MrATG5 recombinant protein and its preparation method and application

Abstract

The present application discloses a MrATG5 recombinant protein and its preparation method and application, which relates to the field of recombinant protein technology, and aims to solve the technical problem that there is a lack of a drug that can prevent and treat Macrobrachium rosenbergii diseases in the prior art. The nucleotide sequence of the MrATG5 recombinant protein is MrATG5‑pET‑32a(+) recombinant protein as shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2. The present application cloned and identified the Macrobrachium rosenbergii MrATG5 gene, and by constructing the MrATG5‑pET‑32a(+) prokaryotic expression vector, the recombinant expression active protein of MrATG5 was successfully induced and purified, and its binding and antibacterial activity to bacteria was analyzed. And based on the experimental results, it is shown that MrATG5 can inhibit the proliferation of pathogens in the body by binding to bacteria, thereby enhancing the antibacterial immunity of Macrobrachium rosenbergii. The results of this application will provide data support for in-depth research on the immune defense function of Macrobrachium rosenbergii, and provide a certain theoretical basis for the prevention and control of Macrobrachium rosenbergii diseases.

Description

Technical Field

[0001] The present application relates to the field of recombinant protein technology, and in particular to MrATG5 recombinant protein and a preparation method and application thereof. Background Art

[0002] Autophagy is a highly conserved catabolic pathway in the body. The double-membrane autophagosome encapsulates the molecular substances and cell components that need to be decomposed, and then carries them to the lysosome for degradation. As a protective mechanism for the body to cope with external stimuli, autophagy plays an important role in the body's immune regulation process. When external bacteria, viruses and other pathogens invade cells, they will induce and activate cell autophagy. The autophagosome will encapsulate the invading pathogens and then combine with the lysosomes to eliminate the pathogens.

[0003] The occurrence of autophagy is inseparable from the complex regulation of many autophagy-related proteins (ATGs). Studies in humans and mammals have found that autophagy genes can participate in autophagy alone or cooperate with each other to form protein complexes to participate in autophagy after immune stimulation. After Salmonella stimulated human SW-480 cells, the expression of autophagy gene LC3-II increased significantly, and inhibition of de novo sphingolipid synthesis inhibited the membrane recruitment of ATG16L1 and inhibited the expression of autophagy protein LC3-II induced by Salmonella. Sphingolipids and autophagy genes have the function of defending against invading pathogens. Autophagy-related protein ATG10s can promote the formation of autophagosomes to cause lysosomal degradation of Hepatitis C virus (HCV) subgenomic replicons and inhibit the replication of HCV virus particles in human Huh-7.5 cells. Inhibition of the expression of the Gallus domesticus ATG5 gene can change the dynamic expression of lipid metabolism-related genes in chicken fibroblasts DF-1. The researchers knocked down ATG3 and ATG5 by RNA interference, which reduced the survival rate of mealworms infected with Listeria monocytogenes, proving that ATG3 and ATG5 autophagy genes may play a role in the immune process of eliminating L. monocytogenes. In aquaculture animals, researchers have also found that autophagy genes play an important role in the regulation of innate immunity and adaptive immune responses induced by pathogen infection. For example, the expression of ATG5 and ATG12 genes in grass carp (Ctenopharyngodon idella) was significantly reduced after stimulation with Grass Carp Reovirus (GCRV). Further studies found that overexpression of ATG5 and ATG12 in HEK-293T cells can induce autophagy, indicating that ATG5 and ATG12 participate in the antiviral response of grass carp through autophagy regulation. ATG16L1 in black carp (Mylopharyngodon piceus) interacts with stimulator of interferon genes (STING) and inhibits STING oligomerization, negatively regulating STING-mediated antiviral responses. ATG10 in large yellow croaker (Larimichthys crocea) can participate in the body's antiviral immune response by promoting the formation of autophagosomes.The expression of ATG13, an autophagy-related gene in Litopenaeus vannamei, was significantly upregulated after stimulation by polyinosinic acid-polycytidylic acid (PolynosinlcAcid-Polycyttdylic Acld, poly(I:C)) and Vibrio harveyi (V.harveyi), while ATG5 and LC3 (homologs of yeast ATG8 in mammalian cells) can also participate in the immune response after stimulation by White Spot Syndrome Virus (WSSV). In addition, ATG14 in Procambarus clarkii blocks autophagy by inhibiting the fusion of autophagosomes and lysosomes, thereby promoting the replication of WSSV in the body.

[0004] However, the expression of autophagy-related genes in Macrobrachium rosenbergii and their functions in the body are still unclear. Macrobrachium rosenbergii, also known as freshwater long-armed prawn, is one of the important freshwater shrimp aquaculture varieties in my country. The aquaculture production areas are mainly distributed in Guangdong Province, Jiangsu Province and Zhejiang Province in my country. According to the Food and Agriculture Organization of the United Nations (FAO), the total aquaculture production of Macrobrachium rosenbergii in China accounts for more than 50% of the global aquaculture production. With the expansion of aquaculture scale, infectious diseases caused by bacterial infections are increasing, especially diseases such as star disease and black gill disease caused by Aeromonas genus, which cause large-scale deaths of Macrobrachium rosenbergii and cause serious economic losses to the Macrobrachium rosenbergii industry. Studies have shown that cell autophagy plays an important regulatory role in the antibacterial immune response of Macrobrachium rosenbergii, and activating cell autophagy can significantly improve the survival rate of Macrobrachium rosenbergii after pathogen infection.

