Use of the mf1-01441 gene, proteases, recombinant strains

Abstract

The application belongs to the technical field of biology, and discloses application of mf1-01441 gene, protease and recombinant strain; specifically, application of mf1-01441 gene in preparation of protease and application of mf1-01441 gene in preparation of recombinant strain capable of producing protease, wherein the nucleotide sequence of the mf1-01441 gene is shown as SEQ ID NO. 1; the application successfully screens a novel protease mf1-01441 gene by constructing a Fosmid library of intestinal tract microorganisms of gibel carp; the library not only breaks through the limitation of traditional culture method, captures genetic information of a large number of uncultured microorganisms in the intestinal tract of gibel carp, lays a foundation for exploring microbial resources in the unique habitat, but also provides high-quality materials for screening of functional genes such as protease, and is expected to provide a new idea for improving feed utilization rate of fish and reducing breeding cost, and promote sustainable development of green breeding.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically the application of the mf1-01441 gene, proteases, and recombinant strains. Background Technology

[0002] Fishmeal and plant protein are important protein sources in fish feed. Plant protein sources in feed often contain anti-nutritional factors, which not only reduce protein digestibility but may also inhibit growth. Protein is the most critical component of fish feed, and the efficient absorption and conversion of protein by fish directly impacts feed costs, improves growth performance, and reduces nitrogen emissions from water bodies. Using exogenous proteases is one of the most common methods to improve feed protein utilization. For example, prior art CN114921369B discloses a protease-producing strain and its application. This strain can produce a large amount of protease, thereby improving the fish's ability to break down protein and increasing feed utilization. Prior art CN117721057A discloses an engineered bacterium heterologously expressing the E0TYP4 gene and its application in the production of fish feed proteases. The fish intestinal microbial protease heterologously expressed by this engineered bacterium has good adaptability to different feed formulations and fish species, and is characterized by small dosage and simple operation, making it suitable for different needs in fish farming. When these engineered bacteria are used as feed additives, they can not only colonize the digestive tract of fish and continuously secrete active proteases to make up for the lack of endogenous digestive enzymes, but also improve the intestinal microecological environment by taking advantage of the probiotic characteristics of the host strain, thereby further promoting nutrient absorption.

[0003] However, it is worth noting that the screening of protease genes is a core prerequisite for the application of heterologous expression technology, and the process faces multiple technical challenges: First, natural protease gene resources are scattered in various microorganisms (bacteria, fungi, archaea) and plants and animals, and their numbers are vast. Complex techniques such as metagenomic sequencing and functional gene library construction are required for their discovery, and the functions of most uncharacterized genes are unknown, resulting in extremely low screening efficiency. Second, screening must simultaneously meet the dual requirements of "highly efficient catalysis" and "environmental adaptability." The protease expressed by the target gene must have high specific activity, be able to rapidly degrade feed proteins, and be tolerant of the low temperature, fluctuating pH, and protease inhibitor environment of the fish digestive tract. Naturally occurring protease genes often fail to meet these characteristics and require modification through directed evolution, site-directed mutagenesis, and other techniques, which are technically challenging and time-consuming. Furthermore, the screened protease genes must be compatible with the expression system of the host strain to avoid problems such as codon mismatch and misfolding of expression products, further increasing the complexity and uncertainty of the screening. Therefore, successfully selecting suitable genes to prepare engineered bacteria or expression plasmids that effectively improve the absorption of feed nutrients by fish requires a tremendous amount of work and presents significant technical difficulties. Summary of the Invention

[0004] One of the objectives of this invention is to provide the application of the mf1-01441 gene in the preparation of proteases.

[0005] Another object of the present invention is to provide the application of the mf1-01441 gene in the preparation of recombinant strains capable of producing proteases.

[0006] Another object of the present invention is to provide the use of a protease based on the expression of the mf1-01441 gene in the preparation of feed additives or food additives or pharmaceuticals that act on the intestine.

[0007] Another object of the present invention is to provide the use of a recombinant strain capable of producing protease prepared based on the mf1-01441 gene in the preparation of feed additives or food additives or pharmaceuticals acting on the intestine.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] Application of the mf1-01441 gene in the preparation of proteases.

[0010] Application of the mf1-01441 gene in the preparation of recombinant strains capable of producing proteases.

[0011] Application of proteases based on mf1-01441 gene expression in the preparation of feed additives, food additives, or drugs that act on the intestines.

[0012] Application of recombinant strains that produce proteases, prepared based on the mf1-01441 gene, in the preparation of feed additives, food additives, or drugs that act on the intestines.

