Metallothionein recombinant vector, recombinant bacterium and construction method and application of metallothionein recombinant vector

Abstract

The invention provides a metallothionein recombinant vector, recombinant bacteria and a construction method and application thereof, and belongs to the technical field of protein engineering. The present invention provides a recombinant vector into which a nucleic acid molecule encoding a fusion protein is inserted; the fusion protein comprises CBM and metallothionein which are sequentially connected from the C end to the N end; the metallothionein comprises MTT1, MTT5, TM1 or TM2; and the skeleton plasmid of the recombinant vector comprises pET-28a-SUMO, pGEX-6P-1 or pGEX-4T-1. The invention further discloses a preparation method of the recombinant vector. According to the method, the expression quantity of the fusion protein can be increased by selecting the appropriate skeleton plasmid of the recombinant vector, and heavy metal ions can be removed by combining the fusion protein with cellulose.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering, and in particular relates to a metallothionein recombinant vector, a recombinant bacterium, and a construction method and application thereof. Background Art

[0002] Carbohydrates are biomolecules involved in many life processes. The region in carbohydrate-active enzymes that provides carbohydrate binding affinity is called the carbohydrate binding module (CBM), and the CBM is an excellent model system to study the mechanism of protein-carbohydrate recognition.

[0003] Metallothioneins (MTs) are widely present in various organisms. They are a class of low molecular weight proteins rich in cysteine ​​(Cys) and can be induced to express by metals such as cadmium, copper, zinc, lead or mercury.

[0004] The fusion protein formed by the combination of CBM and metallothionein can be used to construct a cellulose-based composite heavy metal adsorbent. However, the fusion protein of CBM and metallothionein is prepared by genetic engineering, and the expression level needs to be further improved. Summary of the invention

[0005] The purpose of the present invention is to provide a metallothionein recombinant vector, a recombinant bacterium, a construction method and an application thereof. The recombinant vector of the present invention can increase the expression level of a fusion protein of CBM and metallothionein.

[0006] The invention provides a recombinant vector, into which a nucleic acid molecule encoding a fusion protein is inserted; the fusion protein comprises a CBM and a metallothionein connected in sequence from the C-terminus to the N-terminus; the metallothionein comprises MTT1, MTT5, TM1 or TM2; the amino acid sequence of the MTT1 is shown in SEQ ID NO.1; the amino acid sequence of the MTT5 is shown in SEQ ID NO.2; the amino acid sequence of the TM1 is shown in SEQ ID NO.3; the amino acid sequence of the TM2 is shown in SEQ ID NO.4; the amino acid sequence of the CBM is shown in SEQ ID NO.5; the backbone plasmid of the recombinant vector comprises pET-28a-SUMO, pGEX-6P-1 or pGEX-4T-1.

[0007] Preferably, when the metallothionein is MTT1, the backbone plasmid of the recombinant vector includes pGEX-6P-1; when the metallothionein is MTT5, the backbone plasmid of the recombinant vector includes pET-28a-SUMO; when the metallothionein is TM1 and / or TM2, the backbone plasmid of the recombinant vector includes pET-28a-SUMO and / or pGEX-4T-1.

[0008] Preferably, the insertion site of the nucleic acid molecule on the recombinant vector includes restriction endonuclease BamHI and XhoⅠ restriction sites.

[0009] The present invention also provides a host cell comprising the recombinant vector described in the above scheme.

[0010] Preferably, the original cell of the host cell comprises Escherichia coli.

[0011] The present invention also provides a method for preparing a fusion protein, which is characterized in that it comprises the following steps: inducing and culturing the host cells described in the above scheme to obtain an induced culture product; the fusion protein comprises a CBM and a metallothionein connected sequentially from the C-terminus to the N-terminus; the metallothionein comprises MTT1, MTT5, TM1 or TM2; the amino acid sequence of the MTT1 is shown in SEQ ID NO.1; the amino acid sequence of the MTT5 is shown in SEQ ID NO.2; the amino acid sequence of the TM1 is shown in SEQ ID NO.3; the amino acid sequence of the TM2 is shown in SEQ ID NO.4; the amino acid sequence of the CBM is shown in SEQID NO.5.

[0012] Preferably, the induction agent for the induction culture includes IPTG; the working concentration of the IPTG includes 0 to 0.2 mMol / mL.

[0013] Preferably, the induction culture temperature is 16-37°C.

[0014] The present invention also provides the use of the recombinant vector, the host cell or the preparation method described in the above scheme in the preparation of a cellulose-based composite heavy metal adsorbent.

[0015] The present invention also provides the use of the recombinant vector, the host cell or the preparation method described in the above scheme in heavy metal adsorption or heavy metal pollution control.

[0016] The present invention provides a recombinant vector, into which a nucleic acid molecule encoding a fusion protein is inserted; the fusion protein comprises a CBM and a metallothionein connected sequentially from the C-terminus to the N-terminus; the metallothionein comprises MTT1, MTT5, TM1 or TM2; the backbone plasmid of the recombinant vector comprises pET-28a-SUMO, pGEX-6P-1 or pGEX-4T-1. The present invention can increase the expression amount of the fusion protein of CBM and metallothionein by selecting a suitable backbone plasmid of the recombinant vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 Sequence modules, cysteine ​​cluster arrangement characteristics and amino acid sequences of metallothioneins MTT1, TM1, TM2 and MTT5. The arrangement pattern of cysteine ​​residues is marked in red.

[0019] Figure 2 Design of fusion protein CBM-MTT1, CBM-TM1, CBM-TM2 and CBM-MTT5 sequences; (a): fusion protein CBM-MTT1 sequence; (b): fusion protein CBM-MTT5 sequence; (c): fusion protein CBM-MTT1 sequence; (d): fusion protein CBM-MTT5 sequence; the blue part represents the CBM module sequence, the light brown and orange parts represent the MTT1, TM1 and TM2 and MTT5 component sequences respectively, and the highlighted part is the flexible linker GSAGSA;

[0020] Figure 3 is the plasmid map of the recombinant plasmid pET28a-CBM-MTT1;

[0021] Figure 4 is the plasmid map of the recombinant plasmid pET28a-CBM-MTT5;

[0022] Figure 5 This is a plasmid identification diagram of the recombinant plasmid pET28a-CBM-MTT1; wherein, M: Trans 2KplusⅡDNAMarker; Lane 1: recombinant plasmid pET28a-CBM-MTT1; Lane 2: BamHI and Xho I double enzyme digestion product of the recombinant plasmid pET28a-CBM-MTT1;

[0023] Figure 6 This is the identification diagram of the recombinant plasmid pET28a-CBM-MTT5; where M: Trans 2KplusⅡDNAMarker; Lane 1: recombinant plasmid pET28a-CBM-MTT5; Lane 2: BamHI and Xho I double enzyme digestion products of the recombinant plasmid pET28a-CBM-MTT5;

[0024] Figure 7 is the recombinant plasmid map of the recombinant plasmid pSUMO-CBM-MTT1;

[0025] Figure 8 is the recombinant plasmid map of the recombinant plasmid pSUMO-CBM-MTT5;

[0026] Fig. 9 This is a plasmid identification diagram of the recombinant plasmid pSUMO-CBM-MTT1; wherein, M: Trans 2KplusⅡDNAMarker; Lane 1: recombinant plasmid pSUMO-CBM-MTT1; Lane 2: BamHI and Xho I double enzyme digestion product of the recombinant plasmid pSUMO-CBM-MTT1;

[0027] Fig.10 This is an identification diagram of the recombinant plasmid pSUMO-CBM-MTT5; where M: Trans 2KplusⅡDNAMarker; Lane 1: recombinant plasmid pSUMO-CBM-MTT5; Lane 2: BamHI and Xho I double enzyme digestion products of plasmid pSUMO-CBM-MTT5;

[0028] Fig.11 is the plasmid map of the recombinant plasmid pGST-CBM-MTT1;

[0029] Fig.12 This is a plasmid identification diagram of the recombinant plasmid pGST-CBM-MTT1; wherein, M: Trans 2KplusⅡDNAMarker; Lane 1: recombinant plasmid pGST-CBM-MTT1; Lane 2: BamHI and Xho I double enzyme digestion product of the recombinant plasmid pGST-CBM-MTT1;

[0030] Fig.13The results of expression of CBM-MTT1 and CBM-MTT5 in E. coli BL21 and Western blotting verification are shown; (a) is the expression of CBM-MTT1 in E. coli BL21, Lane M: protein marker; Lane 1: whole E. coli BL21 / pET-28a; Lane 2: whole E. coli BL21 / pET28a-CBM-MTT137℃IPTG uninduced whole bacteria; Lane 3-5: whole E. coli BL21 / pET28a-CBM-MTT137℃0.1mM IPTG induced whole bacteria, supernatant and precipitate; Lane 6-8: whole E. coli BL21 / pET28a-CBM-MTT116℃0.1mM IPTG induced whole bacteria, supernatant and precipitate; (b) is the expression of CBM-MTT5 in E. coli BL21, Lane M: protein marker; Lane 1-2: whole cells and supernatant of E. coli BL21 / pET-28a; Lane 3-4: whole cells and supernatant of E. coli BL21 / pET28a-CBM-MTT5 without 37℃IPTG induction; Lane 5-6: whole cells and supernatant of E. coli BL21 / pET28a-CBM-MTT5 induced with 37℃0.1mM IPTG; Lane 7-8: whole cells and supernatant of E. coli BL21 / pET28a-CBM-MTT5 induced with 16℃0.1mM IPTG; (c) is Western blotting verification of protein CBM-MTT5, Lane M: protein marker; Lane 1: supernatant of E. coli BL21 / pET28a-CBM-MTT5 induced with 37℃0.1mM IPTG;

