WO2021258637A1 - 一种胰蛋白酶样丝氨酸蛋白酶基因、编码的蛋白质和应用 - Google Patents

一种胰蛋白酶样丝氨酸蛋白酶基因、编码的蛋白质和应用 Download PDF

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WO2021258637A1
WO2021258637A1 PCT/CN2020/132289 CN2020132289W WO2021258637A1 WO 2021258637 A1 WO2021258637 A1 WO 2021258637A1 CN 2020132289 W CN2020132289 W CN 2020132289W WO 2021258637 A1 WO2021258637 A1 WO 2021258637A1
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pmtls
protein
trypsin
gene
serine protease
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French (fr)
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梁海鹰
何军军
申铖皓
房晓宸
卢金昭
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广东海洋大学
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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21004—Trypsin (3.4.21.4)
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21106—Hepsin (3.4.21.106)

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  • the invention belongs to the technical field of genetic engineering, and specifically relates to a trypsin-like serine protease gene, encoded protein and application.
  • the purpose of the present invention is to provide a protein encoded by a trypsin-like serine protease gene and an application.
  • the protein encoded by the gene provided by the present invention can inhibit four microorganisms.
  • the present invention provides a trypsin-like serine protease gene, and the nucleotide sequence of the trypsin-like serine protease gene is shown in SEQ ID No. 1.
  • the present invention also provides the protein encoded by the trypsin-like serine protease gene described in the above technical solution, and the amino acid sequence of the protein is shown in SEQ ID No.2.
  • the present invention also provides the application of the protein described in the above technical scheme in the preparation of drugs for inhibiting microorganisms.
  • the microorganisms include one or more of Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio parahaemolyticus, and Vibrio harveyi.
  • the present invention provides a trypsin-like serine protease gene, characterized in that the nucleotide sequence of the trypsin-like serine protease gene is shown in SEQ ID No. 1.
  • the amino acid sequence of the protein encoded by the gene provided by the present invention is shown in SEQ ID No. 2, and the protein can inhibit Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio parahaemolyticus and Vibrio harveyi.
  • Figure 1 is a schematic diagram of the cloning site of pMD19-T Vector
  • Figure 2 shows the prediction of the PmTLS domain
  • FIG. 1 shows the homology comparison of PmTLS
  • Figure 4 shows the construction of the PmTLS phylogenetic tree
  • Figure 5 shows the expression distribution of PmTLS gene in each tissue of Pinctada martensii, M: mantle, B: blood cells, Gi: gills, Go: gonads, A: adductor muscle, He: hepatopancreas;
  • Figure 6 shows the sequential expression of PmTLS in blood cells after PAMPs stimulation
  • Figure 7 shows the temporal expression of PmTLS in blood cells after PAMPs stimulation
  • Figure 8 shows the sequential expression of PmTLS in blood cells after PAMPs stimulation
  • Figure 9 shows the verification of PmTLS prokaryotic expression recombinant plasmid
  • Figure 10 shows the induced expression of rPmTLS (SDS-PAGE), PC1: BSA (1 ⁇ g); PC2: BSA (2 ⁇ g); M1: Marker; NC: no induced whole bacteria; 1: 15°C induced whole bacteria; 2: 37°C Induction of whole bacteria; NC1: no induction supernatant; 3: 15°C induction supernatant; 4: 37°C induction supernatant; NC2: no induction precipitation; 5: 15°C induction precipitation; 6: 37°C induction precipitation;
  • Figure 11 shows the induced expression of rPmTLS (Western Blot), M2: Marker; NC: no induced whole bacteria; 1: 15°C induced whole bacteria; 2: 37°C induced whole bacteria; NC1: no induced supernatant; 3: 15°C Induced supernatant; 4: 37°C induced supernatant; 5: 15°C induced precipitation; 6: 37°C induced precipitation;
  • Figure 12 is rPmTLS purification analysis (SDS-PAGE); M1: Marker; 1: BSA (2 ⁇ g); 2: Target protein after renaturation (2 ⁇ g); 3: Target protein before renaturation (2 ⁇ g)
  • Figure 13 is the determination of the antibacterial activity of rPmTLS (Pseudomonas aeruginosa);
  • Figure 14 is the determination of the antibacterial activity of rPmTLS (Aeromonas hydrophila);
  • Figure 15 is the determination of the antibacterial activity of rPmTLS (Vibrio parahaemolyticus).
  • Figure 16 shows the antibacterial activity determination of rPmTLS (Vibrio harveyi).
  • Figure 17 shows the morphological changes of rPmTLS on bacteria
  • A, E, I are the PBS control group of Pseudomonas aeruginosa, Aeromonas hydrophila, and Vibrio parahaemolyticus, respectively
  • B, C, and D are the effects of rPmTLS on aeruginosa Pseudomonas experimental group
  • F, G, H are the experimental groups where rPmTLS acts on Aeromonas hydrophila
  • J and K are the experimental groups where rPmTLS acts on Vibrio parahaemolyticus.
  • the present invention provides a trypsin-like serine protease gene, the nucleotide sequence of the trypsin-like serine protease gene is shown in SEQ ID No. 1, specifically as follows:
  • the present invention also provides the protein encoded by the trypsin-like serine protease gene according to the above technical solution, the abbreviation of the protein is PmTLS, which is the trypsin-like serine protease of Pinctada martensii, and the amino acid sequence of the protein is as SEQ ID As shown in No. 2, the details are as follows:
  • the present invention also provides the application of the protein described in the above technical scheme in the preparation of drugs for inhibiting microorganisms.
  • the microorganism preferably includes one or more of Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio parahaemolyticus, and Vibrio harveyi.
  • the specific primers of the TLS gene are designed using Primer Premier 5.0.
  • the 5'-end and 3'-end specific primers are designed according to the principle of RACE amplification, and the full-length cDNA of the gene is obtained by comparison and splicing.
  • the primer sequence is as follows.
  • a trace nucleic acid quantifier measures the concentration and purity of total RNA.
  • the total RNA obtained is stored in an ultra-low temperature refrigerator at -80°C for later use.
  • PrimeSTARHS high-fidelity enzyme amplification obtains the target fragment.
  • the PCR reaction system is as follows:
  • the purified PCR product was ligated to the pMD-19T Vector vector ( Figure 1).