[0013] Preferably, the nucleotide sequence of the mf1-01441 gene is shown in SEQ ID NO.1.

[0014] Preferably, the feed additive is a fish feed additive; the food additive is a fish food additive; the medicine is a fish medicine; the fish feed additive, fish food additive, or fish medicine are all used to improve the fish's ability to digest and absorb nutrients from feed.

[0015] Preferably, the recombinant strain is a recombinant lactococcus that can express the mf1-01441 gene.

[0016] In addition, the present invention also discloses a protease, wherein the gene encoding the protease is the mf1-01441 gene, and its nucleotide sequence is shown in SEQ ID NO.1.

[0017] In addition, the present invention also discloses a recombinant vector, wherein the recombinant vector is linked with an mf1-01441 gene fragment, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0018] Finally, the present invention also discloses a protease recombinant expression strain, wherein the protease recombinant expression strain is a recombinant lactococcus that can express the mf1-01441 gene.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] This invention successfully screened the novel protease gene mf1-01441 by constructing a Fosmid library of mandarin fish gut microbiota. This library not only breaks through the limitations of traditional culture methods and captures the genetic information of a large number of unculturable microorganisms in the mandarin fish gut, laying the foundation for exploring the microbial resources of this unique habitat, but also provides high-quality materials for screening functional genes such as proteases and digestive enzymes. It is expected to provide new ideas for improving fish feed utilization and reducing aquaculture costs, and promote the sustainable development of green aquaculture. Attached Figure Description

[0021] Figure 1 Figure showing the results of constructing recombinant plasmids for Fosmid libraries;

[0022] Figure 2 Figure showing the results of DNA extraction and pCC1FOS plasmid preparation;

[0023] Figure 3 Figure showing the results of Fosmid library construction and protease clone screening;

[0024] Figure 4 Figure showing the KOG database analysis results of mf1 sequencing results;

[0025] Figure 5 Figure showing the KEGG database analysis results of mf1 sequencing results;

[0026] Figure 6 The figure shows the GO database analysis results of mf1 sequencing results;

[0027] Figure 7 This is a diagram showing the multiple sequence alignment results for mf1-01441;

[0028] Figure 8 The result image is of Lactococcus lactis NZ9000;

[0029] Figure 9 Figure showing the results of constructing the plasmid pMG36e-p32-usp45-mf1-01441 / mf1-02918 for the protease recombinant expression strain;

[0030] Figure 10 A diagram showing the results of protease gene construction;

[0031] Figure 11 The figure shows the functional verification results of the mf1-01441 protease recombinant expression strain;

[0032] Figure 12 The image shows the protease activity results of the protease recombinant expression strain.

[0033] Figure 13 Figure showing the analysis results of how recombinant expression of Lactococcus lactis improves the growth performance of largemouth bass. Detailed Implementation

[0034] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Materials and product information:

[0036] Mandarin fish (body weight 100±5 g) cultured with artificial compound feed were purchased from Guangdong Shunan Fengtai Aquatic Technology Co., Ltd. for the extraction of total DNA from intestinal microorganisms;

[0037] pCC1FOS vector was purchased from Shanghai Newp Biotechnology Co., Ltd.

[0038] EPI300 electrocompetent cells, MC1061 chemocompetent cells and SOC culture medium were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0039] EPI300 supercompetent cells, CopyCutter induction solution (200×) and rapid end-flattening kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0040] pMG36e vector and NZ9000 were both purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0041] It should be noted that the technical solution of this application can be achieved by purchasing other commercially available raw materials, and is not limited to the aforementioned raw material suppliers.

[0042] Example 1: Construction of Fosmid library of intestinal microbial genome from mandarin fish

[0043] 1. Extraction of total genomic DNA from the intestinal microorganisms of mandarin fish

[0044] Wipe the surface of the mandarin fish with alcohol swabs, and remove the intestines under aseptic conditions. Gently scrape the inner wall of the intestine and add it along with the contents to 10 mL of 40% PEG8000 solution and mix well; centrifuge at 1000×g for 15 min, collect the supernatant; centrifuge at 12,000×g for 8 min, discard the supernatant, and obtain the intestinal microbial sample. Extract the genomic DNA of the intestinal microorganisms using the CTAB-SDS method described by Zuo Rui et al. in "Comparison of Different Methods for Extracting Metagenomic DNA of Intestinal Microorganisms". Wash the DNA precipitate twice with 500 µL of 70% ethanol, and centrifuge at 12,000×g for 10 min at room temperature. Air dry naturally, add 100 µL of preheated TE buffer to dissolve the DNA, which is the total genomic DNA of the mandarin fish intestinal microorganisms, and store at -80℃ for later use.

