CN102643762B - Brevibacillus parabrevis as well as method and application of protein series prepared thereby - Google Patents

Abstract

The present invention discloses a method and application of Bacillus brevius and the protein series prepared therefrom. Its classification name is Bacillus brevis ( Brevibacillus parabrevis ) GYZC01, CCTCC NO: M2011461, and its 16SrRNA has the sequence of SEQ ID NO: 1. The protein series prepared by Bacillus pseudobrevius GYZC01 strain can be used to prepare thrombolytic drugs, the protein series can dissolve thrombus slowly, the effect is long-lasting, and the EC ₅₀ is 200

, and can effectively dissolve fresh thrombus, EC ₅₀ is 400 , for old thrombus, the activity of crude protein of GYZC01 strain is obviously stronger than that of urokinase, EC ₅₀ is 800

.

Description

A kind of Bacillus brevius and the method and application of protein series prepared therefrom

technical field

The present invention relates to a kind of Bacillus brevius and the method and application of the protein series prepared therefrom.

Background technique

The mortality rate of cardiovascular and cerebrovascular diseases has always been in the second place, among which thrombotic diseases have the highest morbidity, disability and mortality. Thrombotic disease refers to a type of disease in which thrombus forms in the circulating blood and the thrombus falls off from the local area and flows into the front blood vessel, blocking or partially blocking the vascular lumen, leading to thromboembolism. For thrombotic diseases, there are currently three clinical therapies: surgery, antithrombotic therapy, and thrombolytic therapy. Surgical treatment must identify the location of embolism, which is difficult and dangerous to implement; antithrombotic therapy is to use drugs to control thrombus re-formation, but it is ineffective for already formed thrombus; thrombolytic therapy is to use drugs to dissolve already formed thrombus, so that Blocked blood vessels are reopened. Thrombolytic therapy is currently the focus of research.

Thrombolytic therapy is the use of thrombolytic agents to directly dissolve thrombus or by activating the conversion of plasminogen in blood into plasmin to catalyze the hydrolysis of thrombus fibrin, the main matrix of thrombus. Thrombolytic agents that have been officially approved for clinical use are roughly divided into three generations, the first generation: streptokinase (Streptokinase), urokinase (Urokinase); the second generation: Anistreplase (Anistreplase), recombinant tissue plasminogen activation (Alteplase, recombinant human tissue-type plasminogen activator); third generation: Reteplase, Tenecteplase, Lanoteplase, Staphylokinase.

The above-mentioned drugs have achieved remarkable results in the treatment of thrombosis, but there is still a certain distance from ideal thrombolytic drugs. The disadvantages are that the specificity of thrombolysis is not high, the half-life is short, and it is easy to cause internal bleeding. An ideal thrombolytic drug should be safe, effective, convenient to administer, strong specificity, long half-life, capable of dissolving old thrombus, low recurrence rate, no side effects such as bleeding, and reasonable price.

Contents of the invention

The technical problem to be solved in the present invention is: to overcome the shortcomings of the existing antithrombotic drugs, such as low thrombolytic specificity, short half-life, and easy to cause internal bleeding, the present invention provides a class of Bacillus brevis GYZC01, with a molecular weight greater than The 7000D protein series can be used to prepare thrombolytic drugs with good effect. the

Another object of the present invention is to provide a method for producing protein series by Bacillus pseudobrevius.

A kind of Bacillus brevis of the present invention, which is classified as Bacillus brevius ( Brevibacillus parabrevis ) GYZC01, is preserved in the China Type Culture Collection Center, and the preservation address is: Wuhan University, Wuhan, China, and the preservation number is: CCTCC NO : M 2011461, date of deposit: December 11, 2011. Brevibacillus parabrevis (Brevibacillus parabrevis) GYZC01 of the present invention is a kind of Gram-positive bacterium that exists in the soil, is to isolate a strain of bacterium from the soil of Guizhou University Caijiaguan Campus, can Growth, the optimum culture temperature is 30°C, the colony is grayish yellow, the length of the bacteria is 3-4 microns, the width is 0.5-0.8 microns, aerobic, can use citrate, ammonium salt, can not use nitrate, can hydrolyze casein , Gelatin, can utilize glucose, fructose, maltose to produce acid, produce gas, can not decompose lactose, its protein series has the effect of thrombolysis, after identification, bacterial strain GYZC01 is class Bacillus brevibacillus (Brevibacillus parabrevis).

