CN107163148A - Recombinant protein of pulmonary surfactant protein B propetides and its preparation method and application - Google Patents
Recombinant protein of pulmonary surfactant protein B propetides and its preparation method and application Download PDFInfo
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- CN107163148A CN107163148A CN201710340260.7A CN201710340260A CN107163148A CN 107163148 A CN107163148 A CN 107163148A CN 201710340260 A CN201710340260 A CN 201710340260A CN 107163148 A CN107163148 A CN 107163148A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C12N15/09—Recombinant DNA-technology
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Abstract
The present invention relates to genetic engineering field, recombinant protein of pulmonary surfactant protein B propetides and its preparation method and application is disclosed.Specifically, the recombinant protein has SEQ ID NO：Amino acid sequence shown in 1, or in SEQ ID NO：1 amino terminal and/or carboxyl terminal are connected with the amino acid sequence of label.The present invention obtains proSP B recombinant proteins using the method for genetic engineering, and the cytotoxicity of the proSP B recombinant proteins is low, and shows good antibacterial activity, with good actual application prospect.
Description
Technical field
The present invention relates to genetic engineering field, and in particular to a kind of recombinant protein of pulmonary surfactant protein B propetides, coding
The nucleic acid of the recombinant protein, a kind of recombinant plasmid, a kind of recombinant bacterial strain, a kind of restructuring for preparing pulmonary surfactant protein B propetides
The method of albumen and their application.
Background technology
Since antibiotic is found, great effect is played during human disease treatment, many has been cured
The infectious diseases that the mankind feel simply helpless, has saved the life of many people, greatly improves the average life span of the mankind.But by
In their a large amount of uses, abuse and it is lack of standardization the reason such as use, cause substantial amounts of antibody-resistant bacterium to occur, propagate, cause infection
Property disease treatment complicate, medical expense increase, the incidence of disease, the death rate increase so that the drug resistance of infectious bacteria turn into one
Unavoidable problem.And these traditional anti-infectives also have ototoxicity, renal toxicity, hepatotoxicity wind agitation, neurotoxicity, the heart
Dysentery, allergy and to hemopoietic system into cause damage etc. many obvious toxic side effects, so as to be caused to patient
Unpredictalbe influence, in addition entail dangers to patient life.Due to many unfavorable factors of conventional antibiotic medicine, therefore find
Effective substitute of antibiotic, developing the antibacterials that new toxicity is low and effect is good has the meaning of reality.
And antibacterial peptide, as the new antibacterials of a class, development in recent years is very swift and violent.Wherein, lung surface active
Material due to be as expressed by human body own cells, secretion material, thereby increases and it is possible to anti-inflammatory, antibacterial activity, therefore, with lung table
A class antibacterials are developed based on the active material of face has substantial promise.Pulmonary surfactant protein (surfactant
Protein, SP) it is used as one of material mostly important in pulmonary surfactant, the antibacterial of SP-B albumen particularly therein
Effect, is increasingly paid close attention to by people.But, the research such as Marnie finds that the antibacterial action of ripe SP-B albumen only occurs
In external, in body, ripe SP-B and Phospholipids are interrelated, and Phospholipids and SP-B combination are relatively stable, it is impossible to so that
SP-B acts on external bacterium to play its antibacterial functions, and ripe SP-B it is non-selective act on phospholipid molecule
Layer, cell lysis film shows non-selective dissolving body cell, particularly red blood cell, causes the body injuries such as haemolysis.Cause
This, sight is transferred on pulmonary surfactant protein B propetides (prosurfactant protein-B, proSP-B) by people again,
But, proSP-B is because with stronger hydrophobicity, it is difficult to be obtained by chemical synthesis mode, which increase to such material
Further R and D.Therefore, it is badly in need of finding a kind of method for obtaining the proSP-B with good antibacterial activity at present,
Think to develop and can both overcome many defects of conventional antibiotic, the novel antibacterial medicine with good antibacterial effect establishes base again
Plinth.
The content of the invention
The invention aims to the above mentioned problem for overcoming prior art presence, there is provided the restructuring egg of proSP-B a kind of
In vain, encode the nucleic acid of the recombinant protein, recombinant plasmid, recombinant bacterial strain and prepare proSP-B recombinant protein method and it
Application.The method provided using the present invention, successfully obtains proSP-B recombinant proteins, the proSP-B recombinant proteins
Cytotoxicity it is low, and show good antibacterial activity.
To achieve these goals, in a first aspect, the invention provides a kind of restructuring egg of pulmonary surfactant protein B propetides
In vain, wherein, the recombinant protein has SEQ ID NO：Amino acid sequence shown in 1, or in SEQ ID NO：1 amino end
End and/or carboxyl terminal are connected with the amino acid sequence of label.
Second aspect, present invention also offers the nucleic acid that can encode above-mentioned recombinant protein.
The third aspect, present invention also offers a kind of recombinant plasmid, the recombinant plasmid contains above-mentioned nucleic acid.
Fourth aspect, present invention also offers a kind of recombinant bacterial strain, the recombinant bacterial strain contains above-mentioned recombinant plasmid.
5th aspect, present invention also offers a kind of method for the recombinant protein for preparing pulmonary surfactant protein B propetides, its
In, this method comprises the following steps：
(1) by above-mentioned recombinant plasmid transformed into expressive host bacterium, to obtain recombinant bacterial strain；
(2) recombinant bacterial strain carries out Fiber differentiation after culture in the presence of derivant, to obtain nutrient solution；
(3) nutrient solution that will be obtained by step (2) carries out purifying and label is removed, to obtain before pulmonary surfactant protein B
The recombinant protein of peptide.
The recombinant protein that above-mentioned recombinant protein, nucleic acid, recombinant plasmid, recombinant bacterial strain and above-mentioned method are prepared
Applied in antimicrobial is prepared；Preferably, the antimicrobial is anti-Staphylococcus aureus (Staphylococcus
Aureus), Escherichia coli (Escherichia coli), Microsporum gypseum (Microsporum gypseum), white
Candida albicans (Monilia albican), Trichophyton rubrum (Trichophyton rubrum), pseudomonas aeruginosa
(Pseudomonas aeruginosa) and methicillin-resistant staphylococcus aureus (Methicillin-resistant
Staphylococcus aureus) at least one of medicine.
ProSP-B is because with stronger hydrophobicity, it is difficult to be obtained by chemical synthesis mode, the present invention uses gene work
Cheng Fangfa, especially by the specific expression vector of use (pGEX4T-1) and expressive host bacterium (e. coli bl21 (DE3)
PLysS bacterial strains), the higher proSP-B recombinant proteins of concentration are successfully obtained, also, by by pGEX4T-1 carriers
GST labels are merged with proSP-B, because GST labels have hydrophily, can significantly improve the water-soluble of proSP-B recombinant proteins
Property, expand its application.
In addition, the proSP-B recombinant proteins that the present invention is provided show good broad spectrum antibiotic activity, it is to clinically normal
Bacterium, the fungi seen is respectively provided with stronger inhibitory action, particularly to staphylococcus aureus, Escherichia coli, the small spore of gypsum sample
Daughter bacteria, Candida albicans, Trichophyton rubrum, pseudomonas aeruginosa and methicillin-resistant staphylococcus aureus are shown significantly
Inhibitory activity.Further investigation revealed that, proSP-B recombinant proteins play its antibacterial action may be by influence bacterium
Membrane passage, so as to suppress the normal growth of bacterium.Meanwhile, the cell for the proSP-B recombinant proteins that the present invention is provided
Toxicity is smaller, even if protein concentration is brought up into 1mg/mL (concentration is far longer than its MIC value to bacterium), the increasing of cell
Grow ability and be still higher than 59%, the proSP-B recombinant proteins that this explanation present invention is provided have higher security.
