CN110041421B - Procambarus clarkii C-type lectin gLecB gene and gLecB protein coded by same - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a procambarus clarkia C-type lectin gLecB gene and a gLecB protein coded by the same, wherein the sequence of the procambarus clarkia C-type lectin gLecB gene is SEQ ID NO: 1; the amino acid sequence of the gLecB protein is SEQ ID NO: 2; the expression vector is pGEX-5X-1-B-Lectin. The invention mainly separates a new C-type agglutinin gLecB in the procambarus clarkia body and has antiviral effect.

Description

Procambarus clarkii C-type lectin gLecB gene and gLecB protein coded by same

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a procambarus clarkia C-type lectin gLecB gene and a gLecB protein coded by the same.

Background

Currently, the current state of the art commonly used in the industry is such that:

due to the lack of specific immune mechanisms based on "antibody-antigen recognition", invertebrates such as shrimps, crabs rely solely on germline gene-encoded pattern recognition receptors PRRs and related immune regulatory systems to protect against pathogenic attack. Immune recognition is the first step of immune response, and invertebrates mainly rely on pattern recognition receptors PRRs (pattern recognition receptors) to recognize invading pathogens, i.e., the aforementioned pathogen-associated molecular patterns PAMPs can be recognized by the pattern recognition receptors PRRs as ligands. The PRRs can directly kill and eliminate pathogenic bacteria on one hand, can activate corresponding downstream signal paths on the other hand, and then can induce and express corresponding antibacterial peptides to resist the invasion of the pathogenic bacteria.

Pattern recognition receptors PRRs can be classified into three major classes, membrane receptors, secretory receptors, and cytoplasmic receptors. Wherein the membrane receptors include Toll-like receptors (Toll-like receptors) and Scavenger Receptors (SRs), etc.; secretory receptors include peptidoglycan binding protein (peptidoglycan), lectin (lectin), and lipopolysaccharide binding protein (lipoglycan and b-1,3-glucan binding protein), and the like; representative of cytoplasmic receptors are Nod-like receptors.

Lectins are a very important class of secretory receptors, each of which contains a carbohydrate recognition domain crd (carbohydrate recognition domain) having the property of binding carbohydrates, and are involved in various physiological processes including protein transport, signal transduction, recognition of pathogens or as immune effector molecules, and CRDs of different types of lectins have different structures. Intracellular lectins are primarily involved in protein screening and trafficking, and extracellular lectins are primarily involved in cell signaling and pathogen recognition^[1]. The lectin involved in protein transport in the cell includes L-type lectin and the like^[2-3]Extracellular proteins involved in cell signaling and recognition of disease origin include C-type lectins^[4]. Currently, 7 lectins have been identified from prawns, respectively C-type lectins, L-type lectins, P-type lectins, M-type lectins, fibrinogen-like domain lectins, galectins and calnexin/calreticulin.

C-type lectins are a superfamily of proteins containing calcium ion dependent carbohydrate recognition domains CRDs, which are highly conserved in structure in vertebrates, but exhibit some diversity in invertebrates. The current research reports and related literature on C-type lectins of shrimp are particularly abundant:the C-type lectins isolated from Penaeus chinensis include Fclectin, Fc-hsl, Fc-Lec2, Fc-Lec3, Fc-Lec4 and Fc-Lec5^[4-9](ii) a LvLT, LvLec, LvCTL1, LvCTL and LvCTLD of Penaeus vannamei^[10-13]And MjLecA, MjLecB and MjLecC of Japanese marsupenaeus japonicas^[14]In particular, the study of MjCC-CL, a C-type lectin from marsupenaeus japonicus, demonstrated that the lectin not only acts as a pattern recognition receptor, but also has the effect of an effector molecule^[15]. For Procambrus clarkii, PcLT was the first reported C-type lectin, followed by PcLec1, PcLec2, PcLec3, PcLec4, PcLec5, and PcLec6, which were reported in sequence^[16-21]. Wherein, researchers find that PcLec4 is not only a pattern recognition receptor, but also an immune signal switch molecule, provides a new basis for explaining the phenomena of signal crossing and conduction delay in immune regulation, and proposes the hypothesis that C-type lectin participates in immune reaction as a molecular switch^[19]。

The first report on the participation of L-type lectin in shrimp immune response was that MjLTLl, a L-type lectin distributed in blood cells, heart, liver pancreas, cheek, stomach and intestine, was found in the bodies of Japanese marsupenaeus japonicus^[22]. In vitro experiments prove that rMjLTLl has the activity of binding and agglutinating bacteria, and MjLTLl can enhance the capability of prawn in clearing in vivo bacteria by promoting phagocytosis of blood cells in prawn bodies. For procambarus clarkia, there are researchers^[23]The molecular mechanism of the L-type lectin PcL-lectin possibly participating in the antiviral immune response is systematically researched.

