Exploring the ability of TAT, a cell-penetrating peptide, to deliver proteins in a non-fused form / 药学学报

Accumulating evidence has shown that the cell-penetrating peptide TAT can be applied to deliver different types of drug molecules, including nucleic acids, proteins and small molecule drugs. Usually TAT delivers cargoes on the basis of their covalent bonds or non-covalent interactions. However, there are few reports on the delivery of proteins by TAT in a non-covalent manner, and no quantitative comparisons have been made on the protein delivery ability of TAT in fusion and non-fusion manners. In order to explore the ability of TAT to deliver proteins in non-fusion manner, here we used fluorescence microscopy and flow cytometry to investigate the ability of TAT to deliver enhanced green fluorescent protein (EGFP) into non-small cell lung cancer cells A549 in a non-fusion manner. It was found that TAT could deliver EGFP into A549 cells, and its delivery ability was positively correlated with its concentration. In addition, the fusion protein TAT-EGFP was overexpressed and purified, and its permeability across cell membrane was also investigated. In this paper, based on quantitative comparison, we found that the delivery of EGFP by TAT in fusion manner is significantly efficient than that of TAT in non-fusion manner. This is the report that TAT can deliver EGFP in a non-fusion manner. Although its delivery efficiency remains to be improved as compared with the fusion manner, the non-fusion manner has shown incomparable advantages in ease of operation, suggesting that it is also a candidate for delivery strategy in the future.

Cell-penetrating Effects and Repair of Cell Oxidative Damage of TAT-metallothionein Fusion Proteins / 中国生物化学与分子生物学报

The human Immunodeficiency Virus Transactivator (TAT) protein transduction peptide is a trans-transcription activator encoded by HIV-1. It is rich in basic amino acids, and capable of efficiently mediating the passage of exogenous macromolecules through a variety of membrane structures, such as the cytoplasmic membrane and the blood-brain barrier. Metallothionein (MT) is a protein with low molecular weights and rich cysteine contents. It plays important roles in maintaining the dynamic balance of metal contents in the body, in the detoxification of heavy metals and in defense against oxidative stress. Based on the full-length MT cDNA previously cloned from Sinopotamon henanense, we aim to prepare a TAT-mediated recombinant fusion protein that can cross the membrane and enter the cell by means of genetic engineering. The hydroxyl radical scavenging rate and total antioxidant capacity of TAT-MT were measured in vitro. An immunofluorescence technique was used to detect the transmembrane activity. An MTT assay was used to study the repair effect of H

Fusion expression with catalase improves the stability of multicopper oxidase and its efficiency in degrading biogenic amines / 生物工程学报

Some enzymes belonging to the multicopper oxidase (MCO) family can degrade the hazardous biogenic amine (BA) present in food. However, the oxidation of MCO in the process of degrading BAs may reduce its activity and stability, resulting in decreased catalytic efficiency. In this work, an MCO from Lactobacillus fermentum (MCOF) was fused with a Bacillus subtilis catalase (CAT) using different strategies and the fusion enzymes were respectively expressed in Escherichia coli BL21(DE3). The tolerance of eight fused MCOFs to H2O2 increased by 51%-68%, and the stability of CAT&MCOF increased by 17%, compared to the wild type MCOF. Using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a substrate, the substrate affinity (Km), the catalytic efficiency (kcat/Km) and the molar specific activity of CAT&MCOF increased by 1.0-fold, 1.7-fold and 1.2-fold than those of MCOF, respectively. The stability of CAT&MCOF under acidic conditions (pH 2.5-4.5) and moderate temperatures (35-55 °C) also improved. Moreover, the degradation rates of putrescine, cadaverine and histamine catalyzed by CAT&MCOF reached 31.7%, 36.0% and 57.8%, respectively, which increased by 132.5%, 45.7% and 38.9% compared to that of MCOF. The improvement on the stability and catalytic efficiency of MCOF by fusion expression with CAT provides a good example for improving the applicability of enzymes through molecular modifications.