Brief description of the drawings
The result that PCR primer is identified in embodiments of the invention 1 is shown in Fig. 1；
The qualification result of recombinant plasmid pET30-proSP-B in embodiments of the invention 1 is shown in Fig. 2；
The qualification result of recombinant plasmid pGEX4T-1-proSP-B in embodiments of the invention 1 is shown in Fig. 3；
The SDS-PAGE results of proSP-B recombinant proteins induced expression in embodiments of the invention 1 are shown in Fig. 4；
The Western-blot knots of proSP-B recombinant proteins induced expression in embodiments of the invention 1 are shown in Fig. 5
Really；
Fig. 6 be shown the proSP-B recombinant proteins of induced expression in embodiments of the invention 1 it is purified after SDS-
PAGE and SDS-PAGE；
The result that the antimicrobial spectrum of proSP-B recombinant proteins in embodiments of the invention 3 is determined is shown in Fig. 7.
Embodiment
The end points and any value of disclosed scope are not limited to the accurate scope or value herein, these scopes or
Value should be understood to comprising the value close to these scopes or value.For number range, between the endpoint value of each scope, respectively
It can be combined with each other between the endpoint value of individual scope and single point value, and individually between point value and obtain one or more
New number range, these number ranges should be considered as specific open herein.
In a first aspect, the invention provides a kind of recombinant protein of pulmonary surfactant protein B propetides, wherein, the restructuring
Albumen has SEQ ID NO：Amino acid sequence shown in 1, or in SEQ ID NO：1 amino terminal and/or carboxyl terminal
It is connected with the amino acid sequence of label.
SEQ ID NO：Amino acid sequence shown in 1 is： AGANDLCQECEDIVHLLTKMTKEDAFQDTIRKFLEQEC
DILPLKLLVPRC RQVLDVYLPLVIDYFQGQIKPKAICSHVGLC。
In the present invention, described " amino acid sequence for being connected with label " refers to using the common label in this area to SEQ
ID NO：Amino acid sequence shown in 1 is added and modified.There is stronger hydrophobicity in view of proSP-B, preferred
In the case of, the label has hydrophily.It is highly preferred that the label is GST labels.
In the present invention, GST (Glutathione S-transferase, the glutathione S-transferase) label can
To be GST labels commonly used in the art, it can be introduced by expression vector.In situations where it is preferred, the GST labels
No. GenBank of amino acid sequence be：AMQ34028.1 (is specifically referred tohttps://www.ncbi.nlm.nih.gov/ protein/1005883217)。
Second aspect, the nucleic acid of above-mentioned recombinant protein can be encoded present invention also offers a kind of.
According to the present invention, the nucleic acid has SEQ ID NO：Nucleotide sequence shown in 2, or in SEQ ID NO：2
Amino terminal and/or carboxyl terminal are connected with the nucleotide sequence of label；Preferably, the label has hydrophily；It is highly preferred that
The label is GST labels.
SEQ ID NO：Nucleotide sequence shown in 2 is： gcaggagctaatgacctgtgccaagagtgtgaggatattg
tccacctcctcacaaagatgaccaaggaagacgcttt ccaggacacgatccggaagttcctggaacaagaatgtga
tatcctacccttgaagctgcttgtgccccggtgtcgcc aagtgcttgatgtctacctgcccctggttatcgactac
ttccagggccagattaaacccaaagccatctgcagtcatgt gggcctgtgc。
In the present invention, because the nucleic acid that the present invention is provided has stronger hydrophobicity, it is difficult to be obtained by artificial synthesized method
, therefore, the nucleic acid that the present invention is provided generally is obtained using gene engineering method, for example, can pass through PCR
(PCR) TRAP is obtained.Specifically, those skilled in the art can be according to nucleotide sequence provided by the present invention, can be very
Template and corresponding primer are readily obtained, carrying out amplification using PCR obtains relevant sequence.Once relevant nucleotide sequence is obtained,
Relevant amino acid sequence just can be obtained with recombination method is large batch of.Gained nucleotide sequence is generally cloned into expression vector, then
It is transferred in expressive host bacterium, it is then isolated about nucleotide sequence from the expressive host bacterium after propagation by conventional method.
In the present invention, described " nucleotide sequence for being connected with label " refers to using the common label in this area to SEQ ID
NO：Nucleotide sequence shown in 2 is added and modified.There is stronger hydrophobicity in view of proSP-B, in preferred situation
Under, the label has hydrophily.It is highly preferred that the label is GST labels.
In the present invention, the GST labels can be GST labels commonly used in the art, and it can be carried by expressing
Body is introduced, and is preferably introduced by pGEX4T-1 plasmids.In situations where it is preferred, the nucleotide sequence of the GST labels is GenBank
Number：Sequence shown in the 258th to the 959th of KU312308.1.
The third aspect, present invention also offers a kind of recombinant plasmid, the recombinant plasmid contains above-mentioned nucleic acid.
In the present invention, the plasmid that the recombinant plasmid is used is preferably pET30a plasmids and/or pGEX4T-1 plasmids
(can be obtained by commercially available means).The recombinant plasmid, which can be used, to have cleavage site in vector multiple cloning site
Various endonucleases carry out digestion obtain linear plasmid, then with using identical nucleic acid inscribe cleavage nucleic acid fragment connect
Connect, obtain recombinant plasmid.For example, for pET30a plasmids, Xhol I, Not I, Eag I, Hind III, EcoR can be used
The restriction endonucleases such as I, BamH I, Nco I, Kpn I, BgI II, Nde I；For pGEX4T-1 plasmids, can use Xhol I,
The restriction endonucleases such as Not I, EcoR I, BamH I, Sma I.
The present invention it is a kind of preferred embodiment in, the recombinant plasmid be pGEX4T-1 plasmids BamH I and
SEQ ID NO are inserted between Xho I restriction enzyme sites：Expression vector obtained from nucleotide sequence shown in 2.
Fourth aspect, present invention also offers a kind of recombinant bacterial strain, wherein, the recombinant bacterial strain contains above-mentioned recombinant plasmid；
Preferably, the recombinant bacterial strain is to contain above-mentioned recombinant plasmid e. coli bl21 (DE3) pLysS bacterial strains
The present invention can be entered recombinant plasmid transformed in expressive host bacterium using method commonly used in the art, to obtain
Obtain recombinant bacterial strain.It is, for example, possible to use heat shock method, calcium chloride chemical transformation etc., preferably heat shock method.
In the present invention, the expressive host bacterium can be any bacterium for being adapted as host commonly used in the art
Or fungi, for example, can be Escherichia coli.Preferably, for preserving the e.colistraindh5α of recombinant plasmid, for expressing
E. coli bl21 (DE3) pLysS bacterial strains.The expressive host bacterium that the present invention is used can be obtained by conventional commercially available means
.
5th aspect, present invention also offers a kind of method for the recombinant protein for preparing pulmonary surfactant protein B propetides, its
In, this method comprises the following steps：
(1) by above-mentioned recombinant plasmid transformed into expressive host bacterium, to obtain recombinant bacterial strain；
(2) recombinant bacterial strain carries out Fiber differentiation after culture in the presence of derivant, to obtain nutrient solution；
(3) nutrient solution that will be obtained by step (2) carries out purifying and label is removed, to obtain before pulmonary surfactant protein B
The recombinant protein of peptide.