[0031] Fig.14The figure shows the expression of SUMO-CBM-MTT1 in E. coli BL21 and the results of Western blotting verification; wherein, (a) shows the expression of SUMO-CBM-MTT1 in E. coli BL21, Lane M: protein marker; Lane 1-2: E. coli BL21 / pET-28a-SUMO 37℃ uninduced whole bacteria and supernatant; Lane 3-4: E. coli BL21 / pET-28a-SUMO 37℃ 0.1mM IPTG induced whole bacteria and supernatant; Lane 5-6: E. coli BL21 / pSUMO-CBM-MTT137℃ uninduced whole bacteria and supernatant; Lane 7-8: E. coli BL21 / pSUMO-CBM-MTT137℃ 0.1mM IPTG induced whole bacteria and supernatant; Lane 9-10: E. coli BL21 / pSUMO-CBM-MTT116℃ 0.1mM Whole cells and supernatant induced by IPTG; (b) Expression of SUMO-CBM-MTT1 in E. coli BL21 at 24℃, Lane M: Protein Marker; Lane 1-2: Whole cell lysates of E. coli BL21 / pET-28a-SUMO at 24℃ without induction and induced with 0.1mM IPTG; Lane 3-4: Whole cells and supernatant induced by E. coli BL21 / pSUMO-CBM-MTT at 124℃ with 0.01mM IPTG; Lane 5-6: Whole cells and supernatant induced by E. coli BL21 / pSUMO-CBM-MTT at 124℃ with 0.05mM IPTG; Lane 7-8: Whole cells and supernatant induced by E. coli BL21 / pSUMO-CBM-MTT at 124℃ with 0.1mM IPTG; (c) Western blotting verification of protein SUMO-CBM-MTT1, Lane M: Protein Marker; Lane Lane 1: E. coli BL21 / pET-28a-SUMO induced supernatant at 37°C; Lane 2: E. coli BL21 / pSUMO-CBM-MTT uninduced supernatant at 137°C; Lane 3: E. coli BL21 / pSUMO-CBM-MTT induced whole bacteria at 137°C with 0.1 mM IPTG;

[0032] Fig.15: The expression result of GST-CBM-MTT1 in Escherichia coli BL21; wherein, (a) is the expression induced by Escherichia coli BL21 / pGST-CBM-MTT at 137℃ and 16℃, Lane M: protein marker; Lane 1-2: Escherichia coli BL21 / pGEX-6P-137℃ uninduced whole bacteria and supernatant; Lane 3-4: Escherichia coli BL21 / pGEX-6P-137℃ 0.1mM IPTG induced whole bacteria and supernatant; Lane 5-6: Escherichia coli BL21 / pGST-CBM-MTT137℃ uninduced whole bacteria and supernatant; Lane 7-8: Escherichia coli BL21 / pGST-CBM-MTT137℃ 0.1mM IPTG induced whole bacteria and supernatant; Lane 9-10: Escherichia coli BL21 / pGST-CBM-MTT116℃ 0.1mM IPTG induced whole cells and supernatant; (b) Expression of GST-CBM-MTT1 in E. coli BL21 under 37℃ IPTG concentration gradient conditions, Lane M: Protein Marker; Lane 1-2: E. coli BL21 / pGEX-6P-1 uninduced whole cells and supernatant at 37℃; Lane 3-4: E. coli BL21 / pGEX-6P-1 induced with 0.1mM IPTG at 37℃, supernatant; Lane 5-6: E. coli BL21 / pGST-CBM-MTT1 uninduced whole cells and supernatant at 137℃; Lane 7-8: E. coli BL21 / pGST-CBM-MTT induced with 0.05mM IPTG at 137℃, supernatant; Lane 9-10: E. coli BL21 / pGST-CBM-MTT induced with 0.1mM IPTG at 137℃, supernatant; Lane 11-12: E. coli BL21 / pGST-CBM-MTT induced with 0.2 mM IPTG at 137°C, whole bacteria, supernatant;

[0033] Fig.16The figure shows the expression of SUMO-CBM-MTT5 in E. coli BL21 and the results of Western blotting verification; wherein, (a) shows the expression of SUMO-CBM-MTT5 in E. coli BL21, Lane M: protein marker; Lane 1-2: E. coli BL21 / pET-28a-SUMO uninduced whole bacteria and supernatant at 37℃; Lane 3-4: E. coli BL21 / pET-28a-SUMO induced whole bacteria and supernatant at 37℃ 0.1mM IPTG; Lane 5-6: E. coli BL21 / pSUMO-CBM-MTT5 uninduced whole bacteria and supernatant at 37℃; Lane 7-8: E. coli BL21 / pSUMO-CBM-MTT5 induced whole bacteria and supernatant at 37℃ 0.1mM IPTG; Lane 9-10: E. coli BL21 / pSUMO-CBM-MTT5 induced whole bacteria and supernatant at 16℃ 0.1mM IPTG induced whole bacteria and supernatant; (b) Analysis of protein expression ratio of SUMO-CBM-MTT5 at 16℃, Lane M: Protein Marker; Lane 1-2: E. coli BL21 / pET-28a-SUMO 37℃ uninduced and induced samples; Lane 3: E. coli BL21 / pSUMO-CBM-MTT5 37℃ uninduced sample; Lane 4: E. coli BL21 / pSUMO-CBM-MTT5 16℃ 0.1mM IPTG induced 10-fold whole bacteria; Lane 5: E. coli BL21 / pSUMO-CBM-MTT5 16℃ 0.1mM IPTG induced 10-fold supernatant; (c) Western blotting verification of protein SUMO-CBM-MTT5, Lane M: Protein Marker; Lane 1: E. coli BL21 / pET-28a-SUMO 37℃ induced supernatant; Lane Lane 2: E. coli BL21 / pSUMO-CBM-MTT5 uninduced supernatant at 37°C; Lane 3: E. coli BL21 / pSUMO-CBM-MTT5 induced supernatant at 37°C with 0.1 mM IPTG;

[0034] Fig.17 The results of PCR amplification of CBM-TM1 and CBM-TM2 products; M: Trans 2KplusⅡDNAMarker; Lane 1: CBM-TM1 PCR product; Lane 2: CBM-TM2 PCR product;

[0035] Fig.18 is the plasmid map of the recombinant vector pSUMO-CBM-TM1;

[0036] Fig.19This is a plasmid identification diagram of the recombinant vector pSUMO-CBM-TM1; wherein, M: Trans 2KplusⅡDNAMarker; Lane 1: recombinant vector pSUMO-CBM-TM1; Lane 2: PCR product of the recombinant vector pSUMO-CBM-TM1; Lane 3: BamHI and Xho I double restriction products of the recombinant vector pSUMO-CBM-TM1;

[0037] Fig. 20 is the plasmid map of the recombinant vector pGST-CBM-TM1;

[0038] Fig.21 This is a plasmid identification diagram of the recombinant vector pGST-CBM-TM1; wherein, M: Trans 2KplusⅡDNAMarker; Lane 1: recombinant vector pGST-CBM-TM1; Lane 2: PCR product of the recombinant vector pGST-CBM-TM1; Lane 3: BamHI and Xho I double restriction product of the recombinant vector pGST-CBM-TM1;

[0039] Fig. 22 is the plasmid map of the recombinant vector pSUMO-CBM-TM2;

[0040] Fig.23 This is a plasmid identification diagram of the recombinant vector pSUMO-CBM-TM2; wherein, M: Trans 2KplusⅡDNAMarker; Lane 1: recombinant vector pSUMO-CBM-TM2; Lane 2: PCR product of the recombinant vector pSUMO-CBM-TM2; Lane 3: BamHI and Xho I double restriction products of the recombinant vector pSUMO-CBM-TM2;

[0041] Fig.24 is the plasmid map of the recombinant vector pGST-CBM-TM2;

[0042] Fig.25 This is a plasmid identification diagram of the recombinant vector pGST-CBM-TM2; wherein, M: Trans 2KplusⅡDNAMarker; Lane 1: recombinant vector pGST-CBM-TM2; Lane 2: PCR product of the recombinant vector pGST-CBM-TM2; Lane 3: BamHI and Xho I double restriction products of the recombinant vector pGST-CBM-TM2;

[0043] Fig.26The expression results of recombinant protein SUMO-CBM-TM1 and GST-CBM-TM1 in Escherichia coli BL21 are shown in FIG. (a) shows the expression of recombinant protein SUMO-CBM-TM1 in Escherichia coli BL21, Lane M: protein marker; Lane 1: Escherichia coli BL21 / pET-28a-SUMO 37°C uninduced sample; Lane 2: Escherichia coli BL21 / pET-28a-SUMO 37°C 0.1mM IPTG induced sample; Lanes 3-4: Escherichia coli BL21 / pSUMO-CBM-TM137°C uninduced whole bacteria and supernatant; Lanes 5-6: Escherichia coli BL21 / pSUMO-CBM-TM137°C 0.05mM IPTG induced whole bacteria and supernatant; Lanes 7-8: Escherichia coli BL21 / pSUMO-CBM-TM137°C 0.1mM IPTG induced whole bacteria and supernatant; Lane Lanes 9-10: whole cells and supernatant of E. coli BL21 / pSUMO-CBM-TM induced at 116℃ with 0.05mM IPTG; Lanes 11-12: whole cells and supernatant of E. coli BL21 / pSUMO-CBM-TM induced at 116℃ with 0.1mM IPTG; (b) is the expression of recombinant protein GST-CBM-TM1 in E. coli BL21, Lane M: protein marker; Lane 1: uninduced E. coli BL21 / pGEX-4T-137℃; Lane 2: uninduced E. coli BL21 / pGEX-4T-137℃ with 0.1mM IPTG; Lanes 3-4: whole cells and supernatant of E. coli BL21 / pGST-CBM-TM induced at 137℃; Lanes 5-6: whole cells and supernatant of E. coli BL21 / pGST-CBM-TM induced at 137℃ with 0.1mM IPTG IPTG induced whole bacteria, supernatant; Lane 7-8: E. coli BL21 / pGST-CBM-TM 137℃ 0.2mM IPTG induced whole bacteria, supernatant; Lane 9-10: E. coli BL21 / pGST-CBM-TM 116℃ 0.1mM IPTG induced whole bacteria, supernatant; Lane 11-12: E. coli BL21 / pGST-CBM-TM 116℃ 0.2mM IPTG induced whole bacteria, supernatant;

[0044] Fig. 27The results of the protein expression ratio analysis of SUMO-CBM-TM1 and GST-CBM-TM1; (a) is the protein expression ratio analysis of SUMO-CBM-TM1 at 16°C, Lane M: protein marker; Lane 1: E. coli BL21 / pET-28a-SUMO induced at 37°C; Lane 2: E. coli BL21 / pSUMO-CBM-TM1 uninduced at 37°C; Lane 3: E. coli BL21 / pSUMO-CBM-TM1 induced at 16°C with 0.1mM IPTG and 5-fold dilution of whole bacteria; Lane 4: E. coli BL21 / pSUMO-CBM-TM1 induced at 16°C with 0.1mM IPTG and 5-fold dilution of supernatant; (b) is the protein expression ratio analysis of GST-CBM-TM1 at 37°C, Lane M: protein marker; Lane Lane 1: E. coli BL21 / pGEX-4T-137℃ induced sample; Lane 2: E. coli BL21 / pGST-CBM-TM137℃ uninduced sample; Lane 3: E. coli BL21 / pGST-CBM-TM137℃ 0.1mM IPTG induced 5-fold dilution of whole bacteria; Lane 4: E. coli BL21 / pGST-CBM-TM137℃ 0.1mM IPTG induced 5-fold dilution of supernatant;

