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1.
目的快速制备大量具有生物活性的独特型纳米抗体F7。方法构建pET22b-F7原核表达载体,转化至大肠杆菌E.coliBL21(DE3)中进行诱导表达,对诱导温度、诱导剂浓度和诱导时间进行优化。结果独特型纳米抗体F7表达量有所增加但主要以包涵体形式存在。经过包涵体的溶解、复性获得了具有生物活性的抗AFB1独特型纳米抗体,ELISA证实复性后的蛋白依然存在对鼠源AFB1抗体的结合特性。结论为后续抗体的性质研究和改造奠定了基础。  相似文献   

2.
抗黄曲霉毒素B_1单链抗体的表达载体的比较   总被引:1,自引:0,他引:1  
目的:比较4种不同的pET载体在表达抗黄曲霉毒素B1(AFB1)的单链抗体(scFv)克隆的特点,确定用以表达scFv-H4的合适载体。方法:将目的基因H4分别克隆到载体pET20b、pET22b、pET28a和pET32a上,分别转入大肠杆菌BL21(DE3)或Origami(DE3)中,比较诱导过程中细胞的生长状况和诱导结束后细胞破碎液中scFv-H4的生物活性以及细胞各部分包涵体。结果:pET20b和pET22b能表达具有生物活性的scFv-H4,这些具有生物活性的蛋白主要存在于周质中;pET28a和pET32a不能表达有生物活性的scFv-H4,而pET32a仅能在细胞质中产生大量的包涵体。结论:pET22b可能是表达AFB1的单链抗体较优的表达载体。  相似文献   

3.
以抗黄曲霉毒素B1(AFB1)单克隆抗体的F(ab')2片段为抗原免疫兔子,得到AFB1抗独特型抗体。经过酶联免疫吸附法(ELISA)条件的优化,建立AFB1抗独特型抗体最佳竞争抑制曲线。该曲线与AFB1竞争抑制曲线相比较可知,AFB1抗独特型抗体与AFB1之间存在指数增长关系,且相关性系数R为0.999 8。以AFB1抗独特型抗体浓度与其相应的抑制率作标准曲线,ELISA法测定花生中AFB1添加回收率,范围在90.4%~100.2%之间。综上,AFB1抗独特型抗体可以作为AFB1无毒替代标准品。  相似文献   

4.
目的:原核表达抗克伦特罗重组单链抗体,对表达的包涵体蛋白进行提取和纯化,并鏊定重组抗体特性.方法:通过温度诱导大肠杆菌表达抗克伦特罗单链抗体,超声破碎菌体细胞,采用不同洗涤溶液提取包涵体.以不同变性剂溶解包涵体,Ni-NTA亲和柱纯化后,经脉冲稀释法将其复性.超滤及透析浓缩蛋白复性液,经Ni-NTA亲和柱二次纯化,得到纯化重组目的蛋白.通过SDS-PAGE电泳分析重组目的蛋白纯度,采用Western bloning和间接竞争EUSA对所制备的抗克伦特罗重组单链抗体特异性进行鉴定.结果:以大肠杆菌Es-cherichia coli BL21(DE3)为宿主菌诱导表达重组单链抗体,产率最高,占菌体蛋白的31%.以1 mol/L尿素洗涤包涵体,6 mol/L盐酸胍为变性剂,经Ni-NTA亲和柱纯化,所得复性重组单链抗体蛋白的纯度达96%.Western blotting分析显示,纯化并复性后的单链抗体可与鼠抗His标签蛋白单克隆抗体发生特异性的结合反应.间接竞争ELSIA结果表明,该重组抗体IC50值为3.35 ng/mL,与沙丁胺醇等结构类似物无交叉反应.结论:原核表达制备的抗克伦特罗重组单链抗体具有较好的抗原结合活性及特异性,为进一步开展克伦特罗的免疫快速检测研究奠定基础.  相似文献   

5.
表达和纯化抗黄曲霉毒素B1(Aflatoxin B1,AFB1)纳米抗体(G8),并分析纳米抗体的热稳定性及生物学活性,建立基于纳米抗体检测AFB1的ELISA方法。将编码抗AFB1纳米抗体的基因亚克隆至原核表达载体,转化至大肠杆菌BL21(Rosetta,DE3),IPTG诱导表达后,SDS-PAGE分析蛋白表达情况;采用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)分析纳米抗体的热稳定性、有机溶剂耐受性、盐离子耐受性及耐酸碱性。结果显示,在大肠杆菌中成功表达可溶性的抗AFB1纳米抗体,表达量为80 mg/L,在25~65℃之间,抗体具有良好的热稳定性。在5%甲醇、p H7.4、10 mmol/L PBS条件下,建立了G8-ELISA检测AFB1的方法,该方法的半抑制浓度(IC50)为4.61 ng/m L,线性范围为0.95~42.45 ng/m L。  相似文献   