  • the system is as follows:
  • Reagent dose Solution I 5 ⁇ L Destination segment 4.5 ⁇ L pMD19-T Vector 0.5 ⁇ L Total 10 ⁇ L
  • connection procedure is: connect at 16°C for about 16 hours.
  • DH5 ⁇ competent cells Take the DH5 ⁇ competent cells out of the ultra-low temperature refrigerator, place them on ice until they are slightly soluble, and then add the ligation product to a tube containing 100 ⁇ L DH5 ⁇ competent cells, gently pipetting evenly; place on ice for 30 minutes, heat shock at 42°C for 60 to 90 seconds, Place on ice for 2 to 3 minutes; then add 890 ⁇ L of LB liquid medium preheated at 37°C in advance, and culture at 37°C with 200 rpm shaking for 1 h; then at 4000 rpm for 2 minutes, discard the supernatant, leaving about 100 ⁇ L, gently blow off the suspended cells, and Coat on the LA (Amp+) solid plate; finally, incubate upright for half an hour at 37°C, then inverted for overnight incubation.
  • Nested PCR uses 3', 5'specific primers combined with universal primers (NUP and UPM) for amplification.
  • PCR gel cutting recovery/product purification, connection, transformation and colony PCR detection refer to steps (2) to (5) of 5.
  • NCBI blastx for sequence homology and similarity analysis
  • DNAMAN 6.0 software sequence search, splicing and comparison ExPASy online prediction of its theoretical molecular weight (MW) and isoelectric point (pI); use SignalP 4.0 Server for signal peptide sequence prediction ; TMHMM Server v.2.0 analyzes the transmembrane domain of the sequence; MEGA 6 software uses the NJ method to construct a phylogenetic tree; SoftBerry Psite predicts the functional site of its deduced amino acid sequence; SOPMA predicts the protein secondary structure.
  • Tissue quantification Take 10 scallops with the same size and good vigor, and collect tissues including gonads, gills, mantle, hemolymph, adductor muscle and hepatopancreas.
  • PAMPs stimulation 320 healthy Pinctada martensii kept for 1 week and randomly divided into 4 groups: LPS stimulation group (experimental group), PGN stimulation group (experimental group), PolyI:C stimulation group (experimental group) ), PBS group (control group), 80 shells each. Adopting the method of adductor muscle injection, the experimental group was injected with 100 ⁇ L of LPS, PGN, PolyI:C (10 ⁇ g/mL) each, and 100 ⁇ L of PBS was injected as the control group. At 0, 3, 6, 12, 24, 48, 72, 96 hours after injection, 10 shellfish were randomly selected from each group to take the whole tissue for use.
  • the hemolymph was centrifuged at 4°C, 800 ⁇ g, and the supernatant was discarded.
  • Add 1mL Trizol shake gently to make the suspension uniform, extract the total RNA and reverse transcribed into cDNA for subsequent quantitative analysis (Note: PBS is sterilized, LPS, PGN, PolyI:C are prepared with sterilized PBS ; During the experiment, no bait was fed and no deaths occurred).
  • the primers used in the fluorescence quantitative PCR reaction are shown in Table 1.
  • the extraction method of total RNA is the same as steps (1) to (4) in 3.
  • Fluorescence quantitative data was analyzed by the 2- ⁇ ct method. The test results were all expressed as mean ⁇ standard deviation, and SPSS17.0 software was used to perform one-way ANOVA, and Duncan’s multiple comparison was used to test the significance of differences in the mean.
  • Gene synthesis is performed according to the existing target gene sequence, which is used as a template for subsequent subcloning, and the restriction sites for introducing restriction enzymes are NcoI and HindIII.
  • Recombinant PmTLS ORF amplification, PCR system 10 ⁇ L of Premix Ex Taq, 0.5 ⁇ L of synthetic template, 0.4 ⁇ L of Forward Primer and Reverse Primer, and ddH 2 O to make up the system to 20 ⁇ L.
  • the procedure is: denaturation at 94°C for 5min; 98°C for 10s, 55°C for 30s, 72°C for 60s, 30 cycles.
  • the reaction products were detected by gel electrophoresis.
  • Plasmid extraction refers to the instructions of the Easy Pure Plasmid Mini Prep Kit Silica Gel Membrane Spin Column Plasmid DNA Mini Extraction Kit.
  • Double digestion of the empty plasmid pET-28a(+) and the extracted recombinant plasmid with MluI, XhoI and HindIII endonucleases The system is 5.0 ⁇ L of 10 ⁇ FD Buffer, MluI endonuclease 3.0 ⁇ L, 20 ⁇ L of plasmid, and ddH 2 O to make up the system to 50 ⁇ L. Mix gently, and incubate at 37°C for 2h.
  • step 2.2.5(2) for the purification of cut gum.
  • the recovered product purified by enzyme digestion is connected with pET-28a(+) vector through the action of T4 ligase to construct a recombinant expression plasmid pET28a-PmTLS.
  • the ligation system is: 6 ⁇ L of the recovered enzyme digestion purified product, 2 ⁇ L of pET-28a(+), 10 ⁇ T4 buffer is 1 ⁇ L, 1 ⁇ L T4 ligase, the total system is 10 ⁇ L. Mix lightly, overnight at 16°C.
  • Plasmid extraction Refer to step (3) of 9.2 to obtain the pET28a-PmTLS recombinant plasmid and store it at -20°C for later use.
  • Supernatant and debris of cell lysate Centrifuge the remaining 200 ⁇ L of cell lysate at 15,000 rpm for 10 minutes, collect the supernatant and cell debris of the cell lysate, and mix 90 ⁇ L of 5 ⁇ loading buffer with 180 ⁇ L of supernatant. As a sample. Supernatant of cell lysate. Resuspend all pellets with 150 ⁇ L of 5 ⁇ loading buffer as a sample of cell lysate fragments. The sample was heated at 100°C for 10 min, and centrifuged at 15,000 rpm for 5 min before loading the sample.
  • Binding buffer with 5 times the column bed volume to clean the column with a flow rate of 5 m L/min.
  • sample preparation sample loading.
  • the primary antibody is rabbit anti-his label, antibody company: Sangon Biotech, number: D110002, diluted 1:500, and shaken slowly at 37°C for 60 minutes.
  • the secondary antibody is goat anti-rabbit, antibody company: Sangon Biotech, number: D110058, diluted 1:8000, and shaken slowly at 37°C for 60 minutes.