The 16S rRNA of the brevibacillus parabrevis ( Brevibacillus parabrevis ) GYZC01 has the sequence of SEQ ID NO: 1, and the resulting sequence is compared with the nucleic acid data in GenBank through the Blast program, and its series is similar to that of Brevibacillus parabrevis M3; AB215101 strain 99% similarity and between 97-98.5% similarity to other Brevibacillus parabrevis strains. Therefore, it was judged that GYZC01 belonged to a new strain. Further use of MEGA5.0 to establish a phylogenetic tree also proved that GYZC01 was a new strain.

The method for preparing the protein series by Bacillus brevius includes freezing the bacterial fermentation liquid containing Bacillus brevis GYZC01 overnight, and thawing at room temperature to obtain a frozen lysate, which is centrifuged in a centrifuge to collect the supernatant Liquid and supernatant were gradually added with ammonium sulfate powder until saturated, the precipitate was collected overnight, and the precipitate was dialyzed with a dialysis bag to obtain protein series.

In the method for preparing a protein series from Bacillus brevus, the molecular weight of the protein series is greater than 7000D.

The application of the protein series in thrombolytic drugs.

The Brevibacillus parabrevis GYZC01 strain of the present invention uses 27f and 1492r as primers for PCR amplification to obtain a 16S rRNA base size of 1392 bp. See the nucleotide or amino acid sequence list at the end of the specification for the sequence.

The beneficial effect that the present invention reaches:

The thrombolytic effect of the Brevibacillus parabrevis series proteins with a molecular weight greater than 7000D obtained in the present invention: an in vitro thrombolytic test method is adopted, and it can be produced by fermentation. The protein series can dissolve thrombus slowly and have a long-lasting effect. The _EC50 is 200?g/ml. In vivo experiments showed that this protein series can effectively dissolve fresh thrombus (EC ₅₀ is 400?g/kg), but for old thrombus, the activity of the crude protein of GYZC01 strain is significantly stronger than that of urokinase, with EC ₅₀ of 800?g/kg. ml, the EC ₅₀ of urokinase is 1600??g/ml. Studies have shown that the thrombolytic active ingredient of Brevibacillus parabrevis series proteins with a molecular weight greater than 7000D is metalloproteinase, which can directly dissolve thrombus and cannot activate plasminogen, so it will not cause side effects such as bleeding after in vivo use. It has extremely great application value.

Instructions attached

Figure 1 establishes a phylogenetic tree for MEGA5.0.

Fig. 2 is the electrophoresis analysis map of the Bacillus brevus GYZC01 genome of the present invention.

Fig. 3 is the electrophoresis profile of the PCR product of Bacillus brevus GYZC01 of the present invention.

Figure 4 is the thrombolytic time-effect curve of GYZC01 crude protein, in which the concentration of GYZC01 crude protein (□) is 800??g/ml, and the concentration of urokinase (◆) is 200??g/ml.

Fig. 5 is the chicken embryo test of in vivo thrombolysis (fresh thrombus), where ▲ is urokinase, ◆ is GYZC01, and ■ is normal saline.

Figure 6 is the in vivo thrombolysis (old thrombus) chicken embryo test. ▲ is GYZC01, ◆ is urokinase, and ■ is saline.

Detailed ways

Embodiment 1: Bacillus brevis GYZC01 16S rRNA sequence determination

1. Genome extraction and electrophoresis detection

1. Genome extraction process:

Extract according to the instructions of Sanko SK1201-UNIQ-10 Column Bacterial Genomic DNA Extraction Kit.

