CN104987377B

CN104987377B - Natural anti-infective antitumor bifunctional polypeptides ECCT and its gene and application

Info

Publication number: CN104987377B
Application number: CN201510341844.7A
Authority: CN
Inventors: 王义鹏; 于海宁; 许国强
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2015-06-19
Filing date: 2015-06-19
Publication date: 2018-03-06
Anticipated expiration: 2035-06-19
Also published as: CN104987377A

Abstract

The present invention relates to a kind of natural anti-infective antitumor bifunctional polypeptides ECCT and its encoding gene and application, ECCT is made up of 30 amino acid residues, for straight-chain polypeptide, molecular weight 3662.7Da, isoelectric point 12.33, the encoding gene of ECCT precursors is straight chain nucleotide sequence of the length for 720 bases, and polypeptide ECCT has the beneficial features such as molecular weight is small, chemical synthesis process is simple, strong, the antitumor activity broad-spectrum high efficacy of antibacterial action broad-spectrum high efficacy, anti-inflammatory activity, is with a wide range of applications.

Description

Natural anti-infective antitumor bifunctional polypeptides ECCT and its gene and application

Technical field

The invention belongs to field of biomedicine technology, specifically a kind of natural anti-infective antitumor bifunctional polypeptides ECCT and its gene and application.

Background technology

Microorganism infection can cause serious locally or systemically diseases associated with inflammation, such as pneumonia, gastritis, peritonitis and pyemia Deng the extensive abuse of conventional antibiotic causes increasingly severe pathogenic microorganism resistance problems in recent years, to human health Bring huge threat.The measure for clinically tackling drug-resistant microorganism infection is to use new or alternative antibiotic, because This this just need the new anti-microbial infection medicine of Persisting exploitation.

Tumour is body under the effect of various carcinogenic factors, and the cell of local organization loses on gene level to be grown to it Normal regulation, the neoformation for causing its clonal abnormality hyperplasia and being formed.Tumour is generally divided into benign and malignant two major class, its Middle malignant tumour is the high a kind of disease of the death rate, and its fatal rate is in second in all diseases of the mankind.Malignant tumour Treatment method, mainly have three kinds of operation, radiotherapy and chemotherapy at present, but operative treatment is only applicable to early stage entity Knurl, radiotherapy side effect is big, and chemotherapy one side side effect is big, and another aspect tumour easily produces drug resistance.Therefore Need to develop more tumor therapeuticing methods, including the research and development of new type antineoplastic medicine.

Polypeptide is a kind of macromolecular substances being formed by connecting by different aminoacids residue by amido link condensation, at present from The largely peptide molecule with different structure and function is found in the various organisms of nature, these peptide molecules show various Biological activity, such as antibacterial, anti-inflammatory, immunological regulation, it is antitumor and promote wound reparation.

Antibacterial peptide is a kind of natural small molecule polypeptide of organism gene code, is a kind of important of organism immune system Molecule, direct killing action is respectively provided with to bacterium, fungi, virus even protozoon.In addition, some antibacterial peptides also have suppression Inflammatory factor secretion processed, adjusts the effect of immune response.

Natineoplaston is a kind of polypeptide for having and suppressing tumor cell proliferation or killing activity of tumor cells, never at present With finding tens of kinds of natineoplastons in organism.The natineoplaston mechanism of action is various, such as induced tumor membranolysis, induction ROS generations, induced tumor meronecrosis and apoptosis etc..Polypeptide generally have molecular weight is small, simple in construction, pharmacological activity is strong, The advantages that mechanism of action is various, toxicity is low and is not easy to cause drug resistance, there is great potentiality to be exploited in new drug development field.

The content of the invention

It is an object of the invention to provide a kind of natural anti-infective antitumor bifunctional polypeptides ECCT and its gene and application.

To achieve the above object, the technical solution adopted by the present invention is：

One of the object of the invention is to provide a kind of polypeptide ECCT, the straight-chain polypeptide being made up of 30 amino acid residues, molecule Amount 3662.7Da, isoelectric point 12.33, the primary structure of its amino acid sequence are O.1 shown by SEQ ID N：

Ser¹Arg²Lys³Leu⁴Lys⁵Lys⁶Phe⁷Phe⁸Arg⁹Lys¹⁰Val¹¹Lys¹²Lys¹³Gly¹⁴Il e¹⁵Lys¹⁶Lys¹⁷Val¹⁸Phe¹⁹Lys²⁰Lys²¹Val²²Val²³Lys²⁴Ala²⁵Ile²⁶Phe²⁷Arg²⁸Leu²⁹Leu³⁰。

The two of the object of the invention are to provide a kind of gene of polypeptide ECCT precursors, are made up of 720 nucleotides, from 5 ' ends extremely The base sequence at 3 ' ends is as shown in SEQ ID NO.2：

The three of the object of the invention are to provide a kind of polypeptide ECCT preparation method, and this method is carried out successively according to the following steps：

1) total serum IgE of derived tissues is extracted；

2) construction cDNA library；

3) synthetic pcr primer thing is designed, enters performing PCR amplification ECCT coding gene sequences.