[1]KILPATRICK D C.Animal lectins:a historical introduction and overview[J].Biochim Biophys Acta,2002,1572(2):187-197.

[2]NEVE E P,SVENSSON K,FUXE J,et al.VIPL,a VIP36-like membrane protein with a putative function in the export of glycoproteins from the endoplasmic reticulum[J].Experimental cell research,2003,288(1):70-83.

[3]NUFER O,MITROVIC S,HAURI H P.Profile-based data base scanning for animal L-type lectins and characterization of VIPL,a novel VIP36-like endoplasmic reticulum protein[J].Journal of biological chemistry,2003,278(18):15886-15896.

[4]ZHANG X W,XU W T,WANG X W,et al.A novel C-type lectin with two CRD domains from Chinese shrimp Fenneropenaeus chinensis functions as a pattern recognition protein[J].Molecular immunology,2009,46(8):1626-1637.

[5]LIU Y C,LI F H,DONG B,et al.Molecular cloning,characterization and expression analysis of a putative C-type lectin(Fclectin)gene in Chinese shrimp Fenneropenaeus chinensis[J].Molecular immunology,2007,44(4):598-607.

[6]SUN Y D,FU L D,JIA Y P,et al.A hepatopancreas-specific C-type lectin from the Chinese shrimp Fenneropenaeus chinensis exhibits antimicrobial activity[J].Molecular immunology,2008,45(2):348-361.

[7]WANG X W,XU W T,ZHANG X W,et al.A C-type lectin is involved in the innate immune response of Chinese white shrimp[J].Fish&shellfish immunology,2009,27(4):556–562.

[8]WANG X W,ZHANG X W,XU W T,et al.A novel C-type lectin(Fc-Lec4)facilitates the clearance of Vibrio anguillarum in vivo in Chinese white shrimp[J].Developmental&comparative immunology,2009,33(9):1039–1047.

[9]XU W T,WANG X W,ZHANG X W,et al.A new C-type lectin(Fc-Lec5)from the Chinese white shrimp Fenneropenaeus chinensis[J].Amino acids,2010,39(5):1227-1239.

[10]MA T H,TIU S H,HE J G,et al.Molecular cloning of a C-type lectin(LvLT)from the shrimp Litopenaeus vannamei:early gene down-regulation after WSSV infection[J].Fish&shellfish immunology,2007,23(2):430-437.

[11]ZHAO Z Y,YIN Z X,XU X P,et al.A novel C-type lectin from the shrimp Litopenaeus vannamei possesses anti-white spot syndrome virus activity[J].Virology,2009,83(1):347-356.

[12]ZHANG Y,QIU L,SONG L,et al.Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei[J].Fish&shellfish immunology,2009,26(1):183-192.

[13]LUO Z,ZHANG J Q,LI F H,et al.Identification of a novel C-type lectin from the shrimp Litopenaeus vannamei and its role in defense against pathogens infection[J].Chinese journal of oceanology and limnology,2011,29(5):942-951.

[14]SONG K K,LI D F,ZHANG,M C,et al.Cloning and characterization of three novel WSSV recognizing lectins from shrimp Marsupenaeus japonicus[J].Fish&shellfish immunology,2010,28(4):596-603.

[15]SUN J J,LAN J F,ZHAO X F,et al.Binding of a C-type lectin's coiled-coil domain to the Domeless receptor directly activates the JAK/STAT pathway in the shrimp immune response to bacterial infection[J].PLoS pathogens,2017,13(9):e1006626.https://doi.org/10.1371/journal.ppat.1006626

[16]CHEN D D,MENG X L,XU J P,et al.PcLT,a novel C-type lectin from Procambarus clarkii,is involved in the innate defense against Vibrio alginolyticus and WSSV[J].Developmental&comparative immunology,2013,39(3):255-264.

[17]ZHANG X W,LIU Y Y,MU Y,et al.Overexpression of a C-type lectin enhances bacterial resistance in red swamp crayfish Procambarus clarkii[J],Fish&shellfish immunology,2013,34(5):1112-1118.

[18]ZHANG X W,REN Q,ZHANG H W,et al.A C-type lectin could selectively facilitate bacteria clearance in red swamp crayfish,Procambarus clarkii[J].Fish&shellfishimmunology,2013,35(5):1387-1394.

[19]ZHANG X W,WANG X W,SUN C,et al.C-type lectin from red swamp crayfish Procambarus clarkii participates in cellular immune response[J],Archives of insect biochemistry&physiology,2015,76(3):168-184.

