Outer membrane vesicle, recombinant vector, cell and application thereof

Abstract

The invention discloses an outer membrane vesicle, a recombinant vector, a cell and application thereof, and relates to the field of bioengineering. The preparation method of the outer membrane vesicle comprises the following steps: 1) reducing the stability of an escherichia coli cell membrane; 2) the secretion efficiency of escherichia coli outer membrane vesicles is improved; (3) displaying the surface of the tail tube protein A from the vibrio parahaemolyticus bacteriophage on the secreted outer membrane vesicles to realize targeted modification of the outer membrane vesicles; 4, Lsyqdvp001 endolysin coded by the ed1060 gene from the phage QDVP001 is wrapped on the modified outer membrane vesicles, the prepared outer membrane vesicles have the function of rapidly killing vibrio parahaemolyticus, can kill 1 / 10 <-3 > CFU / mL vibrio parahaemolyticus within 5 min, are suitable for departments such as farms and aquatic product technology promotion stations, and are used for solving the problems of AHPND treatment and control in the prawn breeding process.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to an outer membrane vesicle, a recombinant vector, a cell and uses thereof. Background Art

[0002] Litopenaeus vannamei ( Litopenaeus vannamei ), also known as Penaeus vannamei, is the largest economic shrimp farming variety in the aquaculture industry. In order to meet the growing demand for Penaeus vannamei, Penaeus vannamei farming has ushered in a booming development, and intensive high-density farming has emerged. With the continuous expansion of the scale of intensive high-density farming, problems such as deterioration of the farming environment, slowed growth rate, and the breeding of infectious pathogens have occurred frequently, resulting in increasingly serious shrimp diseases, causing huge economic losses to farmers. At present, the common outbreaks of Penaeus vannamei are mainly classified into three categories: viral diseases such as white spot disease (WSD) and yellow head disease (YHD); bacterial diseases such as acute hepatopancreasnecrosis syndrome (AHPND), red leg disease (RAD), and crustacean rot disease (SUD); parasitic diseases such as hepatopancreatic cysts and fixed ciliates. The breeding of shrimp diseases has caused huge economic losses to the shrimp farming industry, among which acute hepatopancreasnecrosis syndrome (AHPND) is one of the most influential diseases in the past two years.

[0003] Acute hepatopancreatic necrosis syndrome (AHPND) is a disease that often occurs within 30 to 35 days after the shrimp fry are released and can be observed as a sudden and massive death of shrimp fry. It is also called early mortality syndrome (EMS). Penaeus monodon , also known as Asian tiger shrimp ( Asian tiger shrimp )) and Penaeus vannamei ( Litopenaeus vannamei ) are more susceptible. AHPND has the characteristics of rapid transmission, wide spread and high mortality. Therefore, once the disease occurs, the losses caused are extremely heavy.

[0004] In 2013, the Global Aquaculture Association (GAA) and the Food and Agriculture Organization (FAO) reported that the causative agent of acute hepatopancreatic necrosis syndrome (AHPND) in shrimp was Vibrio parahaemolyticus. Vibrio parahaemolyticus ), a halophilic Gram-negative bacterium, is one of the most common pathogens of shrimp diseases. The main treatment method is to use antibiotics to kill bacteria. However, with the emergence of problems such as overuse of antibiotics and drug resistance, it is necessary to develop a safe and effective method to treat AHPND. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems in the prior art and provide an outer membrane vesicle, a recombinant vector, a cell and its use. The present invention includes the efficient excretion of outer membrane vesicles, the construction of outer membrane vesicles targeting Vibrio parahaemolyticus and the further encapsulation of Lsyqdvp001 endolysin in the vesicles. The prepared outer membrane vesicles have the function of rapidly killing Vibrio parahaemolyticus and can kill 1 / 10 of the Vibrio parahaemolyticus within 5 minutes. -3 CFU / mL Vibrio parahaemolyticus, suitable for farms, aquatic technology extension stations and other departments for the treatment and control of AHPND in shrimp farming.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A method for reducing the stability of Escherichia coli cell membranes, using RNA interference technology to inhibit the secretion of Escherichia coli lipoprotein Lpp, the gene sequence for inhibiting the secretion of Escherichia coli lipoprotein Lpp is shown in SEQ ID NO:1.

