CN1274008A - Exoteric expression of hay bacillus cecropi and its method - Google Patents

Abstract

Using the method of biotechnology, according to the amino acid sequence of cecropin LCI, the DNA gene fragment of LCI was artificially synthesized using the codons favored by plants. The constructed plasmid pBVAB16 highly expressed LCI in Escherichia coli DH5α, accounting for 8.7% of the total protein content of Escherichia coli. Escherichia coli can be purified LCI by centrifugation, ultrasonic disruption, heating precipitation, ion exchange and hydrophobic interaction liquid chromatography. LCI can specifically kill Xanthomonas oryzae strain G, and the minimum effective concentration is 2.5μg/ml. It is preliminarily deduced that LCI enters the bacteria, inhibits the transcription of RNA and causes the death of bacteria. The gene is expected to be used in plant disease resistance breeding and production of special bacterial fertilizer.

Description

In vitro expression and method of Bacillus subtilis cecropin

The in vitro expression of bacillus subtilis cecropin and the method thereof belong to the field of modern biotechnology.

current technology:

As early as 1960, people conducted extensive physiological and biochemical research on the antagonism of microorganisms, classified various antagonistic substances, and made great contributions to the development of medicine. In recent years, people have expanded the mechanism of action and molecular genetics of peptide antibiotics isolated from Lactobacillus very extensively and deeply (review articles include Klaenhammer, T.R. (1993). Genetics of bacteriaocins produced by lactic acid bacteria. FEMS Microbiol Rev 12, 39-86. and Jack, R.W., Tagg, J.R. & Ray, B. (1995). Bacteriocins of gram-positive bacteria. Microbiol Rev 59, 171-200.). Because they are different from other antibiotics in that they hardly cause microbial resistance, such as nisin (nisin) has been used as a food preservative for fifty years, but no spoilage bacteria have been found to produce resistance. sexual phenomenon. Therefore peptide antibiotics will have huge application prospect (Breukink, E., Wiedemann, I., van Kraaij, C., Kuipers, O.P., Sahl, H.G.& de Kruijff, B. (1999) .Use of the Cell Wall Precursor Lipid II by a Pore-Forming Peptide Antibiotic. Science 287, 2361-2364.).

Cecropin LCI is a basic polypeptide isolated from the culture solution of Bacillus subtilis AO14 in our laboratory, with a molecular weight of 5.4 kDa. It is effective against Xanthomonas oryzae pv.oryzaestrain G., Xanthomonas campestris pv.oryzea S2, Pseudomonas solanacearum POl, Pseudomonassolanacearum T62, T64 and pepper solanacearum blight bacteria (Pseudomonas solanacearum PE1) has obvious inhibitory effect. Its full amino acid sequence is:

AIKLVQSPNG NFAASFVLDG TKWIFKSKYY DSSKGYWVGI YEVWDRK

By searching the international gene and protein databases (Genbank and Swiss-port), no protein or gene similar in structure to the cecropin sequence was found. The results have been published (Liu Jinyuan, Chen Zhangliang, Primary Structure of Antibacterial Peptide LCI, Advances in Natural Science, 1994, 4(3): 365-367).

Bacillus subtilis has a wide range of antagonistic properties, and the antagonistic substances are varied. In our country in recent years, the research on the antibacterial protein of Bacillus subtilis has also been reported from time to time, but the research on molecular biology is not yet in-depth (such as Xin Yucheng, Qin Shulian, Jin Jing, Zhang Yingchun, Xu Kun, apple moldy heart disease biocontrol strains Preliminary report on the purification and partial properties of antibacterial proteins, Journal of Laiyang Agricultural College, 1999, 16 (1). Sun Jian, The inhibitory effect of Bacillus subtilis B-903 strain antibiotics on plant pathogenic fungi, Acta Phytopathology, 1995, 25( 1): 69~72).

purpose of the invention

While extensive research has been conducted on the use of plant, animal and insect-derived anti-plant pathogenic bacteria and disease and insect pest genes for crop disease-resistant breeding, and many achievements have been made, there are still relatively few studies on disease-resistant genes derived from antagonistic microorganisms and applied to plant genetic engineering. .

