Method for expressing bacteriophage lyase by using chlorella vulgaris

Abstract

The invention discloses a method and an expression system for expressing bacteriophage lyase by using chlorella vulgaris. The microalgae vector takes chlorella vulgaris as an expression vector, and recombinant plasmids are electrically transferred into cell nucleus genes of the chlorella vulgaris. The method is simple, safe and low in cost. The carrier is high in microalgae system conversion rate, and the expressed lyase has good activity to bacteria. The chlorella vulgaris is an edible alga, and has a wide prospect in the subsequent application of oral administration or perfusion. Meanwhile, the fluorescence imaging performance of the microalgae system is outstanding, and possibility is provided for non-invasive tracking and real-time monitoring of the lyase in vivo. The unique characteristics of chlorella vulgaris are utilized, and a feasible thought and method are provided for efficient expression of the lyase.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for expressing bacteriophage lytic enzymes by utilizing Chlorella vulgaris. Background Art

[0002] Phage lytic enzymes are new antibacterial molecules encoded by the phage genome. Their bactericidal mechanism is to hydrolyze the cell wall peptidoglycan to cause bacterial lysis and death. In the severe situation of "drug-resistant bacterial infections are incurable" caused by the lack of safe green antibiotics, phage lytic enzymes, as potential new antibacterial drugs, have attracted great attention from scientists from all over the world. LysBT1 is a chimeric broad-spectrum phage lytic enzyme with the characteristics of safety, high efficiency, strong specificity and wide cleavage spectrum. However, as a protein drug, it will be degraded by proteases in the body, unstable and with high purification costs. Based on this, it is more urgent to develop and study a safer and more efficient expression system. In recent years, the rapid development of genetic engineering has provided a feasible solution for the improvement of microalgae species, and also provided a new idea for building a new expression platform as a delivery carrier for protein drugs to achieve safe, efficient and stable expression.

[0003] Chlorella is a kind of freshwater green microalgae widely found in nature. It is spherical, subspherical or ellipsoidal, without flagella, with a diameter of 2 to 10 μm and a cell wall thickness of 100 to 200 nm. It is a unicellular eukaryotic green algae. It is widely found in soil, lakes, oceans, Arctic and Antarctic ecosystems and desert systems. It is the first microalgae to be artificially cultivated by humans. Currently, the main widely cultivated ones are Chlorella vulgaris, Chlorella pyrenoidosa, Chlorella protozoa and Chlorella ellipsoidea. Chlorella is now widely used in various fields such as medicine, energy, aquaculture and wastewater treatment. It has a history of more than 20 years to synthesize target proteins through Chlorella. Chlorella vulgaris is one of the most widely cultivated algae species of Chlorella. It is rich in protein and various nutrients, with more than 50% protein. It has important commercial value and is also one of the human foods currently studied and certified. Due to its fast growth rate, easy cultivation and high plasticity, it is often used as the first choice for exogenous gene expression platform. Summary of the invention

[0004] The purpose of the present invention is to provide a safe, efficient and simple method for expressing exogenous lytic enzymes in microalgae.

[0005] The present invention first constructs a plasmid carrying a lytic enzyme gene fragment, and introduces the plasmid into competent common Chlorella cells by means of electroporation, and successfully achieves expression by utilizing the characteristics of common Chlorella, such as safety, rapid growth and reproduction, and high protein content. The transformation process is simplified, and the lytic enzyme produced and transformed by the microalgae expression system has obvious and good bactericidal or antibacterial effects.

[0006] The gene transfer technique is electroporation (also known as electroporation);

[0007] The exogenous expression system microalgae comprises the following steps:

[0008] 1) constructing a plasmid carrying a lytic enzyme gene fragment;

[0009] 2) Conduct cloning transformation verification;

[0010] 3) Establish a Chlorella expression system and perform electroporation;

[0011] 4) Fluorescence microscopy

[0012] 5) Conduct enzyme activity experiments;

[0013] 6) Minimum bactericidal test.

[0014] Compared with the prior art, the present invention has the following advantages and effects:

[0015] The homologous recombination sequence of the present invention enables the expression vector to be introduced into the genome of Chlorella vulgaris by electroporation, simplifies the operation of introducing the vector into the nuclear genome, and greatly reduces the conversion cost, providing technical support for the use of Chlorella vulgaris as a phage lytic enzyme bioreactor to produce high value-added products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : 21-day growth curve of Chlorella vulgaris;

[0017] Figure 2 : Tre li efTM5α competent cells carrying the target plasmid;

[0018] Figure 3 :Nucleic acid electrophoresis verification;

[0019] Figure 4 : Fluorescence verification after plasmid introduction;

[0020] Figure 5 : The left side shows the supernatant sample after direct centrifugation after recovery, and the right side shows the sample sample after centrifugation after algae fragmentation; the selected strain is Escherichia coli ATCC 25922.

