Probiotics with adhesiveness as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses a probiotic with adhesiveness and a preparation method and application thereof, and the preparation method comprises the following steps: obtaining a target protein expression sequence; the recombinant plasmid pHSP02-VP28-Lp0237 is obtained by using the recombinant plasmid as a template; the method comprises the following steps: obtaining a pHSP02-VP28-Lp023-pLH01-WCFS1 bacterial strain; and sequentially removing the pLH01 plasmid and the recombinant plasmid pHSP02-VP28-Lp023-pLH01-WCFS1 in the step 3, so as to obtain the new bacterial strain. The pLH01 plasmid and the recombinant plasmid pHSP02-VP28-Lp0237 in the pHSP02-VP28-Lp0237 in the pHSP02-VP28-Lp0237 in the The probiotics have high adhesiveness and can be adhered to intestinal tracts of aquatic animals, envelope protein VP28 generated by the probiotics and prawn white spot syndrome viruses are competitively combined to cells of prawns, the competitive inhibition effect of the envelope protein VP28 and the prawn white spot syndrome viruses is achieved, and therefore infection of the prawn white spot syndrome viruses to the aquatic animals is blocked.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to an adhesive probiotic and a preparation method and application thereof. Background Art

[0002] With the rapid development of science and technology, the aquaculture industry has gradually entered a mature stage, and the aquaculture output and scale have been continuously improved. However, the intensive aquaculture process with high density and high feeding not only leads to low quality of aquaculture products, but also high incidence of nutritional metabolic diseases and even large-scale deaths, causing huge economic losses. Among them, White spot syndrome virus (WSSV) causes acute and fatal infectious diseases in farmed shrimps worldwide, causing huge economic losses to the shrimp farming industry.

[0003] The use of probiotics by humans has gradually become popular, but their development and application in the aquaculture industry is still in its infancy. Existing aquaculture probiotics are generally single strains with single functions, resulting in poor efficacy of probiotics in treating white spot syndrome virus in aquatic animals and inability to safely prevent and control aquatic pathogens.

[0004] Therefore, there is an urgent need for a new type of engineered probiotic and its use method to effectively improve the adhesion of probiotics and reduce the impact of white spot syndrome virus on aquatic animals in aquaculture. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an adhesive probiotic and a preparation method and application thereof. The probiotic of the present invention has high adhesion and can adhere to the intestines of aquatic animals. The envelope protein VP28 produced by the probiotic competitively binds to the cells of the shrimp with the white spot syndrome virus of the shrimp, exerts its competitive inhibition effect, and thus blocks the infection of the white spot syndrome virus of the shrimp to the aquatic animals.

[0006] The present invention provides a method for preparing probiotics with adhesion, comprising the following steps: Step 1: The N20 sequence, the lower homologous arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1, the Lp_0237 gene of Lactobacillus acidophilus, the linker sequence, the gene of the envelope protein VP28, and the upper homologous arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 were sequentially connected by the PCR method to obtain the target protein expression sequence, i.e., AB1-VP28-Lp_0237-AB2-N20sgRNA; Step 2: Cut the pHSP02 plasmid with an enzyme to obtain a plasmid backbone; recover the solution containing the plasmid backbone and clone and assemble it with the solution containing the AB1-VP28-Lp_0237-AB2-N20 sgRNA in step 1, and react to obtain the recombinant plasmid pHSP02-VP28-Lp_0237; Step 3: transform the pLH01 plasmid into competent cells of Lactobacillus plantarum WCFS1, culture once, and obtain the pLH01-WCFS1 strain; transform the recombinant plasmid pHSP02-VP28-Lp_0237 into competent cells of the pLH01-WCFS1 strain, culture twice, and screen to obtain the pHSP02-VP28-Lp_023-pLH01-WCFS1 strain; Step 4: Discard the pLH01 plasmid and the recombinant plasmid pHSP02-VP28-Lp_0237 in the pHSP02-VP28-Lp_023-pLH01-WCFS1 strain in step 3 in sequence to obtain adhesive probiotics.

[0007] Furthermore, the Lactobacillus plantarum WCFS1 was purchased from Beijing Biobo Biotechnology Co., Ltd. with a platform number of bio-03564 and another number of ATCC BAA-793.

[0008] Furthermore, the Lactobacillus acidophilus was purchased from Ningbo Testo Biotechnology Co., Ltd. with the product number TS279066.

[0009] Furthermore, the method for preparing the N20 sequence includes: designing an N20 sequence targeting the Lp_0237 gene of Lactobacillus plantarum WCFS1.

[0010] Furthermore, the N20 sequence is shown as SEQ ID NO.1.

[0011] Furthermore, the Lp_0237 gene of Lactobacillus acidophilus is shown as SEQ ID NO.2.

[0012] Furthermore, the amino acid sequence of the envelope protein VP28 is SEQ ID NO.3, or an amino acid sequence in which the 135th amino acid threonine in the amino acid sequence SEQ ID NO.3 is mutated to an amino acid alanine, or an amino acid sequence in which the 152nd amino acid serine in the amino acid sequence SEQ ID NO.3 is mutated to a lysine, or an amino acid sequence in which the 182nd amino acid alanine in the amino acid sequence SEQ ID NO.3 is mutated to an aspartic acid, or an amino acid sequence in which the 196th amino acid valine in the amino acid sequence SEQ ID NO.3 is mutated to a methionine.

[0013] Furthermore, the gene of the envelope protein VP28 is a gene that can encode the amino acid sequence of the envelope protein VP28.

[0014] Furthermore, the gene sequence encoding the envelope protein VP28 with the amino acid sequence of SEQ ID NO.3 is shown as SEQ ID NO.18.

[0015] Furthermore, the sequence SEQ ID NO.18 is.

[0016] Furthermore, the linker sequence is shown as SEQ ID NO.10.

[0017] Further, the preparation method of the upper homologous arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 and the lower homologous arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 comprises: The whole genome of Lactobacillus plantarum WCFS1 was extracted, and the upper homologous arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 was obtained by PCR using the upstream primer AB1-F and the downstream primer AB1-R; the lower homologous arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 was obtained by PCR using the upstream primer AB2-F and the downstream primer AB2-R.

[0018] Furthermore, the sequence of the upstream primer AB1-F is shown in SEQ ID NO.4.

[0019] Furthermore, the sequence of the downstream primer AB1-R is shown in SEQ ID NO.5.

[0020] Furthermore, the sequence of the upstream primer AB2-F is shown in SEQ ID NO.6.

[0021] Furthermore, the sequence of the downstream primer AB2-R is shown in SEQ ID NO.7.

[0022] Furthermore, the method for preparing the Lp_0237 gene of Lactobacillus acidophilus includes: extracting the whole gene of Lactobacillus acidophilus, and obtaining the Lp_0237 gene of Lactobacillus acidophilus by PCR method using upstream primer Lp_0237-F and downstream primer Lp_0237-R.

[0023] Furthermore, the sequence of the upstream primer Lp_0237-F is shown in SEQ ID NO.8.

[0024] Furthermore, the sequence of the downstream primer Lp_0237-R is shown in SEQ ID NO.9.

[0025] Furthermore, in step 2, the enzymes used are ApaⅠendonuclease and XbaⅠendonuclease (purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.), wherein the CAS number of ApaⅠendonuclease is 85270-15-1, and the CAS number of XbaⅠendonuclease is 81295-42-3.

[0026] Furthermore, in step 2, the plasmid backbone is 13000 bp.

[0027] Furthermore, in step 2, the volume ratio of the solution containing the plasmid backbone to the solution containing AB1-VP28-Lp_0237-AB2-N20sgRNA is 1:3.