2. Genome electrophoresis analysis map: see appendix 2 of the manual

2. PCR reaction

1. PCR system establishment (50ul):

Template (genome) 10pmol

Primer up (10uM) 1ul

Primer down (10 uM) 1ul

dNTP mix (10Mm each) 1ul

10*Taq reaction Buffer 5ul

Taq (5u/ul) 0.25 ul

Add water to 50ul

PCR program setting

Pre-denaturation 98℃ 5mim ;

Cycle 95°C 35S , 55°C 35S , 72°C 1min 30s , 35 cycles, extension 8min.

3. Electrophoretic pattern of PCR products

4. Primer sequence:

27f 5’AGAGTTTGATCCTGGCTCAG 3’ 20bp

1492r 5’ GGTTACCTTGTTACGACTT 3’ 19bp

3. DNA agarose cutting gel purification

According to the electrophoresis results of the PCR products, the target band of the desired DNA was cut.

4. DNA Sequencing

TTTTAGGACCCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTTTCAGACCGGGATAACATAGGGAAACTTATGCTAATACCGGATAGGTTTTTGGATTGCATGATCCGAAAAGAAAAGATGGCTTCGGCTATCACTGGGAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCTGTTGTCAGGGACGAACACGTGCCGTTCGAATAGGGCGGTACCTTGACGGTACCTGACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGCGCGCAGGCGGCTATGTAAGTCTGGTGTTAAAGCCCGGAGCTCAACTCCGGTTCGCATCGGAAACTGTGTAGCTTGAGTGCAGAAGAGGAAAGCGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGGTTTCAATACCCTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGCTGACCGCTCTGGAGACAGAGCTTCCCTTCGGGGCAGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCG TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTTAGTTGCCAGCATTCAGTTGGGCACTCTAGAGAGACTGCCGTCGACAAGACGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGGGATGCTACCTCGCGAGGGGACGCCAATCTCTGAAAACCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCGCAAGGAGCCAGCCGCCGAAA

Example 2: Preparation of protein series with a molecular weight greater than 7000D of Bacillus brevus strain GYZC01

1. Experimental materials

Brevibacillus parabrevis GYZC01 strain, peptone, sodium chloride, glucose, dialysis bag, ammonium sulfate.

2. Experimental method

2.1 Bacterial culture

Composition of bacterial culture medium: peptone 1g, sodium chloride 0.5g, glucose 1.5g, distilled water 100ml, autoclaved for later use. Bacteria were inoculated in 100ml of culture solution and cultured at 30°C for 48h to obtain seeds. According to the ratio of 1:100, add the seed bacteria solution into the culture solution, co-cultivate 5L, and culture at 30°C for 96-120h for later use.

Bacterial fermentation broth (5L) was frozen overnight, thawed at room temperature, and then frozen overnight, and this was repeated 3 times to obtain a frozen lysate, which was centrifuged in a centrifuge at 5000-10000r/min for 10-15min, the supernatant was collected, and gradually added ammonium sulfate powder until saturated, let it stand overnight, and collected the precipitate (20g). The precipitate was repeatedly dialyzed with a 7000D dialysis bag to obtain protein series with a molecular weight greater than 7000D. The 3500D dialysis bag was used to desalt the crude protein solution, freeze-dried, and the dried product (5g) was stored in the refrigerator (4°C).

The bacterial strain GYZC01 grew well at 30°C, and the number of bacteria reached the peak when it was cultured for 120 hours. Previous experiments have confirmed that the protein with thrombolytic activity exists both extracellularly and intracellularly. Therefore, the bacteria were lysed by freezing and thawing. After dialysis, desalination and freeze-drying, a total of 5 g of crude protein with a molecular weight greater than 7000D was obtained.

