Synthesis of ligand-specific phage-display ScFv against the herbicide picloram by direct cloning from hyperimmunized mouse

Immunoglobulin genes were directly isolated from the splenocytes of a BALB/C mouse hyperimmunized with the auxinic herbicide picloram conjugated to bovine serum albumin. Variable light and heavy domain DNA were joined to produce single-chain Fv (scFv) DNA, which was cloned into phage vector fd-tet-GIIID to display multiple copies of scFv on the filamentous phage minor coat protein gIIIp. The phage-display scFv library (10(4) clones) was selected against picloram conjugated to ovalbumin. After five rounds of panning, individual clones were analyzed. ScFv with different affinities to picloram (IC(50) values ranging from 20 ppb to 10 ppm) were detected in the final enriched pool. The increased avidity of the phage vector enhanced the selection (i.e., panning) of multiple picloram-specific recombinant antibodies. Stringent selection was required to isolate the clones with the highest affinity. Nucleotide sequence analysis of six isolated clones revealed that all of the V(L) belonged to the V kappa 9A family joined to J kappa 2 segments. All of the V(H) belonged to the V(H)()7183 family and joined to two different J segments (i.e., J(H)()2 or J(H)()4). Different from the immune response to large molecular weight molecules (MW > 10,000 Da), which requires both VDJ segment rearrangement and somatic hypermutations, production of high-affinity antibodies to picloram, a small ligand having a formula weight of 241.5 Da, predominantly requires somatic hypermutations.     

Development of a sensitive enzyme-linked immunosorbent assay for the determination of ochratoxin A

Polyclonal antibodies for ochratoxin A (OTA) were generated from rabbits after the animals had been immunized with either OTA-gamma-globulin or OTA- keyhole limpet hemocyanin (KLH). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of OTA in various agricultural commodities. The antibody titers in the serum of rabbits immunized with OTA-gamma-globulin were considerably higher than those in rabbits immunized with OTA-KLH. The antibodies from the rabbits immunized with OTA-gamma-globulin were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of OTA-horseradish peroxidase to the antibodies by OTA, ochratoxin B (OTB), and ochratoxin C (OTC) were found to be 0.90, 110, and 0.54 ng/mL, respectively. When 10 to 250 ng/g of standard OTA was spiked to soybean samples and then extracted with 50% aqueous methanol, the recovery rate of OTA was found to be 85.9% in the cdELISA. Analysis of OTA in various agricultural commodities showed that 12 of the 20 examined samples were contaminated with OTA at levels from 16 to 160 ng/g. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method.     

Development of a monoclonal antibody against domoic acid and its application in enzyme-linked immunosorbent assay and colloidal gold immunostrip

A monoclonal antibody (mAb) specific to domoic acid was produced from a stable hybridoma cell line, 9F1F11, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with domoic acid--keyhole limpet hemocyanin. The 9F1F11 mAb belongs to the immunoglobulin G1 (kappa-chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. In the cdELISA, the concentration causing 50% inhibition (IC50) of binding of domoic acid-horseradish peroxidase to the antibody by domoic acid was found to be 0.58 ng/mL. A sensitive and rapid mAb-based colloidal gold immunostrip was also developed. The immunostrip assay, which has a detection limit of 5 ng/mL for domoic acid, can be completed in 10 min. Analysis of domoic acid in blue mussel samples revealed that data obtained from immunostrip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunostrip assay established in this study were sensitive and accurate for rapid screening of domoic acid in shellfish samples.     

抗速灭威单链抗体基因在巴斯德毕赤酵母中的表达及性质研究

选用巴斯德毕赤酵母（Pichia pastoris）系统表达特异性抗速灭威单链抗体（scFv）基因,以为速灭威特异性抗体的大量制备奠定基础。设计引物扩增阳性克隆scFv基因,亚克隆至表达载体pPICZαC,获得重组酵母表达质粒pPICZαC-scFv,线性化pPICZαC-scFv并高效电转P.pastoris（X-33）,对转化子进行抗性梯度筛选得到一株高效表达的X-33-Pp-SMW-12-6菌株。对获得的菌株先后进行表达条件的优化、优化条件下的诱导表达及单链抗体性质研究。结果表明：P.pastoris-scFv的质量接近其亲本E.coli-scFv的质量,X-33-Pp-SMW12-6在优化条件下的表达产量达28mg/L,比未优化前的产量提高了约8mg/L,经纯化后抗体纯度可达85%以上。因此,利用P.pastoris表达系统制备抗速灭威单链抗体比细菌表达系统更有效、更经济。     