[20]ZHANG X W,YUE W,WANG X W,et al.A C-type lectin with an immunoglobulin-like domain promotes phagocytosis of hemocytes in crayfish Procambarus clarkii[J].Scientific reports,2016,6:29924.https://doi.org/ 10.1038/srep29924

[21]WANG X W,ZHANG H W,LI X A,et al.Characterization of a C-type lectin(PcLec2)as an upstream detector in the prophenoloxidase activating system of red swamp crayfish[J].Fish&shellfish immunology,2011,30(1):241-247.

[22]WU C L,CHAROENSAPSRI W,NAKAMURA S,et al.An MBL-like protein may interfere with the activation of the proPOsystem,an important innate immune reaction in invertebrates[J].Immunobiology,2013,218(2):159-168.

[23]DAI Y,WANG Y,ZHAO L,et al.A novel L-type lectin was required for the multiplication of WSSV in red swamp crayfish(Procambarus clarkii)[J].Fish&shellfish immunology,2016,55:48-55.https://doi.org/10.1016/j.fsi.2016.05.020

In summary, the problems of the prior art are as follows:

in the prior art, C-type lectins are not proposed as molecular switches to participate in immune reactions to explain the phenomena of signal crossing and conduction delay in immune regulation.

The difficulty and significance for solving the technical problems are as follows:

the previous studies have shown that CTLs are an important component of the innate immune system, play an important role in immune responses, disease responses, and biological responses, and are capable of clearing invading pathogens and viruses, promoting phagocytosis, nodule formation, encapsulation, darkening, cell adhesion and aggregation, and opsonizing various biological responses. The study of shrimp immune responses to pathogenic infections has become more important. Meanwhile, researchers hope to provide necessary basis for developing green prevention and control products by carrying out functional analysis on identified related genes and proteins of immune response reaction of shrimps on pathogen infection. Innate immunity is somewhat adaptive, and it is believed that the innate immune response network will become clearer as technology advances and researchers make efforts. However, extensive analysis of genomic information about shrimp is lacking, and support for cell lines is lacking in shrimp-crab basic research. Further discussion of the immune response of shrimp to pathogenic infections from these 3 aspects of immune recognition, signaling and effector molecules is an essential means for the prevention and control of disease. Subsequent research results indicate synergy or antagonism between invertebrate immune signaling pathways. The method discloses the innate immunity regulation mechanism of shrimps and crabs from a more deep visual angle, and is a very challenging and meaningful subject.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a Procambrus clarkii C-type lectin gLecB gene and a gLecB protein coded by the same. The invention mainly separates a new C-type agglutinin gLecB in the procambarus clarkia body and has antiviral effect.

The invention is realized in such a way that the sequence of the Procambrus clarkii C-type lectin gLecB gene is SEQ ID NO: 1.

another object of the present invention is to provide a glegcb protein encoded by the proclarch C-type lectin gLecB gene, wherein the amino acid sequence of the glecbprotein is SEQ ID NO: 2.

the invention also aims to provide an expression vector constructed by using the gLecB protein, wherein the expression vector is pGEX-5X-1-B-Lectin.

Another object of the present invention is to provide a method for constructing the expression vector, which comprises:

first step, RNA extraction and reverse transcription:

selecting three procambarus clarkii, drawing blood, dissecting, taking tissue samples such as hemolymph, heart, liver, branchia, stomach and intestine, and taking total RNA by a TRIzol method. Detecting the purity and concentration of RNA by using Nano-Drop 300, and analyzing the quality of the total RNA by using a Bio-Rad gel imager through agarose gel electrophoresis detection; synthesizing cDNA by reverse transcription according to the operation steps of the reverse transcription instruction;

secondly, amplifying the B-Lectin full-length coding region gene:

designing a specific primer for sequence amplification, wherein an upstream primer gLecB-EcoR I: 5 '(TACTCAGAATTCGCCAAATCCACGTGC) 3', downstream primer Pc-B-Lectin-Xho I: 5 '(TACTCACTCGAGTTA CTGTGCTTGCTTGGG) 3'; the length of the amplified fragment is 942 bp; using cDNA obtained by reverse transcription as a template for PCR amplification; establishing a 25 mu L PCR reaction system, wherein the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 10s, annealing at 54 ℃ for 20s, extension at 72 ℃ for 40s, and extension at 72 ℃ for 8min after 35 cycles; detecting the amplified product by using 1.0% agarose gel electrophoresis, and recovering the PCR product of 942bp by using a gel recovery kit; recovering and purifying PCR amplification product with glue to obtain target segment;