[0045] 2. End repair of total genomic DNA

[0046] Take 50 µL of the prepared genomic DNA and mechanically cleave it by repeatedly pipetting. Fragmentation is then performed using nucleic acid electrophoresis, and fragments of 20–40 kb are recovered using a gel extraction kit (U-Rayland). The recovered DNA fragments are then end-modified using a rapid end-trimming kit, and the DNA is purified and recovered.

[0047] 3. Preparation of pCC1FOS vector

[0048] According to the pCC1FOS vector instructions, pCC1FOS was heat-shocked and transformed into EPI300 supercompetent cells, and then plated on LB agar plates containing chloramphenicol. Single colonies were picked and added to 4 ml of LB liquid medium containing 1% chloramphenicol resistance, and cultured overnight at 37°C and 200 rpm to obtain a stock solution. 250 μL of the stock solution was added to 25 mL of LB medium containing 1% chloramphenicol and 1% CopyCutter induction solution (200×), and cultured overnight at 37°C and 200 rpm. The pCC1FOS plasmid was then extracted using a plasmid extraction kit (Tiangen Biotech).

[0049] The obtained pCC1FOS plasmid was digested with Eco72Ⅰ (Thermo Fisher Scientific). Following the instructions for FastAP heat-sensitive alkaline phosphatase (Thermo Fisher Scientific), the digestion product was dephosphorylated. After the reaction, the plasmid was incubated at 80°C for 20 min to inactivate the alkaline phosphatase. The linearized pCC1FOS plasmid was then purified and recovered using a PCR cleaning kit (Ultralandi).

[0050] 4. Ligation of vector and genomic DNA

[0051] The prepared linearized pCC1FOS vector was ligated with the processed genomic DNA of mandarin fish gut microbiota according to the instructions of T4 DNA ligase (Takara Bio Inc.). The reaction system was placed in a 16℃ metal bath overnight for ligation, yielding the ligation product, recombinant plasmid pCC1FOS-Intestinal microbiota DNA. A desalted gel was prepared according to Lü Liyuan's "Construction of BAC Library and Identification and Transcriptional Regulation of Olfactory Gene Cluster in Sole". The ligation product was transferred to the desalted gel, placed in a 4℃ refrigerator for 2 h of desalting, and then transferred to a new centrifuge tube for subsequent electroporation library construction.

[0052] 5. Electroconversion to construct Fosmid library

[0053] Following the EPI300 electroporation instructions for competent cells, the ligation product was mixed with the competent cells and transferred to a 0.1 cm electroporation cuvette. The electroporation settings were: C=25 μF, PC=200 Ω, V=1.8 kV, τ=4 ms. Immediately after electroporation, preheated SOC medium (37°C) was added, transferred to a centrifuge tube, and SOC medium was added to a final volume of 10 mL. The cells were incubated at 37°C and 225 rpm for 90 min. After resuspending, 150 μL of the resuspended cells was plated onto a blue-white screening plate containing chloramphenicol antibiotic. The plate was incubated overnight at 37°C.

[0054] 6. Preservation of the Fosmid Library

[0055] White transformants from the blue-white transformation plate were transferred to 96-well plates. 600 μL of LB liquid medium containing chloramphenicol was added to each well of the 96-well plate. The plates were labeled and incubated at 37°C for 24 h to serve as the mother library. 200 μL of 80% glycerol was added to the mother library, and the plates were stored at -80°C. For subsequent experiments, the plates were thawed and activated at 4°C in advance to avoid repeated freeze-thaw cycles.

[0056] 7. Screening of proteases

[0057] Fosmid clones were inoculated onto chloramphenicol-resistant (Chl 25 mg / mL) protease selection plates, labeled, and inverted at 37°C. Observations were made every 4 hours; the appearance of a clear zone indicated the clone possessed potential protease hydrolysis function, which was then verified through rescreening. Protease-producing clones were inoculated onto 25 mL of chloramphenicol-containing (25 mg / mL) LB broth and cultured overnight. This seed culture was then inoculated at a 1% inoculation rate onto 25 mL of chloramphenicol-containing (25 mg / mL) LB broth for expansion. The culture was incubated at 37°C and 180 rpm for 12 hours. Cells were collected by centrifugation and sent to Beijing Qingke Biotechnology for sequencing analysis.

