山葡萄VaCIPK18原核表达与多克隆抗体制备     

吴楠  张宁波  郑巧玲  陈卫平  徐伟荣 《核农学报》2020,34(7):1387-1396

类钙调磷酸酶B亚基蛋白(CBLs)互作蛋白(CIPKs) 作为类丝氨酸/苏氨酸蛋白激酶,在植物响应非生物胁迫信号转导中起着重要作用。基于前期山葡萄响应低温胁迫转录组测序结果,发现低温胁迫引发的早期伤害及感知阶段的激酶基因中涉及CBL-CIPK 信号通路的VaCIPK18表达显著上调。为进一步研究山葡萄(Vitis amurensis)VaCIPK18激酶参与低温胁迫的功能,采用同源克隆获得了VaCIPK18基因,其开放阅读框为1 320 bp,编码439个氨基酸。基于对VaCIPK18蛋白生物信息学分析,获取胞外结构域中抗原表位丰富的肽段,并将其C端调控结构域(230~439 aa)构建到原核表达载体pET28a-SUMO。将重组表达载体转化至大肠杆菌(E. coli Rosetta)中,经0.8 mmol·L^-1 IPTG、37℃诱导4 h表达出大小为42 kDa的包涵体蛋白。将重组蛋白作为抗原免疫日本大耳白兔,获得anti-VaCIPK18多克隆抗体,经检测具有高效价及特异性。Western Blot结果表明,该抗体可以与葡萄内源CIPK18特异性结合,且在50 kDa位置出现与预期一致的条带。同时,CIPK18在低温胁迫后葡萄叶片中蛋白表达水平与室温下相一致,但两种状态下均存在可能的磷酸化与泛素化修饰现象。本研究结果为进一步探究VaCIPK18的蛋白定位、表达及其功能奠定了基础。  相似文献   

Comparison of dot blot and PCR diagnostic techniques for detection of white spot syndrome virus in different tissues of Penaeus monodon     

M.S. Shekhar  I.S. Azad  P. Ravichandran 《Aquaculture (Amsterdam, Netherlands)》2006,261(4):1122-1127

Comparison of PCR and dot blot diagnostic techniques for detection of white spot syndrome virus (WSSV) was made on different tissues of infected Penaeus monodon including eye stalk, eye stalk with eye, gills, cuticle, pleopod, periopods, uropods and telson. Dot blots of crude DNA extracted from infected tissue samples showed positive reactions with all the samples; however, the sensitivity of the dot blot was reduced with the purification of DNA samples extracted from pleopod, telson and uropod. PCR was found to be more sensitive when compared to dot blot. Both crude DNA and purified DNA samples extracted from all the tissues except for eye stalk with eye showed single step nested PCR positive reaction. The amplification of all or either of the three bands of 941 bp, 525 bp and 204 bp size varied with the tissues analysed. The severity of infection assessed by PCR amplification was found to be maximum in cuticle and telson followed by gill. Other tissues such as eye stalk, pleopod, periopods and uropod were observed to have mild infection. The maximum intensity of the PCR product was for the smallest amplified product of 204 bp followed by 525 bp and the weakest intensity was observed for the 941 bp size. The limitation of PCR due to inhibiting factors present in tissues could be overcome with the use of dot blot which gave positive reaction from the DNA extracted from eye stalk including the eye but yielded no amplification by PCR.  相似文献   

显微注射生长激素基因导入中国对虾（Penaeus chinensis）受精卵的研究   总被引：4，自引：0，他引：4  

刘萍  孔杰  王清印  李健  杨丛海  梁利群  孙孝文 《中国水产科学》1996,3(4):35-38

本实验采用显微微量注射方法，交羊生长激素基因导入中轻地虾受精卵，受精卵发育到蚤状幼体第三期后采样检测。ＰＣＲ检测结果表明：７个样品共９３尾幼体，有３个样品呈出阳性信号，基因转移比率至少在３％以上。同时斑点杂交结果也证明有两个明显阳性斑点。  相似文献   

Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans     

Yoganandhan K  Syed Musthaq S  Narayanan RB  Sahul Hameed AS 《Journal of fish diseases》2004,27(9):517-522

The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp.  相似文献   

Purification and analyses of the specificity of two putative diagnostic antigens for larval cyathostomin infection in horses     

Dowdall SM  Proudman CJ  Love S  Klei TR  Matthews JB 《Research in veterinary science》2003,75(3):223-229

Cyathostomins are important equine gastrointestinal parasites. Mass emergence of mucosal stage larvae causes a potentially fatal colitis. Mucosal stages are undetectable non-invasively. An assay that would estimate mucosal larval stage infection would greatly assist in treatment, control and prognosis. Previously, we identified two putative diagnostic antigens (20 and 25 kDa) in somatic larval preparations. Here, we describe their purification and antigen-specific IgG(T) responses to them. Western blots confirmed the purity of the antigens and showed that epitopes in the 20 kDa complex were specific to larval cyathostomins. No cross-reactive antigens appeared to be present in Parascaris equorum or Strongyloides westeri species. Low levels of cross-reactivity were observed in Strongylus edentatus and Strongylus vulgaris species. Use of purified antigens greatly reduced background binding in equine sera. These results indicate that both antigen complexes may be of use in a diagnostic assay.  相似文献   

