Development of Competitive Enzyme-Linked Immunosorbent Assays for Antibody Detection Based on Bluetongue Virus Monoclonal Antibodies.

Bluetongue is a ruminant infectious disease that is characterized by hyperpyrexia, leukocyte decrease and mucosal ulcerative inflammation changes. In this study, three segments of Bluetongue virus (BTV)-8 VP2 protein (BTV-8A, 8B, and 8C) were cloned into pET-28a (+) and pMAl-c5X vectors and expressed in Escherichia coli BL21 (DE3) as histidine (His)-tagged (His-8A/8B/8C) and maltose-binding protein (MBP)-tagged (MBP-8A/8B/8C) fusion proteins. After purification, His-8A/8B/8C were used to immunize BALB/mice and MBP-8A/8B/8C were used to screen for monoclonal antibody (mAb)-secreting hybridomas. Two mAbs (8B-5D4 and 8B-3G11) that could recognize BTV-8 were obtained. Two competitive enzyme-linked immunosorbent assays with good specificity and sensitivity using mAbs 8B-5D4 or 8B-3G11 as competitive antibody were established. With the joint reaction of these methods, serum samples containing anti-BTV-7 or anti-BTV-8 antibody could be screened out, suggesting that these methods would be useful for the diagnosis and epidemiological studies of BTV-8.

Effects of different resin core materials on microleakage for post-core restorations / 西安交通大学学报(医学版)

Objective To compare the effects of different composite resins as core material on the degree of microleakage in post-core repairation.Methods A total of 46 recently extracted single-rooted mandibular premolars were distributed into different groups according to different core material including ParaCore,AP-X and Ceramage.Then we used direct or indirect forming method to make post-core restorations.All of the specimens were submerged in dyes.Then,they were demineralized,dehydrated and processed to be transparent.The extent of the dye leakage was examined under a stereomicroscope.Results The microleakage value was significantly higher in Group of direct-mold-cement with ParaCore (4.94± 1.71)mm than in Group of indirect-mold-secondary-cement with ParaCore (0.91 ± 0.33) mm,Group of indirect-mold-secondary-cement with AP-X (0.87 ± 0.27) mm,and Group of indirect-mold-secondary-cement with Ceramage (1.02 ± 0.34)mm.Conclusion Different methods of building and cementing FRC post-core restorations,but not different composite resins as core material,have significant effects on the extent of microleakage in post-core repair.

Effects of different fabricating methods on the microleakage for preformed fiber posts / 实用口腔医学杂志

To compare the microleakage of fiber post and resin-core system fabricated by different methods.The roots of 36 recently extracted single-rooted mandibular premolars were undergone endodontic treatment.Fiber posts and Paracore flowable resin composite were used for fabricating post-core restorations.Microleakage was examined by dye penetration method.The microleakage value was significantly higher in Direct-Mold-Cement-Method group(4.94 ± 1.71) mm compared to Direct-Mold-Secondary-Cement-Method group(0.91 ±0.33) mm and Indirect-Mold-Secondary-Cement-Method group (0.87 ± 0.27) mm (P ＜ 0.05).

Identification of epitope recognized by a monoclonal antibody against VP2 protein of bluetongue virus serotype 8 / 生物工程学报

To confirm the B cell epitope recognized by monoclonal antibody (MAb) 3G11 of bluetongue virus type 8 (BTV-8) VP2 protein prepared in our laboratory, antigen epitopes recognized by 3G11 were screened and identified by phage display technology. KLLAT sequence was found by sequencing of blue spot after four rounds panning and 283LL284 of common short peptide sequence was obtained after comparison to amino acid sequence of BTV-8 VP2 protein. The peptide sequences KLLAA, KALAT, KLAAT and KLLAT were synthesized and identified by indirect ELISA. KLLAA and KLLAT bound strongly with supernatant and as cites of 3G11 cells and reacted specifically with BTV-8 positive standard sera. Further sequence analysis showed that amino acid sequence 283LL284 was conserved among different serotypes of BTV-8 strains, and283LL284 was the key amino acids of antigen epitopes recognized by 3G11. This study laid the foundation to establish type 8 BTV specific immunological detection methods.

