植物源性转基因食品PCR检测技术研究

〔目的〕建立简便、快速、灵敏、特异的植物源性转基因食品DNA检测技术。〔方法〕针对转基因植物技术中载体上常用花椰菜花叶病毒的 3 5S启动子和根农杆菌的NOS终止子特异基因序列 ,设计合成特异的引物对 3 5s -f/ 3 5s-r、Nos -f/Nos -r ,利用基因PCR扩增技术检测植物源性转基因食品。〔结果〕引物对 3 5s -f/ 3 5s -r、Nos -f/Nos -r分别成功从转基因大豆中扩增出 195bp和 180bp特异的DNA带。采用引物对 3 5s -f/ 3 5s -r进行PCR时检测灵敏度为每反应 10 4分子 ;采用Nos -f/Nos -r时检测灵敏度为 2× 10 4分子。最低可以对 0 .1μg转基因大豆进行检测。〔结论〕基于 3 5S启动子和NOS终止子基因序列的PCR扩增技术是一种简便、快速、灵敏、特异的植物源性转基因食品DNA检测技术     

基于GADD153启动子的环境致癌物快速筛选系统研究

[目的]构建快速检测环境中痕量致癌物DNA损伤效应的重组人肝细胞株。[方法]构建含生长抑制与DNA损伤基因153(GADD153)启动子和萤光素酶报告基因的重组稳定转染人肝肿瘤HepG2细胞;发光检测不同致癌物对稳定转染细胞的DNA损伤效应;同时RT-PCR检测内源性GADD153mRNA的表达;彗星试验(Cometassay)检测DNA的损伤效应。[结果]稳定转染GADD153-LUC细胞株可检出多环芳烃、芳香胺、重金属、农药等已知环境致癌物以及实际水样中的含量。检测结果与彗星试验结果相比,大多数检测下限均低于彗星试验的检测限,其中对苯并芘、氯化镉和2-氨基芴的检测灵敏度分别高于彗星试验1000、300和100倍;对Aems试验不敏感的重金属、四氯化碳、氟化钠等重组细胞株具有较高的检测灵敏性。10μmol/L的氯化镉可诱导内源性GADD153mRNA和萤光素酶活性表达增加,相关分析表明两者具有良好的相关性(r2=0.9539)。[结论]重组细胞株GADD153-LUC细胞可快速检测环境中痕量污染物的DNA损伤效应。     

泛素水解酶22调控丝裂原活化蛋白激酶激酶6基因的转录活性

目的：克隆丝裂原活化蛋白激酶激酶6(mitogen-activated protein kinase kinase 6,MKK6)基因启动子,研究泛 素水解酶22(ubiquitin specific peptidase 22,USP22)对MKK6转录活性的调控作用。方法：采用PCR扩增MKK6基因启动 子片段,并以该片段为模板对USP22结合位点进行定点突变,将野生型与突变型启动子片段定向插入荧光素酶表达 载体pGL3-Basic;用重组载体与内参质粒pRL-TK共转染HeLa细胞,行双荧光素酶活性检测以确定其转录活性;利用 染色质免疫共沉淀(chromatin immunoprecipitation,ChIP)实验观察USP22蛋白与MKK6启动子是否存在直接的结合;下 调USP22表达后,检测MKK6转录活性的变化。结果：成功扩增MKK6启动子及其突变体并构建荧光素酶表达载体; USP22结合位点的突变导致该启动子活性在HeLa细胞中明显降低(P<0.05);USP22与MKK6启动子在细胞中存在直接结 合;抑制USP22的表达导致MKK6转录水平明显下降(P<0.05)。结论：在HeLa细胞中,USP22有效地调控MKK6基因的 转录。     

HLA-DQA1 gene expression profiling in oligoarticular JIA

Polymorphisms in the upstream regulatory region of the HLA class II DQA1 gene are currently defined by 10 different alleles. Two of them carrying a Y-box mutation are associated with susceptibility to oligoarticular juvenile idiopathic arthritis (OA-JIA). We investigated allele-dependent differences in HLA-DQA1 gene expression in OA-JIA patients. In cells from affected joints compared to peripheral blood, gene expression of HLA-DRA as well as total HLA-DQA1 was significantly upregulated. Differential analyses of HLA-DQA1 allelic expression showed DQA1*02 and *04 to be comparatively increased. Intra-articular upregulation of HLA-DQA1 was predominantly observed for the OA-JIA associated allele HLA-DQA1*04. Nevertheless, the Y-box mutation of the disease-associated allele DQA1*0401 was not a common denominator for expression behaviour.     

Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

Primary objective. Heparin-binding EGF-like growth factor (HB-EGF) protects the intestine from damage in animals. Future clinical trials of HB-EGF may involve administration of repeated doses of HB-EGF. Since HB-EGF activates EGF receptors which have been implicated in tumor development, we examined the effects of HB-EGF overexpression in the intestine.

Research design. We generated transgenic (TG) mice in which the human HB-EGF gene is driven by the villin promoter to overexpress HB-EGF along the crypt-villous axis from the duodenum to the colon.

Results. HB-EGF TG mice have increased enterocyte proliferation balanced by increased enterocyte apoptosis. Despite prolonged overexpression of HB-EGF, no evidence of intestinal hyperplasia or tumor formation occurs. Although HB-EGF TG mice have no significant phenotypic alterations under basal conditions, they have increased resistance to intestinal injury.

Conclusions. Prolonged intestinal HB-EGF overexpression results in no significant phenotypic alterations under basal conditions, but confers protection against intestinal injury.     

Bacillus spp. as direct-fed microbial antibiotic alternatives to enhance growth,immunity, and gut health in poultry

The increasing occurrence of antibiotic-resistant bacteria combined with regulatory pressure and consumer demands for foods produced without antibiotics has caused the agricultural industry to restrict its practice of using antibiotic growth promoters (AGP) in food animals. The poultry industry is not immune to this trend, and has been actively seeking natural alternatives to AGP that will improve the health and growth performance of commercial poultry flocks. Bacillus probiotics have been gaining in popularity in recent years as an AGP alternative because of their health-promoting benefits and ability to survive the harsh manufacturing conditions of chicken feed production. This review provides an overview of several modes of action of some Bacillus direct-fed microbials as probiotics. Among the benefits of these direct-fed microbials are their production of naturally synthesized antimicrobial peptides, gut flora modulation to promote beneficial microbiota along the gastrointestinal tract, and various immunological and gut morphological alterations. The modes of action for increased performance are not well defined, and growth promotion is not equal across all Bacillus species or within strains. Appropriate screening and characterization of Bacillus isolates prior to commercialization are necessary to maximize poultry growth to meet the ultimate goal of eliminating AGP usage in animal husbandry.     

Oncolytic adenoviruses for treatment of brain tumours

Standard therapies are not capable of curing patients with malignant glioma; more than 90% of patients die within 2 years after diagnosis. Gene therapy appeared as a promising new approach for this disease. However, results of clinical trials with replication deficient viral vectors were disappointing. The main reasons being poor transduction efficiency of adenovirus towards glioma cells and limited spread and distribution of the vector in the tumour. With the increasing knowledge of viral genetics and its functions, an attractive alternative tool to kill malignant glioma cells has been developed: Replicating adenovirus as an oncolytic agent. This type of therapy, also referred to as virotherapy, has the potential to overcome some of the limitations connected with replication deficient adenoviral vectors. In this review the authors describe the latest developments in strategies that are being used to create a tumour- or glioma- selective replicating adenovirus. Special attention is given to the methods of viral delivery to an infiltrating tumour in the brain, regarding optimal dose and toxicity. Furthermore, the role of conventional antitumour treatments, such as irradiation and chemotherapy, in enhancing the effect of virotherapy is being emphasised.     

A rice chloroplast transit peptide sequence does not alter the cytoplasmic localization of sheep serotonin N‐acetyltransferase expressed in transgenic rice plants

Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin‐rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N‐acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild‐type plants, but lower activity than transgenic rice plants expressing the wild‐type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild‐type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed‐specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared with seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter.     

Sanger sequencing versus INNO-LiPA® HBV PreCore assay for routine detection of precore and basal core promoter mutations in hepatitis virus B chronically infected patients

We compared the Sanger sequencing and the commercial INNO-LiPA® HBV assay for the routine detection of precore (PC) and basal core promoter (BCP) mutations of hepatitis B virus in chronically infected patients. The overall agreement rate between assays was 94.2% and 98.8% for the detection of PC and BCP mutations, respectively.