Long oyster CgVg functional domain DUF1943 and VWD recombinant protein and its application

Abstract

The present invention belongs to the field of molecular biology technology, and in particular relates to a recombinant protein of the CgVg functional domain DUF1943 and VWD of the long oyster, as well as a preparation method and application thereof. The amino acid sequence of the recombinant protein of the CgVg functional domain DUF1943 of the long oyster is shown in SEQ ID NO.1. The amino acid sequence of the recombinant protein of the CgVg functional domain VWD of the long oyster is shown in SEQ ID NO.2. The recombinant proteins of the CgVg functional domain DUF1943 and VWD of the long oyster obtained by the present invention have a wide range of binding activity against microorganisms, among which the DUF1943 recombinant protein has the activity of agglutinating bacteria and fungi, and the VWD recombinant protein has a broad spectrum of activity in inhibiting bacterial growth, and can be applied to the research and development of antibacterial drugs, immunopotentiators and feed additives for marine organisms.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a recombinant protein of the CgVg functional domain DUF1943 and VWD of a long oyster, and a preparation method and application thereof. Background Art

[0002] Vitellogenin (Vg) is a high molecular weight protein rich in sugar, phosphorus, lipids, etc. It belongs to the large lipid transfer protein superfamily (LLTP) together with apolipoprotein B (ApoB) and microsomal triglyceride transfer protein (MTTP). The main function of Vg is to provide the main nutrients for the growth and development of embryos and larvae. In recent years, Vg has been shown to have immune-related functions in fish, crustaceans and mollusks. It can be used as a type of pattern recognition molecule that can specifically identify bacteria and as an opsonin to enhance the phagocytic ability of macrophages. In addition, Vg also has inhibitory and killing effects on bacteria. Vg mainly relies on its structural domain to exert its function, which mainly contains three conserved functional domains, namely, Vitellogenin at the N-terminus, - N, a domain of unknown function (DUF1943), and a VWD domain located at the C-terminus. While the functional role and mechanisms of Vg in vertebrate immune defense are well understood, research in invertebrates, particularly mollusks, is relatively lacking. There are no reports on the preparation of recombinant proteins containing the DUF1943 and VWD domains of the long oyster Cg, or their use as agglutination and antibacterial agents. Summary of the Invention

[0003] The present invention aims to solve the above-mentioned problems by utilizing the existing technology, and provides a preparation method and application of a long oyster CgVg functional domain DUF1943 and VWD recombinant protein.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] A long oyster CgVg functional domain DUF1943, the long oyster CgVg functional domain DUF1943 recombinant protein amino acid sequence is shown in SEQ ID NO.1.

[0006] Method for constructing the functional domain DUF1943 of the long oyster CgVg:

[0007] a. PCR amplification of the coding region of the oyster CgVg functional domain DUF1943 using specific primer pair P1;

[0008] Primer pair P1

[0009] 5'-CGGGGTACCCAAATGTCCAACAGTACTTAC-3';

[0010] 5'-CCCAAGCTTTTCTCTTGGCGAAGGATA-3';

[0011] b. The PCR amplification product was digested with Kpn I and Hind III and then ligated with T4 ligase, transformed, and the recombinants were identified by sequencing;

[0012] c. The recombinant was transformed into an Escherichia coli Transetta (DE3) expression strain for induction culture, followed by purification and renaturation to obtain a recombinant protein of the long oyster CgVg functional domain DUF1943.

[0013] A recombinant protein of the long oyster CgVg functional domain VWD, the amino acid sequence of the recombinant protein of the long oyster CgVg functional domain VWD is shown in SEQ ID NO.2.

[0014] The method for constructing the recombinant protein of the long oyster CgVg functional domain VWD:

[0015] a. PCR amplification of the coding region of the VWD domain of the long oyster CgVg using specific primer pair P2;

[0016] Primer pair P2

[0017] 5'-CGGGGTACCGACCTCAGGAGAAACAACAGA-3';

[0018] 5'-CCCAAGCTTTGGTTTACACTGACTCGGACA-3';

[0019] b. The PCR amplification product was digested with Kpn I and Hind III and then ligated with T4 ligase, transformed, and the recombinants were identified by sequencing;

[0020] c. The recombinant was transformed into an Escherichia coli Transetta (DE3) expression strain for induction culture, followed by purification and renaturation to obtain a recombinant protein of the long oyster CgVg functional domain VWD.

