Inhibition of porcine circovirus type 1 and type 2 production in PK-15 cells by small interfering RNAs targeting the Rep gene

Porcine circovirus type 1 (PCV1) and type 2 (PCV2) are two genotypes of porcine circovirus. Both of them are presumed to be widespread in the swine population. Currently, there is no specific treatment for their infections. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), which represents a possible therapeutic application for the treatment of viral infections. In this study, three siRNA expression plasmids (pS-RepA, pS-RepB and pS-RepC) were generated to target three different coding regions of the Rep protein (Rep) of PCV. These siRNAs were used to inhibit PCV production in a porcine kidney cell line, PK-15 cells. Our results revealed that Rep gene expression was inhibited by pS-RepA, pS-RepB and pS-RepC to different degrees. Moreover, our study also showed that the production of PCV1 and PCV2 was reduced by these siRNAs. pS-RepC, which targets the middle region of Rep gene, proved to be the most efficient siRNA for inhibition of Rep expression and viral production. Taken together, our data suggest that RNAi could be investigated as a potential treatment for PCV infection.     

Evaluation of immunogenicity and protective efficacy of Actinobacillus pleuropneumoniae HB04C(-) mutant lacking a drug resistance marker in the pigs

Previously, we reported the construction and characterization of a genetically defined Actinobacillus pleuropneumoniae (A. pleuropneumoniae) apxIIC gene mutant, HB04C(-), which conferred protection to mice against infection with A. pleuropneumoniae. In this study, we further evaluated HB04C(-) for safety and its ability to elicit protective immunity in pigs. It was demonstrated that a dose of 2 x 10(8) CFU HB04C(-) was safe to the pigs via intranasal or intramuscular injection. Immunization with a dose of 2 x 10(8) HB04C(-) by both intranasal and intramuscular routine could yield equal protective efficacy and elicited significant protection against experiment challenge with homologous or heterologous serotypes of a virulent A. pleuropneumonia. Taken together, HB04C(-) might serve as a promising vaccine candidate against infection with A. pleuropneumoniae.     

特色蔬菜芥蓝高产栽培技术

芥蓝属十字花科芸薹属甘蓝类蔬菜,为一年生或二年生草本植物,以食用菜薹为主,是中国广东的特色蔬菜。芥蓝以蔬菜基地种植为主。在广东,芥蓝主要种植区域集中在惠州、广州花都、增城、从化、清远这几个地方的蔬菜基地。     

副猪嗜血杆菌的分离培养和血清型鉴定

从全国十多个省市送检的疑似患多发浆膜炎与关节炎猪的病料中分离到32株细菌,进行了细菌形态观察、培养特性和生化特性鉴定;根据副猪嗜血杆菌16S rRNA序列设计引物进行PCR扩增,将822bp扩增片段连入T-载体后测序,再与GenBank(M75065)中的序列进行比对,表明其与国外副猪嗜血杆菌菌株16S rRNA序列的同源性为97％以上,确定为副猪嗜血杆菌(Haemophilus parasuis,Hps)。将其中的15株按Kieletein-Rapp-Gabriedson(KRG)琼脂扩散血清分型方法进行血清型鉴定,结果为血清5型3株、血清4型4株、血清13型2株、血清12型2株,另外有4株不能进行分型。该结果表明副猪嗜血杆菌在我国广泛存在。     

姬松茸固体饮料的研究

姬松茸是一种正待开发的药食兼用真菌,因其含有抗肿瘤活性多糖而备受关注。利用现代食品工程高新技术———超微粉技术将姬松茸子实体粉碎,使其组织细胞高度破坏,提高了多糖的溶出率。通过正交实验确定姬松茸固体饮料配方为蔗糖量10%,柠檬酸,糖酸比为33.3∶1。     

