Ni-NTA Resin Research Articles

Variations of binding, washing, and concentration of imidazole on purification of recombinant Fim-C Protein Salmonella typhi with Ni-NTA Resin

Typhoid fever is an endemic disease in Indonesia. Prevention of typhoid fever can be done by administering vaccines. It is known that one of the raw materials for vaccines is a recombinant protein. This study aims to obtain information on the optimum conditions for purification of Salmonella typhi Fim-C recombinant protein with Ni-NTA resin as vaccine raw material. The three main stages of the purification process in this study were binding, washing, and elution of S. typhi Fim-C recombinant proteins. The binding and washing variations of recombinant proteins were carried out twice, four times and six times, while the elution process was carried out at imidazole concentrations of 200 mM, 250 mM, and 300 mM. Purification with a binding process four and six times gave almost the same intensity of S. typhi Fim-C protein bands. Whereas protein was elution at an imidazole concentration of 300 mM showed higher band instability. The results of characterization using SDS-PAGE and analysis using software ImageJ gel analysis showed that the longer the incubation time and the repetition of the binding process, the more protein bound to the resin. Furthermore, the more washing processes are obtained the purer proteins. Based on the data obtained it can be concluded that the purification of the S. typhi Fim-C recombinant protein was optimum at a four-time binding process, six times washing and a 300 mM imidazole concentration. These results are expected to be the basis for recombinant protein refining on a pilot scale and industry scale in better vaccine preparation.

High-efficiency Ni2+-NTA/PAA magnetic beads with specific separation on His-tagged protein.

To effective capture and universal enrichment of His-tagged protein, polyacrylic acid (PAA) brushes were used to encapsulate Fe3O4 nanoparticles, connect NTA, and Ni2+ to prepare magnetic beads. These materials provide many advantages, such as excellent stability, tuneable particle size, and a surface for further functionalisation with biomolecules. His-tagged green fluorescence protein (GFP) was separated efficiently, and the binding capacity of Fe3O4/MPS@PAA/NTA-Ni2+ was 93.4 mg/g. Compared with High-Affinity Ni-NTA Resin and Ni-NTA Magnetic Agarose Beads, Fe3O4/MPS@PAA/NTA-Ni2+ nanocomposites exhibited higher separation efficiency and binding capacity towards His-tagged GFP. Moreover, the selectivity and recyclability of them for the target proteins were maintained well after six cycles. This study would widen the application of PAA in constructing multifunctional nanocomposites for biomedical fields.

The Effects of Temperature and Culture Medium Composition on Protein Recombinant JSU-pET Expression

There are several factors influencing the protein expression. The study was aimed to increase the yield of protein recombinant JSU-pET by applying combination of temperature and medium culture composition. This research was composed in a 2x2 factorial design with two level temperatures of 37<sup>o</sup>C and 25<sup>o</sup>C and 2 medium compositions of LB and mLB (modified). <em>Escherichia coli</em> bearing JSU-pET plasmid were grown in two applied media and incubated in two applied temperatures. The cells were harvested by centrifugation the pellets were then lysed. The lysed cells were centrifuged, the supernatant then purified by Ni-NTA Resin Agarose. The purified JSU protein recombinant was characterized and quantified by spectrophotometer. The Result showed that the JSU protein recombinant was successfully expressed on the right size (37kDa) in all treatments even though the JSU-pET showed sharper band in m LB at 37<sup>o</sup>C. Statistically the protein yield was not significant different (P>0.05), however culture temperature of 25<sup>o</sup>C showed higher yield (0.618±0.095 vs 0.704±0.094) than cultured at 37<sup>o</sup>C (0.598±0.137 vs 0.553±0.041), respectively either cultured in LB or mLB. This finding suggests that a lower culture temperature could increase protein yield both either in LB or modified LB.

