A potential hyphal fusion protein complex with an important role in development and virulence interacts with autophagy-related proteins in Fusarium pseudograminearum

Hyphal fusion(anastomosis)is a common process serving many important functions at various developmental stages in the life cycle of ascomycetous fungi.However,the biological roles and molecular mechanisms in plant pathogenic fungi were widely unknown.In this study,a hyphal fusion protein FpHam-2 was screened from a T-DNA insertion mutant library of Fusarium pseudograminearum,and FpHam-2 interacts with another 2 hyphal fusion protein homologues FpHam-3 and FpHam-4.Each of these 3 genes deletion mutant revealed in similar defective phenotypes compared with the WT and complemented strains,including reduction in growth rate,defects in hyphal fusion and conidiation,more sensitive for cell membrane,cell wall and oxidative stress responses,and decreased in virulence.The yeast two-hybrid assay was used to identify that FpHam-2 interacts with 3 autophagy-related proteins,including FpAtg3,FpAtg28 and FpAtg33.Furthermore,FpHam-2-deletion mutant showed decreased accumulation of autophagic bodies in hypha.In conclusion,FpHam-2,FpHam-3 and FpHam-4 have an essential role for hyphal fusion and regulating the growth,conidiation and virulence in F.pseudograminearum.

Affinity Chromatography Purification of Recombinant Human Interleukin-6 from Its Fusion Protein with GST

Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both in the supernatant and inside the bacterial cell,but the insoluble protein had no biological activity and could not be refolded. The rotative speed of the shaker and the temperature of induction were optimized to maximize the expression of the soluble fusion protein. From the supernatant of the cell sonicates Glutathion Sephrose 4B affinity column chromatography was employed to isolate the fusion protein which could be purified to >80 0 0 in a single step. The yield of soluble GST IL 6 was about 10 mg per liter culture. The GST was site specifically cloven by 6 hours of treatment with thrombin and from the thrombin digest mixture IL 6 was purified by Q high performance ion exchange chromatography. From 1 liter of E.coli culture 2 mg refined IL 6 was obtained. The purified IL 6 had a purity of more than 95 0 0 and a biological activity of 1.02×10 8 IU/mg.

Induced Expression and Optimal Expression Conditions of Recombinant Alpha-bungarotoxin Gene Fusion Protein

[Objective] This study aimed to obtain recombinant alpha-bungarotoxin （a-BG-0 gene fusion protein with biological activity and investiagte its fusion expression. [Method] The plasmid pGEX-a-BGT was transformed into E coil BL21 （DE3） and BL21 （DE3） plysS host bacteria to identify the optimal engineering strain. Fusion expression of the optimal engineering strain was induced, in order to optimize the induced expression conditions of the soluble fusion protein. [Result] JP-a-BGT was identified as the optimal engineering strain, which could express fusion protein after induced by IPTG. The optimal induced expression conditions of the soluble fusion protein were investigatect JP-a-BGT was incubated at 37 ℃ for 2.5 h and induced with 0.50 mmol4. IPTG for 4 h at 22 ℃, and the expression level of the soluble fusion protein reached 18.42%. [Conclusion] This study laid a solid foundation for the subsequent purification of fusion proteins and the separation and purification of a-BGT.

Preparation of Recombinant Islet Cell Autoantigen 69 kD Fusion Protein

To get recombinant antigen (Is/et Cell Autoantigen 69)ICA69 which was expressed in Escherichia coli strains (E.coli) by means of the gene engineering technique so that it can be used for early diagnosis of and screening in type Ⅰ diabetes mellitus, the cDNA fragment of human ICA69 was amplified by PCR, and then cloned into pSPORT 1 vector. After DNA sequencing, it was inserted into pGEX-2T between the sites of EcoR Ⅰ and Sma Ⅰ, then recombinant plasmid p2T-ICA69 was constructed and introduced into E.coli. The GST-ICA69 fusion protein was expressed by the induction of IPTG. The recombinant ICA69 proteins were used to detect the antibodies against hICA69 in 100 healthy subjects and type Ⅰ diabetic serum by the use of indirect ELISA. The sequence analysis showed that the amplified fragments contained 1449 bp, encoded 483 amino acids, and had been correctly inserted into pGEX-2T vector. The recombinant proteins expressed in the prokaryotic cells had immunogenicity and could be used to detect antibodies against ICA69 in type Ⅰ diabetic serum. Finally it can be concluded in this paper that the expression products obtained by the method of gene engineering are recombinant ICA69 antigen and may be used to improve the forecast rate and the diagnostic rate of type Ⅰ diabetes in combination with other tests.

