In order to extend the detection range of super ̄sensitive DNA damage responded yeast HUG1 ̄yEGFP biosensor which was constructed by our laboratory, and to accumulate more relevant data for practical application, 26 kinds of chemicals which were widely distributed in the environment and human lives were detected and 8 kinds of genotoxic compounds were screened out. Compared with three existed microbiological whole ̄cell sensing sys ̄ tems, the detection results in this study matched over 50% of the data from Ames test and SOS colorimetric test 第 1 期 汤花梅等:应用超敏感酵母 HUG1 ̄yEGFP 生物传感器筛选基因毒性化合物 195 based on bacterial cell, and over 85% of the data from the GSA test based on yeast cell. The differences of the re ̄ sults between the test systems based on eukaryotic yeast cell and prokaryotic bacteria cells reflected the different mechanisms of stress resistance. In this study, three kinds of genotoxicity ̄positive compounds, mancozeb, malachite green and acrylamide which were reported as potential carcinogens, were specifically detected by super ̄sensitive yeast HUG1 ̄yEGFP biosensor. The results of the study indicated that super ̄sensitive yeast HUG1 ̄yEGFP biosen ̄ sor can be used to effectively detect genotoxic compounds which were related to environmental pollution. There ̄ fore, they are valuable in environmental health risk assessment for chemical pollutant as a complementary method for other existed detection systems.

利用野生型酿酒酵母菌株及七基因缺失突变型酵母菌株,研究了不同基因型酵母株在不同的活性状态下对重金属Co2+和Cd2+的富集。结果显示,在磷酸盐缓冲液（PBS）中,酵母处于生长基本停止状态,两种酵母菌株都对Co2+和Cd2+表现出比较强的被动吸附能力,但野生型酵母的富集效率优于七基因缺失突变酵母株。说明酵母在PBS中对重金属的富集主要是依靠被动吸附机制,且与细胞的表面结构有关。在限制性培养基中,酵母的生长状态较好,野生型酵母对Co2+表现出了较好的主动富集现象,并且在暴露96 h时达到富集高峰,而七基因缺失突变酵母株中的含量随时间推移变化不大。在对Cd2+的富集过程中,七基因缺失突变型酵母表现出较野生型酵母更快速富集的效果。研究结果为利用酵母富集环境中重金属的实际应用提供了新的参考数据,提示对于不同环境下的不同毒性的重金属离子,可以采取不同的富集方法,针对性地设计应用方案。

基于酵母DNA损伤响应机制,本实验室构建了超敏感型酵母HUG1-yEGFP生物传感器,检测方法主要依靠流式细胞仪.为了简化检测方法,实现高通量检测,本研究通过摸索和测试各种条件,建立了优化的、更适合于96孔板的荧光酶标仪的检测方法.结果显示:F1培养基可以作为一种本底荧光较低的培养基用于荧光酶标仪的检测,使用96孔透明底黑色细胞培养板的检测菌液体积为100 μL,初始菌液浓度OD600nm值为0.05.在此优化检测条件下,超敏感酵母HUG1-yEGFP生物传感器与典型的遗传毒性化合物甲磺酸甲酯(MMS)可以建立良好的剂效和时效关系,说明该生物传感器已具备了实际应用的实验基础.

Vitellogenin (VTG), the precursor of yolk proteins, is a sensitive biomarker of estrogenic contamination in aquatic environments. Traditionally, VTG was believed to be synthesized under the control of estrogen in the livers of mature females and then secreted into the blood, before being taken up by the ovaries and cleaved into lipovitellin and phosvitin, which provide nutrition for developing embryos. However, recent studies have reported that the liver is not the only tissue to express VTG and this has led to questions over the precise tissue distribution of VTG in zebrafish. Moreover, studies in zebrafish on the expression of the VTG protein are rare. Using Western blotting and reverse-transcriptase polymerase chain reaction, this present study reports that the VTG protein and VTG1 mRNA were detected not only in the liver, but also in various extrahepatic tissues, including the heart, spleen, kidney, skin, muscle, gill, eye and brain tissues, of female and 17α-ethinylestradiol (EE2)-treated male zebrafish. Due to the high expression levels of VTG and the ease of taking samples, skin and eye tissues were chosen to evaluate the effects of varying doses and exposure times of EE2 on male zebrafish. The VTG gene and protein were induced by EE2 exposure in liver, skin and eye tissues of male zebrafish in dose- and time-dependent patterns. Therefore, we suggest that zebrafish skin and eye tissues may be alternatives to plasma and liver tissues for VTG biomarker analysis.

