Influence of Repair Welding on the Fatigue Behavior of S355J2 T-Joints.

This paper investigated the effect of repair welding on the microstructure, mechanical properties, and high cycle fatigue properties of S355J2 steel T-joints in orthotropic bridge decks. The test results found that the increase in grain size of the coarse, heat-affected zone decreased the hardness of the welded joint by about 30 HV. The tensile strength of the repair-welded joints was reduced by 20 MPa compared to the welded joints. For the high cycle fatigue behavior, the fatigue life of repair-welded joints is lower than that of the welded joints under the same dynamic load. The fracture positions of toe repair-welded joints were all at the weld root, while the fracture positions of the deck repair-welded joints were at the weld toe and weld root, with the same proportion. The fatigue life of toe repair-welded joints is reduced more than that of deck repair-welded joints. The traction structural stress method was used to analyze fatigue data of the welded and repair-welded joints, and the influence of angular misalignment on was considered. The fatigue data with and without AM are all within the ±95% confidence interval of the master S-N curve.

Effect of Glucose Contents on Electrochemical Corrosion Behavior of Ti/ZrO2 Brazing Joint in SBF.

The effect of glucose content on the electrochemical corrosion behavior of the Ti/ZrO2 brazing joint in simulated body fluid (SBF) was researched by the means of SEM morphologies, electrochemical and XPS analyses. Herein, pitting is observed to be a dominating corrosion model under the investigated glucose content. The pitting corrosion of the joint in 200 mg/dL SBF is minimal. In addition, the joint in 200 mg/dL SBF manifests the best corrosion resistance by electrochemical analyses, which indicates that glucose content has a bidirectional effect on corrosion of the Ti/ZrO2 brazing joint. Additionally, the corrosion current value and impedance of titanium and brazing joint are close, which indicates that their corrosion resistance is similar. Finally, the OH-, Cl-, Sn2+/Sn4+ and -COOH on the joint surface are found by XPS analysis, and the mechanism of Ti/ZrO2 brazing joint corrosion is elucidated. The study provides a novel understanding of the corrosion behavior and relevant corrosion mechanism of the Ti/ZrO2 brazing joint in body fluids with different glucose content.

Contact Reactive Brazing of TC4 Alloy to Al7075 Alloy with Deposited Cu Interlayer.

The brazing of Titanium alloy to Aluminum alloy is of great significance for lightweight application, but the stable surface oxide film limits it. In our work, the surface oxide film was removed by the ion bombardment, the deposited Cu layer by magnetron sputtering was selected as an interlayer, and then the contact reactive brazing of TC4 alloy to Al7075 alloy was realized. The microstructure and joining properties of TC4/Al7075 joints obtained under different parameters were observed and tested, respectively. The results revealed that the intermetallic compounds in the brazing seam reduced with the increased brazing parameters, while the reaction layer adjacent to TC4 alloy continuously thickened. The shear strength improved first and then decreased with the changing of brazing parameters, and the maximum shear strength of ~201.45 ± 4.40 MPa was obtained at 600 °C for 30 min. The fracture path of TC4/Al7075 joints changed from brittle fracture to transgranular fracture, and the intergranular fracture occurred when the brazing temperature was higher than 600 °C and the holding time exceeded 30 min. Our work provides theoretical and technological analyses for brazing TC4/Al7075 and shows potential applications for large-area brazing of titanium/aluminum.

A Simple High Power, Fast Response Streaming Potential/Current-Based Electric Nanogenerator Using a Layer of Al2O3 Nanoparticles.

Harvesting energy from ambient moisture and natural water sources is currently of great interest due to the need for standalone self-powered nano/micro-systems. In this work, we report on the development of a cost-effective nanogenerator based on a carbon paper-Al2O3 nanoparticle layer-carbon paper (CAC) sandwich structure, where the 3D Al2O3 layer is deposited via vacuum filtration. This type of device can produce an open-circuit voltage (UOC) of up to 4 V and a short-circuit current (ISC) of ∼18 µA with only an 8 µL water droplet applied. To our knowledge, this is the highest voltage yet reported from a single moisture/water-induced electricity nanogenerator using solid oxides and carbon-based materials. A remarkable output power of 14.8 µW can be reached with an optimized resistive load. An LED with a working voltage of 3-3.2 V can operate for a short time with the power from a single CAC device exposed to one 8 µL water droplet. Furthermore, a CAC generator adsorbing as little as 2 µL water droplets every 3 min can also give a UOC of 3.63 V. We show that CAC devices provide a robust electrical output over more than 200 wet-dry cycles without any deterioration in performance. These units demonstrate much promise as cost-effective electricity generators for harvesting energy from natural sources like rainwater, tap water, snow runoff, and dew. The response time of CAC devices can be as fast as 10-100 ms, making them ideal for applications as self-powered water detectors. The generation of power in this device arises from the streaming current. To assist in the optimization of these devices, we have analyzed how their response is related to such factors as layer thickness, time interval between application of water droplets, and the volume of each water droplet.