Bacterial expression of 183-227aa region of HER3 extracellular domain I and preparation and identification of its polyclonal antibodies / 南方医科大学学报

OBJECTIVE@#To prepare the recombinant peptide MVF-HER3 I composed of the 183-227aa peptide segment of human epidermal growth factor receptor 3 (HER3 I) and the measles virus protein 288-302 peptide segment (MVF), and prepare polyclonal antibodies (PcAb) against this recombinant peptide.@*METHODS@#The MVF-HER3 I gene was synthesized chemically and subcloned into pET21b or pET32a plasmid containing Thioredoxin (Trx) tag gene. The recombinant plasmids were identified by endonuclease digestion. MVF-HER3 I was expressed in BL21(DE3) cells under an optimal bacterial expression condition. The fusion protein Trx-MVF-HER3 I was purified using nickel ion affinity chromatography, and the purified protein was digested by enterokinase to remove Trx tag. The digested mixture underwent further nickel ion affinity chromatography to obtain purified MVF-HER3 I. The purified MVF-HER3 I was used to immunize SD rats subcutaneously for preparing anti-MVF-HER3 I PcAb. The titer of PcAb was determined using ELISA. The bindings of anti-MVF-HER3 I PcAb to MVF-HER3 I, native HER3 and MCF7 cells were analyzed using immunoblotting, immunoprecipitation and laser confocal microscopy. The growth inhibition effect of the antibodies on MCF7 cells cultured in the absence or presence of NRG was assessed using sulforhodamine B.@*RESULTS@#The recombinant peptide gene could not be expressed alone, but could be efficiently expressed after fusion with Trx gene under optimized conditions. The fusion peptide MVF-HER3 I was successfully prepared from Trx-MVF-HER3 I. The anti-MVF-HER3 I PcAb, with a titer reaching 1: 512 000, specifically bound to MVF-HER3 I, recognized native HER3 and bound to the membrane of MCF7 cells. The obtained PcAb could dose-dependently inhibit the growth of MCF7 cells irrespective of the presence or absence of NRG.@*CONCLUSIONS@#We successfully obtained the recombinant peptide MVF-HER3 I and prepared its PcAb, which can facilitate further functional analysis of HER3 signaling pathway.

Construction and application of mCherry red fluorescent protein fusion expression system in lactic acid bacteria / 生物工程学报

In order to enrich the library of domestic research about new red fluorescent marker in lactic acid bacteria (LAB), we described a new fusion expression system in Lactobacillus plantarum WCFS1 based on the pSIP vector. This system contained red fluorescent protein mCherry as a marker and bile salt hydrolase gene (bsh) as a reporter gene. Moreover, in this study, four different promoters (PsppA, PldhL, P32 and PslpA) were used to regulate the expression of the fusion protein mCherry-BSH, completing the inducible and constitutive expression in lactic acid bacteria. The recombinant protein mCherry-BSH presented activity of red fluorescence and bile salt hydrolase (BSH). The successful construction of the fusion expression system in LAB using a red fluorescent protein mCherry provides favorable conditions for the distribution, intestinal colonization and survival rate of lactic acid bacteria, so as to reveal the function mechanism of its probiotic characteristics; and the system also could lay the foundation for researches on protein expression, cellular localization and properties identification of active protein in lactic acid bacteria.

Expression, subcellular localization and characterization of dammarenediol-Ⅱ synthase of Panax ginseng in Saccharomyces cerevisiae / 药学学报

To study the expression and subcellular localization of recombinant dammarenediol-Ⅱ synthase (DS) in Saccharomyces cerevisiae, the dammarenediol-Ⅱ synthase gene ds was cloned from Panax ginseng, and the gene ds was fused with the gene of green fluorescent protein to obtain the fusion gene ds-gfp. The recombinant expression plasmids pESC-HIS-DS and pESC-HIS-DS-GFP were constructed and transformed into S. cerevisiae INVSc1 to obtain recombinant strains INVSc1-DS and INVSc1-DS-GFP. Microsomes of recombinant strains were prepared by differential centrifugation and observed by fluorescence microscope. The green fluorescence was only detected in INVSc1-DS-GFP microsomes, which indicated that DS was a membrane protein. It was also proved that dammarenediol-Ⅱ was produced from the cyclization of 2, 3-oxidosqualene catalyzed by DS through in vitro enzymatic reaction. In addition, our results revealed that the fusion expression of ds with gfp significantly improved the production of dammarenediol-Ⅱ from 7.53 mg·g-1 to 12.24 mg·g-1. This study provides a new strategy in the optimization of the pathway of ginsenosides biosynthesis in S. cerevisiae.

Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli

Human enterokinase (synonym: enteropeptidase, EC 3.4.21.9) light chain (hEKL) gene was designed and artificially synthesized with built-in codon blas towards Escherichia coli codon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3). Recombinant hEKL protein with a maltose binding protein (MBP) tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (> 95%) by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 1:5000 (enzyme:substrate). Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen). From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104 U/mg.

Study of fusion expression effect on conformation and immune activity of polyepitope protein,M.RCAg-1 / 中华微生物学和免疫学杂志

ObjectiveTo explore the effects of vector fusion peptides on the conformation and immune reactivity of recombinant polyepitope antigens,M.RCAg-1.MethodsWe subcloned polyepitope artificial antigen gene,M.RCAg-1,into prokaryotic expression vectors,pDS-ex,that contain different fusion tags at the N-terminus or no any tag by different restriction enzyme cutting site.Three recombinant proteins expressed by these vectors,named P312-1,P312-2,and P312-3,were purified and purity is greater than 95％.Then BALB/c mice were vaccinated with the three proteins formulated with Freund's adjuvant through intranasal.Serum were collected to detect specific antibodies of M.RCAg-1 and individual epitope by ELISA ; natural parasite antigen was recognized by indirect immunofluorescence assay; mouse specific T lymphocyte activation was detected by enzyme-linked immunosorbent spot test (ELISPOT) ; and the growth of Plasmodium falciparum in vitro to evaluate by growth inhibition assay(GIA),and analyze secondary and tertiary structures of recombinant proteins from different expression vectors by bioinformatics and circular dichroism technique.ResultsThe P312-1,P312-2 have almost the same amino acid sequence,and the three proteins have the same immunogenicity in animal models(P＞0.05),however,the different proteins elicited various T-cell responses,the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assaysin vitro ( respectively,93.9％,14.7％,54.3％ ).The significant differences of secondary and tertiary structures were shown in recombinant proteins from different expression vectors,analyzed by bioinformatics and circular dichroism technique,which demonstrated the change of protein molecule spaces conformation,and the obviously change of some epitope locations.ConclusionThese results suggest that the expressed polyepitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and,subsequently,the epitope exposure.Thus,these proteins are able to induce both distinct humoral and cellular immune responses in animal models,and they affect the efficacy of immune inhibition against the parasite,the enhancement or suppresses.

Expression of scFv SA3 against hepatoma fused with enhanced green fluorecsent protein and its targeted ability in vivo / 中南大学学报(医学版)