Expression, Purification, and Refolding of Recombinant Fusion Protein hIL-2/mGM-CSF

Objective To study the activities of interleukin （IL）-2 and granulocyte-macrophage colony-stimulating factor （GM-CSF） （hlL-2/mGM-CSF）. Methods SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli （E. coil） BL21 （DE3） in inclusion body （IB） form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. Results The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein/L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4×10^6 IU/mg and 7.1×10^6 IU/mg, respectively. Conclusion This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hlL-2/mGM-CSF used in immune therapy.

Soluble Expression and Rapid Quantification of GFP-hepA Fusion Protein in Recombinant Escherichia coli

To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C terminus of a green fluorescent protein mutant （GFPmutl）. As a result, not only was the functional recombinant expression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluorescence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.

Identification of the immunogenic domains in HBsAg preS1 region using overlapping preS1 fragment fusion proteins

AIM: The incorporation of hepatitis B virus (HBV) preS1 region into epitope-based vaccines against HBV has been accepted widely, but the incorporate site and size of preS1 sequence is controversial. Therefore our purpose was to further investigate its immunogenic domains for the epitopebased hepatitis B vaccine design.METHODS: Eight GST fusion proteins containing overlapping preS1 fragments in preS1 (21-119) region were expressed in E.coli. Using these purified fusion proteins, the immunogenic domains in preS1 region were identified in detail in mice and humans by Western blot analysis and ELISA.RESULTS: The results in mice showed that the immunogenic domains mainly existed in preS1 (21-59) and preS1 (95-109). Similarly, these fragments had strong immunogenicity in humans; whereas the other parts except for preS1 (60-70) also had some immunogenicity.More importantly, a major immunogenic domain, preS1 (34-59), which has much stronger immunogenicity, was identified. Additionally, the antibodies against some preS1 fragments, especially preS1 (34-59), were speculated to be virus-neutralizing.CONCLUSION: Eight GST fusion proteins containing overlapping preS1 fragments were prepared successfully. They were used for the study on the immunogenic domains in preS1 (21-119) region. The preS1 (34-59) fragments were the major immunogenic domains in the preS1 region, and the antibodies against these fragments were speculated to be virus-neutralizing. Therefore, the incorporation of preS1 (34-59) fragments into epitopebased HBV vaccines may be efficient for enhancement of immune response. Additionally, the results also imply that there are more complex immune responses to preS1 region and more abundant immunogenic domains in humans.

A fusion protein containing murine vascular endothelial growth factor and tissue factor induces thrombogenesis and suppression of tumor growth in a colon carcinoma model

Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we construct and express a fusion protein containing vascular endothelial growth factor (VEGF) and tissue factor (TF) to explore whether this fusion protein has the capability of inhibiting tumor growth in a colon carcinoma model. The murine cDNA of VEGF A and TF were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned into prokaryotic expression plasmid pQE30 with a linker. The expression product recombinant VEGF-TF (rVEGF-TF) was purified and proved to have comparable enzyme activity to a commercial TF and the capability of specific binding to tumor vessels. Significant decrease of tumor growth was found in the mice administered with rVEGF-TF on Day 6 after initiated rVEGF-TF treatment (P<0.05), and the tumor masses in 2 of 10 mice were almost disappeared on Day 14 after the first treatment. In addition, valid thrombogenesis and tumor necrosis were observed in the tumor tissues injected with rVEGF-TF. Our results demonstrate that occlusion of tumor vasculature with rVEGF-TF is potentially an effective approach for cancer therapy.

Construction, expression and characterization of human interferon α2b-(G4S)n-thymosin α1 fusion proteins in Pichia pastoris

AIM:Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Ta1 linked by different lengths of (G4S)n(n = 1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex?75 gel filtration and analyzed by SDS-PAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFNa2b-(G4S)n-Tα1 (n= 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex?75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNa2b monoclonal antibody and Tal polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFNa2b-(G4S)n-Tα1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNa2b and immunomodulatory activity of Tal in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.

