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.

Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and their potential use for male fertility regulation

Rat protein DE is an androgen-dependent cysteine-rich secretory protein （CRISP） synthesized by proximal epididymal regions. DE, also known as CRISP-1, is localized on the equatorial segment of acrosome-reacted spermatozoa and participates in gamete fusion through binding to egg complementary sites. Immunization of rats with DE inhibits fertility and sperm fusion ability, suggesting that DE represents a good epididymal contraceptive target. Recombinant DE fragments and synthetic peptides revealed that DE binds to the egg via a 12-amino acid region of an evolutionarily conserved motif, Signature 2 （S2）. The ability of other CRISP to bind to the rat egg was correlated with their S2 amino acid sequences. Although testicular protein Tpx- 1 （CRISP-2） was capable of binding to rodent eggs, human epididymal AEG-related protein （ARP） and helothermine （from lizard saliva） were not. The S2 region presented only two substitutions in Tpx-1 and four in ARP and helothermine, compared with the DE S2, suggesting that this amino acid sequence was relevant for egg interaction. Studies with Tpx- 1 and anti-Tpx- 1 revealed the participation of this protein in gamete fusion through binding to complementary sites in the egg. In competition studies, DE reduced binding of Tpx- 1 dose-dependently, indicating that both CRISP share the egg complementary sites. That anti-DE and anti-Tpx-1 inhibit sperm-egg fusion while recognizing only the corresponding proteins, suggests functional cooperation between these homologous CRISP to ensure fertilization success. These results increase our understanding of the molecular mechanisms of gamete fusion and contribute to the development of new and safer fertility regulating methods. （Asian J Androl 2007 July; 9： 528-532）

A Sensitive and Specific IgM-ELISA for the Serological Diagnosis of Human Leptospirosis Using a rLipL32/1-LipL21-OmpL1/2 Fusion Protein

Objective To construct a lipL32//1-1ipL21-OmpL1//2 fusion gene and its prokaryotic expression system, and to establish an enzyme-linked immunosorbent assay （ELISA） using the rLipL32/1-LipL21-OmpL1/2 fusion antigen of Leptospira interrogans for sensitive and specific detection of IgM in the serum of patients with leptospirosis. Methods lipL32/1-1ipL21-OmpL1/2 fusion genes were constructed using a primer-linking PCFI. The target recombinant protein antigens, rLipL32/1, rLipL21, rOmpL1/2 and rLipL32/1-LipL21-OmpL1/2, were expressed and the purified antigens were then immobilized to the surface of microplate wells for ELISA-based detection of IgM in the sera of leptospirosis patients; Results Of 493 acute leptospirosis patients, 95.7% and 97.8% were positive by rLipL32/1-LipL21- OmpL1/2-1gM-ELISA using different serum dilutions, which was higher than the rLipL32/1-1gM-ELISA （93.1% and 90.3%）, rLipL21-1gM-ELISA （90.3% and 87.0%）, and rOmpLI-lgM-ELISA （85.6% and 81.1%） （P〈0.01）. All IgM-ELISAs tested negative against 56 non-leptospirosis patients with typhoid fever, hemorrhagic fever or dengue fever. Conclusion Trigeminal fusion antigen increases ELISA sensitivity and the rLipL32/1-LipL21-OmpL1/2- IgM-ELISA is a sensitive and specific serological diagnostic method for clinical leptospirosis.

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.

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.

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.

Combination immunotherapy with Survivin and luteinizing hormone-releasing hormone fusion protein in murine breast cancer model