[0058] The results are as follows Figure 1 , Figure 2 and Figure 3 As shown; Construction of the recombinant plasmid of the present invention: pCC1FOS-Intestinal microbiota DNA ( Figure 1 Total genomic DNA of the intestinal microorganisms of mandarin fish was extracted using a slightly modified CTAB-SDS method, and the extracted samples were detected by agarose gel electrophoresis. The DNA length was greater than 20 kb. Figure 2 (a) can be used for Fosmid library construction. The pCC1FOS vector, after electrophoresis verification, is approximately 8 kb in size. Figure 2 The position of b in the image corresponds to the actual fragment size. Enzyme digestion of pCC1FOS ( Figure 2 The enzyme digestion results (c) showed a single band with no impurities, indicating good digestion efficiency and complete plasmid cleavage. The linearized vector pCC1FOS plasmid was successfully prepared. The experiment successfully constructed a Fosmid library of the intestinal microorganisms of mandarin fish (Pyrus pyrifos). Figure 3 (a) The recombinant clones in the library were screened using blue-white screening plates, and white positive recombinant clones were precisely selected and stored at -80℃. A function-dependent screening method was used to screen clones in the library for protease-positive clones, and a clone with high protease activity, mf1, was successfully screened. Figure 3 (b) In the rescreening of this clone, a clear transparent zone was observed. Figure 3 (c) The hydrolysis ability is relatively stable and reliable. Cloner mf1, when strewn into culture, shows a large transparent hydrolysis zone and strong protease production ability. Figure 3 (d in the text)

[0059] Figure 1 Figure showing the results of constructing recombinant plasmids for Fosmid libraries;

[0060] Figure 2 The figure shows the results of DNA extraction and pCC1FOS plasmid preparation. In the figure, M1: 15000 bp Maker; M2: 10000 bp Maker.

[0061] Figure 2 Lanes 1 and 2 of swim 'a': Total genomic DNA extracted from the gut microbiota of mandarin fish;

[0062] Figure 2 Lane 1 of b: pCC1FOS vector extraction;

[0063] Figure 2 Lane 1 of c: pCC1FOS plasmid;

[0064] Figure 2 Lanes 2 and 3 of c in the sample: pCC1FOS digestion (Eco72I);

[0065] Figure 3 The image shows the results of Fosmid library construction and protease clone screening. Figure 3 a: Fosmid library construction of genomic DNA from the gut microbiota of mandarin fish and white clones obtained through blue-white co-screening;

[0066] Figure 3 b in the text: The protease-producing clone mf1 was screened using a screening medium with skim milk powder as the substrate;

[0067] Figure 3 c: mf1 clone point for rescreening;

[0068] Figure 3 The d:mf1 was purified by streaking and culture.

[0069] Example 2: Identification of the protease gene

[0070] The selected protease clone plasmid fragments were 30 kb in size. After the DNA samples passed the initial screening, the extracted DNA was randomly fragmented using a Covaris ultrasonic disruptor. The DNA then underwent end repair, tail addition (A), sequencing adapter addition, purification, and PCR amplification to complete the library preparation. Once the library passed the initial screening, it was sequenced using Illumina sequencing. Sequencing by Synthesis (BSB) technology on the Illumina HiSeqX high-throughput sequencing platform yielded a large amount of high-quality raw data. BLAST software was used to align all genes to databases such as Nr, Swiss-Prot, GO, eggNOG / COG, KOG, KEGG, and Pfam to obtain gene function annotation information. Genes capable of hydrolyzing proteases were screened using these databases. BLAST alignment of genes with protease hydrolytic ability was performed, and genes with lower consistency were selected for heterologous expression.

[0071] The results are as follows Figures 4-7 As shown in Table 1; the sequencing and gene function annotation results of the mf1 recombinant plasmid were obtained using the Illumina HiSeqX high-throughput sequencing platform. Figures 4-6 According to the KOG database, a total of 3167 genes were annotated for the predicted coding genes, including 706 genes with unknown functional pathways. Among these, 360 genes were annotated for amino acid transport and metabolism, and 201 genes were annotated for carbohydrate transport and metabolism. The abundance values ​​of these genes were among the highest. Figure 4The clone ligation product had a large sequence, further indicating the high quality of the genomic DNA prepared from the intestinal microorganisms of mandarin fish. Annotation of the predicted coding genes using the KEGG database revealed six major functional categories: 472 genes involved in carbohydrate metabolism and 422 genes involved in amino acid metabolism. A count of 117 hydrolase genes [EC 3.-] was found, with carboxypeptidase [EC 3.4.16.4] being the most abundant with 31 genes. Figure 5 Analysis and comparison using the GO database revealed that 481 genes functionally annotated to mf1 possess this ability during metabolism. Figure 6 A total of 27 genes with protease hydrolysis capabilities were screened from the above databases. Based on BLAST database alignment, 6 candidate genes with coding sequence identity of 90%-99% were obtained (Table 1), and their protease hydrolysis functions need further investigation. Among them, mf1-01441 was subjected to multiple sequence alignment using MEGA (Molecular Evolutionary Genetics Analysis) software. Figure 7 The results showed that the protein sequence differed from that of Bacillus sp. FSL_M8-0063, which had the highest known sequence similarity. The nucleotide sequence of mf1-01441 is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; the nucleotide sequence of mf1-02918 is shown in SEQ ID NO.3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.4.