[0021] An application of the recombinant protein, wherein the long oyster CgVg functional domain DUF1943 or VWD recombinant protein is used in the preparation of marine antibacterial preparations, immunopotentiators or feed additives.

[0022] The long oyster CgVg functional domain DUF1943 and VWD recombinant protein have binding activity with a variety of microorganisms;

[0023] The long oyster CgVg functional domain DUF1943 and VWD recombinant protein interact with bacteria through LTA and LPS;

[0024] The long oyster CgVg functional domain DUF1943 and VWD recombinant protein have agglutination effects on various microorganisms;

[0025] The long oyster CgVg functional domain DUF1943 and VWD recombinant protein have an inhibitory effect on bacterial growth.

[0026] The advantages of the present invention are:

[0027] The present invention uses in vitro recombinant expression technology to obtain recombinant proteins of the long oyster vitellogenin CgVg functional domain DUF1943 and VWD. The recombinant proteins rDUF1943 and rVWD have a wide range of binding activity against a variety of microorganisms such as Staphylococcus aureus (S.aureus), Micrococcus luteus (M.luteus), Escherichia coli (E.coli), Vibrio splendidus (V.splendidus) and Pichia pastoris (P.pastoris). In addition, the recombinant protein rDUF1943 also has a strong agglutination effect on the above five microorganisms; and the recombinant protein rVWD has a significant inhibitory effect on the growth of Staphylococcus aureus (S.aureus), Escherichia coli (E.coli) and Vibrio splendidus (V.splendidus). The long oyster vitellogenin CgVg functional domain DUF1943 and VWD of the present invention are effective immune recognition and immune effector molecules, and have application value in the preparation of antibacterial drugs, new immune preparations and feed additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a purification diagram of the long oyster CgVg functional domain DUF1943 and VWD recombinant protein provided in the embodiments of the present invention.

[0029] Figure 2 This is a diagram showing the detection effect of the binding activity of the long oyster CgVg functional domain DUF1943 and VWD recombinant protein with microorganisms in an embodiment of the present invention.

[0030] Figure 3 This is a diagram showing the detection effect of the binding activity of the long oyster CgVg functional domain DUF1943 and VWD recombinant protein to LTA and LPS in an embodiment of the present invention.

[0031] Figure 4 This is a diagram showing the effect of detecting the microbial agglutination activity of the long oyster CgVg functional domain DUF1943 and VWD recombinant protein in an embodiment of the present invention.

[0032] Figure 5This is a diagram showing the effect of inhibiting bacterial growth (growth curve method) by the long oyster CgVg functional domain DUF1943 and VWD recombinant protein in an embodiment of the present invention. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are further described below with reference to examples. It should be noted that the specific embodiments described here are only for illustrating and explaining the present invention, and are not intended to limit the present invention.

[0034] The present invention uses in vitro recombination technology to express and purify the long oyster CgVg functional domain DUF1943 and VWD recombinant proteins, which have binding activity to multiple pathogenic microorganisms and can inhibit their growth, and also have an agglutination effect on pathogenic microorganisms. They can be used as preparations to replace antibiotics and are applied to the research and development of antibacterial drugs, immunopotentiators and feed additives.

[0035] Experimental Example 1: In vitro prokaryotic expression and purification of the long oyster CgVg functional domain DUF1943 and VWD recombinant proteins

[0036] 1. Construction of recombinant vector

[0037] The recombinant vector used in the present embodiment is the Novagen pET-30a(+) prokaryotic expression vector. PCR was used to amplify the oyster CgVg functional domain DUF1943 or VWD using primer pairs P1 or P2 with Kpn I and Hind III restriction enzyme sites added to the 5' and 3' ends, respectively.