Objective To express and purify the human scFv antibody,SA3,against the hepatoma fused to enhanced green fluorecsent protein,and to observe the targeted capacity of fusion protein EGFP-SA3 in vivo.Methods SA3 and EGFP genes were cloned into plasmid pET-25b( + )to construct the recombinant plasmid EGFP-SA3/pET-25b ( + ),followed by DNA sequencing.Then it was transformed into E.coli BL21 ( DE3 ) and induced for fusion expression of EGFP-SA3with IPTG.The expressed fusion protein EGFP-SA3 was purified and detected with SDS-PAGE.HepG2 cells were incubated with the fusion protein EGFP-SA3 in vitro,and the binding bioactivity was observed under the fluorecsent microscope.Further more,we injected the EGFP-SA3 by caudal vein into nude mice planted by hepatoma and observed the whole body fluorescence image of EGFP.Results SA3 and EGFP genes were successfully cloned into pET-25b( + ),which was confirmed by restriction enzyme Nco I-Xho I or Nco I-Eco RI.A band migrated at the position 750 bp,same to EGFP gene,emerged when recombinant plasmid was digested by restriction enzyme Nco I-Eco RI.Similarly,a band,about 1 500 bp,emerged when digested by Nco I-Xho I.The open-reading frame was confirmed by DNA sequencing.Fusion protein EGFP-SA3 was expressed as inclusion body.After purification and refolding,the result of immunofluorecsence detection verified that EGFP-SA3could specifically bind to HepG2 cells and maximum tumor penetration was at 24 h after the injection.Conclusion The purified fusion protein EGFP-SA3 has strong binding capacity to HepG2cells,indicating the scFv SA3 has a potential value as a targeting molecule for diagnosis and targeted therapy for liver cancer.

Prokaryotic Expression and Potential Application of the Truncated PCV-2 Capsid Protein / 中国病毒学·英文版

Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a(+) vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta(DE3) E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % (73/264). Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-2 ORF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.

Fusion expression of the extracellular carbohydrate recognition domain of mouse Dectin-1 and its recognition of β-glucans in the cell wall of Candida albicans / 中华微生物学和免疫学杂志

Objective To clone, express and purify the extracellular carbohydrate recognition do-main(CRD) of Dectin-1 in mouse peritoneal macrophages and to further investigate its ability to recognize and bind to β-glucans. Methods The Dectin-1 CRD gone was amplified by RT-PCR from RNA of mouse peritoneal macrophages and cloned into prokaryotic expression vector pET28a (+), the constructed pET-CRD recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and the fusion protein was in-duced to express. After affinity purification and renaturation, the fusion protein was incubated with Candida albicans yeast and its ability to recognize and bind to β-glucans in the cell wall of fungi. Results The fu-sion protein could recognize β-glucans in the fungal cell wall. Conclusion The recombinant expression plasmid pET28a-CRD was successfully constructed and the fusion protein was induced. The fusion protein is able to recognize and bind to β-glucans in the fungal cell wall, thus laying a good foundation for fungal de-tection and the exploration of the biological role of β-glucans.

Cloning of the antibacterial peptide cecropin gene of Musca domestica larvae and its fusion expression in Escherichia coli / 中国人兽共患病学报

In the present study, the total RNA was extracted from three instar larvae of Musca domestica, the cDNA sequence encoding the ORF of cecropins was amplified by RT-PCR, and the target fragment was further sequenced after being cloned into T vector pUCm-T. Then, the cDNA sequence of the mature cecropins was amplified by PCR with recombinant plasmid pUCm-T/cecropin as template, the N-terminal rare codon GGA of E. coli was changed to the favorable codon GGC,and a Asn codon AAC was added in front of the stop coden TAA in the C- terminus. This mutant gene designated as mCecropin was then ligated with the fusion expression vector pGEX-4T-1. After restriction analysis and DNA sequencing, the positive recombinant plasmid pGEX-4T-1/mCecropin was transformed to different strains of E. coli cells and the fusion protein was expressed after IPTG induction. The fusion protein was assayed by SDS-PAGE and the E. coli BL21(DE3) cell was chosen as the host cell for the expression of the fusion protein. The expressed fusion protein GST-mCecropin was purified by GSTrap affinity coloum and the GST marker was then cleaved by thrombin. In this way, the fusion protein mCecropin with antibacterial activity was obtained after purification with HiTrap benzamidine column.