  • the purified target protein was renatured by dialysis, and finally replaced into a soluble buffer (PBS, 10% glycerol, 1ML-arginine, pH 7.4), and compared with the purified protein by SDS-PAGE and Western blot. The purity before sex (precipitation) and after refolding (dissolution).
  • PBS 10% glycerol, 1ML-arginine, pH 7.4
  • the purified protein has antibacterial activity against Escherichia coli, Micrococcus luteus, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio harveyi, Bacillus subtilis and Staphylococcus aureus.
  • each type of bacteria was cultured in 2216E liquid medium to the logarithmic growth phase. The bacteria were then centrifuged (3,000 ⁇ g, 10 min), washed 3 times with phosphate buffered saline (1 ⁇ PBS), and then resuspended in PBS.
  • PBS was used instead as a control.
  • the purified protein and bacteria mixture was centrifuged at 3000 rpm for 10 min at room temperature for precipitation, and then washed with PBS three times to remove impurities, and then the bacterial pellet was fixed in 200 ⁇ L 3% glutaraldehyde overnight.
  • 200 ⁇ L of 2% sodium phosphotungstate aqueous solution was added to the bacterial suspension, and then it was dropped on the copper net.
  • Use filter paper to remove residual water, and after 5 minutes, air dry the sample. Observe under standard operating conditions with a JEM-1400 (Japan Electronics Corporation) microscope.
  • the full length of the PmTLS gene (SEQ ID No.1) cloned by RACE technology is 778bp, which contains 420bp of 5'UTR, 97bp of 3'UTR, and 261bp of ORF box, encoding a total of 86 amino acids (SEQ ID No.2) .
  • the molecular weight of PmTLS protease is 9.5KDa, the theoretical isoelectric point is 4.22, and its hydrophilicity is predicted. It is found that the highest continuous hydrophobicity occurs at positions 51 and 52, with an index of 2.011, and the highest hydrophilicity occurs at position 63. Its index is -2.156, and the total average hydrophilicity coefficient is -0.172, which is a hydrophilic protein.
  • the total number of negatively charged residues (Asp+Glu) is 9, and the total number of positively charged residues (Arg+Lys) is 3. Therefore, the PmTLS protease is generally negatively charged.
  • the prediction of transmembrane structure revealed that it has no transmembrane domain.
  • DNAMAN was used to compare the amino acid sequence of PmTLS with the corresponding amino acids of other invertebrates. The analysis found that the similarity between PmTLS and Pacific oyster was the highest, reaching 69.77%, and the consistent similarity between species reached 41.42% (Figure 3).
  • the existing full-length cDNA sequence of the PmTLS gene predict the open reading frame ORF and analyze it to obtain the ORF sequence that needs to be recombined. Design and add histidine gene sequence to the recombination ORF sequence, then proceed to gene synthesis, and then design prokaryotic expression primers . After PCR amplification, the PmTLS gene fragment was about 290bp; then the PmTLS gene was ligated with the pMD19-T vector, transformed, and a single colony was picked. Colonies of positive clones were identified by colony PCR, and plasmid extraction kits were used for plasmids.
  • the recombinant expression vector pET28-PmTLS was transformed into Escherichia coli, and induced with 0.5mM IPTG at 15°C for 16h and 37°C for 4h respectively.
  • the negative control was no induction with IPTG. It can be seen from Figure 9 that the target bands did not appear in lanes NC (whole bacteria), NC1 (supernatant) and NC2 (precipitation) in the negative control group; at the same time, no target bands were seen in the supernatant lanes 3 and 4 at 15°C and 37°C.
  • the pET28-PmTLS expression strain was cultured with 1L expression amount, and induced at 0.5mM IPTG at 37°C for 4h.
  • the results showed that a clear and single band appeared at about 10KDa, indicating that rPmTLS with higher purity was successfully obtained, and the molecular weight of the recombinant protein did not change before and after renaturation, indicating that the purity after renaturation was higher ( Figure 12).
  • the concentration of rPmTLS was determined by Bradford protein assay, and the concentration was 1.07mg/mL, and a total of 8.02mg protein of rPmTLS was obtained.
  • the control group of Pseudomonas aeruginosa (A in Figure 17) has clear and complete edges, and the contents are dense and uniform; while the experimental group (B in Figure 17) ) It can be observed that the bacterial body of Pseudomonas aeruginosa swells, the content of the substance shrinks, and the internal structure is also significantly changed compared with the control group. At the same time, the cell wall seems to begin to dissolve, the cytoplasm separates from the cell wall, and the phenomenon of plasmolysis occurs (C-D in Figure 17), indicating that the cell wall has collapsed and the local contents of the cell are released.
  • E in Figure 17 is the control group of Aeromonas hydrophila, which has long and slender cells, clear edges and uniform contents; while in the experimental group (F in Figure 17), the inside of Aeromonas hydrophila can be observed The substance-containing part is lost and the internal structure changes. The two ends of the bacteria are enlarged and then observed. It is found that the edges of the bacteria are very irregular, and the cell wall is dissolved (GH in Figure 17).
  • I in Figure 17 is the control group of Vibrio parahaemolyticus, the cells are short rod-shaped, homogeneous and full; while the experimental group (J in Figure 17) lacks content and the cells swell in the middle, further enlargement and observation, It was found that the cytoplasm was separated from the cell wall and the cytoplasm was separated (K in Figure 17).