In the present invention, in step (1), recombinant plasmid transformed can be entered using method commonly used in the art
In expressive host bacterium, to obtain recombinant bacterial strain.It is, for example, possible to use heat shock method, calcium chloride chemical transformation etc., preferably heat shock
Method.
In the present invention, in step (1), the expressive host bacterium can arbitrarily be suitable to work for commonly used in the art
For the bacterium or fungi of host, for example, can be Escherichia coli.Preferably, the expressive host bacterium for preserving recombinant plasmid is
E.colistraindh5α, the expressive host bacterium for expression is e. coli bl21 (DE3) pLysS bacterial strains.The present invention is used
Expressive host bacterium can pass through conventional commercially available means and obtain.
In the present invention, in step (2), there is no particular limitation for species of the present invention to the derivant, Ke Yiwei
The conventional selection of this area, for example, the derivant can be isopropyl-β-d- thiogalactosides (IPTG), and described is lured
The concentration for leading agent is 0.5-2mmol/L, preferably 0.8-1.5mmol/L.
In the present invention, in step (2), there is no particular limitation for condition of the present invention to the Fiber differentiation, as long as
The purpose of induction expression of recombinant proteins can be reached.In situations where it is preferred, the condition of the Fiber differentiation includes：Temperature
For 28-37 DEG C, preferably 28-35 DEG C, more preferably 28-32 DEG C；Time is more than 3h, more preferably preferably 3-24h, 12-
24h。
In the present invention, in step (3), the mode that the present invention is removed to the purifying and label is not limited particularly
It is fixed, as long as the purpose that protein purification and label are removed can be realized, for example, it is pure albumen can be carried out by way of absorption
Change, protease can be used to enter row label removal.In a kind of specific embodiment of the present invention, for pGEX4T-1 matter
Grain, using the prepacked columns of GSTrap FF 16/10 (can be obtained by conventional commercially available means) carry out recombinant protein purifying and
Label is removed.
6th aspect, present invention also offers above-mentioned recombinant protein, above-mentioned nucleic acid, above-mentioned recombinant plasmid, above-mentioned recombinant bacterium
The application of strain, the recombinant protein prepared by the above method in antimicrobial is prepared；Preferably, the antimicrobial is anti-golden yellow
Color staphylococcus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Microsporum gypseum
(Microsporum gypseum), Candida albicans (Monilia albican), Trichophyton rubrum (Trichophyton
Rubrum), pseudomonas aeruginosa (Pseudomonas aeruginosa) and methicillin-resistant staphylococcus aureus
The medicine of at least one of (Methicillin-resistant Staphylococcus aureus)；It is highly preferred that described
Antimicrobial is anti-Staphylococcus aureus, Escherichia coli, Microsporum gypseum, Candida albicans, Trichophyton rubrum and resistance to first
The medicine of at least one of oxygen XiLin staphylococcus aureus；It is further preferred that the antimicrobial is anti-golden yellow grape
Coccus and/or the medicine of methicillin-resistant staphylococcus aureus.
In application of the present invention, above-mentioned recombinant protein, above-mentioned nucleic acid, above-mentioned recombinant plasmid, above-mentioned recombinant bacterial strain,
The pH value that the recombinant protein prepared by the above method is used in the antimicrobial is 3-8, preferably 5-6, is more preferably
5.4-6。
The present invention will be described in detail by way of examples below.
In the examples below：
SD rats are purchased from aseptic sursery research institute of great Ping hospitals Experimental Animal Center；
Bacillus coli DH 5 alpha is purchased from Shanghai Ang Yu bio tech ltd；E. coli bl21 (DE3) plysS is purchased from
Ocean biotech firm of Beijing CHMC, Escherichia coli, staphylococcus aureus, Microsporum gypseum, Candida albicans, red hair tinea
Bacterium, pseudomonas aeruginosa are purchased from Beijing North Na Chuanlian Bioteknologisk Institut, methicillin-resistant staphylococcus aureus (numbering
ATCC 43300) it is purchased from Guangdong Province's Culture Collection；
CCL-149 cell lines grind biotechnology company purchased from upper sea valley；PGEX4T-1 has purchased from the friendly section's biotechnology in Shanghai
Limit company；BamH I, Hind III, Xho I are purchased from NEB companies of the U.S.；T4 ligases, total RNA extraction reagent box, converse turn
Record kit is purchased from GeneCopoeia companies of the U.S.；DNA glue reclaims kit, DNA Loading Buffer,
DNAMarker is purchased from Japanese Takara companies；Propidium iodide (PI) is purchased from Shanghai Xiang Sheng bio tech ltd；LDH is tried
Agent box is purchased from green skies bio tech ltd.Remaining experiment reagent and consumptive material can be obtained by conventional commercially available means
.
Embodiment 1
The present embodiment is used for the preparation method for illustrating the proSP-B recombinant proteins that the present invention is provided.
1st, lung tissue of rats Total RNAs extraction
(1) lung tissue of rats total serum IgE is extracted using total RNA extraction reagent box (being purchased from GeneCopoeia companies of the U.S.)；
(2) RNA electrophoresis detection
Electrophoresis, gel imager detection and analysis RNA mass are carried out using the gel electrophoresis liquid newly configured.
2nd, the acquisition and amplification of proSP-B gene cDNAs
(1) cDNA fragments are obtained
Reverse transcription reaction system：
The μ g of total serum IgE 1
Oli(dT)18 1μL
Free RNase Water to 13 μ L
In reacting 10min at 65 DEG C, then in preservation at 4 DEG C.
Reaction system：
Reaction condition：In reacting 60min at 42 DEG C, 5min is reacted at 85 DEG C, then in 4 DEG C of preservations.Finally will be anti-
The product of transcription is placed on -80 DEG C of refrigerators and preserved for a long time.
(2) performing PCR amplification is entered by template of cDNA
(a) primer sequence is designed
According to pET30a plasmid maps and BamH I and the restriction enzyme sites of Hind III, set with the softwares of Primer Premier 5.0
Upstream and downstream primer is counted, its primer sequence is as follows.
proSP-B-F 5'-AAAGGATCCGCAGGAGCTAATGACCTG-3'(SEQ ID NO：3)
proSP-B-R 5'-TTTAAGCTTTTAGCACAGGCCCACATG-3'(SEQ ID NO：4)
(b) reaction solution is prepared：
(c) PCR reaction conditions：95 DEG C of reaction 5min；95 DEG C of denaturation 30s, 64 DEG C of annealing 30s, 72 DEG C of extension 45s, totally 39
Individual circulation；72 DEG C of extension 6min, 4 DEG C of preservations.
(d) PCR primer is identified
After PCR reactions terminate, enter on row agarose gel electrophoresis detection, gel imaging system and observe band, analysis result.
As a result as shown in figure 1, occurring band at about 250bp as shown in Figure 1, size meets target gene size, preliminary proof reversion
Record successfully.
(e) QIAquick Gel Extraction Kit of being purified with PCR primer carries out the purifying of PCR primer.
(3) structure of pET30-proSP-B expression vectors, identification
Double digestion, and gel extraction digestion products are carried out to pET30 and proSP-B fragments using BamH I, Hind III,
4 DEG C of T4DNA ligases are connected overnight.
(a) BamH I, Hind III's double digestion reaction system：
Reaction condition：Thermostat water bath, 37 DEG C, 8h, agarose gel electrophoresis detection.
(b) carrier, purpose fragment gel extraction
The gel extraction of product and purifying are carried out by DNA purifying QIAquick Gel Extraction Kit specifications.