thirdly, constructing pGEX-5X-1-B-Lectin expression vector:

the gLecB PCR product after pGEX-5X-1 vector and gel recovery is respectively subjected to double enzyme digestion by EcoRI and XhoI, electrophoresis detection and gel recovery and purification are respectively carried out, then T4DNA ligase is used for connecting the gLecB PCR product at 16 ℃ in a constant temperature water bath for overnight, the connecting product is transformed into escherichia coli DH5 alpha, the culture is carried out for 2h at 37 ℃, the product grows on LB culture medium containing 100 mug/mL ampicillin (Amp), a single colony is selected to be subjected to PCR identification by using primer-B-Lectin primer, positive clone is subjected to amplification culture according to the proportion of 1 percent, plasmid is extracted after amplification, and finally the plasmid is sent to the company for sequencing verification.

Further, after construction of the pGEX-5X-1-B-Lectin expression vector, the following steps are carried out:

inducible expression of GST-gLecB fusion protein: coli BL21 competent cells were transformed with the correctly sequenced recombinant plasmid, and a single colony was picked up and cultured at 37 ℃ to OD in LB liquid medium containing 100. mu.g/mL ampicillin (Amp)₆₀₀When the concentration is 0.6, adding IPTG with the final concentration of 1mmol/L, carrying out induction expression at 16 ℃ overnight, and collecting thalli; resuspending the thallus with PBS, ultrasonically crushing for 6min at 4 ℃, centrifuging the ultrasonically crushed bacteria liquid for 20min at 10000 r/min at 4 ℃, and separating supernatant and precipitate; 10. mu.L of each of the supernatant and the precipitate after the ultrasonic treatment was subjected to SDS-PAGE.

Further, after construction of the pGEX-5X-1-B-Lectin expression vector, purification of the fusion protein is required:

the expression of GST-gLecB fusion protein is induced under the optimum induction condition, the supernatant of the induced thallus ultrasonication is collected, chromatography is carried out according to the instruction of GST affinity resin, and 12% SDS-PAGE electrophoresis is carried out.

Further, the method for purifying the fusion protein specifically comprises the following steps:

(1) residual alcohol in the column was washed off using 5 column volumes of PBS, 140mM NaCl,2.7mM KCl,10mM Na2HPO4,1.8mM KH2PO4, pH 7.9;

(2) adding 3 times of 1 × PBS to the column, and balancing the column bed;

(3) adding a protein sample to be purified into the equilibrium column, and filtering the sample by using a 0.45 mu m microporous filter membrane before adding the sample;

(4) adding Bingding buffer (1 × PBS) with the volume of 5-10 times of the column volume into the column, and washing off mycoprotein which is not combined with the resin;

(5) finally, adding Elution Buffer with 5 times of column volume to elute the target protein, collecting eluent which possibly contains the purified target protein, and finally detecting the purity by SDS-PAGE; elution Buffer 10mM reduced glutathione in 50mM Tris, pH 8.0.

In summary, the advantages and positive effects of the invention are:

the invention mainly separates a new C-type agglutinin gLecB in the procambarus clarkia body and has antiviral effect.

In the experiments of the present invention, fig. 2 shows that the experiments provided by the examples of the present invention are divided into 3 groups, a gLecB protein injection group, a tag protein injection group, and a blank group. WSSV was injected simultaneously in 3 groups 1h after protein injection. Total protein was extracted 24h after WSSV infection and WSSV replication was examined. beta-Actin was used as a control, and the results showed that the injection of the gLecB protein significantly inhibited the level of WSSV replication in shrimp compared to the control group.

Drawings

FIG. 1 is an SDS-PAGE analysis of recombinant proteins of gLecB provided in an embodiment of the present invention.

FIG. 2 shows the experimental groups of 3 groups, gLecB-injected proteome, tag-injected proteome, and blank group. WSSV was injected simultaneously in 3 groups 1h after protein injection. Total protein was extracted 24h after WSSV infection and WSSV replication was examined. beta-Actin was used as a control, and the results showed that the injection of the gLecB protein significantly inhibited the level of WSSV replication in shrimp compared to the control group.

FIG. 3 is a graph comparing survival rates for three cases provided by the example of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the prior art, C-type lectins are not proposed as molecular switches to participate in immune reactions to explain the phenomena of signal crossing and conduction delay in immune regulation.

To solve the above problems, the present invention will be described in detail with reference to specific embodiments.

The sequence of the procambarus clarkia C-type lectin gLecB gene provided by the embodiment of the invention is SEQ ID NO: 1.

the invention provides a gLecB protein encoded by the Procambrus clarkii C-type lectin gLecB gene, wherein the amino acid sequence of the gLecB protein is SEQ ID NO: 2.

the invention provides an expression vector constructed by using the gLecB protein, wherein the expression vector is pGEX-5X-1-B-Lectin.