[0028] Furthermore, in step 2, the reaction temperature is 50° C. and the reaction time is 1 h.

[0029] Furthermore, in step 3, the method for preparing competent cells of Lactobacillus plantarum WCFS1 comprises: Lactobacillus plantarum WCFS1 was inoculated into MRS broth medium containing glycine and cultured at 37°C for 12 h; then transferred to SGMRS broth medium containing glycine and sorbitol at a 2% inoculum volume and cultured at 37°C for 3 h; 5 μL of 50 μg / mL ampicillin was added and cultured for 1 h until OD 600 End the culture at 0.5-0.6; centrifuge the bacterial solution at 4°C and 4000 rpm, remove the supernatant, collect the bacterial precipitate, resuspend the bacterial precipitate with 30 mL of SM buffer and mix it by blowing; centrifuge again at 4°C and 4000 rpm, remove the supernatant, collect the bacterial precipitate, resuspend it with SM buffer and mix it by blowing (the volume of SM buffer is 1% of the volume of the bacterial solution when centrifuged again at 4°C and 4000 rpm), and divide it into 1.5 mL centrifuge tubes after resuspending, and store 100 μL in each tube.

[0030] Furthermore, the mass concentration of glycine in the MRS broth medium is 3 g / 100 mL.

[0031] Furthermore, the mass concentration of glycine in the SGMRS broth culture medium is 3 g / 100 mL.

[0032] Furthermore, the mass concentration of sorbitol in the SGMRS broth culture medium is 3 g / 100 mL.

[0033] Further, the method of transforming the plasmid into competent cells includes: Add 1.5 μg of plasmid to the prepared competent cells, let them stand on ice for 30 min, then add them to the electroporation cup, and perform electric shock for 5 ms at 2.0 kV. Immediately after the electric shock, add 100 μL of incubation medium and incubate in a 37°C incubator for 3 h.

[0034] Furthermore, in step 3, the one-time culturing method includes: First, the pLH01 plasmid was transformed into competent cells of Lactobacillus plantarum WCFS1, spread on MRS solid medium containing chloramphenicol, and cultured at 37°C for 12 h. The cultured strain was prepared for competence again. During the competent preparation process, when the cells were cultured to OD 600 When the molar concentration of the inducing peptide was 0.2-0.3, ampicillin (Amp) was added to the incubation medium and cultured at 37°C for another 1 h until the OD 600 was 0.5, and the pLH01-WCFS1 strain was obtained.

[0035] Furthermore, the volume ratio of chloramphenicol in the MRS solid culture medium is 10%.

[0036] Furthermore, the mass concentration of ampicillin in the incubation medium is 100 μg / mL.

[0037] Furthermore, the volume ratio of the inducing peptide in the incubation medium is 1%.

[0038] Furthermore, in step 3, the secondary culture method includes: First, take 1.5 μg of the recombinant plasmid PHSP02-VP28-Lp_0237 and transform it into PLH01-WCFS1 competent cells at 2.0 kV and 5 ms, then add it to 1 mL of incubation medium, culture it at 37 °C for 3 h, spread it on MRS solid medium containing chloramphenicol (50 mg / mL) and erythromycin (50 mg / mL), and culture it at 37 °C for 12 h to obtain the pHSP02-VP28-Lp_0237-pLH01-WCFS1 strain.

[0039] Furthermore, the method for removing the pLH01 plasmid specifically includes: inoculating the pHSP02-VP28-Lp_0237-pLH01-WCFS1 strain into an MRS broth medium containing erythromycin (molar concentration of 10 mM in MRS broth medium) and chloramphenicol (molar concentration of 10 mM in MRS broth medium), shaking and culturing at a speed of 220 rpm and a temperature of 37°C for 12 hours, diluting and spreading on an MRS solid medium containing erythromycin (10 μg / mL), culturing at 37°C for 12 hours, and inoculating on an MRS solid medium containing erythromycin (10 μg / mL) and chloramphenicol (10 μg / mL), and culturing at 37°C for 12 hours.

[0040] Furthermore, verification is required after the pLH01 plasmid is removed. The verification method includes: inoculating the strain in MRS solid culture medium containing chloramphenicol (10 μg / mL) and culturing at 37°C for 12 hours.

[0041] Furthermore, the method for removing the recombinant plasmid pHSP02-VP28-Lp_0237 specifically includes: inoculating the strain from which the pLH01 plasmid has been removed into MRS broth medium and shaking and culturing at 37°C and 220 rpm for 12 hours, and then spreading the strain on MRS solid medium and culturing at 37°C for 12 hours.

[0042] Furthermore, verification is required after the removal of the recombinant plasmid pHSP02-VP28-Lp_0237. The verification method includes: inoculating the strain in MRS solid culture medium containing erythromycin (10 μg / mL) and culturing at 37°C for 12 hours.

[0043] The invention also provides plant lactobacillus obtained by the method.

[0044] The invention also provides application of the plant lactobacillus in cultured aquatic products.

[0045] The present invention also provides the use of the plant lactobacillus in preparing a medicine for treating and / or preventing white spot syndrome of shrimp.

[0046] The embodiments of the present invention have the following technical effects: 1. The present invention uses probiotic Lactobacillus plantarum WCFS1 as the starting strain, and transforms into a new probiotic containing the envelope protein VP28 and the adhesion protein Lp_0237 provided by the present invention. The adhesion protein Lp_0237 can enhance the adhesion ability of the probiotic, so that the probiotic can be colonized in the intestine, thereby continuously generating the envelope protein VP28 in the body, blocking the target virus, and thus preventing the effect of white spot syndrome of shrimp. In addition, due to the adhesion effect of the adhesion protein Lp_0237, it can establish the first line of defense at the mucosal invasion point to prevent the WSSV virus from invading the body, effectively solving the problem of inconvenience of ordinary vaccine administration and unsatisfactory duration of drug efficacy.

[0047] 2. The present invention provides a new type of engineered probiotics, which is added to feed and fed to whiteleg shrimp. The effects of the probiotics on intestinal adhesion and the protection of whiteleg shrimp against viral infection are analyzed, providing a theoretical and application basis for the immune prevention of white spot syndrome virus in shrimp. The probiotics can serve the majority of farmers and enhance the rural economy, and have important economic and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 It is a process diagram provided by an embodiment of the present invention.

[0050] Figure 2 It is the pHSP02 recombinant plasmid provided in the embodiment of the present invention.

[0051] Figure 3 1 is an adhesion index diagram provided by the embodiments and comparative examples of the present invention.

[0052] Figure 4 2 is a graph showing the adhesion rates of the embodiments of the present invention and the comparative examples.

[0053] Figure 5 It is a graph showing the antibacterial zone diameter results of the embodiments of the present invention and the comparative examples. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0055] In a first aspect, some embodiments of the present invention provide a method for preparing probiotics with adhesion, comprising the following steps: Step 1: The N20 sequence, the lower homologous arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1, the Lp_0237 gene of Lactobacillus acidophilus, the linker sequence, the gene of the envelope protein VP28, and the upper homologous arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 were sequentially connected by the PCR method to obtain the target protein expression sequence, i.e., AB1-VP28-Lp_0237-AB2-N20sgRNA; Step 2: Cut the pHSP02 plasmid with an enzyme to obtain a plasmid backbone; recover the solution containing the plasmid backbone and clone and assemble it with the solution containing the AB1-VP28-Lp_0237-AB2-N20 sgRNA in step 1, and react to obtain the recombinant plasmid pHSP02-VP28-Lp_0237; Step 3: transform the pLH01 plasmid into competent cells of Lactobacillus plantarum WCFS1, culture once, and obtain the pLH01-WCFS1 strain; transform the recombinant plasmid pHSP02-VP28-Lp_0237 into competent cells of the pLH01-WCFS1 strain, culture twice, and screen to obtain the pHSP02-VP28-Lp_023-pLH01-WCFS1 strain; Step 4: Discard the pLH01 plasmid and the recombinant plasmid pHSP02-VP28-Lp_0237 in the pHSP02-VP28-Lp_023-pLH01-WCFS1 strain in step 3 in sequence to obtain adhesive probiotics.

