Ni-NTA Purification System Research Articles

Fusion Protein Consisting of Hemagglutinin Small Subunit and Truncated Nucleoprotein as a Universal Influenza Vaccine Candidate: Starting In-Silico Evaluation Toward In Vitro Expression.

Influenza virus is a respiratory pathogen, which causes high degree of mortality and morbidity during seasonal epidemics and sporadic pandemics. By selecting conserved antigenic proteins, for example, hemagglutinin small subunit (HA2) and nucleoprotein (NP), we aimed to develop a vaccine based on a fusion protein leading to both cellular and humoral responses that are the most challenging aspects in designing a universal vaccine. The bioinformatics analysis was performed for HA2-NP structure and function prediction. Primers for the antigenic part of NP were designed using bioinformatics tools. The desired product was amplified via polymerase chain reaction using the designed primers, which was then penetrated into T vector, followed by insertion into pET28a vector in order to construct pET28a/NP. The pET28a/HA2, previously generated in our lab, was digested with the same restriction enzymes as pET28a/NP (HindIII/Xhol). Then, NP was inserted to the downstream region of HA2 to construct pET28a/HA2. The generated pET28a/HA2-NP was transformed into Escherichia coli BL21 (DE3). The expression was induced by isopropyl β-d-l-thiogalactopyranoside. The results showed that the antigenic segment of NP was successfully cloned into pET28a/ HA2. The protein band of HA2-NP was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis, confirmed by Western blotting and purified with Ni-NTA purification system (QIAGEN, Germany). As currently available vaccines can cause some allergic reactions, using a chimer protein based on the bioinformatics analysis is continual, safe, and affordable, thus stimulating both cellular and humoral immunity systems. Our construct could potentially provide a basis for a universal vaccine candidate.

Prokaryotic Expression and Monoclonal Antibody Preparation of Rabies Virus Phosphoprotein

Background: Rabies is a zoonotic infectious disease that infects the human and animal central nervous system. Worldwide, especially in low-income countries, this disease is still a burden for public health. Among the rabies virus proteins, phosphoprotein plays a very important role in viral infection, and this research found that immunization of rabies virus vaccines could widely induce antibody responses against phosphoprotein, therefore rabies virus phosphoprotein may be a useful target for development of rapid and low cost serological diagnosis test, and therapeutic drugs. Objectives: The aim of this study was to prepare anti-rabies virus phosphoprotein monoclonal antibody, which is used for rapid detection and diagnosis of rabies virus infection. Methods: The phosphoprotein gene was amplified by the polymerase chain reaction (PCR), and sub-cloned in a his-tagged prokaryotic expression vector to generate a recombinant plasmid named pET-32a-RABV-P. Recombinant RABV-P was induced by isopropyl-β-D-Thiogalactopyranoside (IPTG), and then purified by the Ni-NTA purification system. Immunization of BALB/c mice with the recombinant protein was performed, and the spleen cells of the immunized mice and SP2/0 myeloma cells were fused together to obtain the monoclonal cell strains, and then identification of the characteristics of the antibody by the enzyme linked immunosorbent assay (ELISA), western blotting, and indirect immunofluorescence assay was done. Results: The prokaryotic expression vector of pET-32a-RABV-P was successfully constructed. The fusion protein was expressed in Escherichia coli Rosetta and purified. After cell fusion, a hybridoma cell line 1A4 was successfully obtained. The antibody titer of the anti-RABV-P ascites reached 256,000. The results of western blotting and indirect immunofluorescence assay indicated that 1A4 hybridoma cell line was able to produce specific antibodies against rabies virus phosphoprotein. Conclusions: Recombinant rabies virus phosphoprotein could be successfully expressed in E. coli. A specific monoclonal antibody against rabies virus phosphoprotein has been successfully prepared.

Specific Monoclonal Antibodies Recognizing the Endogenous Chicken High Mobility Group Box 1 Protein.

High mobility group box 1 (HMGB1) is a key member of the "danger associated molecular patterns" (DAMPs), which can localize in various compartments of the cell, and plays important roles in systemic inflammation. In the present study, monoclonal antibodies (MAbs) specifically against chicken HMGB1 were generated. The open reading frame of chicken HMGB1 was amplified by RT-PCR and cloned into the prokaryotic expression vector pET-28a to construct a recombinant plasmid pET-chHMGB1. The recombinant chicken HMGB1 protein was expressed in Escherichia coli Rosetta under IPTG induction and then purified by Ni-NTA Purification System. BALB/c mice were immunized with the purified recombinant HMGB1 protein, and three strains of hybridoma cells named 1F10, 8C11, and 4D8 secreting MAbs of chicken HMGB1 were obtained by hybridoma technique. Western blot and indirect immunofluorescence assays showed that the endogenous HMGB1 in various cell lines and glycosylated HMGB1 could both be specifically recognized by the prepared MAbs. This work indicated that the MAbs against chicken HMGB1 would be a valuable tool for further studies of HMGB1-mediated signaling in virus-infected cells and investigates the role of HMGB1 in avian virus pathogenesis.