[0038] The PCR reaction conditions were as follows: first, pre-denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 2 min, with a final extension at 72°C for 10 min. The amplified fragments were purified and recovered by agarose gel electrophoresis, and the two recovered fragments were ligated into the pMD19-T vector respectively. After transformation, positive clones were screened, plasmids were extracted, and the plasmids were double-digested with Kpn I and Hind III. The recovered target fragments were ligated into the expression vector pET-30a(+) digested with Kpn I and Hind III to complete the construction of the recombinant plasmids.

[0039] 2. Expression of the Long Oyster CgVg Functional Domain DUF1943 and VWD Recombinant Protein

[0040] The constructed different recombinant plasmids were transformed into Escherichia coli Transetta (DE3) and single clones were picked and inoculated into 200 mL LB liquid medium and cultured at 150 rpm and 37 °C until the logarithmic growth phase, i.e., OD 600=0.4-0.6; IPTG (final concentration 1 mmol / L) was added, and the cells were cultured at 16°C for 12 h, then centrifuged at 10,000 rpm for 10 min at 4°C to collect the cells and freeze them at -80°C for later use; at the same time, 1 mL of the bacterial solution was centrifuged, the supernatant was discarded, 80 μL of water and 20 μL of 5× loading buffer were added, and the cells were boiled at 99°C for 10 min, centrifuged briefly, and the expression products were detected by SDS-PAGE.

[0041] 3. Purification and refolding of the long oyster CgVg functional domain DUF1943 and VWD recombinant protein

[0042] The different expression products obtained above were purified using nickel-agarose gel FF columns to obtain denatured recombinant proteins, which were then dialyzed with dialysis buffer for refolding. The specific steps are as follows:

[0043] (1) Nickel Sepharose FF column, 1.6 × 20 cm, bed volume 10 mL;

[0044] (2) Equilibrate 2–5 bed volumes with buffer I (50 mM / L Tris-HCl buffer, pH 7.4, 50 mM / L NaCl, 8 mol / L urea) at a flow rate of 2 mL / min;

[0045] (3) The IPTG-induced expression cells were resuspended in buffer I, ultrasonically disrupted at 150W for 30 min, and centrifuged at 12,000 × g at 4°C for 30 min. The supernatant was filtered through a 0.45 μM filter membrane and passed through a column at a flow rate of 1 mL / min.

[0046] (4) Wash again with buffer I for 2 to 5 bed volumes at a flow rate of 2 mL / min;

[0047] (5) Wash again with 50 mM / L imidazole buffer I for 2–5 column volumes at a flow rate of 2 mL / min;

[0048] (6) Elute the target protein with buffer I containing 400 mM / L imidazole and collect;

[0049] (7) Detect fusion protein expression using SDS-PAGE;

[0050] (8) Wash 5 column volumes with pure water, and then wash 3 column volumes with 20% ethanol at a flow rate of 2 mL / min. Store the column at 4°C. Purify the recombinant protein in a denatured state by dialyzing in a refolding buffer to remove urea so that the protein can be properly refolded and restored to the correct conformation. The denatured purified product was dialyzed against 2 mM reduced glutathione, 0.4 mM oxidized glutathione, 1 mM EDTA, 50 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 1% glycine and a gradient of urea for refolding. The urea concentration started at 6 M and was gradually replaced with 4 M, 3 M, 2 M, 1 M, and 0 M. No glycerol was added when the dialysate was dialyzed to no urea for the last time. Each dialyzation was performed at 4°C for 12 h to obtain the recombinant protein of the long oyster CgVg functional domain DUF1943 or VWD. The results are shown in FIG. Figure 1 shown.

[0051] Experimental results:

[0052] The results of SDS-PAGE showed that the molecular weight of the purified recombinant protein of the oyster CgVg functional domain DUF1943 was approximately 44 kDa, which was consistent with the predicted molecular weight ( Figure 1 A) The molecular weight of the purified long oyster CgVg functional domain VWD recombinant protein is approximately 39 kDa, which is consistent with the predicted molecular weight.