[0056] In some embodiments, the method for preparing the N20 sequence includes: designing an N20 sequence targeting the Lp_0237 gene of Lactobacillus plantarum WCFS1.

[0057] In some embodiments, the N20 sequence is shown as SEQ ID NO.1.

[0058] In some embodiments, the Lp_0237 gene of Lactobacillus acidophilus is as shown in SEQ ID NO.2.

[0059] In some embodiments, the amino acid sequence of the envelope protein VP28 is SEQ ID NO.3, or an amino acid sequence in which the 135th amino acid threonine in the amino acid sequence SEQ ID NO.3 is mutated to alanine, or an amino acid sequence in which the 152nd amino acid serine in the amino acid sequence SEQ ID NO.3 is mutated to lysine, or an amino acid sequence in which the 182nd amino acid alanine in the amino acid sequence SEQ ID NO.3 is mutated to aspartic acid, or an amino acid sequence in which the 196th amino acid valine in the amino acid sequence SEQ ID NO.3 is mutated to methionine.

[0060] In some embodiments, the gene of the envelope protein VP28 is a gene that can encode the amino acid sequence of the envelope protein VP28.

[0061] In some embodiments, the gene sequence encoding the envelope protein VP28 with the amino acid sequence of SEQ ID NO.3 is shown as SEQ ID NO.30.

[0062] In some embodiments, the sequence SEQ ID NO.30 is.

[0063] In some embodiments, the linker sequence is shown as SEQ ID NO.10.

[0064] In some embodiments, the preparation method of the upper homology arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 and the lower homology arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 comprises: The whole genome of Lactobacillus plantarum WCFS1 was extracted, and the upper homologous arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 was obtained by PCR using the upstream primer AB1-F and the downstream primer AB1-R; the lower homologous arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 was obtained by PCR using the upstream primer AB2-F and the downstream primer AB2-R.

[0065] In some embodiments, the sequence of the upstream primer AB1-F is shown as SEQ ID NO.4.

[0066] In some embodiments, the sequence of the downstream primer AB1-R is shown as SEQ ID NO.5.

[0067] In some embodiments, the sequence of the upstream primer AB2-F is shown as SEQ ID NO.6.

[0068] In some embodiments, the sequence of the downstream primer AB2-R is shown as SEQ ID NO.7.

[0069] In some embodiments, the method for preparing the Lp_0237 gene of Lactobacillus acidophilus includes: extracting the whole gene of Lactobacillus acidophilus, and obtaining the Lp_0237 gene of Lactobacillus acidophilus by PCR using an upstream primer Lp_0237-F and a downstream primer Lp_0237-R.

[0070] In some embodiments, the sequence of the upstream primer Lp_0237-F is shown as SEQ ID NO.8.

[0071] In some embodiments, the sequence of the downstream primer Lp_0237-R is shown in SEQ ID NO.9.

[0072] In some embodiments, in step 2, the enzymes used are ApaⅠendonuclease and XbaⅠendonuclease.

[0073] In some embodiments, in step 2, the plasmid backbone is 13000 bp.

[0074] In some embodiments, in step 2, the volume ratio of the solution containing the plasmid backbone to the solution containing AB1-VP28-Lp_0237-AB2-N20 sgRNA is 1:3.

[0075] In some embodiments, in step 2, the reaction temperature is 50° C. and the reaction time is 1 h.

[0076] In some embodiments, in step 3, the method for preparing competent cells of Lactobacillus plantarum WCFS1 includes: Lactobacillus plantarum WCFS1 was inoculated into MRS broth medium containing glycine and cultured at 37°C for 12 h; then transferred to SGMRS broth medium containing glycine and sorbitol at a 2% inoculum volume and cultured at 37°C for 3 h; 5 μL of 50 μg / mL ampicillin was added and cultured for 1 h until OD 600End the culture at 0.5-0.6; centrifuge the bacterial solution at 4°C and 4000 rpm, remove the supernatant, collect the bacterial precipitate, resuspend the bacterial precipitate with 30 mL of SM buffer and mix it by blowing; centrifuge again at 4°C and 4000 rpm, remove the supernatant, collect the bacterial precipitate, resuspend it with SM buffer and mix it by blowing (the volume of SM buffer is 1% of the volume of the bacterial solution when centrifuged again at 4°C and 4000 rpm), and divide it into 1.5 mL centrifuge tubes after resuspending, and store 100 μL in each tube.

[0077] In some embodiments, the mass concentration of glycine in the MRS broth medium is 3 g / 100 mL.

[0078] In some embodiments, the mass concentration of glycine in the SGMRS broth culture medium is 3 g / 100 mL.

[0079] In some embodiments, the mass concentration of sorbitol in the SGMRS broth culture medium is 3 g / 100 mL.

[0080] In some embodiments, the method of transforming a plasmid into a competent cell comprises: Add 1.5 μg of plasmid to the prepared competent cells, let them stand on ice for 30 min, then add them to the electroporation cup, and perform electric shock for 5 ms at 2.0 kV. Immediately after the electric shock, add 100 μL of incubation medium and incubate in a 37°C incubator for 3 h.

[0081] In some embodiments, in step 3, the method of culturing once comprises: First, the pLH01 plasmid was transformed into competent cells of Lactobacillus plantarum WCFS1, spread on MRS solid medium containing chloramphenicol resistance, and cultured at 37°C for 12 h. The cultured strain was prepared for competence again. During the competent preparation process, when the cells were cultured to OD 600 When the molar concentration of the inducing peptide is 0.2-0.3, add ampicillin to the incubation medium and culture at 37°C for another 1 h until the OD 600 was 0.5, and the pLH01-WCFS1 strain was obtained.

[0082] In some embodiments, the volume ratio of chloramphenicol in the MRS solid culture medium is 10%.

[0083] In some embodiments, the mass concentration of ampicillin in the incubation medium is 100 μg / mL.

[0084] In some embodiments, the volume ratio of the inducing peptide in the incubation medium is 1%.

[0085] In some embodiments, in step 3, the secondary culturing method includes: First, take 1.5 μg of the recombinant plasmid pHSP02-VP28-Lp_0237 and electrotransform it into PLH01-WCFS1 competent cells at 2.0 kV and 5 ms, then add it to 1 mL of incubation medium, culture it at 37 °C for 3 h, spread it on MRS solid medium containing chloramphenicol (50 mg / mL) and erythromycin (50 mg / mL), and culture it at 37 °C for 12 h to obtain the pHSP02-VP28-Lp_0237-pLH01-WCFS1 strain.

[0086] In some embodiments, the method for removing the pLH01 plasmid specifically includes: inoculating the pHSP02-VP28-Lp_0237-pLH01-WCFS1 strain into an MRS broth medium containing erythromycin (10 mM) and chloramphenicol (10 mM), shaking and culturing at a speed of 220 rpm and a temperature of 37°C for 12 hours, diluting and spreading on an MRS solid medium containing erythromycin (10 μg / mL), culturing at 37°C for 12 hours, and inoculating on an MRS solid medium containing erythromycin (10 μg / mL) and chloramphenicol (10 μg / mL), and culturing at 37°C for 12 hours.