GST Fusion Protein Based Specific Polyclonal Antibody Preparation of Mouse Aquaporin 1

Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.

Contruction of the Genetic Engineering Strain Expressed Nontoxic ST_1-LT_B Fusion Protein Against Enterotoxigenic Eschenichia coli

Thermostable enterotoxinⅠ(ST1) mutant genes and thermolabile enterotoxin B subunit (LTB)genes were amplified by PCR from plasmids of Eschenichia coli C83902. The recombinantexpression plasmid pZST3LTB containing ST1-LTB fusion gene was constructed by recombinantDNA technique and then transformed into Escherichia coli BL21(DE3). The ST1-LTB fusionprotein was highly expressed in recombinant strain BL21(DE3)(pZST3LTB) and the fusionprotein was about 38.53% of total cellular protein by SDS-PAGE and thin-layer gelscanning analysis. More important, mice immunized with crude preparation containing thefusion protein inclusion bodies or inactivated recombinant strain produced antibodiesthat were able to recognize ST1 in vitro. These sera antibodies were able to neutralizethe biological activity of native ST1 in the suckling mouse assay. Hence the ST1-LTBfusion protein was nontoxic and immunogenic, the constructed recombinant strain BL21(DE3)(pZST3LTB) could be used as a candidate of vaccine strain.

Enhanced Anti-tumor Effect of an IFN-γ-EGF Fusion Protein

The novel fusion proteins harbering human or mouse interferon combined with epidermalgrowth factor receptor binding domain were constructed using methods of genetic and proteinengineering. The fusion proteins were assayed to retain complete antiviral activities. The EGFreceptor binding moiety of the fusion proteins exhibited competitive binding saainst 125 I-EGFfor EGF receptbrs on A431 cells. The fusion proteins were shown to be more potent in in-hibiting the growth of cultured target carcinoma cells than interferon-y alone. Experimentaldata derived from rnouse Bl6 malignant melanoma models indicates that the weight of tumorin mice treated with IFN fusion proteins was significantly smaller than that of mice treatedwith interferon-y alone. The work here is unprecedented in the world and provides a reliableevidence to supPOrt the upcoming clinical employment of a class of interferons that specificallytarget tumor cell

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c(Hsp70_(359–610)) fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.

Construction of Expression Vector for Porcine Gastrin-releasing Peptide Fusion Protein

[Objectives]This study was conducted to obtain porcine gastrin-releasing peptide(GRP)fusion protein.[Methods]The constructed pET32a(+)-GRP plasmid was transformed into Escherichia coli BL21(DE3)competent cells to obtain the pET32a(+)-GRP-BL21(DE3)fusion protein expression strain,which was induced with 0.5 mM IPTG at 25℃and 150 r/min for 12 h,and the His-tagged GRP fusion protein was detected by SDS-Page gel electrophoresis and Western Blot.[Results]After optimizing the IPTG-induced expression conditions,it was confirmed that the porcine GRP fusion protein was obtained,and the porcine GRP fusion protein was soluble,stable and highly active.[Conclusions]This study lays a foundation for the subsequent preparation of anti-pig GRP antibodies.

Function of IgD on lymphocyte activation and effect of hIgD-Fc-Ig fusion protein on human PBMC proliferation