Since 1990, our laboratory has isolated several Bacillus subtilis with strong antagonistic ability from the field, and isolated and purified several polypeptides and protein. These disease-resistant genes are further cloned, and the possible synergistic effect produced by their different mechanisms of action is used to introduce them into plants at the same time. It is hoped that the transgenic plants will not only obtain broad-spectrum antibacterial ability but also enhance resistance to single pathogens. Peptide antibiotics with broad-spectrum antibacterial and non-toxic to humans and animals can also be used in the food and pharmaceutical industries based on biogenetic engineering.

Separation and purification of specific antibacterial peptides, research and analysis of their physiological and biochemical characteristics, structure and function, mainly elucidation of the antagonistic mechanism, which has important theoretical and application value in the fields of plant genetic engineering and plant pathology. Clarification of the structure and function of the polypeptide depends to a large extent on the cloning and in vitro expression of the polypeptide gene, thereby providing a molecular biological basis for cloning other related genes of the polypeptide, studying gene expression and regulation, and at the same time providing a basis for obtaining Transgenic antibacterial crops have laid a material foundation.

The content and scheme of the invention

1. Synthesize its gene according to the existing LCI amino acid sequence.

2. Cloning the LCI gene into the intermediate vector pBC7.

3. Cloning the LCI gene into the expression vector pBVAB16.

4. Induced expression of LCI cecropin in Escherichia coli.

5. Separation and purification of expressed LCI.

6. Detection of the bactericidal activity of the expressed LCI.

7. Inhibitory effect of expressed LCI on transcription in vitro.

Advantages and effects

The recombinant expression vector pBV AB16 was transferred into Escherichia coli DH5α, induced by high temperature at 42°C, and the expression level of LCI reached the highest in 5 hours, and the expression level of LCI accounted for the total protein content of Escherichia coli by scanning analysis of the electrophoresis gel. 8.7%.

Cecropin LCI is a ribosome-synthesized peptide chain encoded by a plasmid released extracellularly, with low molecular weight, positive charge and thermal stability.

Purified LCI can specifically kill rice bacterial blight (Xanthomonas oryzae pv.oryzae) strain G, the minimum effective concentration of LCI is 2.5μg/ml, and the survival rate of the bacteria is 10 ^-3 under the treatment of 0.13mg/ml LCI .

LCI contains a conserved sequence structure similar to other bacteriocins, which are mainly the second class of bacteriocins of lactic acid bacteria. According to the presumed secondary structure of LCI and the in vitro transcription inhibitory activity of LCI, it can be preliminarily inferred that LCI enters the bacteria and inhibits the transcription activity of RNA to cause the death of bacteria. Unlike other bacteriocins that can only kill Gram-positive bacteria, the feature that LCI can kill Gram-negative bacteria will greatly facilitate the application of ribosome-synthesized bacteriocins in genetic engineering disease-resistant breeding. Firstly, it is used in plant disease resistance breeding and production of special bacterial fertilizer.

Description of drawings

Figure 1. LCI gene sequence and its corresponding amino acid sequence. A: Alanine, I: Isoleucine, K: Lysine, L: Leucine, V: Valine, Q: Glutamine, S: Serine, P: Proline, N: Day Paragine, G: Glycine, F: Phenylalanine, D: Aspartic Acid, T: Threonine, W: Tryptophan, Y: Tyrosine, E: Glutamic Acid, R: Arginine . The underlined regions are chemically synthesized fragments.

Figure 2. The inhibitory effect of LCI on the growth of rice bacterial blight (Xanthomonas oryzaepv.oryzae) strain G at different concentrations. The concentration of LCI was 0 μg/ml (●); 0.6 μg/ml (O); 2.5 μg/ml (Δ); 4.9 μg/ml (▲); 9.8 μg/ml (■).

Figure 3. Determination of LCI's ability to kill Xanthomonas oryzae pv. oryzae strain G. The two concentrations of LCI were 0.13 mg/ml (•) and 0.066 mg/ml (■).

Figure 4. Effects of different concentrations of LCI on in vitro transcription. The concentration of LCI: the first lane is the control without LCI, the second lane is 0.11 mg/ml, the third lane is 0.22 mg/ml, and the fourth lane is 0.39 mg/ml.