[0087] In some embodiments, verification is required after the pLH01 plasmid is removed. The verification method includes: inoculating the strain in MRS solid culture medium containing chloramphenicol (10 μg / mL) and culturing at 37° C. for 12 hours.

[0088] In some embodiments, the method for removing the recombinant plasmid pHSP02-VP28-Lp_0237 specifically includes: inoculating the strain from which the pLH01 plasmid has been removed into MRS broth medium and shaking and culturing at 37° C. and 220 rpm for 12 hours, and then spreading the strain on MRS solid medium and culturing at 37° C. for 12 hours.

[0089] In some embodiments, verification is required after the removal of the recombinant plasmid pHSP02-VP28-Lp_0237. The verification method includes: inoculating the strain in MRS solid culture medium containing erythromycin (10 μg / mL) and culturing at 37° C. for 12 hours.

[0090] In a second aspect, some embodiments of the present invention also provide Lactobacillus plantarum obtained by the method.

[0091] In a third aspect, some embodiments of the present invention also provide the use of the Lactobacillus plantarum in farmed aquatic products.

[0092] In a fourth aspect, some embodiments of the present invention also provide use of the Lactobacillus plantarum in the preparation of a drug for treating and / or preventing white spot syndrome in shrimp.

[0093] The following is described in conjunction with specific embodiments: Embodiment 1, as Figure 1 and Figure 2 As shown: In the present invention, Lactobacillus plantarum WCFS1 was purchased from Beijing Biobo Biotechnology Co., Ltd., with a platform number of bio-03564 and other numbers of ATCC BAA-793. Lactobacillus acidophilus was purchased from Ningbo Testo Biotechnology Co., Ltd., with a product number of TS279066. Both pHSP02 plasmid and pLH01 plasmid were purchased from Shanghai Hewu Biotechnology Co., Ltd.

[0094] The nucleotide sequence of the signal peptide PelB is shown in SEQ ID NO.21: ggccatcgccggctgggcagcgaggagcagcagaccagcagcggtcggcagcaggtatttcat.

[0095] (1) Design of pHSP02 recombinant plasmid: The complete sequence of Lp_0237 gene of Lactobacillus plantarum WCFS1 was obtained from KEGG, and the N20 sequence targeting Lp_0237 gene was designed online through the chopchop website. The N20 sequence with the highest comprehensive score was selected for subsequent assembly into the upstream of sgRNA of target plasmid pHSP02 (i.e. Figure 2 at the g RNA scaffold in the Among them, the N20 sequence is shown in SEQ ID NO.1, and SEQ ID NO.1 is 5'-GCTTACATCATGGGTGAACA-3'.

[0096] (2) Obtaining the lower homologous arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 and the upper homologous arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1: First, the whole genome of Lactobacillus plantarum WCFS1 was extracted as follows: 2 mL of Lactobacillus plantarum WCFS1 bacterial solution was added to a centrifuge tube, centrifuged at 8000 rpm for 2 min, and the bacteria were collected. Add 200µL digestion buffer, then add 30µL protease solution, pipette to mix, incubate at 56℃ for 1h until the bacteria are fully lysed, add 300µL BDBuffer, invert to mix, and incubate at 70℃ for 10min; add 200µL anhydrous ethanol, invert to mix; add the solution to the adsorption column, let it stand for 5min, centrifuge at 12000rpm for 1min, and discard the supernatant; add 500µL buffer solution (SolutionBuffer), centrifuge at 12000rpm for 1min, and discard the supernatant; add 500µL buffer solution (SolutionBuffer), centrifuge at 12000rpm for 1min, and discard the supernatant; centrifuge at 12000rpm for 2min, add 100µL CE Buffer, let it stand for 5min, centrifuge at 12000rpm for 2min, collect the DNA solution, and store the extracted DNA at -20℃. Then, the upper homology arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 was obtained by PCR using the upstream primer AB1-F and the downstream primer AB1-R, and the lower homology arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 was obtained by PCR using the upstream primer AB2-F and the downstream primer AB2-R. Both the upper homology arm AB1 and the lower homology arm AB2 are 1000 bp.

[0097] PCR reaction conditions: According to the instructions of Taq enzyme reagent, the PCR system was prepared on ice. The volume of Taq Mix is ​​125μL, the volume of ddH2O is 105μL, the volume of upstream primers (AB1-F / AB2-F) is 10μL, and the volume of downstream primers (AB1-R / AB2-R) is 10μL. Add the above PCR system to the eight-tube concatenation. Add the collected DNA solution on the clean bench, put it into the eight-tube concatenation, shake it several times, cover it, and put the eight-tube concatenation into the PCR instrument for reaction.

[0098] Among them, the sequence of the upstream primer AB1-F is shown as SEQ ID NO.4; the sequence of the downstream primer AB1-R is shown as SEQ ID NO.5; the sequence of the upstream primer AB2-F is shown as SEQ ID NO.6; and the sequence of the downstream primer AB2-R is shown as SEQ ID NO.7.

[0099] SEQ ID NO.4 is: 5'-ATTAGTTGCCGACTACGCGG-3'.

[0100] SEQ ID NO. 5 is: 5'-GCTTGACAAAATAAGTCATCCTCTCGTA-3'.

[0101] SEQ ID NO.6 is: 5'-TCATTTCATACGATTAAATGTATGAT-3'.

[0102] SEQ ID NO.7 is: 5'-CTGAGAATTAGGTCGTGAGGAATC-3'.

[0103] (3) Obtaining the Lp_0237 gene of Lactobacillus acidophilus: Take 2 mL of Lactobacillus acidophilus bacterial solution, add it to a centrifuge tube, centrifuge at 8000 rpm for 2 minutes, and collect the bacteria. Add 200µL digestion buffer, then add 30µL protease solution, pipette to mix, incubate at 56℃ for 1h until the bacteria are fully lysed, add 300µL BD Buffer, invert to mix, and incubate at 70℃ for 10min; add 200µL anhydrous ethanol, invert to mix; add the solution to the adsorption column, let it stand for 5min, centrifuge at 12000rpm for 1min, and discard the supernatant; add 500µL buffer solution (Solution Buffer), centrifuge at 12000rpm for 1min, and discard the supernatant; add 500µL buffer solution (Solution Buffer), centrifuge at 12000rpm for 1min, and discard the supernatant; centrifuge at 12000rpm for 2min, add 100µL CEBuffer, let it stand for 5min, centrifuge at 12000rpm for 2min, collect the DNA solution, and store the extracted DNA at -20℃. Then, the upstream primer Lp_0237-F and the downstream primer Lp_0237-R were used to obtain the Lp_0237 gene of Lactobacillus acidophilus by PCR. The Lp_0237 gene of Lactobacillus acidophilus is 1300 bp.

[0104] PCR reaction conditions: According to the instructions of Taq enzyme reagent, prepare the PCR system on ice. The volume of Taq Mix is ​​125μL, the volume of ddH2O is 105μL, the volume of upstream primer (Lp_0237-F) is 10μL, and the volume of downstream primer (Lp_0237-R) is 10μL. Add the above PCR system to the eight-tube. Add the collected DNA solution on the clean bench, put it into the eight-tube, shake it several times, cover it, and put the eight-tube into the PCR instrument for reaction.

[0105] Among them, the Lp_0237 gene of Lactobacillus acidophilus is shown as SEQ ID NO.2; the sequence of the upstream primer Lp_0237-F is shown as SEQ ID NO.8; and the sequence of the downstream primer Lp_0237-R is shown as SEQ ID NO.9.