[0008] A method for improving the secretion efficiency of Escherichia coli outer membrane vesicles, overexpressing mepS encoding a peptidase gene in the above-mentioned Escherichia coli to improve the secretion efficiency of the outer membrane vesicles, wherein the sequence of the mepS encoding the peptidase gene is shown in SEQ ID NO: 2.

[0009] A method for targeting and binding to the outer membrane vesicle of Vibrio parahaemolyticus, wherein the tail tube protein A (TTPA) from the Vibrio parahaemolyticus phage is displayed on the outer membrane vesicle to achieve targeted modification of the outer membrane vesicle. The gene sequence for surface display of the tail tube protein A (TTPA) is shown in SEQ ID NO: 3.

[0010] An outer membrane vesicle that targets and kills Vibrio parahaemolyticus, wherein the Lsyqdvp001 endolysin encoded by the edl060 gene in the bacteriophage QDVP001 is encapsulated on the outer membrane vesicle, and the edl060 gene sequence is shown in SEQ ID NO:5.

[0011] A recombinant vector comprises at least one of the following: a gene sequence for inhibiting the secretion of Escherichia coli lipoprotein Lpp, mepS encoding a peptidase gene, a gene sequence of tail tube protein A (TTPA) for surface display, and an edl060 gene sequence from bacteriophage QDVP001. In the present invention, various vectors known in the art, such as plasmids, bacteriophages, and retroviruses, can be used.

[0012] A recombinant strain comprises the recombinant vector and obtains corresponding functions through vector introduction.

[0013] A host cell, comprising the recombinant vector, is used to carry and express the relevant gene. The recombinant vector can be introduced into the host cell by methods well known in the art, including: calcium chloride heat shock method, point transformation method, PEG-mediated method, gene gun method, etc.

[0014] A method for targeted killing of Vibrio parahaemolyticus using outer membrane vesicles: first, outer membrane vesicles that can target and bind to Vibrio parahaemolyticus are obtained, and then the edl060 gene carried by bacteriophage QDVP001 is encapsulated in the outer membrane vesicles, and then Vibrio parahaemolyticus is added to the culture medium, which can quickly and effectively kill Vibrio parahaemolyticus.

[0015] Furthermore, the working concentration of the outer membrane vesicles is 1-4 mg / mL; the reaction conditions are 30-37°C, and the concentration of the Vibrio parahaemolyticus liquid is 1 / 10 -3 ~1 / 10 -6 CFU / mL; time is 5 min. The outer membrane vesicles are gene sequences that have not been found in other microorganisms and have the ability to kill Vibrio parahaemolyticus in a targeted manner.

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0017] 1. The present invention inhibits the expression of lipoprotein LPP by RNA interference (RNAi), thereby reducing the stability of Escherichia coli cell membranes. Based on this, the peptidase encoding gene mepS is further overexpressed in Escherichia coli to obtain a recombinant Escherichia coli engineered strain capable of efficiently expressing outer membrane vesicles E. coil Lpp-mepS-pSB1C3 (carrying I0500-OmpA 5'UTR-Lpp 1.0-hfqbinding sequence-B0015_pSB1C3 and I0500-B0034-mepS-B0015_pSB1C3 plasmids) broadens the resources for the application of outer membrane vesicles.

[0018] 2. The present invention obtains a recombinant Escherichia coli engineered strain capable of secreting and binding to the outer membrane vesicles of Vibrio parahaemolyticus by displaying the tail tube protein A (TTPA) from the Vibrio parahaemolyticus phage on the surface of the outer membrane vesicles. E. coil Lpp-mepS-TTPA-pSB1C3 (carrying I0500-OmpA 5'UTR-Lpp 1.0-hfq binding sequence-B0015_pSB1C3, I0500-B0034-mepS-B0015_pSB1C3 and I0500-B0034-INPNC-TTPA-his-B0015_pSB1C3 plasmids) achieves targeted binding of outer membrane vesicles to Vibrio parahaemolyticus in the environment.

