibacterial peptides and Bombina orientalis Bombintoro of Japanese Rana guentheri
Rientalis Bombinin H antibacterial peptides are respectively provided with very strong hemolytic activity.Therefore, the skin secrete polypeptide of amphibian animal
It is the active material of a class diversity extremely abundant and function highly significant, great researching value and wide application prospect.
Bufo gargarizans Cantor skin secretion is referred to as the dried venom of toads after drying, is China's traditional Chinese medicine, long-term in China's history
For purposes such as cardiac stimulant, anesthesia, anti-inflammation, cough-relievings, and then find that the dried venom of toads has effects that to suppress tumour.Modern pharmacology is ground
Studying carefully the cardiac stimulant steroidal compounds for finding to contain in the dried venom of toads and indole alkaloids compound on tumor cell has stronger cell
Toxicity and inhibitory activity, and it is found that Bufalin, cinobufagin etc. have the monomeric compound of antitumor activity successively.In the recent period,
It is reported to be derived from the cathelicidin-Bg of bufo gargarizans Cantor with resisting gram-positive bacterial activity, this is to report first
The active antibacterial polypeptide of source bufo gargarizans Cantor.
The content of the invention
To solve the above problems, the invention provides a kind of bufo gargarizans Cantor lysozyme, the lysozyme recombination expression product
Suppression to two kinds of gram-positive bacteriums, two kinds of gramnegative bacteriums and two kinds of fungi growths.
To achieve the above object, the technical scheme taken of the present invention is：
A kind of bufo gargarizans Cantor lysozyme, it has SEQ ID NO：Nucleotide sequence shown in 1.
The lysozyme recombination expression product is to two kinds of gram-positive bacteriums, two kinds of gramnegative bacteriums and two kinds of fungies
Growth is inhibited.
Wherein, the lysozyme recombination expression product is synthesized by following steps：
S1, with TRlzol reagents method extract bufo gargarizans Cantor gland tissue total serum IgE, with ReverTra Ace qPCR
Total serum IgE reverse transcription is cDNA by RT Kit (TOYOBO, Japan) reverse transcription reagent box；
S2, the primer with addition restriction enzyme site
5 '-CCGGAATTCAAGTATGAAAAATGCGAACTTGCTA-3 ' and
5 '-CCCTTCGAATTAAAGTTTGCATCCTTGAGTCCA-3 ' are primer；
The cDNA synthesized using above step is template amplification bufo gargarizans Cantor lysozyme functional domain coding sequence fragment；
S3, cloned sequence purified with DNA purification kits, DNA fragmentation and pET-28 empty plasmids after purification is equal
Double digestion is carried out with EcoR I and Hind III, reaction condition is 37 DEG C of placement 8h；Reaction uses digestion products after terminating again
DNA purification kits are purified, and the fragment of DNA fragmentation after purification and pET-28 empty plasmids is entered with T4 DNA ligases
Row connection.
The invention has the advantages that：
The lysozyme recombination expression product is to two kinds of gram-positive bacteriums, two kinds of gramnegative bacteriums and two kinds of fungies
Growth is inhibited.
Brief description of the drawings
Fig. 1 is the bacteriostatic experiment result of lysozyme in the embodiment of the present invention.
Embodiment
In order that objects and advantages of the present invention are more clearly understood, the present invention is carried out with reference to embodiments further
Describe in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this hair
It is bright.
In following examples, used bufo gargarizans Cantor (100-130g) collection is from Hangzhou Linan, by skin after ear
Gland carries out surface sterilization with 75% ethanol, then dissects gland tissue therein.
Embodiment
The total serum IgE of the gland tissue of bufo gargarizans Cantor is extracted with TRlzol reagents method, with ReverTra Ace qPCR RT
Total serum IgE reverse transcription is cDNA by Kit (TOYOBO, Japan) reverse transcription reagent box；
With the primer of addition restriction enzyme site
5 '-CCGGAATTCAAGTATGAAAAATGCGAACTTGCTA-3 ' and
5 '-CCCTTCGAATTAAAGTTTGCATCCTTGAGTCCA-3 ' are primer,
The cDNA synthesized using above step is template amplification bufo gargarizans Cantor lysozyme functional domain coding sequence fragment.
Cloned sequence is purified with DNA purification kits, DNA fragmentation after purification is used with pET-28 empty plasmids
EcoR I and Hind III carry out double digestion, and reaction condition is 37 DEG C of placement 8h.Digestion products are used DNA by reaction again after terminating
Purification kit is purified, and the fragment of DNA fragmentation after purification and pET-28 empty plasmids is carried out with T4 DNA ligases
Connection.
Recombinant plasmid transformed e. coli bl21 (DE3) after connection, is plated on the LB solids for adding kanamycin sulfate
Culture medium flat plate, 37 DEG C are placed after 12h, random picking single bacterium spot, and being detected with PCR has the positive colony bacterial plaque of Insert Fragment.
It is OD630=0.2 by BL21 (DE3) bacterium solution adjustment concentration containing recombinant plasmid, and adds final concentration of 1mM's
IPTG, put on shaking table 28 DEG C, 210rpm shake bacterium and induction bacteriolyze expression of enzymes 8h.
Bacterium solution 8000rpm after induced expression is centrifuged into 5min, supernatant is abandoned, precipitates and uses PBS resuspension, and with instead
The method cracking bacterial cell of multiple freeze thawing ultrasonic degradation simultaneously, by the supernatant after cracking handled with nickel element affinity column with
Purify the fusion protein wherein containing six poly- His fusion tags albumen.