(c) T4DNA ligases reaction system：
(d) conversion and identification of recombinant plasmid：
10 μ L connection products are all added in 100 μ L bacillus coli DH 5 alpha competence, turned with PCR instrument
The plasmid import operation of change, process is as follows：0 DEG C of reaction 30min, then at 42 DEG C of reaction 30s, then in 0 DEG C of preservation.
Then toward adding 300 μ L LB liquid mediums, 37 DEG C, 150r/min cultures 1h in product.Then it is coated on containing 50 μ
On that solid LB flat boards of g/mL cards, 37 DEG C of incubated overnights, then picking colony progress bacterium colony PCR identifications, are accredited as the positive
Bacterium colony carries out the upgrading grain that spreads cultivation, plasmid PCR and double digestion identification.The recombinant plasmid for being accredited as the positive is named as pET30-
ProSP-B, send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced.Sequencing result shows, the of the recombinant plasmid sequence
187-429 and SEQ ID NO：Sequence is consistent shown in 2.
PCR results are as shown in Figure 2.Specifically, by connection product conversion enter DH5 α competent cells in, through containing card that
Resistant panel is screened, shown in its bacterium colony PCR qualification results such as Fig. 2 (a), bacterium colony shaking flask plasmid agarose electrophoresis result such as Fig. 2 (b)
It is shown, shown in bacterium colony plasmid PCR qualification result such as Fig. 2 (c), shown in bacterium colony plasmid double digestion result such as Fig. 2 (d), as seen from the figure,
PCR results are single band, and its size about 250bp, are coincide with target gene proSP-B sizes, double digestion figure is displayed that
There is single band at 250bp, position is correct, meets with proSP-B sizes, preliminary proof recombinant plasmid pET30-proSP-B
Successfully construct.
Also, the result through the BLAST in NCBI understands that sequencing result and the proSP-B of positive plasmid match, this
Show the pET30-proSP-B vector constructions success built in experimentation.
(4) structure of pGEX4T-1-proSP-B expression vectors, identification
Double digestion and gel extraction digestion are carried out to pGEX4T-1 and pET30-proSP-B plasmids using BamH I, Xho I
Product, 4 DEG C of T4DNA ligases are connected overnight.
(a) endonuclease reaction system：
Double digestion reaction condition：Thermostat water bath, 37 DEG C, 8h, agarose gel electrophoresis detection.
(b) T4DNA ligases reaction system：
(c) conversion and identification of recombinant plasmid：
Connection product is transformed into bacillus coli DH 5 alpha competence, and carries out bacterium colony PCR identifications.To being accredited as the positive
Bacterium colony carries out the upgrading grain that spreads cultivation, plasmid PCR and double digestion identification.The recombinant plasmid for being accredited as the positive is named as pGEX4T-1-
ProSP-B, send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced.Sequencing result shows, the of the recombinant plasmid sequence
609-851 and SEQ ID NO：Sequence is consistent shown in 2.
PCR results are as shown in Figure 3.Specifically, connection product conversion is entered in DH5 α competent cells, through the benzyl containing ammonia
Resistant panel is screened, shown in its bacterium colony PCR qualification results such as Fig. 3 (a), bacterium colony shaking flask plasmid agarose electrophoresis result such as Fig. 3 (b)
It is shown, shown in bacterium colony plasmid PCR qualification result such as Fig. 3 (c), shown in bacterium colony plasmid double digestion result such as Fig. 3 (d), as seen from the figure,
PCR results are single band, and its size about 250bp, are coincide with target gene proSP-B sizes, double digestion figure is displayed that
There is single band at 250bp, position is correct, meets with proSP-B sizes, preliminary proof recombinant plasmid pGEX4T-1-
ProSP-B is successfully constructed.
Also, the result through the BLAST in NCBI understands that sequencing result and the proSP-B of positive plasmid match, this
Show the pGEX4T-1-proSP-B vector constructions success built in experimentation.
(5) proSP-B recombinant proteins induced expression and SDS-PAGE detections
Sequencing result is shown that correct recombinant plasmid pGEX4T-1-proSP-B is converted to e. coli bl21 (DE3)
In plysS.In 37 DEG C, 250r/min incubated overnights, next day is with 1:100 inoculum concentration is transferred in the fresh of the μ g/mL containing Amp 50
In LB fluid nutrient mediums, 37 DEG C, 250r/min shaken cultivations, when bacterium solution OD600 is about 0.8, draw part bacterium solution as luring
Leading is compareed, and 12000r/min is collected by centrifugation；Bacterium solution, which adds IPTG inductions, makes its final concentration of 1.0mmol/L, respectively at 30 DEG C
With 37 DEG C, 250r/min shaken cultivations, and 3h, 6h, 9h, 12h, 24h collect thalline, tested and analyzed with SDS-PAGE.
SDS-PAGE matches somebody with somebody colloid system：
SDS-PAGE conditions：
Constant pressure carries out electrophoresis, wherein concentration glue is 80V, separation gel is 120V, treats that albumen Marker is completely separable and occupies
Single file glue 3/4 when stop electrophoresis.
Dyeing：The complete glue of electrophoresis is removed into concentration glue, separation gel is placed in coomassie brilliant blue R250 dyeing liquor, room temperature water
1h is dyed on yawing bed；Then decolourized with destainer, coomassie brilliant blue R250 blue portion on glue is purified completely, take
Go out, take pictures.
As a result as indicated at 4.Specifically, the SDS-PAGE that Fig. 4 (A) is shown after being induced respectively at 37 DEG C and 30 DEG C is run
Cementing fruit.Wherein, the protein expression result of the thalline without induction is shown in band 1；Band 2, which is shown at 37 DEG C, to be induced
Thalline protein expression result；The protein expression result of the thalline induced at 30 DEG C is shown in band 3；What band 4 was shown
It is the protein expression result that 30 DEG C of hypothalluses crush supernatant；Band 5 is albumen Marker.From Fig. 4 (A), in 37 DEG C of conditions
Protein content obtained by lower induction is big without the amount obtained by induction under the conditions of 30 DEG C, and the protein content and whole cell that thalline supernatant contains
Amount it is basically identical, when thus illustrating 30 DEG C, recombinant protein exists in the form of soluble protein, and in induction at 30 DEG C of albumen
It is more suitable.
The situation of protein expression after induction different time at 30 DEG C is shown in Fig. 4 (B).Wherein, band 1 is albumen
Marker；The protein expression result of the thalline without induction is shown in band 2；Band 3 is shown after induction 3h on thalline
Clear protein expression result；The protein expression result of thalline supernatant after induction 6h is shown in band 4；Band 5, which is shown, to lure
Lead the protein expression result of thalline supernatant after 9h；The protein expression result of thalline supernatant after induction 12h is shown in band 6；Bar
With the 7 protein expression results that thalline supernatant after induction 24h is shown.From Fig. 4 (B), under the conditions of 30 DEG C, 3- is induced
24h, the expression quantity of destination protein is increased over time and gradually increased, to 12h after increase trend slow down.
(6) Western Blot detect proSP-B expression
After albumen is separated through SDA-PAGE, GST-proSP-B regions are cut according to albumen Marker positions, are turned
On print to pvdf membrane, Constant Electric Current turns, 250mA, 1h.1h is closed with the TBS-T containing 5% skimmed milk power；Carry out rabbit-anti rat
4 DEG C of refrigerator overnights of proSP-B polyclonal antibodies (1: 10000 dilution) are incubated；After second day has been washed with TBS-T, marked with HRP
Goat anti-rabbit igg secondary antibody (1: 5000 dilution) incubation at room temperature 1h, then Chemiluminescent is added dropwise after being washed through TBS-T
HRP Substrate luminescent solutions, are imaged with day energy luminescence imaging work station.
As a result as shown in figure 5, wherein, the protein expression result of the thalline without induction is shown in band 1；Band 2-7
Respectively it is shown that induction 3h, 6h, 9h, 12h and after 24 hours thalline supernatant protein expression result.As shown in Figure 5, it is of the invention
Obtained proSP-B recombinant proteins band is single band, and specific band occurs nothing but, and albumen size is with being expected unanimously,
The albumen of this explanation expression is proSP-B recombinant proteins.
(7) purifying of GST-proSP-B recombinant proteins
By 1:Recombinant bacterial strain pGEX4T-1-proSP-B is inoculated in the liquid LB of the 50ug/mL containing Amp by 100 inoculum concentration
In culture medium, thalline is collected by centrifugation after 30 DEG C of induction 12h.Bacterial sediment is resuspended in PBS (containing 0.1 mmol/LPMSF), ultrasonically treated
10min, 12000r/min centrifugation 20min collect supernatant, then with 0.45 μm of filtering with microporous membrane.Albumen protein purification system
Purified, wherein purification column is the prepacked columns of GSTrap FF 16/10.SDS-PAGE, Western Blot inspections are carried out after purification
Analysis is surveyed, and with BCA protein quantification kit measurement protein concentrations.After measured, the concentration of recombinant protein is about 1mg/mL.
SDS-PAGE, Western Blot testing results after purification are as shown in Figure 6.Specifically, Fig. 6 (A) is shown
SDS-PAGE results after albumen is purified, wherein, band 1 is albumen Marker；Band 2 is shown in induction at 30 DEG C
The result of thalline supernatant (before purification) after 12h；Band 3 is shown purified in the thalline supernatant after induction 12h at 30 DEG C
Result afterwards.Fig. 6 (B) be shown albumen it is purified after Western Blot results, wherein, band 1 be albumen marker；
The result for not inducing thalline supernatant is shown in band 2；Band 3 is shown in the thalline supernatant warp after induction 12h at 30 DEG C
Result after purification.Fig. 6 (A) and Fig. 6 (B) result show that can obtain the higher recombinant protein of expression quantity after purification (has
SEQ ID NO：Amino acid sequence shown in 1).
Embodiment 2
The present embodiment is used for the cytotoxicity for illustrating the proSP-B recombinant proteins that the present invention is provided
(1) the R/MINI1640 medium culture CCL-149 cells containing 10%FBS are used.
(2) stable CCL-149 cells will be cultivated, with trypsin digestion cell, cell is collected and dilutes regulation with culture medium
Cell concentration, makes it there are about 5000 cells in 100 μ L, 100 μ L CCL-149 is inoculated with the hole of 96 orifice plate the 1st~10 thin
Born of the same parents, add 100 μ L culture mediums, 37 DEG C, 5%CO in the 11st hole224h is cultivated in cell culture incubator.
(3) successively toward 96 orifice plates the 1st~9 hole add 10 μ L concentration be 1000 μ g/mL, 500 μ g/mL, 250 μ g/mL,
125 μ g/mL, 62.5 μ g/mL, 21.25 μ g/mL, 15.63 μ g/mL, 7.81 μ g/mL, the 3.91 μ g/mL step in embodiment 1
(7) obtain albumen after purification, the 10th, 11 holes add 10 μ L sterile purified waters.
(4) 96 orifice plates are placed in 37 DEG C, 5%CO212h is cultivated in cell culture incubator.
(5) 10 μ L CCK-8 solution are added toward every hole in 96 orifice plates.
(6) by 96 orifice plates in 37 DEG C, 5%CO2It is incubated 4 hours in cell culture incubator.
(7) absorbance is determined at 450nm with ELIASA.
(8) cell viability is calculated by following equation：
AC：The absorbance that cell is treated by proSP-B recombinant proteins solution, i.e. experimental group；
A0：Not celliferous absorbance, i.e. blank group；
AS：There are cell, but the absorbance that cell is not treated by proSP-B recombinant proteins solution, i.e. control group.
The result of cytotoxicity experiment is as shown in table 1.
Table 1
The IC for the proSP-B recombinant proteins that the present invention is provided can be calculated by the result of table 150=1595.68 μ g/mL,
Specifically, when the concentration of proSP-B recombinant proteins is 1mg/mL, its cell viability is 59.62%, illustrates that proSP-B is recombinated
The cytotoxicity of albumen is less, according to Cytotoxic evaluation standard-RGR toxicity grading methods, when cell is with respect to proliferation rate
(RGR) when >=75%, it is possible to determine that drug toxicity be 0 grade or 1 grade, when RGR is at 50%~74%, judge drug toxicity as
2 grades；Wherein 2 grades and the following toxicity for all illustrating medicine are acceptable, are safe.It is dense when proSP-B recombinant proteins
When degree is improved to 1595 μ g/mL, its cell proliferation rate is also above 50%, and it is 1595 μ g/ to illustrate proSP-B recombinant proteins concentration
During mL, it is low toxicity, can be used for drug development, and the treatment metering of general medicine is below 1595 μ g/mL, therefore is originally ground
The object proSP-B recombinant proteins studied carefully are entirely safe as class antibacterials exploitation.
Embodiment 3
The present embodiment is used for the antibacterial activity for illustrating the proSP-B recombinant proteins that the present invention is provided
1st, the activation of strain and the preparation of bacteria suspension
(1) staphylococcus aureus is cultivated：Staphylococcus aureus is activated on LB agar plates with method of scoring, 37
DEG C, 12h it is incubated, after single bacterium colony is grown, picking single bacterium colony is seeded in 10mL LB fluid nutrient mediums, 37 DEG C, 12h,
Bacteria suspension is made in 150r/min, culture, the distilled water that liquid-transfering gun is drawn bacterium solution and sterilized, and adjusts its concentration, is made 106cfu
Bacteria suspension.
(2) culture of Escherichia coli：Escherichia coli are activated on LB agar plates with method of scoring, 37 DEG C, 12h constant temperature
Culture, after single bacterium colony is grown, picking single bacterium colony is seeded in 10mL LB fluid nutrient mediums, 37 DEG C, 12h, 150r/min, training
Support, liquid-transfering gun draws bacterium solution and bacteria suspension is made with sterilized distilled water, adjusts its concentration, be made 106Cfu bacteria suspension.
(3) Trichophyton rubrum is cultivated：Prepare SDA culture mediums, pour into teat glass, formed inclined-plane, inclined-plane it is upper in
Lower three positions are inoculated with Trichophyton rubrum, 28 DEG C of incubated 10d, then toward adding sterilized distilled water in every test tube
2mL, makes the Trichophyton rubrum in pipe be scattered in liquid, adjusts its concentration, is made 106Cfu bacteria suspension.
(4) culture of Microsporum gypseum：Prepare SDA slant medium flat boards, three positions of upper, middle and lower on inclined-plane
Microsporum gypseum is inoculated with, 28 DEG C of incubated 10d, then toward sterilized distilled water 2mL is added in every test tube, make pipe
In Microsporum gypseum be scattered in liquid, adjust its concentration, be made 106Cfu bacteria suspension.
(5) Candida albicans：With method of scoring by Candida albicans work on LB agar plates, 28 DEG C, 10d it is incubated,
After single bacterium colony is grown, picking single bacterium colony is seeded in 10mL LB fluid nutrient mediums, 37 DEG C, 12h, 150r/min, is cultivated, and is moved
Liquid rifle draws bacterium solution and bacteria suspension is made with sterilized distilled water, adjusts its concentration, is made 106Cfu bacteria suspension.
(6) pseudomonas aeruginosa：Pseudomonas aeruginosa is activated on LB agar plates with method of scoring, 37 DEG C, 12h it is permanent
Temperature culture, after single bacterium colony is grown, picking single bacterium colony is seeded in 10mL LB fluid nutrient mediums, 37 DEG C, 12h, 150r/min,
Culture, liquid-transfering gun draws bacterium solution and bacteria suspension is made with sterilized distilled water, adjusts its concentration, is made 106Cfu bacteria suspension.
(7) methicillin-resistant staphylococcus aureus：With method of scoring by the methicillin-resistant staphylococcus aureus of preservation
Activate on LB agar plates, 37 DEG C, 12h it is incubated, after single bacterium colony is grown, picking single bacterium colony is seeded in 10mL LB liquid
In body culture medium, 37 DEG C, 12h, 150r/min, culture, liquid-transfering gun draw bacterium solution and bacteria suspension are made with sterilized distilled water, adjust
Its whole concentration, is made 106Cfu bacteria suspension.
2nd, the optimization of proSP-B recombinant proteins bacteriostatic experiment-pH value
(1) the albumen buffer solution prepared is adjusted to different pH value, different albumen buffer solutions are prepared into.
(2) preparation of antibacterial filter paper：Prepare about 6mm circular filter paper piece, 121 DEG C of sterilizing 20min, electric heating air blast
Drying box dries 2h.The gripping of filter paper aseptic nipper is put into the 500 μ g/mL albumen that step (7) is obtained in embodiment 1
Soaked in liquid, the filter paper after immersion is respectively put into sterile petri dish, dry excess surface liquid, to obtain the filter for soaking medicine
The scraps of paper；And using sterile saline as negative control, by positive control of penicillin or Terbinafine, (positive control of bacterium is
Penicillin, the positive control of fungi is Terbinafine).
(3) coating of strain：Liquid-transfering gun draws each μ L of bacteria suspension 50 for diluting and preparing, and glass spreading rod is uniformly smeared
In on each solid plate.
(4) pastille filter paper aseptic nipper will be soaked to filter paper, negative control filter paper, the positive control filter paper of medicine
It is affixed in flat board, mark is carried out at the culture dish back side with marking pen.
(5) thalline culture：The Escherichia coli of filter paper, staphylococcus aureus, pseudomonas aeruginosa, resistance to methoxy will have been pasted
XiLin staphylococcus aureus is inverted in 37 DEG C of bacteria bio incubator culture 12h；Trichophyton rubrum, Microsporum gypseum,
Candida albicans flat board is inverted in 28 DEG C of bacteria bio incubator culture 5d.
(6) the inhibition zone size (unit mm) of each flat board is measured with ruler, as a result as shown in table 2.Statistical analysis, screening is closed
Suitable pH value.
Table 2
As shown in Table 2, proSP-B recombinant proteins are respectively provided with certain antibacterial activity in pH 3-8, illustrate anti-
Bacterium peptide all has certain patience to faintly acid and alkalescent.Wherein, under weakly acidic condition, the antibacterial of proSP-B recombinant proteins
Activities present must become apparent from, and when pH is 5.6, its fungistatic effect is most strong, works as pH>When 5.6, its bacteriostatic level with pH value increase
And reduce, this may have relation with the conformation change of albumen, and its molecular conformation under alkaline environment changes, and have impact on anti-
The biological activity of bacterium peptide, causes the reduction of its antibacterial activity.
3rd, the antimicrobial spectrum of proSP-B recombinant proteins is determined
(1) preparation of antibacterial filter paper：Prepare about 6mm circular filter paper piece, 121 DEG C of sterilizing 20min, electric heating air blast
Drying box dries 2h.The gripping of filter paper aseptic nipper is put into the 500 μ g/mL albumen that step (7) is obtained in embodiment 1
Soaked in liquid, the filter paper after immersion is respectively put into sterile petri dish, dry excess surface liquid, to obtain the filter for soaking medicine
The scraps of paper；And using sterile saline as negative control, by positive control of penicillin or Terbinafine, (positive control of bacterium is
Penicillin, the positive control of fungi is Terbinafine).
(2) coating of strain：Liquid-transfering gun draws each μ L of bacteria suspension 50 for diluting and preparing, and glass spreading rod is uniformly smeared
In on each solid plate.
(3) pastille filter paper aseptic nipper will be soaked to filter paper, negative control filter paper, the positive control filter paper of medicine
It is affixed in flat board, mark is carried out at the culture dish back side with marking pen.
(4) thalline culture：The Escherichia coli of filter paper, staphylococcus aureus, pseudomonas aeruginosa, resistance to methoxy will have been pasted
XiLin staphylococcus aureus is inverted in 37 DEG C of bacteria bio incubator culture 12h；Trichophyton rubrum, Microsporum gypseum,
Candida albicans flat board is inverted in 28 DEG C of bacteria bio incubator culture 5d.
(5) with the inhibition zone size of each flat board of ruler measurement, (unit mm), as a result such as Fig. 7, (A is blank control, and B is sun
Property control) and table 3 shown in.Specifically in the figure 7, the result of staphylococcus aureus is shown in Fig. 7 (a)；Fig. 7 (b) displays
It is the result of Escherichia coli；The result of Candida albicans is shown in Fig. 7 (c)；The knot of Trichophyton rubrum is shown in Fig. 7 (d)
Really；The result of Microsporum gypseum is shown in Fig. 7 (e)；The result of pseudomonas aeruginosa is shown in Fig. 7 (f)；Fig. 7 (g)
It is shown that the result of methicillin-resistant staphylococcus aureus.
Then statistical analysis is carried out, the sensitivity of each bacterium is calculated, the criterion of drug sensitive experiment is as shown in table 4.
Table 3
Table 4
| Antibacterial circle diameter (mm) | More than 20 | 15-10 | 10-14 | Less than 10 | Less than 6 |
| Susceptibility | It is extremely quick | Gao Min | In it is quick | Muting sensitive | It is insensitive |
From table 3 and Fig. 7, proSP-B recombinant protein to staphylococcus aureus, methicillin-resistant staphylococcus grape ball
Bacterium, Escherichia coli, Trichophyton rubrum, Microsporum gypseum, Candida albicans are respectively provided with stronger inhibitory action, wherein to gold
Staphylococcus aureus, methicillin-resistant staphylococcus aureus inhibitory action it is most strong, its inhibition zone size is respectively reached
20.6mm, 20mm, so staphylococcus aureus, methicillin-resistant staphylococcus aureus are 500 μ g/mL for protein concentration
When be all extremely quick；And the inhibitory action to Escherichia coli is stronger, its inhibition zone size reaches 15.3mm, so Escherichia coli
It is Gao Min when being 500 μ g/mL for protein concentration；Trichophyton rubrum, Microsporum gypseum, the inhibition zone of Candida albicans
Size is respectively 12.3mm, 14mm, 13mm, therefore Trichophyton rubrum, Microsporum gypseum, Candida albicans are for protein concentration
All it is Gao Min during for 500 μ g/mL；And the inhibition zone size of pseudomonas aeruginosa only has 8mm, thus pseudomonas aeruginosa for
Protein concentration is all muting sensitive when being 500 μ g/mL.
4th, the measure of proSP-B recombinant proteins minimal inhibitory concentration (MIC)
(1) the bacteria suspension addition 100 μ L cultures prepared will be diluted in 96 orifice plates, and added successively in the 1st~9
The protein liquid that the step in embodiment 1 (7) of 50 each concentration of μ L is obtained, the 10th hole as blank control, the 11st, 12 holes be used as sun
Property control (bacterium be penicillin and Cefixime, fungi be Terbinafine and ketoconazole, concentration is 200 μ g/mL).
(2) Escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus are in life
Change after 37 DEG C of incubated 12h of incubator, 96 porocyte culture plates are taken out, be distributed at 600nm wavelength and determined with ELIASA
OD values, calculate albumen to staphylococcus aureus, Escherichia coli, methicillin-resistant staphylococcus aureus, pseudomonas aeruginosa
Minimal inhibitory concentration.
(3) Microsporum gypseum, Trichophyton rubrum, Candida albicans be after incubated 4 days of 30 DEG C of biochemical cultivation case,
96 porocyte culture plates are taken out, measure OD values are distributed at 600nm wavelength with ELIASA, calculating albumen to Trichophyton rubrum,
The minimal inhibitory concentration of Microsporum gypseum, Candida albicans.As a result it is as shown in table 5.
Table 5
| Bacterium name | Minimal inhibitory concentration (MIC) μ g/mL |
| Staphylococcus aureus | 20 |
| Escherichia coli | 30 |
| Candida albicans | 60 |
| Trichophyton rubrum | 30 |
| Microsporum gypseum | 30 |
| Pseudomonas aeruginosa | 125 |
| Methicillin-resistant staphylococcus aureus | 20 |
As shown in Table 5, proSP-B recombinant proteins are to bacterium class such as staphylococcus aureus, Escherichia coli, methicillin-resistant
Staphylococcus aureus is respectively provided with higher fungistatic effect, suppress they growth proSP-B recombinant protein concentration it is relatively low, respectively
Only need 20 μ g/mL, 30 μ g/mL, 20 μ g/mL；And proSP-B recombinant proteins also show obvious bacteriostasis to fungi,
Minimal inhibitory concentration such as Candida albicans, Microsporum gypseum, Trichophyton rubrum is respectively 60 μ g/mL, 30 μ g/mL, 30 μ g/
ML, it is also relatively very low, illustrate that the proSP-B recombinant proteins for only needing to low concentration can suppress the growth of various bacteriums, fungi,
So as to illustrate that proSP-B recombinant proteins have very high sterilization, bacteriostatic activity；But proSP-B recombinant proteins are false to verdigris single
The minimal inhibitory concentration of born of the same parents bacterium is 125 μ g/mL, and relative is higher with other six kinds of bacterium.This experiment and then proof proSP-B weights
Histone is to common G+、G-The fungi such as bacterium and saccharomycete has stronger inhibition.
5th, PI determines influence of the proSP-B recombinant proteins to aureus cell membrane permeability
(1) the μ L of staphylococcus aureus suspension 100 cultures that dilution is prepared are sequentially added in 96 orifice plates 1~7 row,
Totally 4 row.100 μ L sterile purified water is added in the 8th row, is added in then being arranged successively the 1st~4 different amounts of by embodiment 1
The proSP-B recombinant protein liquids that middle step (7) obtains, make its final concentration of protein be followed successively by 20 μ g/mL, 30 μ g/mL, 60 μ g/mL,
120 μ g/mL, the 5th~8 row sequentially add penicillin, Triton X-100, sterile purified water, proSP-B recombinant protein liquids, made
Final concentration of 50 μ g/mL, the Triton X-100 of its penicillin final concentration of 0.1%, proSP-B recombinant proteins it is final concentration of
120 μ g/mL, are eventually adding a certain amount of LB culture mediums, it is ensured that be 200 μ L per pore volume.
(2) 96 orifice plates are placed in biochemical cultivation case 37 DEG C of culture 12h, then take out, add PI, make its final concentration of 50
μg/mL。
(3) 30min is incubated after adding PI, fluorescence OD values are determined on multi-function microplate reader, its excitation wavelength is 488nm,
Launch wavelength is 630nm.
(4) using TritonX-100 result as 100% transmitance for calculating analysis bacterial cell membrane.
As a result it is as shown in table 6.
Table 6
As shown in Table 6, compared with negative control and blank control, the fluorescence OD values of experimental group are significantly higher, its cell it is penetrating
Property is stronger；Relative to positive control, 20 μ g/mL proSP-B recombinant proteins can reach to the penetrating of bacterial cell membrane
12.68%, and then the destruction of intracellular environment is destroyed, so as to influence it to grow, so proSP-B recombinant proteins can be by changing
Membrane passage influences the growing state of bacterium.
6th, cell membrane extravasation property experiment
(1) the μ L of staphylococcus aureus bacteria suspension 100 prepared are added sequentially in 96 orifice plates, and in 1~4 according to
Secondary to add a certain amount of proSP-B recombinant protein liquids that step (7) is obtained in embodiment 1, it is respectively 20 μ g/ to make its final concentration
ML, 30 μ g/mL, 60 μ g/mL, 120 μ g/mL, sequentially added in 5~8 penicillin, Triton X-100, sterile purified water,
ProSP-B recombinant protein liquids, wherein the 8th hole is not added with staphylococcus aureus, make the final concentration of 50 μ g/mL of its penicillin,
The final concentration of 120 μ g/mL of Triton X-100 final concentration of 0.1%, proSP-B recombinant proteins, are eventually adding a certain amount of
LB culture mediums, it is ensured that per pore volume be 200 μ L.
(2) 96 orifice plates are placed in biochemical cultivation case 37 DEG C of culture 12h, then taken out, according to LDH kit specifications according to
It is secondary to add each reagent, it is incubated at room temperature 5min.
(3) then take out, its OD value at 450nm is determined on multi-function microplate reader.
(4) using TritonX-100 result as 100% release rate for calculating analysis LDH.
As a result it is as shown in table 7.
Table 7
As shown in Table 7, under normal cell, intracellular environment is stable, and its intracellular foreign object quality guarantee is held relative steady
It is fixed, and in the case where damaged membrane is hindered, its cell transit rate substantially increases, intracellular macromolecular substances can be thin by what is be damaged
After birth is discharged, and obvious change whether can occur with side light membrane passage by detection bacterium nutrient solution.Breast
Acidohydrogenase (LDH) is the one of which macromolecular substances of intracellular, and its detection is fairly simple, and his extravasation can be reflected directly
The change of intracellular environment, so as to reflect the physiological status change of cell.It is right with feminine gender it can be seen from the result determined
Compared according to, blank control, the staphylococcus aureus after proSP-B recombinant proteins processing effect, the obvious increase of its LDH extravasations,
Illustrating the permeability of cell membrane of thalline is increased；And compared with positive control,
120 μ g/mL proSP-B recombinant proteins influence bacterium thin with regard to the LDH extravasation rates of cell can be made to reach 31%
The stabilization of born of the same parents, influences it to grow, and illustrates that proSP-B recombinant proteins can play the effect for changing Bacterial Physiological state.
Generally speaking, the present invention uses gene engineering method it can be seen from above example 1-3 result, particularly
By using specific expression vector (pGEX4T-1) and expressive host bacterium (e. coli bl21 (DE3) pLysS bacterial strains), success
Ground obtains the higher proSP-B recombinant proteins of concentration, also, by by the GST labels and proSP-B on pGEX4T-1 carriers
Fusion, because GST labels have hydrophily, can significantly improve the water solubility of proSP-B recombinant proteins, expand it and apply model
Enclose.