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Abstract

提供了一种胰蛋白酶样丝氨酸蛋白酶基因、编码的蛋白质和应用，该胰蛋白酶样丝氨酸蛋白酶基因的核苷酸序列如SEQ ID No.1所示，该胰蛋白酶样丝氨酸蛋白酶基因编码的蛋白质的氨基酸序列如SEQ ID No.2所示。上述基因编码的蛋白质能够抑制铜绿假单胞菌、嗜水气单胞菌、副溶血弧菌和哈维氏弧菌。

Description

一种胰蛋白酶样丝氨酸蛋白酶基因、编码的蛋白质和应用
本申请要求于2020年06月24日提交中国专利局、申请号为202010589361.X、发明名称为“一种胰蛋白酶样丝氨酸蛋白酶基因、编码的蛋白质和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于基因工程技术领域，具体涉及一种胰蛋白酶样丝氨酸蛋白酶基因、编码的蛋白质和应用。
背景技术
近年来，由于海洋环境污染严重，水产养殖动物病害频发，传统抗生素的滥用导致越来越严重的病原微生物的耐药性问题，给水产养殖动物和人类的健康带来巨大威胁，迫切需要开发新的抗菌药物。而海洋是世界上物种最为丰富的聚集地，从海洋无脊椎动物中探索出具有抗菌活性的物质，具有极大的潜力。为了解决抗生素滥用和耐药性问题，从水产养殖动物自身出发，探究接近天然的生物抗生素，提高抗菌广谱性和耐药性。
发明内容
基于上述原因，本发明的目的在于提供一种胰蛋白酶样丝氨酸蛋白酶基因编码的蛋白质和应用，本发明提供的基因编码的蛋白能够抑制四种微生物。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种胰蛋白酶样丝氨酸蛋白酶基因，所述胰蛋白酶样丝氨酸蛋白酶基因的核苷酸序列如SEQ ID No.1所示。
本发明还提供了上述技术方案所述的胰蛋白酶样丝氨酸蛋白酶基因编码的蛋白质，所述蛋白质的氨基酸序列如SEQ ID No.2所示。
本发明还提供了上述技术方案所述的蛋白质在制备抑制微生物的药物中的应用。
优选的，所述微生物包括铜绿假单胞菌、嗜水气单胞菌、副溶血弧菌和哈维氏弧菌中的一种或多种。
本发明提供了一种胰蛋白酶样丝氨酸蛋白酶基因，其特征在于，所述胰蛋白酶样丝氨酸蛋白酶基因的核苷酸序列如SEQ ID No.1所示。本发明提供的基因编码的蛋白质的氨基酸序列如SEQ ID No.2所示，所述蛋白质能够抑制铜绿假单胞菌、嗜水气单胞菌、副溶血弧菌和哈维氏弧菌。
附图说明
图1为pMD19-T Vector克隆位点示意图；
图2为PmTLS结构域预测；
图3为PmTLS同源性比对；
图4为PmTLS系统进化树构建；
图5为PmTLS基因在马氏珠母贝各组织的表达分布，M：外套膜，B：血细胞，Gi：鳃，Go：性腺，A：闭壳肌，He：肝胰腺；
图6为PAMPs刺激后PmTLS在血细胞的时序表达；
图7为PAMPs刺激后PmTLS在血细胞的时序表达；
图8为PAMPs刺激后PmTLS在血细胞的时序表达；
图9为PmTLS原核表达重组质粒验证；
图10为rPmTLS的诱导表达(SDS-PAGE)，PC1：BSA(1μg)；PC2：BSA(2μg)；M1：Marker；NC：无诱导全菌；1：15℃诱导全菌；2：37℃诱导全菌；NC1：无诱导上清；3：15℃诱导上清；4：37℃诱导上清；NC2：无诱导沉淀；5：15℃诱导沉淀；6：37℃诱导沉淀；
图11为rPmTLS的诱导表达(Western Blot)，M2：Marker；NC：无诱导全菌；1：15℃诱导全菌；2：37℃诱导全菌；NC1：无诱导上清；3：15℃诱导上清；4：37℃诱导上清；5：15℃诱导沉淀；6：37℃诱导沉淀；
图12为rPmTLS纯化分析(SDS-PAGE)；M1：Marker；1：BSA(2μg)；2：复性后的目的蛋白(2μg)；3：复性前的目的蛋白(2μg)
图13为rPmTLS的抗菌活性测定(铜绿假单胞菌)；
图14为rPmTLS的抗菌活性测定(嗜水气单胞菌)；
图15为rPmTLS的抗菌活性测定(副溶血弧菌)；
图16为rPmTLS的抗菌活性测定(哈维氏弧菌)；
图17为rPmTLS作用于细菌的形态学变化；A、E、I分别为铜绿假单胞菌、嗜水气单胞菌、副溶血弧菌的PBS对照组，B、C、D为rPmTLS作用铜绿假单胞菌的实验组，F、G、H为rPmTLS作用嗜水气单胞菌的实验组，J和K为rPmTLS作用副溶血弧菌的实验组。
具体实施方式
本发明提供了一种胰蛋白酶样丝氨酸蛋白酶基因，所述胰蛋白酶样丝氨酸蛋白酶基因的核苷酸序列如SEQ ID No.1所示，具体如下：
ACATGGGCTAATAAATGGATCGTGACAGCCGCCCACTGTATCGTGCGTTTTCCCGAGAAATTCCACGAATTGTTCCACCCCTCTAAGGTCACCCTTATTATTGGTACAGAGCAGTGTAGCGGATATGACGGCCAAATCGTGGACATCGAGTCATATGTTGTGCATCCTAGATTTGCAGAAAGGGCTCCATACGACCATGATATAGCTTTGATAGAACTTCGTCAAGATTTAAACTTTACAGAACGTGTACAACCAATATGTCTCAAGCAGCCGGATTACGTGAATACTGCTTTCCTTCATCGCAAAGTCGGGCGTAAGGCAGGGAGGGTTGTAGGGTGTGGTCAATTGTATGAAAATGTAGATGCTATACCCACGGAGCTACATGACGTTTTCGTACCAACAGTGACTAGGGAGAAATGT A TGGAGGCGGACATAGGGCGAGGAAATTTCACTGACACTATGTTCTG CGCAGGGTATGACAGGGCTTTATTCGGAGATGCTTGTTATGGTGATA GTGGTGGCTCTTTGGCGATGAATGACTCCCCATTTGACCCCTGGGTC CTTGTGGGCGTGGTGTCATGGGGAGTTGGGTGTGACCGACAAGGAC ATTATGGATACTATACAAATATAGCTCACTTTTATAACTGGATACAAA ATGTCACAAATGTTTTAAATAATTAGGATTGAAACAATAAAGAGATATAGATCTTAATTTATACTATTGAGACACAATTAAAAAAAGTTTAACCCTAAAAAAAAAAAAAAAAAAAAAAAAAAA。下划线为胰蛋白酶样丝氨酸蛋白酶基因开放阅读框ORF。