[0106] SEQ ID NO.2 is atgaactcattcaagaacccacttatgacaacaccctatctgctagccataatcgccacactgacgatgctagttagtccactgacaacggttgttatatcagtactgatatggttgtcgggaattacgctggtgtggttacaccgccagcaacgatggct ccgcttcttctacgcacccgtgtttttgtacacttttttactagaacaaaggtatttctccccatcaacccagccactgacagaaacgattctcattaacctcgcaattcttggcattgccattttcagcacatgggcagcttatcaattacgtcataacacgtaa.

[0107] SEQ ID NO.8 is: 5'-ATGAAGAAAAATTTAAGAATCGT-3'.

[0108] SEQ ID NO.9 is: 5'-TTATCTAAAGTTTGCAACCTTAACGTAA-3'.

[0109] (4) Obtain the target protein expression sequence: The N20 sequence, the lower homologous arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1, the Lp_0237 gene of Lactobacillus acidophilus, the linker sequence, the gene of the envelope protein VP28, and the upper homologous arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 were connected in sequence by the PCR method, i.e., AB1-VP28-Lp_0237-AB2-N20 sgRNA. The PCR connection products were detected by agarose gel electrophoresis, and the solution containing the AB1-VP28-Lp_0237-AB2-N20 sgRNA sequence was recovered.

[0110] Among them, the amino acid sequence of the envelope protein VP28 is SEQ ID NO.3, and the linker sequence is shown in SEQ ID NO.10.

[0111] SEQ ID NO.3 is: mdlsftlsvvsailaitaviavfivifryhntvtktiethtgnietnmdenlripvtaevgsgyfkmtdvsfdsdtlgkikirngksdaqmkeedadlv itpvegralevtvgqnltfegtfkvwnntsrkinitgmqmvpkinpskafvgssntssftpvsidedevgtfvcgttfgapiaataggnlfdmyvhvtysgtete.

[0112] SEQ ID NO. 10 is 5'-GGCGGAGGTGGTTCTGGCGGTGGAGGTTCAGGCGGTGGTGGAAGT-3'.

[0113] Table 1 Templates for amplifying target genes

[0114] Table 2 Reaction system and reaction conditions for the first round of PCR

[0115] Template 1 and Template 2 refer to different templates in Table 1.

[0116] For example, the lower homology arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 is connected to the Lp_0237 gene of Lactobacillus acidophilus, and two templates of the lower homology arm AB2 and Lp_0237 of Lactobacillus acidophilus are added and connected through the first round of PCR reaction conditions. Among them, the N20 sequence and the linker sequence are directly added without the need for a template.

[0117] The reaction conditions of the first round of PCR were as follows: pre-denaturation at 95°C for 5 min: unwinding at 95°C for 30 s, annealing at 60°C for 1 min, extension at 72°C for 2 min, 10 cycles, and final extension at 72°C for 10 min.

[0118] Table 3 Reaction system and reaction conditions for the second round of PCR

[0119] The reaction conditions of the second round of PCR were as follows: pre-denaturation at 95°C for 5 min; unwinding at 95°C for 30 s, annealing at 60°C for 1 min, extension at 72°C for 2 min, 20 cycles, and final extension at 72°C for 10 min.

[0120] (5) Construction and identification of pHSP02 recombinant plasmid: The pHSP02 plasmid was extracted and double-digested with ApaⅠendonuclease and XbaⅠendonuclease, and subjected to agarose gel electrophoresis. The plasmid backbone size was 13000 bp, and the gel was cut and recovered to obtain the corresponding solution containing the plasmid backbone.

[0121] After recovering the solution containing the backbone, Gibson Clone ligation was performed with the solution containing the AB1-VP28-Lp_0237-AB2-N20 sgRNA sequence at a volume ratio of 1:3, and the reaction was carried out at 50°C for 1 hour to obtain the plasmid pHSP02-VP28-Lp_0237. The ligation product was transformed into the competent E. coli DH5α and coated on LB solid medium (commercially available) containing kanamycin, wherein the mass concentration of kanamycin in the LB solid medium was 100 μg / mL; cultured at 37°C overnight. The single colony grown was verified by colony PCR for pHSP02-VP28-Lp_0237 using primers pHSP-TF and pHSP-TR.

[0122] After preliminarily proving that the pHSP02-VP28-Lp_0237 plasmid was constructed correctly, the PCR product was sequenced to further verify the correctness of the plasmid assembly, and the correct single bacteria were cultured and the plasmid was extracted for subsequent genetic recombination.

[0123] The primer pHSP-TF is shown as SEQ ID NO.11, and the primer pHSP-TR is shown as SEQ ID NO.12.

[0124] SEQ ID NO. 11 is 5'-CAATGTTGCCTAACCCAACGATCATTTTC-3'.

[0125] SEQ ID NO. 12 is 5'-TACGTCGACAGCGCTATAGTT-3'.

[0126] (6) Transform the pLH01 plasmid into competent cells of Lactobacillus plantarum WCFS1: 1.5 μg of plasmid pLH01 was added to the competent cells of Lactobacillus plantarum WCFS1, and the cells were placed on ice for 30 minutes before being added to an electroporation cup. Electric shock was performed at 2.0 kV for 5 ms. 100 μL of incubation medium was immediately added after the electric shock and the cells were placed in a 37°C incubator for incubation for 3 hours to obtain the pLH01-WCFS1 strain. The pLH01-WCFS1 strain was spread on MRS solid culture medium containing chloramphenicol resistance and cultured at 37°C overnight. Single colonies grown on the culture medium were identified by colony PCR using primers pLH01-F and primers pLH01-R to verify that the pLH01 plasmid was successfully transferred into the Lactobacillus plantarum WCFS1 strain.

[0127] The primer pLH01-F is shown as SEQ ID NO.13, and the primer pLH01-R is shown as SEQ ID NO.14.

[0128] SEQ ID NO. 13 is: 5'-CATGCAGTTTAAATTCGGTC-3'.

[0129] SEQ ID NO. 14 is: 5'-ATATTTAAGTTAAACCCAGTAAATG-3'.

[0130] Among them, the preparation of competent cells: The fresh overnight culture of Lactobacillus plantarum WCFS1 was inoculated into 10 mL MRS broth containing glycine at a 2% inoculation rate and incubated at 37°C for 12 h (the mass concentration of glycine in MRS broth was 3 g / 100 mL); then the inoculation rate was transferred to 100 mL SGMRS broth containing glycine and sorbitol (the mass concentration of glycine and sorbitol in SGMRS broth was 3 g / 100 mL) and incubated at 37°C for 3 h; 5 μL of 50 μg / mL ampicillin was added and the culture was continued for 1 h until the OD 600 to 0.5-0.6; centrifuge the bacterial solution at 4°C and 4000rpm, remove the supernatant, collect the bacterial precipitate, resuspend the bacterial precipitate with 30mL of SM buffer and mix it by blowing; centrifuge again at 4°C and 4000rpm, remove the supernatant, collect the bacterial precipitate, resuspend it with SM buffer and mix it by blowing (the volume of SM buffer is 1% of the volume of the bacterial solution when centrifuged again at 4°C and 4000rpm), resuspend and divide into 1.5mL centrifuge tubes, and store 100μL in each tube to obtain competent cells of Lactobacillus plantarum WCFS1.