[0005] Therefore, there is an urgent need for a drug that can prevent and treat Macrobrachium rosenbergii diseases. Summary of the invention

[0006] The present application provides MrATG5 recombinant protein and its preparation method and application, aiming to solve the technical problem that there is a lack of a drug capable of preventing and treating diseases of Macrobrachium rosenbergii in the prior art.

[0007] To solve the above technical problems, the embodiments of the present application provide: a MrATG5 recombinant protein, wherein the nucleotide sequence of the MrATG5-pET-32a(+) recombinant protein is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0008] On the other hand, the present invention provides a method for preparing the MrATG5 recombinant protein as described above, comprising the following steps:

[0009] Obtaining a target tissue sample of Macrobrachium rosenbergii; extracting target RNA from the target tissue sample, and reverse transcribing the target RNA into target cDNA;

[0010] Using the target cDNA as a template, PCR amplification is performed to obtain a recombinant expression vector;

[0011] The plasmid of the recombinant expression vector is extracted, and induction treatment, purification treatment and dialysis treatment are performed to obtain the MrATG5 target recombinant protein.

[0012] As some optional embodiments of the present application, the step of performing PCR amplification using the target cDNA as a template to obtain a recombinant expression vector includes:

[0013] The target cDNA is used as a template to perform gene-specific amplification to obtain a first PCR product; the conditions of the gene-specific amplification are: 94° C., 5 min; 94° C., 30 s; 55° C., 30 s; 72° C., 30 s, 35 cycles; 72° C., 10 min;

[0014] The PCR products were detected and separated by agarose gel electrophoresis, and the separated products were connected to the pMD-18T vector to obtain the connection products; the connection conditions were: 16°C, 2h;

[0015] The ligation product was transformed into DH5α competent cells using a heat shock method to obtain transformed competent cells; the transformation conditions were: first ice bath for 30 minutes, then placed in a 42°C water bath for 45 seconds, and then placed on ice for 5 minutes;

[0016] Add 500 μL of antibiotic-free LB liquid culture medium to the transformed competent cells and culture at 37° C., 200 rpm for 1 hour; then spread 200 μL of the culture product on LB solid culture medium with ampicillin resistance and culture at 37° C. for 12 hours;

[0017] Using a sterile inoculation loop to randomly pick a single colony on the LB solid medium, and perform PCR amplification, and perform agarose gel electrophoresis separation and verification on the amplified product to obtain ATG5-specific primers; the ATG5-specific primers include ATG5-F and ATG5-R;

[0018] Based on the ATG5-specific primers, a recombinant expression vector was constructed.

[0019] As some optional embodiments of the present application, the step of constructing a recombinant expression vector based on the ATG5-specific primers includes:

[0020] pET-32a(+) and pEGFP-N1-GFP were selected as the prokaryotic expression vector and eukaryotic expression vector of MrATG5;

[0021] Based on the ATG5-specific primers, the MrATG5 gene and the expression vector are connected by a double enzyme digestion method to obtain recombinant expression vector primers; the recombinant expression vector primers are recombinant prokaryotic expression vector primers: ATG5-BamH IF and ATG5-Xho IR.

[0022] Based on the recombinant expression vector primers, a gene-specific amplification process is performed to obtain a second PCR product; the conditions of the gene-specific amplification process are: 94°C, 5 min; 94°C, 30 s; 55°C, 30 s; 72°C, 30 s, 35 cycles; 72°C, 10 min;

[0023] The second PCR product was connected and transformed into the pMD 18-T vector, and spread on LB solid medium with corresponding resistance. After overnight culture at 37°C, a single colony was picked for expansion and testing;

[0024] After the sequencing is correct, the retained bacterial solution is expanded and the plasmid is extracted to obtain the plasmid of the recombinant expression vector;

[0025] The MrATG5 gene primer with the restriction site is double-digested with the plasmid of the recombinant expression vector to obtain the MrATG5 gene gel recovery product after restriction digestion and the recombinant expression vector plasmid after restriction digestion;

[0026] The MrATG5 gene gel recovery product after the enzyme digestion was connected with the recombinant expression vector plasmid after the enzyme digestion at 16°C for 5h, and then transformed into competent cells. After overnight culture at 37°C in LB solid medium with ampicillin resistance, a single colony was picked for sequencing analysis;

[0027] After correct sequencing, MrATG5-pET-32a(+) recombinant expression vector and MrATG5-pEGFP-N1 recombinant expression vector were obtained.

[0028] As some optional embodiments of the present application, the steps of extracting the plasmid of the recombinant expression vector, performing induction treatment, purification treatment and dialysis treatment to obtain the MrATG5 target recombinant protein include:

[0029] The plasmid of MrATG5-pET-32a(+) recombinant expression vector was extracted and transformed into Transetta(DE3) competent cells. The bacteria were expanded and cultured until the bacterial solution concentration reached the logarithmic growth phase. Isopropyl-β-D-thiogalactoside with a final concentration of 1 mM was added, and the expression was induced at 16°C and 130 rpm for 12 h to obtain the induced product.