[0072] SEQ ID NO.1:

[0073] ATGAAACCTAACTTTTCAAAAGGACTATTACCAGCAGTTGTAATTGAAGAAGATACAAAAGAAGTTTTTAATGCTAGCTTATATGAATGAAGAGGCATATGAAAAAACAATAGAAACGAAAAGAACATGGTTTTATTCTCGTTCAAGAAGGTCG TTATGGAATAAAGGAGAAACATCAGGTAATGTCCAACATGTGCAATCTCTTTATTTAGATTGTGATCAAGATGCAATTGTTGTTGTCGTAAAGCAAGTAGGGCCTGCTTGCCATACGGGAGAAAAAACGTGTTTTCATTACAAAATTATATAG;

[0074] SEQ ID NO.2:

[0075] MKPNFSKGLLPAVVIEEDTKEVLMLAYMNEEAYEKTIETKRTWFYSRSRRSLWNKGETSGNVQHVQSLYLDCDQDAIVVVVKQVGPACHTGEKTCFHYKII;

[0076] SEQ ID NO.3:

[0077]

[0078] SEQ ID NO.4:

[0079] MKKVGTAFLTTLFIFSSFTSANAEEKKDSKAFIDVSAATLWTTPDSLRPIDAPSATNPVDLWKWTKSMTLDEKLWLTSANKLETQALLGQEVTVVDKKGDWVKVLVHGQPTPRNEEGYPGWMPEKQLTYNQEFADKTNEPFVLVTKPTAILYINPSEKHKSLEVSYN TRLPLLSEDTISYRVLLPNGQKAWLRKHDGTVYRSQNDIPTPTADDLINTGKMFLGLPYIWAGTSGFGFDCSGFTHTIYKSHGITIPRDSGPQSRNGVAVDKEHLQKGDLIFFAHDQGKGSVHHVGMYIGDGNMIHSPRAERSVEIIPLNTPGYIEEYAGARRYLP.

[0080] Table 1. Proteolytic enzymes predicted by mf1

[0081] Gene number Query Coverage E value consistency Login ID Function Description mf1_01441 100% 2e-65 94.06% WP_340980239.1 hydrolase cyclohydrolase mf1 03673 100% 0.0 96.17% WP_000745120.1 neutral protease NprB mf1_00667 100% 0.0 98.09% WP_065222749.1 M42 family metallopeptidase mf1_02566 100% 0.0 96.92% WP_431181288.1 S1C family serine protease mf1_02918 100% 0.0 94.89% WP_098682708.1 C40 family peptidase mf1_00241 100% 0.0 96.99% WP_100060975.1 SpoIVB peptidase

[0082] Figure 4 Figure showing the KOG database analysis results of mf1 sequencing results;

[0083] Figure 5 Figure showing the KEGG database analysis results of mf1 sequencing results;

[0084] Figure 6 The figure shows the GO database analysis results of mf1 sequencing results;

[0085] Figure 7 The image shows the multiple sequence alignment results for mfi-01441. In the image, * indicates the same amino acid, and * and * indicate similar amino acids.

[0086] Example 3: Expression of the protease gene

[0087] 1. Preparation of electrocompetent cells of Lactococcus lactis NZ9000

[0088] The electroporation of NZ9000 competent cells was carried out according to the method described in the literature by Li Linfang et al., "Screening of goat rumen-derived ester hydrolase genes and verification of their heterologous expression based on Fosmid library".

[0089] 2. Preparation of linear carrier pMG36e

[0090] Following the instructions for the pMG36e vector, single colonies were picked and cultured overnight in LB broth containing 25 μg / mL erythromycin at 37°C and 180 rpm. The culture was expanded at a 1% inoculum, and pMG36e was extracted using a plasmid kit (Tiangen Biotech). Double digestion with XbaⅠ and SacⅠ (Takara Bio Inc.) was performed according to the instructions. The mixture was incubated at 37°C for 20 min; the enzymes were then inactivated at 70°C for 15 min. The linear vector pMG36e was purified and recovered using a PCR cleaning kit (U-Landi).