[0053] CgVg:

[0054] MLLLLAVSTALVAAAAQTPSQAYESNKEYLYEYETQALTGIPMGSTIYS

[0055] GMKMKSDVRIQFRSRSSATLKMDKLTFAKINDPIESVDPTQQQVPAEM

[0056] FQPLTGRDAEQMLTDLSRPINFRYVRGNVKDIHHEADDPEWSVNVKKG

[0057] LLSMLEMNLEKRKELRKSSAIPQVLRPQSSDEGSMFTVMEPSIVGECET

[0058] LYRISPWTSTSANPWMHITKVRNYHHCLDRPKYFASMFHLRQCAECVR

[0059] EQSEPLRSASQIRYTLRGNMRQFQIQSAIAESQHVFTPYSAKGGHVATY

[0060] LNQTLNLVKEEDVKTSLSEPQSPTKVKSGLQYTSRELELRDSSSILQQSS

[0061] NSLSTDKSSPESPQHSSSSNHHPSPQPPSPQPKTTSNQAADRIRKLLKML

[0062] EVFMKPTIQPEAGPLLMSLLEEIRTADSESLRAVYREMFQTAKTDSKQF

[0063] KMLMDIIPSAGTTSATEVLVDSILNDQLPQSEAVVALGILSLSARPDVLI

[0064] AKKLLDLTRSSQVSKDRYLKRAAFLCLGSVAGMLREEGWRRSREISRQ

[0065] EEIVKTLISQESRSQTRNELKSKKRELEDLKKREHSINTKIKQEIVKELQ

[0066] MLMRSTVFDDKILSFKTIGNAGLWEMIPTIRTYIQDKSQPQMLRTQAIY

[0067] SLRKLARHYPDDIQTTLLPLYFDQSEKEEVRIGSYLMMTFTEPSRQLLE

[0068] MVAQSLHRERNPHVGTFVYTHLEQMSNSTYPCLMSWAKNASFAMRF

[0069] AKKFSPMYHYSRFMHLSGFNEMHKMGAAAELGLVTTPEEFIPRAGAV

[0070] NLHTYVFGRSVNFAEIGFNTEGLQTLVSKLVGPLGELTKGKSLVDVLK

[0071] QRVRRSAESSQPSDSISQIHKQLKVSPRTSPTPKGHMYMKMMGNELQYI

[0072] TLDGTLVDTLLREGKLLSGVSEQDLKTGLNVEVHRSTMPLDAEIMIPSE

[0073] CGLPLRLKLHGTAAIKVTGKVGVTGMPSIFEINRPGKQAKELSFNFELR

[0074] PSVLFQLRGEMEMDAEYFKMGVALKTMAHMEAPLSLTASANLPKAKF

[0075] YTKFNVEKLSEKIARLEVSPYTYFKEDPSEITKYPSPRETQEISVAKNAK

[0076] VFPMSMAYGKQTLGLEIKLSGQAVVRDFEMDVPYYPMIGKQEMIVTLS

[0077] PGTDPQKYVEIQFQIMKRMSQSKQQPTSEESSSSTGLLSWVSSLIGSDQS

[0078] DQSSPSSSSSSSTSPPKDIRSKDTSLKDLLEHLKESQSVHPDGSVVTRNNS

[0079] MGLIFNVIGIDQSRSIKRHFHISMAAGMNPSSKTTSILLRMNRSPIPTMET

[0080] KPWNMDVAVNVNLPSRLADPSELLSDAYQRELEQQIEVYVRRHGESD

[0081] YHRWIMLEEPRWESSVLQRLLSLSDTQEVIDEVMNSELSKSEESGTPED

[0082] RNIIDLIRKQKQILKEARKIWQRSYDSLQVQDIKAKLSSLIKRSESVRKD

[0083] LEKDSASVSKSEKSDALRKYCALLQQLIDQRLDEAVRSSRRSLSSKERQ

[0084] DIAKSLESSKESLRKILQQQHRSPSKVDSSNSPPSSVQIDSSSVSESDSEEI

[0085] KESKKPEQKDKSQQQQQPSQQSQDTVSSNQPSRVSSSSSLSKSVSDLLQ