[0030] The induced product was purified and dialyzed to obtain the MrATG5 target recombinant protein.

[0031] As some optional embodiments of the present application, the step of purifying and dialysis-treating the induced product to obtain the MrATG5 target recombinant protein includes:

[0032] Using BeyoGold TM The induced product was purified by His-tag Purification Resin; after balancing the Ni column with the lysis solution, 8 times the column volume of the protein supernatant was loaded onto the column, the protein flow-through was recovered and repeatedly loaded onto the column 5 times, the nickel column was eluted with imidazole-free Wash Buffer, and then the protein was eluted with imidazole-containing Wash Buffer to obtain the purified product;

[0033] The purified product was dialyzed to obtain the MrATG5 target recombinant protein.

[0034] As some optional embodiments of the present application, the step of dialyzing the purified product to obtain the MrATG5 target recombinant protein comprises:

[0035] A protein refolding dialysate containing urea at a gradient concentration is prepared; the protein refolding dialysate containing urea at a gradient concentration includes a protein refolding dialysate containing urea at concentrations of 0 M, 1 M, 2 M, 3 M, 4 M and 6 M, and a pH of 7.4-8.0;

[0036] The purified product is placed in the protein renaturation dialysate containing the stepwise concentration of urea, and the urea concentration is placed in the order from high to low for protein dialysis renaturation at 4°C, and the dialysate is replaced every 12 hours;

[0037] After dialysis was completed, the MrATG5 target recombinant protein was obtained.

[0038] As some optional embodiments of the present application, the steps of obtaining a target tissue sample; extracting a target RNA from the target tissue sample, and reverse transcribing the target RNA into a target cDNA include:

[0039] The in vitro tissue sample of Macrobrachium rosenbergii was mixed with an equal amount of anticoagulant and then centrifuged. After the centrifugation, the supernatant was removed and ground into powder in liquid nitrogen;

[0040] The target RNA in the target tissue sample is extracted, and the target RNA is reverse transcribed into target cDNA.

[0041] On the other hand, an embodiment of the present application provides an application of the MrATG5 recombinant protein as described above, for preparing a drug for preventing and treating diseases of Macrobrachium rosenbergii.

[0042] As some optional embodiments of the present application, when the MrATG5 recombinant protein is used to prepare a drug for preventing and treating diseases of Macrobrachium rosenbergii, its prevention and treatment mechanism is: inhibiting the proliferation of A.hydrophila bacteria and C.freundii bacteria in the body to enhance the antibacterial immunity of Macrobrachium rosenbergii.

[0043] This application cloned and identified the MrATG5 gene of Macrobrachium rosenbergii, and successfully induced and purified the recombinant expression active protein of MrATG5 by constructing the MrATG5-pET-32a(+) prokaryotic expression vector, and analyzed its binding and antibacterial activity against bacteria. Based on the experimental results, it was shown that MrATG5 can inhibit the proliferation of pathogens in the body by binding to bacteria, thereby enhancing the antibacterial immunity of Macrobrachium rosenbergii. The results of this application will provide data support for in-depth research on the immune defense function of Macrobrachium rosenbergii, and provide a certain theoretical basis for the prevention and control of diseases of Macrobrachium rosenbergii. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific implementation of the present application or the technical solution in the prior art, the following is a brief introduction to the drawings required for the specific implementation or the prior art description. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale.

[0045] Figure 1 This is a diagram of the MrATG5 gene identification results involved in the examples of this application;

[0046] Figure 2 This is a diagram showing the PCR amplification results of the MrATG5 gene involved in the examples of the present application;

[0047] Figure 3 The purification and Western blot identification diagram of MrATG5-pET-32a(+) recombinant protein involved in the examples of the present application;

[0048] Figure 4 This is a graph showing the binding activity of the MrATG5 recombinant protein to bacteria involved in the examples of the present application;

[0049] Figure 5 This is a graph showing the effect of rMrATG5 on bacterial proliferation involved in the examples of the present application.

[0050] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0052] Autophagy plays an important regulatory role in cellular immunity, metabolism and survival. The occurrence of autophagy involves the interaction of many autophagy-related genes (ATGs). However, the specific molecular regulatory mechanism of ATGs in Macrobrachium rosenbergii is still unclear. In this study, MrATG5 was cloned and identified from Macrobrachium rosenbergii. Bioinformatics analysis showed that the MrATG5 open reading frame (ORF) was 807 bp, predicted to encode 269 amino acids, with a predicted protein molecular mass of 31.04 kDa and an isoelectric point of 5.68, with a typical APG5 domain. The prokaryotic recombinant active protein of MrATG5 was obtained by constructing a prokaryotic recombinant expression vector and inducing protein. Through bacterial binding and antibacterial activity experiments, it was found that the prokaryotic recombinant expression active protein of MrATG5 can produce binding activity with four bacteria, Staphylococcus aureus, Streptococcus iniae, Aeromonas hydrophila and Citrobacter freundii, and inhibit the growth of A.hydrophila and C.freundii. In summary, MrATG5 may participate in the antibacterial immune function of Macrobrachium rosenbergii by binding to pathogenic bacteria and inhibiting their proliferation. In this application, ATG5 (MrATG5), a key molecule for autophagy regulation, was cloned and identified in Macrobrachium rosenbergii. First, the molecular characteristics of MrATG5 were analyzed by bioinformatics technology, and the role of MrATG5 in the antibacterial immunity of Macrobrachium rosenbergii was verified by the binding and antibacterial experiments of MrATG5 prokaryotic recombinant expression protein with bacteria. The research results of this application can provide important insights into the relationship between autophagy genes and immune defense in invertebrates, and also provide an important theoretical basis for clarifying the antibacterial immune regulation mechanism of Macrobrachium rosenbergii.