Figure 5. Secondary structure of LCI and comparison with other bacteriocin polypeptide sequences. Boxes, underlines, shading, and boldface in the figure all represent conserved regions. CF: Chou-Fasman; GOR: Garnier-Osguthorpe-Robson.

Example

1. Chemical synthesis and cloning of LCI gene

Cecropin LCI is produced in the culture fluid of Bacillus subtilis A014. It consists of 47 amino acids in total, containing 6 lysines, 1 arginine and 3 tryptophans. According to the amino acid sequence of LCI, the DNA gene fragment of LCI was artificially synthesized by using the codons favored by plants, plus the start and stop codons and the two restriction sites of ClaI and Hind III (see Figure 1.). The four underlined nucleic acid fragments were synthesized by the solid-phase phosphoramidite method using a DNA synthesizer ABI381A (LKB):

1. 5′atatcgatggctatcaagctggtgcagtccccaaacggcaacttcgccgct 3′

2. 5′acttgaagatccacttagtaccgtccagcacgaaggaagcggcgaagttgc 3′

3. 5′taagtggatcttcaagtccaaatactatgactccagcaagggctactgggt 3′

4. 5′cgaagcttacttgcggtccccaacacctcgtagatgccgacccagtagccctt 3′

Each fragment contains 51 nucleotide residues. They were purified by electrophoresis on a 16% acrylamide gel containing 8M urea. The gel segment containing DNA fragments was immersed in 0.5M NaCl solution overnight, and then the supernatant was further purified by DEAE-Sepharose, desalted, and freeze-dried. Each DNA fragment was mixed in equal amounts of 200 pmol, heated in a water bath at 85°C for 5 minutes, and then gradually cooled to room temperature. Then add dNTP and T4 DNA polymerase, and carry out the filling reaction at 37°C.

2. Construction of LCI expression vector

The synthesized LCI gene fragment was double digested with Cla I and Hind III, connected to the pBluescript II KS plasmid treated with the same two enzymes with T4 DNA ligase, and transformed into Escherichia coli DH5α and screened to obtain The cloning plasmid pBC7 of the LCI gene. This plasmid was confirmed by sequence analysis to contain the correct LCI gene.

After the pBC7 plasmid was digested with Cla I enzyme, it was filled with DNA polymerase I Klenow large fragment, then digested with BamH I, and the LCI gene fragment was recovered with 2% agarose. Plasmid pBV220 was digested with EcoRI and filled in with the large fragment of DNA polymerase I Klenow, then digested with BamH I. After linking the LCI gene fragment into the pBV220 plasmid, it was transformed into Escherichia coli DH5α and screened to make the plasmid pBVAB16.

3. Extraction and Purification of Overexpressed LCI

Escherichia coli DH5α containing the plasmid pBVAB16 was activated in LB liquid medium, in which the content of ampicillin was 50 mg/l, inserted into fresh same medium at 1% inoculum size, cultured at 30°C for 2.5 hours, and then cultured at 42°C The induction culture was carried out for 5 hours, and the rotational speed was 200 rpm.

The induced expression of Escherichia coli was recovered by centrifugation at 4000rpm for 10 minutes, washed twice with 50mM Tris-HCl, pH7.8 buffer solution, ultrasonically broken at 400W for 30 times in an ice bath, each time for 15 seconds, with an interval of 10 seconds, and heated at 60°C for 20 Minutes, centrifuged at 10000rpm for 20 minutes to remove the precipitate. Then it was eluted by ion exchange liquid chromatography CM-Sepharose Fast Flow C26/20 with a linear gradient of 0-0.6M NaCl. Add 4M ammonium sulfate to the obtained active chromatographic peak to adjust to 2M, add 0.5M, pH6.5 potassium dihydrogen phosphate to adjust to 50mM, and use hydrophobic interaction liquid chromatography Phenyl-Superose HR5/5 to adjust to 1.7～0M ammonium sulfate Perform linear elution, and the main peak obtained is the purified LCI. The LCI sample was desalted with PD-10Sephadex G-25M (Pharmacia), freeze-dried, and stored at -20°C for future use. The purity of LCI was determined by Tris-tricine SDS-PAGE electrophoresis. The concentration of LCI was determined by 280nm ultraviolet absorption method and Lowry method. The expression level of LCI was determined by SDS-PAGE gel analysis stained with Coomassie brilliant blue by TLC scanner.