[0019] 3. The present invention encapsulates the Lsyqdvp001 endolysin encoded by the edl060 gene in the bacteriophage QDVP001 in a recombinant Escherichia coli engineering strain E. coil A recombinant Escherichia coli engineered strain capable of secreting outer membrane vesicles that target and kill Vibrio parahaemolyticus was obtained from the outer membrane vesicles secreted by Lpp-mepS-TTPA-pSB1C3 E. coil Lpp-mepS-TTPA-edl060-pSB1C3 (carrying I0500-OmpA 5'UTR-Lpp 1.0-hfq binding sequence-B0015_pSB1C3, I0500-B0034-mepS-B0015_pSB1C3, I0500-B0034-INPNC-TTPA-his-B0015_pSB1C3 and I0500-B0034-his-linker-edl060-B0015_pUC57-Simple plasmids), this strain has the function of efficiently secreting outer membrane vesicles that target and kill Vibrio parahaemolyticus. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the efficiency diagram of secretion of outer membrane vesicles by the recombinant E. coli engineering strain E. coil Lpp-mepS-pSB1C3, where: Figure 1 a in the figure is the concentration of secreted outer membrane vesicles and bacterial growth value of the engineered strain E. coil Lpp-pSB1C3. Figure 1 Middle b shows the secretion outer membrane vesicle concentration and bacterial growth value of the engineered strain E. coil Lpp-mepS-pSB1C3.

[0021] Figure 2 This is a picture of outer membrane vesicles taken by transmission electron microscopy.

[0022] Figure 3 This is the immunofluorescence reaction diagram of the outer membrane vesicles secreted by the recombinant Escherichia coli engineered strain E. coil Lpp-mepS-TTPA-pSB1C3 and the targeted binding of the Vibrio parahaemolyticus receptor Vp0980.

[0023] Figure 4 This is a diagram showing the effect of the outer membrane vesicles secreted by the recombinant E. coli engineering strain E. coil Lpp-mepS-TTPA-edl060-pSB1C3 targeting and killing Vibrio parahaemolyticus, in which: Figure 4 Figure a shows the growth of three bacteria (Escherichia coli, Vibrio parahaemolyticus and Vibrio natriuresis) after EDTA and water treatment. Figure 4 Figure b shows the growth of three bacteria (Escherichia coli, Vibrio parahaemolyticus and Vibrio natrii) after alkali treatment. Figure 4(c) shows the growth of three bacteria (Escherichia coli, Vibrio parahaemolyticus and Vibrio natriureticus) after treatment with EDTA and outer membrane vesicles containing endolysin Lsyqdvp001 over time.

[0024] Figure 5 This is a diagram showing the effect of adding arabinose inducer to induce the recombinant Escherichia coli engineered strain E. coil Lpp-mepS-TTPA-edl060-pSB1C3 to secrete functional outer membrane vesicles to target and kill Vibrio parahaemolyticus. DETAILED DESCRIPTION

[0025] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and Examples. If specific techniques or conditions are not indicated in the embodiments, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.

[0026] The Escherichia coli E. coil BL21 (DE3) involved in the following examples was purchased from Shanghai Biotechnology Co., Ltd.; Vibrio parahaemolyticus CICC 23924 was purchased from China Industrial Microbiological Culture Collection Center; I0500-OmpA 5'UTR-Lpp 1.0-hfq binding sequence-B0015_pSB1C3, I0500-B0034-mepS-B0015_pSB1C3, I0500-B0034-INPNC-TTPA-his-B0015_pSB1C3 and I0500-B0034-his-linker-edl060-B0015_pUC57-Simple plasmids were synthesized by GenScript Corporation.