Research of Fusion Expression of Human β-Defensin-3 in Escherichia coli / 中国比较医学杂志

Objective To achieve the fusion expression of the entire human beta-defensin-3(hBD-3) gene. Method We synthesized two oligonucleotide primers accor ding to the codon preference of Escherichia coli. The gene was cloned into p GEX -4T-2 to establish the pGEX-4T-2-hBD-3 as the fusion expression vector by PCR. Transformed into E.coli strain DH5α, the express vector was induced an d ex pressed by IPTG. The fusion protein GST-hBD-3 was obtained by repeated cycles of freezing and thawing, cut by thrombin to attain the recombinant hBD-3 protei n. Result The result of the antibacterial peptide agarose diffu sion assay shows the antibacterial activity of the rhBD-3 against the S.aureu s exists, and it reaches 0.843U. Conclusion The fusion expr ession of the hBD-3 gene is successful.

Gene Coloning,Expression and Enzymatic Assay of Human sPLA2-IIA / 中国生物工程杂志

Objective: To clone the cDNA of human sPLA2-IIA,construct the engineered Escherischia coli expressing human sPLA2-IIA and identify the expressed human sPLA2-IIA. Methods: Total RNAs were purified from human fetal spleen. The cDNA of human sPLA2-IIA was cloned by RT-PCR and inserted into plasmid pET32a(+) between NcoI and EcoRI sites for expressing the recombinant human sPLA2-IIA in Escherischia coli BL21(DE3). The recombinants were screened by SDS-PAGE. The engineered Escherischia coli expressing trxA-human sPLA2-IIA fusion protein was established. The expressed human sPLA2-IIA exists in the form of inclusion body and accounts for about 25% of the total proteins of Escherischia coli BL21(DE3). Conclusion: the engineered E. coli methods are suitable for preparing plenty of human sPLA2-IIA which has laid base for the large-scale expression,purification and basic studies of human sPLA2-IIA.

Expression and Purification of Human Parathyroid Hormone Peptide(1-34) in Escherichia coli / 中国生物工程杂志

Human parathyroid hormone peptide1-34(hPTH1-34) was highly expressed in Escherichia coli by inserting the synthesized hPTH1-34 cDNA into pThioHis, the prokaryotic expression vector. The expressed hPTH1-34 was purified by chelating sepharose immobilized metal ion affinity, reverse and filter chromatographic steps. Its purity was verified above 95% by HPLC. The quality was identified by N-terminal sequencing and MALDI-TOF-MS analysis. In vitro analysis showed the adenylate cyclase of ROS 17/2.8 cells was activated by hPTH1-34.

Research Advance in Recombinant Expression of Microbial Lipases / 中国生物工程杂志

Microbial lipases have been widely used in traditional industries,as well as in emerging biocatalysed areas owing to their ability to catalyze a variety of reactions in aqueous and non-aqueous media.Therefore,it is very important to enhance amount of lipase production by recombinant overexpression for meeting market demand.A critical review of main and novel strategies which have been employed for recombinant expression of microbial lipases are presented,including codon optimization,fusion and co-expression,dual expression system based on hybrid promoters,homologous overexpression,cell surface displaying and high-throughput screening based on gene library of expression.These new technologies are gradually coming to the forefront in the recombinant expression of lipase,especially for cell surface displaying and high-throughput screening based on gene library of expression.Meanwhile,several recombinant expressions for representative microbial lipases were also introduced and discussed,which are available for consultation when attempting to overexpress any lipases by scientists and industrialists.

