6株猪O型口蹄疫病毒VP1基因的克隆与序列分析

根据口蹄疫病毒（FMDV）VP1基因的序列，设计并合成了2对用于扩增VP1基因的引物。从组织中提取总RNA，首先用P1、P2引物对6株猪。型口蹄疫病毒进行RT—PCR扩增，获得1000bp的片段；再用P3、P4引物进行巢式PCR扩增，结果获得850bp的片段。将850bp的片段克隆到pMD18—-T载体中，通过PCR鉴定，将阳性重组质粒进行测序并分析。结果发现6株FMDV的核苷酸同源性为80．2％～99．4％，其推导的氨基酸序列同源性为86．9％～99．5％；构建遗传发生树，发现6株FMDV属于两个不同的基因型，其中的Shunde00、Sihui01、Shenzhen99、Fushan01株属一个基因型（与Hongkong93、广东86分离株属同一基因型）；Guangzhou99、Shenzhen00株属另一个基因型（与UKG-12—2001株、JPN2000株属同一基因型）。通过对口蹄疫病毒VP1基因的测序与分析，了解其变异情况，为科学地防控FMD提供分子水平的依据。     

3株新城疫病毒广西分离株L基因的克隆与序列分析

根据GenBank登陆的新城疫病毒L基因序列,设计了3对引物(L1和L2、L3和L4、L5和L6)。用RT-PCR技术对3株新城疫病毒广西分离GX7/02、GX9/03、GX11/03的L基因进行了分段扩增和克隆,并对克隆出来的3个片段进行序列测定,用DNAstar软件比较分析后进行拼接,得到长约为6.8 kb、包含有L基因全长的核苷酸序列。L基因的RNA全长为6 704 bp,拥有一个6 615 bp的开放阅读框,推导其编码的氨基酸数为2 204个。氨基酸同源性分析表明广西分离株之间同源性为98.6%~98.7%;与ZJ1株同源性为98.8%~98.9%;与La-Sota、B1、F48E9、HB92同源性为92.0%~94.2%。     

Expression of MHC Ⅱ on rat corneal keratocytes is inhibited by special siRNA targeting CⅡTA

AIM: To investigate the feasibility to inhibit the expression of MHCⅡ by special siRNA targeting class Ⅱ major histocompatibility complex (MHC Ⅱ) transactivator (CⅡTA), which might regulate MHC Ⅱexpression for suppressing immune rejection. METHODS: Five different siRNA were designed, synthesized and transfected into freshly isolated rat corneal keratocytes. At 24 hours posttransfection, the changes of MHC Ⅱexpression were detected by flowcytometry, and the mRNA abundance of CⅡTA and MHC Ⅱ was measured by FQ-PCR after inducing with recombinant rat interferon-gamma (IFN-γ). RESULTS: Different siRNA showed different reduction in MHC Ⅱ and CⅡTA expression compared with the control (P<0.01). Among the five groups, the siRNA-4 was the most efficient. The mRNA content of CⅡTA and MHC Ⅱ were reduced by 95.10%±1.25% and 82.70%±1.95% respectively and the expression of MHC Ⅱ was inhibited by over 80% in siRNA-4 group at 24 hours posttransfection. CONCLUSIONS: The special siRNA targeting to CⅡTA inhibits CⅡTA mRNA and further inhibits its regulation of MHC Ⅱ molecular expression. The blockade of MHC Ⅱ by siRNA may be useful for further studying allogeneic corneal limbal transplantation.     

猪圆环病毒2型吉林地方株ORF2基因的克隆、测序及其在原核细胞中的表达

根据GenBank中发表的猪圆环病毒2型（PCV2）ORF2基因序列，设计合成1对特异性引物，用PCR方法从接种PCV2吉林株（JL01）PK-15细胞中，扩增出PCV2毒株的ORF2基因。将扩增片段克隆于pMD18-T载体，进行序列测定。结果表明，PCV2JL01毒株的ORF2基因核苷酸长度为702bp。将重组质粒用Sal Ⅰ和Xho Ⅰ酶切后与同样处理的pET-32a载体连接，转化BL21细胞后，挑取阳性克隆。经PCR和酶切鉴定后，用IPTG诱导，细菌裂解液经SDS—PAGE和Western—blot分析，表明ORF2基因在大肠杆菌中得到了表达，并能被PCV2阳性血清所识别。     

牛瑟氏泰勒虫P33表面蛋白基因的克隆与序列分析

为分析吉林省流行的牛瑟氏泰勒虫基因序列，根据GenBank上发表的牛瑟氏泰勒虫P33表面蛋白基因序列设计合成一对引物，用PCR方法扩增出牛瑟氏泰勒虫的基因片段，并成功地将该基因纯化后克隆到pGEM-TEasy载体上，将经EcoRⅠ酶切鉴定和PCR鉴定为阳性的重组质粒进行测序。结果表明克隆的基因片段长度为868bp，编码283个氨基酸，有2个潜在的糖基化位点。核苷酸同源性分析表明，该基因片段与韩国株（AF521557）、日本株（AB016280）、俄罗斯株（AB016279）的核苷酸序列同源性分别为99．4％、88.0％、88．1％。     