Functional assembly of a microbial consortium with autofluorescent and mineralizing activity for the biodegradation of organophosphates

Organophosphorus pesticides (OPs) cause serious environmental problems, and bioremediation using bacterial enzymes may provide an efficient and economical method for their detoxification. Green fluorescent protein (GFP) is a stable and easily detectable marker in monitoring genetically engineered microorganisms (GEMs) in the environment. In our research, the methyl parathion hydrolase gene (mpd) and enhanced green fluorescent protein gene (egfp) were successfully coexpressed using pETDuet vector in E. coli BL21 (DE3). The coexpression of methyl parathion hydrolase (MPH) and enhanced green fluorescent protein (EGFP) were confirmed by determining MPH activity and fluorescence intensity. The recombinant protein MPH showed high enzymatic degradative activity of several widely used OP residues on vegetables determined by GC analysis. Subsequently, a dual-species consortium comprising engineered E. coli and a natural p-nitrophenol (PNP) degrader Ochrobactrum sp. strain LL-1 for complete mineralization of dimethyl OPs was studied. It could completely mineralize methyl parathion (MP) via MP through PNP and hydroquinone and eventually through the TCA cycle as determined by HPLC analysis. The accumulation of PNP in suspended culture was prevented. The consortium could be further utilized for complete mineralization of PNP-substituted OPs in a laboratory-scale bioreactor and easily monitored by fluorescence of EGFP for its activity and fate.     

Production of a monoclonal antibody against ochratoxin A and its application to immunochromatographic assay

A monoclonal antibody (Mab) against ochratoxin A (OTA) was produced from the hybridoma cell line C7G25, which was established by the fusion of Sp2/0-Ag14 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with the OTA-bovine serum albumin conjugate. This Mab belongs to the IgG(2a) heavy-chain subclass with a kappa-type light chain. The level of 50% inhibition concentration was 1.20 ng/mL in a competitive direct enzyme-linked immunosorbent assay (cdELISA), and the detection limit was 0.12 ng/mL. This antibody is specific for OTA but also shows cross-reactivity with ochratoxin B (31.7%) in a cdELISA. On the basis of the sandwich format using the produced Mab against OTA, a rapid immunochromatographic assay was developed to efficiently detect OTA. This method was able to detect up to 500 ng/mL of OTA in <10 min.     

Development of a sensitive enzyme-linked immunosorbent assay for the determination of domoic Acid in shellfish

Polyclonal antibodies for domoic acid were generated from rabbits after the animals had been immunized with either domoic acid-keyhole limpet hemocyanin (KLH) or domoic acid-bovine serum albumin (BSA). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of domoic acid in blue mussels and clams. The antibody titers in the serum of rabbits immunized with domoic acid-KLH were considerably higher than those in rabbits immunized with domoic acid-BSA. The antibodies from the rabbits immunized with domoic acid-KLH were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of domoic acid-horseradish peroxidase to the antibodies by domoic acid and a domoic acid analogue, kainic acid, were found to be 0.75 and 200 ng/mL, respectively. In the presence of blue mussel matrix, the detection limit of domoic acid was <25 ng/g. The overall analytical recovery of domoic acid (25-500 ng/g) added to the blue mussels and then extracted with 50% aqueous methanol in the cdELISA was found to be 81.1%. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method. Analysis of domoic acid in shellfish samples showed that 10 of the 15 shellfish examined were contaminated with domoic acid at levels of <50 ng/g.     