[0045] Fig.28The expression results of recombinant protein SUMO-CBM-TM2 and GST-CBM-TM2 in Escherichia coli BL21 are shown in FIG. (a) shows the expression of recombinant protein SUMO-CBM-TM2 in Escherichia coli BL21, Lane M: protein marker; Lane 1: Escherichia coli BL21 / pET-28a-SUMO 37°C uninduced sample; Lane 2: Escherichia coli BL21 / pET-28a-SUMO 37°C 0.1mM IPTG induced sample; Lanes 3-4: Escherichia coli BL21 / pSUMO-CBM-TM2 37°C uninduced whole bacteria and supernatant; Lanes 5-6: Escherichia coli BL21 / pSUMO-CBM-TM2 37°C 0.05mM IPTG induced whole bacteria and supernatant; Lanes 7-8: Escherichia coli BL21 / pSUMO-CBM-TM2 37°C 0.1mM IPTG induced whole bacteria and supernatant; Lane Lanes 9-10: whole cells and supernatant of E. coli BL21 / pSUMO-CBM-TM2 induced at 16℃ with 0.05mM IPTG; Lanes 11-12: whole cells and supernatant of E. coli BL21 / pSUMO-CBM-TM2 induced at 16℃ with 0.1mM IPTG; (b) is the expression of recombinant protein GST-CBM-TM2 in E. coli BL21, Lane M: protein marker; Lane 1: uninduced E. coli BL21 / pGEX-4T-1 at 37℃; Lane 2: induced E. coli BL21 / pGEX-4T-1 at 37℃ with 0.1mM IPTG; Lanes 3-4: whole cells and supernatant of E. coli BL21 / pGST-CBM-TM2 at 37℃ with 0.1mM IPTG; Lanes 5-6: whole cells and supernatant of E. coli BL21 / pGST-CBM-TM2 at 37℃ with 0.1mM IPTG IPTG induced whole bacteria, supernatant; Lane 7-8: E. coli BL21 / pGST-CBM-TM2 37℃ 0.2mM IPTG induced whole bacteria, supernatant; Lane 9-10: E. coli BL21 / pGST-CBM-TM2 16℃ 0.1mM IPTG induced whole bacteria, supernatant; Lane 11-12: E. coli BL21 / pGST-CBM-TM2 16℃ 0.2mM IPTG induced whole bacteria, supernatant;

[0046] Fig.29The results of the protein expression ratio analysis of SUMO-CBM-TM2 and GST-CBM-TM2 at 16°C and 37°C are shown in the figure; wherein, (a) is the results of the protein expression ratio analysis of SUMO-CBM-TM2 at 16°C and 37°C, Lane M: protein marker; Lane 1: E. coli BL21 / pET-28a-SUMO induced at 37°C; Lane 2: E. coli BL21 / pSUMO-CBM-TM2 uninduced at 37°C; Lane 3: E. coli BL21 / pSUMO-CBM-TM2 induced at 16°C with 0.1 mM IPTG and diluted 5 times the whole bacteria; Lane 4: E. coli BL21 / pSUMO-CBM-TM2 induced at 16°C with 0.1 mM IPTG and diluted 5 times the supernatant; (b) is the results of the protein expression ratio analysis of GST-CBM-TM2 at 16°C and 37°C, Lane M: protein marker; Lane Lane 1: E. coli BL21 / pGEX-4T-137℃ induced sample; Lane 2: E. coli BL21 / pGST-CBM-TM137℃ uninduced sample; Lane 3: E. coli BL21 / pGST-CBM-TM116℃ 0.2mM IPTG induced 5-fold dilution of whole bacteria; Lane 4: E. coli BL21 / pGST-CBM-TM116℃ 0.2mM IPTG induced 5-fold dilution of supernatant;

[0047] Fig.30 Cellulose-GST-CBM-MTT1 reacts with heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption capacity analysis results; (a): cellulose-GST-CBM-MTT1 for heavy metal ions (Pb 2+ 、Cd 2+ ) removal rate; (b): cellulose-GST-CBM-MTT1 for heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption amount;

[0048] Fig.31 The cellulose-SUMO-CBM-TM1 reacts with heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption capacity analysis results; (a): cellulose-SUMO-CBM-TM1 for heavy metal ions (Pb 2+ 、Cd 2+ ) removal rate; (b): removal rate of heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption amount;

[0049] Fig.32 The cellulose-SUMO-CBM-TM2 reacts with heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption capacity analysis results, where (a) cellulose-SUMO-CBM-TM2 for heavy metal ions (Pb 2+ 、Cd 2+ ) removal rate; (b): removal rate of heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption amount;

[0050] Fig.33 The effect of cellulose-SUMO-CBM-MTT5 on heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption capacity analysis results, where (a) cellulose-SUMO-CBM-MTT5 for heavy metal ions (Pb 2+ 、Cd 2+ ) removal rate; (b): removal rate of heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption amount. DETAILED DESCRIPTION

[0051] The invention provides a recombinant vector, into which a nucleic acid molecule encoding a fusion protein is inserted; the fusion protein comprises a CBM and a metallothionein connected in sequence from the C-terminus to the N-terminus; the metallothionein comprises MTT1, MTT5, TM1 or TM2; the amino acid sequence of the MTT1 is shown in SEQ ID NO.1; the amino acid sequence of the MTT5 is shown in SEQ ID NO.2; the amino acid sequence of the TM1 is shown in SEQ ID NO.3; the amino acid sequence of the TM2 is shown in SEQ ID NO.4; the amino acid sequence of the CBM is shown in SEQ ID NO.5; the backbone plasmid of the recombinant vector comprises pET-28a-SUMO, pGEX-6P-1 or pGEX-4T-1.

[0052] In the present invention, the amino acid sequence of MTT1 is shown in SEQ ID NO.1, specifically: MDKVNSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCKPQANCC CGVNAKPCCFDPNSGCCCVSKTNNCCKSDTKECCTGTGEGCKCTSCQCCKPVQQGCCCGDKAKAC CTDPNSGCCCSNKANKCCDATSKQECQTCQCCK.

[0053] In the present invention, the amino acid sequence of MTT5 is shown in SEQ ID NO.2, specifically: MDKISGESTKICSKTEEKWCCCPSETQNCCNSDDKQCCVGSGEGCIYVCCKCCKVQAECKCGPNAK YCCIDPNTGNCCVCKTKFCSKSDSKECCPGGSC.

[0054] In the present invention, the amino acid sequence of TM1 is shown in SEQ ID NO.3, specifically: MDKVNSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCK.

[0055] In the present invention, the amino acid sequence of TM2 is shown in SEQ ID NO.4, specifically: MDKVNSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCKPQANCC CGVNAKPCCFDPNSGCCCVSKTNNCCKSDTKECCTGTGEGCKCTSCQCCK.

[0056] In the present invention, the amino acid sequence of the CBM is shown in SEQ ID NO.5, specifically: MDLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGS YNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSAS QFVEWDQVTAYLNGVLVWGKEPG.

[0057] In the present invention, the amino acid sequences of metallothionein MTT1 and MTT5 are obtained from the Tetrahymena genome database (http: / / www.ciliate.org). In the present invention, the MTT1 sequence is truncated, and only the first sequence module of MTT1 is retained as TM1, and the first and second sequence modules are retained as TM2.

[0058] In the present invention, the CBM and metallothionein are preferably connected by a flexible linker; the amino acid sequence of the flexible linker is shown in SEQ ID NO.6, specifically: GSAGSA. In the present invention, the use of a flexible linker can ensure that the metallothionein (MTT1, MTT5) and carbohydrate CBM components in the fusion protein can be correctly folded and effectively function.

[0059] In one embodiment of the present invention, the fusion protein comprises CBM and MTT1 connected sequentially from the C-terminus to the N-terminus, named CBM-MTT1, and the amino acid sequence is as shown in SEQ ID As shown in NO.7, MDLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEPGGSAGSAMDKV NSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCKPQANCCCGVNAKPCCFDPNSGCCCVSKTNNCCKSDTKECCTGTGEGCKCTSCQCCKPVQQGCCCGDKAKACCTDPNSGCCCSNKANKCCDATSKQECQTCQCCK, with a predicted size of approximately 34.57kDa.

[0060] In another embodiment of the present invention, the fusion protein includes CBM and MTT5 connected sequentially from the C-terminus to the N-terminus, named CBM-MTT5, and the amino acid sequence is shown in SEQ ID NO.8, specifically: MDLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEPGGSAGSAMDKISGESTKICSKTEEKWCCCPSETQNCCNSDDKQCCVGSGEGCIYVCCKCCKVQAECKCGPNAKYCCIDPNTGNCCVCKTKFCSKSDSKECCPGGSC, with a predicted size of approximately 28.41 kDa.

[0061] In another embodiment of the present invention, the fusion protein includes CBM and TM1 connected sequentially from the C-terminus to the N-terminus, named CBM-TM1, and the amino acid sequence is shown in SEQ ID NO.9, specifically: MDLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEPGGSAGSAMDKVNSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCK.

[0062] In another embodiment of the present invention, the fusion protein includes CBM and TM2 connected sequentially from the C-terminus to the N-terminus, named CBM-TM2, and the amino acid sequence is shown in SEQ ID NO.10, specifically: MDLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEPGGSAGSAMDKVNSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCKPQANCCCGVNAKPCCFDPNSGCCCVSKTNNCCKSDTKECCTGTGEGCKCTSCQCCK.

[0063] In the present invention, the C-terminus of the CBM is preferably modified with a tag; the tag preferably includes SUMO or GST. In the present invention, the tag helps to screen and obtain a sufficient amount of protein components.