Prokaryotic expression of a multi-epitope gene of Xinjiang hemorrhagic fever virus and development of an indirect ELISA for virus detection

Objective To express and purify the multi-epitope peptide of Xinjiang hemorrhagic fever virus (XHFV) in a prokaryotic expression system and to further use it as diagnostic antigen to establish an indirect ELISA for the detection of XHFV in animal sera. Methods A double copy multi-epitope gene (MEPX2) segment with repeated tandem including six highly conservative and dominant B cell epitopes was designed based on the analysis of hydrophilicity and antigenic determinant sites in amino acid sequences of nucleoprotein and glycoprotein from XHFV strain YL04057. The multi-epitope gene MEPX2 was synthesized by chemical method and then inserted into the prokaryotic expression vector pET-32a (+ ) to construct the prokaryotic expression plasmid pET-32a (+ )-MEPX2. The recombinant expression plasmid was transformed into E.coli BL21 (DE3) to induce the expression of rMEPX2 by IPTG. The expressed products were purified by Ni-NTA purification system. The antigenicity of rMEPX2 was identified by Western blot assay. The purified rMEPX2 was used as coating antigen to establish an indirect ELISA by checkerboard for the detection of XHFV in animal serum samples. The results of virus detection were compared with those by using commercial ELISA kit. Results The recombinant expression plasmid pET-32a (+ )-MEPX2 was constructed successfully as confirmed by restriction enzyme digestion and DNA sequencing analysis. Results of the SDS-PAGE and Western blot assay confirmed that the rMEPX2 was expressed correctly with a relative molecular mass (Mr) of about 49×103. The rMEPX2 was recognized specifically by His-tag mouse monoclonal antibody and by positive serum samples of sheep naturally infected with XHFV. The indirect ELISA for the detection of XHFV IgG antibody was established by using the purified rMEPX2 as antigen. Compared with the commercial ELISA kit, the established indirect ELISA showed good specificity for the detection of XHFV in 108 sheep serum samples. Conclusion The recombinant multi-epitope protein rMEPX2 with good antigenicity was obtained successfully. The established indirect ELISA based on the rMEPX2 could be used for the detection of specific antibodies against XHFV. Key words: Xinjiang hemorrhagic fever virus; Multi-epitope peptide; Prokaryotic expression; Indirect ELISA

Prokaryotic expression and antibody preparation of truncated glycoprotein of Crimean-Congo hemorrhagic fever virus

Objective To express and purify truncated glycoprotein(Gn, Gc1, Gc2) of Crimean-Congo hemorrhagic fever virus(CCHFV) strain 79121 from E. coli cells, and to prepare their polyclonal antibodies respectively. Methods The genes encoding Gn, Gc1 and Gc2 from CCHFV strain 79121 were amplified by RT-PCR, and cloned into prokaryotic expression vector pET-28a (+ ) to construct recombinant plasmids. The three recombinant plasmids were then transformed into E. coli BL21(DE3) to obtain the Gn-His, Gc1-His and Gc2-His fusion proteins through IPTG induction. The fusion proteins were purified by Ni-NTA purification system, and then analyzed by using SDS-PAGE. To prepare antisera, the purified Gn-His, Gc1-His and Gc2-His proteins were employed to immunize New Zealand White Rabbit (NZW), respectively. The titer and specificity of the antisera to each truncated glycoprotein were analyzed by using ELISA and Western blot, respectively. Results The recombinant expression vectors of pET-28a-Gn, pET-28a-Gc1 and pET-28a-Gc2 were constructed successfully by restriction enzyme analysis and DNA sequencing. The relative molecular mass(Mr) of Gn-His, Gc1-His and Gc2-His were approximately 25 000, 33 000 and 34 000, and the titers of the corresponding polyclonal antibodies were above 1∶25 600, 1∶6400 and 1∶25 600, respectively. The antisera were able to bind to the corresponding fusion protein specifically. Conclusion The recombinant expression of truncated glycoprotein (Gn, Gc1, Gc2) of CCHFV strain 79121 and the preparation of their high titer polyclonal antibodies laid foundation for further studying the biological function of glycoprotein and the rapid detection of CCHFV. Key words: Crimean-Congo hemorrhagic fever virus; Glycoprotein; Prokaryotic expression; Polyclonal antibody

Heterologous expression of the antifungal β-chitin binding protein CBP24 from bacillus thuringiensis and its synergistic action with bacterial chitinases.