[0021] Figure 6 :MIC experiment of concentrated and purified protein. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with the following drawings and embodiments, but the present invention is not limited to the following embodiments.

[0023] Example 1. Cultivation of algae species and determination of growth curves

[0024] The culture solution of Chlorella vulgaris at the end of logarithmic growth was inoculated into a 100 mL conical flask containing 50 mL of culture medium at a 10% inoculation rate, and placed in a light incubator for static culture. The culture conditions were: illumination 2640 Lux, humidity 72%, light / dark cycle 12h / 12h, and culture temperature (28.0±0.5°C). The absorbance of the algae solution at OD=560nm was measured regularly to detect the growth rate of Chlorella vulgaris.

[0025] Example 2. Recombinant cloning and transformation

[0026] Take two 100μL tubes of Tre li efTM5α competent cells melted on ice, add 10μL of the constructed recombinant plasmid pCAMBIA1301-BT1 respectively, gently blow and mix, and let stand on ice for 30min; heat shock in a 42℃ water bath for 45-60s, quickly transfer to an ice bath, and let stand for 2min. Add 700μL of non-resistant LB culture medium to the centrifuge tube, and resuscitate at 37℃200r / min for 60min; then take 80μL of the resuscitation solution and evenly spread it on the LB plate with 50μg / mL kanamycin, and invert the plate in a 37℃ incubator for overnight culture; add 5mL of LB culture medium to a 10mL EP tube, pick a single colony on the resistant plate, stir it in the culture medium, and culture it overnight.

[0027] Example 3. Extraction and verification of plasmid

[0028] 1. Add 250 μL Buffer BL to the adsorption column (the adsorption column is placed in the collection tube in advance), centrifuge at 12000 r / min for 1 min to activate the silica gel mold;

[0029] 2. Take 1-4 mL of overnight cultured bacterial solution, centrifuge at 12000 r / min for 1 min, collect the bacteria, and remove the supernatant as much as possible;

[0030] 3. Add 250 μL Buffer S1 (RNase A has been added) to resuspend the bacterial pellet and vortex until there is no sterile block;

[0031] 4. Add 250 μL Buffer S2 and gently turn upside down 6-8 times to fully lyse the bacteria;

[0032] 5. Add 350 μL Buffer S3, gently invert up and down 6-8 times, mix thoroughly (white flocculent precipitate will appear at this time), and centrifuge at 12000 r / min for 10-15 min;

[0033] 6. Carefully aspirate the supernatant and transfer it into the adsorption tube (without aspirating the precipitate), centrifuge at 12000r / min for 1min, discard the waste liquid, and put the adsorption column back into the empty collection tube;

[0034] 7. Add 700 μL Buffer W2 (with a specified volume of anhydrous ethanol) to the adsorption column, centrifuge at 12000 r / min for 1 min, and discard the waste liquid;

[0035] 8. Repeat step 7;

[0036] 9. Place the adsorption column back into the empty collection tube and centrifuge at 12000r / min for 2min;

[0037] 10. Remove the adsorption tube, place it in a clean 1.5mL centrifuge tube, and let it stand at 20-25℃ for 2-5min to allow the residual ethanol to evaporate. Add 30-50μLE luent (preheated at 60-65℃) to the middle part of the adsorption column, let it stand at 20-25℃ for 2min, and centrifuge at 12000r / min for 2min;

[0038] 11. Take 4 μL of extracted plasmid DNA and mix it with 2 μL of Master for nucleic acid verification.

[0039] Example 4. Electrotransformation of Chlorella

[0040] 1. The culture medium selected for Chlorella vulgaris is BG11 medium, with a light intensity of 2640 Lux, a humidity of 72%, a light / dark cycle of 12 h / 12 ​​h, and a culture temperature of (28.0 ± 0.5 ° C);

[0041] 2. Take Chlorella in the logarithmic growth period, collect about 1×108 Chlorella cells by centrifugation, and wash them three times with BG11 at 5000r / min for 5min;

[0042] 3. Add 100 μL 0.1 mol / L mannitol and 100 μL 0.1 mol / L sorbitol to suspend the Chlorella vulgaris cells;

[0043] 4. Add HEPES buffer (2.38 g dissolved in 9 mL) and suspend for later use;

[0044] 5. Take 100 μL of the algae cell solution prepared in step (4) and transfer it to a 0.2 cm electric shock cup, add 10 μL of the constructed plasmid and mix well, and place on ice for 30 min;

[0045] 6. Wipe the water off the electric shock cup and place it in an electroporator (Bio-RAD, USA). The electric field strength is 1.5 kV, the pulse distance is 2 mm, and the pulse time is 0.2 ms.