6.
目的构建一株高效表达人骨形成蛋白2(hBMP-2)的基因工程菌株,并通过复性、纯化获得有生物活性的rhBMP-2。方法采用基因工程法将hBMP-2基因片段与pET-28a(+)质粒载体相连,构建大肠杆菌表达系统。工程菌发酵后,超声破碎收集包涵体,包涵体经裂解后通过稀释复性法进行复性,SDS-PAGE检测复性情况,并采用HiTrap Heparin HP纯化,C2C12细胞检测活性。结果 rhBMP-2在大肠杆菌中以无活性的包涵体形式表达,1 L发酵罐高密度发酵rhBMP-2包涵体湿重可达20 g/L。采用稀释法复性,蛋白复性率大于40%,亲和层析纯化后,二聚体蛋白纯度达95%以上,且能诱导C2C12细胞碱性磷酸酶的表达,具有同市售产品相同的细胞活性。结论成功构建了高效表达rhBMP-2的基因工程菌株,复性方法简单且无需对包涵体进行纯化,蛋白复性浓度高,纯化方法简便,大大降低了工业化生产成本。  相似文献   

7.
绿色无毒黄曲霉毒素B_1免疫检测方法   总被引:1,自引:0,他引:1       下载免费PDF全文
建立了一种基于抗独特型抗体的绿色无毒的ELISA方法,并用于检测面粉中黄曲霉毒素B1。通过酶解2种抗黄曲霉毒素小鼠单克隆抗体(11A9和1G3)制备F(ab’)2片段,并将其免疫新西兰大白兔制备抗独特型抗体。最终得到两种抗独特型抗体,并对抗独特型抗体和AFB1进行相关性分析。通过实际样品添加回收,其批内回收率为115.60%~121.88%,变异系数在5%以内;而批间回收率为111.89%~126.98%,变异系数低于8%。分析结果准确可靠,表明此无毒绿色ELISA是一种安全可靠的AFB1分析方法。  相似文献   

8.
西维因单链抗体基因克隆、表达及活性分析   总被引:1,自引:0,他引:1  
利用兼并引物从西维因单克隆杂交瘤细胞的cDNA中克隆得到抗体基因的轻链可变区(VL)和重链可变区(VH),长度分别为324bp和360bp,将其分别连接入pGEM-T-Easy载体进行测序;根据测序结果设计特异性引物和连接序列(Gly4Ser)3,利用重叠延伸PCR扩增得到长度为729bp的单链抗体基因片段(scFv),亚克隆至原核表达载体pET30a(+),并转化感受态细胞B121(DE3)进行诱导表达;聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示,重组表达单链抗体形成包涵体,分子量大小约为33kDa,变性后利用不同条件进行复性,结果显示最佳复性时间为48h,最佳起始蛋白浓度为2001xg/mL,含有精氨酸的复性液效果最佳;利用亲和层析纯化scFv,并利用竞争ELISA检测其活性.结果显示该scFv对西维因具有良好的亲和性和特异性.  相似文献   

9.
通过研究重组几丁质酶在大肠杆菌表达系统中诱导表达的浓度、时间和温度,获得表达的最佳条件。表达产物除了可溶性目的蛋白以外,仍存在于包涵体中。采用透析复性和Ni-NTA亲和层析柱上复性两种方法对包涵体中的目的蛋白进行复性,并比较对目的蛋白产率和比酶活的影响。并考察了亲和层析柱上复性时的上样量、洗脱速率和温度对酶活的影响。结果发现,表达的几丁质酶可以采用透析和亲和层析柱上复性,亲和层析柱上复性的比酶活为1347.7 U/mg,明显高于透析复性,但产率明显低于透析复性。对1 m L的Ni-NTA亲和层析柱,较低浓度的蛋白复性液在0.4 m L·min-1洗脱速率下,降低上样量和温度,可以提高复性效率。复性后的几丁质酶对荧光底物具有较高活性,反应的最适温度为37℃,最适p H为3.8。  相似文献   

10.
对创伤弧菌重组外膜蛋白VuuA进行表达纯化及复性,以获得可溶性VuuA蛋白。构建重组工程菌Escherichia coli BL21(DE3)-pET-28a-vuuA,利用IPTG诱导表达重组蛋白VuuA,利用亲和层析分离纯化蛋白并对包涵体进行复性。VuuA蛋白主要以包涵体形式存在于重组工程菌细胞内,经复性后和亲和层析纯化后,蛋白浓度达到28.2mg/L发酵液,有效提高可溶性蛋白的浓度。VuuA蛋白的纯化及复性为创伤弧菌新型疫苗的制备奠定了基础。  相似文献   

11.
A study was undertaken to evaluate the total aflatoxin content in naturally contaminated eggs. Two pools of eggs from laying hens were collected after 2 and 7 days of treatment with aflatoxin B1 (AFB1). An HPLC method has been developed for the determination of AFB1, aflatoxin M1 (AFM1), aflatoxin B2a (AFB2a) and aflatoxicol (Ro) both in free form and after release from water-soluble conjugates. The bound form was cleaned up after acid hydrolysis of the aqueous phase. Eggs collected after 2 days of treatment revealed residues of AFB1, AFB2a and Ro in the organic phase, but none in the aqueous portion. After 7 days of treatment both AFB1 and its hydroxy derivative were found in the organic phase but the aqueous portion showed only hydroxylated metabolites accounting for 35% of the total aflatoxin content.  相似文献   

12.
The aim was to evaluate the excretion of residues of aflatoxin B(1) (AFB(1)), aflatoxin M(1) (AFM(1)), aflatoxin B(2a) (AFB(2a)) and aflatoxicol (AFL) in eggs of laying Japanese quail fed rations with low levels of aflatoxin B(1) for 90 days. The quail were randomly assigned into four experimental groups and given prepared rations containing either 0 (controls), 25, 50 or 100 microg AFB(1) kg(-1) feed. Thirty-two eggs per treatment were collected on days 1-7, 10, 20, 30, 60 and 90 of the aflatoxin treatment period, and submitted to aflatoxin analysis by high-performance liquid chromatography. Average egg production and feed consumption were not affected ( p > 0.05) by AFB(1). Egg weight was significantly lower ( p<0.05) only for groups exposed to 100 microg AFB(1) kg(-1). Residues of aflatoxins were detected in eggs at levels that ranged from 0.01 to 0.08 microg kg(-1) (AFB(1)), 0.03-0.37 microg kg(-1) (AFM(1)), 0.01-1.03 microg kg(-1)(AFB(2a)) and 0.01-0.03 microg kg(-1) (AFL). Results indicate that the excretion of aflatoxin residues in quail eggs might occur at relatively low concentrations under conditions of long-term exposure of quail to low levels of AFB(1).  相似文献   

13.
目的 建立了聚多巴胺磁性分子印迹聚合物 用于食用油中黄曲霉毒素B的吸附富集方法。方法 以多巴胺为功能单体和交联剂,5,7-二甲氧基香豆素为模拟模板分子,一步法在Fe3O4 NPs表面制备磁分子印迹聚合物。采用透射电子显微镜和红外光谱仪对该分子印迹材料形态和结构进行表征,研究了其对两种黄曲霉毒素B吸附性能。最终以该印迹材料作为分散固相萃取剂,对影响黄曲霉毒素B吸附的条件进行了优化。结果 该分子印迹材料对两种黄曲霉毒素B的吸附模式符合Langmuir理论,对黄曲霉毒素B1和黄曲霉毒素B2最大吸附容量分别为0.46 mg/g和0.047 mg/g。在最佳萃取条件(材料用量(10 mg)、溶液pH(7.0)、超声时间(5 min)和1 mL(解吸2次,每次0.5 mL)3%乙酸/乙腈作为解析液下,结合配有大体积流通池的超高效液相色谱-荧光检测法定量检测,黄曲霉毒素B1和黄曲霉毒素B2浓度分别在0.005-0.500 ng/mL(R=0.9989)和0.001-0.100 ng/mL(R=0.9992)范围内与对应的峰面积呈现良好的线性相关性,检出限分别为0.0024 ng/mL和0.00038 ng/mL,食用油样品加标回收率为93.2%~104.0%,相对标准偏差5.8%~7.7%,且可重复使用至少7次。结论 该吸附富集方法具有快速、低廉、选择性高、稳定性好等特点,适用于食用油中黄曲霉毒素B的前处理。  相似文献   