AIM To investigate the therapeutic potential of two recombinant proteins, Survivin and luteinizing hormone-releasing hormone (LHRH) fusion protein [LHRH(6 leu)-LTB] for immunotherapy of breast cancer.METHODS Murine 4 T-1 breast cancer model was used to evaluate the efficacy of recombinant proteins in vivo. Twenty four Balb/c mice were divided into 4 groups of 6 mice each. Recombinant Survivin and LHRH fusion protein, alone or in combination, were administered along with immunomodulator Mycobacterium indicus pranii (MIP) in Balb/c mice. Unimmunized or control group mice were administered with phosphate buffer saline. Each group was then challenged with syngeneic 4 T-1 cells to induce the growth of breast tumor. Tumor growth was monitored to evaluate the efficacy of immune-response in preventing the growth of cancer cells.RESULTS Preventive immunization with 20 μg recombinant Survivin and MIP was effective in suppressing growth of 4 T-1 mouse model of breast cancer (P = 0.04) but 50 μg dose was ineffective in suppressing tumor growth. However, combination of Survivin and LHRH fusion protein was more effective in suppressing tumor growth (P = 0.02) as well as metastasis in vivo in comparison to LHRH fusion protein as vaccine antigen alone.CONCLUSION Recombinant Survivin and MIP suppress tumor growth significantly. Combining LHRH fusion protein with Survivin and MIP enhances tumor suppressive effects marginally which provides evidence for recombinant Survivin and LHRH fusion protein as candidates for translating the combination cancer immunotherapy approaches.

Recombinant Mycobacterium smegmatis expressing Hsp65-hIL-2 fusion protein and its influence on lymphocyte function in mice

Objective:To coastruct a strain of recombinant Mycobacterium smegmatis expressing the heat shock protein 65(Hsp65) and human interleukin 2(IL-2) fusion protein(rMS-Hsp65/IL-2) and to explore the effect of this construct on lymphocyte function in mice.Methods:The fusion gene encoding Hsp65-hIL-2 was cloned into shuttle vector pSMT3.The recombinant plasmid pSMT3- Hsp65-hIL-2 was transferred to Mycobacterium smegmatis by electroporation.Positive clones were selected by hygromycin and identified by PCR.The expression of fusion protein Hsp65- hIL-2 was verified using indirect immunofluorescence staining.Mice were immunized for two times by subcutaneously injection with 1×10~6 CFU rMS-Hsp65/IL-2 at a three-week interval.Two weeks after the second immunization,mice were sacrificed and the serum samples were collected for determination of anli-Hsp65 specific IgG.Splenic lymphocytes were isolated and treated with the rMS-Hsp65/IL-2 to determine lymphocytic proliferation activity by MTT assay.IFN-γand IL-2 in the medium of the treated cells were also determined by ELISA.Results:Successful construction of rMS-Hsp65/IL-2 was verified by PCR and immunofluorescence staining.Compared to the splenic lymphocytes isolated from mice immunized with Bacille Calmette-Guerin or mice immunized with Mycobacterium smegmatis alone,the splenic lymphocytes isolated from mice immunized with rMS-Hsp65/IL-2 showed a marked increase in the proliferation of lymphocytes, together with an increased production of important cytokines such as IFN- 7 and IL-2. Conclusions:rMS-Hsp65/IL-2 markedly enhances lymphocyte function.Therefore,the fusion protein generated by rMS-Hsp65/lL-2 may be of potential value in generating an effective vaccine against tuberculosis.

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.

An efficient fusion protein system for expression of Bacillus anthracis protective antigen as immunogenic and diagnostic antigen

Objective:To produce high quantities of recombinant protective antigen(rPA) for human vaccine and diagnosis.Methods:The PA gene was amplified by PCR with pXO1 plasmid as template. The PCR product was cloned into pMAL-c2X vector using the BamH1 and SalI restriction enzymes.The recombinant plasmid was transformed into Escherichia coli DH5 a strain and then screened for transformation.The expression of protective antigen was analyzed by SDS-PAGE and Western blotting after isopropyl β-D-thiogalactopyranoside(IPTG) induction.Results: The full-length PA gene(2.2 kb) was cloned into pMAL vector system.The recombinant vector was confirmed by restriction enzyme and PCR analysis.The expression of cytoplasmic maltose-binding protein-protective(MBP-P) antigen fusion protein was detected by SDS-PAGE and Western blotting,and obtained a 125 kDa protein band,which was similar to expected size of fusion protein.Conclusions:This expression system can be used in the high production of rPA. After purification and immunization studies,the purified rPA may be used in the development of the human recombinant anthrax vaccine and also in diagnosis of anthrax disease.