Chemical Vapor Deposition Growth of Degenerate p-Type Mo-Doped ReS2 Films and Their Homojunction.

Substitutional doping of transition metal dichalcogenide two-dimensional materials has proven to be effective in tuning their intrinsic properties, such as band gap, transport characteristics, and magnetism. In this study, we realized substitutional doping of monolayer rhenium disulfide (ReS2) with Mo via chemical vapor deposition. Scanning transmission electron microscopy demonstrated that Mo atoms are successfully doped into ReS2 by substitutionally replacing Re atoms in the lattice. Electrical measurements revealed the degenerate p-type semiconductor behavior of Mo-doped ReS2 field effect transistors, in agreement with density functional theory calculations. The p-n diode device based on a doped ReS2 and ReS2 homojunction exhibited gate-tunable current rectification behaviors, and the maximum rectification ratio could reach up to 150 at Vd = -2/+2 V. The successful synthesis of p-type ReS2 in this study could largely promote its application in novel electronic and optoelectronic devices.

IgG, IgM and IgA antibodies against the novel polyprotein in active tuberculosis.

BACKGROUND: The present study was aimed to evaluate whether IgG, IgM and IgA antibodies levels detected against a novel Mycobacterium tuberculosis polyprotein 38 F-64 F (with 38 F being the abbreviation for 38kD-ESAT6-CFP10 and 64 F for Mtb8.4-MPT64-TB16.3-Mtb8) are suitable for diagnosing active tuberculosis, and for monitoring the efficacy of chemotherapy on TB patients. METHODS: In this study, a total of 371 active TB patients without treatment were selected and categorized into S+/C+group (n=143), S-/C+group (n=106) or S-/C- group (n=122). A series of serum samples were collected from 82 active TB patients who had undergone anti-TB chemotherapy for 0-6 months at one month interval. Humoral responses (IgG, IgM and IgA) were determined for the novel Mycobacterium tuberculosis polyprotein using indirect ELISA methods in all of serum samples. RESULTS: For S+/C+, S-/C+and S-/C- active tuberculosis patients before anti-TB chemotherapy, the sensitivities of tests based on IgG were 65.7%, 46.2% and 52.5% respectively; the sensitivities based on IgM were 21.7%, 24.5% and 18.9%; and the sensitivities based on IgA were 25.2%, 17.9% and 23.8%. By combination of three isotypes, for all active tuberculosis patients, the test sensitivity increased to 70.4% with the specificity being 91.5%. After anti-TB chemotherapy, there were no significant differences between groups with different courses of anti-TB chemotherapy. CONCLUSIONS: The novel Mycobacterium tuberculosis polyprotein 38 F-64 F represents potential antigen suitable for measuring IgG, IgM and IgA antibodies. However, the serodiagnostic test based on the 38 F-64 F polyprotein appears unsuitable for monitoring the efficacy of chemotherapy.

Welding and Joining of Titanium Aluminides.

Welding and joining of titanium aluminides is the key to making them more attractive in industrial fields. The purpose of this review is to provide a comprehensive overview of recent progress in welding and joining of titanium aluminides, as well as to introduce current research and application. The possible methods available for titanium aluminides involve brazing, diffusion bonding, fusion welding, friction welding and reactive joining. Of the numerous methods, solid-state diffusion bonding and vacuum brazing have been most heavily investigated for producing reliable joints. The current state of understanding and development of every welding and joining method for titanium aluminides is addressed respectively. The focus is on the fundamental understanding of microstructure characteristics and processing-microstructure-property relationships in the welding and joining of titanium aluminides to themselves and to other materials.