[0086] KQKDVVQDISRSKESEGLKSVDEKKIKEALLLAKEVTEKIRQENSGPSP

[0087] QNIERERQRLMKSVIQQQQIMKCLEQRVKKPQTASGKSEDQIKEIQRIM

[0088] QEIKQENNRLSKIAATSSQQKREKEEATIRHSQSEQDTLGRLRQVHMSQ

[0089] IIAAQRLAVEEQGSKTDQSKQIQQIREVMNTLGQTEIQIMSIVSQKSVAE

[0090] HPVDVVRKLVEVSHMQHSLLGLVAECARDESVRSVSRSEAKDSEIETL

[0091] KKRAESKQVDCLQMLRSIMTSRSLKRTVSSLSGSERQSLKRLHGEVEDI

[0092] RSKQIEIEKNVRKIDQDKEKSDKKPDSATIKDLVSKSKAVQDKVQKIES

[0093] DIRSLKLHQKVADTTIEPQQRIEVQKNIVEKIKKMIPDSSMSSQEKESAK

[0094] VSVTSQLSKLEEVKRKLQELKREESQEKVASPNQSSRQIQSPVTSPPSSRI

[0095] QNLEPSARGELRAETGREVCISIKAEYGLEGEKKKLFDVQILGTPSLEQI

[0096] LWERSQRSPLERSSITKRYLERKHNGSEEVTSDYLRLRLEMNMIRHLHV

[0097] RAHWEKNELPRVLEEYVWRTHEAFKAMMYGQMYTEYPHSFRPKDRL

[0098] EILLDLRRNNRQYDTELKLPEESNFIRGMWMPYFGPFMFESVQSIRDGV

[0099] HRSFPESTSSITSRLPGQCEMKHSQIRTMDGKEYSIPDTKECEVVMAMD

[0100] CSSSKHFAIKTKMDSSDSTKRVVEVHIEGKKLEIVPVNSSLVVRVDGHI

[0101] RQVPEESSITISQSTGSIPSTIKVSRSQRIVSVLASQKGVRVSSDGRKVSL

[0102] KITPFYYSHVCGLCGNFDGKQGNEFQSPSRTDRSDSSCLVLDYLVPDSK

[0103] CDSQSIRKECQQPHTSSSGCQLESKTIRRTRLHKGESQLCLSQEPVKSCP

[0104] SQCKPVDPKSTPVRMACFPHDSAKAKELERDSFKRPLDLKAQQADYTE

[0105] YVQVPRSCGEM

[0106] DUF1943 shown in SEQ ID NO.1:

[0107] KMGAAAELGLVTTPEEFIPRAGAVNLHTYVFGRSVNFAEIGFNTEGLQTLVSKLVGPLGELTKGKSLVDVLKQRVRRSAESSQPSDSISQIHKQLKVSPRTSPTPKGHMYMKMMGNELQYITLDGTLVDTLLREGKLLSGVSEQDLKTGLNVEVHRSTMPLDAEIMIPSECGLPLRLKLHGTAAIKVTGKVGVTGMPSIFEINRPGKQAKELSFNFELRPSVLFQLRGEMEMDAEYFKMGVALKTMAHMEAPLSLTASANLPKAKFYTKFNVEKLSEKIARLEVSPYTY

[0108] VWD shown in SEQ ID NO.2:

[0109] SITSRLPGQCEMKHSQIRTMDGKEYSIPDTKECEVVMAMDCSSSKHFAIKTKMDSSSDSTKRVVEVHIEGKKLEIVPVNSSLVVRVDGHIRQVPEESSITISQSTGSIPSTIKVSRS QRIVSVLASQKGVRVSSDGRKVSLKITPFYYSHVCGLCGNFDGKQGNEFQSPSRTDRSDSSCLVLDYLVPDSKCDSQSIRKECQQPHTSSSGCQLESKTIRRTRLHKGESQLCLSQ

[0110] Primer pair P1 (for amplification of the functional domain of DUF1943)

[0111] 5'-CGGGGTACCCAAATGTCCAACAGTACTTAC-3';

[0112] 5'-CCCAAGCTTTTCTCTTGGCGAAGGATA-3'.