[0064] In the present invention, the nucleotide sequence of the nucleic acid molecule CBM-MTT1 encoding the CBM-MTT1 is shown in SEQ ID NO.11, specifically:

[0065] atggacctgaaagtggagttctacaactccaatccgtccgacaccaccaactccatcaatccgcagttcaaagttaccaacaccggtagcagcgcgatcgatctgtctaaactgacgctgcgttactactacactgtggatggtcagaaagaccagaccttctggtgcgatcacgcggcaatcatcggttccaacggtagctacaacggcatcacctccaacgtgaaaggcaccttcgttaagatgtcttcttctaccaacaacgctgatacctacctggaaatcagcttcaccggcggtactttggaaccaggcgcgcacgttcagatccagggtcgctttgcgaagaacgactggagcaactacacgcagagcaacgactactctttcaaatccgcttctcagttcgtggaatgggaccaggtgactgcctacctgaacggtgttctggtgtggggcaaagaaccgggcggttctgcaggctccgcaatggataaagttaactcttgctgctgtggtgtgaacgctaaaccgtgctgcactgacccgaactctggttgctgttgtgtgtccaagaccgataactgctgcaaatccgacaccaaagaatgttgcaccggcaccggtgaaggttgcaaatgcgtgaactgtaaatgctgcaaaccgcaggctaactgctgttgcggcgttaacgcaaagccgtgctgcttcgatccaaactccggttgttgttgcgttagcaagaccaacaactgctgtaaatccgataccaaagaatgctgtacgggcaccggcgaaggttgtaaatgcaccagctgtcagtgctgtaaaccagtacagcagggctgctgctgcggcgacaaagctaaagcatgctgcaccgacccgaacagcggctgctgctgctccaacaaagcgaacaaatgctgcgacgctacctctaaacaagaatgccagacttgccagtgctgcaag, The fragment size is 975 bp.

[0066] In the present invention, the nucleotide sequence of the nucleic acid molecule CBM-MTT5 encoding the CBM-MTT5 is shown in SEQ ID NO.12, specifically:

[0067] , the fragment size is 786bp.

[0068] In the present invention, the nucleotide sequence of the nucleic acid molecule CBM-TM1 encoding the CBM-TM1 is shown in SEQ ID NO.13, specifically:

[0069] atggacctgaaagtggagttctacaactccaatccgtccgacaccaccaactccatcaatccgcagttcaaagttaccaacaccggtagcagcgcgatcgatctgtctaaactgacgctgcgttactactacactgtggatggtcagaaagaccagaccttctggtgcgatcacgcggcaatcatcggttccaacggtagctacaacggcatcacctccaacgtgaaaggcaccttcgttaagatgtcttcttctaccaacaacgctgatacctacctggaaatcagcttcaccggcggtactttggaaccaggcgcgcacgttcagatccagggtcgctttgcgaagaacgactggagcaactacacgcagagcaacgactactctttcaaatccgcttctcagttcgtggaatgggaccaggtgactgcctacctgaacggtgttctggtgtggggcaaagaaccgggcggttctgcaggctccgcaatggataaagttaactcttgctgctgtggtgtgaacgctaaaccgtgctgcactgacccgaactctggttgctgttgtgtgtccaagaccgataactgctgcaaatccgacaccaaagaatgttgcaccggcaccggtgaaggttgcaaatgcgtgaactgtaaatgctgcaaa。

[0070] In the present invention, the nucleotide sequence of the nucleic acid molecule CBM-TM2 encoding the CBM-TM2 is as shown in SEQ ID NO.14, specifically:

[0071] atggacctgaaagtggagttctacaactccaatccgtccgacaccaccaactccatcaatccgcagttcaaagttaccaacaccggtagcagcgcgatcgatctgtctaaactgacgctgcgttactactacactgtggatggtcagaaagaccagaccttctggtgcgatcacgcggcaatcatcggttccaacggtagctacaacggcatcacctccaacgtgaaaggcaccttcgttaagatgtcttcttctaccaacaacgctgatacctacctggaaatcagcttcaccggcggtactttggaaccaggcgcgcacgttcagatccagggtcgctttgcgaagaacgactggagcaactacacgcagagcaacgactactctttcaaatccgcttctcagttcgtggaatgggaccaggtgactgcctacctgaacggtgttctggtgtggggcaaagaaccgggcggttctgcaggctccgcaatggataaagttaactcttgctgctgtggtgtgaacgctaaaccgtgctgcactgacccgaactctggttgctgttgtgtgtccaagaccgataactgctgcaaatccgacaccaaagaatgttgcaccggcaccggtgaaggttgcaaatgcgtgaactgtaaatgctgcaaaccgcaggctaactgctgttgcggcgttaacgcaaagccgtgctgcttcgatccaaactccggttgttgttgcgttagcaagaccaacaactgctgtaaatccgataccaaagaatgctgtacgggcaccggcgaaggttgtaaatgcaccagctgtcagtgctgtaaa。

[0072] In the present invention, when the metallothionein is MTT1, the backbone plasmid of the recombinant vector preferably includes pGEX-6P-1; when the metallothionein is MTT5, the backbone plasmid of the recombinant vector preferably includes pET-28a-SUMO; when the metallothionein is TM1 and / or TM2, the backbone plasmid of the recombinant vector preferably includes pET-28a-SUMO and / or pGEX-4T-1.

[0073] In the present invention, the insertion site of the nucleic acid molecule on the recombinant vector includes restriction endonuclease BamHI and XhoⅠ restriction sites.

[0074] In the present invention, the recombinant vector is preferably constructed by Qingke Biotechnology Co., Ltd.

[0075] In one embodiment of the present invention, the backbone plasmid of the recombinant vector is pET28a, into which the coding gene of CBM-MTT1 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pET28a-CBM-MTT1.

[0076] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pET28a, into which the coding gene of CBM-MTT5 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pET28a-CBM-MTT5.

[0077] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pET-28a-SUMO, into which the coding gene of CBM-MTT1 is inserted, and the insertion sites are restriction endonuclease BamHⅠ and XhoⅠ restriction sites; the recombinant vector is named pSUMO-CBM-MTT1.

[0078] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pET-28a-SUMO, into which the coding gene of CBM-MTT5 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pSUMO-CBM-MTT5. In the present invention, the SUMO tag can promote the soluble expression of CBM-MTT5.

[0079] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pGEX-6P-1, into which the coding gene of CBM-MTT1 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pGST-CBM-MTT1. The GST fusion tag can promote the soluble expression of CBM-MTT1.

[0080] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pET-28a-SUMO, into which the coding gene CBM-TM1 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pSUMO-CBM-TM1. The SUMO fusion tag promotes the soluble expression of CBM-TM1.

[0081] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pET-28a-SUMO, into which the CBM-TM2 coding gene CBM-TM2 is inserted, and the insertion sites are restriction endonuclease BamHⅠ and XhoⅠ restriction sites; the recombinant vector is named pSUMO-CBM-TM2.

[0082] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pGEX-4T-1, into which the CBM-TM1 coding gene CBM-TM1 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pGST-CBM-TM1. The GST fusion tag promotes the soluble expression of CBM-TM1

[0083] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pGEX-4T-1, into which the CBM-TM2 coding gene CBM-TM2 is inserted, and the insertion sites are restriction endonuclease BamHⅠ and XhoⅠ restriction sites; the recombinant vector is named pGST-CBM-TM2.

[0084] The present invention also provides a host cell comprising the recombinant vector described in the above scheme.

[0085] The present invention has no particular limitation on the method for constructing the host cell, and conventional methods in the art may be used.

[0086] In the present invention, the original cell of the host cell includes Escherichia coli; the Escherichia coli is preferably Escherichia coli BL21 (DE3).

[0087] The present invention also provides a method for preparing the fusion protein, comprising the following steps:

[0088] The host cells described in the above scheme are induced and cultured to obtain an induced culture product; the fusion protein includes a CBM and a metallothionein connected sequentially from the C-terminus to the N-terminus; the metallothionein includes MTT1, MTT5, TM1 or TM2; the amino acid sequence of the MTT1 is shown in SEQ ID NO.1; the amino acid sequence of the MTT5 is shown in SEQ ID NO.2; the amino acid sequence of the TM1 is shown in SEQ ID NO.3; the amino acid sequence of the TM2 is shown in SEQ ID NO.4; the amino acid sequence of the CBM is shown in SEQ ID NO.5.

[0089] In the present invention, the induction agent for the induction culture preferably includes IPTG; the working concentration of the IPTG preferably includes 0-0.2 mMol / mL, more preferably 0, 0.05 mMol / mL, 0.1 mMol / mL or 0.2 mMol / mL; the temperature of the induction culture is preferably 16-37°C, more preferably 16°C, 24°C or 37°C. In the present invention, when the temperature of the induction culture is 16°C, the time of the induction culture is preferably 12-16h; when the temperature of the induction culture is 37°C, the time of the induction culture is preferably 4h; when the temperature of the induction culture is 24°C, the time of the induction culture is preferably 12h.

[0090] In one embodiment of the present invention, the host cell is obtained by transferring the recombinant vector pET28a-CBM-MTT1 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0, 0.05, 0.1 or 0.2 mM, and the temperature of the induction culture is 16 or 37° C. In this embodiment, the CBM-MTT1 protein is successfully expressed, the expression amount of the target protein CBM-MTT1 is low, and the expressed fusion protein is mostly present in the form of inclusion bodies.

[0091] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant vector pET28a-CBM-MTT5 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0, 0.05, 0.1 or 0.2 mM, and the temperature of the induction culture is 16 or 37° C. In this embodiment, CBM-MTT5 protein is successfully expressed, and more soluble protein is expressed at 16° C.

[0092] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant vector pSUMO-CBM-MTT1 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0, and the temperature of the induction culture is 24° C. In this embodiment, the fusion protein SUMO-CBM-MTT1 is successfully expressed.

[0093] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant vector pSUMO-CBM-MTT5 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0.1 mM, the induction culture temperature is 16° C., and the induction culture time is 16 h. In this embodiment, the fusion protein SUMO-CBM-MTT5 is successfully expressed.

[0094] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant vector pGST-CBM-MTT1 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0.2 mM, the temperature of the induction culture is 37° C., and the time of the induction culture is 4 hours. In this embodiment, the fusion protein GST-CBM-MTT1 is successfully expressed, and more soluble protein can be obtained under the above conditions.

[0095] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant vector pSUMO-CBM-TM1 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0.05, 0.1 or 0.2 mM, preferably 0.1 mM; the temperature of the induction culture is 16 or 37°C, preferably 16°C; when the temperature of the induction culture is 37°C, the time of the induction culture is 4h; when the temperature of the induction culture is 16°C, the time of the induction culture is 16h. In this embodiment, the fusion protein SUMO-CBM-TM1 is successfully expressed, and more soluble protein can be obtained by induction at 16°C and 0.1mM IPTG for 16h.