Apple polyphenols extend the mean lifespan of Drosophila melanogaster

Apple polyphenols (AP) are an excellent source of dietary antioxidants. The present study investigated the effect of AP on the lifespan of fruit flies and their interaction with gene expressions of superoxide dismutase (SOD), catalase (CAT), methuselah (MTH), Rpn11, and cytochrome c oxidase (CcO) subunits III and VIb. Results showed the mean lifespan was significantly extended by 10% in fruit flies fed the AP diet. This was accompanied by up-regulation of genes SOD1, SOD2, and CAT and down-regulation of MTH in the aged fruit flies. Paraquat and H(2)O(2) challenge tests demonstrated that AP prolonged the survival time only for Oregon R wild type flies but not for SOD(n108) or Cat(n1) mutants, in which either SOD or CAT was knocked out. Chronic paraquat exposure could shorten the maximum lifespan from 68 to 31 days and reduce the climbing ability by 60%, whereas AP could partially reverse the paraquat-induced mortality and decline in climbing ability. AP could up-regulate Rpn11 at day 30, whereas it appeared to have no significant effect on gene expression of ubiquitinated protein, CcO subunits III and VIb. These AP-induced changes were unlikely associated with caloric restriction as the gustatory assay found no difference in average body weight and stomach redness index between the control and AP fruit flies. It was therefore concluded that the antiaging activity of AP was, at least in part, mediated by its interaction with genes SOD, CAT, MTH, and Rpn11.     

Kinetics and products of photo-Fenton degradation of triazophos

Triazophos is a contaminant of wastewater at manufacturing facilities, and remediative treatment may be needed. While toxicity and persistence limit the effectiveness of biological and physicochemical methods, photo-Fenton processes are promising. UV-Fenton and solar-Fenton processes were applied to degrade triazophos. The optimum parameters were 50 mmol/L H(2)O(2), 0.3 mmol/L FeSO(4), and pH 3.0. The decomposition of triazophos by a photo-Fenton process followed first-order kinemics. At 30 degrees C, the half-life of triazophos in a UV-Fenton process ranged from 9.1 min at 2.0 x 10(5) Lx to 27.3 min at 1.0 x 10(5) Lx. At 35 degrees C and with solar irradiation luminance, it ranged within 1.0 x 10(5) -1.2 x 10(5) Lx; the half-life of triazophos in the solar-Fenton process was 11.2 min. Five major degradation products, O,O-diethyl phosphorothioic acid, monoethyl phosphorothioic acid, phosphorothioic acid, 1-phenyl-3-hydroxy-1,2,4-triazole, and phenylsemicarbazine, were tentatively identified as their corresponding trimethylsilyl derivatives with a gas chromatography-mass spectrometer. The possible degradation pathway of triazophos was proposed. The results indicate the potential use of a solar-Fenton treatment for triazophos-contaminated water.     

Thin layer chromatography of parathion as paraoxon with cholinesterase inhibition detection.

A simple, sensitive, and rapid method is described for the quantitative estimation of ng amounts of parathion (O,O-diethyl O-p-nitrophenyl phosphorothioate) as paraoxon (O,O-diethyl O-p-nitrophenyl phosphate) on thin layer chromatograms. Paraoxon is detected by inhibition, using p-nitrobenzenediazonium fluoroborate as the chromogenic reagent. This chromogenic reagent is more sensitive than Fast Blue B or indoxyl acetate; 0.1 ng may be detected and amounts from 5 to 50 ng may be estimated. The method is a viable alternative to gas chromatographic analysis for parathion.     

Development of a sensitive ELISA for the determination of microcystins in algae

Polyclonal antibodies for microcystin-leucine-arginine (MCYST-LR) were generated from rabbits after immunizing the animals with MCYST-LR conjugated with gamma-globulin. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of the toxin in algal cultures and dietary supplements. The concentrations causing 50% inhibition (IC(50)) of binding of MCYST-horseradish peroxidase (MCYST-HRP) to the solid-phase antibodies by MCYST-LR, MCYST-arginine-arginine variant (MCYST-RR), MCYST-tyrosine-arginine variant (MCYST-YR), and nodularin (NODLN) in the cdELISA were found to be 0.10, 0.12, 0.14, and 0.20 ng/mL, respectively. In the presence of algae matrix, the detection limit is less than 10 ppb. The overall analytical recovery of MCYST-LR (25 to 500 ng/g) added to the algal dietary supplements and then extracted with 0.1 M ammonium bicarbonate in the cdELISA was found to be 83.7%. Analysis of MCYSTs in algal cultures and dietary supplements showed that six of eleven cultures produce MCYSTs, and five of the algal cultures were not MCYST producers. Eight of eleven tested commercial algal dietary supplements contained MCYSTs at a level lower than 100 ppb. The presence of MCYST-LR in the Microcystis aeruginosa culture was confirmed by high-performance liquid chromatography.     