Embodiment 3: In vitro thrombolysis test of Bacillus brevus strain GYZC01 molecular weight greater than 7000D protein series

1. Experimental materials

  Crude protein, capillary, urokinase, plate of Bacillus brevis GYZC01 strain with a molecular weight greater than 7000D; 2. Experimental methods

Disinfect the ring finger with alcohol, collect blood by acupuncture, inhale the blood with a capillary, place it horizontally, and the blood will coagulate in about 10 minutes. Dissolve the crude protein of Bacillus brevius GYZC01 strain with normal saline to make sample solutions with series concentrations. Urokinase is used as positive control and normal saline is used as negative control. The above samples, positive control and negative control solutions are placed in 60mm plates respectively. 1. Cut the capillaries with blood clots into 5mm lengths, put them into petri dishes, 5 capillaries per dish, make them immersed in the liquid, put the capillaries on the petri dishes, put them at room temperature, and start timing. After 24 hours, observe and record the dissolving of the blood clot in the capillary;

3. Results

时能100%地溶解血栓，EC₅₀为200

。 Table 1 shows the results of the thrombolytic test in vitro. It can be seen that the thrombolytic activity of urokinase is the strongest, and the GYZC01 crude protein is

It can dissolve thrombus 100% of the time, and the EC ₅₀ is 200

.

GYZC01 crude protein has a milder effect on thrombus dissolution, and can dissolve all thrombus within 24 hours, while urokinase can dissolve all thrombus within 12 hours.

From the experimental results, it can be seen that the GYZC01 protein series has good thrombolytic activity in vitro, and can be produced by fermentation. The characteristics of thrombolysis are: mild dissolution and long-lasting effect.

Embodiment 4: Thrombolysis test in chicken embryo of protein series of Bacillus brevis GYZC01 strain with molecular weight greater than 7000D

1. Experimental materials

Crude protein with a molecular weight greater than 7000D of Bacillus brevis GYZC01 strain, urokinase, 8-day-old chicken embryos, normal saline, ferric chloride, and qualitative filter paper.

2. Experimental method

Open the egg shell from the end of the air cell of the 8-day-old chicken embryo to expose the allantoic membrane, separate the blood vessel with a hook needle, and then use a filter paper strip containing ferric chloride solution to contact the blood vessel of the chicken embryo for 15-20 seconds, and a thrombus will form at the contact point. Visible to the naked eye. About 2 hours after thrombus formation (fresh thrombus) or 6 hours later (old thrombus), start administration (urokinase as a positive control, normal saline as a negative control), that is, drop the drug solution on the thrombus formation site, or inject the drug solution For blood vessels or allantoic cavity, seal the opening with sterile transparent adhesive tape. Thrombolysis was observed at 2, 8, 16, 20, and 24 hours after administration.

3. Results

，尿激酶EC₅₀为100

，但是针对陈旧性血栓，GYZC01菌株粗蛋白的活性强于尿激酶，EC₅₀为800

，尿激酶EC₅₀为1600

。 From the experimental results, it can be seen that the thrombolytic activity (fresh thrombus) of the crude protein with a molecular weight greater than 7000D of the GYZC01 strain is not as good as that of urokinase, and the EC ₅₀ is 400

, urokinase EC ₅₀ is 100

, but against old thrombus, the activity of GYZC01 strain crude protein is stronger than that of urokinase, EC ₅₀ is 800

, urokinase EC ₅₀ is 1600

.

Nucleotide or Amino Acid Sequence Listing SEQ ID NO: 1

<110> Guizhou University

<120> A kind of Bacillus brevus and the method and use of the protein series prepared therefrom