[0096] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant vector pSUMO-CBM-TM2 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0.05, 0.1 or 0.2mM, preferably 0.05 or 0.1mM; the temperature of the induction culture is 16 or 37°C, preferably 16°C; when the temperature of the induction culture is 37°C, the time of the induction culture is 4h; when the temperature of the induction culture is 16°C, the time of the induction culture is 16h. In this embodiment, the fusion protein SUMO-CBM-TM2 is successfully expressed, and more soluble protein can be obtained at 16°C, 0.05 or 0.1mM IPTG induction for 16h, and the amount of soluble protein under 0.05mM IPTG induction is slightly higher than that under 0.1mM IPTG induction.

[0097] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant vector pGST-CBM-TM1 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0.05, 0.1 or 0.2 mM, preferably 0.1 mM; the temperature of the induction culture is 16 or 37°C, preferably 37°C; when the temperature of the induction culture is 37°C, the time of the induction culture is 4h; when the temperature of the induction culture is 16°C, the time of the induction culture is 16h. In this embodiment, the fusion protein GST-CBM-TM1 is successfully expressed, and more soluble protein can be obtained by induction at 37°C and 0.1mM IPTG for 4h.

[0098] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant vector pGST-CBM-TM2 into Escherichia coli BL21 (DE3) cells, the working concentration of the IPTG is 0.05, 0.1 or 0.2 mM, preferably 0.1 or 0.2 mM; the temperature of the induction culture is 16 or 37 ° C, preferably 16 ° C; when the temperature of the induction culture is 37 ° C, the time of the induction culture is 4 h; when the temperature of the induction culture is 16 ° C, the time of the induction culture is 16 h. In this embodiment, the fusion protein GST-CBM-TM2 is successfully expressed, and more soluble protein can be obtained at 16 ° C, 0.1 or 0.2 mM IPTG induction for 16 h, and the amount of soluble protein under 0.2 mM IPTG induction is slightly higher than that under 0.1 mM IPTG induction.

[0099] The present invention also provides the use of the recombinant vector, the host cell or the preparation method described in the above scheme in the preparation of a cellulose-based composite heavy metal adsorbent.

[0100] The present invention also provides the use of the recombinant vector, the host cell or the preparation method described in the above scheme in heavy metal adsorption or heavy metal pollution control.

[0101] To further illustrate the present invention, the metallothionein recombinant vector, recombinant bacteria, and construction methods and applications thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0102] Example 1

[0103] 1. Fusion protein sequence design

[0104] The amino acid sequences of metallothionein MTT1 and MTT5 were obtained from the Tetrahymena genome database (http: / / www.ciliate.org). The amino acid sequence of MTT1 is shown in SEQ ID NO.1, and the amino acid sequence of MTT5 is shown in SEQ ID NO.2. According to literature reports, the CBM GenBank accession number was consulted, and the carbohydrate binding module CBM was determined by GenBank search and Blast comparison, and the amino acid sequence is shown in SEQ ID NO.5. In order to ensure that both the CBM and metallothionein components can be correctly and stably folded and function, the CBM and the metallothionein MTT1 and MTT5 components were connected using a flexible linker GSAGSA.

[0105] Metallothionein MTT1 and MTT5 belong to the 7a subfamily and contain the same motif. The MTT1 sequence is longer and has more repeating modules, mainly composed of type 1 and type 2 submodules. The sequence of type 1 submodule is CCCXXXXXXCCXXXXXX (SEQ ID NO.15), while the complete type 2 submodule can be expressed as CCXXXXXXCXCXXCXCCK (SEQ ID NO.16). Its cysteine ​​(Cys) arrangement has a typical regular modular distribution feature. The latter motif (type 2 submodule) only exists at the c-terminus of each module, but not in module 2 of MTT5 ( Figure 1 ). Metallothionein MTT1 is composed of three repeated sequence modules and contains a large amount of cysteine. In order to compare with the fusion protein CBM-MTT1 containing the full-length sequence of MTT1, the MTT1 sequence was truncated, retaining only the first sequence module of MTT1 as TM1, and retaining the first and second sequence modules as TM2 ( Figure 1 ).

[0106] In the sequence design of the fusion proteins CBM-MTT1 and CBM-MTT5, the N-terminal connection of the CBM module was used to introduce a flexible linker GSAGSA to ensure that the fusion protein metallothionein (MTT1, MTT5) and the carbohydrate CBM component can be correctly folded and effectively function, see Figure 2 (a) and (b) in FIG. 3 . The fusion proteins CBM-TM1 and CBM-TM2 are also linked by the flexible linker GSAGSA, see Figure 2 (c) and (d) in .

[0107] 2. Obtaining pET28a-CBM-MTT1 and pET28a-CBM-MTT5 plasmids and constructing pSUMO-CBM-MTT1, pSUMO-CBM-MTT5 and pGST-CBM-MTT1 recombinant vectors

[0108] 1) Based on the gene sequences of CBM-MTT1 and CBM-MTT5, restriction endonuclease BamHⅠ and XhoⅠ restriction sites were designed and introduced. The prokaryotic expression vector pET-28a was selected as the vector and delivered to Qingke Biotechnology Company for synthesis of pET28a-CBM-MTT1 and pET28a-CBM-MTT5 plasmids. The results are shown in Figure 3 and Figure 4 After obtaining the plasmid, a small amount of restriction enzyme digestion was performed using restriction enzymes BamHI and XhoⅠ (Table 1) and detected by 1% agarose gel electrophoresis. The results are shown in Figure 5 and Figure 6 The obtained pET28a-CBM-MTT1 and pET28a-CBM-MTT5 plasmids were successfully verified by enzyme digestion.

[0109] Table 1 Identification of small amount of enzyme digestion

[0110]

[0111] 2) In order to further obtain more soluble protein expression, recombinant vectors pSUMO-CBM-MTT1 and pSUMO-CBM-MTT5 with SUMO-tagged proteins were constructed.

[0112] Plasmids pET28a-CBM-MTT1, pET28a-CBM-MTT5 and prokaryotic expression vector pET-28a(+)-SUMO were double-digested with restriction endonucleases BamHⅠ and XhoⅠ, respectively, in a 37°C water bath for 8 h. The double-digestion system is shown in Table 2.

[0113] Table 2 Double enzyme digestion system (large amount)

[0114]

[0115] The target fragments were recovered by 1% agarose gel electrophoresis and connected overnight at 16°C using T4 DNA ligase. The CBM-MTT1 and CBM-MTT5 sequence fragments were respectively integrated into the pET-28a-SUMO vector. The connection system is shown in Table 3.

[0116] Table 3 T4 DNA Ligase ligation system

[0117]

[0118]

[0119] The ligation product was transformed into E. coli DH5ɑ competent cells and cultured overnight for 12-16 hours. 8-10 monoclonal colonies with good growth were picked on the LB (containing Kana) plate and positive monoclonal strains were screened with Cracking buffer. The selected strains were transferred to 5mL LB (containing Kana) liquid culture medium and cultured at 37°C and 180rpm for 12 hours to amplify the plasmid. The plasmid was extracted with a plasmid extraction kit of Biotech. A small amount of enzyme digestion (Table 1) was performed to identify whether the obtained recombinant vector successfully cloned the target fragment.

[0120] Schematic diagrams of the SUMO-tagged recombinant vector pSUMO-CBM-MTT1 and the SUMO-tagged recombinant vector pSUMO-CBM-MTT5 are shown in Figure 7 and Figure 8 After successful plasmid extraction, enzyme digestion verification and 1% agarose gel electrophoresis were performed. The test results are shown in Fig. 9 and Fig.10 Theoretically, the sizes of CBM-MTT1 and CBM-MTT5 gene fragments are 978 bp and 789 bp, respectively. The actual results are consistent with the predictions, indicating that the plasmids are constructed correctly and can be used for the expression of target proteins.

[0121] 3) Construct a recombinant vector pGST-CBM-MTT1 with a GST-tagged protein, and use restriction endonucleases BamHⅠ and XhoⅠ to double-digest the plasmid pET28a-CBM-MTT1 and the prokaryotic expression vector pGEX-6P-1, respectively, in a 37°C water bath for 8 h. The double-digestion system is the same as in Table 2. Recover the target fragments, and use T4 DNA ligase to connect overnight at 16°C to integrate the CBM-MTT1 sequence fragments into the pGEX-6P-1 vector, respectively. The connection system is the same as in Table 3.

[0122] The ligation product was transformed into E. coli DH5ɑ competent cells, cultured overnight for 12-16 hours, 8-10 monoclonal colonies with good growth on the LB (containing Kana) plate were picked, and positive monoclonal strains were screened with Cracking buffer. The selected strains were transferred to 5mL LB (containing Kana) liquid culture medium, cultured overnight at 37°C and 180rpm for 12 hours, and the plasmid was extracted using a plasmid extraction kit of the Biotech Group. A small amount of enzyme digestion (Table 1) was performed to identify whether the obtained recombinant vector was recombined with the target fragment.

[0123] The schematic diagram of the recombinant vector pGST-CBM-MTT1 with GST-tagged protein is shown in Fig.11 After successful plasmid extraction, enzyme digestion verification and 1% agarose gel electrophoresis were performed. The test results are shown in Fig.12Theoretically, the size of the CBM-MTT1 gene fragment is 978 bp, and the actual result is consistent with the prediction, indicating that the plasmid is constructed correctly and can be used for the expression of the target protein.

[0124] 3. Expression of CBM-MTT1, CBM-MTT5 and fusion proteins SUMO-CBM-MTT1, SUMO-CBM-MTT5 and GST-CBM-MTT1 in Escherichia coli

[0125] The recombinant vectors pET28a-CBM-MTT1, pET28a-CBM-MTT5, pSUMO-CBM-MTT1, pSUMO-CBM-MTT5 and pGST-CBM-MTT1 as well as pGEX-6P-1 and pET-28a-SUMO vectors (control) were transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB (containing Kana or Amp) solid plates, and cultured in a constant temperature incubator at 37°C for 12 to 16 hours. Take 5 mL of LB liquid culture medium and add Kana or Amp at a final concentration of 100 μg / mL. Pick 3 to 5 single colonies respectively, place them in LB liquid culture medium, and shake and culture them at 37°C in a constant temperature shaker for 12 hours. Transfer 50 μL of bacterial solution to the corresponding 5 mL of LB liquid culture medium, and shake and culture the bacterial solution at 37°C in a constant temperature shaker. OD 600 To 0.4-0.6, add IPTG with a final concentration of 0.1mM / mL, and induce culture at 37℃ for 4h. Centrifuge at 12,000r / min for 1min to collect bacteria, wash the bacteria once with PBS buffer (pH=7.4), centrifuge at 12,000r / min for 1min, resuspend with PBS buffer, and use ultrasonic cell disruptor to ultrasonically disrupt the bacteria (power 100W, on 4s off 6s, ultrasonic 4min). Take the whole broken bacteria solution, centrifuge at 12,000rpm for 15min, take the supernatant solution, add 5× protein loading buffer and heat at 100℃ for 10min for sample preparation, and perform expression analysis by 12% SDS-PAGE gel electrophoresis (voltage 90V, 30min, 120V, 1h).