Use of immuno-dominant epitope derived from genotype 4 as a diagnostic reagent for detecting the antibodies against Hepatitis E Virus.

BACKGROUND: Despite the genotype 4 has become the dominant cause of hepatitis E disease in China, none antigen derived from genotype 4 of hepatitis E virus (HEV) was used in current commercial anti-HEV immunoassay, and the serological reactivity of antigen derive from genotype 4 is not well-charactered. METHODS: We expressed and purified the 4 main immuno-dominant epitopes derived from genotype 1 and 4 including ORF2 (410-621aa) of genotype 4, ORF3 (47-114aa) of genotype 4, ORF2 (396-606aa) of genotype 1 and ORF3 (56-123aa) of genotype 4. RESULTS: The ORF2 of genotype 4 displayed good diagnostics performance according to ROC analysis using in-house panel, and the immunoassays based the ORF2 of genotype 4 was then developed to detect the anti-HEV IgG antibodies and evaluated further in 530 anti-HEV IgG positive specimens and 380 negative specimens. The sensitivity and the specificity is 98.1% (520/530) and 94.7% (360/380) for immunoassay based on ORF2 of genotype 4, 96.6% (512/530) and 92.6% (352/380) for commercial immunoassay based on genotype 1. It is noted that all of the positive samples will be detected by combing two assays together. The anti-HEV immunoassays based on genotype 4 are in accordance with Chinese anti-HEV national standard,and show an good agreement of 95.8% with commercial assay (kappa=0.913, P=0.014). CONCLUSIONS: The immunoassay based on ORF2G4 displays good performance, and combining assay based on genotype 1 together with genotype 4 will benefit the HEV diagnosis in large scale samples.

Enhanced serodiagnostic utility of novel Mycobacterium tuberculosis polyproteins.

OBJECTIVES: The detection of Mycobacterium tuberculosis specific antibodies in human sera has been a rapid and important diagnostic aid for tuberculosis (TB) control and prevention. However, any single antigen is not enough to be used to cover the antibody profiles of all TB patients. METHODS: Seven single antigens (38 kDa, ESAT-6, CFP10, Mtb8.4, MPT64, TB16.3 and Mtb8) were evaluated serodiagnostically. Two novel M. tuberculosis polyproteins, 38kD-ESAT6-CFP10 (38F) and Mtb8.4-MPT64-TB16.3-Mtb8 (64F), were expressed and the novel 38F-64F indirect ELISA assay used to analyze antibody responses to polyproteins in serum samples. RESULTS: The sensitivity of the novel 38F-64F indirect ELISA alone was much higher than that of the sputum culture test (86.91% vs. 50.62%) and that of the sputum smear test (78.64% vs. 47.57%). The novel 38F-64F indirect ELISA had a sensitivity of 74.16% with sera from extrapulmonary TB patients and a sensitivity of 37.14% with sera from LTBI. The specificity of the novel 38F-64F indirect ELISA was 90.36% with the sera from healthy blood donors and 94.15% with the sera from non-TB patients. CONCLUSIONS: The novel 38F-64F indirect ELISA assay had effective diagnostic performance and would make meaningful contribution to the diagnosis of TB disease in developing countries.

Hepatitis E virus ORF3 antigens derived from genotype 1 and 4 viruses are detected with varying efficiencies by an anti-HEV enzyme immunoassay.

The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein product remains unclear but it is able to induce a strong antibody response following HEV infection. Therefore, it has been used in some enzyme immunoassays (EIAs) for detecting anti-HEV antibody. In order to evaluate the difference in antigenicity of HEV ORF3 polypeptides derived from genotypes 1 and 4, two EIAs were developed, based on ORF3 polypeptides from genotypes 1 and 4 HEV. Serial weekly serum samples from two rhesus monkeys vaccinated with ORF3 antigens derived from the genotype 4 ORF3 protein and nine rhesus monkeys experimentally infected with genotypes 1 and 4 HEV were tested for anti-HEV using the assays. HEV ORF3 antigens derived from viruses of genotypes 1 and 4 showed different patterns of reactivity with sera obtained from monkeys immunized with ORF3 antigens or infected experimentally with HEV. The genotype 1 ORF3 polypeptide exhibited stronger reactivity with the sera from monkeys infected with genotype 1 than the genotype 4 ORF3 polypeptide. The genotype 4 ORF3 polypeptide demonstrated stronger reactivity with the sera from monkeys infected with genotype 4 than did the genotype 1 ORF3 polypeptide. The HEV ORF3 polypeptide contains genotype-specific antigens and the antigen-antibody reactions between the same genotypes were stronger than those between different genotypes.

Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody.

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies.

A double-antigen sandwich ELISA was developed a detection of HCV antibodies by a recombinant multi-epitope HCV antigen and a biotin-streptavidin amplification system. Three plasma specimens from 1708 individuals who were suspected previously to be HCV-positive using an HCV antibody diagnostic kit (Chuangxin, Xiamen, China) displayed negative results when using the ELISA. These results were validated by a recombinant immunoblotting assay (two were negative, and one was indeterminate). Among 889 blood specimens donated for clinical evaluation, 246 were positive and 630 were negative using the ELISA. The sensitivity and specificity of the ELISA were 98.7% and 100%, respectively. In 43 donors and 14 patients with chronic hepatitis C, the detectable rates for HCV IgM by both ELISA and the HCV anti-IgM detection reagents (Huimin, Shenyang, China) were 100%, and the detectable rate for HCV IgG using an indirect HCV-antibody detection kit (GWK, Beijing, China) was 98.3%. Thus, the double-antigen sandwich ELISA exhibits strong specificity and sensitivity and has been approved by the China State Food and Drug Administration (SFDA). The performance of the double-antigen sandwich ELISA was similar to the Ortho ELISA 3.0. It did not give false-negative results otherwise IgM was undetectable using an indirect HCV-antibody detection kit. This ELISA provides another method for the detection of HCV antibodies.

Evaluation of cross-reactive antibody response to HVR1 in chronic hepatitis C.

AIM: To evaluate the presence and cross-reactive antibodies against hypervariable region 1 (HVR1) in hepatitis C virus (HCV) infected patients and its relationship with the progression of the disease. METHODS: Sixteen representative HVR1 proteins selected from a unique set of 1600 natural sequences were used to semiquantitate the cross-reactivity of HVR1 antibodies in the sera of HCV patients. Fifty-five chronic HCV patients including 23 with asymptomatic mild hepatitis, 18 with chronic hepatitis and 16 with liver cirrhosis patients were studied. RESULTS: The degree of the cross-reactivity of anti-HVR1 antibodies in 23 patients with mild asymptomatic hepatitis was 3.09 ± 2.68, which was significantly lower than in those with chronic hepatitis (5.44 ± 3.93, P < 0.05) and liver cirrhosis (7.44 ± 3.90, P < 0.01). No correlation was observed between the broadness of the cross-reactivity anti-HVR1 antibodies and patient's age, infection time, serum alanine aminotransferase activity, or serum HCV-RNA concentration. It was the breath of cross-reactivity rather than the presence of anti-HVR1 antibody in HCV sera that was associated with the progression of liver disease. CONCLUSION: The broadly cross-reactive HVR1 antibodies generated in natural HCV patients can not neutralize the virus, which results in persistent infection in patients with chronic hepatitis.

Immunogenicity and efficacy of a bacterially expressed HEV ORF3 peptide, assessed by experimental infection of primates.

A 12.4-kDa peptide, corresponding to the entire ORF3 protein of hepatitis E virus (HEV), derived from human HEV genotype 4 and expressed in Escherichia coli as a fusion protein with a 17.5-kDa fragment of interleukin (IL)-1beta at the N-terminus, was recognized by HEV-reactive sera. Eight monkeys were immunized with the purified peptide, and seven were used as non-immunized controls. All 15 monkeys were challenged with HEV genotype 1 or 4. All control animals developed infection and hepatitis, and all but one vaccinated monkey became infected. Nevertheless, the vaccine was effective in reducing the virus titer and shortening the duration of viremia and fecal shedding. Furthermore, the vaccine provided some protection against hepatitis (1 of 2 monkeys in the two-dose regimen and 4 of 6 in the three-dose regimen did not develop severe hepatitis) compared to the controls. These results suggest that immunization with the bacterially expressed peptide may partially prevent experimental hepatitis, and even infection, in primates, following intravenous challenge with high doses of two HEV genotypes.

Varying abilities of recombinant polypeptides from different regions of hepatitis E virus ORF2 and ORF3 to detect anti-HEV immunoglobulin M.