Seroprevalence of Neospora caninum infection following an abortion outbreak in a dairy cattle herd     

Atkinson RA  Cook RW  Reddacliff LA  Rothwell J  Broady KW  Harper P  Ellis JT 《Australian veterinary journal》2000,78(4):262-266

OBJECTIVE: To investigate the seroprevalence of Neospora caninum infection in a commercial dairy cattle herd, 15 months after detection of an abortion outbreak. PROCEDURE: Sera from the whole herd (n = 266) were examined for N caninum antibodies by indirect fluorescent antibody test (IFAT) and immunoblot analysis. Herd records were reviewed to collate serological results with abortion history, proximity to calving, and pedigree data. RESULTS: The seroprevalence of N caninum infection was 24% (63/266) for IFAT titre > or = 160, 29% (78/266) for immunoblot positive (+ve), and 31% (82/266) for IFAT > or = 160 and/or immunoblot +ve; 94% (59/63) of animals with IFAT > or = 160 were immunoblot +ve. The association between seropositivity (IFAT > or = 160 and/or immunoblot +ve) and history of abortion was highly significant (P < 0.001); the seroprevalence was 86% (18/21) in aborting cows, compared with 30% (50/164) in non-aborting animals. The abortion rate for seropositive cows was 26% (18/68) compared with 3% (3/117) for seronegative animals. IFAT titres of infected cows were higher within 2 months of calving than at other times (P < 0.001). The association between seropositivity in dams and daughters was highly significant (P = 0.009). CONCLUSIONS: The abortions were associated with N caninum infection and there was evidence of reactivation of latent infection close to calving and congenital transmission of infection. Immunodominant antigens identified by immunoblots may prove useful for improved diagnostic tests.  相似文献   

堆型艾美球虫3-1E基因在大肠杆菌中的可溶性表达及鉴定     

吴德铭  于康震  毕英佐  曹永长  马静云 《中国兽医学报》2006,26(5):504-505,582

将堆型艾美球虫（E．acervulina）3-1E基因克隆至pET-32a（＋）载体中。转化大肠杆菌BL21，在37℃下，用终浓度为1.0mmol／L的IPTG诱导表达，得到相对分子质量约为38500的重组蛋白。Western blot检测证实，该重组蛋白可以与特异性抗血清结合。在20℃条件下，以终浓度分别为2．0、1．0、0．5mmol／L的IPTG进行诱导．当IPTG终浓度为0．5mmol／L时，以可溶性形式存在的目的蛋白含量最高。  相似文献   

地高辛标记cDNA探针检测苹果茎痘病毒   总被引：3，自引：0，他引：3  

杨俊玲  董雅凤  李世访  张尊平  何峻 《植物病理学报》2006,36(5):470-472

 Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP. The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization. The results showed that the probe was sensitive and specific. The probe couldn't hybridize with total RNA of Apple stem grooving virus, Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control, only hybridized with that extracted from dormant shoot infected with ASPV. The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.  相似文献   

猪蛔虫感染期幼虫抑制消减cDNA文库的构建     

黄翠琴  陈宁  张新高  林瑞庆  宋慧群  朱兴全 《畜牧兽医学报》2007,38(8):832-836

为筛选与线虫感染性相关的基因,本研究以猪蛔虫为对象,构建猪蛔虫感染期幼虫差异表达消减cDNA文库,为研究线虫期特异性发育的分子机制奠定基础。分别提取感染期幼虫和其它各期幼虫及成虫的总RNA,纯化mRNA后,采用Clontech公司PCR-selectTM试剂盒进行反转录合成cDNA并进行抑制消减杂交(SSH),构建猪蛔虫感染期幼虫差异表达的消减cDNA文库,并采用Southern斑点杂交进行消减效率的检测。随机从文库中抽取45个克隆进行测序及在线BLAST分析。试验结果表明,感染期幼虫差异表达的消减cDNA文库具有较强的特异性;在得到的41个表达序列标签(ESTs)中,有40个ESTs与已报道的基因有较高的相似性,主要代表猪蛔虫第三期幼虫基因和成虫头部基因,有1个cDNA片段可能代表新基因。猪蛔虫感染期幼虫差异表达消减cDNA文库的成功构建,为进一步研究幼虫发育差异表达基因的功能奠定了基础。  相似文献   

甘蔗黄叶病毒的TBIA快速检测     

李文凤  ;黄应昆  ;王晓燕  ;陈学宽  ;卢文洁  ;吴才文  ;罗志明 《中国甜菜》2009,(3):22-23

利用从法国引进的甘蔗黄叶病毒（SCYLV）标准抗原抗体，研究建立了TBIA快速检测SCYLV的方法。通过对田间采集的样本进行检测，同时以DAS—ELISA检测法印证，检测结果一致，表明TBIA能简便、快速、准确、有效地检测出SCYLV，且检测效率高，适合于田间大批量样品快速检测。为甘蔗黄叶病的诊断和防治、脱毒种苗的生产、对外甘蔗品种／材料交换检疫检测提供了技术支撑。  相似文献