4) ECCT coding gene sequences are expanded according to PCR and derives ripe peptide amino acid sequence；

5) with automatic Peptide synthesizer synthesis polypeptide ECCT complete sequences；

6) HPLC reversed phase column chromatography desalinations are passed through.

Polypeptide ECCT of the present invention can be used for preparing antibacterials, anti-inflammatory drug, antineoplastic, suppression bacterium life Long medicine, preservative, animal feed or cosmetic additive agent.

Polypeptide ECCT of the present invention base sequence can be applied to polypeptide recombination expression, transgenic animals, plant, plant In thing part, zooblast or plant cell.

The present invention is detected by experiment to ECCT antibacterial activities, sterilization speed, anti-inflammatory activity and antitumor activity, institute Stating polypeptide ECCT has that molecular weight is small, chemical synthesis process is simple, antibacterial action broad-spectrum high efficacy, anti-inflammatory activity are strong, antitumor work The beneficial features such as property broad-spectrum high efficacy, are with a wide range of applications.

Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention, And can be practiced according to the content of specification, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.

Brief description of the drawings

Fig. 1 is that ECCT suppresses the TNF-α that LPS is induced in Turnover of Mouse Peritoneal Macrophages, IL-1 β, IL-6 and iNOS genes turn Record.**P<0.01.

Embodiment

With reference to the accompanying drawings and examples, the embodiment of the present invention is described in further detail.Implement below Example is used to illustrate the present invention, but is not limited to the scope of the present invention.

Embodiment 1

The clone of natural anti-infective antitumor bifunctional peptide ECCT encoding genes：

1) water snake (Enhydris chinensis) lung Total RNAs extraction：

1. taking 250mg water snake lung tissues, it is put into mortar and adds liquid nitrogen grinding into powder, be transferred in EP pipes, add Enter 1m1 total RNA extraction reagents (Trizol, U.S.'s Invitrogen Products), fully mix, and after 4 DEG C, 12000rpm Centrifuge 10min.

2. centrifuging and taking supernatant, adding 0.2ml chloroformic solutions, acutely mixing, room temperature is placed 10 minutes, then with 4 DEG C, 12000rpm is centrifuged 10 minutes, reject precipitation.

3. supernatant adds isometric isopropanol, room temperature is placed 10 minutes, and with 4 DEG C, 12000rpm is centrifuged 10 minutes, is collected Precipitation is washed once with 75% (V/V) ethanol, is dried, ttom of pipe sediment is water snake lung total serum IgE.

2) water snake lung cDNA library is built：Using CLONTECH companies SMART^TM cDNA Library Construction Kit are built.

(1) chains of cDNA first synthesis (mRNA reverse transcriptions)：

1. through DEPC processing (DEPC processing is in the water soaked overnight containing 0.1% (V/V) DEPC, autoclaving, drying) Centrifuge tube in add 3 μ l water snake lungs total serum IgEs, 1 μ l SMART IV Oligonucleotide and 1 μ l CDS III/3 ' PCR Primer, of short duration centrifugation (2000rpm, 30s), centrifuges and is incubated 2 minutes after 72 DEG C after mixing；Then by centrifuge tube in ice It is upper to place 2 minutes.

2. following reagent (being to build in the kit of storehouse to be equipped with), 2.0 5 × First- of μ l are added in above-mentioned centrifuge tube strand Buffer、1μl 20mM DTT、1.0μl 10mM dNTP Mix、1.0μl SMARTScribe MMLV Reverse Transcriptase, reagent and of short duration centrifugation (2000rpm, 30s) in centrifuge tube are mixed, 60min is incubated at 42 DEG C, will centrifuge Pipe is placed in the synthesis for stopping the first chain on ice.Take the chains of cDNA first synthesized by 2 μ l standby from centrifuge tube.

(2) using the second chain of long end polymeric PCR (LD-PCR) method amplification, (agents useful for same is to build storehouse examination It is equipped with agent box)

1. by 2 μ l First-strand cDNA, 80 μ l Deionized H₂O、10μl 10×Advantage2PCR Buffer, 2 μ l 50 × dNTP Mix, PCR Primer of 2 μ l 5 ', 2 μ l CDS III/3 ' PCR Primer and 2 μ l 50 × Mixed in the PCR pipe of Advantage 2Polymerase Mix preheatings at 95 DEG C.