[0053] Example 1

[0054] 1.1 Experimental Materials

[0055] The Macrobrachium rosenbergii used in this experiment was purchased from Guangdong Haixingnong Group Co., Ltd. (Guangzhou, China), with a size of 10±0.50 g. The purchased Macrobrachium rosenbergii was placed in a continuous aerated water body with a culture water temperature of 28±2℃ and a pH value of 7.5-8.0 for 2 weeks. During the temporary culture period, commercial feed was fed 3 times a day.

[0056] The strains and vectors involved in the experiment: Aeromonas hydrophila, Staphylococcus aureus, Streptococcus iniae, and Citrobacter freundii were all identified and preserved by the Aquatic Disease and Immunity Laboratory, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0057] 1.2 Tissue sample extraction and reverse transcription synthesis of cDNA

[0058] Ten healthy shrimps were randomly selected, and a sterile syringe pre-filled with anticoagulant was used to extract shrimp hemolymph in an amount equal to the anticoagulant. The extracted hemolymph was placed at 4°C and centrifuged at 1000g for 5 min. The supernatant was removed and the blood cells were collected and placed in liquid nitrogen. The heart, eyestalk, intestine, stomach, hemolymph, hepatopancreas, muscle and gill tissues were extracted using sterile tweezers and placed in liquid nitrogen. All tissue samples were then transferred to -80°C for storage. The collected tissue samples were ground into powder in liquid nitrogen, and RNA was extracted according to the instructions of the RNA extraction kit (Tiangen, China), and the RNA concentration and quality were detected using a microspectrophotometer to determine the integrity of the RNA quality. According to PrimeScript TM According to the instruction manual of II 1st Strand cDNA Synthesis Kit (Takara, Japan), the extracted RNA was reverse transcribed into cDNA and stored at -20°C.

[0059] 1.3 Analysis of MrATG5 molecular characteristics

[0060] The isoelectric point and molecular weight of the amino acid sequence of MrATG5 were predicted using SWISS-PROT (https: / / web.expasy.org / cgi-bin / compute_pi / pi_tool). The phosphorylation sites and glycosylation sites were predicted using NetNGlyc (http: / / www.cbs.dtu.dk / services / ). The signal peptide and protein domain of MrATG5 were predicted using Signal P 5.0 Server (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0) and SMART (http: / / smart.embl-heidelberg.de / smart / set_mode.cgi), respectively.

[0061] The results showed that the open reading frame (ORF) of MrATG5 gene was 807 bp, which was predicted to encode 269 amino acids, with a protein relative molecular mass of 31.04 kDa and an isoelectric point of 5.68. The nucleotide sequence was shown in SEQ ID No.1, and the amino acid sequence was shown in SEQ ID No.2.

[0062] SEQ ID No.1:

[0063] ATGGCAGAAGACAGGGAAATTTTACGGAAATATGGGATGGTCGTGTCCCAGTATGTGCCCAGCTTGCAAGTGAAGATTGCAACACTCTTAGTGCTCCTGATCCATATTACTTGATGTGTTCCAAGGCTGTCGTATTTCCCACTAGTTATGGATAAGGTGAGGAAACATTTGCTACGGTTTATTCCTCCTGATCTTCAGGAA GCAGAAATGTGGTTTGAATGTGATGGATATCCTTTAAAATGGCATTATCCAGTAGGAGTATTATTTGATCTTCATTGTGGTGGAACAGCTTTGCCATGGACTATTACTGCACATTTTTCTCATTTTCCTGAAAAAGAATTAATAAGATGTCCTTCAAGGGAAGTAGTAGAGTCACAGTTTATGTCATCCTTGAAAGAAGCCG ATGGATTGAAGCACCGTGGACAAGTCATAACGAACATGCAAAGTAAAGATCATAAACAGTTGTGGATGGGTCTTTGCAATGATAAATTTGACCAATTTTGGGCTATTAATAGAAAACTTATGGAATGTTGTTCTGATGAGGGATTTAAACACATTCCATTCCGTCTTCACACACTTTACCGACCACCCATACAAAAACTTAT AAAACCCCTCACTGATGATGGGAGGAAAGTCACCTTGGCAGATCTCCTTCATGATGCCTTACCAGATACCATAGGGGAAGGTGACCGAATCGTGATCCAAGGTATTGAAACTCCCCTAGATACACCAGTACAATGGCTCAGTGAACATCTCAGCCATCCTGACAACTTTCTTCATATAAGTTATATTCCTGCTCAAGCCTGA

[0064] SEQ ID No.2:

[0065] MAEDREILREIWDGRVPVCAQLASEDCNTLSAPDPYYLMVPRLSYFPLVMDKVRKHLLRFIPPDLQEAEMWFECDGYPLKWHYPVGVLFDLHCGGTA LPWTITAHFSHFPEKELIRCPSREVVESQFMSSLKEADGLKHRGQVITNMQSKDHKQLWMGLCNDKFDQFWAINRKLMECCSDEGFKHIPFRLHTLYRPPIQKLIKPLTDDGRKVTLADLLHDALPDTIGEGDRIVIQGIETPLDTPVQWLSEHLSHHPDNFLHISYIPAQA

[0066] The analysis was performed using online software. Figure 1 As shown, there are N-glycosylation sites at amino acid positions 28, 146, 161, 171 and 257, serine sites at amino acid positions 24, 31, 44, 107, 119, 125, 129, 130, 253 and 262, and tyrosine sites at amino acid positions 37 and 77. The predicted polypeptide contains an APG5 domain (amino acid positions 81 to 264) without a signal peptide.

[0067] in, Figure 1 In the figure, A indicates the black panes representing the start codon (ATG) and the stop codon (TGA); the red panes represent the N-glycosylation sites; the blue arrows represent serine; the green arrows represent tyrosine; and B indicates the MrATG5 protein domain predicted by SMART.

[0068] 1.4 MrATG5 gene cloning

[0069] PrimerPremier 5 software was used to design specific primers for amplifying the ATG5 gene ORF, namely:

[0070] ATG5-F:5′-ATGGCAGAAGACAGGGAAATTTTA-3′,

[0071] ATG5-R: 5′-GGCTTGAGCAGGAATATAACTTATA-3′.

[0072] Gene-specific amplification (PCR amplification) was performed using cDNA from various tissues of healthy shrimp as a template. The PCR amplification conditions were: 94°C, 5min; 94°C, 30s; 55°C, 30s; 72°C, 30s, 35 cycles; 72°C, 10min. Agarose gel electrophoresis was used to detect and separate the PCR products, and the products were connected to the pMD-18T vector (Takara, Japan) under the conditions of 16°C, 2h. The ligation products were transformed into DH5α competent cells using the heat shock method. The transformation conditions were: first ice bath for 30min, then placed in a 42°C water bath for 45s, and finally placed on ice for 5min. 500μL of antibiotic-free LB liquid medium (Huankai Microbiological Technology Co., Ltd., China) was added to the transformed competent cells and cultured at 37°C, 200rpm for 1h. Then, 200μL of the culture product was spread on LB solid medium with ampicillin resistance (Huankai Microbiological Technology Co., Ltd., China) and cultured at 37°C for 12h. Use a sterile inoculation loop to randomly pick a single colony on the LB plate medium, perform PCR amplification, and separate and verify the amplified product by agarose gel electrophoresis. Finally, send the verified colony to a biological company for sequencing to verify the correctness of the MrATG5 sequence.

[0073] The results showed that the MrATG5 gene was amplified by PCR and then detected and separated by agarose gel electrophoresis. Figure 2 As shown, the results showed an obvious single band in the target gene lane, and further sequencing analysis showed that it was consistent with the MrATG5 gene sequence, which indicated that specific primers for the ATG5 gene ORF had been successfully obtained using the cDNA of Macrobrachium rosenbergii tissue as a template.

[0074] 1.5MrATG5 recombinant protein expression and purification

[0075] pET-32a(+) was selected as the prokaryotic expression vector of MrATG5. The appropriate restriction sites on the vector, namely BamH I (GGATCC) and Xho I (CTCGAG), were found by using Snap Gene software, and primers of MrATG5 gene containing restriction sites were designed, namely:

[0076] ATG5-BamH IF:5′-GGATCCATGGCAGAAGACAGGGAAATTTTA-3′,

[0077] ATG5-Xho IR:5′-CTCGAGGGGCTTGAGCAGGAATATAACTTAT-3′;

[0078] The PCR procedure and system are the same as 1.4.

[0079] The PCR products were detected and separated by agarose gel electrophoresis and ligated into pMD-18T vector (Takara, Japan). The ligation, transformation and colony PCR steps were the same as in 1.4.

[0080] The bacterial solution verified by sequencing and the bacterial solution containing only the pET-32a(+) empty vector were expanded and cultured at the same time, and plasmid DNA was extracted using a plasmid extraction kit (Tiangen, China) according to the product instructions. Subsequently, the MrATG5 gene and the plasmid DNA of the expression vector were double-digested, and the digestion system and conditions were: 1 μL each of BamH I and Xho I, 1 μg of plasmid DNA, 5 μL of 10X QuickCut Buffer, and sterile H2O was added to the system to 50 μL. The digestion products were separated by agarose gel electrophoresis and recovered by gel cutting, and the two products were connected and transformed, and colony PCR verification was performed, with the same steps as 1.4. Finally, the verified colonies were sent to a biological company for sequencing verification. After correct sequencing, the recombinant expression vector: MrATG5-pET-32a(+) was successfully created.

[0081] The plasmid of MrATG5-pET-32a(+) recombinant expression vector was extracted and transformed into Transetta(DE3) competent medium (Beijing Quanshijin Biotechnology Co., Ltd., China). The bacteria were cultured to make the concentration of the bacterial solution reach the logarithmic growth phase (OD 600 =0.4-0.6), add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1mM, induce expression for 12h at 16°C and 130rpm, and set the group without IPTG as the control group. Use an ultrasonic disruptor to break the induced bacteria, centrifuge and retain the supernatant and precipitate of the bacterial liquid for polyacrylamide gel electrophoresis (SDS-PAGE) verification.