Soluble expression, purification and biochemical characterization of a C-7 cholesterol dehydrogenase from Drosophila melanogaster

The C-7 cholesterol dehydrogenase (NVD), which converts cholesterol to 7-dehydrocholesterol (7-DHC) by 7,8-dehydrogenation, plays a pivotal role in the metabolism of cholesterol and steroid intermediates. The NVD protein was successfully expressed in insect Sf9 cells. To reduce the production cost for industrial application, the NVD gene was cloned into E. coli BL21(DE3). However, the His-tagged NVD protein showed poor binding to Ni-NTA resin, mainly due to the formation of inclusion bodies. Consequently, the expression and solubility of NVD were optimized by respectively fusing it with maltose-binding protein (MBP), glutathione S-transferase (GST), and the nonspecific DNA binding protein from Sulfolobus solfataricus (Sso7d) as solubility tags. The NVD fusion with MBP at the N-terminus and His-tag at the C-terminus achieved a high yield of the soluble enzyme. It was further purified by ion-exchange chromatography with 95.4% purity and with a 10.4% yield. The product 7-DHC, which is produced in a reaction catalyzed by NVD and ferredoxin reductase KshB, was initially identified by GC–MS. An analysis of the amino acid sequence alignment revealed a distinct Rieske-type iron-sulfur cluster and non-heme Fe2+-binding domain, which are evolutionarily conserved among NVD family enzymes.

Role of Molecular Interactions and Oligomerization in Chaperone Activity of Recombinant Acr from Mycobacterium tuberculosis.

Background: The chaperone activity of Mycobacterium tuberculosis Acr is an important function that helps to prevent misfolding of protein substrates inside the host, especially in conditions of hypoxia.Objectives: The aim of this study was to establish the correlation of structure and function of recombinant Acr proteins both before and after gel filtration chromatography. The aim was also to find the oligomeric conformation of these samples and use this information to explain differences in activit.Material and Methods: M. tuberculosis acr gene was cloned with an N-terminal His-tag in pET28a and expressed with IPTG induction in BL2 (DE3) competent Escherichia coli. The activity of a recombinant Acr without gel filtration was checked by preventing thermal aggregation of citrate synthase at 45°C and the chaperone activity against insulin B chain aggregation at 60°C and 37°C. On further purification using gel filtration chromatography, the protein was again tested for chaperone activity using insulin as substrate at 37°C with two types of samples without and with gel filtration designated A and B respectively. The effects of pre–heat treatment at 60 °C on chaperone activity of both A and B samples were studied by performing the chaperone assay at 37°C.Results: The level of expression was 40 to 50 mg /l. The protein was expressed in a soluble form at 37°C and subsequently purified by a 3 step gradient of imidazole using Ni-NTA resin. Gel filtration chromatography showed recombinant Acr to be a mixture of 9 to 15-mers, whereas Native-PAGE analysis showed a large proportion of 5 and 7 mers in the non gel-filtered sample, while non gel –filtered samples showed more proportions of higher size oligomers. The chaperone activity of non gel-filtered (A) samples was less than gel-filtered (B) samples at 37°C with 24 µM required of A for complete inhibition as compared to 6 µM of B. The chaperone activity of non gel–filtered samples at 60°C showed complete inhibition of activity at a concentration of 44 µM. Molecular interaction studies showed influence of size of oligomers on molecular coverage of insulin B chain. Pre-heat treatment improved the activity only after the gel filtration.Conclusions: The larger proportion of monomers in the non gel-filtered sample could explain the difference in activity as compared to the gel-filtered samples in terms of molecular interaction with insulin. Increased oligomer size favorably affected secondary structure, a finding not reported so far, and warranting further investigation. A molecular level interaction of inhibition was predicted using Avogadro number of molecules and oligomer size. The difference in activity after pre–heat treatment seemed to indicate an important role for oligomerization.

Cloning and prokaryotic expression of CHI in Chrysanthemum morifolium cv.'Hangju'