[0046] 7. Immediately after the electric shock, add 100 μL of pre-cooled BG11 liquid culture medium, transfer the mixture into a sterile centrifuge tube, continue to add 0.9 mL of BG11 culture medium, and continue to recover the culture under light conditions for 48 hours.

[0047] Example 5 Fluorescence microscopy

[0048] Take 20 μL of the recovered algae solution from the recovered culture medium and observe it under a fluorescence microscope. Observe and compare the Chlorella under a fluorescence microscope under different conditions of no light and light.

[0049] Example 6. Phage Lytic Enzyme Activity Assay

[0050] After 24 hours of recovery, the Chlorella was placed in a centrifuge and centrifuged at 12000r / min for 2 minutes, and the supernatant was collected. The precipitated algae was suspended with BG11, and then placed in an ultrasonic crusher and crushed at 300W for 15 minutes, and the supernatant was collected by centrifugation. The activity of the enzyme in the supernatant was tested by spotting a double layer (the bottom layer was 1.2% LB, and the upper layer was a mixture of 0.6% LB and bacterial solution). The strain selected this time was Escherichia coli ATCC25922.

[0051] 1. Inoculate a single colony of ATCC25922 into LB liquid culture medium and culture for about 4-6 hours until the logarithmic phase;

[0052] 2. Wash three times with PBS, adjust to OD600 of 0.6, and then dilute with LB medium at 1:100 for later use;

[0053] 3. Adjust the concentration of the isolated, purified and concentrated phage lytic enzyme to 512μg / mL, add 200μL lytic enzyme to the first column of the first three rows of the 96-well plate, add 100μL PBS to each of the 2nd to 12th columns, draw 100μL lytic enzyme from the first well and dilute it to the 10th well in turn, add 100μL diluted bacterial solution to wells 1-11, and add 100μL LB to well 12. Set up three groups of controls for each gradient lytic enzyme concentration, and set up blank controls and positive controls;

[0054] Incubate the culture in a 37°C incubator for 24 hours, and take the minimum inhibitory concentration (MIC) as the concentration of lytic enzyme that does not allow any bacterial growth.

Claims

1. An expression vector microalgae, characterized in that: The expression vector consists of common chlorella and a recombinant plasmid carrying a lytic enzyme.

2. The expression vector microalgae according to claim 1, characterized in that: The lytic enzyme was successfully expressed and had good lytic enzyme activity.

3. A method for expressing bacteriophage lytic enzyme using Chlorella vulgaris, characterized in that: The following steps are involved: (1) Inoculating 10% of the Chlorella vulgaris in the late logarithmic growth stage into a culture container filled with BG11 culture medium and culturing the culture in a light incubator; (2) Transform the recombinant plasmid encoding the bacteriophage lytic enzyme gene into TreliefTM5α competent cells, spread on LB plates containing resistance, and pick a single colony for overnight culture; (3) Extracting the recombinant plasmid and performing nucleic acid verification; (4) collecting the Chlorella in the logarithmic growth phase by centrifugation, washing, and then suspending in mannitol, sorbitol, and HEPES buffer; (5) Mix the algae cell solution with the recombinant plasmid, add BG11 medium to resume culture after electroporation; (6) Observe the transformed Chlorella under a fluorescence microscope; (7) collecting the transformed Chlorella, disrupting it with ultrasonic waves, and collecting the supernatant by centrifugation; (8) Detect the activity of bacteriophage lytic enzyme in the supernatant.

4. The method according to claim 3, characterized in that The culture conditions of the BG11 medium in step (1) are: light intensity 2640 Lux, humidity 72%, light / dark cycle 12h / 12h, and culture temperature 28.0±0.5°C.

5. The method according to claim 3, characterized in that: The recombinant plasmid in step (2) is pCAMBIA1301-BT1, and the LB plate contains 50 μg / mL kanamycin.

6. The method according to claim 3, characterized in that In step (4), the concentrations of mannitol and sorbitol are both 0.1 mol / L, and the amount of HEPES buffer used is 2.38 g / 9 mL.

7. The method according to claim 3, characterized in that The electroporation conditions in step (5) are: electric field strength 1.5 kV, pulse distance 2 mm, pulse time 0.2 ms, and recovery culture for 48 h.

8. The method according to claim 3, characterized in that The ultrasonic wave crushing conditions in step (7) are 300W and crushing for 15 minutes.

9. The method according to claim 3, characterized in that: In step (8), the activity of the bacteriophage lytic enzyme is detected by a double-layer agar spot method, the indicator bacteria is Escherichia coli ATCC25922, and the enzyme activity is determined based on the minimum inhibitory concentration.

10. An application of the expression vector microalgae according to any one of claims 1-2, characterized in that: Includes one or more of the following: (1) Used as an expression vector for other lytic enzymes; (2) Preparation of oral medications; (3) preparing bactericidal or bacteriostatic feed; (4) Preparation of other bactericidal and bacteriostatic products.

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