[0082] After protein mass induction of the recombinant expression vector MrATG5-pET-32a(+), BeyoGold TM Protein purification was performed using His-tag Purification Resin (denaturation-resistant formulation). After balancing the Ni column with the lysate, 8 times the column volume of the protein supernatant was loaded onto the column. After recovering the protein flow-through and loading the column 5 times, the nickel column was first eluted with imidazole-free Wash Buffer, followed by protein elution with Wash Buffer containing different concentrations of imidazole (10 / 20 / 60 mM imidazole, pH = 7.4). The eluted protein was verified using SDS-PAGE.

[0083] Prepare protein refolding dialysate containing different concentrations of urea (0M / 1M / 2M / 3M / 4M / 6M urea, pH = 7.4-8.0), put the purified MrATG5-pET-32a(+) recombinant active protein (rMrATG5) into the pretreated dialysis bag, and place it at 4°C for protein dialysis refolding in the order of urea concentration from high to low, and replace the dialysate every 12 hours. The dialyzed MrATG5-pET-32a(+) recombinant active protein was freeze-dried in a vacuum dryer and stored at -80°C.

[0084] The accuracy of rMrATG5 recombinant protein was identified by Western blot. 80uL rMrATG5 recombinant protein was mixed with 20uL 5X SDS-PAGE protein loading buffer and placed in a 100℃ metal bath for 10min. The protein bands were separated by polyacrylamide gel electrophoresis, and the bands in the gel after electrophoresis were transferred to a hydrophilic polyvinylidene fluoride (PVDF) blotting membrane with a pore size of 0.45μm. The PVDF membrane was blocked with 5% skim milk at room temperature for 1h. The membrane was washed 3 times with TBS-T, each time for 5min, and then incubated with His-tagged mouse monoclonal antibody (dilution ratio of 1:1000, Beyotime, China) at 4℃ overnight. The membrane was washed three times with TBS-T for 5 min each time, and then incubated with goat anti-mouse IgG (H+L) antibody labeled with alkaline phosphatase (AP) (dilution ratio of 1:1000, Bio-Tech, China) at room temperature for 1.5 h. Finally, the membrane was washed five times with TBS-T for 3 min each time and developed with BCIP / NBT alkaline phosphatase color development kit (Sangon, China).

[0085] The results showed that the molecular weight of the empty vector protein of pET-32a(+) was about 22 kDa, and the molecular weight of the predicted protein of MrATG5 was about 31.04 kDa. The molecular weight of the protein after adding the two together was about 52 kDa. The results of SDS-PAGE electrophoresis showed that ( Figure 3 A), after induction, rMrATG5 showed a specific band between 43kDa and 55kDa, which was consistent with the predicted results, indicating that the induction expression of rMrATG5 recombinant active protein was successful. Compared with the unpurified protein band, the protein band after purification by Ni column had a high concentration, a clear and single specific band, and no other mixed bands. Western blot results showed ( Figure 3 B), rMrATG5 can specifically bind to mouse anti-His-tag monoclonal antibody, and a clear single specific band appears between 43kDa and 55kDa, indicating that the purified and renatured protein is MrATG5 recombinant active protein.

[0086] in, Figure 3 In the figure, M represents the protein molecule standard, 1 represents MrATG5-pET-32a(+) without induction, 2 represents MrATG5-pET-32a(+) with induction, 3 represents MrATG5-pET-32a(+) purification, and 4 represents Western blot identification.

[0087] 1.6 Binding activity of MrATG5 recombinant protein to bacteria

[0088] After the four bacteria Aeromonas hydrophila (A.hydrophila), Staphylococcus aureus (S.aureus), Streptococcus iniae (S.iniae), and Citrobacter freundii (C.freundii) were expanded and cultured, they were centrifuged at 1000rpm and 4℃ for 5min, and the supernatant culture medium was discarded to collect the bacteria. After the collected bacteria were washed 3 times with pre-cooled TBS, PBS was added to resuspend the bacteria, and the OD value of the bacterial solution was adjusted to 1.0. Take 50uL of the bacterial solution after the above adjusted concentration, add 100uL of recombinant protein MrATG5, and incubate at 4℃ for 1h. The control group was incubated with 100uL of recombinant protein rTrx and 50uL of bacterial solution. After the incubation, the bacteria were washed with TBS and repeated 3 times. Finally, resuspend the bacteria in TBS, take 80uL of the resuspended solution and add 20uL of loading buffer and heat in a 100℃ metal bath for 10min to obtain the loaded protein sample. Use SDS-PAGE and Western blot to detect the binding activity of the recombinant protein to bacteria. The experimental steps are the same as 1.5.

[0089] The results showed that a specific band appeared between 43 kDa and 55 kDa, which was consistent with the position of the purified rMrATG5 protein band ( Figure 4 ), while no specific protein bands appeared in the rTrx protein of the control group, indicating that the rMrATG5 protein can produce binding activity with S. aureus, S. iniae, A. hydrophila and C. freundii.