2. expanded in PCR instrument by following procedure：95 DEG C, 1min；18 circulations：95 DEG C, 15sec, 68 DEG C, 6min.Follow After ring terminates, -80 DEG C of the cDNA double-strands that will be synthesized in centrifuge tube preserve.

(3) ECCT encoding genes are cloned：

Engineer synthesizes forward primer and enters performing PCR amplification, and its sequence is 5'-ATGGAGGGCTGTTTCTGGAAGACC- 3'(SEQ ID NO:3), another amplimers of PCR are the 3 '-PCR primer built in the kit of storehouse, its sequence is 5 '- CTAGAGGCCGAGGCGGCCGACATG-3’(SEQ ID NO:4).PCR reactions are carried out under the following conditions：94 DEG C of 5min, 94 DEG C 30sec, 58 DEG C of 30sec and 72 DEG C of 1min, 30 circulations.After the completion of amplification mesh is carried out with glue reclaim kit (Tiangeng biology) Fragment recovery.The purpose fragment of recovery is connected to pMD19-T carriers (Takara, Dalian), is transformed into CaCl₂-MgCl₂Method The DH5 α competent cells prepared.Coated plate simultaneously carries out ampicillin and blue hickie Double Selection, and picking single bacterium colony is drawn with M13 Thing PCR detects Insert Fragment size.Picking positive bacterium colony, bacterium extraction plasmid is shaken, uses Applied Biosystems DNA Sequencer, model ABI PRISM 377 carry out nucleotide sequencing.

Measurement result：

The gene of coding ECCT precursors shown in SEQ ID NO.2 is held to 3 ' terminal sequences from 5 '：

It is 469-558 positions nucleotides wherein to encode ECCT.

The encoding gene nucleotides sequence list of ECCT precursors is：Sequence length is 720 bases, sequence type：Nucleic acid, chain Number：It is single-stranded, topology：Straight-chain, sequence species：CDNA, source：Water snake lung.

Embodiment 2

ECCT chemical synthesis：

Ith, ECCT chemical synthesis process：The ripe peptide amino acid sequence derived according to gene, with automatic Peptide synthesizer (433A, Applied Biosystems) synthesizes its complete sequence, passes through HPLC reversed phase column chromatography desalinations.

IIth, molecular weight determination uses MALDI-TOF-MS (MALDI-TOF).

IIIth, the ECCT of purifying identifies its purity with high-efficient liquid phase chromatogram HPLC method, and molecular weight determination uses Matrix-assisted MALDI-TOF-MS (MALDI-TOF), isoelectric focusing electrophoresis measure isoelectric point, with automatic Protein Sequencer Determine amino acid sequence structure.

ECCT is a kind of straight-chain polypeptide of ECCT gene codes, containing 30 amino acid residues, molecular weight 3662.7Da, etc. Electricity point 12.33, its SEQ ID NO.1 amino acid sequence are：

Ser¹Arg²Lys³Leu⁴Lys⁵Lys⁶Phe⁷Phe⁸Arg⁹Lys¹⁰Val¹¹Lys¹²Lys¹³Gly¹⁴Ile¹⁵Lys¹⁶Lys¹⁷Val¹⁸Phe¹⁹L ys²⁰Lys²¹Val²²Val²³Lys²⁴Ala²⁵Ile²⁶Phe²⁷Arg²⁸Leu²⁹Leu³⁰。

Embodiment 3

ECCT antibacterial activities detect：

(1) test strain that picking is stored on inclined-plane respectively is spread evenly across MH solid mediums and (is purchased from Qingdao Hai Bo Bioisystech Co., Ltd) on flat board, the filter paper of the 0.5cm diameters by sterilizing is placed in media surface, dissolving is added dropwise In the 2mg/ml of the sterile deionized water μ l of ECCT sample solutions 10, culture 18-20 hours are inverted in 37 DEG C, observe inhibition zone Whether formed.If sample has antibacterial activity, Clear ＆ Transparent inhibition zone, the bigger table of inhibition zone can be formed around filter paper Bright sample antibacterial activity is stronger.

(2) ECCT minimal inhibitory concentrations (Minimum Inhibitory Concentration) measure (2 times of dilution methods)：

The bacterial strain for having inhibition zone in step experiment in selection carries out MIC determination experiments.Test strain is inoculated into the training of MH liquid Support in base (Qingdao Hai Bo Bioisystech Co., Ltd), 37 DEG C of shaken cultivations to exponential phase, then trained with fresh MH liquid Support base and culture to the nutrient solution of exponential phase is diluted to 2 × 10⁵Cfu/ml is stand-by.