Establishment of two competitive ELISAs for specific detection of bluetongue virus serotype 4 / 生物工程学报

To develop a clinical diagnosis technique for bluetongue virus infection, we established serotype-specific methods to detect serotype 4 of bluetongue virus (BTV-4). Two monoclonal antibodies (mAbs) against VP2 protein of BTV-4, named 4A-1G7 and 4B-1B6, were used as competitive antibodies in the competitive enzyme-linked immunosorbent assays (C-ELISA). We detected 50 negative serum samples from sheep, goats and cattle by C-ELISA. The cut-off values of 4A-1G7 and 4B-1B6 mAbs were 49% and 40%, respectively. The results of the sensitivity, specificity and repeatability by detecting standard positive serum, were consistent with the general standard of Office International Des Epizooties. Furthermore, serum samples of BTV-4, BTV-18 and BTV-20 infection could be screened out through the combined C-ELISAs by 4A-1G7 and 4B-1B6 mAbs. Thus, this technique may diagnose BTV-4, BTV-18 and BTV-20 infections.

Development of an antigen capture enzyme-linked immunosorbent assay for virus detection based on porcine epidemic diarrhea virus monoclonal antibodies.

Porcine epidemic diarrhea virus (PEDV), a coronavirus, can cause acute diarrhea and dehydration in pigs. In the current study, two positive monoclonal cell lines (5D7 and 3H4) specific for PEDV were established, and the immunoreactivity of the monoclonal antibodies was confirmed by immunofluorescence and dot-immunobinding assays. A method, termed antigen capture enzyme-linked immunosorbent assay (AC-ELISA), which used the monoclonal antibody 5D7 as the detecting antibody and rabbit antiserum of PEDV protein S as the capture antibody, was developed. Compared with the reverse transcription polymerase chain reaction method of detecting PEDV in fecal samples, AC-ELISA showed similar sensitivity and specificity. These results suggested that AC-ELISA would be useful for the diagnosis and epidemiological studies of PEDV.

Angiotensin Ⅱ type 1 receptor agonistic antibodies enhance acute ischemic tubular injury in patients subject to kidney transplantation / 中华器官移植杂志

Objective To evaluate the impact of autoantibodies to angiotensin Ⅱ type 1 receptor AT1-AA on clinic outcomes of delayed graft function (DGF) grafts.Method We reviewed the records of all 139 consecutive adult recipients who received single kidney transplantation and clinical management between Jan.2010 and Dec.2012 in our centre.The serum levels of AT1-AA were measured by a streptavidin-enzyme-linked immunosorbent assay.All patients with DGF were enrolled in this study and divided into two groups:(1) AT+ DGF group (serum AT1-AA positive,11 cases) ;(2) AT-DGF group (serum AT1-AA negative,23 cases).All clinical and laboratory data were recorded in our transplant database system at each visit.Result 139 recipients were enrolled.The overall presence of DGF was 24.5％ (34/139).The incidence of DGF in patients with high binding AT1-AA was significantly higher than that in those with low binding of AT1-AA (11/24 vs.23/115,45.8％ vs.20.0％,P＜0.05).In addition,longer duration of renal replacement therapy (59 ± 32 vs.47 ± 26 months,P＜0.05),higher resistance index (0.80 ± 0.10 vs.0.72 ± 0.10,P＜0.05) of allografts and more severe acute tubular injury (2.7 ± 0.5 vs.1.8 ± 1.1,P＜0.05)/acute tubular necrosis (0.9 ± 0.5 vs.0.5 ± 0.3,P＜0.05) were observed in AT + DGF group than in AT-DGF group.One-year graft survival and death censored graft survival were similar between two groups (90.9％ vs.95.7％,P＞0.05).Conclusion Presence of high binding anti-AT1 receptor had detrimental impacts on initiation and development of DGF.