Because the pBV AB16 plasmid has the following characteristics, LCI can be expressed efficiently in E. coli: 1. The plasmid contains SD sequence, followed by the multiple cloning site, if it is located 3 to 11 nuclei upstream of the start codon AUG It is complementary to the 3' end sequence of 16s rRNA, which can promote efficient translation of proteins; 2. The two promoters of _PR and _PL are connected in series, and _PL is inhibited by CI protein, which can be induced by temperature changes 3. The transcription termination signal of the rrB ribosomal gene from Escherichia coli has a strong termination effect, can avoid codon read-through, and is conducive to the stability of the plasmid-host system.

Utilizing the heat-stable properties of LCI, the overexpressed LCI in E.coli can be extracted from the cell suspension sonicated, and most of the proteins are thermally deformed and precipitated by means of a hot water bath, so that it is easy to use CM -Sepharose Fast Flow and Phenyl-Superose HR5/5 or MonoS liquid chromatography purification to obtain electrophoretic pure LCI. The expression level of LCI was determined by TLC scanning to be 8.7% of the total protein content of Escherichia coli. The apparent molecular weight of the expressed LCI electrophoresis is 3.5kDa.

It should be noted that both the cell debris removed by centrifugation and the precipitate produced by heat treatment at 60°C had antibacterial activity, that is to say, a considerable part of LCI was not recovered. If these precipitates were washed two more times with buffer and the washes were combined, it is hoped that the yield could be improved.

4. Detection of antibacterial activity of LCI

1. Determine the antibacterial specific activity of LCI by 96-well plate turbidity colorimetry:

Use Xanthomonas oryzae pv.oryzea strain G as indicator bacteria, activate it with LB liquid medium overnight (200rpm, 28℃), take 20μl, add 100μl fresh LB medium, 20μl different concentrations LCI aqueous solution (starting from 1.1mg/ml LCI, diluted eight times by two-fold method), added to a 96-well plate, and repeated twice. The no-inhibition control was 20 μl LCI solution replaced with 20 μl deionized water, and the blank control was 120 μl LB medium and 20 μl deionized water. The 96-well plate was cultured at 28°C in an incubator. Every hour, the optical density of each sample well was measured under the light of 540 nm on a micro-volume spectrophotometer (Microplate Autoreader EL310, BIO-TEK INSTRUMENTS).

From the different growth curves of the pathogenic bacteria Xanthomonas oryzae pv.oryzae G, it can be determined that the minimum concentration of LCI is 2.5 μg/ml (see Figure 2.).

2. Analyze the antibacterial spectrum of LCI by plate diffusion method:

On the LB medium plate, pour 1ml of activated test bacteria (including Xanthomonas oryzaepv.oryzea strain G) culture suspension, spread evenly, remove excess liquid after 3 minutes, and dry in the air. Add 5 μl of LCI solution dropwise on different positions of the plate, and 5 μl of the corresponding buffer as a control, incubate overnight at 28°C, and observe the size of the inhibition zone. Another method is to drop 5μl LCI solution and 5μl corresponding buffer solution on the same line of the plate as a control. After the droplet disappears, use an inoculation loop to dip the activated bacterial solution along the two liquid Draw a straight line at the location of the drop. One plate can measure several different bacteria at the same time. Incubate overnight at 28°C and observe the inhibition zone. Experiments found that LCI has a special inhibitory effect on Xanthomonas oryzae pv.oryzae G.

3. Bactericidal ability test of LCI

The overnight activated X. oryzae pv. oryzae strain G was diluted to 0.035OD 600nm with LB liquid medium. The microfiltration-sterilized LCI sample (0.53 mg/ml) was diluted once twice, mixed with 100 μl of diluted bacterial solution and 300 μl of the diluted bacteria solution, and stored in a microcentrifuge tube at room temperature. At regular intervals, 20 μl was taken out and mixed in 980 μl LB liquid medium, and then 100 μl was taken out and spread on an LB agar plate, and cultivated overnight at 28°C. The bactericidal ability of LCI can be determined by the number of colonies formed on the plate. From Figure 3. It can be seen that LCI does not kill bacteria immediately. The number of bacteria begins to decline about half an hour after adding LCI, and the decline process lasts until eight hours. Rice bacterial blight (X.oryzaepv.oryzae strain G) showed roughly the same sensitivity trend to two concentrations of LCI, 0.13mg/ml (●) and 0.066mg/ml (■), at high LCI concentrations, The survival rate of the bacteria is about 10 ^-3 .

LCI expressed in E. coli may not have post-transcriptional modifications, but native LCIs may, and such modifications tend to confer more active characteristics on cecropins. It is also difficult to say whether adding methionine at the N-terminus would affect the bactericidal properties of LCI.

X.oryzae pv.oryzae G is a gram-negative bacterium, and the bacteriocin produced by gram-positive bacteria only has an effect on gram-positive bacteria under normal conditions. Most of the currently known plant pathogenic bacteria are Gram-negative bacteria, so the feature that LCI can kill Gram-negative bacteria will greatly facilitate the application of ribosome-synthesized bacteriocins in genetically engineered disease-resistant breeding.

5. Inhibition experiment of LCI on in vitro transcription

The possible transcriptional repressive activity of LCI was determined using Riboprobe Combination System (Promega) and SP6 polymerase. In vitro transcription analysis contains: 40mM Tris-HCl, pH7.5, 6mM MgCl ₂ , 2mM spermidine, 10mM NaCl, 10mM DTT, 500μM each ATP, GTP, CTP and UTP, 40 U Recombinant RNasin Ribonuclease in a 20μl reaction system Inhibitor, 0.5μg pGEMExpress Positive Control Template, 20 U SP6 polymerase, react at 37℃ for 80 minutes. Different amounts of LCI were added in different reactions. After the reaction product was subjected to electrophoresis on 1% agarose (containing 0.5 μg/ml ethidium bromide), the synthesis amount of RNA was observed under 254 nm ultraviolet light. As shown in Figure 4, the three different concentrations of LCI: 0.11 mg/ml in lane 2, 0.22 mg/ml in lane 3 and 0.39 mg/ml in lane 4 all exhibited relative to the control without LCI (lane 1). No effect on RNA synthesis, and at the concentration of 0.39mg/ml, the synthesis of RNA was almost completely inhibited.

6. Sequence analysis of LCI

The sequence of LCI was searched in "nr" database through http://www.ncbi.nlm.nih.gov website with BLAST software, but no similar polypeptide or gene was found. The secondary structure of LCI was determined by GCG software using Chou-Fasman (CF) and Garnier-Osguthorpe-Robson (GOR) methods. The calculated molecular weight of LCI is 5595.12 Da and its calculated isoelectric point pI=10.25. As shown in Figure 5, according to the predicted secondary structure and comparison with the amino acid sequences of other bacteriocins, it can be found that the peptide segment of tryptophan from the N-terminus of LCI to the 23rd position may contain a βsheet structure, while in After this tryptophan, the C-terminus of the peptide chain may form an amphipathic structure that can cross the plasma membrane, such as bacteriocin piscicocins V1. The complementarity of the peptide structure in bacteriocin is very important for its activity, just as the activity of lactococcin G, plantaricin A and plantaricin S requires the cooperation of its alpha and beta chains.

The primary structure of cecropin LCI has three similarities with many other classes of bacteriocins, including lantibiotics. The conserved sequence YGNGV of the first and second bacteriocins is incomplete in LCI, and it only has NG at the same position. Referring to the position of tryptophan in the peptide chain, it can be found that the glycine at the tenth position is very conserved, as shown in the box in Figure 5 . It is true in many bacteriocins, such as lactacinF and lacticin 481, among others. Second, the number of tryptophan residues in bacteriocins varies from 1 to 4, most of them contain 2 to 3, but their distribution is regular, although not very strict. Any tryptophan in bacteriocin divercin V41 will lose its activity through N-bromosuccinyl amidation, so it is speculated that the three tryptophans in LCI also play an important role in its bactericidal activity. Third, the KYY structure at the 28th to 30th positions in the LCI peptide chain also exists in the bacteriocin I (enterocin L50A), and the bacteriocin enterocin L50B contains a similar structure KFY. KYY and its similar structures are also found in some other bacteriocins, but they are not in the middle of the peptide chain, but in the N-terminal region, and the isoleucine residue replaces the KYY structure at this position.

According to these characteristics, it is inferred that LCI is most similar to the bacteriocins of the pediocin family of the second type of bacteriocins in lactic acid bacteria, which are curvacin A (sakacin A), divercin V41, enterocin I (L50A), enterocin L50B, pediocin PA-1 (AcH ) and sakacin P.

Claims (7)

1. In vitro expression and method thereof of a Bacillus subtilis cecropin, characterized in that said method comprises: (1) chemical synthesis of cecropin LCI gene and cloning thereof; (2) construction of cecropin LCI expression vector; 3) Extraction and purification of overexpressed cecropin LCI; (4) Antibacterial activity detection of cecropin LCI; (5) Inhibition experiment of cecropin LCI on in vitro transcription; (6) Sequence analysis of cecropin LCI. 2. According to the chemical synthesis and cloning of the cecropin LCI gene described in claim 1.(1), it is characterized in that the DNA gene of cecropin LCI is artificially synthesized, and the gene sequence and its corresponding amino acid sequence diagram are as follows: Cla I startcodon A - I - K - L - V - Q - S - P - N - G atatcgatg gct atc aag ctg gtg cag tcc cca aac ggc tatagctac cga tag ttc gac cac gtc agg ggt ttg ccg N - F - A - A - S - F - V - L - D - G aac ttc gcc gct tcc ttc gtg ctg gac ggt ttg aag cgg cga agg aag cac gac ctg cca T - K - W - I - F - K - S - K - Y - Y ac t aag tgg atc ttc aag tcc aaa tac tat tga ttc acc tag aag ttc a gg ttt atg ata D - S - S - K - G - Y - W - V - G - I gac tcc agc aag ggc tac tgg gt c ggc atc ctg agg tcg ttc ccg atg acc cag ccg tag stop Y - E - V - W - D - R - K codon tac gag gtg tgg gac cgc aag taagcttcg atg ctc cac acc ctg gcg ttc attcgaagc ▲ Hind III 3. According to the construction of the cecropin LCI expression vector described in claim 1.(2), it is characterized in that the synthetic LCI gene fragment is double digested with Cla I and Hind III, and it is connected to the vial with T4 DNA ligase. On the same pBluescript II KS plasmid treated with these two enzymes, after transforming Escherichia coli DH5α and screening, the cloning plasmid pBC7 containing the LCI gene was obtained, which was confirmed by sequence analysis to contain the correct LCI gene. After digestion, use the large fragment of DNA polymerase I Klenow to fill in, then digest with BamH I, use 2% agarose to reclaim the LCI gene fragment, and use the large fragment of DNA polymerase I Klenow to fill in the plasmid pBV220 after EcoR I digestion, and then use After BamH I digestion, the LCI gene fragment was connected to the pBV220 plasmid, and then transformed into Escherichia coli DH5α and screened to make plasmid pBVAB16. 4. According to the extraction and purification of the overexpressed cecropin LCI described in claim 1. (3), it is characterized in that: Escherichia coli DH5α containing the plasmid pBVAB16 was activated in LB liquid medium, in which the content of ampicillin was 50 mg/l, inserted into fresh same medium at 1% inoculum size, cultured at 30°C for 2.5 hours, and then cultured at 42°C Induction culture was carried out for 5 hours, and the rotation speed was 200rpm; The induced expression of Escherichia coli was recovered by centrifugation at 4000rpm for 10 minutes, washed twice with 50mM Tris-HCl, pH7.8 buffer solution, ultrasonically broken at 400W for 30 times in an ice bath, each time for 15 seconds, with an interval of 10 seconds, and heated at 60°C for 20 Minutes, centrifuged at 10,000rpm for 20 minutes to remove the precipitate, and then eluted by ion-exchange liquid chromatography CM-Sepharose Fast Flow C26/20 with a linear gradient of 0-0.6M NaCl, the active chromatographic peak obtained was adjusted to 2M by adding 4M ammonium sulfate, and added 0.5M, pH 6.5 potassium dihydrogen phosphate adjusted to 50mM, after microfiltration, use hydrophobic interaction liquid chromatography Phenyl-Superose HR5/5 to linearly elute with 1.7-0M ammonium sulfate, and the main peak obtained is the purified LCI. The LCI sample was desalted with PD-10 Sephadex G-25M, freeze-dried, stored at -20°C for later use, and the purity of LCI was determined by Tris-tricine SDS-PAGE electrophoresis. The