[0131] (7) Transform the pHSP02 recombinant plasmid into competent cells of the pLH01-WCFS1 strain: Add 1.5 μg of pHSP02 recombinant plasmid to the competent cells of the pLH01-WCFS1 strain, let it stand on ice for 30 minutes, then add it to the electroporation cup, and perform electric shock at 2.0 kV for 5 ms. Immediately after the electric shock, add 100 μL of incubation medium and incubate it in a 37°C incubator for 3 hours to obtain the target strain, add 1 mL of incubation medium, incubate it at 37°C for 3 hours, spread it on MRS solid culture medium containing chloramphenicol (50 mg / mL) and erythromycin (50 mg / mL), and place it in a 37°C incubator for overnight culture. The single colony that grows theoretically completes the insertion of the target protein gene expression cassette. The grown PHSP02-VP28-Lp_023-PLH01-WCFS1 strain (abbreviated as WCFS1-VP28-Lp_0237) was screened to see whether the target protein gene expression cassette was successfully integrated into its genome. The strain was identified by colony PCR using specific primers WCFS1-GF and WCFS1-GR. In order to verify the strain, we performed sequencing to further confirm that the sequence of the target protein was indeed integrated into the genome.

[0132] Among them, the primer WCFS1-GF is shown as SEQ ID NO.15, and the primer WCFS1-GR is shown as SEQ ID NO.16.

[0133] SEQ ID NO. 15 is: 5'-TAGCAAGGGCTTTGGCCTCTG-3'.

[0134] SEQ ID NO. 16 is: 5'-TAGCAAGGGCTTTGGCCTCTG-3'.

[0135] Among them, the preparation method of competent cells of pLH01-WCFS1 strain is: The PLH01-WCFS1 strain was inoculated into 10 mL MRS broth containing glycine at a 2% inoculum and incubated at 37°C for 12 h (the mass concentration of glycine in MRS broth was 3 g / 100 mL); then the inoculum was transferred to 100 mL SGMRS broth containing glycine and sorbitol at a 2% inoculum (the mass concentration of glycine and sorbitol in SGMRS broth was 3 g / 100 mL), incubated at 37°C for 3 h; 5 μL of 50 μg / mL ampicillin was added and the culture was continued for 1 h until the OD 600 When the OD value was 0.2-0.3, an inducing peptide with a final concentration of 10 mM was added to induce homologous recombination of λ-Red protein, and then 5 μL of ampicillin was added and cultured at 37°C for another 1 h until the OD value was 0. 600 was 0.5, and competent cells of the pLH01-WCFS1 strain were obtained.

[0136] (8) Discarding the working plasmid The target strain was inoculated into 2 mL of MRS broth containing erythromycin (10 mM) and chloramphenicol (10 mM), and cultured overnight with shaking at 220 rpm for 10 -7 The strains were diluted and spread on MRS solid medium containing erythromycin (10 μg / mL), cultured at 37°C overnight, and then inoculated on MRS solid medium containing chloramphenicol (10 μg / mL) and MRS solid medium containing erythromycin (10 μg / mL) and chloramphenicol (10 μg / mL). The strains sensitive to chloramphenicol indicated that the plasmid pLH01 had been eliminated. Then the pHSP02 recombinant plasmid was further eliminated, and the above single colonies were further inoculated in MRS broth medium without antibiotics and cultured at 37°C and 220 rpm overnight. Then 100 μL of 10 -7 The diluted bacterial solution was spread on non-resistant MRS solid culture medium and MRS solid culture medium with erythromycin (10 μg / mL). After culturing at 37°C overnight, the strains sensitive to erythromycin resistance successfully eliminated the pHSP02 recombinant plasmid and the pLH01 plasmid, thus obtaining the modified Lactobacillus plantarum WCFS1-VP28-LP_0237 strain integrated with the target protein gene.

[0137] (9) Target protein extraction and Western Blot identification The recombinant strain was inoculated into MRS broth medium at a 2% inoculation rate and cultured at 37°C for 12h. 50mL of the recombinant bacterial solution was centrifuged at 8000rpm for 10min, and the bacteria and the precipitate were separated. The supernatant was collected, and the bacterial precipitate was evenly blown with 30mL of 0.1M PBS solution, centrifuged at 4°C, 8000rpm, and the supernatant was discarded. Repeated twice. The bacterial precipitate was resuspended with 10mL of PBS solution, boiled in boiling water for 10min, and then quickly placed in a -80°C refrigerator for 25min, and the boiling and freezing were repeated 5 times. Centrifuged at 4°C, 8000rpm, and the supernatant and precipitate were collected respectively. The precipitate was evenly blown with 5mL of PBS solution for Western Blot detection.

[0138] Embodiment 2: Compared with Example 1, the difference is that the 135th amino acid threonine in the amino acid sequence encoding the envelope protein VP28 (SEQ ID NO: 3) is mutated to alanine (abbreviated as WCFS1-VP28-Lp_0237 / A).

[0139] The amino acid sequence of the envelope protein VP28 of Example 2 is shown in SEQ ID NO.17.

[0140] SEQ ID NO.17 is: mdlsftlsvvsailaitaviavfivifryhntvtktiethtgnietnmdenlripvtaevgsgyfkmtdvsfdsdtlgkikirngksdaqmkeedadlv itpvegralevtvgqnltfegtfkvwnntsrkiniagmqmvpkinpskafvgssntssftpvsidedevgtfvcgttfgapiaataggnlfdmyvhvtysgtete.

[0141] Embodiment 3: Compared with Example 1, the difference is that the 152nd amino acid serine in the amino acid sequence encoding the envelope protein VP28 (SEQ ID NO: 3) is mutated to lysine (abbreviated as WCFS1-VP28-Lp_0237 / K).

[0142] The amino acid sequence of the envelope protein VP28 of Example 3 is shown in SEQ ID NO.18.

[0143] SEQ ID NO.18 is: mdlsftlsvvsailaitaviavfivifryhntvtktiethtgnietnmdenlripvtaevgsgyfkmtdvsfdsdtlgkikirngksdaqmkeedadlv itpvegralevtvgqnltfegtfkvwnntsrkinitgmqmvpkinpskafvgksntssftpvsidedevgtfvcgttfgapiaataggnlfdmyvhvtysgtete.

[0144] Embodiment 4: Compared with Example 1, the difference is that the 182nd amino acid alanine in the amino acid sequence encoding the envelope protein VP28 (SEQ ID NO: 3) is mutated to aspartic acid (abbreviated as WCFS1-VP28-Lp_0237 / D).

[0145] The amino acid sequence of the envelope protein VP28 of Example 4 is shown in SEQ ID NO.19.

[0146] SEQ ID NO.19 is: mdlsftlsvvsailaitaviavfivifryhntvtktiethtgnietnmdenlripvtaevgsgyfkmtdvsfdsdtlgkikirngksdaqmkeedadlv itpvegralevtvgqnltfegtfkvwnntsrkinitgmqmvpkinpskafvgssntssftpvsidedevgtfvcgttfgapidataggnlfdmyvhvtysgtete.

[0147] Embodiment 5: Compared with Example 1, the difference is that the 196th amino acid valine in the amino acid sequence encoding the envelope protein VP28 (SEQ ID NO: 3) is mutated to methionine (abbreviated as WCFS1-VP28-Lp_0237 / M).

[0148] The amino acid sequence of the envelope protein VP28 of Example 5 is shown in SEQ ID NO.20.

[0149] SEQ ID NO.20 is: mdlsftlsvvsailaitaviavfivifryhntvtktiethtgnietnmdenlripvtaevgsgyfkmtdvsfdsdtlgkikirngksdaqmkeedadlv itpvegralevtvgqnltfegtfkvwnntsrkinitgmqmvpkinpskafvgssntssftpvsidedevgtfvcgttfgapiaataggnlfdmyvhmtysgtete.

[0150] Comparative Example 1: Compared with Example 1, the difference is that the strain is wild-type Lactobacillus plantarum WCFS1, and the starting strain is not changed.