The genome sequence analysis of Bacillus thuringiensis serovar konkukian S4 has shown to contain two chitinases (Chi74, Chi39) and two chitin-binding proteins (CBP50 and CBP24). The Chi74, Chi39 and CBP50 have been characterized previously. The chitin-binding protein CBP24 was cloned and heterologously expressed in Escherichia coli. The recombinant protein was purified using a Ni-NTA purification system. The purified protein was used to study its substrate binding activity using crystalline chitin variants as substrates. The Bmax and Kd values have shown that it preferably binds to β-type of the crystalline chitin at a range of pH with peak activity between 5.5-7.5. To elucidate the role of CBP24 in the chitin degradation system of S4, the purified chitinases Chi74, Chi39 along with the ChiA from Serratia proteamcualans were used in different combinations with the CBP24 and chitinolytic activity was assayed. It was shown that the CBP24 acts synergistically with chitin degradation activity of bacterial chitinases non-specifically. Moreover, the CBP24 has shown antifungal activity against plant pathogenic fungi Fusarium oxysporum and Rhizoctonia solani. The present study will lead us to develop a technology for environmental friendly conversion of chitin to valuable products.

Transgenic Expression of a Bacterial Thermophilic Amylase in the Chlamydomonas reinhardtii Chloroplast to Facilitate Algal Biofuel Production

This study investigated expression of a thermophilic amylase (arAmyBH) from hyperthermophilic bacterium Thermotoga neapolitana into Chlamydomonas reinhardtii chloroplast to facilitate in vivo starch hydrolysis for algal biofuel production. The expression and accumulation of the recombinant protein in the algal chloroplast were verified by Western blot analysis. The recombinant arAmyBH was purified using Ni-NTA purification system. The optimum pH and temperature of arAmyBH were 5.5 and 60 °C, respectively. The enzymatic hydrolysis of the algal starch demonstrated that with supplemental commercial alpha-amylase, the transformant completely converted starch into reducing sugars in vivo at an elevated temperature.

Expression and functional analysis of polygalacturonase gene Pcipg5 from the plant pathogen Phytophthora capsici

As one of the cell wall-degrading enzymes, polygalacturonase involves in pathogenicity and virulence in a number of host pathogen interactions. In the polygalacturonase multigene family of Phytophthora capsici , Pcipg5 was screened from the genomic library. The Pcipg5 gene contains three putative active sites (179Asp, 200Asp, 201Asp) and one potential N-glycosylation site (252Asn). PCIPG5, four active-site mutagenesis proteins and N-glycosylation mutation were expressed in Pichia pastoris and purified by Ni-NTA Purification System. Pcipg5 functions were determined by Western blot, RT-PCR and Northern blot. Transmission electron microscopy discovery individually in treated pepper leaves was used to determine the virulence of PCIPG5. The results show that Pcipg5 can encode a pathogenesis-related protein during P. capsici infection of pepper leaves and degrade cell walls to produce necrotic lesions in treated pepper leaves. Meanwhile, the N-glycosylation mutagenesis protein decreased the activity compared with PCIPG5. It indicates that the existence N-glycosylation site in PCIPG5 plays a partial role in the activity of this enzyme. Key words : Phytophthora capsici, Pcipg5, host-pathogen interactions, N-glycosylation.

Molecular cloning and characterization of a HSP70 gene from Schistosoma japonicum