Vigilin, a highly conserved protein from yeast to mammals, is a multifunctional protein in eukaryotic organisms. One biological function of vigilin is to stabilize the expression level of vitellogenin (VTG). This study aimed to develop vigilin as a new estrogen-inducible biomarker that correlates with the widely applied estrogen-inducible biomarker VTG and expand the ability to detect it in various species. Here, a recombinant monoclonal antibody with high specificity against the conserved C-terminal region of vigilin from zebrafish (Danio rerio) was successfully isolated from a phage display antibody library and found to recognize vigilin proteins from multiple vertebrate species. The effect of 17α-ethinylestradiol (EE2) on vigilin expression in the liver of zebrafish and juvenile crucian carp (Carassius auratus) was investigated. Although vigilin mRNA was expressed in all tissues analyzed from male zebrafish, vigilin protein was detected exclusively in the testis of male zebrafish, as well as the liver of female zebrafish and juvenile crucian carp at a lower level without exposure to EE2. Significant induction of vigilin mRNA by exposure to EE2 was observed in the liver and testis of male zebrafish, even at a low dose of 6.25 ng/L (21.09 pmol/L). In Hela cells, the expression of vigilin coincided with high protein synthesis activity but not dose-dependently by EE2 exposure. Therefore, the recombinant antibody may be used as a detection tool to screen for mammalian cell lines or organs with estrogen-inducible expression of vigilin.

Grass carp reovirus (GCRV) is the main cause of severe outbreaks of hemorrhagic disease in fingerling and yearling grass carp. The virus outer capsid proteins, VP5 and VP7, have been shown to play critical roles in the invasion of host cells. In our study, prokaryotic-expressed VP7, VP5, and the N and C terminals of VP5 protein were used as targets to select the desired antibodies from phage-displayed scFv library. After 3 rounds of panning, we selected 7 an-tibodies against the prokaryotic-expressed antigens-- VP7, VP5, VP5N and VP5C. Furthermore, we found that 2 anti-bodies against VP7 out of the total 7 were able to recognize native GCRV particles. Our study should provide an insight into the interactions between GCRV and its host cells.

Grass carp (Ctenopharyngodon idella) is an important species of freshwater aquaculture fish in China. However, grass carp reovirus (GCRV) can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic capacity of the virus’s structural proteins for preparing an effective vaccine has not been available. In this study, some single-chain variable fragment antibodies (scFv), which could specifically recognize grass carp IgM, were selected from a constructed mouse naïve antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from virus-infected grass carp was more than two times higher than that of the control group at 5–7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.

Antibody-displaying phage library was selected after three rounds of panning against spring viraemia of carp virus (SVCV) by phage display technology. Eight positive clones which could produce soluble single-chain fragment variable (scFv) antibody induced by isopropyl-beta-d-thiogalactopyranoside (IPTG) were obtained. Dot blot results showed that the eight scFv antibodies could recognize SVCV. The soluble scFv antibodies showed a molecular weight 29 kD by Western blot. All scFv antibodies could recognize SVCV proteins specifically without cross-reaction with other virus proteins by ELISA. Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the SVCV-infected cells. These scFv antibodies will be useful tools to establish immunological detection methods for SVCV.

Human exposure to particulate matter emitted from on-road motor vehicles includes complex mixtures of heavy metals from tyres, brakes, part wear, and resuspended road sediment. The purpose of this study was to determine the concentrations of 14 platinum-group and other traffic-related heavy metals in road sediment within the metropolitan area of Guangzhou, China, with a view to identifying their sources and assessing the extent of anthropogenic influence on heavy metal contamination of road sediment.