[0091] 3. Cloning of the protease gene

[0092] Primer sequences mf1-01441F, mf1-01441R, and mf1-02918F, mf1-02918R containing the signal peptide usp45 and the target gene were designed. PCR amplification of the target genes (mf1-01441, mf1-02918) was performed. The reaction system was prepared according to the PrimeSTARMax Premix(2x) (Takara Bio Inc.) instructions. The PCR program was set up. After amplification, the gel was run for verification, and the gel was cut and recovered using a gel recovery kit (U-Landi). The recovered products were double-digested with XbaI and SacI (Takara Bio Inc.) as described above. The linear target genes mf1-01441 and mf1-02918 were recovered using a PCR cleaning kit (U-Landi).

[0093] The gene sequence of the mf1-01441F primer is as follows:

[0094] GCGAGCTCGCATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAGCCCCGTTGTCAGGTGTTTACGCTATGAAACCTAACTTTTC (SEQ ID NO. 5);

[0095] The gene sequence of the mf1-01441R primer is as follows:

[0096] GCTCTAGACTATATAATTTTGTAATGAAA (SEQ ID NO. 6);

[0097] The gene sequence of primer mf1-02918F is as follows:

[0098] GCGAGCTCGCATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAGCCCGTTGTCAGGTGTTTACGCTATGAAAAAAGTAGGAACTGC (SEQ ID NO. 7);

[0099] The gene sequence of the mf1-02918R primer is as follows:

[0100] GCTCTAGATTAAGGTAAGTAACGACGGGC (SEQ ID NO.8);

[0101] The recovered linear target gene mf1-01441 has the following nucleotide sequence:

[0102] GCATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAGCCCCGTTGTCAGGTGTTTACGCTATGAAACCTAACTTTTCAAAAGGACTATTACCAGCAGTTGTAATTGAAGAAGATACAAAAGAAGTTTTTAATGCTAGCTTATATGAATGAAGAGGCATATGAAAAAACAATAGA AACGAAAAGAACATGGTTTTATTCTCGTTCAAGAAGGTCGTTATGGAATAAAGGAGAAACATCAGGTAATGTCCAACATGTGCAATCTCTTTATTTAGATTGTGATCAAGATGCAATTGTTGTTGTCGTAAAGCAAGTAGGGCCTGCTTGCCATACGGGAGAAAAAACGTGTTTTCATTACAAAATTATATAG (SEQ ID NO.9);

[0103] The recovered linear target gene mf1-02918 has the following nucleotide sequence:

[0104]

[0105] 4. Connection and Electrical Conversion

[0106] The linear vector pMG36e was ligated with the recovered linear target gene mf1-01441 or mf1-02918 using T4 DNA ligase (Takara Bio Inc.), following the same ligation procedure as in Part 4 of Example 1. The reaction system was mixed and incubated at 16°C for 4 h. The ligation product was then mixed with NZ9000 electroporation competent cells using the same desalting gel method as in Part 4 of Example 1, and transferred to a pre-cooled 0.1 cm electroporation cuvette (C=25 μF, PC=200 Ω, V=2.0 kV). Resuscitation medium (refer to the literature Li Linfang et al., "Screening of goat rumen-derived ester hydrolase genes and their heterologous expression verification based on Fosmid library") was added, and the cells were incubated at 30°C for 2 h. 100 μL of the resuspended cells was plated onto GM17 plates containing erythromycin (25 ug / mL) and incubated upside down at 30°C for 36-48 h to obtain the recombinant strain. Colony PCR was performed on single clones using primers for the target gene. The correctly identified single clones were sent to Guangzhou Tianyi Huiyu Gene Technology Co., Ltd. for sequencing to verify the success of the transformation.

[0107] 5. Functional verification and enzyme activity detection

[0108] Successfully transformed positive clones were inoculated into GM17 liquid medium containing erythromycin (25 μg / mL) and cultured at 30℃. The protease activity was detected according to the national standard GB / T 23527.1-2023 "Determination of protease activity - Folin method". The function of the protease recombinant expression strain was verified by screening plates with skim milk powder.