[0027] Example 1: Construction of recombinant Escherichia coli engineered bacteria that secrete outer membrane vesicles targeting Vibrio parahaemolyticus

[0028] The lipoprotein Lpp of Escherichia coli can connect the bacterial outer membrane to the peptidoglycan layer and plays an important role in maintaining the stability of the membrane. However, the high stability of the membrane is not conducive to the large-scale secretion of outer membrane vesicles. In order to avoid the influence of endogenous omp, the present invention uses RNAi to eliminate the influence of this gene. The I0500-OmpA 5'UTR-Lpp 1.0-hfq binding sequence-B0015_pSB1C3 plasmid (nucleic acid sequence is shown in SEQ ID NO: 1) was constructed to reduce the expression of lipoprotein Lpp.

[0029] Specifically, the I0500-OmpA 5'UTR-Lpp 1.0-hfq binding sequence-B0015_pSB1C3 plasmid was transformed into Escherichia coli. Plasmid transformation: Take a tube of BL21 (DE3) competent cells from the -80 °C refrigerator and place it on ice to melt, add 10 μl of the target plasmid, mix gently and place it on ice, incubate for 30 min; then take it out and place it in a 42 °C water bath for 45 s; then quickly transfer it to ice and place it for 2 min, add 500 μl of sterile LB medium to the centrifuge tube, place it in a shaker at 37 °C 200 rpm and culture it for 1 h, then take 100 μl and spread it on the LB agar plate with kanamycin (1‰, v / v) resistance, and culture it in a constant temperature incubator at 37 °C for 12-16 hours. Pick 5 single colonies on the plate and perform colony PCR to determine the positive clone strain.

[0030] The purification method of outer membrane vesicles (OMV) was as follows: Escherichia coli carrying pI0500-OmpA 5'UTR-Lpp 1.0-hfq binding sequence-B0015_pSB1C3 was added to 10 mL LB medium containing 34 μg / mL chloramphenicol at a 1% inoculum and cultured at 37°C, 200 rpm for 12 hours. 1 mL of the overnight bacterial culture was transferred to 50 mL LB medium containing the same antibiotics and continued to incubate at 37°C, 200 rpm in a shaker until the OD 600 When the p-value reached 0.6-0.8, 20% arabinose was added for induction, and then the cells were transferred to 28°C and 200 rpm for further cultivation for 16 hours. The bacterial solution was collected and centrifuged at 4°C and 10,000 × g for 15 minutes to remove the cells. Then, the supernatant was first filtered with a 0.45 µm filter and then filtered again with a 0.22 µm filter. The cell-free supernatant was ultracentrifuged at 4°C and 140,000 × g for 2 hours to extract OMVs. The OMV pellet was suspended in 100 μL of buffer.

[0031] For quantification of outer membrane vesicles, OMVs were lysed with 1% (w / v) SDS (final concentration) at room temperature for 10 minutes, and 4 μL of lysate was taken to determine the protein concentration of each group of OMVs according to the instructions of the BCA protein assay kit. The protein concentration was calculated based on the standard curve. The results are shown in Figure 1 As shown in middle a, after eliminating the influence of lipoprotein Lpp, the concentration of outer membrane vesicles secreted by the recombinant Escherichia coli engineered bacteria E. coilLpp-pSB1C3 was 4.8 times that of the original strain.

[0032] MepS is a PG endopeptidase that is associated with the secretion of large amounts of OMVs. Therefore, the same method was used to transform the I0500-B0034-mepS-B0015_pSB1C3 plasmid (the nucleic acid sequence is shown in SEQ ID NO: 2) into the recombinant E. coli E. coilLpp-pSB1C3 to obtain the recombinant E. coli engineered bacteria E. coil Lpp-mepS-pSB1C3, and its outer membrane vesicle secretion efficiency was tested. The results were as follows: Figure 1 As shown in middle b, after overexpression of mepS enzyme, the secretion of outer membrane vesicles increased significantly, which was 1.9 times higher than that of E. coilLpp-pSB1C3. Negative staining of OMV samples was performed, and transmission electron microscopy (TEM) was used to observe that recombinant E. coli secreted a large number of outer membrane vesicles ( Figure 2 ).

[0033] In order to improve the specificity of OMV, the present invention uses INPNC to display the tail tube protein A (TTPA) from Vibrio parahaemolyticus phage on the surface of OMV, and the tail tube protein TTPA can target the receptor Vp0980 on Vibrio parahaemolyticus. The I0500-B0034-INPNC-TTPA-his-B0015_pSB1C3 (nucleic acid sequence is shown in SEQ ID NO: 3) plasmid is introduced into Escherichia coli E. coil Lpp-mepS-pSB1C3 to obtain recombinant Escherichia coli engineered bacteria E. coil Lpp-mepS-TTPA-pSB1C3.

[0034] The receptor Vp0980 of Vibrio parahaemolyticus is a membrane protein. Vp0980 was linked to pet28a(+) via NcoI and XhoI to obtain the his-Vp0980_pet28a(+) recombinant plasmid (the nucleic acid sequence is shown in SEQ ID NO:4). The plasmid was synthesized by GenScript Biotech Co., Ltd. The his-Vp0980_pet28a(+) plasmid was transformed into Escherichia coli. Plasmid transformation: Take a tube of BL21 (DE3) competent cells from the -80 °C refrigerator and place it on ice to melt, add 10 μL of the target plasmid, mix gently and place it on ice, incubate for 30 min; then take it out and place it in a 42 °C water bath for 45 seconds; then quickly transfer it to ice and place it for 2 min, add 500 μL of sterile LB medium to the centrifuge tube, place it in a shaker at 37 °C 200 rpm and culture it for 1 hour, then take 100 μL and spread it on the LB agar plate with kanamycin (1‰, v / v) resistance, and culture it in a constant temperature incubator at 37 °C for 12-16 hours. Pick 5 single colonies on the plate and perform colony PCR to determine the positive clone strain.

[0035] E. coli carrying his-Vp0980_pet28a(+) was added to 10 mL LB medium containing 40 μg / mL kanamycin at 37°C and 200 rpm to culture until OD600 reached 0.6-0.8, and 100 μmol / L IPTG was added to induce Vp0980 expression. After 24 hours of induction, the bacterial solution was collected and centrifuged at 4°C and 6500 rpm for 15 min. The supernatant was discarded, and the bacteria were washed with PBS and centrifuged under the same conditions. After the end, the bacteria were resuspended in binding buffer for disruption. The resuspended bacterial solution was placed on ice and the bacteria were disrupted using an ultrasonic disruptor. The ultrasonic disruptor probe was about 1 cm below the liquid. Note that the probe could not touch the wall of the centrifuge tube. The ultrasonic power was 220 W, the working time was 3 seconds, the interval time was 6 seconds, and the number of ultrasonic times was 90. The whole process was kept on ice. After the disruption, the bacterial liquid was centrifuged at 4 °C and 10,000 rpm for 20 min to obtain the crude enzyme, and the supernatant was filtered using a 0.45 µm filter membrane and placed on ice. The protein was purified using a HisTrap column produced by GE. The nickel ions in the column filler can bind to the 6×His tag on the target protein, thereby achieving protein purification.

[0036] Immunofluorescence reaction verified that tail tube protein A could target the receptor Vp0980 on Vibrio parahaemolyticus. The plasmid I0500-B0034-INPNC-TTPA-his-B0015_pSB1C3 (nucleic acid sequence is shown in SEQ ID NO: 3) was introduced into Escherichia coli E. coil (BL21). The E. coli carrying I0500-B0034-INPNC-TTPA-his-B0015_pSB1C3 was added to 10 mL LB medium containing 34 μg / mL chloramphenicol at a 1% inoculation rate and cultured at 37°C and 200 rpm until OD600 reached 0.6-0.8. Then, 0.2% (v / v) arabinose was added and the culture was continued at 37°C and 200 rpm for 2-4 hours.

[0037] (1) Pipette 1 mL of bacterial culture into a centrifuge tube and centrifuge at 6500 rpm for 3 min at room temperature. Discard the supernatant.

[0038] (2) Add 1 mL of PBS buffer, mix well, centrifuge at 6500 rpm for 3 min at room temperature, and discard the supernatant.