草鱼(Ctenopharyngodon idellus)是我国重要的淡水经济鱼类之一.为了制备有效的抗各种病原体的疫苗,必须对草鱼的免疫应答分子进行分析.IgM是大多数真骨鱼类血清中都存在的一种免疫球蛋白,是主要的介导体液免疫应答的分子,也是有颌类脊椎动物进化过程中最先出现的免疫球蛋白[1].1965年Hodgins发现真骨鱼类鲑鱼体内的抗体为四聚体构型[2].近些年研究表明,硬骨鱼的IgM和哺乳动物类似,由两条重链和两条轻链形成一个IgM单体,通过二硫键连接.通常硬骨鱼IgM以四聚体存在,但是也有其他的多聚体形式,这可能是硬骨鱼产生抗体多样性的一种机制[3].在哺乳动物中,5个IgM单体由二硫键相互连接,并通过二硫键与连接链连接形成五聚体[4].而硬骨鱼IgM缺乏连接链[1,5],单体常常通过非共价键结合成四聚体[3,6],空间结构可能为四面体结构[7].不同鱼类,它们的单体间二硫键的形成也是不确定的[7-10].

Various polyclonal and monoclonal antibodies have been developed for vitellogenin (Vtg) bioassays in different aquatic species. Preparation of these reagents is time-consuming and expensive. In the present study, a phage-displayed, recombinant, single-chain variable fragment (scFv) format antibody library was constructed using splenic mRNA from non-immunized mice. After 3 rounds of panning, 3 scFv antibodies with specificity for the highly conserved N-terminal region of cyprinid fish Vtg were isolated. One of these, antibody H4, bound purified Vtg from common carp Cyprinus carpio, zebrafish Danio rerio and Chinese rare minnow Gobiocypris rarus with similar affinities and detected Vtg in zebrafish plasma samples. This study provides a simple, low cost Vtg bioassay for plasma samples from a variety of cyprinid fish.

White spot syndrome virus (WSSV) is one of the most important pathogens in shrimp farm throughout the world. Many researches on WSSV have been done, but no efficient approach has been gained to protect and cure the disease. In this study, we constructed a single-chain fragment variable (scFv) antibody library displayed on phage using spleen cells from mice immunized with denatured WSSV. After several rounds of panning respectively against purified intact WSSV virions and purified VP28 expressed in Escherichia coli, five novel scFv antibodies specifically against WSSV were selected, one of which, clone P75E8, recognized a linear epitope. The location in virions of the epitopes recognized by the five scFv clones was determined by immunoelectron microscopy. This study provides a new way to obtain more different antibodies specifically binding to WSSV, and especially provides a new strategy to obtain scFvs against linear epitopes.

Monoamine transporters play key roles in controlling monoamine levels and modulating monoamine reuptake. The objective of the present study was to identify monoamine transporter inhibitors from herbal sources. We discovered that bakuchiol analogs isolated from Fructus Psoraleae inhibited monoamine transporter uptake to differing degrees. The bakuchiol analog, Δ3,2-hydroxybakuchiol was the most potent and efficacious reuptake blocker and was thus selected as the candidate target. Monoamine transporter inhibition by Δ3,2-hydroxybakuchiol was more selective for the dopamine transporter (DAT) (IC50=0.58±0.1μM) and norepinephrine transporter (NET) (IC50=0.69±0.12μM) than for the serotonin transporter (SERT) (IC50=312.02±56.69μM). Δ3,2-Hydroxybakuchiol exhibited greater potency (pEC50 for DAT and NET) than bupropion and exhibited similar efficacy (Emax for DAT and/or NET) to bupropion and GBR12,935. Pharmacokinetically, Δ3,2-hydroxybakuchiol competitively inhibited DAT and NET with partial reversibility and occupied cocaine binding sites. Moreover, Δ3,2-hydroxybakuchiol counteracted 1-methyl-4-phenylpyridinium-induced toxicity in cells expressing DAT with similar efficacy to GBR12,935. In vivo studies showed that Δ3,2-hydroxybakuchiol increased the activity of intact mice and improved the decreased activity of reserpinized mice. In the conditioned place preference test, preference scores in intact mice were unaffected by Δ3,2-hydroxybakuchiol treatment. Bakuchiol analogs, especially Δ3,2-hydroxybakuchiol, are monoamine transporter inhibitors involved in regulating dopaminergic and noradrenergic neurotransmission and may have represented potential pharmacotherapies for disorders such as Parkinson's disease, depression, and cocaine addiction.