[0109] The results are as follows Figures 8-12 As shown; Lactococcus lactis strain NZ9000 exhibits smooth, raised, milky-white colonies on GM17 solid plates. Figure 8 (a) NZ9000 is a Gram-positive bacterium with a thick cell wall; it stains as a purple coccus with hematoxylin and eosin (HE). Figure 8 (b) The plasmids of the protease recombinant expression strain were neatly arranged, uniform in size, and of high purity. The plasmids of the strain were constructed as pMG36e-p32-usp45-mf1-01441 / mf1-02918. Figure 9 ).

[0110] Two novel proteolytic enzymes predicted in Table 1 were selected. Primers were designed using Oligo7 software to target the protease gene and bind to the restriction sites of the pMG36e vector. mf1-01441 and mf1-02918 were amplified from the mf1 plasmid. The expected sizes of mf1-01441 and mf1-02918, as verified by agarose gel electrophoresis, were 306 bp and 1002 bp, respectively. Figure 10(b, c) The above genes and the linearized pMG36e vector ( Figure 10 (a) The DNA fragment was ligated using T4 DNA ligase. The ligation product was then electroporated into a prepared NZ9000, and the corresponding primers were used to verify the successful insertion of the target fragment. Figure 10 (d) The test results showed that the target gene sequence was inserted correctly.

[0111] The recombinant strains successfully transformed with NZ9000 were functionally verified, among which the mf1-01441 recombinant strain showed a clear zone ( Figure 11 The presence of protease hydrolysis ability indicates the successful construction of the recombinant NZ9000 strain containing this protease gene, demonstrating the successful expression of the protease gene in the NZ9000 strain, which possesses the ability to hydrolyze proteins. The mf1-02918 strain, however, cannot produce protease and therefore cannot express protease hydrolysis ability. Enzyme activities of the two recombinant strains expressing proteases were measured using casein as a substrate. The extracellular protease activity of the mf1-01441 recombinant strain was 35.6 U / mL, and the intracellular protease activity was 6.97 U / mL. The protease activity of the successfully expressed mf1-01441 protease gene was significantly different from that of the empty vector pMG36e (P < 0.001). This recombinant strain exhibits high protease activity and stable secretion. Figure 12 ).

[0112] Figure 8 This is a result image of Lactococcus lactis NZ9000. Figure 8 a: Lactococcus lactis NZ9000 was streaked on a GM17 plate; Figure 8 b: HE staining of Lactococcus lactis NZ9000;

[0113] Figure 9 Figure showing the results of constructing the plasmid pMG36e-p32-usp45-mf1-01441 / mf1-02918 for the protease recombinant expression strain;

[0114] Figure 10 The image shows the results of protease gene construction. In the image, M2 represents a 15000 bp Maker, and M3 represents a 2000 bp Maker.

[0115] Figure 10 Lane 1 of a: pMG36e plasmid;

[0116] Figure 10 Lane 2 of a: pMG36e plasmid was double-digested with XbaII and SacI;

[0117] Figure 10 Lane 1 of b: mf1-01441 amplified with high-fidelity enzyme;

[0118] Figure 10 Lanes 1-2 of c in the image: mf1-02918 amplified with high-fidelity enzyme;

[0119] Figure 10 Lanes 1-6 of the d-type bacteria: Colony PCR verification of recombinant lactic acid bacteria mf1-01441.

[0120] Figure 11 The image shows the functional verification results of the mf1-01441 protease recombinant expression strain.

[0121] Figure 12 This is a graph showing the protease activity results of the recombinant protease expression strain. Figure 12 In the figure, 'a' represents the detection of extracellular enzyme activity in recombinant bacteria. Figure 12 b in the figure: Detection of intracellular enzyme activity in recombinant bacteria.

[0122] Example 4: Feeding experiment of recombinant protease expression strain

[0123] 1. Cultivation and feeding of recombinant expression strains

[0124] The NZ9000 strain transformed with the pMG36e vector and the recombinant strain NZ9000 transformed with the pMG36e-usp45-mf1-01441 plasmid were respectively inoculated into GM17 liquid medium and cultured at 30℃ with constant temperature shaking at 180 r / min for 12-16 h. The bacterial count was performed by plate counting (the viable count was ≥ 10^6). 10 CFU / mL). Adjust the viable count to 10. 9 CFU / mL, take 20 mL of bacterial culture, centrifuge at 5000 r / min for 20 min, discard the supernatant, put the bacterial sludge into 20 mL of sterile PBS buffer containing 0.2 g sodium alginate, mix well, and then stir thoroughly with 200 g of feed. After drying, store at 4℃.