Three Chrysanthemum-chalcone-isomerase genes( CmCHI) were successfully cloned by PCR from the database of Chrysanthemum transcriptome and named CmCHI1,CmCHI2 and CmCHI3,respectively. Bioinformatics analysis showed that the base numbers of CmCHI1-3 open reading frame were 708,633 and 681 bp,encoding 235,210 and 226 amino acids,respectively. Three fusion proteins of about 30 kDa were successfully induced by prokaryotic expression technology,and the corresponding recombinant fusion proteins were isolated and purified by Ni-NTA resin column. Clustering analysis showed that the 3 CmCHI were homologous with Compositae plants,and CmCHI1 and CmCHI3 belonged to type Ⅰ CHI. CmCHI2 belongs to type Ⅳ CHI. Using β-actin as an internal reference gene,RT-qPCR was used to detect and analyze the expression of CmCHI1-3 genes in Hangju. The results showed that the expression levels of CmCHI1 and CmCHI3 were higher,while the expression levels of CmCHI2 were lower. It was concluded that CmCHI1 and CmCHI3 were the main chalcone isomerase genes involved in the synthesis of flavonoids in Hangju,and CmCHI2 was a helper gene. Flooding treatment significantly promoted the expression of CmCHI1 and CmCHI3 genes,but had no regulatory effect on CmCHI2. The above results provided a basis for further study of the molecular regulation mechanism of CHI gene in the metabolism of flavonoids in Hangju,which laid a foundation for improving the content of flavonoids in Hangju and finally improving the medicinal quality of Hangju.

Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Using Polyclonal Antibody Raised against Recombinant VP3 Capsid Protein Expressed in Escherichia coli

In Korea, sacbrood virus infecting Apis cerana (AcSBV) is a small RNA virus belonging to the family Iflaviridae. AcSBV causes larval death, and even the collapse of entire bee colonies and its viral capsid is composed of four structural proteins (VP1, VP2, VP3 and VP4). In this study, we synthesized VP3 gene based on genomic sequences to insert into a His-tag expression plasmid of pET-28a. The resultant recombinant vector was then transformed into Escherichia coli BL21 (DE3) to express the recombinant VP3 protein (rVP3). The rVP3 protein was strongly expressed in an insoluble fraction, with a molecular size of around 26 kDa. Purification of the rVP3 protein using an affinity Ni-NTA resin, followed by SDS-PAGE separation, resulted in a high purity of the protein and used as an antigen to raise the polyclonal antibodies. Mice were then immunized with the purified rVP3 protein and the antiserum collected at 4SUPth/SUP week and 6SUPth/SUP week was able to detect the rVP3 at a concentration of 500 ng and 250 ng. Furthermore, IgG antibody levels reacted with wild-type larva crude protein extract, AcSBV-infected larva crude protein extracts and rVP3 protein were quantitatively analyzed by ELISA and qualitatively analyzed by western blotting. This is the first report on production of polyclonal antibodies directed against the rVP3 protein of AcSBV and its use for detection and diagnosis of virus from field samples.

Expression and purification of recombinant coat protein of sugarcane mosaic virus from Indonesian isolate as an antigen for antibody production

Sugarcane mosaic virus (SCMV, genus Potyvirus, family Potyviridae) is a prominent pathogen of sugarcane (Saccharum sp. hybrids). It can cause losses in susceptible varieties, in crop as well as sugar production, economically. Although it has been studied in major sugar-producing countries, research on the definement of SCMV from Indonesian isolates based on molecular study has been very limited. This study aimed to obtain a proper recombinant antigens emanating from coat protein of SCMV from Indonesian isolate in order to produce polyclonal antibodies that cann be used for immunodiagnosis assays in a subsequent study. A gene-encoding coat protein of SCMV (CP-SCMV) was amplified using RT-PCR and cloned into vector pJET1.2. The cDNA was inserted into 6X His-tag expression plasmid of pET28a(+) and over-expressed in Escherichia coli BL21(DE3) to produce a recombinant protein. The highest expression was found in 0.1M IPTG induction media for 5 h at 37oC. SDS-PAGE analysis clarified that the recombinant CP-SCMV remained as an insoluble fraction. Purifications was carried out by the affinity Ni-NTA resin, followed by electroelution to obtain a highly purified protein. To meet the quality requirements of a proper antigen, the highly purified protein was concentrated. A molecular weight of the rCP-SCMV (approximately 40 kDa) was clearly observed by 10% SDS-PAGE at the concentration of 16.184 mg/mL.