本发明还提供了上述技术方案所述的胰蛋白酶样丝氨酸蛋白酶基因编码的蛋白，所述蛋白的简称为PmTLS，是马氏珠母贝胰蛋白酶样丝氨酸蛋白酶，所述蛋白的氨基酸序列如SEQ ID No.2所示，具体如下：
Figure PCTCN2020132289-appb-000001
本发明还提供了上述技术方案所述的蛋白质在制备抑制微生物的药物中的应用。在本发明中，所述微生物优选包括铜绿假单胞菌、嗜水气单胞菌、副溶血弧菌和哈维氏弧菌中的一种或多种。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
1 马氏珠母贝免疫效应分子筛选
从在线抗菌肽数据库(APD3，http://aps.unmc.edu/AP/main.php)，PubMed(https://www.ncbi.nlm.nih.gov/pubmed/)和国家生物技术信息中心(NCBI)数据库(https://www.ncbi.nlm.nih.gov/)获取所有具有先前验证的抗微生物活性的氨基酸序列，随后，构建了一个本地参考AMP数据库，根据马氏珠母贝基因组数据进行比对搜索。收集比对率最高的基因序列，再运用NCBI数据库中的在线Blast进行分析，预测所收集的免疫效应分子PmTLS基因的类型。
2 PmTLS基因相关引物设计
TLS基因的特异性引物使用Primer Premier 5.0设计。5'端和3'端特异性引物依据RACE扩增原理设计，经过比对拼接获得基因的cDNA全长。引物序列如下。
表1 引物
引物 序列(5'-3') 序号 用途
PmTLS-3'-inner TATGGAGGCGGACATAGGGCG SEQIDNo.3 3'-RACE
PmTLS-3'-outer CATCGCAAAGTCGGGCGTAAG SEQIDNo.4 3'-RACE
PmTLS-5'-inner TGTCCGCCTCCATACATTTCTCC SEQIDNo.5 5'-RACE
PmTLS-5'-outer ATGACACCACGCCCACAAGG SEQIDNo.6 5'-RACE
M13-F CGCCAGGGTTTTCCCAGTCACGAC SEQIDNo.7 菌落PCR检测
M13-R GAGCGGATAACAATTTCACACAGG SEQIDNo.8 菌落PCR检测
PmTLS-RT-F AGAAATGTATGGAGGCGGAC SEQIDNo.9 荧光定量
PmTLS-RT-R ACCATAACAAGCATCTCCGAAT SEQIDNo.10 荧光定量
GAPDH-F GCAGATGGTGCCGAGTATGT SEQIDNo.11 内参基因
GAPDH-R CGTTGATTATCTTGGCGAGTG SEQIDNo.12 内参基因
3总RNA提取
(1)获取马氏珠母贝全组织样品，加Trizol并研磨各组织，依照Trizol法原理提取总RNA。
(2)1％琼脂糖凝胶电泳检测RNA完整性和质量。
(3)微量核酸定量仪测定总RNA的浓度与纯度。
(4)所获总RNA于-80℃超低温冰箱保存备用。
4 cDNA第一链的合成
依照Reverse Transcriptase M-MLV试剂盒操作制备所需模板。具体程序如下：
(1)RNA与引物结合，PCR程序为70℃，10min，冰上放置2～3min。加样体系如下：
表2 体系
试剂 剂量
总RNA 1ng～1μg
Oligo(dT)(50uM) 1μL
RNase-freewater 补加至8μL
(2)向上述RNA体系中加入以下试剂：
表3 试剂
试剂 剂量
5×M-MLV Buffer 2.0μL
dNTP Mixture(10mM) 0.5μL
RNase Inhibitor 0.25μL
RTase M-MLV(RNase H-) 0.25～1μL
RNase-free water 补至12μL
(3)离心混匀数秒，进行PCR，程序为：42℃，60min；70℃，15min。
5中间片段克隆
(1)PrimeSTARHS高保真酶扩增获得目的片段。PCR反应体系如下：
表4 PCR反应体系
试剂 体积
PrimeSTARHS 5μL
模板 0.4μL
上游引物 0.4μL
下游引物 0.4μL
ddH 2O 3.8μL
Total 10μL
采用3步法扩增。PCR反应程序如下：
Figure PCTCN2020132289-appb-000002
(2)凝胶检测及目的片段回收
吸1μL PCR产物用1％琼脂糖电泳检测，扩大目的片段体系，参照PCR产物回收试剂盒(Gene JET Gel ExtractionKit)说明书回收PCR产物。
(3)目的片段连接
将纯化后的PCR产物连接到pMD-19T Vector载体上(图1)。体系如下：
表5 体系
试剂 剂量
Solution I 5μL
目的片段 4.5μL
pMD19-T Vector 0.5μL
Total 10μL
连接程序为：16℃连接约16h。
(4)转化
将DH5α感受态细胞从超低温冰箱取出，放冰上至微溶，再将连接产物加入含100μL DH5α感受态细胞的管子中，轻轻吹打均匀；冰上放置30min，42℃热激60～90s，冰上放置2～3min；接着加入890μL提前37℃预热好的LB液体培养基，37℃200rpm振荡培养1h；然后4000rpm 2min，弃上清，留下约100μL，轻轻吹散悬浮细胞，并涂于LA(Amp+)固体平板上；最后，37℃先正置培养半小时后，再倒置培养过夜。
(5)菌落PCR检测
挑取单菌落接种于LA(含Amp+)液体培养基中，37℃，220rpm振荡培养6h左右。M13通用引物鉴定阳性克隆，经1％琼脂糖凝胶电泳检 测的阳性菌落送到广州生工测序。具体体系与程序如下：
Figure PCTCN2020132289-appb-000003
6 3′和5′端RACE扩增