[0125] 2. Experimental grouping and feeding

[0126] Largemouth bass were purchased from Guangdong Bairong Aquatic Seed Group and temporarily held for 7 days after transport, during which time they were fed conventional feed. Forty-five largemouth bass with an initial weight of (7±0.5 g) were randomly selected and randomly divided into three groups, with three replicates per group, for a total of nine net cages (45 bass / cage). The control group was fed conventional feed; the pMG36e group was fed feed containing NZ9000 transformed with the pMG36e vector (prepared in Part 1 of Example 4); and the mf1-01441 group was fed feed containing NZ9000 transformed with the pMG36e-usp45-mf1-01441 plasmid. Feeding was done twice daily at 09:00 and 17:00 until satiation, with an experimental period of 56 days.

[0127] 3. Data Collection and Analysis

[0128] After the aquaculture experiment, the fish were fasted for 24 hours. The total number and weight of the fish in each net cage were recorded after being anesthetized with eugenol. This data was used to calculate the weight gain rate (WG), feed conversion ratio (FC), and specific growth rate (SGR). Nine fish were randomly selected from each group and their weight was measured.

[0129] The calculation formula is as follows:

[0130] Weight gain rate (WG, %) = 100 × [final weight (g) - initial weight (g)] / initial weight (g);

[0131] Feed conversion ratio (FC) = Total feed intake (g) / [Final body weight (g) - Initial body weight (g)];

[0132] Specific growth rate (SGR, % / d) = 100 × [Ln final body weight (g) - Ln initial body weight (g)] / culture time;

[0133] Statistical analysis was performed on the experimental data. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0134] After feeding largemouth bass with recombinant protease-expressing Lactococcus lactis (mf1-01441), the weight gain rate and specific growth rate were 757.9% and 3.84%, respectively, which were 46.4% and 18.1% higher than those of the group fed with ordinary feed (P<0.05), and 22.7% and 9.1% higher than those of the group fed with Lactococcus lactis containing the pMG36e empty vector (P<0.05). Figure 13 The feed conversion ratio of the mf1-01441 group was 0.58, which was 36.9% lower than that of the group fed with ordinary feed (P<0.05) and 23.7% lower than that of the group fed with Lactococcus lactis containing the pMG36e empty vector (P<0.05). This indicates that the protease-recombinant Lactococcus lactis (mf1-01441) provided by this invention can improve the digestibility and absorption of nutrients from feed by largemouth bass.

[0135] Figure 13 Figure showing the analysis results of how recombinant expression of Lactococcus lactis improves the growth performance of largemouth bass.

[0136] In summary, this invention successfully screened a novel protease gene, mf1-01441, by constructing a Fosmid library of mandarin fish gut microbiota. This gene was then used to transform NZ9000 strain to obtain a recombinant strain that successfully expressed the mf1-01441 gene and possessed the ability to hydrolyze proteins. The recombinant strain exhibited high extracellular protease activity of 35.6 U / mL and intracellular protease activity of 6.97 U / mL, demonstrating stable protease secretion. Feeding this recombinant strain to largemouth bass improved their ability to digest and absorb nutrients from feed.

[0137] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. Application of the mf1-01441 gene in the preparation of protease.

2. Application of the mf1-01441 gene in the preparation of recombinant strains capable of producing proteases.

3. Application of proteases based on mf1-01441 gene expression in the preparation of feed additives, food additives, or drugs acting on the intestine.

4. Application of recombinant strains that produce proteases based on the mf1-01441 gene in the preparation of feed additives, food additives, or drugs that act on the intestines.

5. The application according to any one of claims 1 to 4, characterized in that, The nucleotide sequence of the mf1-01441 gene is shown in SEQ ID NO.

1.

6. The application according to any one of claims 3 or 4, characterized in that, The feed additive is a fish feed additive; the food additive is a fish food additive; the medicine is a fish medicine; the fish feed additive, fish food additive, or fish medicine are all used to improve the fish's ability to digest and absorb nutrients from feed.

7. The application according to any one of claims 2 or 4, characterized in that, The recombinant strain is a recombinant lactococcus that can express the mf1-01441 gene.

8. A protease, characterized in that, The gene encoding the protease is the mf1-01441 gene, whose nucleotide sequence is shown in SEQ ID NO.1 and amino acid sequence is shown in SEQ ID NO.

2.

9. A recombinant vector, characterized in that, The recombinant vector is linked to the mf1-01441 gene fragment, the nucleotide sequence of which is shown in SEQ ID NO.

1.

10. A recombinant bacterial strain, characterized in that, The recombinant strain is a recombinant lactococcus that can express the mf1-01441 gene.

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