[0090] 1.7 Antibacterial activity of MrATG5 recombinant protein

[0091] The concentration of purified rMrATG5 and rTrx was adjusted to 0.2 mg / mL. 600 =0.4-0.6) were centrifuged, washed with TBS and resuspended to adjust the concentration to OD 600=0.2. The blank group was composed of 50ul of each bacterial suspension mixed with an equal volume of TBS, the control group was composed of 50ul of each bacterial suspension mixed with an equal volume of rTrx, and the experimental group was composed of 50ul of each bacterial suspension mixed with an equal volume of rMrATG5. 100ul of the mixed solution of each group was added to a 96-well ELISA plate and then 100ul of HBI liquid culture medium (Solebo, China) was added. The ELISA plate was placed in an ELISA instrument and cultured at 28°C with shaking. The OD value was recorded every half an hour. After culturing for 20 hours, the OD value at each time point was counted to draw the bacterial growth curve, and the effect of rMrATG5 protein on the proliferation of each bacteria was detected. The experimental data were sorted and analyzed using GraphPad Prism v 8.0.2 software, and the experimental data of each group were analyzed by one-way analysis of variance (ANOVA). The experimental data are expressed as mean ± standard error. Significant differences are marked with "*" (P<0.05), and extremely significant differences are marked with "**" (P<0.01).

[0092] Results: The effect of rMrATG5 protein on the proliferation of four bacteria, S. aureus, S. iniae, A. hydrophila and C. freundii, was detected by measuring the bacterial growth curve. An experimental group with rMrATG5 protein added and a control group with rTrx protein added were set up. Figure 5 As shown, starting from 0h, the OD of the experimental groups of the four bacteria 600 The values ​​were greater than the OD of the control group 600 Values, OD of the control group in A.hydrophila, S.iniae and C.freundii groups at 14 h 600 The values ​​were higher than those of the experimental group OD 600 Numerical values ​​show that growth inhibition occurred in the experimental groups of A.hydrophila and C.freundii. The OD 600 The value was significantly higher than that of the experimental group (P<0.05), which indicated that MrATG5-pET-32a(+) recombinant protein had no inhibitory effect on S.iniae and S.aureus, but had inhibitory effect on A.hydrophila and C.freundii.

[0093] Autophagy is a ubiquitous cell decomposition and recycling process in eukaryotic cells. It is used to maintain the body's homeostasis by regulating the body's immunity, metabolism, etc. when the body is subjected to exogenous stimuli. The autophagy process is inseparable from the regulation of many autophagy-related proteins (ATGs). More than 30 ATGs and interacting proteins have been identified, some of which can participate in the body's immune response independently of the autophagy process. In vertebrates, ATG5 is a key protein for the occurrence of autophagy. It can combine with ATG12 to form a protein complex to regulate the formation of autophagosomes. However, the function and molecular mechanism of action of ATG5 in invertebrates need to be improved. In this application, the ATG5 gene was identified in Macrobrachium rosenbergii, and its immune function in Macrobrachium rosenbergii infected with pathogens was explored.

[0094] In vertebrates, ATG5 can inhibit the development of the disease through phosphorylation. In human gastrointestinal stromal tumor (GIST) cells, the activation of a serine / threonine protein kinase (P21-activated kinase1, PAK1) can promote the phosphorylation reaction of ATG5 to participate in the treatment of GIST. Protein phosphorylation usually occurs at serine and tyrosine sites, which can promote biological processes such as protein signal transduction, transcriptional regulation and regulation of cell apoptosis. Glycosylation can affect the activity and stability of proteins and also has an important influence on subcellular localization. In this application, bioinformatics analysis found that there are multiple N-glycosylation sites and serine and tyrosine sites in the amino acid sequence of MrATG5, which indicates that MrATG5 can participate in the physiological and biochemical reactions of Macrobrachium rosenbergii through post-translational modification. More and more studies have shown that ATGs can recognize and bind to pathogens that invade the body, and promote the encapsulation of pathogens by autophagosomes until they are degraded in lysosomes. In the present application, rMrATG5 can produce binding activity with Gram-negative bacteria A.hydrophila, C.freundii and Gram-positive bacteria S.aureus, S.iniae and inhibit the proliferation of A.hydrophila and C.freundii. These results show that rMrATG5 has a broad spectrum of binding to most microorganisms and participates in the antibacterial immune response of Macrobrachium rosenbergii by specifically inhibiting the invasion of pathogens to the body.

[0095] This application cloned and identified the MrATG5 gene of Macrobrachium rosenbergii, and by constructing the MrATG5-pET-32a(+) prokaryotic expression vector, successfully induced and purified the recombinant expression active protein of MrATG5, and analyzed its binding and antibacterial activity against bacteria. The above experiments show that MrATG5 can inhibit the proliferation of pathogens in the body by binding to bacteria, thereby enhancing the antibacterial immunity of Macrobrachium rosenbergii. The results of this application will provide data support for in-depth research on the immune defense function of Macrobrachium rosenbergii, and provide a certain theoretical basis for the prevention and control of diseases of Macrobrachium rosenbergii.