OBJECTIVE This study aimed to investigate the influence of IgD on T/B cell activation and construct h IgD-Fc-Igfusion protein to competitive inhibition IgD binding with IgDR.METHODS T/B cells were sorted by magnetic cell sorting.The differences of m IgD and IgD-R level between different T/B cell subtypes were detected by FCM.Serum IgD level was detected by ELISA.Human IgD-Fc-IgG1-Fc sequence was amplified by cross-PCR and then subcloned into PET28 a(+) empty vector.After prokaryotic expression through escherichia coli,we obtained the h IgD-Fc-Igfusion protein by affinity chromatograph.Western blot was used to identify the h IgD-Fc-Igfusion protein.Human peripheral blood monouclear cells(PBMC) and fibroblast like synoviocytes(FLS) proliferation were detected using a cell counting kit-8(CCK-8).RESULTS The percentage of CD3^+/CD4^+,CD3^+/IgD^+,CD3^+/CD4^+/IgD^+,CD3^+/IgD-R+and CD3^+/CD4^+/IgD-R+cells increased significantly in RA patients comparing to healthy people.IgD can stimulate PBMC proliferation.IgD(1,3,10,30 μg·mL^(-1)) stimulate PBMC proliferation significantly after 24 h.We obtained stable and active h IgD-Fc-Igfusion protein.The h IgD-Fc-Igfusion protein showed no effect on PBMC proliferation.But it could downregulate human IgD protein promoting proliferation effects in human PBMC.CONCLUSION This result suggests that IgD and IgDR play an important role on T/B cell activation in RA patients and the h IgD-Fc-Igfusion protein may competitively inhibit IgD′s function and may play an therapeutic role in autoimmune diseases.

Cloned s-Lap Gene Coding Area, Expression and Localization of s-Lap/GFP Fusion Protein in Mammal Cells

s-Lap is a new gene sequence from pig retinal pigment epithelial(RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B_~16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. ~s-Lap/GFP fusion protein can be expressed in CHO and B_~16 cells with a high rate expression in the nuclei.

Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E.coli

In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.

Diphtheria Toxin/Human B-Cell Activating Factor Fusion Protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study

Objective： This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor （DT388sBAFF） in Escherichia coli （E. coli） and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells （BALL-1）. Methods： A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction （PCR） and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside （IPTG）. The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot, and then purified by Ni2＋-NTA affinity chromatography. The expression level of B cell-activating factor receptor （BAFF-R） on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide （MTT） assay. Results： The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion： The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

An Exploration of the Optimal Induction Conditions for Recombinant Fusion Protein Expression

[ Objective] This study aimed to investigate the optimal induction conditions for expression of recombinant fusion protein. [ Method] The optimal ini- tial OD600, induction dose, induction duration, induction temperature, ampicillin concentration, liquid volume and other culture conditions were explored by using parallel fermentation experiments with different technical indicators, to improve the expression level of the recombinant fusion protein of IMPACT-CN pTYB11 vector. [Result] SDS-PAGE and western-blotting analysis show that the recombinant fusion protein has a high immunogenicity. [Conclusion] This study laid the foundation for further protein purification in diagnosis, production of diagnostic kits with protein and clinical application.

Promotion of Chondrogenesis of Marrow Stromal Stem Cells by TGF-β3 Fusion Protein In Vitro

The purpose of this study was to investigate the repair of the osteoarthritis（OA）-induced car- tilage injury by transfecting the new TGF-β3 fusion protein （LAP-MMP-mTGF-β3） with targeted ther- apy function into the bone marrow-derived mesenchymal stem cells （MSCs） in rats. The recombinant of plRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting site and eukaryotic expression vector plRES-EGFP. LAP and mTGF-β3 fragments were obtained from rat em- bryos by RT-PCR and inserted into the upstream and downstream of MMP from plRES-EGFP-MMP respectively, so as to construct the recombinant plasmid ofplRES-EGFP-LAP-MMP-mTGF-β3, plRES- EGFP-LAP-MMP-mTGF-β3 was transfected into rat MSCs. The genetically modified MSCs were cul- tured in medium with MMP-1 or not. The transfected MSCs were transplanted in the rat OA models. The OA animal models were surgically induced by anterior cruciate ligament transaction （ACLT）. The pathological changes were observed under a microscope by HE staining, Alcian blue, Safranin-fast Gre- en and graded by Mankin＇s scale, plRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed by means of enzyme cutting and sequencing, and the mTGF-β3 fusion protein （39 kD） was certified by Western blotting. Those genetically modified MSCs could differentiate into chondrocytes induced by MMP and secrete the relevant-matrix. The transfected MSCs could promote chondrogenesis and matrix production in rat OA models in vivo. It was concluded that a new fusion protein LAP-MMP-mTGF-β3 was constructed successfully by gene engineering, and could be used to repair the OA-induced cartilage injury.