100 μ l MH fluid nutrient mediums are previously added in sterile each hole of 96 orifice plate, 100 μ l are then added in the first hole The certain density ECCT sample solutions through 0.22 μm of hole membrane filtration are diluted to MH fluid nutrient mediums, 100 μ are taken after mixing L adds the 2nd hole, successively doubling dilution (referring to table 1), and suctioning out 100 μ l from the 9th hole discards, the 10th hole system control tube.

Table .1 dilution process

37 DEG C of placement slowly vibrating culture 18 hours, determines light absorbs at 600nm wavelength after above-mentioned each pipe is mixed.Most Small Mlc is the minimum sample concentration of invisible bacterial growth.As a result it is as shown in table 2.

From table 2, ECCT is to going out very strong antibacterial activity, and including largely pathogenic bacteria are clinically separated, MIC value is in 1.17-37.5 μ g/ml scope.

The ECCT antibacterial activities of table 2

MIC：Minimal inhibitory concentration, result above repeat laboratory mean values to be independent three times.

Embodiment 4

ECCT sterilization speeds determine

Escherichia coli ATCC25922 cultivates 12 with MH fluid nutrient mediums (Qingdao Hai Bo Bioisystech Co., Ltd) at 37 DEG C Hour, then it is diluted to 10 with fresh MH fluid nutrient mediums⁶CFU/ml bacteria suspension.It will be dissolved in the deionized water of sterilizing ECCT samples be added in bacteria suspension, make final concentration of 5 × MIC (93.75 μ g/ml).The bacterium solution for adding ECCT samples is put Concussion and cultivate in 37 DEG C of incubators is placed in, took 50 μ l bacterium solutions to dilute at 0,5,10,20,30,45,60,90 and 120 minute respectively 1000 times, the bacterium solution for then taking 50 μ l to dilute is applied on MH solid mediums, and bacterium colony counts after 37 DEG C of incubator overnight incubations. The experiment is by the use of Meropenem as positive control, and the deionized water of sterilizing is as negative control.

As a result as shown in table 3, ECCT bactericidal actions are rapid, and all Escherichia coli can be killed in 10 minutes ATCC25922 cells.And positive control Meropenem needs 120 minutes could kill all bacterial cells.Illustrate killing for ECCT Bacterium speed is faster than conventional antibiotic.

The ECCT sterilization speeds of table 3

Embodiment 5

ECCT anti-inflammatory activities determine

Brewer thioglycollate medium (Sigma-Aldrich, USA) is expelled in C57 mouse peritoneals, is located after 3 days Dead mouse, collect Turnover of Mouse Peritoneal Macrophages.The Turnover of Mouse Peritoneal Macrophages of collection (contains 10% tire with RPMI-1640 nutrient solutions Cow's serum, 100U/ml penicillin, 100 μ g/ml streptomysins) culture, plate is planted after cell count, is made in 96 orifice plates per hole cell number For 1 × 10⁴It is individual.After cell attachment, upper cell nutrient solution is sucked, fresh RPMI-1640 nutrient solutions is added and (contains 10% tire ox blood Clearly, 100U/ml penicillin, 100 μ g/ml streptomysins).Add ECCT samples (various concentrations gradient) and Helicobacter pylori lipopolysaccharide LPS (final concentration of 100ng/ml), cell culture incubator culture 6h, is collected by centrifugation cell, using Trizol reagents (Life tech, The U.S.) extraction cell total rna, utilize Takara PrimeSript 1st Strand cDNA Synthesis Kit (Takara, Japan) Reverse Transcriptase kit reverse transcription synthesis chains of cDNA mono-, utilize SYBR Premix Ex Taq^TM II(Tli RNaseH Plus) two-step qRT-PCR Kit (Takara, Japan) carry out qRT-PCR experiment detection ECCT samples to deep and remote The influence of the proinflammatory factor gene expression of Helicobacter pylori LPS inductions.

As a result as shown in figure 1, ECCT can significantly inhibit what helicobacter pylori LPS in Turnover of Mouse Peritoneal Macrophages was induced INOS genes and proinflammatory factor IL-1 β, IL-6, TNF-α gene transcription, show that ECCT has stronger anti-inflammatory activity.