Schistosoma japonicum is the pathogen responsible for schistosomiasis japonica, one of the major infectious diseases targeted for prevention nationally in China. Expression of heat shock proteins (HSPs) following stress plays a very important biological role in many organisms including S. japonicum. Among the HSP family, the 70-kDa HSPs are most responsible for intracellular chaperone and extracellular immunoregulatory functions. Based on the published sequences in GenBank/EMBL (AF044412.1), open reading frame belonging to HSP70 protein corresponds to a full-length cDNA containing an open reading frame of 1,947 bp encoded of 648 amino acids was identified as HSP70 from schistosome. In this study, the coding region that we named rSj648/hsp70 was amplified from S. japonicum adult worm cDNA library, and the recombinant protein was expressed in vector pET32a(+) and purified using a Ni-NTA purification system. The target protein rSj648/hsp70 was determined by matrix-assisted laser desorption/ionization mass spectrometer after thrombin digestion and dialysis. Reverse transcriptase polymerase chain reaction and Western blotting analysis confirmed that Sj648/hsp70 could be expressed in the eggs, normal cercariae, ultraviolet-attenuated cercariae (UVAC), and adult worms of S. japonicum. Real-time quantitative PCR analysis indicated that Sj648/hsp70 was expressed significantly higher in eggs than that in cercariae and adult worms, and the expression in UVAC was higher than that in normal cercariae. A thermotolerance assay showed that rSj648/hsp70 could protect Escherichia coli cells from heat damage. The detection of specific antibody levels by indirect enzyme-linked immunosorbent assay demonstrated that mice immunized with rSj648/hsp70 induced higher level of specific anti-rSj648/hsp70 IgG1 compared with those vaccinated with adjuvant alone, indicating that rSj648/hsp70 was able to elicit Th2-type bias immune response. Our results suggest that Sj648/hsp70 might be an important molecule in parasite-host interaction and display potential roles in mice immunoregulation system.

Construction and expression of a prokaryotic expression vector for human TREM-1 and preparation of a polyclonal antibody

Objective To study the expression of a prokaryotic expression vector for human triggering receptor expressed on myeloid cell ( TREM-1 ) and preparation of a polyclonal antibody. Methods Human TREM-1 gene was amplified with specific primers. The polymerase chain reaction (PCR) product was digested with enzymes and then linked into the recombinant prokaryotic expression vector pET28a ( + ). The expression vector was transformed into E. coli BL21 (DE3) and induced with IPTG to express human TREM-1 fusion protein that was then purified with Ni-NTA purification system and analyzed with SDS-PAGE. The purified fusion protein was used to prepare polyclonal antibody in rabbits. Results The sequence analysis confirmed that this recombinant expression vector contained human TREM-1 coding sequence that was identical with the published sequence in Genbank. The plasmid expressed a 21.80 kDa protein. After purification with Ni-NTA purification system, the resulted protein was analytically pure according to the analysis with SDS-PAGE. The titer of the antiserum was 1 : 16 and 1 : 25 600 detected by double diffusion test and ELISA, respectively. Western blotting analysis demonstrated that the anti-TREM-1 antibody bound specifically human TREM-1 recombinant protein. Conclusion The prokaryotic expression vector and the polyclonal antibody against human TREM-1 were prepared successfully. Key words: TREM-1 ; Prokaryotic expression vector; Sepsis; Polyclonal antibody

Expression ofMycobacterium tuberculosisFerric Uptake Regulator A Gene inEscherichia coliand Generation of Monoclonal Antibodies to FurA

Ferric uptake regulator A of Mycobacterium tuberculosis (MTB), which belongs to the Fur superfamily, is situated immediately upstream of katG encoding catalase-peroxidase, a major virulence factor that also activates the pro-drug isoniazid. The feature and role of FurA in oxidative stress contribute to research on the pathogenesis of mycobacteria. In this study, four novel mouse monoclonal antibodies were generated using the prokaryotically expressed FurA protein as immunogen. The furA gene of M. tuberculosis H37Rv was inserted into a bacterial expression vector of pRSET-A and effectively expressed in Escherichia coli BL21(DE3). The expressed fusion protein existed as soluble form in cell lysates and was purified via Ni-NTA purification system. Using the fusion protein to immunize BALB/c mice, four monoclonal antibodies (H9H6, H9E12, H10H6, and H10H8) were produced. As shown by Western blot analysis and cell fluorescence microscopy assay, the four antibodies could recognize the FurA protein, respectively. Then we assessed the effect of iron on the expression of FurA in MTB H37Rv and we concluded that iron does not affect FurA expression. These results suggest that the antibodies against FurA may provide a powerful tool for elucidating FurA biofunctions and regulation mechanism in the pathogenesis of tuberculosis.

Effects of Micrococcus luteus Rpf and Rpf domain protein on growth of Mycobacterium tuberculosis