Coexpression of the superoxide dismutase and the catalase provides remarkable oxidative stress resistance in Lactobacillus rhamnosus

Lactic acid bacteria (LAB) are generally sensitive to oxidative stress caused by reactive oxygen species (ROS). Antioxidant enzymes, especially superoxide dismutase (SOD) and catalase (CAT), can protect against ROS by eliminating superoxide and H(2)O(2), respectively. Lactobacillus rhamnosus is a valuable probiotic starter culture but is deficient in both SOD and CAT, and is thus likely to suffer from oxidative stress in industrial fermentation. To confer high level of oxidative resistance on L. rhamnosus , the SOD gene sodA from Streptococcus thermophilus and CAT gene katA from L. sakei were coexpressed in L. rhamnosus AS 1.2466. The enzyme activities of SOD and CAT were 147.80 ± 1.08 U/mg protein and 2.53 μmol of H(2)O(2) /min/10(8) cfu, respectively, in the recombinant L. rhamnosus CS. After incubation with 10 mM H(2)O(2), the survival ratio of L. rhamnosus CS was 400-fold higher than that of L. rhamnosus CAT. In long-term aerated conditions, viable cells of L. rhamnosus CS remained ～10(6) cfu/mL after incubation for 7 days, while no living cells of the control were detected. These results showed that the cooperation between SOD and CAT could significantly enhance oxidative resistance in L. rhamnosus . To our best knowledge, this is the first report of two synergistic antioxidant genes being coexpressed in the same Lactobacilli.     

Development of competitive direct ELISA for gossypol analysis

Anti-gossypol monoclonal antibody was purified from cell culturing supernatant by ammonium sulfate precipitation and Protein A AffinityPak. The antigen (i.e., gossypol) was labeled with horseradish peroxidase through Schiff-base formation. Both the purified antibody and the enzyme-labeled gossypol were employed to develop a competitive direct enzyme-linked immunosorbent assay (cdELISA) for gossypol analysis. I50 value, the concentration of gossypol causing 50% inhibition of the maximum ELISA signal in the competitive standard curve, was 0.067 microg/mL, whereas the detection limit for gossypol was 0.005 microg/mL. We also observed a good correlation (R2= 0.96, P < 0.05) between the cdELISA method and the AOCS official method for "free" gossypol (extractable gossypol and gossypol derivatives by 70% acetone) analysis of cottonseed meals. This indicates that the newly developed cdELISA could be a valuable and feasible alternative for determination of "free" gossypol, especially when the available sample is limited with relatively low gossypol concentration.     

自由基引起的氧化对牛乳清蛋白凝胶特性的影响

为了深入了解蛋白氧化对凝胶特性的影响,以此探讨乳清蛋白氧化对其功能性质的影响机制,该文主要研究了氧化对乳清蛋白凝胶质地、流变学特性和微观结构变化的影响。试验采用羟基自由基氧化体系,在不同H2O2浓度(1～20mmol/L)及不同FeCl3浓度(0.1～1mmol/L)对乳清蛋白分别氧化3h,通过质构仪、流变仪和扫描电镜对凝胶特性和微观结构进行研究。结果显示:同未氧化乳清蛋白相比,在所有氧化条件下,凝胶硬度降低了90%以上,贮藏模量(G')值降低了17%以上,复合模量(G*)值降低了20%以上;高浓度氧化条件下,弹性降低了20%以上。氧化明显改变了凝胶的微观结构,随着氧化剂的加入,导致了疏松多孔且不规则凝胶的形成。上述结果表明,氧化对蛋白凝胶质地和凝胶形成能力起着很大的破坏作用,并影响着其微观结构。     

抗对硫磷基因工程抗体ScFv的制备及其初步鉴定

用一段45个核苷酸的片段连接抗对硫磷抗体重链和轻链可变区基因片段VH和VL,获得了抗对硫磷单链抗体(single chain variable fragment,scFv)基因,构建了抗对硫磷scFv基因原核表达载体。SDS-PAGE和Western blot分析显示,该单链抗体基因能在大肠杆菌Origami 2中特异性表达,融合蛋白分子量约为28kD。用Ni-NTA金属亲和层析法对可溶性表达产物进行纯化,得到目的蛋白纯度为74.8%;ELISA反应结果证明,该单链抗体可以与对硫磷发生特异性反应。     