The invention provides a construction method of the expression vector, which comprises the following steps:

first step, RNA extraction and reverse transcription:

selecting three procambarus clarkii, drawing blood, dissecting, taking tissue samples such as hemolymph, heart, liver, branchia, stomach and intestine, and taking total RNA by a TRIzol method. Detecting the purity and concentration of RNA by using Nano-Drop 300, and analyzing the quality of the total RNA by using a Bio-Rad gel imager through agarose gel electrophoresis detection; the cDNA was synthesized by reverse transcription according to the procedure described in the reverse transcription instructions.

Secondly, amplifying the B-Lectin full-length coding region gene:

designing a specific primer for sequence amplification, wherein an upstream primer gLecB-EcoR I: 5 '(TACTCAGAATTCGCCAAATCCACGTGC) 3' SEQ ID NO: 3, a downstream primer Pc-B-Lectin-Xho I: 5 '(TACTCACTCGAGTTA CTGTGCTTGCTTGGG) 3' SEQ ID NO: 4; the length of the amplified fragment is 942 bp; using cDNA obtained by reverse transcription as a template for PCR amplification; establishing a 25 mu L PCR reaction system, wherein the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 10s, annealing at 54 ℃ for 20s, extension at 72 ℃ for 40s, and extension at 72 ℃ for 8min after 35 cycles; detecting the amplified product by using 1.0% agarose gel electrophoresis, and recovering the PCR product of 942bp by using a gel recovery kit; and recovering and purifying the PCR amplification product by using glue to obtain a target fragment.

Thirdly, constructing pGEX-5X-1-B-Lectin expression vector:

the gLecB PCR product after pGEX-5X-1 vector and gel recovery is respectively subjected to double enzyme digestion by EcoRI and XhoI, electrophoresis detection and gel recovery and purification are respectively carried out, then T4DNA ligase is used for connecting the gLecB PCR product at 16 ℃ in a constant temperature water bath for overnight, the connecting product is transformed into escherichia coli DH5 alpha, the escherichia coli DH5 alpha is cultured for 2 hours at 37 ℃, the escherichia coli is grown on an LB culture medium containing 100 mu g/mL ampicillin (Amp), a single colony is selected to be subjected to PCR identification by using a primer-B-Lectin primer, a positive clone is subjected to amplification culture according to the proportion of 1 percent, and plasmids are extracted after amplification and finally sent to the company for sequencing verification.

In the embodiment of the invention, after the construction of the pGEX-5X-1-B-Lectin expression vector, the following steps are carried out:

inducible expression of GST-gLecB fusion protein: coli BL21 competent cells were transformed with the correctly sequenced recombinant plasmid, and a single colony was picked up and cultured at 37 ℃ to OD in LB liquid medium containing 100. mu.g/mL ampicillin (Amp)₆₀₀When the concentration is 0.6, adding IPTG with the final concentration of 1mmol/L, carrying out induction expression at 16 ℃ overnight, and collecting thalli; resuspending the thallus with PBS, ultrasonically crushing for 6min at 4 ℃, centrifuging the ultrasonically crushed bacteria liquid for 20min at 10000 r/min at 4 ℃, and separating supernatant and precipitate; 10. mu.L of each of the supernatant and the precipitate after the ultrasonic treatment was subjected to SDS-PAGE.

After construction of the pGEX-5X-1-B-Lectin expression vector, purification of the fusion protein is required:

the expression of GST-gLecB fusion protein is induced under the optimum induction condition, the supernatant of the induced thallus ultrasonication is collected, chromatography is carried out according to the instruction of GST affinity resin, and 12% SDS-PAGE electrophoresis is carried out.

The invention is further described with reference to specific examples.

Example (b):

the construction method of the expression vector provided by the embodiment of the invention comprises the following steps:

RNA extraction

Sucking 0.5ml anticoagulant with 2ml disposable syringe, drawing blood, mixing, collecting into 1.5ml RNA free EP tube, centrifuging at 3000r for 10min at 4 deg.C, removing supernatant, adding 1ml RNAioso Plus, and blowing; the other tissue samples were completely ground using a grinder, to which 1mL of RNAioso Plus had been previously added. Then collected into 1.5mL RNA free EP tubes, all on ice. To the collected sample, 400. mu.l of chloroform was added, gently mixed, and allowed to stand on ice for 5 min. Then 10000 r 15min, 4 ℃ centrifugation, 400 mul supernatant fluid absorption to a new 1.5ml RNA free EP tube, then 600 mul isopropanol, gentle mixing and placing in a refrigerator of-20 ℃ for 2h to make RNA sedimentation; then 10000 r, 10min, centrifuging at 4 ℃, and discarding the supernatant; adding 1ml of 75% ethanol, purging and precipitating, then 7500r, centrifuging for 10min at 4 ℃, removing supernatant, and scraping water on the tube wall; adding 20 μ l DEPC water, dissolving the precipitate to obtain RNA.