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV.

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 ± 13.19 s−1, which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody–enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [–(Gly–Ser)5–] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 μL hemolymph from an infected shrimp and differentiated it from a healthy shrimp.

White spot syndrome virus(WSSV) from shrimp was first found in Taiwan in 1992.Since 1993,white spot syndrome disease of shrimp caused by this virus was widely spread throughout China,Asia and Pan-Pacific Ocean.It can cause 100% cumulative mortality of cultured shrimps in 3—4 days.The virus particles contain five major proteins:VP28,VP26,VP24,VP19 and VP15.The mode of entry and systemic infection of WSSV in shrimp and the funotions of these virion proteins are not clear.VP60B is a kind of minor structure proteins in WSSV particles.Using interproscan to analysis on homology to VP60B,a piece of sequence of amino acid in VP60B was found homology with adenovirus type 5 fiber protein knob domain.In this study,the gene of VP60B had been cloned into a prokaryotic expression system,and induced to express at low temperature.The result showed that the expressed protein was existed mainly as inclusion body in this expression system. Prokaryotic expressed VP60B could not recognize by polyclone antiserum against WSSV,which suggested that this protein might be a weak immunogen.According to the analysis of the sequence of amino acid,a trans-membrane domain was found,which suggested that this protein might locate on the envelop membrane of WSSV.

A fluorescent quantitative PCR (FQ-PCR) assay utilizing SYBR green I dye is described for quantitation of white spot syndrome virus (WSSV) particles isolated from infected crayfish, Cambarus clarkii. For this assay, a primer set was designed which amplifies, with high efficiency and specificity, a 129bp target sequence within ORF167 of the WSSV genome. Conveniently, WSSV particles can be added into the FQ-PCR assay with a simple and convenient method to release its DNA. To establish the basis for an in vitro neutralization test, primary cultures of shrimp cells were challenged with WSSV that had been incubated with a polyclonal anti-WSSV serum or with control proteins. The number of WSSV particles released from the cells after these treatments were assayed by FQ-PCR. This test may serve as a method to screen monoclonal antibody pools or recombinant antibody pools for neutralizing activity prior to in vivo animal experiments.

VP60B是对虾白斑综合症病毒（WSSV）中含量很少的一个结构蛋白。VP60B的一段序列与腺病毒纤维蛋白（Adenovirus type 5 fiber protein）的knob domain的一段序列具有同源性。本试验将VP60B基因克隆到原核表达系统中,在低温条件下,诱导了VP60B蛋白的表达。结果显示VP60B在该系统主要是以包含体的形式存在。原核表达的VP60B不能被鼠抗WSSV多克隆血清所识别,预示该蛋白的免疫原性较弱。通过对VP60B氨基酸序列的分析,发现有一个跨膜区,这预示着该蛋白可能位于WSSV病毒的囊膜上。

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A1 with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A1 scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the preferred selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv.

本实验以pCANTAB 5 E噬菌粒为载体,成功构建了较高容量的噬菌体展示随机十肽库,并将其应用于抗原模拟表位的淘选和鉴定.将一种特异识别对虾白斑综合症病毒(White spot syndrome virus, WSSV)的单链抗体A1对十肽库和十五肽库分别进行淘选,结果得到一系列能与单链抗体A1特异性结合的阳性克隆.将这些阳性克隆所编码的多肽氨基酸序列与已知的单链抗体A1的抗原WSSV388片段氨基酸序列做比对,发现多数阳性多肽序列都与WSSV388片段序列的C端一处K····R··R·QS的氨基酸片段相似,由此推论单链抗体A1的模拟抗原表位可能是由该不连续氨基酸片段所构成的构象表位,而非线性表位.研究结果表明,噬菌体展示随机肽库技术是一种用于研究抗原表位结构的有效方法,有助于进一步探讨WSSV的结构蛋白的构象及功能,以及相应单链抗体与细胞受体相互作用的机理.