concentration of LCI was determined by 280nm ultraviolet absorption method and Lowry method, LCI The expression level of was determined by SDS-PAGE gel analysis stained with Coomassie Brilliant Blue by a TLC scanner; Due to the following characteristics of the pBVAB16 plasmid, LCI can be expressed efficiently in E. coli: 1. The plasmid contains the SD sequence, followed by the multiple cloning site, if it is located 3 to 11 nucleosides upstream of the start codon AUG It is complementary to the 3' end sequence of 16s rRNA, which can promote the efficient translation of protein; 2. The two promoters of _PR and _PL are connected in series, and _PL is inhibited by CI protein, which can be induced by temperature changes 3. The transcription termination signal of the rrB ribosomal gene from E. coli has a strong termination effect, which can avoid codon read-through, and is conducive to the stability of the plasmid-host system; Utilizing the heat-stable properties of LCI, the overexpressed LCI in E.coli can be extracted from the cell suspension sonicated, and most of the proteins are thermally deformed and precipitated by means of a hot water bath, so that it is easy to use CM - Purified by Sepharose Fast Flow and Phenyl-Superose HR5/5 or MonoS liquid chromatography to obtain electrophoretic pure LCI, the expression amount of LCI was determined by TLC scanning to be 8.7% of the total protein amount of Escherichia coli, and the apparent molecular weight of the expressed LCI electrophoresis was 3.5 kDa. 5. According to the antibacterial activity detection of cecropin LCI described in claim 1. (4), it is characterized in that the antibacterial activity detection of cecropin LCI comprises: (1), 96-well plate turbidity colorimetric method to determine the antibacterial specific activity of LCI: Use Xanthomonas oryzae pv.oryzea strain G as indicator bacteria, activate it with LB liquid medium overnight, 200rpm, 28°C, then take 20μl, add 100μl fresh LB medium, 20μl LCI aqueous solution of different concentrations , that is, starting from 1.1mg/ml LCI, diluting eight times by two-fold method, adding to a 96-well plate, and repeating twice; the non-inhibitory control is to replace 20μl LCI solution with 20μl deionized water, and the blank control is 120μl LB medium and 20 μl deionized water, 96-well plate was cultured in an incubator at 28°C, every hour, on a micro-spectrophotometer Microplate AutoreaderEL310, BIO-TEK INSTRUMENTS, measure the optical density of each sample well under the light of 540nm; From the different growth curves of the pathogenic bacteria Xanthomonas oryzae pv.oryzae G, it can be determined that the minimum concentration of LCI is 2.5 μg/ml; (2), plate diffusion method to analyze the antibacterial spectrum of LCI: On the LB medium plate, pour 1ml of activated test bacteria, including Xanthomonas oryzaepv.oryzea strain G, culture the suspension, spread it flat, remove the excess liquid after 3 minutes, dry it, and place it on different positions on the plate Add 5 μl of LCI solution and 5 μl of the corresponding buffer as a control, and culture overnight at 28°C to observe the size of the inhibition zone. Another method is to add 5 μl of LCI solution and 5 μl of the corresponding buffer as a control to the plate respectively. On the same line, after the droplet disappears, use an inoculation loop to dip the activated bacterial solution and draw a straight line along the position of the two droplets. One plate can measure several different bacteria at the same time, cultivate overnight at 28°C, and observe Inhibition zone, the experiment found that LCI has a special inhibitory effect on Xanthomonas oryzaepv.oryzae G; (3) Bactericidal ability test of LCI Dilute the overnight activated X.oryzae pv.oryzae strain G to 0.035OD 600nn with LB liquid medium, and the concentration of the LCI sample sterilized by microfiltration is 0.53mg/ml. Mix it with 300μl of diluted bacterial solution in a microcentrifuge tube, store at room temperature, take out 20μl at regular intervals, mix in 980μl LB liquid medium, and then take out 100μl and spread it on LB agar plate, at 28℃ Overnight culture, the bactericidal ability of LCI can be determined by the number of colonies formed on the plate. From Figure 3. It can be seen that LCI does not kill bacteria immediately. The number of bacteria begins to decline about half an hour after adding LCI, and the decline process continues By eight hours, rice bacterial blight X.oryzae pv.oryzae strain G showed roughly the same sensitivity trend to the two concentrations of LCI 0.13mg/ml (●) and 0.066mg/ml (■). Under the concentration of LCI, the survival rate of the bacteria is about 10 ^-3 . 