[0151] MRS broth medium: weigh 29g of MRS medium solid powder and dissolve it in 1L of pure water, and sterilize it at 115℃ for 30min; MRS solid medium was purchased commercially; SGMRS broth culture medium: weigh 13.65 g sorbitol, 3 g glycine, and 2.9 g MRS medium powder and dissolve them in 100 mL pure water. Sterilize at 115°C for 30 min. SM buffer: weigh 32.5 g of sucrose and 0.033 g of MgCl2 and dissolve them in 100 mL of pure water, and sterilize them at 115 °C for 30 min; Incubation medium: Weigh 17.2 g sucrose, 0.95 g MgCl2, and 2.9 g MRS medium powder and dissolve them in 100 mL pure water. Sterilize at 115°C for 30 min.

[0152] Lactobacillus plantarum culture method: Streak the frozen glycerol bacterial solution on an MRS solid culture medium, incubate it in a 37°C incubator for 12 h, select a single colony and inoculate it into a test tube containing 2 mL of MRS broth medium, and incubate it in an incubator at 37°C, 220 rpm overnight. Inoculate the seed solution into 100 mL of MRS broth medium at a 1% inoculation rate, and incubate it in an incubator at 37°C, 220 rpm for 12 h to obtain fresh bacterial solution.

[0153] The strains and plasmid vectors used in this study are listed in Table 4 below: Table 4 Strains and plasmids

[0154] The embodiments and comparative examples were tested: (1) Cell adhesion experiment of engineered probiotics: Cell culture: IPEC-J2 cells stored in liquid nitrogen were taken out and revived, and then added with DMEM / F12 complete medium containing 1× penicillin-streptomycin resistance (commercially purchased, Jiangsu KeyGen Biotechnology Co., Ltd.), and cultured in a 5% CO2 incubator for 24 hours. When the cell density grew to 80%, 0.25% trypsin-EDTA was used for digestion for 10 minutes and then passaged.

[0155] (2) Microscope counting method The engineered probiotics were inoculated into DMEM / F12 basal medium at a 1% inoculation rate and cultured at 37°C and 220rpm for 12h. IPEC-J2 cells were subcultured and added to 6mL of DMEM / F12 complete medium containing penicillin-streptomycin resistance, and cultured in a 37°C, 5% CO2 incubator for 24h. When the cell density reached 80%, 2mL of trypsin-EDTA was added. After digestion for 10min, 1mL of complete medium was added to terminate the digestion. Centrifuge at 3000rpm for 4min, remove the supernatant, add 1mL of complete medium, and mix the precipitate by blowing. Collect cells for counting, adjust the concentration of cell suspension, drip 10μL PBS buffer into the bottom of the 12-well plate, add the slide, and drip 1mL of cell suspension on each slide to adjust the density of the cells to be tested in the 12-well plate to 2×10 per well. 4 The unmodified strain was used as a control, and each strain sample was repeated 3 times and placed in a 37°C, 5% CO2 incubator for 12 hours. The cells grown to a monolayer on the slide were rinsed 3 times with PBS buffer, and 1 mL of DMEM / F12 basal medium was added to each well to dilute to OD600 The recombinant probiotic suspension with a value of 0.2 was gently shaken to mix, and cultured in a 37°C, 5% CO2 incubator for 1 hour. After taking it out, the cells were washed 3-5 times with PBS buffer to remove the unadhered bacteria. The slides were taken out and dried naturally, fixed with neutral methanol for 20 minutes, and then Gram staining was performed. The adhesion of the strain to the cells was observed under a microscope. Ten fields of view were randomly selected under an oil immersion lens, and the adhesion index of the bacteria was counted and calculated. The adhesion index refers to the average number of bacteria adhered to each IPEC-J2 cell in 10 fields of view under a microscope.

[0156] (3) Spread plate counting method The engineered probiotics were inoculated into DMEM / F12 basal medium at a 1% inoculation rate and cultured at 37°C and 220rpm for 12h. IPEC-J2 cells were subcultured and added to 5mL of DMEM / F12 complete medium containing 1× penicillin-streptomycin resistance, and cultured in a 37°C, 5% CO2 incubator for 24h. When the cell density reached 80%, 2mL of trypsin-EDTA was used for digestion for 10min, and then 1mL of DMEM / F12 complete medium was added to terminate the digestion. The cells were centrifuged at 3000rpm for 4min, the supernatant was removed by centrifugation, and 1mL of DMEM / F12 complete medium was added to mix by blowing. The cells were collected for counting, and the concentration of the cell suspension was adjusted. 1mL was added to each well so that the density of the cells to be tested in the 12-well plate was 2×10 per well. 4 Each strain sample was repeated for 3 wells, and incubated at 37°C with 5% CO2 for 24 h. The cells that had grown to a monolayer were rinsed 3 times with PBS buffer, and then 1 mL of DMEM / F12 basal medium was added to each well to dilute to OD 600 The recombinant probiotic suspension was 0.2, gently shaken to mix, and cultured in a 37°C, 5% CO2 incubator for 1 hour. After taking out, the cells were washed 3 to 5 times with PBS buffer to remove non-adherent bacteria. Then 500 μL of trypsin was added for 30 seconds, and 500 μL of DMEM / F12 complete medium without resistance was added to terminate the digestion. The cells were blown down and diluted to 10 -5 Gradient, spread on the plate, count the single colonies grown on the plate, compare with the blank control, and calculate the adhesion rate; Adhesion rate (%) = (number of adhered bacteria / total number of bacteria added) × 100%.

[0157] (4) MTT toxicity test of the recombinant strain WCFS1-VP28-Lp_0237 on porcine small intestinal epithelial cells IPEC-J2: IPEC-J2 cells (preserved and provided by Professor Huang He's laboratory of Nanjing Normal University) were taken out of liquid nitrogen for cell recovery. The cryovials were taken out of the liquid nitrogen container, directly immersed in 37°C warm water, and shaken from time to time to melt as quickly as possible; the cryovials were taken out from the 37°C water bath, the lid was opened, the cell suspension was aspirated with a pipette, added to a centrifuge tube and 10 times more culture medium was added dropwise, and mixed; centrifuged at 1000 rpm for 4 min; the supernatant was discarded, and the cells were resuspended in DMEM-F12 culture medium containing 10% calf serum (calf serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd., No. 11011-8611 Sijiqing fetal bovine serum; DMED-F12 culture medium was purchased from Jiangsu KeyGen Biotechnology Co., Ltd., No. KGM12500N-500), counted, adjusted the cell density, and inoculated into culture bottles at 37°C, 5% The cells were cultured in a CO2 incubator. The culture medium was replaced the next day and culture was continued for one day. The cells in the logarithmic phase were digested with trypsin (purchased from Jiangsu KeyGen Biotech Co., Ltd., number KGY0012). After termination, the cells were collected by centrifugation to prepare a cell suspension. The cell count was adjusted to a concentration of 5 × 10 4 / mL; 100 μL of cells with adjusted concentration were inoculated into a 96-well cell culture plate and cultured for 24 h; The inoculated cell culture plate was placed in an incubator and cultured for 24 h until the cell monolayer covered the bottom of the well (96-well flat-bottom plate), and the metabolic supernatant of WCFS1-VP28-Lp_0237 with a concentration gradient of 1 / 12.5, 1 / 25, 1 / 50, and 1 / 100 and the metabolic supernatant of Lactobacillus plantarum WCFS1 with the same concentration gradient were added, respectively, and incubated in a 5% CO2, 37°C incubator for 24 h, and the effect of the drug was observed under an inverted microscope; Add 100 μL of MTT solution (0.5 mg / mL, i.e., 0.5% MTT, diluted with DMED / F12 basal medium) to each well and continue culturing for 4 h. Terminate the culture, prepare to dissolve the crystals, remove the MTT, add 150 μL of DMSO to each well, place on a shaker and oscillate at low speed for 10 min to fully dissolve the crystals, and read the OD value of the enzyme-linked immunosorbent assay. 570nm The absorbance value of each well was measured, and the cell survival rate was calculated to determine whether the recombinant strain WCFS1-VP28-Lp_0237 obtained in Example 1-5 was toxic to the cells. The results are shown in Table 6.