Activity determination
Pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, Escherichia coli, Candida albicans and yeast
The microbial strains such as bacterium are purchased from ATCC, and four kinds of bacteriums use LB medium cultures, and two kinds of fungies are with improvement Martin's culture medium
Culture.
Microbial inoculum adjustment concentration be OD630 between 0.1-0.2, be added to 96 holes according to the amount in every 100 microlitres of hole
ELISA Plate, OD630 is determined after adding the polypeptide that concentration is respectively 0,0.1,1,10,100 μ g/ml, 37 DEG C of culture 24h with ELIASA
Value.
As a result with analysis
1 lysozyme maturation peptide sequence analysis
Mature peptide coding nucleotide sequence is
AAGTATGAAAAATGCGAACTTGCTAGGGTTCTGAAAAAGGGAGGACTTGGCGGCTTCAGAGGTTACAGT
CTGGAAAATTGGATTTGTACCGCATACTATGAGAGTGGCTTCAACACAGCCAGCACAAACTACAATCCACCTGACAA
GAGCACTGATTATGGCATTTTTCAAATAAATAGTCGATGGTGGTGCAATGATAACAAGACCCCCGGAAGTAAAAATG
CTTGCAATATCAACTGCAAAGTACTGTTAGGTGGCGATATAAGTCAAGCTATAAAATGTGCAAAGCGTGTTGTGAGT
GATCCCAACGGCATGGGTGCATGGGTTGCATGGAGAAATCACTGTAAGGGCAAAAACTTATCAAAGTGGACTCAAGG
ATGCAAACTT
Peptide section sequence is
MKWQIHILGGLLLLLAIASGKKYEKCELARVLKKGGLGGFRGYSLENWICTAYYESGFNTASTNYNPPDKSTDYGIF
QINSRWWCNDNKTPGSKNACNINCKVLLGGDISQAIKCAKRVVSDPNGMGAWVAWRNHCKGKNL5KWTQGCKL
2 lysozyme recombination expression products are to two kinds of gram-positive bacteriums, two kinds of gramnegative bacteriums and two kinds of fungies
The suppression of growth.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (3)
1. a kind of bufo gargarizans Cantor lysozyme, it has SEQ ID NO：Nucleotide sequence shown in 1.
2. a kind of bufo gargarizans Cantor lysozyme as claimed in claim 1, it is characterised in that lysozyme recombination expression product is to leather
Gram-positive bacteria, gramnegative bacterium and fungi growth are inhibited.
3. a kind of bufo gargarizans Cantor lysozyme as claimed in claim 2, it is characterised in that the lysozyme recombination expression product
Synthesized by following steps：
S1, with TRIzol reagents method extract bufo gargarizans Cantor gland tissue total serum IgE, with ReverTra Ace qPCR RT
Total serum IgE reverse transcription is cDNA by Kit (TOYOBO, Japan) reverse transcription reagent box；
S2, the primer with addition restriction enzyme site
5 '-CCGGAATTCAAGTATGAAAAATGCGAACTTGCTA-3 ' and
5 '-CCCTTCGAATTAAAGTTTGCATCCTTGAGTCCA-3 ' are primer；
The cDNA synthesized using above step is template amplification bufo gargarizans Cantor lysozyme functional domain coding sequence fragment；
S3, cloned sequence purified with DNA purification kits, DNA fragmentation after purification is used with pET-28 empty plasmids
EcoR I and Hind III carry out double digestion, and reaction condition is 37 DEG C of placement 8h；Digestion products are used DNA by reaction again after terminating
Purification kit is purified, and the fragment of DNA fragmentation after purification and pET-28 empty plasmids is carried out with T4 DNA ligases
Connection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710062396.6A CN107043758A (en) | 2017-01-22 | 2017-01-22 | A kind of bufo gargarizans Cantor lysozyme |
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| CN201710062396.6A CN107043758A (en) | 2017-01-22 | 2017-01-22 | A kind of bufo gargarizans Cantor lysozyme |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063145A (en) * | 2007-04-20 | 2007-10-31 | 厦门大学 | Expression carrier for black porgy antibiotic peptide Hepcidin and expression product and constructing preparation method |
| CN102061291A (en) * | 2010-11-05 | 2011-05-18 | 中国科学院海洋研究所 | Lysozyme as well as preparation and application thereof |
| CN104530209A (en) * | 2014-12-17 | 2015-04-22 | 潍坊医学院 | Antibacterial peptides BG-CATH37 and BG-CATH(5-37) of bufo bufo gargarigans and coding gene and application thereof |
-
2017
- 2017-01-22 CN CN201710062396.6A patent/CN107043758A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063145A (en) * | 2007-04-20 | 2007-10-31 | 厦门大学 | Expression carrier for black porgy antibiotic peptide Hepcidin and expression product and constructing preparation method |
| CN102061291A (en) * | 2010-11-05 | 2011-05-18 | 中国科学院海洋研究所 | Lysozyme as well as preparation and application thereof |
| CN104530209A (en) * | 2014-12-17 | 2015-04-22 | 潍坊医学院 | Antibacterial peptides BG-CATH37 and BG-CATH(5-37) of bufo bufo gargarigans and coding gene and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| F. GAO等: ""Characteristics of cathelicidin-Bg, a novel gene expressed in the ear-side gland of Bufo gargarizans"", 《GENETICS AND MOLECULAR RESEARCH》 * |
| 展波等: ""中华大蟾蜍皮肤Cathelicidin 家族新型抗菌肽的鉴定及其抗菌活性"", 《中国中药杂志》 * |
| 张英等: ""中华大蟾蜍的研究进展"", 《中草药》 * |
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Application publication date: 20170815 |
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