Detection and quantification of transition metal leaching in metal affinity chromatography with hydroxynaphthol blue

The widespread use of immobilized metal-affinity chromatography (IMAC) for fast and efficient purification of recombinant proteins has brought potentially toxic transition elements into common laboratory usage. However, there are few studies on the leaching of metal from the affinity resin, such as nickel-nitrilotriacetic acid (Ni-NTA), with possible deleterious impact on the biological activity. This is of particular importance when reducing or chelating eluants stronger than imidazole are used. We present a detailed study of hydroxynaphthol blue (HNB) as an indicator of several divalent metal cations, but with emphasis on Ni2+, clarifying and correcting many errors and ambiguities in the older literature on this dye compound. The assay is simple and sensitive and many metals, notably Ni2+, Zn2+, Cu2+, Pb2+, Fe2+, Co2+, and Al3+, can be readily detected and quantified at concentrations down to 15–50 nM (1–5 ppb) at neutral pH and in most commonly used buffers using spectroscopic equipment available in typical biochemistry research labs. Using this method, we show that significant amounts of Ni2+ (up to 20 mM) are co-purified with a target protein (cytochrome bc1 complex) when histidine is used to elute from Ni-NTA resin.

Expression, purification and characterization of recombinant PLCζ protein in baculovirus-insect cell expression system

PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.

Multifunctional elastin-like polypeptide renders \u03b2-glucosidase enzyme phase transition and high stability

BackgroundIn the enzymatic conversion of biomass, it becomes an important issue to efficiently and cost-effectively degrade cellulose into fermentable glucose. β-Glucosidase (Bgluc), an essential member of cellulases, plays a critical role in cellulosic biomass degradation. The difficulty in improving the stability of Bgluc has been a bottleneck in the enzyme-dependent cellulose degradation. The traditional method of protein purification, however, leads to higher production cost and a decrease in activity. To simplify and efficiently purify Bgluc with modified special properties, Bgluc-tagged ELP and His with defined phase transitions was designed to facilitate the process.ResultsHere, a novel binary ELP and His tag was fused with Bgluc from termite Coptotermes formosanus to construct a Bgluc–linker–ELP–His recombinant fusion protein (BglucLEH). The recombinant plasmid Bgluc expressing a His tag (BglucH) was also constructed. The BglucLEH and BglucH were expressed in E. coli BL21 and purified using inverse transition cycling (ITC) or Ni-NTA resin. The optimum salt concentration for the ITC purification of BglucLEH was 0.5 M (NH4)2SO4 and the specific activity of BglucLEH purified by ITC was 75.5 U/mg for substrate p-NPG, which was slightly higher than that of BglucLEH purified by Ni-NTA (68.2 U/mg). The recovery rate and purification fold of BglucLEH purified by ITC and Ni-NTA were 77.8%, 79.1% and 12.60, 11.60, respectively. The results indicated that purification with ITC was superior to the traditional Ni-NTA. The Km of BglucLEH and BglucH for p-NPG was 5.27 and 5.73 mM, respectively. The Kcat/Km (14.79 S−1 mM−1) of BglucLEH was higher than that of BglucH (12.10 S−1 mM−1). The effects of ELP tag on the enzyme activity, secondary structure and protein stability were also studied. The results showed that ELP tag did not affect the secondary structure or enzyme activity of Bgluc. More importantly, ELP improved the protein stability in harsh conditions such as heating and exposure to denaturant.ConclusionThe Bgluc–linker–ELP–His system shows wide application prospect in maintaining the activity, efficient purification and improving the stability of Bgluc. These properties of BglucLEH make it an interesting tool to reduce cost, to improve the efficiency of biocatalyst and potentially to enhance the degradation of lignocellulosic biomass.

A new vector coupling ligation-independent cloning with sortase a fusion for efficient cloning and one-step purification of tag-free recombinant proteins

We have developed a new ligation independent cloning (LIC) vector – pSrtA9, which can be utilized for one-step purification of recombinant proteins. The new LIC site in the pSrtA9 vector, hosts a DNA sequence centered on a SfoI restriction site and integrates a coding sequence for sortase A (SrtA) recognition. Preceding the LIC site, pSrtA9 incorporates an N-terminal 6xHis-tag and the catalytic core of SrtA from Staphylococcus aureus (SrtAΔ59). Thus, after cloning and protein expression in Escherichia coli, the resultant fusion protein comprises an N-terminal 6xHis-tag, SrtAΔ59, an L-P-E-T-G linker and the protein of interest at the C-terminus. The fusion protein can be captured onto immobilized Ni-NTA resin and any unwanted proteolysis activity of SrtA is suppressed during the purification by optimisation of solution conditions. Upon addition of Ca2+ and triglycine (Gly3), the immobilized fusion protein undergoes on-column SrtA-mediated cleavage at the T-G bond of LPETG linker to selectively release 90% of the protein of interest within 3 h when incubated at room temperature. This new pSrtA9 vector, thus, offers an efficient method for LIC of genes and a one-step purification procedure to obtain a tag-free recombinant protein, and is therefore suitable for the high-throughput proteins production.