[0158] (5) Inhibitory effect of engineered probiotics on common aquatic pathogens: The applicant used Vibrio Parahemolyticus and Vibrio harveyi, which are common in white shrimp farming, as indicator bacteria to detect the antagonistic effect of engineered probiotics on pathogenic bacteria.

[0159] Indicator bacteria were inoculated in nutrient broth medium (beef extract 5g, peptone 10g, sodium chloride 5g, distilled water 1000mL, pH 7.2-7.4) respectively, and 30°C constant temperature culture was used for 24h. Plant lactobacillus was inoculated in MRS broth medium, and 37°C constant temperature culture was used for 24h. The pH value of MRS broth medium was adjusted with lactic acid to make it the same as the pH value of the probiotic composition, which was used as a control. 0.1mL indicator bacteria culture solution was evenly applied to solid MRS medium with a sterile applicator, and a metal pipe was used to evenly punch holes after the plate was dry, with an aperture of 10mm ± 0.1mm, and then agar blocks in the hole were picked out with sterile tweezers, and then the bottom was sealed with a hot glass rod. Engineering probiotics and control solution were added to each well, and 37°C culture was used for 24h, and the presence or absence of antibacterial rings was observed, and the antibacterial diameter of the control and engineering probiotics was measured, and the specific results were shown in Table 7.

[0160] Results and Analysis: Table 5 Cell adhesion of engineered probiotics measured by microscopic counting (x̄±S)

[0161] Table 6 Cell survival rate

[0162] Table 7 Antibacterial effect of probiotic composition

[0163] exist Figure 3-Figure 4 As shown in Table 5, the number and adhesion rate of adhered bacteria counted in each sample field of view were observed under an oil immersion microscope. The adhesion rate of the modified strain WCFS1-VP28-Lp_0237 in Example 1 was about 10% higher than that of the wild-type L. plantarum WCFS1. The adhesion rate was further improved by optimizing the sequence of the envelope protein VP28, especially in Example 4 where the 182nd amino acid alanine was mutated to aspartic acid. Compared with the wild-type L. plantarum WCFS1, the adhesion rate was increased by about 20%, indicating that the fusion-expressed envelope protein VP28 and adhesion protein Lp_0237 were helpful to improve the adhesion of WCFS1 to IPEC-J2 cells.

[0164] The experimental data in Table 6 show that the recombinant strains obtained in Examples 1 to 5 have no active effect on cell growth.

[0165] From Table 7 and Figure 5It can be seen from the data that the engineered probiotics provided by the present invention have a strong inhibitory effect on the pathogenic bacteria Vibrio parahaemolyticus and Vibrio harveyi, and the diameter of the inhibition zone exceeds 1.5 cm. In particular, after the sequence optimization of the envelope protein VP28, its antibacterial effect is further improved. The diameter of the inhibition zone of the recombinant strain of Example 4 exceeds 2.0 cm, and the strain has a stronger inhibitory effect on Vibrio harveyi, achieving unexpected technical effects.

[0166] It should be noted that the terms used in the present invention are only for describing specific embodiments, rather than limiting the scope of the present application. As shown in the present specification, unless the context clearly indicates an exception, the words "one", "a", "a kind of" and / or "the" do not specifically refer to the singular, but may also include the plural. The terms "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method or device including a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method or device. In the absence of more restrictions, the elements defined by the sentence "include one..." do not exclude the presence of other identical elements in the process, method or device including the elements.

[0167] It should also be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention. Unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", etc. should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be a connection between the two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0168] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing probiotics with adhesion, characterized in that: The steps include: Step 1: The N20 sequence, the lower homologous arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1, the Lp_0237 gene of Lactobacillus acidophilus, the linker sequence, the gene of the envelope protein VP28, and the upper homologous arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 were sequentially connected by the PCR method to obtain the target protein expression sequence, i.e., AB1-VP28-Lp_0237-AB2-N20sgRNA; Step 2: Cut the pHSP02 plasmid with an enzyme to obtain a plasmid backbone; recover the solution containing the plasmid backbone and clone and assemble it with the solution containing the AB1-VP28-Lp_0237-AB2-N20 sgRNA in step 1, and react to obtain the recombinant plasmid pHSP02-VP28-Lp_0237; Step 3: transform the pLH01 plasmid into competent cells of Lactobacillus plantarum WCFS1, culture once, and obtain the pLH01-WCFS1 strain; transform the recombinant plasmid pHSP02-VP28-Lp_0237 into competent cells of the pLH01-WCFS1 strain, culture twice, and screen to obtain the pHSP02-VP28-Lp_023-pLH01-WCFS1 strain; Step 4: Discard the pLH01 plasmid and the recombinant plasmid pHSP02-VP28-Lp_0237 in the pHSP02-VP28-Lp_023-pLH01-WCFS1 strain in step 3 in sequence to obtain adhesive probiotics.

2. The method according to claim 1, characterized in that The preparation method of the N20-sgRNA sequence comprises: designing an N20 sequence targeting the Lp_0237 gene of Lactobacillus plantarum WCFS1; The N20 sequence is shown as SEQ ID NO.

1.

3. The method according to claim 1, characterized in that The Lp_0237 gene of the Lactobacillus acidophilus is shown in SEQ ID NO.

2.

4. The method according to claim 1, characterized in that: The amino acid sequence of the envelope protein VP28 is SEQ ID NO.3, or an amino acid sequence in which the 135th amino acid threonine in the amino acid sequence SEQ ID NO.3 is mutated to alanine, or an amino acid sequence in which the 152nd amino acid serine in the amino acid sequence SEQ ID NO.3 is mutated to lysine, or an amino acid sequence in which the 182nd amino acid alanine in the amino acid sequence SEQ ID NO.3 is mutated to aspartic acid, or an amino acid sequence in which the 196th amino acid valine in the amino acid sequence SEQ ID NO.3 is mutated to methionine.

5. The method according to claim 4, characterized in that The gene of the envelope protein VP28 is a gene that can encode the amino acid sequence of the envelope protein VP28.

6. The method according to claim 1, characterized in that The linker sequence is shown as SEQ ID NO.

10.

7. The method according to claim 1, characterized in that The preparation method of the upper homology arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 and the lower homology arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 comprises: The whole genome of Lactobacillus plantarum WCFS1 was extracted, and the upper homologous arm AB1 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 was obtained by PCR using the upstream primer AB1-F and the downstream primer AB1-R; the lower homologous arm AB2 of the Lp_0237 gene of Lactobacillus plantarum WCFS1 was obtained by PCR using the upstream primer AB2-F and the downstream primer AB2-R; The sequence of the upstream primer AB1-F is shown in SEQ ID NO.4; The sequence of the downstream primer AB1-R is shown in SEQ ID NO.5; The sequence of the upstream primer AB2-F is shown in SEQ ID NO.6; The sequence of the downstream primer AB2-R is shown in SEQ ID NO.

7.

8. The method according to claim 1, characterized in that In step 2, the enzymes used are ApaⅠ endonuclease and XbaⅠ endonuclease; In the step 2, the reaction temperature is 50° C. and the reaction time is 1 h.

9. The plant lactobacillus obtained by the method described in any one of claims 1 to 8.

10. Use of the plant lactobacillus according to claim 9 in aquaculture products and / or in the preparation of a medicament for treating and / or preventing white spot syndrome of shrimp.

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