参照SMARTTM RACE cDNA Amptification kit说明书制备3′和5′RACE的cDNA模板。巢式PCR以3′、5′特异性引物结合通用引物(NUP和UPM)扩增。
(1)第一轮RACE反应
表8 体系
试剂 体积
PrimeSTAR HS 5μL
3′/5′cDNA模板 0.4μL
UPM引物 0.4μL
Outer特异性引物 0.4μL
ddH 2O 3.8μL
Total 10μL
PCR反应程序：
Figure PCTCN2020132289-appb-000004
(2)第二轮RACE反应
表9 体系
试剂 体积
Premix Taq 5μL
3′/5′cDNA模板 0.4μL
NUP引物 0.4μL
Inner特异性引物 0.4μL
ddH 2O 3.8μL
Total 10μL
PCR反应程序：
Figure PCTCN2020132289-appb-000005
(3)PCR切胶回收/产物纯化，连接、转化以及菌落PCR检测参考5的步骤(2)～(5)。
7生物信息学分析
利用NCBI blastx进行序列同源性和相似性分析；DNAMAN 6.0软件序列查找、拼接和比对；ExPASy在线预测其理论分子量(MW)和等电点(pI)；采用SignalP 4.0 Server进行信号肽序列预测；TMHMM Server v.2.0分析序列的跨膜结构域；MEGA 6软件以NJ法构建系统进化树；SoftBerry Psite对其推导的氨基酸序列功能位点的预测；SOPMA预测蛋白质二级结构。
8组织表达及PAMPs刺激后时序表达分析
8.1贝的处理及样品采集
(1)组织定量：取10只规格一致、活力较好的贝，采集组织为性腺、鳃、外套膜、血淋巴、闭壳肌和肝胰腺。
(2)PAMPs刺激：取暂养1周健康的马氏珠母贝320只，随机分成4组：LPS刺激组(实验组)、PGN刺激组(实验组)、PolyI:C刺激组(实验组)、PBS组(对照组)，各80只贝。采用闭壳肌注射的方法，实验组每只注射100μL的LPS、PGN、PolyI:C(10μg/mL)，注射100μL PBS作为对照组。注射后的0、3、6、12、24、48、72、96h，各组分别随机抽取10只贝取全组织备用，其中血淋巴于4℃，800×g，离心15min后，舍弃上清，加1mL Trizol，轻轻摇荡使悬浮均匀，提取总RNA反转录成cDNA，用于后面的定量分析(注：PBS进行了灭菌处理，LPS、PGN、PolyI:C由灭菌的PBS配制；实验过程中，不喂饵料，且未出现死亡现象)。
8.2引物设计
荧光定量PCR反应中所用引物见表1。
8.3总RNA的提取
总RNA的提取方法同3中的步骤(1)～(4)。
8.4荧光定量cDNA模板的制备
依照Reverse Transcriptase M-MLV(RNase H)说明书操作。
(1)RNA与引物结合，PCR程序为70℃，10min，冰上放置2～3min。加样体系如下：
表10 加样体系
试剂 体积
总RNA 1ng～1μg
Random Primers(25uM) 1μL
RNase-free water 补加至8μL
(3)向上述RNA体系中加入以下试剂：
表11 试剂
试剂 剂量
5×M-MLV Buffer 2.0μL
dNTP Mixture(10mM) 0.5μL
RNase Inhibitor 0.25μL
RTase M-MLV(RNase H-) 0.25～1μL
RNase-free water 补至12μL
(3)离心混匀数秒，进行PC，程序为：42℃，60min；70℃，15min，所得的cDNA第一链于-20℃保存。
8.5荧光定量PCR
以8.4的反转录cDNA为模板，内参基因选择GAPDH。按3个实验重复设置加样孔。按照以下反应体系和反应条件实时荧光定量PCR：
表12 反应体系
Figure PCTCN2020132289-appb-000006
反应程序：
Figure PCTCN2020132289-appb-000007
8.6数据统计与分析
荧光定量的数据采用2-△△ct法进行分析。试验结果均采用平均值±标准差表示，用SPSS17.0软件进行单因素方差分析(one-way ANOVA)，并用Duncan’s多重比较对均值进行差异显著性检验。
9重组蛋白制备
9.1基因合成
根据已有的目的基因序列进行基因合成，以此作为后续亚克隆的模板，引入酶切位点为NcoI和HindIII。
9.2原核表达载体构建
(1)重组PmTLS ORF扩增，PCR体系为：10μL的Premix Ex Taq，0.5μL合成模板，Forward Primer和Reverse Primer各0.4μL，ddH 2O补足体系至20μL。程序为：94℃变性5min；98℃10s，55℃30s，72℃60s，30个循环。凝胶电泳检测反应产物。
(2)PmTLS与PmKuPI的克隆与测序见步骤5。
(3)质粒抽提参照Easy Pure Plasmid Mini Prep Kit硅胶膜离心柱质粒DNA小量抽提试剂盒说明书提取。
(4)用MluⅠ、XhoI和HindIII内切酶对空质粒pET-28a(+)和抽提的重组质粒进行双酶切。体系为5.0μL的10×FD Buffer，MluⅠ内切酶3.0μL，20μL的质粒，ddH 2O补足体系至50μL。轻微混匀，37℃保温2h。
(5)切胶纯化参照步骤2.2.5(2)。
(6)酶切纯化的回收产物通过T4连接酶的作用与pET-28a(+)载体连接构建重组表达质粒pET28a-PmTLS。连接体系为：回收的酶切纯化产物6μL，2μL的pET-28a(+)，10×T4 buffer为1μL，1μL T4连接酶，总体系为10μL。轻微混匀，16℃过夜。
(7)连接产物的转化入、阳性克隆筛选及测序参照5中的步骤(4)～(5)。
(8)质粒提取参照9.2的步骤(3)，即获得pET28a-PmTLS重组质粒，-20℃保存备用。
9.3重组融合蛋白的诱导表达
(1)转化、筛选阳性克隆
从超低温冰箱取出感受态细胞BL21(DE3)，先冰解；将100ng质粒DNA加入BL21(DE3)菌株并轻轻混合，试管在冰上孵育30min，震 动管在42℃加热90s，不要摇晃，将试管放在冰上放置3min，加入100μL室温LB培养基。摇动并在37℃下于200rpm孵育管60min，在含50μg/mL卡那霉素的LB琼脂平板上铺展，将板倒置在37℃下孵育过夜。筛选阳性克隆并测序。