O型口蹄疫病毒VP1基因的高效可溶表达及抗原性分析

将猪源Ｏ型口蹄疫病毒(Foot-and-mouth disease virus, FMDV)VP1基因克隆到原核表达载体pMBX上，成功地构建重组表达质粒pMBX-VP1。将其转化大肠杆菌(Escherichia coli)BL21(DE3)感受态细胞中，经SDS-PAGE分析，融合蛋白分子量约为58 kD，表达产物占菌体总蛋白的35.5％。 经蛋白可溶性分析，目的蛋白在裂解沉淀中占6.8％，在裂解上清中占31.2％，融合蛋白绝大部分是以可溶形式表达的。经Western blot证实，可溶表达的融合蛋白与FMDV阳性血清具有良好的免疫反应性。将可溶表达的融合蛋白用50%Ni-NTA树脂纯化，用融合蛋白作包被抗原，通过ELISA方法，能特异性地检测出口蹄疫阳性血清。     

绵羊HSPA1L基因的生物信息学分析

为了解绵羊热休克蛋白1L (heat shock protein family A member 1 like，HSPA1L) 基因及其编码产物的基本结构和生物学特征，给绵羊的抗热应激和免疫等方面的研究提供依据，利用生物信息学数据库及软件对绵羊HSPA1L基因进行生物信息学分析。结果表明，该基因最大长度的ORF有1 926 bp，编码641个氨基酸序列。其编码的蛋白质分子式为C3094H4985N859O969S20，理论等电点为5.89，不稳定指数为32.56。该蛋白无信号肽和跨膜结构，为非分泌性蛋白，主要在细胞质中发挥生物学作用。无规则卷曲是构成该蛋白二级结构和三级结构的主要方式。     

烟夜蛾泛素延伸蛋白基因的克隆与表达

应用RT-PCR技术，从烟夜蛾(Helicoverpa assulta)幼虫脂肪体组织总RNA中反转录扩增泛素延伸蛋白基因的cDNA片段，扩增得到的片段全长390bp，编码一个长为129个氨基酸残基的蛋白质，预测分子量14.8kDa。同源性分析表明，此cDNA序列为ubiquitin-53aa extension protein(ubi-53，UBE)基因，在泛素蛋白后融合了一个核糖体L40蛋白(ribosomal L40 protein)。使用Clustal W软件，对cDNA编码的氨基酸序列进行了同源性分析，分析表明：烟夜蛾的泛素延伸蛋白氨基酸序列与其他真核生物泛素延伸蛋白氨基酸序列高度同源（90%－98%），与棉铃虫核多角体病毒(HaSNPV)泛素的同源性为69%。将烟夜蛾的UBE基因克隆到原核表达载体pGEX-4T-2上，转化大肠杆菌BL21(DE3)，经IPTG进行诱导表达，电泳检测到一条约40kDa大小的外源蛋白，用鼠抗GST抗体进行Western blot检测表明原核表达蛋白是目的蛋白。     

Production and characterization of single chain Fv directed against beta2-agonist clenbuterol

The single chain Fv (scFv) directed against beta2-agonist clenbuterol (CBL) was produced by using phage display technology. The heavy chain and light chain variable region genes (VH) VL) were amplified by the polymerase chain reaction (PCR) from CBL specific hybridoma cell lines 5D1 and assembled as a single chain Fv (scFv) fragment with linker peptide (Gly4Ser)3. Then the scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti-CBL antibody libraries were constructed. Phages displaying scFv were enriched by panning with CBL-ovalbumin (CBL-OVA) conjugate. After only one round of panning, antigen-positive recombinant phage clones were successfully selected by ELISA. The positive phage was used to infect Escherichia coli HB2151, and the expression of soluble scFv was then induced by IPTG. The scFv showed an improved sensitivity (with IC50 of 0.78 +/- 0.005 ng/mL (n = 4)) when compared with the parent monoclonal antibody (MAb) (with IC50 of 1.34 +/- 0.006 ng/mL (n = 4)) in competitive indirect ELISA (CI-ELISA). Cross-reactivity studies showed that the specificity of scFv was similar to that of MAb. The recombinant scFv prepared in this study could be potentially used instead of conventional antisera or MAb for development of a rapid and affordable immunoassay for the detection of residual CBL in biological matrices.