6. According to claim 1. (5) Inhibition experiment of cecropin LCI on in vitro transcription, characterized in that: Use Riboprobe Combination System (Promega) and SP6 polymerase to determine the possible transcriptional inhibitory activity of LCI. In vitro transcription analysis contains: 40mM Tris-HCl, pH7.5, 6mM MgCl ₂ , 2mM spermidine, 10mM NaCl in a 20μl reaction system , 10mM DTT, 500μM each ATP, GTP, CTP and UTP, 40 U Recombinant RNasin Ribonuclease inhibitor, 0.5μg pGEMExpress Positive Control Template, 20 U SP6 polymerase, react at 37℃ for 80 minutes, add different amounts of LCI in different reactions , The reaction product was subjected to 1% agarose containing 0.5 μg/ml ethidium bromide, and after electrophoresis, the synthesis amount of RNA was observed under 254 nm ultraviolet light. 7. According to the sequence analysis of the cecropin LCI described in claim 1. (5), it is characterized in that: The sequence of LCI was searched in the "nr" database with BLAST software through http://www.ncbi.nlm.nih.gov, but no similar polypeptide or gene was found; the secondary structure of LCI was obtained through GCG software Chou-Fasman (CF) and Garnier-Osguthorpe-Robson (GOR) two methods of analysis determine that the calculated molecular weight of LCI is 5595.12Da, and its calculated isoelectric point pI=10.25; As shown in Figure 5, according to the predicted secondary The structure and the comparison with the amino acid sequences of other bacteriocins can be found that there may be a βsheet structure in the peptide segment from the N-terminal of LCI to the 23rd tryptophan, and after this tryptophan to the C-terminal of the peptide chain May form an amphipathic structure that can cross the plasma membrane, such as bacteriocin piscicocins V1, the complementarity of the peptide structure in bacteriocin is very important for its activity, just like the activity of lactococcin G, plantaricin A and plantaricin S requires its alpha and beta chain synergy is the same; The primary structure of cecropin LCI has three similarities with many other types of bacteriocins, including lantibiotics; the conserved sequence YGNGV of the first and second types of bacteriocins is incomplete in LCI, and at the same position it Only NG; referring to the position of tryptophan in the peptide chain, it can be found that the tenth glycine is very conservative, as shown in the box in Figure 5, which is the case in many bacteriocins, such as lactacinF and lacticin 481, etc. etc.; secondly, the number of tryptophan residues in bacteriocins varies from 1 to 4, and most of them contain 2 to 3, but their distribution is regular, although not very strict; in Any tryptophan in bacteriocin divercin V41 will lose its activity through N-bromosuccinyl amidation, so it is speculated that the three tryptophans in LCI also play an important role in its bactericidal activity; third, LCI The 28th to 30th KYY structure in the peptide chain also exists in the bacteriocin I, and the bacteriocin L50B contains a similar structure KFY, KYY and similar structures also exist in some other bacteriocins, but not in the peptide chain , but in the N-terminal region, and at the same time replacing the KYY structure at this position is an isoleucine residue; Based on these characteristics, it was inferred that LCI was most similar to the bacteriocins of the pediocin family of the second class of bacteriocins in lactic acid bacteria, which were curvacin A, divercin V41, enterocin I, enterocin L50B, pediocinPA-1, and sakacin P.

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