<160> 1

<170> Patent In Version BLAST 2.1

<210> 1

<211> 1392

<212> RNA

<213> Bacillus brevis ( Brevibacillus parabrevis ) GYZC01

<400> 1

TTTTAGGACC CTAGCGGCGG ACGGGTGAGT AACACGTAGG CAACCTGCCT TTCAGACCGG 60

GATAACATAG GGAAACTTAT GCTAATACCG GATAGGTTTT TGGATTGCAT GATCCGAAAA 120

GAAAAGATGG CTTCGGCTAT CACTGGGAGA TGGGCCTGCG GCGCATTAGC TAGTTGGTGG 180

GGTAACGGCC TACCAAGGCG ACGATGCGTA GCCGACCTGA GAGGGTGACC GGCCACACTG 240

GGACTGAGAC ACGGCCCAGA CTCCTACGGG AGGCAGCAGT AGGGAATTTT CCACAATGGA 300

CGAAAGTCTG ATGGAGCAAC GCCGCGTGAA CGATGAAGGT CTTCGGATTG TAAAGTTCTG 360

TTGTCAGGGA CGAACACGTG CCGTTCGAAT AGGGCGGTAC CTTGACGGTA CCTGACGAGA 420

AAGCCACGGC TAACTACGTG CCAGCAGCCG CGGTAATACG TAGGTGGCAA GCGTTGTCCG 480

GATTTATTGG GCGTAAAGCG CGCGCAGGCG GCTATGTAAG TCTGGTGTTA A AGCCCGGAG 540

CTCAACTCCG GTTCGCATCG GAAACTGTGT AGCTTGAGTG CAGAAGGGA AAGCGGTATT 600

CCACGTGTAG CGGTGAAATG CGTAGAGATG TGGAGGAACA CCAGTGGCGA AGGCGGCTTT 661

CTGGTCTGTA ACTGACGCTG AGGCGCGAAA GCGTGGGGAG CAAACAGGAT TAGATACCCT 720

GGTAGTCCAC GCCGTAAACG ATGAGTGCTA GGTGTTGGGG GTTTCAATAC CCTCAGTGCC 780

GCAGCTAACG CAATAAGCAC TCCGCCTGGG GAGTACGCTC GCAAGAGTGA AACTCAAAGG 840

AATTGACGGG GGCCCGCACA AGCGGTGGAG CATGTGGTTT AATTCGAAGC AACGCGAAGA 900

ACCTTACCAG GTCTTGACAT CCCGCTGACC GCTCTGGAGA CAGAGCTTCC CTTCGGGGCA 960

GCGGTGACAG GTGGTGCATG GTTGTCGTCA GCTCGTGTCG TGAGATGTTG GGTTAAGTCC 1020

CGCAACGAGC GCAACCCCTTA TCTTTAGTTG CCAGCATTCA GTTGGGCACT CTAGAGAGAC 1080

TGCCGTCGAC AAGACGGAGG AAGGCGGGGA TGACGTCAAA TCATCATGCC CCTTATGACC 1140

TGGGCTACAC ACGTGCTACA ATGGTTGGTA CAACGGGATG CTACCTCGCG AGGGGACGCC 1200

AATCTCTGAA AACCAATCTC AGTTCGGATT GTAGGCTGCA ACTCGCCTAC ATGAAGTCGG 1260

AATCGCTAGT AATCGCGGAT CAGCATGCCG CGGTGAATAC GTTCCCGGGC CTTGTACACA 1320

CCGCCCGTCA CACCACGGGA GTTTGCAACA CCCGAAGTCG GTGAGGTAAC CGCAAGGAGC 1380

CAGCCGCCGA AA 1392

the

Claims (4)

1. the short genus bacillus of a kind is characterized in that: the short genus bacillus of its classification called after class ( Brevibacillus parabrevis) GYZC01, being deposited in Chinese typical culture collection center, deposit number is: CCTCC NO:M 2011461, preservation date is: on December 11st, 2011.

2. the short genus bacillus of a kind according to claim 1, it is characterized in that: the 16S rRNA of the short genus bacillus GYZC01 of described class has the sequence of SEQ ID NO:1.

3. utilize claim 1 or the short genus bacillus of 2 described kinds to prepare the method for protein series, it is characterized in that: the ferment product freeze overnight that will contain the short genus bacillus GYZC01 of class, melt in room temperature, obtain freezing solution liquid, freeze and separate liquid to put whizzer centrifugal, collect supernatant liquor, supernatant liquor adds the ammonium sulfate powder gradually, and until saturated, collecting precipitation spends the night, throw out obtains protein series with the dialysis of 7000D dialysis tubing.

4. the short genus bacillus of a kind according to claim 3 prepares the method for protein series, and it is characterized in that: the molecular weight of described protein series is greater than 7000D.

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