In addition, the proSP-B recombinant proteins that the present invention is provided show good broad spectrum antibiotic activity, it is to clinically normal
Bacterium, the fungi seen is respectively provided with stronger inhibitory action, particularly to staphylococcus aureus, Escherichia coli, the small spore of gypsum sample
Daughter bacteria, Candida albicans, Trichophyton rubrum, pseudomonas aeruginosa and methicillin-resistant staphylococcus aureus are shown significantly
Inhibitory activity.Further investigation revealed that, proSP-B recombinant proteins play its antibacterial action may be by influence bacterium
Membrane passage, so as to suppress the normal growth of bacterium.Meanwhile, the cell for the proSP-B recombinant proteins that the present invention is provided
Toxicity is smaller, even if protein concentration is brought up into 1mg/mL (concentration is far longer than its MIC value to bacterium), the increasing of cell
Grow ability and be still higher than 59%, the proSP-B recombinant proteins that this explanation present invention is provided have higher security.
The preferred embodiment of the present invention described in detail above, still, the present invention is not limited thereto.In the skill of the present invention
In art concept, technical scheme can be carried out a variety of simple variants, including each technical characteristic with it is any its
Its suitable method is combined, and these simple variants and combination should equally be considered as content disclosed in this invention, belong to
Protection scope of the present invention.
SEQUENCE LISTING
<110>Chongqing University of Technology
<120>Recombinant protein of pulmonary surfactant protein B propetides and its preparation method and application
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Ala Gly Ala Asn Asp Leu Cys Gln Glu Cys Glu Asp Ile Val His Leu
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20 25 30
Phe Leu Glu Gln Glu Cys Asp Ile Leu Pro Leu Lys Leu Leu Val Pro
35 40 45
Arg Cys Arg Gln Val Leu Asp Val Tyr Leu Pro Leu Val Ile Asp Tyr
50 55 60
Phe Gln Gly Gln Ile Lys Pro Lys Ala Ile Cys Ser His Val Gly Leu
65 70 75 80
Cys
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<223> The sequence is synthesized
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gcaggagcta atgacctgtg ccaagagtgt gaggatattg tccacctcct cacaaagatg 60
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ctacccttga agctgcttgt gccccggtgt cgccaagtgc ttgatgtcta cctgcccctg 180
gttatcgact acttccaggg ccagattaaa cccaaagcca tctgcagtca tgtgggcctg 240
tgc 243
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tttaagcttt tagcacaggc ccacatg 27
Claims (10)
1. a kind of recombinant protein of pulmonary surfactant protein B propetides, it is characterised in that the recombinant protein has SEQ ID NO：
Amino acid sequence shown in 1, or in SEQ ID NO：1 amino terminal and/or carboxyl terminal are connected with the amino acid of label
Sequence.
2. recombinant protein according to claim 1, wherein, the label has hydrophily；Preferably, the label is
GST labels.
3. a kind of can encode the nucleic acid of the recombinant protein described in claim 1 or 2.
4. nucleic acid according to claim 3, wherein, the nucleic acid has SEQ ID NO：Nucleotide sequence shown in 2, or
In SEQ ID NO：2 amino terminal and/or carboxyl terminal are connected with the nucleotide sequence of label；Preferably, the label has
Hydrophily；It is highly preferred that the label is GST labels.
5. a kind of recombinant plasmid, it is characterised in that the recombinant plasmid contains the nucleic acid described in claim 3 or 4.
6. recombinant plasmid according to claim 5, wherein, the recombinant plasmid be pGEX4T-1 plasmids BamH I and
SEQ ID NO are inserted between Xho I restriction enzyme sites：Expression vector obtained from nucleotide sequence shown in 2.
7. a kind of recombinant bacterial strain, it is characterised in that the recombinant bacterial strain contains the recombinant plasmid described in claim 5 or 6；It is preferred that
Ground, the recombinant bacterial strain is to contain the recombinant plasmid e. coli bl21 described in claim 5 or 6（DE3）PLysS bacterial strains.
8. a kind of method for the recombinant protein for preparing pulmonary surfactant protein B propetides, it is characterised in that this method includes following step
Suddenly：
（1）By the recombinant plasmid transformed described in claim 5 or 6 into expressive host bacterium, to obtain recombinant bacterial strain；
（2）The recombinant bacterial strain carries out Fiber differentiation after culture in the presence of derivant, to obtain nutrient solution；
（3）Will be by step（2）Obtained nutrient solution carries out purifying and label is removed, to obtain pulmonary surfactant protein B propetides
Recombinant protein.
9. method according to claim 8, wherein, in step（1）In, the expressive host bacterium is e. coli bl21
（DE3）PLysS bacterial strains；
Preferably, in step（2）In, the derivant is isopropyl-β-d- thiogalactosides；
Preferably, in step（2）In, the condition of the Fiber differentiation includes：Temperature be 28-37 DEG C, preferably 28-35 DEG C, more
Preferably 28-32 DEG C；Time is more than 3h, more preferably preferably 3-24h, 12-24h.
10. the nucleic acid described in recombinant protein, claim 3 or 4, the weight described in claim 5 or 6 described in claim 1 or 2
Group plasmid, the recombinant bacterial strain described in claim 7, the recombinant protein prepared as the method described in claim 8 or 9 are in system
Applied in standby antimicrobial；Preferably, the antimicrobial is anti-Staphylococcus aureus（Staphylococcus aureus）, it is big
Enterobacteria（Escherichia coli）, Microsporum gypseum（Microsporum gypseum）, Candida albicans
（Monilia albican）, Trichophyton rubrum（Trichophyton rubrum）, pseudomonas aeruginosa（Pseudomonas aeruginosa）And methicillin-resistant staphylococcus aureus（Methicillin-resistant Staphylococcus aureus）At least one of medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710340260.7A CN107163148A (en) | 2017-05-15 | 2017-05-15 | Recombinant protein of pulmonary surfactant protein B propetides and its preparation method and application |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1154117A (en) * | 1994-05-31 | 1997-07-09 | 比克·古尔顿·劳姆贝尔格化学公司 | Synthetic peptide analogs of lung surfactant protein SP-C |
| WO2014207585A1 (en) * | 2013-06-24 | 2014-12-31 | Azargen Biotechnologies (Pty) Ltd | Production of human pulmonary surfactant protein b in plants |
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2017
- 2017-05-15 CN CN201710340260.7A patent/CN107163148A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1154117A (en) * | 1994-05-31 | 1997-07-09 | 比克·古尔顿·劳姆贝尔格化学公司 | Synthetic peptide analogs of lung surfactant protein SP-C |
| WO2014207585A1 (en) * | 2013-06-24 | 2014-12-31 | Azargen Biotechnologies (Pty) Ltd | Production of human pulmonary surfactant protein b in plants |
Non-Patent Citations (3)
| Title |
|---|
| ROMANI-PEREZ.M,ET AL.: "Accession No：NP_620197.1，pulmonary surfactant-associated protein B precursor[Rattus norvegicus]", 《GENBANK DATABASE》 * |
| YANG L,ET AL.: "Surfactant protein B propeptide contains a saposin-like protein domain with antimicrobial activity at low pH", 《J IMMUNOL》 * |
| 张潇骏等: "大鼠成熟的肺表面活性蛋白Ｂ的原核表达及纯化", 《动物医学进展》 * |
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