2. Vector construction

Taking 10uL of the ligation product, adding 10uL of engineering bacteria (DH5 alpha, BL21) for ice incubation for 30min, putting the ligation product into a 42 ℃ water bath kettle for heat shock for 75-90s, immediately putting the ligation product on ice for secondary ice incubation for five minutes, adding the mixture into 500uL of liquid LB culture solution for culture at 37 ℃ for 2h, removing the supernatant at the normal temperature of 3500r for 5min, reserving the precipitate, coating the precipitate on LB culture medium containing 100 mu g/mL ampicillin (Amp), and putting the precipitate into a 37 ℃ incubator for culture.

3. Protein purification

(1) The residual alcohol in the column was washed off using 5 column volumes of PBS (140mM NaCl,2.7mM KCl,10mM Na2HPO4,1.8mM KH2PO4) (pH 7.9).

(2) To the column was added 3 column volumes of 1 × PBS to equilibrate the bed.

(3) The protein sample to be purified is added to the equilibrated column, and the sample is filtered using a 0.45 μm microfiltration membrane prior to loading.

(4) A5-10 column volume of Bingding buffer (1 XPBS) was added to the column to wash away the mycoprotein not bound to the resin.

(5) Finally, adding Elution Buffer (10mM reduced glutathione dissolved in 50mM Tris, pH8.0) with 5 times column volume to elute the target protein, collecting eluent which may contain the purified target protein, and finally detecting the purity by SDS-PAGE.

The gene sequence of gLecB SEQ ID NO: 1 is:

ATGACTGCTACTGTGATAAGGACGGTTGTGCTGCTGATGGGCCTGGTGTGTGCTGCCAAATCCACGTGCCCTGAGATTGACCAAATCCACTGTGGAACGAGTGACAGATGTACCCGGATCCGGTACATTTGTGATGGTGACAACGACTGTGGCGACAGCACTGATGAAGAGAGCTCCCTCTGTGCGGTATGGAGGAACAGTGACTGCGAGAGGAACCACGCCAAGTGTACCAGAAGCGGCCGCTCAGACTGTGTCACCATCAGCACCTACTGTACCCTCACTGACCCGCCCTGTGAGGGTACCGTCGACCCTCGTCTCTGTCAGATGCTGAAGGATGGCAAGATTCAGTCCCTGAACGCCATCCAGATGCCCACCCCCCCTGCTCCCACAGAGCCAACTACGATACAGCCATCTATCCACGTGAGGAATGAGAACTGGTCTGAGCAATTCTTGCTGAAGCTCAATAGTACCATTCACCATCCAGACTGTCCAATGCTATACACAAAGGTCGGGGAACACTGCCTCTCCATCTTCTTCATTGGTAATATGAGTTGGATGGAGGCCCGGACCTTCTGCCAGACTATAGGGGGAGACCTGTTCACCATCACCAAGGAGTTTAACATGTTCGCTACTCTACTGCAGCACTTCACCACTCACCAGGTGACTGCAGACTTCTGGATAGGTGGACGCTACATCAACGACACCTTAGGGTGGACCTGGGTTGATGGATCTCCCATGCCTGTGGGCTCTCCTTATTGGGCTGTCAGACATGAGGAGAGGTGCACCAATAGAAAAATCAGCTATGATCTCCTCAACGTCACTTTACCTGCCAATGATGGCGTGTGCTACAACTACGTGCAGGCCCCCAGGGTGCCACCTATGGGTCACTGCAGCTCCCTATCCTACGAGTACTACCACTACATGTCTGACAATAGCTGTTTCATCAAGAAAAGTCCCTTGTGTGTGCTGGTAGGTGAACACCCCAAGCAAGCACAGTAA

gLecB SEQ ID NO: 2 has the amino acid sequence as follows:

MTATVIRTVVLLMGLVCAAKSTCPEIDQIHCGTSDRCTRIRYICDGDNDCGDSTDEESSLCAVWRNSDCERNHAKCTRSGRSDCVTISTYCTLTDPPCEGTVDPRLCQMLKDGKIQSLNAIQMPTPPAPTEPTTIQPSIHVRNENWSEQFLLKLNSTIHHPDCPMLYTKVGEHCLSIFFIGNMSWMEARTFCQTIGGDLFTITKEFNMFATLLQHFTTHQVTADFWIGGRYINDTLGWTWVDGSPMPVGSPYWAVRHEERCTNRKISYDLLNVTLPANDGVCYNYVQAPRVPPMGHCSSLSYEYYHYMSDNSCFIKKSPLCVLVGEHPKQAQ

FIG. 2 is a graph of WSSV inhibition assays performed on crayfish randomly selected and divided into three groups (GST-gLecB, GST-Tag, WSSV) in the experiments provided by the examples of the present invention. In the first group, 50ug of recombinant gLecB protein was first injected from the abdominal cavity of crayfish using a micro-syringe, and 50uL of WSSV was injected 1h after the first injection; injection of 50ug of GST-tagged protein at the same position and 50uL WSSV after 1h in the second group; the third group of crayfish was injected with 50uL of WSSV only. And protein was extracted from shrimp gill tissue 24h after completion of WSSV injection for detection of WSSV replication. The extracted protein sample is processed and then used for Western Blot analysis, and the sample lanes are sequentially arranged as WSSV, WSSV + GST-Tag and WSSV + GST-gLecB (beta-Actin is used as an internal reference). The results show that the level of replication of WSSV in the group injected with recombinant gLecB protein is significantly lower than in the first two groups compared to the group injected with WSSV only and post-injection of the tag protein. We conclude from this that the gLecB protein is effective in inhibiting the level of WSSV replication in crayfish.

FIG. 3 is a graph comparing survival rates for three cases provided by the example of the present invention.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> a Procambrus clarkii C-type lectin gLecB gene and gLecB protein coded by the same

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<213> Artificial Sequence (Artificial Sequence)

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atgactgcta ctgtgataag gacggttgtg ctgctgatgg gcctggtgtg tgctgccaaa 60

tccacgtgcc ctgagattga ccaaatccac tgtggaacga gtgacagatg tacccggatc 120

cggtacattt gtgatggtga caacgactgt ggcgacagca ctgatgaaga gagctccctc 180

tgtgcggtat ggaggaacag tgactgcgag aggaaccacg ccaagtgtac cagaagcggc 240

cgctcagact gtgtcaccat cagcacctac tgtaccctca ctgacccgcc ctgtgagggt 300

accgtcgacc ctcgtctctg tcagatgctg aaggatggca agattcagtc cctgaacgcc 360

atccagatgc ccaccccccc tgctcccaca gagccaacta cgatacagcc atctatccac 420

gtgaggaatg agaactggtc tgagcaattc ttgctgaagc tcaatagtac cattcaccat 480

ccagactgtc caatgctata cacaaaggtc ggggaacact gcctctccat cttcttcatt 540

ggtaatatga gttggatgga ggcccggacc ttctgccaga ctataggggg agacctgttc 600

accatcacca aggagtttaa catgttcgct actctactgc agcacttcac cactcaccag 660

gtgactgcag acttctggat aggtggacgc tacatcaacg acaccttagg gtggacctgg 720

gttgatggat ctcccatgcc tgtgggctct ccttattggg ctgtcagaca tgaggagagg 780

tgcaccaata gaaaaatcag ctatgatctc ctcaacgtca ctttacctgc caatgatggc 840

gtgtgctaca actacgtgca ggcccccagg gtgccaccta tgggtcactg cagctcccta 900

tcctacgagt actaccacta catgtctgac aatagctgtt tcatcaagaa aagtcccttg 960

tgtgtgctgg taggtgaaca ccccaagcaa gcacagtaa 999

<210> 2

<211> 332

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Thr Ala Thr Val Ile Arg Thr Val Val Leu Leu Met Gly Leu Val