Lethal effect of VEGFR2shRNA on HL60 cells in vitro using lentiviral vector

AIM: To look for harmfulless anti-leukemia drug with selective high performance, lethal effect of small hairpin RNA (shRNA) on VEGFR₂ gene expression of tumor cell line HL60 in vitro.METHODS: The most effective VEGFR₂ siRNA was designed and screened. The shRNA oligo was designed and pU6/VEGFR₂ entry clone was constructed. HL60 was transfected transiently and vascular endothelial growth factor receptor 2(VEGFR₂) expression was tested with MTT assay, RT-PCR and Western blotting. The expression clone was constructed and cotransfected with ViraPowerTM Packaging Mix into 293FTTM cells to produce Lentiviral vectors harboring Lenti6/shVEGFR₂. The virion supernatant was added into HL60 cells and VEGFR₂ gene inhibitory effect was determined. RESULTS: The inhibitory rates of VEGFR₂ siRNA c were high. VEGFR₂ expression in HL60 was inhibited by using pU6/VEGFR₂ entry clone constructed with shRNA and pENTRTM/U6. For HL60 cells, the inhibitory rate was 84.9%. The expression of VEGFR₂ mRNA and protein decreased significantly. 48 hours after transfection of pU6/shVEGFR₂ entry clone and transduction of Lenti6/shVEGFR₂ expression clone, the cell inhibitory rates were similar. Cell growth inhibitory rate of entry clone descended rapidly after this time point, the expression clone changed slowly, reaching the peak at 96 hours, dropped slightly, having no significance deviation. CONCLUSION: in vitro, VEGFR₂ shRNA using lentiviral vector blocks VEGF/VEGFR₂ self-secretion in HL60 cells, which inhibits leukemia development.     

Genotypic and phenotypic characterization of lactic acid bacteria isolated from Italian ryegrass silage

Twenty-three lactic acid bacteria (LAB) isolated from three cultivars (Akiaoba, Nagahahikari and Tachiwase) of Italian ryegrass (Lolium multiflorum Lam.) silage were precisely characterized by a combination of phenotypic tests, genotypic 16S ribosomal DNA sequencing and rapid PCR-based analyses, focusing on their useful phenotypes for silage preparation as inoculants. We successfully identified both at the species and subspecies levels: phenotypically novel Lactococcus lactis subsp. lactis, Lactobacillus brevis, Lactobacillus coryniformis subsp. torquens, Lactobacillus curvatus, Lactobacillus plantarum subsp. plantarum, Lactobacillus sakei subsp. carnosus, Leuconostoc mesenteroides subsp. dextranicum and Pediococcus parvulus. This is the first report to elucidate the presence of Lactobacillus coryniformis ssp. torquens and Leuconostoc mesenteroides subsp. dextranicum in Italian ryegrass silages. Physiological and biochemical tests revealed that phenotypic characteristics are different among the different strains of the same species and subspecies, and that the isolates show unique and diverse phenotypes related to fermentation factors, such as available carbohydrates, optimal growth pH and temperature. These results suggest that, for various well-preserved silage preparations, the isolates may be useful in producing novel inoculants corresponding to their optimally climatic and ecological niches.     

RNA干扰的研究进展

RNA干扰(RNA interference,RNAi)现象是1998年在对秀丽线虫的研究中发现的.RNAi利用双链RNA(dsRNA)特异性地降解相应序列的mRNA.从而特异性地阻断相应基因的表达.本文介绍了RNA干扰现象的发现、分子机制、生物学意义及其技术的应用发展.     

生草对关中地区有机猕猴桃园土壤养分及细菌群落的影响

为探究不同生草模式对关中地区有机猕猴桃园土壤养分及细菌群落的影响,试验设置多年生黑麦草+毛苕子（Mode 1）、多年生黑麦草+草木樨（Mode 2）、多年生黑麦草+白三叶（Mode 3）及鼠茅草（Mode 4）,以自然生草处理为对照（CK）,观察草种生长特性、研究生草对果园耕层（0~20 cm）土壤养分影响,采用高通量测序法分析细菌群落结构。结果表明：鼠茅草越冬率最高,样地杂草株数最少。人工生草较自然生草有机质提高了6.46%~38.63%,以多年生黑麦草+毛苕子效果更为明显,且该处理提高土壤脲酶、蔗糖酶和碱性磷酸酶活性最为显著,分别为3.37、44.17和3.46 mg·d^-1·g^-1。同时,与自然生草相比,多年生黑麦草+毛苕子、多年生黑麦草+草木樨和多年生黑麦草+白三叶提高了土壤细菌群落的丰富度和多样性,多年生黑麦草+毛苕子存在最多差异显著的细菌分支。综上,关中地区有机猕猴桃园种植多年生黑麦草+毛苕子在一定程度上有助于提高土壤有机质及养分含量,改善土壤微生态环境。     

全基因组SNP分型技术在畜禽遗传育种研究中的应用

随着畜禽资源分子鉴定、物种进化、全基因组育种等热点领域的逐渐兴起,准确的全基因组SNP分型成为了畜禽基因组研究的关键。基因芯片、重测序、简化基因组测序及靶向捕获测序等全基因组SNP分型技术已广泛应用于畜禽基因组研究中。本文概述了全基因组SNP分型技术的原理及其在全基因组关联分析、选择信号分析和畜禽遗传资源背景分析等方面的应用,以期为畜禽基因组研究和育种应用提供借鉴和参考。