Comparison of expression and antibacterial activities of recombinant porcine lactoferrin expressed in four Lactobacillus species / 生物工程学报

The coding sequence for the mature peptide of porcine lactoferrin (Plf) was synthesized according to the codon usage of lactobacillus, to establish optimized porcine lactoferrin Lactobacillus expression system. The gene was ligated into the Xho I/BamH I site of Lactobacillus expression vector pPG612.1 and the recombinant plasmid pPG612.1-plf was transformed individually into Lactobacillus casei ATCC393, Lactobacillus pentosus KLDS1.0413, Lactobacillus plantarum KLDS1.0344 or Lactobacillus paracasei KLDS1.0652 by electroporation. After induction with xylose, expression of the recombinant proteins was detected by Western blotting and confocal laser scanning microscopy. Secretion of recombinant Plf proteins from four recombinant species was determined quantitatively by ELISA. The antibacterial activities of recombinant proteins were measured by agar diffusion method. The result shows that Plf was correctly expressed in four species of recombinant lactobacillus, with molecular weight of about 73 kDa. The expression levels in recombinant Lactobacillus casei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paracasei were 9.6 μg/mL, 10.8 μg/mL, 12.5 μg/mL and 9.9 μg/mL, respectively. Antimicrobial activity experiment shows that the recombinant proteins were active against E. coli, Staphylococcus aureus, Salmonella typhimurium, Listeria, Pasteurella. The recombinant Plf expressed by recombinant Lactobacillus plantarum showed the best antibacterial activity among all recombinant lactobacillus species. These data represent a basis for the development and application of porcine lactoferrin from recombinant lactobacillus.

Expression and biological activity of porcine interleukin-18 in recombinant Lactococcus lactis / 生物工程学报

To obtain active protein of pIL-18 expression in Lactococcus lactis, and to observe its biological activity, the total RNA was extracted as template from peripheral blood mononuclear cells. Porcine interleukin 18 (pIL-18) was amplified by RT-PCR. The resulting fragment was cloned into pAMJ399 L. lactis vector, and then transformed to L. lactis MG1363 cells by electroporation. Expression of pIL-18 protein was detected by SDS-PAGE and Western-blotting. Bioactivity of the product was tested by pig spleen lymphocyte proliferation test and cytopathogenic effect inhibition assay. The result of Western blotting and bioactivity test shows that the molecular weight of pIL-18 protein was 19 kDa. The react line was observed in both supernatant and precipitated of the recombinant bacteria pAMJ399-pIL18/MG1363. The expressed pIL-18 can promote the proliferation of pig spleen lymphocyte, and significantly inhibit virus multiplication. As conclusion, porcine interleukin-18 was successfully expressed in L. lactis, and the product was biologically active.

High-level expression and antimicrobial activity of recombinant N-terminal porcine lactoferrin / 生物工程学报

Lactoferrin in milk is a multifunctional protein. In addition, lactoferrin has antiviral, antifungal and antiparasitic activity. In this study, the N-terminus from porcine lactoferrin (PLF-N) was designed to express the antimicrobial action of recombinant porcine lactoferrin. We cloned a 1077 bp fragment of the PLF gene from mammary gland tissue of the lactating sow at the third day. Comparing nucleotide sequence with four strains of PLF gene published on GenBank, the homology was more than 99%. With the reference template of the cloned fragment of PLF-N and optimizing codon bias, we synthesized the gene of N-terminus encoding porcine lactoferrin (PLF-NS). The high expression gene of PLF-NS was cloned into the fusion expression vector pET30b and expressed in E. coli BL21 (DE3). After induced with Isopropyl beta-D-1-Thiogalactopyranoside (IPTG), the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. The protein had a molecular weight of 42 kDa and accounted for 32% of the total cellular protein. After purification and renaturation, the purity of the expressed protein was 98%. The expressed PLF-NS protein showed obviously antibacterial activity. This method provides an excellent way for high expression of antimicrobial proteins when optimizing codon bias.