(2)小试培养选择最佳的诱导条件
挑选三个分离良好的单个菌落，分别接种到含有50μg/mL卡那霉素的4mL LB培养基中，将细胞在37℃的摇床上以200rpm摇动孵育，当OD600值达到0.6～0.8时，在三根管中的两根加入IPTG，最后浓度为0.5mM的IPTG分别在15℃诱导16h和37℃诱导4h，第三根试管作为阴性对照；
(3)重组蛋白制备与检测
①从450μL培养物中收获细胞沉淀，用超声仪裂解1分钟。
②全细胞裂解液：将50μL 5×上样缓冲液与100μL细胞裂解液混合，作为全细胞裂解液的样品，在100℃下加热样品10min，然后以15,000rpm离心5min。
③细胞裂解液的上清液和碎片：将剩余的200μL细胞裂解液以15,000rpm离心10min，收集细胞裂解液的上清液和细胞碎片，分别将90μL5×上样缓冲液与180μL上清液混合作为样品。细胞裂解液的上清液。用150μL 5×上样缓冲液重悬所有沉淀，作为细胞裂解液碎片的样品。将样品在100℃下加热10min，并在上样之前以15,000rpm离心5min。
④使用SDS-PAGE和Western blot检测蛋白的表达和溶解度，最后大量诱导表达融合蛋白。
9.4融合蛋白的分离纯化
(1)超声破碎菌体
①将收集的细菌菌体用Buffer(50mM Tris，150mM NaCl，5％甘油pH 8.0)溶解，冰浴中超声破碎菌体，功率350W，30min(超声4s，暂停6s为一个循环)。
②超声完毕，12,000rpm，4℃，离心20min，弃上清，沉淀用破碎Buffer溶解，冰浴中超声破碎，功率350W，30min(超声4s，暂停6s为一个循环)。
③超声完毕，12,000rpm，4℃，离心20min，收集上清进行下一步纯化。
(2)镍琼脂糖亲和层析
①取5mL Ni-NTA，用5倍柱床体积的Binding buffer清洗平衡柱子，流速5mL/min。
②填料和样品孵育1h后上柱，收集穿透液。
③5倍柱床体积的Binding buffer清洗柱子，流速5m L/min。
④Wash buffer洗杂，流速5mL/min，收集洗脱液。
⑤Elution buffer洗脱，流速2mL/min，收集洗脱液。
⑥收集样品SDS-PAGE检测
(3)纯化蛋白SDS-PAGE检测
准备12％的SDS-PAGE，Tris-Gly电泳缓冲液，浓缩胶80V，20min，分离胶120V，60min，凝胶电泳结束后进行考马斯亮蓝染色20min，脱色过夜。
(4)Western blot检测
①制胶：制备聚丙烯酰胺凝胶：浓缩胶5％，分离胶12％
②制样：上样。
③电泳：浓缩胶80V，30min；分离胶120V，60min。
④转膜：湿转，250mA，90min。
⑤封闭：5％的脱脂奶粉，37℃缓慢振荡2h。
⑥孵育一抗：一抗为兔抗his标签，抗体公司：Sangon Biotech，编号：D110002，1:500稀释，37℃缓慢振荡60min。
⑦孵育二抗：二抗为羊抗兔，抗体公司：Sangon Biotech，编号：D110058，1:8000稀释，37℃缓慢振荡60min。
⑧显色：TMB显色。
(5)包涵体蛋白复性
将纯化后的目的蛋白进行透析复性，并最终置换至可溶性的缓冲液中(PBS，10％甘油，1ML-精氨酸,pH 7.4)，并运用SDS-PAGE和Western blot检测对比纯化蛋白复性前(沉淀)和复性后(溶解)的纯度。
10纯化蛋白抗菌活性检测
纯化的蛋白对大肠杆菌，藤黄微球菌，嗜水气单胞菌，铜绿假单胞菌，副溶血弧菌，哈维氏弧菌，枯草芽孢杆菌和金黄色葡萄球菌的抗菌活性。首先，将每种细菌分别在2216E液体培养基中培养至对数生长期。然后将细菌离心(3,000×g，10min)，用磷酸盐缓冲盐水(1×PBS)洗涤3次，然后重悬于PBS中。接下来，用96孔酶标板将50μL的纯化蛋白(200μg/mL)与10μL的每种细菌悬液分别混合，并在室温下孵育2h。PBS用作阴性对照。然后在每个混合孔中加入140μL培养基，在37℃恒温培养箱孵育培养，同时使用酶标仪(EnSpire，PerkinElmer)测定在添加纯化蛋白和PBS后的0、1、2、3、4、5、6、7、8、9、10、11和12小时测量OD600值。每个样品平行进行三个实验。
11透射电镜观察纯化蛋白作用细菌后的形态变化
在37℃下用200μL的纯化蛋白处理200μL的处于指数生长期的细菌2h。PBS代替作为对照。之后在室温下先将纯化蛋白与细菌混合物以3000rpm离心10min进行沉淀，再用PBS洗涤3次去除杂质，然后将细菌沉淀用200μL 3％戊二醛中固定过夜。处理后，将200μL2％磷钨酸钠水溶液添加到细菌悬浮液中，然后将其滴在铜网上。使用滤纸除去残留的水，5min后，将样品风干。在标准操作条件下用JEM-1400(日本电子公司)显微镜进行观察。
12结果
12.1 PmTLS的基因克隆
通过RACE技术克隆得到PmTLS基因全长(SEQ ID No.1)为778bp，其中包含420bp的5′UTR，97bp的3′UTR，261bp的ORF框，共编码86个氨基酸(SEQ ID No.2)。
12.2 PmTLS理化性质分析
PmTLS蛋白酶的分子量为9.5KDa，理论等电点为4.22，预测其亲水性，发现在第51和52位出现连续的最高疏水性，其指数为2.011，在第63位出现最高亲水性，其指数为-2.156，总的平均亲水性系数为-0.172，为亲水性蛋白。带负电荷的残基总数(Asp+Glu)为9，带正电荷的残基总数(Arg+Lys)为3，因此，PmTLS蛋白酶总体带负电荷。跨膜结构预测发现其无跨膜域。SOPMA软件预测二级结构显示，无规则卷曲占整体的39.53％，α-螺旋结构29.07％，延伸链22.09％，β-转角9.30％。SMART分析结果表明，PmTLS氨基酸序列在第1-77位形成一个Trypsin域(图 2)。
12.3 PmTLS同源性分析