Following infection with hepatitis E virus (HEV), anti-HEV immunoglobulin (Ig) M is thought to develop before anti-HEV IgG and to be a better marker for differentiating between the acute and convalescent phases of infection. In order to select polypeptides for improved detection of anti-HEV IgM, six and three overlapping polypeptides from open reading frames (ORFs) 2 and 3, respectively, of HEV genotypes 1 and 4 were expressed as fusion proteins in Escherichia coli. The reactivities of the polypeptides with anti-HEV IgM were evaluated using immunoblotting and enzyme immunoassays (EIAs). The data indicated that polypeptides from the N-terminus of ORF3 and middle region of ORF2 were weakly or not reactive with anti-HEV IgM, while those from the remaining regions of ORF2 and ORF3 contained reactive epitopes. Anti-HEV IgM against the N- or C-terminus of ORF2 appeared earlier and disappeared faster than that against polypeptides from the C-terminus of ORF3, based on serum samples from rhesus monkeys infected experimentally, and from patients infected naturally, with HEV. The N- and C-terminal polypeptides from ORF2 complemented one another in detecting anti-HEV IgM and EIA sensitivity was improved significantly with a combination of these polypeptides. The reactivities of ORF2 polypeptides from genotypes 1 and 4 were similar but that of ORF3 differed with sera from monkeys infected by the two genotypes. Thus, a combination of N- and C-terminal polypeptides of ORF2 from one genotype may be effective in EIAs to detect anti-HEV IgM.

Localization and cellular distribution of CPNE5 in embryonic mouse brain.

CPNE5 is one of the ubiquitous Ca(2+)-dependent, phospholipid-binding proteins that are highly conserved in animals. It was cloned in the fetal human brain with no exact functions identified yet. We have examined the distribution pattern of CPNE5 mRNA and protein in the developing murine brain by using in situ hybridization, western blotting and immunocytochemistry. Expression of CPNE5 mRNA remains high from embryonic day 9.5 (E9.5) to E15.5 in the developing murine brain. Whole-mount in situ hybridization with the E11.5 and E12.5 embryos showed the strong positive signals in the central nervous system. Western-blot analysis showed that CPNE5 protein is expressed in the developing but not in the adult murine brain. In situ hybridization and immunohistochemistry analysis on the embryonic brain sections indicated that both at RNA and protein levels CPNE5 is mainly expressed in frontal cortex, medial nasal prominence, ganglionic eminence and medulla, particularly in the ventricular zones. Further investigation revealed the co-localization of CPNE5 with Tuj1 and Nestin on embryonic brain sections. In addition to the slight expression in primary cultured neural progenitor cells, CPNE5 is found in soma and neurite projections of primary cultured neurons where Tuj1 is co-localized. Our results demonstrate that CPNE5 is expressed in both neural progenitor cells and the differentiated neurons during the neural development, which suggests that CPNE5 might play an important role in the development of murine central nervous system.

Cancerogenic effect of different fragments of the hepatitis C virus core protein.

The hepatitis C virus core protein plays an extremely important role in the hepatocarcinogenesis of hepatitis C virus. Little, however, is known about the oncogenic potency of fragments. Thus, the purpose of the present study is to investigate the cancerogenic effects of the different core protein fragments. Two series of recombinant plasmids containing hepatitis C virus core gene fragments encoding the different-length core protein were constructed using plasmid enhanced green fluorescent protein (pEGFP)-C1 and pcDNA3.1(+), respectively. Human hepatocyte L02 cells transiently transfected with pEGFP-C1-based plasmids were subjected to confocal laser scanning microscopy analysis to determine the localization of the different core protein fragments. The stably transfected L02 cells with the pcDNA3.1(+)-based core protein plasmids were used to investigate the ultrastructural effects of the core protein and the tumorigenicity of L02 cells expressing core protein fragments in athymic nude mice. The full-length core protein and Core130-191 were completely localized in the cytoplasm, while Core1-59 existed exclusively in the nucleus. On the other hand, Core50-140 and Core1-140 were observed in both the nucleus and the cytoplasm. Ultrastructural changes of L02 cells expressing the full-length core protein were comprehensive and included, for example, irregular nuclear, increased nuclear/cytoplasmic ratio and mitochondria swelling. The slight changes were observed in the cells expressing Core50-140 and Core130-191, whereas the ultrastructure of the cells expressing Core1-59 remained normal. All the L02 cells stably expressing different fragments of the core protein, with the exception of the C-terminal truncated fragment Core1-59, could induce the occurrence of tumor in the nude mice. The N-terminal fragment of the core protein, Core1-59, was not oncogenic, while the intermediate and posterior segments of the hepatitis C virus core protein had the cancerogenic potency. In view of the existence of many important immunogenic epitopes in it, the core protein anterior segment might be a safer candidate for the development of hepatitis C virus vaccine.