Induction of immune response after oral inoculation of mice with Lactobacillus casei surface-displayed porcine epidemic diarrhea viral N protein / 生物工程学报

To evaluate the immune responses of recombinant Lactobacillus casei 393 expressing Porcine Epidemic Diarrhea Viral (PEDV) N protein as oral vaccine, n gene of PEDV was subcloned into the expression vector pPG-1, and then transformed into L. casei 393 by electroporation, resulting in recombinant strain pPG-1-n/L, casei 393. The recombinant strains were induced to express interest protein, which was detected by Western blotting, immunofluorescence microscopy and the whole bacteria ELISA. And then BALB/C mice were used as an animal model immunized with recombinant strains by oral administration, and the immune efficacy was analyzed. The recombinant PEDV N protein showed the antigenic specificity, and was located on the bacterial cell walls of pPG-1-n transformed L. casei. The results of ELISA showed that the mice immunized with recombinant strains could produce remarkable special sIgA level in the feces, and high level of anti-PEDV N protein IgG in the serum (P < 0.01), but the induced antibodies in serum did not demonstrated neutralizing effect. Statistical significant difference was observed among the spleen lymphocyte proliferation index (LPI) among the immunization groups of mice and control groups. And there was significant increase. of IFN-gamma and IL-4 contents in the supernatant of spleen cell culture in immunized group. In conclusion, the oral immunizations with recombinant L. casei 393 can induce significant specific mucosal PEDV N-specific IgA response as well as serum IgG responses, and can evoke both mucosal immune and system immune responses.

High-level prokaryotic expression of envelope exterior of membrane protein of porcine epidemic diarrhea virus.

The truncated fragment M' gene, encoding the exterior of the viral envelope protein of PEDV, was subcloned into prokaryotic expression vector pGEX-6p-1. The recombinant plasmid pGEX-6p-M' was constructed and transformed into E. coli BL21(DE3)pLysS for expression. SDS-PAGE analysis showed recombinant truncated M' protein was highly expressed by pGEX-6p-M' and the product fusion protein GST-M' reached 45% in the total bacteria proteins with the analysis of software AlphaImager2200. The preliminary purified recombinant protein was evaluated for its antigenicity and reactivity through Western blotting and indirect enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody against M protein of PEDV and porcine polyclonal anti-PEDV antiserum as the primary antibody. The results indicated the recombinant truncated M' protein should be candidate as a feasible recombinant diagnostic reagent.

Cloning and sequence analysis of the N gene of porcine epidemic diarrhea virus LJB/03.

The nucleocapsid (N) gene of the porcine epidemic diarrhea virus (PEDV) strain LJB/03 which was previously isolated in Heilongjiang province, China, was cloned, sequenced and compared with published sequences of other avian and mammalian coronavirus. The nucleotide sequence encoding the entire N gene open reading frame (ORF) of LJB/03 was 1326 bases long and encoded a protein of 441 amino acids with predicted Mr of 49 kDa. It consisted of 405 adenines (30.5%), 294 cytosines (22.1%), 329 guanines (24.8%) and 298 thymines (22.5%) residues. Sequence comparison with other PEDV strains selected from GenBank revealed that the LJB/03 N gene has a high sequence homology to those of other PEDV isolates, 97.4% with JS2004, 95.6% with chinju99, 96.6% with Br1/87, and 96.8% with CV777. The encoded protein shared 96.4% amino acid identities compared with CV777, 96.1% with Brl/87, 98% with JS2004, 96.90% with chinju99, respectively. The amino acid sequence contained seven potential protein kinase C phosphorylation sites, nine Casein kinase II phosphorylation sites, one Tyrosine kinase phosphorylation site, two cAMP- and cGMP-dependent protein kinase phosphorylation sites.