Embodiment 6

ECCT antitumor cytolytic activities

With mtt assay detection ECCT samples anti tumor activity in vitro, experiment with tumor cell line include prostate cancer PC3, Liver cancer HepG2, colon cancer Colon38 and stomach cancer BGC823.Cell (contains 10% hyclone, 100U/ with the high sugared nutrient solutions of DMEM Ml penicillin, 100 μ g/ml streptomysins) culture, after cell covers with, with 0.25% Trypsin Induced, 2 are washed with nutrient solution Secondary, suspension cell, plate is planted after cell count again, and it is 1 × 10 to make every hole cell number in 96 orifice plates⁵It is individual, per the μ l of pore volume 200. Cell culture incubator culture 3h, adds the ECCT samples of various concentrations gradient after cell attachment, and blank control group adds same volume Long-pending sterile deionized water, continue in cell culture incubator to cultivate 48h.20 μ l MTT solution of every hole addition (5mg/ml, it is molten with PBS Solution), continue to cultivate 4h.Culture is terminated, carefully sucks nutrient solution in hole.150 μ l dimethyl sulfoxide (DMSO)s are added in per hole, liquid-transfering gun blows Beating is completely dissolved purple crystal.ELIASA detects light absorbs, measure wavelength 490nm.Inhibiting rate=A_Blank-A_Sample/A_Blank× 100%, using ECCT concentration as abscissa, mapped by ordinate of inhibiting rate, calculate halves of the ECCT to different tumor cell lines Inhibiting rate IC₅₀Value.

As a result show, ECCT samples show stronger antitumor activity to 4 plants of tumour cells, to PC3, HepG2, Colon38 and BGC823 IC₅₀Value is respectively 18.27,61.61,10.92 and 14.37 μM.

Described above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is some improvement and Modification, these improvement and modification also should be regarded as protection scope of the present invention.

Claims

A kind of 1. natural anti-infective antitumor bifunctional polypeptides ECCT, it is characterised in that：ECCT is by 30 amino acid residue groups Into straight-chain polypeptide, the Da of molecular weight 3662.7, isoelectric point 12.33, the primary structure of its amino acid sequence is SEQ ID NO.1 It is shown.
2. the encoding gene of the natural anti-infective antitumor bifunctional polypeptides ECCT described in claim 1, it is characterised in that：It is described Polypeptide ECCT precursor coding genes are made up of 720 nucleotides, and the base sequence from 5 ' ends to 3 ' ends is SEQ ID NO.2 institutes Show, and encode the ECCT 469-558 positions nucleotides for the SEQ ID NO.2 sequences.
3. natural anti-infective antitumor bifunctional polypeptides ECCT preparation method described in claim 1, it is characterised in that：By following Step is carried out successively：

1）Extract the total serum IgE of water snake lung tissue；

2）Construction cDNA library；

3）Synthetic pcr primer thing is designed, enters performing PCR and expands to obtain ECCT coding gene sequence；

4）Ripe peptide amino acid sequence is derived according to the ECCT coding gene sequences of PCR amplification gained；

5）With automatic Peptide synthesizer synthesis polypeptide ECCT complete sequence；

6）Pass through HPLC reversed phase column chromatography desalinations.
4. natural anti-infective antitumor bifunctional polypeptides ECCT preparation method according to claim 3, it is characterised in that：Step Rapid 3）Middle PCR primer includes forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4.
5. natural anti-infective antitumor bifunctional polypeptides ECCT application described in claim 1, it is characterised in that：It is antibacterial preparing Anti-inflammatory drugs, antineoplastic, preservative, animal feed and cosmetic caused by medicine, germ killing drugs, anti-helicobacter pylori LPS Application in product additive.
6. application according to claim 5, it is characterised in that：Suppress gram-positive bacterium, Gram-negative preparing Application in the medicine of bacterium and fungi
7. application according to claim 5, it is characterised in that：Suppress Escherichia coli, shigella dysenteriae, kerekou pneumonia preparing Primary bacterium, Klebsiella oxytoca, proteus mirabilis, stenotrophomonas maltophilia, pseudomonas aeruginosa, paratyphoid A Salmonella Bacterium, staphylococcus aureus, bacillus cereus, bacillus subtilis, VREF, Nocardia asteroides, Candida albicans and Application in the medicine of Candida glabrata.
8. application according to claim 5, it is characterised in that：Prepare anti-prostate cancer tumour medicine, anti-liver cancer and anti-tumour medicine Application in thing, inhibitor against colon carcinoma cells tumour medicine and anti-gastric cancer tumour medicine.
9. the application of natural anti-infective antitumor bifunctional polypeptides ECCT encoding gene according to claim 2, its feature It is：It is thin that the base sequence of the polypeptide ECCT is applied to polypeptide recombination expression, transgenic animals, plant, plant part, animal In born of the same parents or plant cell.