14.
A study was performed to determine aflatoxin residues in tissues and organs of male broilers and hens that had been fed a diet contaminated with 50 micrograms/kg aflatoxin B1 (AFB1). Residue levels of AFB1, aflatoxicol (Ro), aflatoxin M1 (AFM1) and aflatoxin B2a (AFB2a) were determined by an HPLC method and, with the exception of AFB2a, were detected in the liver, kidney and thigh of both male broilers and hens. The highest levels found were for Ro in liver (1.10 and 0.60 micrograms/kg for male broilers and hens, respectively). On the other hand no detectable amounts of aflatoxins were found in any tissue after withdrawal periods of 14 and 33 days for male broilers and laying hens respectively.  相似文献   

15.
The effect of Saccharomyces cerevisiae RC008 and RC016, previously selected based on their aflatoxin B(1) binding ability and beneficial properties, against Aspergillus parasiticus under different interacting environmental conditions was evaluated. Studies concerning the lag phase, growth rate and aflatoxin B(1) production were carried out in?vitro under different regimes of a (w) (0.95 and 0.99), pH (4 and 6), temperature (25 and 37°C), and oxygen availability (normal and reduced). Both yeast strains showed great antagonistic activity at pH 4, decreasing growth rate compared with the control. The RC008 strain showed the greatest inhibitory activity at all assayed conditions. A. parasiticus produced large amounts of AFB(1) in?vitro. A significant decrease of AFB(1) levels in comparison with the control were observed with yeast interaction. Differences between control and treatment values ranged from 130 to 5400?ng?ml(-1). S. cerevisiae RC008 and RC016 could be considered as effective agents in reducing growth and AFB(1) production at different interacting environmental conditions, related to that found in stored feedstuff. The importance of the present work lies in the search for live strains with both probiotic and biocontrol properties able to prolong the safe storage of feedstuff and exert beneficial properties after animal consumption and which could be included in a novel product for animal feed.  相似文献   

16.
Polyphenols with antimutagenic and anticarcinogenic properties are present in fruits, vegetables and legumes. In this study, the Salmonella typhimurium tester strains TA98 and TA100 were used in the microsuspension assay to examine the antimutagenic effect of phenolic compounds extracted from the common bean (Phaseolus vulgaris) against mutagenicity induced by aflatoxin B 1 (AFB 1 ). A dose-response curve was constructed for AFB 1 ; from which a level of 40ng AFB 1 /tube was selected for all antimutagenicity assays. The AFB 1 and phenolic extract (PE) were not toxic to the bacteria at concentrations tested. In the case of PE, results were similar to the number of spontaneous revertants for TA98 and TA100. The inhibitory effect of PE against AFB 1 mutagenicity was dose-dependent at the lower concentrations tested (2.5, 5, 10, 12.5, 15 and 25 μg-equivalent ( + )-catechin/tube for TA98; 0.5, 1, 1.5, 2.5, 5, 10 and 25 μg-equivalent ( + )-catechin/ tube for TA100). Further, a two-stage incubation procedure was used to investigate the potential interaction between PE and AFB 1 . The greatest inhibitory effect of the PE on AFB 1 mutagenicity occurred when PE and AFB 1 were incubated together. When the bacteria were first incubated with PE followed by a second incubation with AFB 1 , lower inhibition was observed. Lower inhibition was also observed when the bacteria were first incubated with AFB 1 followed by a second incubation with PE. The results suggest that the mechanism of inhibition could involve the formation of a chemical complex between of PE and AFB 1 .  相似文献   

17.
施氏假单胞菌F4能高效降解黄曲霉毒素B1(aflatoxin B1,AFB1)。研究了F4的AFB1降解活性、降解动力学以及蛋白酶K和SDS对其降解性能的影响。F4细胞悬液与毒素共培养72 h后降解率达80.03%;蛋白酶K处理对降解率没有影响,SDS处理的细胞悬液基本丧失了降解活性。不同时间点的降解液上清液仍能有效降解残留AFB1,其中以60 h的降解液上清液活性较好,与残留AFB1继续作用48 h后降解率达84.30%;而经蛋白酶K处理后降解率仅为45.42%。低浓度AFB1诱导对菌体的降解活性没有影响。上述结果提示,F4通过胞内酶作用降解AFB1。高效液相色谱对产物分析表明,F4可将AFB1酶解为至少2种产物。  相似文献   