[0126] Obtain strains that successfully express CBM-MTT1, CBM-MTT5, and fusion proteins SUMO-CBM-MTT1, SUMO-CBM-MTT5, and GST-CBM-MTT1, or express well, and explore and optimize the expression conditions. Use 80% glycerol-preserved strains to streak on solid plates to pick out single clones or directly take 10-50 μL of bacterial solution and add it to 5 mL LB liquid culture medium (containing Kana / Amp), and culture it in a constant temperature shaker at 37°C overnight. Transfer 50 μL of bacterial solution to the corresponding 5 mL LB (containing Kana / Amp) liquid culture medium, and culture the bacterial solution in a constant temperature shaker at 37°C until OD 600 Reach 0.4 to 0.6. Add IPTG at a final concentration of 0.05, 0.1, and 0.2 mM, and culture at 37°C (4h), 16°C (12h / 16h), or 24°C (12h) to induce the expression of target proteins CBM-MTT1, CBM-MTT5, SUMO-CBM-MTT1, SUMO-CBM-MTT5, and GST-CBM-MTT1. Collect the cells by centrifugation at 12,000 rpm for 1 min, wash the collected cells once with PBS buffer, collect the cells after centrifugation and resuspend them in PBS buffer solution, disrupt the cells by ultrasound, and take the whole bacterial solution and supernatant for sample preparation. Analyze the protein expression by 12% SDS-PAGE gel electrophoresis, and determine the protein concentrations of GST-CBM-MTT1 and SUMO-CBM-MTT5 using a protein quantification kit.

[0127] The recombinant vectors pET28a-CBM-MTT1 and pET28a-CBM-MTT5 were respectively transferred into E. coli BL21 (DE3) cells, and IPTG induced the expression of CBM-MTT1 and CBM-MTT5 at 37°C and 16°C, respectively. The predicted sizes of CBM-MTT1 and CBM-MTT5 were about 34.57 kDa and 28.41 kDa. The results of 12% SDS-PAGE gel electrophoresis showed that compared with the control, new protein bands appeared at 35 kDa and 28 kDa, respectively. The protein expression results were consistent with the prediction, see Fig.13 (a) and (b) in the figure show that CBM-MTT1 and CBM-MTT5 proteins were successfully expressed, the target protein CBM-MTT1 was expressed at a low level, and the expressed fusion protein was mostly in the form of inclusion bodies. The target protein CBM-MTT5 expressed more soluble protein at 16°C, but the overall expression level was low, see Fig.13 (c) in .

[0128] The recombinant vectors pSUMO-CBM-MTT1 and pGST-CBM-MTT1 were respectively transferred into E. coli BL21 (DE3) cells, and induced to express fusion proteins SUMO-CBM-MTT1 and GST-CBM-MTT1 at different IPTG concentrations at 37°C, 24°C, and 16°C, respectively. The predicted protein sizes were about 48.4 kDa and 61.4 kDa (the theoretical molecular weight of SUMO tag is about 11.2 kDa, and the theoretical molecular weight of GST tag is about 26 kDa). The results of 12% SDS-PAGE showed that compared with the control, new protein bands appeared at 48 kDa and 61 kDa, respectively, see Fig.14 (a) and Fig.15 (a) in the figure is consistent with the predicted molecular weight of SUMO-CBM-MTT1 and GST-CBM-MTT1, indicating that the fusion proteins SUMO-CBM-MTT1 and GST-CBM-MTT1 were successfully expressed. SUMO-CBM-MTT1 was also detected by Western blotting experiments to further confirm the soluble expression of SUMO-CBM-MTT1 without induction, see Fig.14 (c) in the figure. After analysis, it was found that the fusion protein SUMO-CBM-MTT1 was quite special. It could obtain more soluble protein without IPTG induction. After combining the conditions of no induction and IPTG induction at 37℃ and 16℃, different IPTG concentrations were used for induction at 24℃. The results showed that more soluble protein was obtained without IPTG induction. Fig.14 (b) in the figure; the fusion protein GST-CBM-MTT1 can obtain more soluble protein at 37℃ than at 16℃. Further induction with gradient concentration of IPTG revealed that more soluble protein can be obtained by induction with 0.2mM IPTG for 4h, see Fig.15 (b) in .

[0129] The recombinant vector pSUMO-CBM-MTT5 was transferred into E. coli BL21 (DE3) cells, and the fusion protein SUMO-CBM-MTT5 was expressed under IPTG induction at 37°C and 16°C. The protein molecular weight was predicted to be about 42.2 kDa (the theoretical molecular weight of SUMO tag is about 11.2 kDa). The results of 12% SDS-PAGE showed that compared with the control, a new protein band appeared at 42 kDa, see Fig.16(a) in the figure is consistent with the predicted molecular weight of SUMO-CBM-MTT5, indicating that the fusion protein SUMO-CBM-MTT5 was successfully expressed. SDS-PAGE analysis showed that the optimal induction expression conditions for the fusion protein SUMO-CBM-MTT5 were 0.1 mM IPTG, 16°C, and 16 h. SUMO-CBM-MTT5 was further confirmed to be soluble by Western blotting experiments. Fig.16 (c) in .

[0130] The concentrations of the fusion proteins GST-CBM-MTT1 and SUMO-CBM-MTT5 were determined to be 1.55 mg / mL and 2.06 mg / mL, respectively. To further determine the content of the fusion proteins GST-CBM-MTT1 and SUMO-CBM-MTT5, SUMO-CBM-MTT5 was diluted 10 times and then subjected to 12% SDS-PAGE detection, see Fig.16 In (b), grayscale scanning analysis was performed by ImageJ, and the expression products GST-CBM-MTT1 and SUMO-CBM-MTT5 accounted for 4.46% and 26.78% of the total protein, respectively. After calculation, it was found that the crude fusion protein content in 1L supernatant was approximately 69.2mg and 552mg, respectively. Compared with the fusion proteins of different tags of CBM-MTT1, SUMO-CBM-MTT5 showed a relatively considerable expression amount and solubility.

[0131] In summary, by comparing the expression levels and soluble protein contents of CBM-MTT1, SUMO-CBM-MTT1, and GST-CBM-MTT1 fusion proteins, only the GST fusion tag has a more obvious promoting effect on the soluble expression of CBM-MTT1. For CBM-MTT5, when expressed in E. coli based on the pET-28a vector, although the expression level of the target protein was low overall, it was expressed itself. After fusion with the SUMO tag, the expression of CBM-MTT5 was further promoted and the soluble protein content also increased, indicating that the SUMO tag has more advantages for the soluble expression of CBM-MTT5.

[0132] 4. Cloning of truncated CBM-MTT1 (CBM-TM1, CBM-TM2) gene fragments

[0133] The CBM-MTT1 gene was truncated, and the plasmid pSUMO-CBM-MTT1 was used as a template. The primers CBM-TM1-F / CBM-TM1-R containing the restriction enzyme sites BamHI and XhoⅠ were designed (Table 4) to amplify the CBM-TM1 fragment. The PCR reaction conditions were as follows: 94℃, 5min, 94℃, 30s, 55℃, 30s, 68℃, 40s, 35 cycles, 68℃, 10min. The reaction system is shown in Table 5. The primers CBM-TM1-F / CBM-TM2-R containing the restriction enzyme sites BamHI and XhoⅠ were designed (Table 4) to amplify the CBM-TM2 fragment. The PCR reaction conditions were as follows: 94℃, 5min, 94℃, 30s, 58℃, 30s, 68℃, 50s, 35 cycles, 68℃, 10min. The reaction system is shown in Table 5.

[0134] Table 4 Amplification primers

[0135] PrimerName Sequence(5'-3') RECS CBM-TM1-F cgggatccatggacctgaaagtggagttctac, SEQ ID NO.17 B H CBM-TM1-R ccctcgagtcatttgcagcatttacagttcacgcat, SEQ ID NO.18 XOt CBM-TM2-R ccctcgagtcatttacagcactgacagctggtgc, SEQ ID NO.19 XOt

[0136] Table 5 PCR amplification of CBM-TM1 and CBM-TM2

[0137]

[0138] The PCR products were detected by 1% agarose gel electrophoresis ( Fig.17 Theoretically, the PCR product sizes of and genes should be 831bp and 663bp respectively. The actual electrophoresis detection results are consistent with the predictions, indicating that the size of the target fragment amplified by the gene sequence is correct and can be used for subsequent construction.

[0139] After PCR amplification and identification, the 60 μL system was used to amplify the CBM-TM1 and CBM-TM2 fragments in large quantities, and the PCR products were recovered by 1% agarose gel electrophoresis. The recovered fragments were connected to pMD-19-T at 16°C overnight (Table 6), and the CBM-TM1 and CBM-TM2 fragments were integrated into the pMD19-T vector through the T terminus to facilitate subsequent enzyme cutting and connection. The ligation product was transformed into Escherichia coli DH5ɑ competent state and cultured on LB (containing Amp) solid plates for 12 hours. Pick 8 to 10 monoclonal colonies with good growth on the solid plate, and use Cracking buffer to screen positive monoclonal strains. The 2 to 3 strains that were correctly screened were cultured, and the plasmid was extracted using the Biotech plasmid extraction kit. The obtained plasmid was identified by enzyme cutting to see whether it was successfully inserted into the target fragment. The correct plasmids pMD-19T-CBM-TM1 and pMD-19T-CBM-TM2 were sent to Bioengineering (Shanghai) Co., Ltd. for DNA sequence determination to determine whether base mutations occurred in the CBM-TM1 and CBM-TM2 fragments obtained by PCR amplification and cloning, and whether cloning was successful.

[0140] Table 6 T-A connection system

[0141] Reagents Volume (μL) Target fragment (CBM-TM1, CBM-TM2) 4 Mix 5 T-Vector (pMD-19-T) 1 Total volume 10

[0142] 5. Construction of pSUMO-CBM-TM1, pGST-CBM-TM1, pSUMO-CBM-TM2, pGST-CBM-TM2 recombinant vectors

[0143] After the TA cloning sequencing test was correct, the pSUMO-CBM-TM1, pGST-CBM-TM1 and pSUMO-CBM-TM2, pGST-CBM-TM2 recombinant vectors were constructed. The plasmids pMD-19T-CBM-TM1, pMD-19T-CBM-TM2 and the prokaryotic expression vectors pET-28a-SUMO and pGEX-4T-1 were double-digested with restriction endonucleases BamHⅠ and XhoⅠ, respectively, and incubated in a 37℃ water bath for 8h. The double-digestion system is shown in Table 7.

[0144] Table 7 Double enzyme digestion system (large amount)

[0145]

[0146] The target fragments were recovered by 1% agarose gel electrophoresis and connected overnight at 16°C using T4 DNA ligase. The CBM-TM1 sequence was integrated into the pET-28a-SUMO and pGEX-4T-1 vectors, and the CBM-TM2 sequence was integrated into the pET-28a-SUMO and pGEX-4T-1 vectors, respectively. The connection system is shown in Table 8.

[0147] Table 8 T4 DNALigase ligation system

[0148]

[0149] The ligation product was transformed into E. coli DH5ɑ competent cells, cultured overnight for 12-16 hours, and 8-10 monoclonal colonies with good growth on the LB (containing Kana / Amp) plate were picked, and positive monoclonal strains were screened with Cracking buffer. The selected strains were transferred to 5mL LB (containing Kana / Amp) liquid culture medium, cultured at 37°C and 180rpm for 12 hours to amplify the plasmid, and the plasmid was extracted with a plasmid extraction kit of the Biotech Group. PCR amplification (Table 5) and a small amount of enzyme digestion (Table 1) were performed to identify whether the obtained recombinant vector successfully recombined the target fragment.

[0150] The plasmid map and identification diagram of the recombinant vector pSUMO-CBM-TM1 can be found in Fig.18 and Fig.19The plasmid map and identification diagram of the recombinant vector pGST-CBM-TM1 can be found at Fig. 20 and Fig.21 The plasmid map and identification diagram of the recombinant vector pSUMO-CBM-TM2 (pSUMO-CBM-TM1.1) can be found at Fig. 22 and Fig.23 The plasmid map and identification map of the recombinant vector pGST-CBM-TM2 (pGST-CBM-TM1.1) can be found at Fig.24 and Fig.25 PCR experiments and BamH I and Xho I double enzyme digestion experiments showed that the recombinant vectors pSUMO-CBM-TM1, pGST-CBM-TM1, pSUMO-CBM-TM2, and pGST-CBM-TM2 were successfully constructed.

[0151] 6. Expression of SUMO-CBM-TM1, GST-CBM-TM1, SUMO-CBM-TM2, and GST-CBM-TM2 in Escherichia coli

[0152] The correctly identified recombinant vectors pSUMO-CBM-TM1, pGST-CBM-TM1, pSUMO-CBM-TM2, and pGST-CBM-TM2 were transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB (containing Kana / Amp) solid plates, and cultured in a constant temperature incubator at 37°C for 12 to 16 hours. Take 5mL LB liquid culture medium and add Kana / Amp with a final concentration of 100μg / mL; pick 3 to 5 single colonies, place them in LB liquid culture medium, and shake and culture them at 37°C in a constant temperature shaker for 12 to 16 hours. Transfer 50μL of bacterial solution to the corresponding 5mL LB (containing Kana / Amp) liquid culture medium, and shake and culture the bacterial solution at 37°C in a constant temperature shaker until OD 600 To 0.4-0.6, add IPTG with a final concentration of 0.1mMol / mL, and induce culture at 37℃ and 16℃ for 4h. Centrifuge at 12,000r / min for 1min to collect the bacteria, wash the bacteria once with PBS buffer, centrifuge again at 12,000r / min for 1min, resuspend with PBS buffer, and use ultrasonic cell disruptor to ultrasonically disrupt the bacteria (power 100W, on 4s off 6s, ultrasonic 4min). Take the whole broken bacteria, centrifuge at 12,000rpm for 15min, take the supernatant, add 5× protein loading buffer for sample preparation, and perform expression analysis by 12% SDS-PAGE gel electrophoresis (voltage 90V, 30min, 120V, 1h).

[0153] The protein expression conditions were explored and optimized using the screened BL21 / pSUMO-CBM-TM1, BL21 / pGST-CBM-TM1, BL21 / pSUMO-CBM-TM2 and BL21 / pGST-CBM-TM2 strains. Use the glycerol-preserved strains to streak solid plates to pick single clones or directly take 10-50 μL of bacterial solution and add it to 5 mL LB liquid culture medium (containing Kana / Amp), and shake culture it at 37°C in a constant temperature shaker overnight. Transfer 50 μL of bacterial solution to the corresponding 5 mL LB (containing Kana / Amp) liquid culture medium, and shake culture the bacterial solution at 37°C in a constant temperature shaker. OD 600 To 0.4-0.6. Add final concentrations of 0.05, 0.1, and 0.2 mM IPTG, and culture at 37°C (4h) and 16°C (16h), respectively, to induce the expression of fusion proteins SUMO-CBM-TM1, GST-CBM-TM1, SUMO-CBM-TM2, and GST-CBM-TM2, collect the cells by centrifugation at 12,000 rpm for 1 min, wash the collected cells once with PBS buffer, collect the cells after centrifugation at 12,000 rpm for 1 min and resuspend them in PBS buffer solution, disrupt the cells by ultrasonication, collect the whole cell solution and the supernatant for sample preparation, and analyze the protein expression by 12% SDS-PAGE gel electrophoresis.

[0154] The recombinant vectors pSUMO-CBM-TM1 and pGST-CBM-TM1 were respectively transferred into E. coli BL21 (DE3) cells, and different IPTG concentrations were used to induce the expression of fusion proteins SUMO-CBM-TM1 and GST-CBM-TM1. The predicted protein sizes were about 37.6 kDa and 50.7 kDa (the theoretical molecular weight of SUMO tag is about 11.2 kDa, and the theoretical molecular weight of GST tag is about 26 kDa). The results of 12% SDS-PAGE gel electrophoresis showed that compared with the control, new protein bands appeared at 38 kDa and 51 kDa, respectively. Fig.26 . It is consistent with the predicted molecular weight of SUMO-CBM-TM1 and GST-CBM-TM1, indicating that the fusion proteins SUMO-CBM-TM1 and GST-CBM-TM1 were successfully expressed. After analysis, it was found that the fusion protein SUMO-CBM-TM1 preferred to be induced at 16°C, and more soluble protein could be obtained by inducing at 16°C and 0.1mM IPTG for 16h; while GST-CBM-TM1 preferred to be induced at 37°C, and more soluble protein could be obtained by inducing at 37°C and 0.1mM IPTG for 4h.

[0155] The concentrations of fusion proteins SUMO-CBM-TM1 and GST-CBM-TM1 were determined to be 2.19 mg / mL and 2.42 mg / mL, respectively. To further determine the content of fusion proteins SUMO-CBM-TM1 and GST-CBM-TM1, both were diluted 5 times and then subjected to 12% SDS-PAGE detection ( Fig. 27 ), grayscale scanning analysis was performed by ImageJ, and the expression products SUMO-CBM-TM1 and GST-CBM-TM1 accounted for 16.77% and 27.57% of the total protein, respectively. After calculation, it was found that the crude fusion protein content in 1L supernatant was approximately 367mg and 667mg, respectively. Compared with the fusion proteins of different tags of CBM-MTT1, SUMO-CBM-TM1 and GST-CBM-TM1 showed higher expression levels and better solubility.

[0156] The recombinant vectors pSUMO-CBM-TM2 and pGST-CBM-TM2 were respectively transferred into E. coli BL21 (DE3) cells, and the fusion proteins SUMO-CBM-TM2 and GST-CBM-TM2 were induced to express at different IPTG concentrations at 37°C and 16°C. The predicted protein sizes were about 43.4 kDa and 56.4 kDa (the theoretical molecular weight of SUMO tag is about 11.2 kDa, and the theoretical molecular weight of GST tag is about 26 kDa). The results of 12% SDS-PAGE electrophoresis showed that compared with the control, new protein bands appeared at 43 kDa and 56 kDa, respectively ( Fig.28 ), which is consistent with the predicted molecular weight of SUMO-CBM-TM2 and GST-CBM-TM2, indicating that the fusion proteins SUMO-CBM-TM2 and GST-CBM-TM2 were successfully expressed. After analysis, it was found that the fusion proteins SUMO-CBM-TM2 and GST-CBM-TM2 both preferred to be induced at 16°C, and the amount of soluble protein of SUMO-CBM-TM2 under 0.05mM IPTG induction was slightly higher than that under 0.1mM IPTG induction; while the amount of soluble protein of GST-CBM-TM2 under 0.2mM IPTG induction was slightly higher than that under 0.1mM IPTG induction.

[0157] The concentrations of fusion proteins SUMO-CBM-TM2 and GST-CBM-TM2 were determined to be 1.29 mg / mL and 1.72 mg / mL, respectively. To further determine the content of fusion proteins SUMO-CBM-TM2 and GST-CBM-TM2, both were diluted 5 times and subjected to 12% SDS-PAGE detection ( Fig.29), grayscale scanning analysis was performed by ImageJ, and the expression products SUMO-CBM-TM2 and GST-CBM-TM2 accounted for 10.68% and 8.27% of the total protein, respectively. After calculation, it was found that the crude fusion protein content in 1L supernatant was approximately 137mg and 142mg, respectively. Compared with the fusion proteins of different tags of CBM-MTT1, CBM-TM2 also showed a relatively considerable expression amount and solubility.

[0158] In summary, based on the expression of CBM-TM1 in E. coli by pET-28a-SUMO and pGEX-4T-1, it was found that the fusion of SUMO and GST tags promoted the soluble expression of CBM-TM1, but both had obvious temperature preferences, and lower temperature induction was also more conducive to protein folding. The contents of SUMO-CBM-TM1 and GST-CBM-TM1 in 1L crude protein solution were approximately 367mg and 667mg, respectively. For the expression of pSUMO-CBM-TM2 and pGST-CBM-TM2 in E. coli, both SUMO-CBM-TM2 and GST-CBM-TM2 had higher soluble expression of fusion proteins at 16°C, and the contents of the target proteins obtained were approximately 137mg and 142mg, respectively. Compared with SUMO-CBM-TM1 and GST-CBM-TM1, both the expression level and the soluble protein amount of SUMO-CBM-TM2 and GST-CBM-TM2 decreased, while the soluble protein amount of SUMO-CBM-MTT1 and GST-CBM-MTT1 increased. The protein content of different MT components with the same tag also showed CBM-TM1>CBM-TM2>CBM-MTT1.