A simplified method to remove fusion tags from a xylanase of Bacillus sp. HBP8 with HRV 3C protease

Human rhinovirus 3C protease (HRV 3C protease) is commonly used as a tool to remove fusion tags from recombinant proteins in gene engineering due to its distinguished specificity and high activity at low temperature. This paper is aimed to simplify the strategy of removing epitope tags from target proteins with HRV 3C protease. Fusion proteins composed of a xylanase from Bacillus sp. HBP8 (xylHB) and double tags (MBP/Nus and 6×His, with an HRV 3C protease recognition site between them) were applied as substrates. To perform the cleavage and purification, strains expressing HRV 3C protease and the substrates were mixed before (co-fermentation method) or after (post-fermentation method) inducing with IPTG, followed by cell disruption and incubation at 4℃, overnight for cleavage. The soluble cytoplasmic fraction was added to Ni-NTA resin to recover the cleaved target protein. Because the process was carried out in the cell lysate, it was named as cell lysate purification system based on HRV 3C protease (CLP3C). Our data indicated small number of cells expressing HRV 3C protease was enough to remove the fusion tags efficiently with both co-fermentation and post-fermentation methods. More importantly, the tags were cleaved precisely with no obvious non-specific degradation to the target protein. Hence, active xylanase was recovered easily with this strategy.

Expression, purification, and evaluation of in vivo anti-fibrotic activity for soluble truncated TGF-β receptor II as a cleavable His-SUMO fusion protein.

Excessive production of transforming growth factor-β1 (TGF-β1) and its binding to transforming growth factor-β receptor type II (TGF-βRII) promotes fibrosis by activation of the TGF-β1-mediated signaling pathway. Thus, the truncated extracellular domain of TGF-βRII (tTβRII) is a promising anti-fibrotic candidate, as it lacks the signal transduction domain. In this work, the native N-terminal tTβRII was prepared as a His-SUMO fusion protein (termed His-SUMO-tTβRII) in Escherichia coli strain BL21 (DE3). His-SUMO-tTβRII was expressed as a soluble protein under optimal conditions (6h of induction with 0.5mM IPTG at 37°C). His-SUMO-tTβRII was purified by Ni-NTA resin chromatography, and then cleaved with SUMO protease to release native tTβRII, which was re-purified using a Ni-NTA column. Approximately 12mg of native tTβRII was obtained from a one liter fermentation culture with no less than 95% purity. In vivo studies demonstrated that tTβRII prevented CCl4-induced liver fibrosis, as evidenced by the inhibition of fibrosis-related Col I and α-SMA protein expression in C57BL/6 mice. In addition, tTβRII downregulated phosphorylation of SMAD2/3, which partly repressed TGF-β1-mediated signaling. These data indicate that the His-SUMO expression system is an efficient approach for preparing native tTβRII that possesses anti-liver fibrotic activity, allowing for the large-scale production of tTβRII, which potentially could serve as an anti-fibrotic candidate for treatment of TGF-β1-related diseases.

Molecular cloning, recombinant expression, and purification of osteocalcin in sika deer (Cervus nippon) antler.

Osteocalcin is a major non-collagenous component of the bone extracellular matrix and is considered to be an indicative factor of osteoblast differentiation. In the present study, we detected osteocalcin expression in different antler areas and growth phases by immunohisto- chemistry. Osteocalcin was highly expressed in all areas during the mineralization period and in mesenchymal cell and chondrocyte areas during the rapid growth period. The nucleotide sequence of the osteocalcin gene in sika deer antler was determined. The open reading frame was 303 bp encoding a protein of 100 amino acids. The estimated molecular mass of osteocalcin was 10.38 kDa and the theoretical isoelectric point was 5.37. The osteocalcin gene with a 6× His-tag at the C-terminus was cloned into the pGEX-4T1 vector and expressed in Escherichia coli under optimal conditions. The recombinant soluble protein fused with GST was purified with Ni-NTA resin. The purified osteocalcin protein exhibited a significant increase in HA adhesion and promoted antler chondrocyte proliferation. Osteocalcin is an important factor in regulating the rapid growth and differentiation of deer antlers.