通过DNAMAN将PmTLS氨基酸序列与其它无脊椎动物的相应氨基酸进行多序列比对，分析发现PmTLS与长牡蛎的相似性最高，达到69.77％，各物种间的一致相似性达到41.42％(图3)。
使用MEGA 6构建系统进化树，在PmTLS的进化树分类中发现无脊椎动物聚为一大支，脊椎动物聚为一大支，其中马氏珠母贝与长牡蛎的亲缘关系最近(图4)。
12.4 PmTLS基因mRNA的组织定量分析
利用Real-time PCR检测PmTLS蛋白酶基因mRNA在马氏珠母贝血细胞等6个组织中的表达模式情况。结果发现：PmTLS蛋白酶基因在血细胞中的表达水平最高(p<0.01)，其次是性腺；在外套膜和闭壳肌中的表达量最低，几乎不表达(图5)。
12.5 PAMPs刺激后PmTLS基因mRNA在血细胞的时序表达分析
选择LPS(脂多糖)、PGN(肽聚糖)和PolyI:C(聚肌胞苷酸)等病原相关分子模式(PAMPs)激活马氏珠母贝先天免疫反应，Real-time PCR检测不同时间PmTLS mRNA在马氏珠母贝血淋巴中的时序变化情况。结果发现：与对照组相相比，LPS刺激后，PmTLS基因表达量在3h时上升，随后至24h都处于正常状态，而到48h时突然上升达到最大值(P<0.05)，在72h略微下降，到96h接近正常水平(图6)；PGN刺激后PmTLS基因表达量在24h内无变化，到48h直接达到最大表达水平(P<0.05)，在48h开始下降，96h时又出现上升趋势(图7)；PolyI:C刺激后，PmTLS基因表达量从24h开始上升，并在48h时表达量达到最高(P<0.05)，72h即恢复正常水平(图8)。
12.6 PmTLS重组蛋白的表达
(1)PmTLS原核表达载体的构建
根据已有的PmTLS基因全长cDNA序列，预测开放阅读框ORF并进行分析，获得需要重组的ORF序列，在重组ORF序列中设计添加组氨酸基因序列，然后进行基因合成，再设计原核表达引物。PCR扩增后得到PmTLS基因片段约290bp；随后将PmTLS基因与pMD19-T载体进行连接、转化并挑取单菌落，通过菌落PCR鉴定出阳性克隆的菌落，利用生工质粒抽提试剂盒进行质粒的抽提，然后MluI内切酶消化，并将PmTLS与pET-28a(+)载体相连接、转化并通过菌落PCR鉴定阳性克隆菌落，MluI消化后的PmTLS基因片段约为900bp。随后对有阳性克隆结果的进行质粒抽提，抽提获得的质粒即为pET28-PmTLS的重组质粒，重组蛋白预计分子量分别为10.32KDa。菌落PCR鉴定、MluI消化鉴定(图9)及测序的结果都验证表明pET28-PmTLS载体构建成功。
(2)PmTLS重组蛋白(rPmTLS)的诱导表达
将重组表达载体pET28-PmTLS转化至大肠杆菌，用0.5mM的IPTG，分别在15℃诱导16h和37℃诱导4h，其中阴性对照为不加IPTG诱导。从图9可知，阴性对照组泳道NC(全菌)、NC1(上清)和NC2(沉淀)未出现目的条带；同时15℃和37℃的上清泳道3和4中也未见目的条带；在15℃的泳道1(全菌)和5(沉淀)中也未见目的条带；而37℃的泳道2(全菌)和6(沉淀)中出现清晰的目的条带(图10)。用Western blot进一步验证，结果显示，在37℃诱导的泳道2(全菌)和6(沉淀)中约10KDa处出现清晰单一染色条带，与理论值10.32KDa一致，表明成功 获得了纯度较高的重组蛋白(图11)。结果表明了rPmTLS诱导的最佳温度是37℃，并且主要以包涵体形式表达。
为了获得大量融合蛋白，以1L的表达量培养pET28-PmTLS的表达菌，在0.5mM IPTG，37℃下诱导4h。结果显示，在约10KDa处出现一条清晰单一的条带，表明成功获得了纯度较高的rPmTLS，并且复性前后重组蛋白分子量并未变化，说明复性后的纯度较高(图12)。通过Bradford蛋白测定法测定rPmTLS浓度，其浓度为1.07mg/mL，共获得rPmTLS 8.02mg蛋白。
12.7 rPmTLS的抗菌活性测定
在细菌液体生长曲线抑制实验中，总共测定了8种细菌，其中3种为革兰氏阳性菌，5种为革兰氏阴性菌，rPmTLS蛋白质显著抑制了4种革兰氏阴性菌种的生长(P<0.05)，分别是铜绿假单胞菌、嗜水气单胞菌、副溶血弧菌和哈维氏弧菌(图13-16)，但对革兰氏阳性菌的生长几乎没有影响。
12.8 rPmTLS抗菌作用机制
为了了解马氏珠母贝的TLS的抗菌作用机制，将rPmTLS溶液分别与铜绿假单胞菌、嗜水气单胞菌和副溶血弧菌一起反应2h，滴网，在透射电子显微镜下镜检。结果如图17所示，从图中可以看出，铜绿假单胞菌的对照组(图17中的A)边缘清晰完整，内含物质致密状且均匀；而实验组(图17中的B)可观察到铜绿假单胞菌的菌体膨胀，内含物质收缩，内部结构和对照组相比也发生明显改变。同时，细胞壁似乎开始溶解，细胞质与细胞壁脱离，出现质壁分离的现象(图17中的C-D)，表明其细胞壁发生了崩解导致细胞局部内容物的释放。图17中的E为嗜水气单胞菌的对照组，其菌体细长，边缘清晰，内含物均匀；而实验组(图17中的F)可观察到嗜水气单胞菌内含物质部分丢失，内部结构发生变化，将其两端放大再观察，发现菌体边缘非常不规整，细胞壁出现溶解(图17中的G-H)。图17中的I为副溶血弧菌的对照组，菌体为短杆状，均质饱满；而实验组(图17中的J)内含物缺失，菌体中间膨胀，进一步放大再观察，发现细胞质与细胞壁脱离，出现质壁分离的现象(图17中的K)。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (4)

  1. 一种胰蛋白酶样丝氨酸蛋白酶基因，其特征在于，所述胰蛋白酶样丝氨酸蛋白酶基因的核苷酸序列如SEQ ID No.1所示。
  2. 权利要求1所述的胰蛋白酶样丝氨酸蛋白酶基因编码的蛋白质，其特征在于，所述蛋白质的氨基酸序列如SEQ ID No.2所示。
  3. 权利要求2所述的蛋白质在制备抑制微生物的药物中的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述微生物包括铜绿假单胞菌、嗜水气单胞菌、副溶血弧菌和哈维氏弧菌中的一种或几种。
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