1 5 10 15

Cys Ala Ala Lys Ser Thr Cys Pro Glu Ile Asp Gln Ile His Cys Gly

20 25 30

Thr Ser Asp Arg Cys Thr Arg Ile Arg Tyr Ile Cys Asp Gly Asp Asn

35 40 45

Asp Cys Gly Asp Ser Thr Asp Glu Glu Ser Ser Leu Cys Ala Val Trp

50 55 60

Arg Asn Ser Asp Cys Glu Arg Asn His Ala Lys Cys Thr Arg Ser Gly

65 70 75 80

Arg Ser Asp Cys Val Thr Ile Ser Thr Tyr Cys Thr Leu Thr Asp Pro

85 90 95

Pro Cys Glu Gly Thr Val Asp Pro Arg Leu Cys Gln Met Leu Lys Asp

100 105 110

Gly Lys Ile Gln Ser Leu Asn Ala Ile Gln Met Pro Thr Pro Pro Ala

115 120 125

Pro Thr Glu Pro Thr Thr Ile Gln Pro Ser Ile His Val Arg Asn Glu

130 135 140

Asn Trp Ser Glu Gln Phe Leu Leu Lys Leu Asn Ser Thr Ile His His

145 150 155 160

Pro Asp Cys Pro Met Leu Tyr Thr Lys Val Gly Glu His Cys Leu Ser

165 170 175

Ile Phe Phe Ile Gly Asn Met Ser Trp Met Glu Ala Arg Thr Phe Cys

180 185 190

Gln Thr Ile Gly Gly Asp Leu Phe Thr Ile Thr Lys Glu Phe Asn Met

195 200 205

Phe Ala Thr Leu Leu Gln His Phe Thr Thr His Gln Val Thr Ala Asp

210 215 220

Phe Trp Ile Gly Gly Arg Tyr Ile Asn Asp Thr Leu Gly Trp Thr Trp

225 230 235 240

Val Asp Gly Ser Pro Met Pro Val Gly Ser Pro Tyr Trp Ala Val Arg

245 250 255

His Glu Glu Arg Cys Thr Asn Arg Lys Ile Ser Tyr Asp Leu Leu Asn

260 265 270

Val Thr Leu Pro Ala Asn Asp Gly Val Cys Tyr Asn Tyr Val Gln Ala

275 280 285

Pro Arg Val Pro Pro Met Gly His Cys Ser Ser Leu Ser Tyr Glu Tyr

290 295 300

Tyr His Tyr Met Ser Asp Asn Ser Cys Phe Ile Lys Lys Ser Pro Leu

305 310 315 320

Cys Val Leu Val Gly Glu His Pro Lys Gln Ala Gln

325 330

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tactcagaat tcgccaaatc cacgtgc 27

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tactcactcg agttactgtg cttgcttggg 30

Claims (3)

1. A Procambrus clarkii C-type lectin gLecB gene, which is characterized in that the sequence of the Procambrus clarkii C-type lectin gLecB gene is SEQ ID NO: 1.

2. a glencb protein encoded by the procymus clarkii C-type lectin glegcb gene of claim 1, wherein the amino acid sequence of the glencb protein is SEQ ID NO: 2.

3. a vector for expressing the gLecB protein of claim 2, wherein the vector is pGEX-5X-1-B-Lectin;

the construction method of the vector comprises the following steps:

first step, RNA extraction and reverse transcription:

selecting three procambarus clarkii, drawing blood, dissecting, taking tissue samples such as hemolymph, heart, liver, branchia, stomach and intestine, and taking total RNA by a TRIzol method; detecting the purity and concentration of RNA by using Nano-Drop 300, and analyzing the quality of the total RNA by using a Bio-Rad gel imager through agarose gel electrophoresis detection; synthesizing cDNA by reverse transcription according to the operation steps of the reverse transcription instruction;

secondly, gene amplification of the full-length coding region of gLecB:

designing a specific primer for sequence amplification, wherein an upstream primer gLecB-EcoR I: 5 '(TACTCAGAATTCGCCAAATCCACGTGC) 3', downstream primer Pc-B-Lectin-Xho I: 5 '(TACTCACTCGAGTTA CTGTGCTTGCTTGGG) 3'; the length of the amplified fragment is 942 bp; using cDNA obtained by reverse transcription as a template for PCR amplification; establishing a 25 mu L PCR reaction system, wherein the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 10s, annealing at 54 ℃ for 20s, extension at 72 ℃ for 40s, and extension at 72 ℃ for 8min after 35 cycles; detecting the amplified product by using 1.0% agarose gel electrophoresis, and recovering the PCR product of 942bp by using a gel recovery kit; recovering and purifying PCR amplification product with glue to obtain target segment;

thirdly, constructing pGEX-5X-1-B-Lectin expression vector:

the gLecB PCR product after pGEX-5X-1 vector and gel recovery is respectively subjected to double enzyme digestion by EcoRI and XhoI, electrophoresis detection and gel recovery and purification are respectively carried out, then T4DNA ligase is used for connecting the gLecB PCR product at 16 ℃ in a constant temperature water bath for overnight, the connecting product is transformed into escherichia coli DH5 alpha, the escherichia coli DH5 alpha is cultured for 2 hours at 37 ℃, the escherichia coli is grown on an LB culture medium containing 100 mu g/mL ampicillin (Amp), a single colony is selected to be subjected to PCR identification by using a primer-B-Lectin primer, a positive clone is subjected to amplification culture according to the proportion of 1 percent, and plasmids are extracted after amplification and finally sent to the company for sequencing verification.

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