Development of IgY-Based Sandwich ELISA as a Robust Tool for Rapid Detection and Discrimination of ToxigenicVibrio cholerae

Background The conventional methods for diagnosis of Vibrio cholerae are time consuming, complicated, and expensive. Development of rapid detection tests is critical for prevention and management of cholera. This study aimed to introduce two sensitive sandwich ELISAs based on avian antibodies (IgY) targeting outer membrane protein W (OmpW) and cytotoxin B (CtxB) antigens of V. cholerae. Methods The sequences of ompW and ctxB genes were cloned into pET28a vector. Escherichia coli BL21 (DE3) was transformed with the recombinant vectors, and gene expression was induced by IPTG. The expressed proteins were purified by affinity chromatography using Ni-NTA resins. Two groups of white Leghorn chickens were immunized by recombinant proteins, and the generated antibodies were purified from egg yolks of chickens by PEG precipitation. The antibodies were used for the development of α-OmpW and α-CtxB ELISAs. Results The expression and purification yielded 59 and 38 mg of recombinant OmpW and CtxB, respectively, per one liter of bacterial culture. PEG precipitation and purification of egg yolk antibodies yielded on average (±SD) 66.5 ± 1.80 and 50.9 ± 2.23 mg of purified α-OmpW and α-CtxB per egg, respectively. The analytical sensitivity of α-OmpW ELISA was 103 cfu/mL of V. cholerae and that of α-CtxB ELISA was 33 pg/mL of recombinant cytotoxin B. The two developed ELISAs did not show any cross-reactivity to any tested bacteria grown in common conditions. Discussion The current study is the first report on using IgY for detection of V. cholerae. The developed ELISAs were shown to have considerable analytical sensitivity and specificity. Therefore, the assays can be one of the convenient methods for sensitive and specific detection of toxigenic V. cholerae strains in clinical and environmental samples.

Prokaryotic expression and in vitro enzyme activity analysis of flavonol synthase in Chrysanthemum morifolium cv. 'Hangju'

We cloned flavonol synthase gene (named as CmFLS) by RACE from Chrysanthemum morifolium cv. 'Hangju' based on transcriptome database. Sequencing results showed that 1 235 bp sequence was acquired with the largest open reading frame (ORF) of 1 008 bp, which encoded 335 amino acids. The predicted CmFLS encoded protein had an isoelectric point (pI) of 5.41. The phylogenetic tree analysis indicated that CmFLS was highly homologous to other FLSs, which identified from the species of Compositae. The recombinant fusion protein, with a molecular mass of 43 kDa, was successfully expressed by prokaryotic expression system. Meanwhile, Ni-NTA resin was used to purify the recombinant fusion protein, and the Ni-Native Buffer containing 250 mmol·L⁻¹ imidazole was most favorable for elution. The purified recombinant fusion protein was subjected to in vitro catalytic reaction, and then the products were extracted and analyzed by HPLC. The results showed that the recombinant fusion protein CmFLS was able to catalyze the production of quercetin by dihydroquercetin under specific buffer and reaction conditions, which indicated that the functional protein encoded by CmFLS had dioxygenase activity in the biosynthetic pathway of flavonoids biosynthesis in Ch. morifolium cv. 'Hangju'. The above results laid the foundation for further studying on CmFLS, and provided new ideas for the regulation of flavonoids metabolism from the molecular level and the catalytic synthesis of flavonols in vitro.

Production of a Polyclonal Antibody against the Recombinant Coat Protein of the Sugarcane Mosaic Virus and Its Application in the Immunodiagnostic of Sugarcane

Sugarcane mosaic virus (SCMV) is a mosaic disease that has spread over sugarcane plantations in Indonesia. The important step to overcome the disease is to detect the pathogen as early as possible. Detection of the pathogen can be achieved using the immunodiagnostic method by employing a specific antibody against the viral coat protein. The objective of this research was to produce a polyclonal antibody using the recombinant coat protein of SCMV, and to test its sensitivity for detection of SCMV in the symptomatic plant. The gene encoding of the coat protein was cloned using the RT-PCR Kit and total RNA isolated from symptomatic sugarcane leaves cultivar PS-881. Nucleotide sequences analysis of the cloned cDNA indicated that the cDNA contained 998 nucleotides and named SCMVCp-cDNA. The cDNA was then inserted into a His-tag expression plasmid of pET28a and overexpressed in Escherichia coli BL21 (DE3) to produce a recombinant protein. The recombinant fused protein SCMVCp was strongly expressed in an insoluble fraction, with a molecular size of around 44 kDa, without the addition of an IPTG inducer. Purification of the recombinant protein using an affinity Ni-NTA resin, followed by SDS-PAGE separation, resulted in a high purity of the protein and used as an antigen to raise the polyclonal antibody in a rabbit. The sensitivity of the antiserum determined by western blot analysis showed that the antiserum was able to detect the recombinant protein at a concentration of 10 ng. The western blot analysis also detected a clear single band of 36.7 kDa of the SCMV coat protein in symptomatic sugarcane leaves and not in healthy leaves. Interestingly, when the sample proteins were prepared using low-speed centrifugation, the corresponding coat protein was detected in a soluble fraction by western blot analysis. Thus, the antiserum was successfully used for indirect-ELISA analysis using the soluble protein fraction. The results provide an easy method to detect and diagnose SCMV infection using the immunodiagnostic.

Development of a rapid immunochromatographic assay for detection of antibodies against porcine epidemic diarrhea virus.

An immunochromatographic test strip was developed for the detection of antibodies against porcine epidemic diarrhea virus (PEDV) in porcine serum. In this test, the recombinant nucleocapsid protein of PEDV was expressed in soluble form and purified by using Ni-NTA resin and gel filtration chromatography. The purified recombinant N protein conjugated with colloidal gold was dispensed on a conjugate pad as the detector. Staphylococcal protein A and rabbit anti-N protein IgG were blotted on a nitrocellulose membrane for the test and control lines, respectively. The immunochromatographic test strip specifically detected PEDV antibodies within 10 min and had higher sensitivity (96.0%) and specificity (90.8%) than those of commercial enzyme-linked immunosorbent assay (ELISA) kits. Our newly developed strip has great potential for the early diagnosis of PEDV infection.

Generation and Characterization of Chicken Egg Yolk Antibodies against Bovine Interleukin-6

Abstract. Interleukin-6 (IL-6), a multifocal cytokine, has been widely regarded as a potential biomarker of diagnosis of disease. Thus, the antibody against IL-6 became more important in detecting IL-6. So far, the anti-human, rat, sheep and porcine IL-6 antibody has been reported. We aimed to produce chicken anti-bovine IL-6 immunoglobulin Y (IgY) polyclonal antibody. In the present study, the gene of bovine IL-6 were synthesized by company (Luoyang Laipson Information Technology Co., Ltd). Then the gene was cloned and expressed with pET-32a expression vector, and then followed by purification of recombinant bovine IL-6 (rbIL-6) protein with Ni-NTA resin. Protein mass of about 45 kDa was found with SDS-PAGE. Egg yolk IgY against rbIL-6 was extracted from egg yolk by the method of PEG precipitation after using the purified rbIL-6 to immune chicken. The egg yolk IgY polyclonal antibody was evaluated by indirect ELISA and Western blot followed by Checkerboard titration for optimal dilutions ratio of antigen and IgY/IgG titers. The results showed that the titer of anti-rbIL-6 IgY gradually raised after the first booster immunization. Booster immunizations were administered at 2-week intervals, and IgY titers were monitored to determine the immune response. The peak titer of anti-rbIL-6 IgY was 1:64 000 after the fifth booster immunization. The Western blot showed that the IgY could specific bind bovine IL-6. Conclusion: We successfully prepared anti-bovine IL-6 IgY polyclonal antibody that would contribute to bovine disease diagnostic test development for improving herd sanitary states of cattle and dairy farms. Keywords: Bovine interleukin-6, Egg yolk antibodies, Immunoglobulin Y, Indirect ELISA.