Objective To purify Micrococcus luteus Rpf and Rpf domain fusion protein, and to in-vestigate its effects on growth of Mycobacterium tuberculosis. Methods The recombinant plasmids pPro-EXHT-Rpf and pPro-EXHT-Rpf domain were expressed in E. Coli DHSa and then purified under denaturing condition via Ni-NTA purification system and confirmed by Western blot. The biochemical property of the M. Luteus Rpf and Rpf domain was analyzed by stimulating the resuscitation of M. Tuberculosis H37Ra which were in non-culturable' condition. Results The Rpf and Rpf domain products achieved 95% and 93% pure respectively, and the molecular weight was 30 x 103 and 12 x 103, the yield of purification was about 471 mg/L and 337 mg/L of the culture. The M. Luteus Rpf and Rpf domain from the E. Coli showed activity of stimulating the resuscitation of M. Luteus and M. Tuberculosis H37Ra in non-cuhurable' condition which could be inhibited by monoclonal antibodies of M. Luteus Rpf domain remarkably. Conclusion It was dem-onstrated that the purification of Rpf and Rpf domain have high biological activity for further functional, pharmacological and clinical investigations, and M. Luteus Rpf domain protein is fully active as M. Lateus full-length Rpf. Key words: Micrococcus luteus; Mycobacterium tuberculosis; Resuscitation promoting factor; Domain

Expression profile, localization of an 8-kDa calcium-binding protein from Schistosoma japonicum (SjCa8), and vaccine potential of recombinant SjCa8 (rSjCa8) against infections in mice

Researches on genes expressed in a cercarial stage-specific manner may help us understand the molecular events and functions during schistosome invasion of skin. A genomic clone encoding 8-kDa calcium-binding protein (SjCa8) specifically expressed in cercariae and skin-stage schistosomulum (transformed within 3 h) was obtained from cercariae. Recombinant protein was expressed in vector pET32a (+) and purified using a Ni-NTA purification system. The target protein SjCa8 was determined by matrix-assisted laser desorption/ionization time-of-flight (TOF)/TOF mass spectrometer after thrombin digestion and dialysis. Reverse transcriptase polymerase chain reaction and Western blot revealed SjCa8 can be detected in cercaria and skin-stage schistosomulum but not lung-stage schistosomulum, adult, or egg and was localized to head gland, penetration gland tubes, and penetration glands where Ca(2+) was abundant, and the cercarial tegument (but not tegument of tail) and body-tail junction. Furthermore, SjCa8 was interestingly detected in cercarial secretions. The characterization of SjCa8 indicated that it may undergo structural and physiological modifications, including repair of the surface membrane, changes in permeability that account for the loss of water tolerance, activities of calcium-depending enzymes, and immune signaling, etc. Furthermore, vaccination with rSjCa8 plus adjuvant induced protective effect with 50.39% worm reduction rate and significantly high hepatic and intestine egg reduction rates (54.16%, 50.63%, respectively), which is possibly mediated through an apparent induction of Th1-type immune response for strikingly high level of IgG2a and IgG2b developed in immunized C57BL/6 mice.

Production and verification of human papillomavirus type 18 vaccine in vitro

This study aimed to generate human papillomavirus (HPV) type 18 L1 virus-like particles (VLPs) and investigated the immunity effectiveness in vitro by using the Bac to Bac baculovirus expression system and Ni-NTA purification system to express and purify the VLP. The bio-function and immune response of purified VLP were analyzed by a mouse erythrocyte haemagglutination assay and the IFN-gamma ELISPOT. The proteins of interest, primarily located at the nucleus, formed VLP by self-assembly, and had an effective immune activity in vitro. The level of IFN-gamma in the specific HPV18 positive group was significantly higher than that of the mixed high-risk HPV infected group and non-HPV infection one in vitro. Our study indicated that HPV18 later protein L1 was efficiently expressed in Bac to Bac baculovirus expression system and the HPV18 L1-VLPs showed immunoreactivity and bio-activity in vitro. Furthermore, the ELISPOT results support the hypothesis that the cell-mediated immunity generated by HPV18 L1-VLP may cross-type among high-risk HPV types.

The murine thymic stromal lymphopoietin receptor is partially expressed with an extracellular C-terminus

The gene encoding the murine thymic stromal lymphopoietin receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica Nuclear Polyhedrosis Virus and expressed with an N-terminal poly-histidine tag and a C-terminal FLAG-tag in the Spodoptera frugiperda insect cell line Sf9 during viral infection. Flow cytometer analysis of cells infected with the produced recombinant virus FastBacHisB-mδ1-FLAG demonstrated that a majority of the infected cells expressed the mTSLPR with an extracellular C-terminal end. A similar observation was noticed in COS cells transfected with pSVL-mTSLPR-FLAG. Immunoblotting with monoclonal anti-FLAG or anti-his antibodies indicated that the corresponding receptor protein migrated as an approximately 50 kDa protein. mTSLPR produced in presence of tunicamycin migrated with a molecular weight around 40 kDa. The genetically fused poly-histidine tag was also demonstrated to be functional using a Ni-NTA purification system, indicating this protein otherwise to have normal biochemical properties.