[0159] Example 2 Preparation of Several Fusion Protein Composite Heavy Metal Adsorbents

[0160] 1. Preparation of composite heavy metal adsorbent cellulose-GST-CBM-MTT1

[0161] The recombinant bacteria BL21 / pGST-CBM-MTT1 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein GST-CBM-MTT1. The supernatant of the obtained fusion protein GST-CBM-MTT1 was used as the fusion protein solution (concentration of 1.55 mg / mL); 0.1 g of cellulose was added to 1 mL of the fusion protein solution, and adsorbed at 25°C and 100 rpm for 2 hours. When the timing was over, the supernatant after adsorption was taken and added to 5× protein loading buffer for sample preparation.

[0162] 2. Preparation of composite heavy metal adsorbent cellulose-SUMO-CBM-TM1

[0163] The recombinant bacteria BL21 / pSUMO-CBM-TM1 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein SUMO-CBM-TM1.

[0164] The supernatant of fusion protein SUMO-CBM-TM1 was used as fusion protein solution (concentration was 2.19 mg / mL), 0.05 g cellulose was added to 0.5 mL fusion protein solution and 0.5 mL PBS buffer, and adsorbed at 25°C and 100 rpm for 2 h. The supernatant after adsorption was added to 5× protein loading buffer for sample preparation.

[0165] 3. Preparation of composite heavy metal adsorbent cellulose-SUMO-CBM-TM2

[0166] The recombinant bacteria BL21 / pSUMO-CBM-TM2 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein SUMO-CBM-TM2.

[0167] The preparation of the composite heavy metal adsorbent cellulose-SUMO-CBM-TM2 refers to step 2.

[0168] 4. Preparation of composite heavy metal adsorbent cellulose-SUMO-CBM-MTT5

[0169] The recombinant bacteria BL21 / pSUMO-CBM-MTT5 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein SUMO-CBM-MTT5.

[0170] The supernatant of the obtained fusion protein pSUMO-CBM-MTT5 was used as the fusion protein solution, 0.1 g of cellulose was added to 1 mL of the fusion protein solution, and adsorbed at 25° C. and 100 rpm for 2 h. After adsorption, the supernatant was taken and added to 5× protein loading buffer for sample preparation.

[0171] Example 3 The composite heavy metal adsorbent prepared in Example 1 is used for heavy metal ion Pb 2+ 、Cd 2+ Adsorption

[0172] Dilution of heavy metal ions (Pb 2+ 、Cd 2+ ) mother liquor, with an initial concentration of 40 mg / L. The prepared composite heavy metal adsorbents cellulose-GST-CBM-MTT1, cellulose-SUMO-CBM-TM1, cellulose-SUMO-CBM-TM2 and cellulose-SUMO-CBM-MTT5 were used for Pb 2+ 、Cd2+ Adsorption experiment.

[0173] In order to reduce the influence of PBS buffer and attachments on the adsorption of heavy metal ions, the prepared adsorbent was washed twice with deionized water (the volume of deionized water corresponded to the volume of the fusion protein solution added when preparing the adsorbent), added to 40 mL of a heavy metal ion solution with an initial concentration of 40 mg / L (adjusted to pH ≈ 7 with NaOH), and adsorbed at 25 °C and 100 rpm for 3 h. After adsorption, the adsorbent was collected by centrifugation at 8,000 rpm for 10 min, and 5 mL of the solution was taken for determination of heavy metal ions (Pb 2+ 、Cd 2+ ) concentration. Each single ion adsorption experiment was performed in triplicate. The conical flask used was nitrated with 20% dilute nitric acid solution and reused.

[0174] Composite heavy metal adsorbent for Pb 2+ and Cd 2+ The removal rate (%) and adsorption capacity are calculated according to the following formula:

[0175] Removal rate: R (%) = (C0-C) / C0×100% (C0: initial concentration of heavy metal ions, C: concentration of heavy metal ions in the solution after adsorption)

[0176] Adsorption amount = (C0-C) × V / M DW (C0: initial concentration of heavy metal ions, C: concentration of heavy metal ions in the solution after adsorption, V: volume of heavy metal ion solution)

[0177] All experiments were repeated 3 times. SPSS Statistics 27.0 software was used for statistical analysis. Origin 8.0 was used for graphics. One-way ANOVA was used to analyze the differences between the control group and the experimental group (*p<0.05 indicates a significant difference, **p<0.01 indicates an extremely significant difference). Dry weight (DW) was used to calculate the corresponding parameters.

[0178] The results show:

[0179] Compared with pure cellulose, the composite heavy metal adsorbent cellulose-GST-CBM-MTT1 has significant differences in removal efficiency and adsorption capacity. 2+ The removal rate and adsorption capacity of Pb were 32.6% and 2.61 mg / g, respectively, while pure cellulose 2+ The removal rate and adsorption capacity of Cd were 18.7% and 1.49 mg / g respectively; the composite heavy metal adsorbent cellulose-GST-CBM-MTT1 had a strong adsorption capacity for Cd 2+The removal rate and adsorption capacity of pure cellulose for Cd were 39.2% and 3.13 mg / g, respectively. 2+ The removal rate and adsorption capacity were 22.3% and 1.79 mg / g ( Fig.30 ).

[0180] The composite heavy metal adsorbent cellulose-SUMO-CBM-TM1 has a higher adsorption rate for Pb than pure fiber. 2+ 、Cd 2+ The ability of the composite heavy metal adsorbent cellulose-SUMO-CBM-TM1 to absorb Pb 2+ The removal rate and adsorption capacity of Cd were 59.3% and 4.75 mg / g respectively; the removal rate and adsorption capacity of Cd of composite heavy metal adsorbent cellulose-SUMO-CBM-TM1 were 59.3% and 4.75 mg / g respectively. 2+ The removal rate and adsorption capacity were 52.1% and 4.17 mg / g ( Fig.31 ).

[0181] The composite heavy metal adsorbent cellulose-SUMO-CBM-TM2 has a higher adsorption rate for Pb than pure fiber 2+ 、Cd 2+ There is a significant difference in the ability of Pb 2+ 、Cd 2+ The removal rates of Pb and 2+ 、Cd 2+ The adsorption amounts were 5.69 mg / g and 4.36 mg / g respectively. Fig.32 ).

[0182] Compared with pure fiber, the composite heavy metal adsorbent cellulose-SUMO-CBM-MTT5 adsorbs heavy metal ions, especially Pb 2+ The ability of Pb has been significantly improved. 2+ 、Cd 2+ The removal rates of Pb and 2+ 、Cd 2+ The adsorption amounts were 5.69 mg / g and 3.45 mg / g respectively. Fig.33 ).

[0183] In summary, compared with pure cellulose, it was found that each composite heavy metal adsorbent had a significant effect on the removal of heavy metal ions. The molecular weights of SUMO-CBM-TM2 and SUMO-CBM-MTT5 proteins were similar, and the amount of protein bound to cellulose was similar. Therefore, cellulose-SUMO-CBM-TM2 and cellulose-SUMO-CBM-MTT5 had a significant effect on the removal of heavy metal ions Pb. 2+ The adsorption amount of Cd 2+The adsorption capacity of cellulose-SUMO-CBM-TM2 was slightly higher than that of cellulose-SUMO-CBM-MTT5. 2+ The ability is significantly stronger than Cd 2+ .

[0184] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant vector, characterized in that: A nucleic acid molecule encoding a fusion protein is inserted; the fusion protein comprises a CBM and a metallothionein connected in sequence from the C-terminus to the N-terminus; the metallothionein comprises MTT1, MTT5, TM1 or TM2; the amino acid sequence of the MTT1 is shown in SEQ ID NO.1; the amino acid sequence of the MTT5 is shown in SEQ ID NO.2; the amino acid sequence of the TM1 is shown in SEQ ID NO.3; the amino acid sequence of the TM2 is shown in SEQ ID NO.4; the amino acid sequence of the CBM is shown in SEQ ID NO.5; The backbone plasmid of the recombinant vector includes pET-28a-SUMO, pGEX-6P-1 or pGEX-4T-1.

2. The recombinant vector according to claim 1, characterized in that When the metallothionein is MTT1, the backbone plasmid of the recombinant vector includes pGEX-6P-1; when the metallothionein is MTT5, the backbone plasmid of the recombinant vector includes pET-28a-SUMO; when the metallothionein is TM1 and / or TM2, the backbone plasmid of the recombinant vector includes pET-28a-SUMO and / or pGEX-4T-1.

3. The recombinant vector according to claim 1 or 2, characterized in that The insertion site of the nucleic acid molecule on the recombinant vector includes restriction endonuclease BamHI and XhoⅠ restriction enzyme cutting sites.

4. A host cell comprising the recombinant vector according to any one of claims 1 to 3.

5. The host cell according to claim 4, characterized in that The original cells of the host cell include Escherichia coli.

6. A method for preparing a fusion protein, characterized in that: The following steps are involved: Carrying out induction culture on the host cell according to claim 4 or 5 to obtain an induced culture product; The fusion protein includes a CBM and a metallothionein connected sequentially from the C-terminus to the N-terminus; the metallothionein includes MTT1, MTT5, TM1 or TM2; the amino acid sequence of the MTT1 is shown in SEQ ID NO.1; the amino acid sequence of the MTT5 is shown in SEQ ID NO.2; the amino acid sequence of the TM1 is shown in SEQ ID NO.3; the amino acid sequence of the TM2 is shown in SEQ ID NO.4; and the amino acid sequence of the CBM is shown in SEQ ID NO.

5.

7. The preparation method according to claim 6, characterized in that: The inducing agent for the induction culture includes IPTG; the working concentration of the IPTG includes 0-0.2 mMol / mL.

8. The preparation method according to claim 6, characterized in that: The induction culture temperature is 16-37°C.

9. Use of the recombinant vector according to any one of claims 1 to 3, the host cell according to claim 4 or 5, or the preparation method according to any one of claims 6 to 8 in the preparation of a cellulose-based composite heavy metal adsorbent.

10. Use of the recombinant vector according to any one of claims 1 to 3, the host cell according to claim 4 or 5, or the preparation method according to any one of claims 6 to 8 in heavy metal adsorption or heavy metal pollution control.

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