Detection of hepatitis C virus core antigen for early diagnosis of hepatitis C virus infection in plasma donor in China.

AIM: To evaluate the efficacy of a new hepatitis C virus (HCV) core antigen assay developed in China. METHODS: After the determination of HCV infection, 49 serial samples were selected from 11 regular plasma donors in 5 different plasma stations. To compare the performance of HCV core antigen detection and HCV PCR, these samples were genotyped, and each specimen was analyzed by ELISA for the detection of HCV core antigen and by qualitative HCV PCR. RESULTS: Among all of the sequential samples, the original 13 specimens were HCV RNA-negative, and 36 samples were HCV RNA-positive. Twenty-seven samples (75%) were HCV core antigen-positive from these HCV RNA-positive specimens. Conversely, 27 samples (93.1%) were found HCV RNA-positive in HCV core antigen-positive samples. Intervals between HCV RNA and HCV core antigen-positive, as well as between HCV core antigen-positive and HCV antibody-positive were 36.0 and 32.8 d, respectively. CONCLUSION: This HCV core antigen assay, developed in China, is able to detect much of anti-HCV-negative, HCV RNA-positive preseroconversion window period (PWP) plasma donations.

[Preparation of recombinant IL-1 fusion protein as the substrate for HCV NS3 serine protease].

AIM: To prepare recombinant IL-1 fusion protein as the substrate for the HCV NS3 serine protease. METHODS: NS5A-B gene fragment (2,412-2,427aa) synthesized by PCR was subcloned into prokaryotic expression vector pBVIL1 to fuse with IL-1 gene and the recombinant vector pBVIL1/NS5A-B was transformed into E.coli strain HB101. The fused protein was induced to express at 42degreesCelsius and purified by two-step column chromatography. The proteolysis of the purified IL-1 fusion protein catalyzed by NS3 serine protease was analyzed with SDS-PAGE, Western blot and ELISA. RESULTS: NS5A-B fragment gene was correctly subcloned into pBVIL1 vector and the fusion protein was expressed as inclusion body in transformed HB101 cells. The recombinant fusion protein can be cleaved into smaller fragments by NS3 protease. CONCLUSION: The recombinant fusion protein can be cleaved by NS3 serine protease successfully and specifically, suggesting that it can be used as a surrogate substrate of NS3 serine protease in searching for inhibitors of this protease.

Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli.

Tetramer analysis is a novel technique in immunological research that has dramatically changed our knowledge of the immune response to pathogens, tumors and autoimmune disease. Through the formation of major histocompatibility complex (MHC)-peptide tetrameric complexes, it can provide accurate counts of antigen-specific T-cells and it allows their phenotypical and functional analysis. The tetramer is composed of the human leukocyte antigen (HLA) heavy chain, beta-2 microglobulin (beta-2m), the nominal peptide, and streptavidin. The HLA heavy chain and the beta-2m are expressed in Escherichia coli. But up to now, all laboratories have been expressing these two proteins by using isopropyl beta-d-thiogalactopyranoside IPTG. IPTG is very expensive, and it is tedious and laborious to induce expression protein. So it is difficult to scale up to express the objective protein. To address this problem, extracellular fractions of HLA-A0201 and beta-2m (absent signal peptide) genes were cloned from peripheral blood mononuclear cells (PBMCs) by RT-PCR. DNA coding for a Gly-Ser linker and a BSP (15-amino acid substrate peptide for BirA-dependent biotinylation) was added to the COOH-terminus of the extracellular fraction of HLA-A0201 by PCR, using an HLA-A0201 as the template. Then the HLA-A0201-BSP and beta-2m genes were cloned into pBV220 vector and expressed, respectively. The expressed proteins were purified and detected by ELISA and Western blot analyses. High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs.