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.

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.

Prokaryotic expression and purification of fibronectin leucine rich transmembrane protein 3 C-terminal domain proteins in rats

BACKGROUND： Studies have suggested that fibronectin leucine-rich transmembrane protein 3 （FLRT3） is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood. OBJECTIVE： To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins. DESIGN, TIME AND SETTING： An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008. MATERIALS： Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coil （E. coil） JM109 were purchased from Promega. E. coil BL21 was provided by Novagen. METHODS： FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. coil BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained. MAIN OUTCOME MEASURES： Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods. RESULTS： FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then clones into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass of 56,600. The recombinant protein was observed in the inclusion body, and highly purified recombinant proteins were obtained through a series of methods, such as rinsing, purifying, dissolving, and renaturing. CONCLUSION： From adult Sprague Dawley rats, FLRT3 C-terminal gene fragments were successfully cloned and shown to be effectively expressed in E. coil BL21. Moreover, highly purified GST fusion proteins were obtained.

Expression and Purification of Active Recombinant Human Nerve Growth Factor from Escherichia coli

Introduction Nerve growth factor （NGF） was first discovered and purified by Rita Levi-Montalcini and Stanley Cohen in the 1950s. It represents the first cellular growth factor ever discovered and involved in the gl-owth ,survival, and differentiation of specific nerve cell populations. Although animal tests and trials indicate that rhNGF could be ment for diabetic and HIV-related phase- Ⅱ clinical an effective treatneuropathies , a large-scale phase-Ⅲ clinical trial has failed to give similar result. NGF isolated from the mouse submaxillary gland has been widely used clinically in China for the treatment of peripheral neuropathy,

Refolding with Simultaneous Purification of Recombinant Human Granulocyte Colony-stimulating Factor from Escherichia coli Using Strong Anion Exchange Chromatography

The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration- of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and 43%, respectively.

Expression and purification of recombinant human hemangiopoietin in Escherichia coli

Objective:To express the soluble recombinant hemangiopoietin protein in E.coli BL21(DE3).Methods:Using human fetal live cDNA as a template,a partial cDNA fragment of HAPO coding N-terminal region was subcloned into plasmids pTrc99,pQE60 and pET32c to construct different recombinant prokaryotic expression systems.After selecting,the soluble rhHAPO fusion protein was expressed stably in E.coli BL21(DE3) by vector pET32c-HAPO and further isolated by nickelnitrilotriacetic acid(NTA) affinity chromatography.After cleavage with enterokinase,the rhHAPO protein was applied to Fast Flow SP sepharose column.Results:The rhHAPO protein had a purity of more than 95% and a good bioactivity based on the cell adhesion assay in ECV304 cells.Conclusion:We have established a protein engineering system to produce rhHAPO which may provide the possibility for clinical application.

Expression, purification and bioactivity of human augmenter of liver regeneration

AIM： To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration （hALR） and to study their biological activity. METHODS： hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase （GST） sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation. RESULTS： The product of PCR from plasmid pGEM-T- hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups （P 〈 0.01）.CONCLUSION： Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application.

Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag

Total RNA was isolated from the hybridoma cell line （LC- 1 ）, which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FRl （framework region l） and FR4 conserved regions of LC-1 gene, the variable regions of heavy chain （Vh） and light chain （Vl） were amplified, and the Vh and modified Vl were connected to single chain Fv （ScFv） by SOE-PCR （splice overlap extension PCR）. The modified ScFv was fused with green fluorescent protein （GFP） and introduced into E. coli JM109. The fusion protein induced by lPTG （Isopropylthiogalactoside） was about 57000 on a 10% SDS-PAGE gel （10% Sds Polyacrylamide Gel Electrophoresis）, and primarily manifested as inclusion bodies. The renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-l lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.

Expression, purification and targeting therapy of mscFv_(25)-TNFα against hepatocellular carcinoma

Objective: To Obtain purified genetic eogineering recombinant scFv-fusion protein with potentialities of clinical application. Methods: Mouse anti-hepatocellular carcinoma (HCC) single chain Fv fragment (mscFv25 ) was fused to human TNFa gene, then mscFv25-TNFa was subcloned into prokaryotic GST fusion expression vector pGEX 4T-l, and ex-. pressed in the host E. colt induced by IPTG. Expressed proteins as inclusion bodies were solubilized, solubilized and purified by GST affinity chromatography. The cytotoxity of mscFv25-TNFa was evaluated on SMMC-7721 by MTT, and the targeting therapeutic value was revealed in nude mice bearing HCC xenografts. Results: The specificity and affinity of mscFv25-TNFa were not markedly reduced compared with its parental antibody HAb25 against SMMC-7721 antigen. In vitro target cell SMMC-7721 was insensitive to mscFv25-TNFa. In the mscFv25-TNFa had certain targeting cytotoxicity and caused complete tumor disappearance in 4 of 14 mice, and the side effects of TNFa were much weaker in mscFv25-TNFa group than in group. Conclusion: SMMC-7721 may belong to the TNF-resistant type. While in the trial, mscFv25-TNFa caused complete tumor disappearance in 4 of 14 mice, but no disappearance in TNFa group, suggesting that mscFv25-TNFa had certain tar geting cytotoxicity, and the cytotoxicity of TNFa depended on some other factors in the. It may damage vascular endothelial cell and lead to ischemic necrosis, or induce the tumor cell to apoptosis and some agents to play the the of actinmycin-D in vivo. The targeting of mscFv25 can diminish unspecific cytotoxicity of TNFa, thus attenuate the side effects of TNFa.

cDNA cloning, prokaryotic expression and purification of rat α-synuclein

Objective To clone the cDNA of rat α-Syn gene, investigate its prokaryotic expression and produce purified recombinant rat α-Syn protein. Methods Rat α-Syn cDNA was amplified from the rat brain total RNA by RT-PCR and was cloned into pGEX-4T-1, a prokaryotic expressing vector. The recombinant plasmid containing rat α-Syn gene was transformed into E. Coli BL21 to express a fusion protein with rat α-Syn protein tagged by glutathione-S-transferase （GST）. The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant rat α-Syn protein. The recombinant rat a-Syn protein was further purified using Superdex S200 gel filtration. Results DNA sequencing confirmed that the cloned cDNA contained 420 base pairs encoding 140 amino acids, which was identical to the reported amino acid sequence of rat α-Syn. After transformation, the recombinant plasmid pGEX-ra-Syn expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 18000, which was identical to the reported molecular size of rat α-Syn. Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against α-Syn. Conclusion The rat α-Syn gene was successfully expressed in prokaryotic expression system and highly purified rat α-Syn recombinant protein was produced.

Expression, purification and polyclonal antibody generation of p23, an Hsp90 cochaperone, in the amphioxus Branchiostoma belcheri

The cDNA of amphioxus p23, a highly conserved co-chaperone for Hsp90, was cloned into a bacterial expression vector pGEX - 6P - 1 and the GST-tagged fusion protein was produced in Eschherichia coli cells. The recombinant p23 was purified by affinity purification, and its molecular mass was estimated to be approximately 22 kDa by sodium dedecyl sulfate-polyacrylamide gel electrephoresis. The N-terminus of purified p23 was sequenced, and the resulting amino acid sequence matches exactly the predicted residues deduced from the amphioxus p23 gene. Besides, pelyclonal antibodies against the recombinant p23 were generated, and these antibodies not only recognized specifically the fusion protein GST - p23 from induced E. coli cells, purified GST - p23 and p23 protein, but also reacted with the total protein extracted fi＇om the adult amphioxus and formed a single positive band. These results pave the way for identifying its tissue and subcellular localization, and may open the door to clarifying its structure and mechanisms of biological role.

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.

Immunogenicity and safety of a recombinant fusion protein vaccine(V-01)against coronavirus disease 2019 in healthy adults:a randomized,double-blind,placebo-controlled,phaseⅡtrial

Background:Innovative coronavirus disease 2019(COVID-19)vaccines,with elevated global manufacturing capacity,enhanced safety and efficacy,simplified dosing regimens,and distribution that is less cold chain-dependent,are still global imperatives for tackling the ongoing pandemic.A previous phase I trial indicated that the recombinant COVID-19 vaccine(V-01),which contains a fusion protein(IFN-PADRE-RBD-Fc dimer)as its antigen,is safe and well tolerated,capable of inducing rapid and robust immune responses,and warranted further testing in additional clinical trials.Herein,we aimed to assess the immunogenicity and safety of V-01,providing rationales of appropriate dose regimen for further efficacy study.Methods:A randomized,double-blind,placebo-controlled phaseⅡclinical trial was initiated at the Gaozhou Municipal Centre for Disease Control and Prevention(Guangdong,China)in March 2021.Both younger(n=440;18–59 years of age)and older(n=440;≥60 years of age)adult participants in this trial were sequentially recruited into two distinct groups:two-dose regimen group in which participants were randomized either to follow a 10 or 25 mg of V-01 or placebo given intramuscularly 21 days apart(allocation ratio,3:3:1,n=120,120,40 for each regimen,respectively),or one-dose regimen groups in which participants were randomized either to receive a single injection of 50 mg of V-01 or placebo(allocation ratio,3:1,n=120,40,respectively).The primary immunogenicity endpoints were the geometric mean titers of neutralizing antibodies against live severe acute respiratory syndrome coronavirus 2,and specific binding antibodies to the receptor binding domain(RBD).The primary safety endpoint evaluation was the frequencies and percentages of overall adverse events(AEs)within 30 days after full immunization.Results:V-01 provoked substantial immune responses in the two-dose group,achieving encouragingly high titers of neutralizing antibody and anti-RBDimmunoglobulin,which peaked at day 35(161.9[95%confidence interval[CI]:133.3–196.7]and 149.3[95%CI:123.9–179.9]in 10 and 25 mg V-01 group of younger adults,respectively;111.6[95%CI:89.6–139.1]and 111.1[95%CI:89.2–138.4]in 10 and 25 mg V-01 group of older adults,respectively),and remained high at day 49 after a day-21 second dose;these levels significantly exceed those in convalescent serum from symptomatic COVID-19 patients(53.6,95%CI:31.3–91.7).Our preliminary data showthat V-01 is safe andwell tolerated,with reactogenicity predominantly being absent or mild in severity and only one vaccinerelated grade 3 or worse AE being observed within 30 days.The older adult participants demonstrated a more favorable safety profile compared with those in the younger adult group:with AEs percentages of 19.2%,25.8%,17.5%in older adults vs.34.2%,23.3%,26.7%in younger adults at the 10,25 mg V-01 two-dose group,and 50 mg V-01 one-dose group,respectively.Conclusions:The vaccine candidate V-01 appears to be safe and immunogenic.The preliminary findings support the advancement of the two-dose,10 mg V-01 regimen to a phaseⅢtrial for a large-scale population-based evaluation of safety and efficacy.

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.

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.

Purification and application of C-terminally truncated hepatitis C virus El proteins expressed in Escherichia coli

AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E, coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E, coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chrbmatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli, C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni2+-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 giycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.

Construction of the recombinant expression vector for CD80-IgG fusion gene and its expression in Chinese hamster ovary cells

To construct the recombinant expression functionally in Chinese hamster ovary cells in order vector for CD80-IgG fusion gene and to express it to be used as an effective method to eliminate the immune escape of leukemic cells, the cDNA encoding the signal and extracellular domains of murine CD80 was generated by PCR amplification from plasmid pcDNMB7 containing the full length cDNA of murine CD80 and those of murine IgG1, in which the Fc fragment was obtained through RT-PCR amplification from murine spleen cells. These two cDNAs were then cloned in tandem into eukaryotic expression vector pcDNA3.0 and the resultant recombinant plasmid pcDNA/CD80-IgG was then transfected to Chinese hamster ovary cells with liposome transfection reagent. The cell clones constitutively expressing CD80-IgG fusion protein were obtained by G418 screening. Western blotting and dot ELISA assay were used to detect the expression of the fusion protein in the supernatants of these cells. Meanwhile, the fusion protein expressed was then purified with affinity chromatography, and its biological activity was demonstrated by flow cytometry, MTr colorimetry and ELISA assay. The experimental resuits showed that these two inserts were successfully cloned into plasmid pcDNA3.0, and the highly purified fusion protein was obtained. This fusion protein was proved to be able to upregulate the density of CD80 on leukemic cells, deliberately promote the proliferative reactions of mouse allogenic lymphocytes and increase the killing activity against WEHI-3 cells from 49.7 % up to 84.6 %. In addition, this fusion protein could also enhance the IL-2 secretion from allogenic lymphocytes activated by tumorspecific antigens. It is concluded that the recombinant vector constructed can be functionally expressed in the mammalian cells, thus providing a solid foundation for the further investigation on the mechanism to eliminate the immune escape of leukemic cells in vivo.

Expression,purification and immunocharacteristics of recombination UreB protein of H.pylori

INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat shock protein and vacuolating cytotoxin and so on ,and urease is an important factor in the colinization of the gastric mucosa and suspected to cause damage to the gastric mucosa[10-14].At the same time ,urdase is also one of the important protective antigens .

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.

Use of recombinant human bone morphogenetic protein-2 in spine surgery

Bone morphogenetic proteins are osteoinductive factors which have gained popularity in orthopaedicsurgery and especially in spine surgery. The use of recombinant human bone morphogenetic protein-2 has been officially approved by the United States Food and Drug Administration only for single level anterior lumbar interbody fusion, nevertheless it is widely used by many surgeons with off-label indications. Despite advantages in bone formation, its use still remains a controversial issue and several complications have been described by authors who oppose their wide use.