[0096] The above are only preferred embodiments of the present application, and are not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A use of MrATG5 recombinant protein for preparing a drug for preventing and treating Macrobrachium rosenbergii diseases, characterized in that: Its prevention and control mechanisms are: Inhibit the proliferation of Aeromonas hydrophila and Citrobacter freundii in the body to enhance the antibacterial immunity of Macrobrachium rosenbergii; The MrATG5 recombinant protein is MrATG5-pET-32a(+) recombinant protein, the nucleotide sequence of which is shown in SEQ ID No.1, and the amino acid sequence of which is shown in SEQ ID No.

2.

2. The use of the MrATG5 recombinant protein according to claim 1 for preparing a drug for preventing and treating diseases of Macrobrachium rosenbergii, characterized in that: The MrATG5 recombinant protein is prepared by the following steps: Step S10: The in vitro hemolymph sample of Macrobrachium rosenbergii is mixed with an equal amount of anticoagulant and centrifuged, and after the centrifugation, blood cells are collected and frozen in liquid nitrogen, and the heart, eyestalk, intestine, stomach, hemolymph, hepatopancreas, muscle and gill tissues are extracted using sterile tweezers and quickly frozen in liquid nitrogen, and then all tissue samples are ground into powder; the target RNA in the target tissue sample is extracted, and the target RNA is reverse transcribed into a target cDNA; Step S20: The target cDNA is used as a template to perform gene-specific amplification to obtain a first PCR product; the conditions for the gene-specific amplification are: 94°C, 5 min; 94°C, 30 s; 55°C, 30 s; 72°C, 30 s, 35 cycles; 72°C, 10 min; The PCR products were detected and separated by agarose gel electrophoresis, and the separated products were connected to the pMD-18T vector to obtain the ligation products; the ligation conditions were: 16°C, 2 h; The ligation product was transformed into DH5α competent cells using a heat shock method to obtain transformed competent cells; the transformation conditions were: first ice bath for 30 min, then placed in a 42°C water bath for 45 s, and then placed on ice for 5 min; Add 500 μL of antibiotic-free LB liquid culture medium to the transformed competent cells and culture at 37°C, 200 rpm for 1 h; then spread 200 μL of the culture product on LB solid culture medium with ampicillin resistance and culture at 37°C for 12 h; Using a sterile inoculation loop, randomly picking a single colony on the LB solid medium, and performing PCR amplification, separating and verifying the amplified product by agarose gel electrophoresis, and obtaining ATG5-specific primers; The ATG5-specific primers include ATG5-F and ATG5-R; Select pET-32a (+) as the prokaryotic expression vector of MrATG5; based on the ATG5 specific primer, the MrATG5 gene and the expression vector are connected by a double enzyme digestion method to obtain a recombinant expression vector primer; the recombinant expression vector primer is a recombinant prokaryotic expression vector primer; ATG5-BamH IF and ATG5-Xho IR; based on the recombinant expression vector primers, gene-specific amplification treatment is performed to obtain a second PCR product; the conditions of the gene-specific amplification treatment are: 94°C, 5min; 94°C, 30s; 55°C, 30s; 72°C, 30s, 35 cycles; 72°C, 10min; the second PCR product is connected and transformed into the pMD 18-T vector, coated on an LB solid culture medium with ampicillin resistance, and a single colony is picked for expansion and testing after overnight culture at 37°C; after the sequencing is correct, the retained bacterial liquid is expanded and the plasmid is extracted to obtain the plasmid of the recombinant expression vector; the MrATG5 gene primers with restriction sites are double-enzyme-cut and the plasmid of the recombinant expression vector are double-enzyme-cut to obtain the MrATG5 gene gel recovery product after enzyme cutting and the recombinant expression vector plasmid after enzyme cutting; the MrATG5 gene gel recovery product after enzyme cutting and the recombinant expression vector plasmid after enzyme cutting are double-enzyme-cut ... ℃ connection for 5 h, then transformed into competent cells, cultured overnight at 37 ℃ in LB solid medium with corresponding resistance, and single colonies were picked for sequencing analysis; after correct sequencing, MrATG5-pET-32a (+) recombinant expression vector was obtained; Step S30: The plasmid of MrATG5-pET-32a (+) recombinant expression vector was extracted and transformed into Transetta (DE3) competent cells. The bacteria were expanded and cultured until the bacterial solution concentration reached the logarithmic growth phase. Isopropyl-β-D-thiogalactoside with a final concentration of 1 mM was added, and the expression was induced at 16 °C and 130 rpm for 12 h to obtain the induced product; the induced product was purified by using BeyoGold™ His-tag Purification Resin; after balancing the Ni column with the lysis solution, 8 times the column volume of the protein supernatant was loaded onto the column, and the protein flow-through was recovered and repeatedly loaded onto the column 5 times, and the nickel column was eluted with imidazole-free Wash Buffer, and then the protein was eluted with imidazole-containing Wash Buffer to obtain the purified product; a protein refolding dialysate containing stepwise concentrations of urea was prepared; the protein refolding dialysate containing stepwise concentrations of urea included urea with concentrations of 0 M, 1 M, 2 M, 3 M, 4 M and 6 M, pH = 7.4 - 8.0 protein renaturation dialysate; placing the purified product in the protein renaturation dialysate containing the stepwise concentration of urea, placing it at 4 ° C in the order of urea concentration from high to low for protein dialysis renaturation, and replacing the dialysate every 12 hours; after the dialysis is completed, the MrATG5 target recombinant protein is obtained.

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