[0039] (3) Repeat step (2) twice.

[0040] (4) Add 500 μL~1 mL of 5% skim milk, mix well, and incubate at 37 °C for 0.5~1 h.

[0041] (5) Centrifuge at 6500 rpm for 3 min at room temperature and discard the supernatant.

[0042] (6) Repeat step (2) three times.

[0043] (The following operations (7) to (9) should be performed in a dark place)

[0044] (7) Add 490 µL PBS buffer and mix well. Add FITC anti-6×His antibody (1: 1000) and incubate at 37°C for 1 h.

[0045] (8) Repeat step (5) once and step (2) three times.

[0046] (9) Take 200 μL of the above solution and add it to a 96-well plate. Use a microplate reader to measure the fluorescence intensity (absorption wavelength: 492 nm, emission wavelength: 518 nm) and OD600.

[0047] The results are as follows Figure 3 As shown, tail tube protein A is able to target the receptor Vp0980 on Vibrio parahaemolyticus.

[0048] Example 2: Construction and application of recombinant Escherichia coli engineering bacteria that secrete outer membrane vesicles that target and kill Vibrio parahaemolyticus

[0049] Lsyqdvp001 endolysin, encoded by the edl060 gene in bacteriophage QDVP001, can specifically hydrolyze chitosan on the cell wall of Vibrio parahaemolyticus Vp. In addition, adding the LMT signal peptide in front of the edl060 gene can guide Lsyqdvp001 to the peptidoglycan layer, thereby being encapsulated in the outer membrane vesicle. Therefore, I0500-B0034-his-linker-edl060-B0015_pUC57-Simple (nucleic acid sequence as shown in SEQ ID NO: 5) was transformed into recombinant Escherichia coli E. coil Lpp-mepS-TTPA-pSB1C3 using the same method to obtain recombinant Escherichia coli engineered bacteria E. coil Lpp-mepS-TTPA-edl060-pSB1C3.

[0050] The engineered bacteria E. coil Lpp-mepS-TTPA-edl060-pSB1C3 was inoculated into LB medium with 1 μL / mL ampicillin resistance and 34 μL / mL chloramphenicol resistance, and cultured at 37 °C and 200 rpm until OD 600Reach 0.6. Transfer to 400 mL culture medium at 1% inoculum and culture at 37 °C, 200 rpm for 2-4 h. Then add arabinose with a final concentration of 0.2% (v / v) for induction. Add the same amount of arabinose after 1 hour, and then repeat the induction every 2 hours until the total induction time reaches 12 hours. Purify and obtain outer membrane vesicles. Investigate the targeted killing application of outer membrane vesicles against Vibrio parahaemolyticus.

[0051] (1) Escherichia coli BL21 (DE3) and Vibrio parahaemolyticus CICC 23924 were cultured in LB medium, and the natural Vibrio strain VnDX was cultured in LBv2 medium.

[0052] (2) Take 1 mL of each bacterial solution and incubate with 1 mL of 150 mM EDTA for 30 min.

[0053] (3) Centrifuge at 6500 rpm for 10 min to remove EDTA and discard the supernatant. Then add 1 mL of the corresponding culture medium and resuspend the pellet.

[0054] (4) Repeat step (3) twice.

[0055] (5) Dilute each solution to a concentration of 10 3 , 10 4 , 10 5 , 10 6 .

[0056] (6) Add 1 mg / mL purified outer membrane vesicles and incubate for 5 minutes, then perform an agar spread experiment and culture at 37 °C overnight.