[0113] Primer pair P2 (for amplification of VWD functional domains)

[0114] 5'-CGGGGTACCGACCTCAGGAGAAACAACAGA-3';

[0115] 5'-CCCAAGCTTTGGTTTACACTGACTCGGACA-3'.

[0116] Experimental Example 2: Detection of the binding activity of the long oyster CgVg functional domain DUF1943 and VWD recombinant protein of the present invention with microorganisms

[0117] Western blotting was used to test the binding activity of the recombinant proteins of the CgVg domain DUF1943 and VWD of Crassostrea gigas against Gram-negative bacteria (Vibrio splendidus and Escherichia coli), Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus), and fungi (Pichia pastoris). The strains used were as follows: Vibrio splendidus was purchased from the Beijing Center for Microbial Culture Collection; Escherichia coli was purchased from Beijing Quanshijin Company; Staphylococcus aureus was purchased from the Beijing Center for Microbial Culture Collection; Micrococcus luteus was purchased from the Beijing Center for Microbial Culture Collection; and Pichia pastoris was purchased from the Beijing Center for Microbial Culture Collection.

[0118] The specific operations are as follows:

[0119] (1) The above five microorganisms were cultured overnight: Micrococcus luteus and Escherichia coli were cultured in LB medium at 37°C for 24 h, Staphylococcus aureus was cultured in LB medium at 28°C for 24 h, Vibrio splendens was cultured in 2216E medium at 28°C for 24 h, and Pichia pastoris was cultured in YBD medium at 28°C for 24 h.

[0120] (2) Collect the cells by centrifugation, wash them three times with TBS buffer and resuspend them to adjust the bacterial concentration to 1×10 8 CFU / mL;

[0121] (3) 100 μL of the microbial suspension was mixed with an equal volume of the long oyster CgVg functional domain DUF1943 or VWD recombinant protein obtained in the above example, and incubated with rotation at room temperature for 30 min;

[0122] (4) Collect the cells by centrifugation at 10,000 rpm for 2 min and wash them three times with TBS buffer;

[0123] (5) After washing, collect the cells and resuspend them in 80 μL TBS buffer;

[0124] (6) Add 20 μL of 5× protein electrophoresis buffer, heat at 99°C for 10 min, and separate the protein samples by 12% SDS-PAGE electrophoresis;

[0125] (7) After electrophoresis is completed, remove the gel and cut NC membranes and filter paper of the same size and immerse them in electrophoresis buffer and let them stand for 10 minutes;

[0126] (8) Place the filter paper, NC membrane, gel, and filter paper into the electroporator in order from top to bottom. Set the current according to the size of the gel block and transfer the membrane for 50 minutes.

[0127] (9) Remove the NC membrane and wash it three times with TBST buffer, each time for 5 minutes;

[0128] (10) Wash three times with TBST buffer, 5 min each time;

[0129] (11) Place the NC membrane in 5% skim milk powder (dissolved in TBST) and block at room temperature for 2 h;

[0130] (12) Remove the NC membrane and wash it three times with TBST buffer, each time for 5 min;

[0131] (13) The NC membrane was immersed in a proportionally diluted His-tagged monoclonal antibody solution (purchased from Shanghai Bioengineering) (5% skim milk powder, TBST buffer) and incubated at room temperature for 1 h;

[0132] (14) Remove the NC membrane and wash it three times with TBST buffer, each time for 5 min;

[0133] (15) The NC membrane was placed in a proportionally diluted goat anti-mouse HRP secondary antibody solution (purchased from Shanghai Biotechnology) (5% skim milk powder in TBST buffer) and incubated at room temperature for 1 h.

[0134] (16) Remove the NC membrane and wash it three times with TBST buffer, each time for 10 min;

[0135] (17) ECL method was used for development, and Western blotting results were recorded by imaging instrument, such as Figure 2 shown.

[0136] Experimental results:

[0137] The results showed that the recombinant proteins of the oyster CgVg functional domain DUF1943 and VWD of the present invention exhibited varying degrees of binding activity against Gram-negative bacteria, Gram-positive bacteria, and fungi. Both rDUF1943 and rVWD exhibited strong binding activity against the Gram-negative bacteria Escherichia coli and Vibrio splendens. However, rDUF1943 had weaker binding activity against the fungus Pichia pastoris, while rVWD had stronger binding activity against Pichia pastoris. No obvious bands were observed in the rTrx negative control group.