垄膜沟播与秸秆还田对内蒙古黄土高原玉米农田土壤水分、酶活性及产量的影响

为明确全覆膜垄膜沟播种植和秸秆还田措施下玉米农田土壤保水改土效果及增产效应,在内蒙古黄土高原旱作区对比分析了全覆膜垄膜沟播结合秸秆还田（S+M）、秸秆还田（S）、全覆膜垄膜沟播（M）、秸秆不还田不覆膜（CK）4种不同耕作方式下土壤水分、酶活性变化规律及玉米产量形成特性。结果表明：全覆膜垄膜沟播种植和秸秆还田均能提高玉米各生育阶段0—100 cm土层土壤含水量,尤以0—20 cm土层土壤含水量差异最为显著,且全覆膜垄膜沟播种植提升效果优于秸秆还田,两者耦合后土壤含水量表现为最大;垄膜沟播和秸秆还田均可显著提高土壤过氧化氢酶、脲酶、蔗糖酶和碱性磷酸酶活性,且随玉米生育期推移,土壤酶活性呈先增加后降低趋势,在大喇叭口期达到峰值;同时,垄膜沟播和秸秆还田均能提高玉米干物质量和产量,由高到低次序为S+M＞M＞S＞CK,与CK相比,S、M、S+M处理玉米产量分别提高11.84%、37.83%、45.13%,全覆膜垄膜沟播结合秸秆还田明显优于其他处理,可作为内蒙古黄土高原旱作区玉米节水增效增产栽培模式。     

基于西门利克森林病毒复制子的"自杀性"DNA疫苗载体的构建及其体外表达特性

对西门利克森林病毒(SFV)复制子质粒型表达载体pSfV1进行改造，即在SFV非结构蛋白(nSPs)上游插入依赖于RNA聚合酶Ⅱ的人巨细胞病毒立即早期启动子／增强子序列(hCMV IE Pro／Enh)，同时在pS-FV1的多克隆位点下游插入SV40 poly(A)终止信号序列，获得可以完全在DNA水平上操作并具有自主复制功能的表达载体pSFV-DNA。为了进一步探讨改造载体的转染效率、表达能力以及是否能诱导细胞凋亡等特性，将报告基因EGFP插入pSFV-DNA中亚基因组启动子下游，构建的重组表达质粒pSFV-EGFP转染BHK-21细胞，转染后12h即可观察到荧光，但转染效率明显低于直接由hCMV IE Pro／Enh驱动的EGFP真核表达质粒pEGFP-C1；提取转染后48h的细胞基因组DNA，电泳发现pSFV-EGFP转染细胞均呈典型的DNA断裂(ladder)，而pEGFP-C1转染细胞未观察到这种现象。上述结果表明：改造的载体不仅可完全在DNA水平上操作，而且能正确表达外源基因和诱导细胞凋亡，为开展各种病原微生物的“自杀性”DNA疫苗研究提供了良好的工具。     

带状留茬间作减轻旱作农田土壤风蚀的生态效应研究

针对阴山北麓农牧交错带干旱少雨,风大风多,风蚀沙化严重的现状,深入分析了影响农田风蚀沙化的各种因素,研究提出了防沙型带状留茬间作轮作技术。结果表明:采用带状留茬间作轮作技术,冬春风蚀季节可以降低留茬带风蚀量幅度基本为68.5%～88.7%,由于留茬带的保护作用,降低间作裸露带风蚀量幅度为42.4%～68.3%。在减轻风蚀的同时,带状留茬间作轮作保持了由于秋季耕翻松土的多蓄降水作用,降低了冬春季节近地面风速和水分蒸发,同时还拦截了冬雪,播种时有留茬带保护的间作裸露带0～1m土层贮水量提高15～41.5mm。由于带状留茬间作轮作的集水、保墒和边际效应,增产作用明显,增产幅度为12%～75%。     

Development of enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection of antibodies to avian influenza virus

During the avian influenza outbreak of 2003-04 in Southeast Asia, two avian influenza viruses (AIV), one of H5N1 subtype and the other H9N2 subtype, were isolated and identified from local farms. The nudeoprotein (NP) gene of the H5N1 AI isolate was cloned, and the segment encoding amino acid 47-384, which covers its major antigenic domains, was subcloned and expressed in E. coli. Subsequently, the NP (47-384) expression product was purified and used as the diagnostic antigen to develop a NP-based type-specific indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to AI from chicken sera. The ELISA is shown to be specific for AIV and does not cross-react with chicken sera that has antibodies to other avian viruses. The NP(47-384)-ELISA was compared with a hemagglutination inhibition test and a commercial AIV ELISA kit in evaluating 150 sera samples from experimentally AIV-infected or vaccinated specific-pathogen-free (SPF) chickens. Our NP(47-384)-ELISA was more sensitive than the two tests and showed an 82% agreement ratio with the HI test and an 80.67% agreement ratio with the commercial kit. The NP(47-384)-ELISA and the commercial AIV ELISA were used to evaluate 448 field sera samples from diseased chickens or vaccinated chickens during the 2003-04 AI outbreak in China. The two ELISA tests had a 95% agreement ratio. We conclude that the NP(47-384)-ELISA developed in our laboratory was specific and sensitive and it has great application potential in China's long-term prevention and control of AI.