18.
The aflatoxins were discovered in toxic peanut meal causing "turkey X" disease, which killed large numbers of turkey poults, ducklings and chicks in the UK in the early 1960s. Extracts of toxic feed induced the symptoms in experimental animals, and purified metabolites with properties identical to aflatoxins B(1) and G(1) (AFB(1) and AFG(1)) were isolated from Aspergillus flavus cultures. Structure elucidation of aflatoxin B(1) was accomplished and confirmed by total synthesis in 1963. AFB(1) is a potent liver carcinogen in rodents, non-human primates, fish and birds, operating through a genotoxic mechanism involving metabolic activation to an epoxide, formation of DNA adducts and, in humans, modification of the p53 gene. Aflatoxins are unique among environmental carcinogens, in that elucidation of their mechanisms of action combined with molecular epidemiology provides a foundation for quantitative risk assessment; extensive evidence confirms that contamination of the food supply by AFB(1) puts an exposed population at increased risk of developing hepatocellular carcinoma (HCC). Molecular biomarkers to quantify aflatoxin exposure in individuals were essential to link aflatoxin exposure with liver cancer risk. Biomarkers were validated in populations with high HCC incidence in China and The Gambia, West Africa; urinary AFB(1)-N (7)-Guanine excretion was linearly related to aflatoxin intake, and levels of aflatoxin-serum albumin adducts also reflected aflatoxin intake. Two major cohort studies employing aflatoxin biomarkers identified their causative role in HCC etiology. Results of a study in Shanghai men strongly support a causal relationship between HCC risk and the presence of biomarkers for aflatoxin and HBV infection, and also show that the two risk factors act synergistically. Subsequent cohort studies in Taiwan confirm these results. IARC classified aflatoxin as a Group 1 human carcinogen in 1993, based on sufficient evidence in humans and experimental animals indicating the carcinogenicity of naturally occurring mixtures of aflatoxins, aflatoxin B(1), G(1) and M(1). Aflatoxin biomarkers have also been used to show that primary prevention to reduce aflatoxin exposure can be achieved by low-technology approaches at the subsistence farm level in sub-Saharan Africa. Also, in residents of Qidong, China, oral dosing with chlorophyllin, a chlorophyll derivative, prior to each meal led to significant reduction in aflatoxin-DNA biomarker excretion, supporting the feasibility of preventive measures to reduce HCC risk in populations experiencing unavoidable aflatoxin exposure. The systematic, comprehensive approach used to create the total aflatoxin database justifies optimism for potential success of preventive interventions to ameliorate cancer risk attributable to aflatoxin exposure. This strategy could serve as a template for the development, validation and application of molecular and biochemical markers for other carcinogens and cancers as well as other chronic diseases resulting from environmental exposures.  相似文献   

19.
To determine whether pozol, a nixtamalized maize-based food was contaminated with aflatoxins, samples of non-fermented pozol were collected during the period November 2002 to April 2003 from local markets at Comitan in Chiapas, Mexico. The samples were analyzed for the presence of aflatoxins. Nineteen out of one hundred and eleven samples were contaminated with aflatoxin B2 (AFB2) and traces of aflatoxin B1 (AFB1). The percentage of samples contaminated with AFB2 in pozol prepared with white maize was 5.4%. Pozol mixed with toasted cacao paste had a contamination rate of 41.5%. No aflatoxins were detected in pozol prepared with yellow maize. It was found that only 1 of 19 contaminated samples had aflatoxin concentrations above 20 ppb.  相似文献   

20.
Traditional nixtamalization and an extrusion method for making the dough ( masa ) for corn tortillas that requires using lime and hydrogen peroxide were evaluated for the detoxification of aflatoxins. The traditional nixtamalization process reduced levels of aflatoxin B 1 (AFB 1 ) by 94%, aflatoxin M 1 (AFM 1 ) by 90% and aflatoxin B 1 -8,9-dihydrodiol (AFB 1 -dihydrodiol) by 93%. The extrusion process reduced levels of AFB 1 by 46%, AFM 1 by 20% and AFB 1 -dihydrodiol by 53%. Extrusion treatments with 0, 0.3 and 0.5% lime reduced AFB 1 levels by 46, 74 and 85%, respectively. The inactivation of AFB 1 , AFM 1 and AFB 1 -dihydrodiol in the extrusion process using lime together with hydrogen peroxide showed higher elimination of AFB 1 than treatments with lime or hydrogen peroxide alone. The extrusion process with 0.3% lime and 1.5% hydrogen peroxide was the most effective process to detoxify aflatoxins in corn tortillas, but a high level of those reagents negatively affected the taste and aroma of the corn tortilla as compared with tortillas elaborated by the traditional nixtamalization process.  相似文献   

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