[0138] Experimental Example 3: Detection of the binding activity of the long oyster CgVg functional domain DUF1943 and VWD recombinant protein of the present invention with LPS and LTA

[0139] The binding activity of the long oyster CgVg functional domain DUF1943 and VWD recombinant proteins to LPS, a surface characteristic molecule of Gram-negative bacteria, and LTA, a surface characteristic molecule of Gram-positive bacteria, was detected based on the Western blotting method.

[0140] The specific operations are as follows:

[0141] (1) 100 μL of LTA (100 μg / mL), LPS (100 μg / mL), and TBS (pH 7.4) were mixed with 100 μL of DUF1943 or VWD, respectively, and incubated at room temperature for 1 h.

[0142] (2) Then Staphylococcus aureus (1×10 7 CFU / mL) were added to LTA and TBS, and Vibrio splendens (1×10 7 CFU / mL) were added to LPS and TBS. Incubate at room temperature for 1 h;

[0143] (3) Collect the cells by centrifugation at 5000 × g for 10 min. Wash three times with TBS (pH 7.4), resuspend in 100 μL TBS (pH 7.4), add 5× protein electrophoresis buffer, heat at 99°C for 10 min, and separate the protein samples by 12% SDS-PAGE electrophoresis.

[0144] The binding effect of LTA and LPS was detected by Western blotting method in Experimental Example 2. The experimental results are as follows: Figure 3 shown.

[0145] Experimental results:

[0146] The results showed that after incubation of LTA with recombinant proteins DUF1943 and VWD, the binding activity of DUF1943 and VWD to bacteria was significantly reduced, but after incubation of the recombinant proteins with LPS, there was no significant effect on the binding activity of bacteria.

[0147] Experimental Example 4: Detection of bacterial agglutination activity of the long oyster CgVg functional domain DUF1943 and VWD recombinant protein of the present invention

[0148] Fluorescence microscopy was used to examine the agglutination activity of the long oyster CgVg domain DUF1943 and VWD recombinant proteins against Gram-negative bacteria (Vibrio splendens and Escherichia coli), Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus), and fungi (Pichia pastoris). The experimental steps are as follows:

[0149] 1. Fluorescent labeling of microorganisms

[0150] (1) The five microorganisms mentioned above were cultured overnight using the same culture method as in Experimental Example 2(1);

[0151] (2) The cells were collected by centrifugation and then inactivated and fixed with formaldehyde for 10 min;

[0152] (3) After washing three times with 0.1 M NaHCO3, the cells were resuspended in a NaHCO3 solution (0.1 mol / L, pH 9.0) containing 1 mg / mL fluorescein isothiocyanate (FITC) and incubated overnight at 4°C in the dark.

[0153] (4) Wash three times with TBS and adjust the microbial concentration to 1×10 8 CFU / mL and store it at 4°C until use.

[0154] 2. Agglutination Activity Detection

[0155] (1) 10 μL of fluorescently labeled microorganisms were incubated with an equal volume of recombinant protein DUF1943 or VWD (rTrx as a negative control, TBS as a blank control) at 25°C for 20 min;

[0156] (2) Take 10 μL of the above incubation mixture and apply it on a glass slide, observe and take pictures with a fluorescence microscope. Figure 4 shown.

[0157] Experimental results:

[0158] The results showed that the long oyster CgVg functional domain DUF1943 recombinant protein of the present invention had strong agglutination activity against Gram-negative bacteria, Gram-positive bacteria and fungi, while the long oyster CgVg functional domain VWD recombinant protein had only weak agglutination activity against Pichia pastoris, and had no obvious agglutination activity against Vibrio splendens, Escherichia coli, Staphylococcus aureus and Micrococcus luteus.

[0159] Experimental Example 5: Antibacterial activity (growth curve method) detection of the long oyster CgVg functional domain DUF1943 and VWD recombinant protein of the present invention

[0160] The long oyster CgVg functional domain DUF1943 or VWD recombinant protein were subjected to antibacterial experiments against three bacteria (Escherichia coli, Vibrio splendens and Staphylococcus aureus), and then the antibacterial activity of the long oyster CgVg functional domain DUF1943 and VWD recombinant protein was detected using a microplate reader (Tecan Infinite M1000 PRO).

[0161] The sources of the strains are as above.

[0162] The specific operations are as follows:

[0163] (1) The bacteria cultured overnight were collected by centrifugation, washed three times with TBS solution, and then diluted with TBS solution to a concentration of about 1×10 6 CFU / mL of bacterial suspension;

[0164] (2) 50 μL of recombinant protein DUF1943 or VWD (final concentration 1 mg / mL) was incubated with an equal volume of bacterial suspension at room temperature for 2 h;

[0165] (3) Take 20 μL of the mixture of the above-mentioned recombinant protein DUF1943 or VWD and the bacterial suspension, add it to the wells of a 96-well plate, and add 200 μL of the corresponding culture medium to the wells. At the same time, set up negative control wells (rTrx) and blank control wells (TBS);

[0166] (4) After mixing the recombinant protein DUF1943 or VWD with the test bacteria, place it in a microplate reader and culture it at an appropriate temperature for 12-14 hours until it reaches the plateau phase. Read the OD every hour. 600 .

[0167] The DUF1943 recombinant protein had no significant inhibitory effect on the growth of the tested bacteria ( Figure 5 A).

Claims

1. A long oyster Cg The functional domain of Vg, DUF1943, is characterized by: Long oysters Cg The amino acid sequence of the recombinant protein of the Vg functional domain DUF1943 is shown in SEQ ID NO.

1.

2. The long oyster according to claim 1 Cg The method for constructing the functional domain DUF1943 of Vg is characterized by: a. Use specific primer pair P1 to identify long oysters Cg The coding region fragment of the functional domain DUF1943 of Vg was amplified by PCR; Primer pair P1 5 ， -CGGGGTACCCAAATGTCCAACAGTACTTAC-3 ， ; 5 ， -CCCAAGCTTTTCTCTTGGCGAAGGATA-3 ， ; b. The PCR amplified product was mixed with the pET30a vector. Kpn I and Hind After digestion with III enzyme, the recombinants were ligated with T4 ligase, transformed, and sequenced to identify the recombinants; c. Transforming the recombinant into Escherichia coli Transetta (DE3) expression strain, and then purified and renatured to obtain the long oyster with the amino acid sequence shown in SEQ ID NO.

1. Cg Recombinant protein of the functional domain of Vg DUF1943.

3. A long oyster Cg The functional domain of Vg is a recombinant protein of VWD, characterized by: Long oysters Cg The amino acid sequence of the recombinant protein of the Vg functional domain VWD is shown in SEQ ID NO.

2.

4. The long oyster according to claim 3 Cg The method for constructing a recombinant protein of the Vg functional domain VWD is characterized by: a. Use specific primer pair P2 to identify long oysters Cg The coding region fragment of the VWD functional domain of Vg was amplified by PCR; Primer pair P2 5 ， -CGGGGTACCGACCTCAGGAGAAACAACAGA-3 ， ; 5 ， -CCCAAGCTTTGGTTTACACTGACTCGGACA-3 ， ; b. The PCR amplified product was mixed with the pET30a vector. Kpn I and Hind After digestion with III enzyme, the recombinants were ligated with T4 ligase, transformed, and sequenced to identify the recombinants; c. Transforming the recombinant into Escherichia coli Transetta (DE3) expression strain, and then purified and renatured to obtain the long oyster with the amino acid sequence shown in SEQ ID NO.

2. Cg Functional domain of Vg recombinant protein of VWD.

5. A use of the recombinant protein according to claim 3, characterized in that: The long oyster Cg The application of recombinant proteins of Vg functional domain and VWD in the preparation of marine antibacterial preparations; The bacteria are Escherichia coli, Vibrio splendens and Staphylococcus aureus.

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