In-fusion Expression of Xylanase Genes XynA and XynB From Aspergillus sulphureus in E. coli and Characterization of The Recombinant Enzyme / 生物化学与生物物理进展

Two sets of primers were designed according to the sequences of xynA and xynB from Aspergillus sulphureus, and the DNA fragments composed of 574 bp and 594 bp were amplified by polymerase chain reaction (PCR), respectively. The two PCR products respectively digested with EcoRⅠ/BamHⅠ and BglⅡ/HindⅢ were ligated into multiple cloning sites of pET-28a(+). The resulting plasmid is pET-xynAB, in which xylanase A and B are ligated by a 7-amino acid peptide (GlyGlyGlySerGlyGlyGly). E. coli BL21 transformed with pET-xynAB was induced by IPTG, and a special protein band about 50 ku was detected by SDS-PAGE. The protein purified with Ni-NTA column was denatured by 8 mol/L urea and dialyzed for refolding. The recombinant xylanase showed optimal activity at 50℃ and pH 4.4. The enzyme retained above 75% of its activity at the range of pH 2.4～5.4. The xylanase displayed about 50% retained acitivity after incubating at 80℃ for 30 min. Various metal ions have different effects on activity of the recombinant xylanase.

Expression and Purification of Hi-lys Peptide,a Recombinant Relevant to Hirudin / 中国生物化学与分子生物学报

A new fusion expression vector, pED-P8-Hi-lys was designed and constructed. It includes four parts, a 20 peptide sequence of hirudin that can maintain anticoagulant activity, the C-terminus of asparaginase as a fusion partner, basic octopeptide (KRKRKKSR) that makes the fusion partner easy to remove, and the unique acid-labile aspartyl-prolyl bond. It was transformed into E. coli BL-21 and the fusion protein (AnsB-C-P8-Hi-lys) was expressed effectively as inclusion bodies after inducing by lactose. The objective peptide Hi-lys was purified by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, and DEAE-cellulose 52 column chromatography. The antithrombin activity of the purified Hi-lys peptide was about 50 ATU/mg by thrombin activity assays.

Construction and prokaryotic expression of SLC / 第三军医大学学报

Objective To construct an expression vector pET32a(+)/SLC and express the fusion protein TrxA SLC. Methods Total RNA from human inflammatory tonsil was extracted and its cDNA was generated with reverse transcription. Mature secondary lymphoid tissue chemokine (SLC) gene was amplified by RT PCR and cloned into plasmid pET32a(+) with Nco Ⅰ and Eco RⅠ sites added to the 5′ and 3′ ends respectively. E. coli DH5? was transformed with the recombinant plasmid, and the grown clones were selected. The inserted DNA was verified by enzyme digestion and DNA sequencing. The 3 amino acids between enterokinase site and target gene were deleted with mutation and the new vector was verified by sequencing. Expression of SLC was analysed by SDS PAGE. The fusion protein was purified by metal affinity chromatography and weak cation exchange chromatography, which was analysed by SDS PAGE and Western blotting. Results Trx SLC fusion protein expression vector was successfully construced, and the fusion protein was expressed with solubility. The purified fusion protein displayed the ability of binding the goat anti human SLC polyclonal antibody. Conclusion The SLC fusion protein can be expressed with stability and solubility and primary purification is performed with metal affinity chromatography and weak cation exchange chromatography.

Construction and expression of prokaryotic vector of a novel candidate gene IDD01 associated with hepatocellular carcinoma / 第三军医大学学报

Objective To construct the prokaryotic vector and to express a novel candidate gene IDD01 associated with hepatocellular carcinoma (HCC) for its functional study. Methods The open reading frame (ORF) of IDD01, amplified from HCC tissue by RT PCR, was inserted into the expression vector pMAL c2X. The recombinant vector was transformed into E. coli TB1, and the expression products were analyzed by SDS PAGE. Results The length of PCR product was about 770 bp. The restriction enzyme digestion and sequencing conformed that the gene segment was correctly inserted into the vector. SDS PAGE and density scanning indicated that the protein was expressed as a fusion protein with 28 KD of molecular weight and the fusion protein possessed 30.4% of the total bacterial protein. Conclusion The recombinant vector is constructed successfully and IDD01 can be expressed at a high level, which lays a foundation for the further research on the functions of IDD01 gene.