[0057] The results of the agar spread experiment are as follows Figure 4 As shown in a, the negative control group was prepared by adding EDTA and sterile water to three bacterial solutions from different sources (Escherichia coli, Vibrio parahaemolyticus and Vibrio natrii). The results showed that EDTA and sterile water did not affect the normal growth of the three bacteria. Then a positive control experiment was carried out by adding alkaline solution to the three bacterial solutions. The results are shown in Figure 4 As shown in b, the bacteria could not grow normally after being treated with strong alkaline solution. Finally, EDTA and outer membrane vesicles containing endolysin Lsyqdvp001 were added to the three bacterial solutions. Figure 4 As shown in Figure c, when the three bacterial cultures were treated with outer membrane vesicles containing endolysin, Escherichia coli and Vibrio natrii could still grow normally, while Vibrio parahaemolyticus basically did not grow on the plate. This shows that the endolysin Lsyqdvp001 has no killing effect on other bacteria, but is highly specific to Vibrio parahaemolyticus, and the killing results are consistent with those of alkaline lysis treatment. The bactericidal effect of outer membrane vesicles before and after arabinose induction was further investigated, and the results are shown in Figure 5. Figure 5 As shown in Figure 2, the outer membrane vesicles secreted by E. coli without the addition of arabinose inducer cannot express proteins that target and kill Vibrio parahaemolyticus, so the final growth concentration of the bacteria is 2x10 8 CFU / mL, the outer membrane vesicles secreted by Escherichia coli added with arabinose inducer can completely kill Vibrio parahaemolyticus in the environment. Therefore, the AHPND treatment method designed by the present invention has broad application prospects for subsequent killing of Vibrio parahaemolyticus.

[0058] In summary, the present invention targets pathogenic Vibrio parahaemolyticus Vp, uses modified OMV to deliver recombinant plasmids, and the plasmids will express endolysin in the cells, thereby specifically killing Vibrio parahaemolyticus Vp and curing sick shrimp. This method can quickly and effectively kill bacteria, thereby achieving the effect of treating AHPND, and is expected to be used for the treatment of sick shrimp in farms.

[0059] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A method for preparing outer membrane vesicles, characterized in that: The following steps are involved: 1) Reduce the stability of E. coli cell membrane; 2) Improve the secretion efficiency of E. coli outer membrane vesicles; 3) The tail tube protein A from the Vibrio parahaemolyticus phage is displayed on the outer membrane vesicles secreted in step 2) to achieve targeted modification of the outer membrane vesicles; 4) Encapsulate the Lsyqdvp001 endolysin encoded by the edl060 gene from bacteriophage QDVP001 onto the outer membrane vesicles transformed in step 3).

2. A method for preparing outer membrane vesicles as claimed in claim 1, characterized in that: In step 1), RNA interference technology is used to inhibit the secretion of Escherichia coli lipoprotein Lpp to reduce the stability of the Escherichia coli cell membrane. The gene sequence for inhibiting the secretion of Escherichia coli lipoprotein Lpp is shown in SEQ ID NO:

1.

3. A method for preparing outer membrane vesicles as claimed in claim 1, characterized in that: In step 2), mepS encoding a peptidase gene is overexpressed in the Escherichia coli treated in step 1) to improve the secretion efficiency of outer membrane vesicles. The sequence of the mepS encoding a peptidase gene is shown in SEQ ID NO:

2.

4. A method for preparing outer membrane vesicles as claimed in claim 1, characterized in that: In step 3), the gene sequence for surface display of tail tube protein A is shown in SEQ ID NO:

3.

5. A method for preparing outer membrane vesicles as claimed in claim 1, characterized in that: In step 4), the edl060 gene sequence is shown in SEQ ID NO:

5.

6. An outer membrane vesicle, characterized in that: Obtained by any preparation method of claims 1 to 5.

7. A recombinant vector, characterized in that: The invention comprises at least one of the following gene sequences: the gene sequence for inhibiting the secretion of Escherichia coli lipoprotein Lpp according to claim 2, the mepS sequence encoding the peptidase gene according to claim 3, the gene sequence of tail tube protein A for surface display according to claim 4, and the edl060 gene sequence according to claim 5.

8. A recombinant strain, characterized in that: The invention comprises the recombinant vector as described in claim 7, and the corresponding function is obtained by introducing the vector.

9. A host cell, characterized in that: The recombinant vector according to claim 7 is used to carry and express relevant genes.

10. The use of an outer membrane vesicle according to claim 6, characterized in that: Used for targeted killing of Vibrio parahaemolyticus.

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