人乳头瘤病毒特异性T细胞系细胞冻存后细胞的存活率及功能

研究人乳头瘤病毒特异性T细胞系细胞冻存后细胞的存活率及功能。应用包含10%二甲基亚砜、90%小牛血清的冻存液冻存6个T细胞系(5个CD4 T细胞系,1个CD8 T细胞系)细胞,液氮中冻存32~54个月后复苏,台盼蓝染色法检测复苏后T细胞系细胞的存活率,用酶联免疫斑点法(enzyme-linked immunospot assay,ELISPOT)检测复苏后T细胞系细胞的功能。结果显示,6个T细胞系细胞液氮冻存解冻后细胞的存活率为24.7%~93.5%,过夜培养后细胞的存活率为2.5%~72.2%。CD8 T细胞系细胞的存活率高于CD4 T细胞系细胞。6个复苏后的T细胞系细胞在PHA诱导后均能分泌IFN-γ。人乳头瘤病毒特异性T细胞系细胞冻存复苏后能够保持较好的存活率和功能。     

人MDM2基因真核表达载体的构建及其与p53的相互作用

目的:构建带Flag标签的MDM2真核表达载体,并检测MDM2与p53的相互作用。方法:从人乳腺文库中PCR扩增MDM2编码序列,将其插入pcDNA3.0-Flag载体,转染293T细胞后用Western印迹检测其在293T细胞中的表达,并通过免疫共沉淀实验检测MDM2与p53的相互作用。结果:双酶切和测序结果表明,Flag-MDM2真核表达载体构建成功,转染293T细胞后成功表达;免疫共沉淀实验证明Flag-MDM2与p53存在相互作用。结论:构建了带Flag标签的人MDM2真核表达载体,并检测了MDM2与p53之间的相互作用,为研究MDM2的功能奠定了基础。     

Evolutionarily-conserved gene CKAP2,located in region 13q14.3 of the human genome, is frequently rearranged in various tumors

The human CKAP2 gene, which is involved in diffuse large B-cell lymphomas, was localized via screening the GeneBridge 4 somatic cell radiation hybrid panel by means of the polymerase chain reaction (PCR). The CKAP2 gene was mapped between the WI-15460 and WI-3673 markers at the boundary between regions 13q14.3 and 13q21.1, at the distance of 14.39 cR (with 4.8 cR per cM) from the WI-5867 framework marker (lod score > 2.26). The human CKAP2 gene displayed high homology to mouse and rat expressed orthologs, A CKAP2-like sequence was found in human chromosome 14 and assumed to be a pseudogene resulting from duplication and subsequent mutations of the CKAP2 gene on chromosome 13. A possible role of the CKAP2 gene in oncogenesis associated with deletions and rearrangements of region 13q14.3-21.1 is discussed.     

Genetic Mechanisms of Resistance and Susceptibility to Leukemia in Ayrshire and Black Pied Cattle Breeds Determined by Allelic Distribution of Gene BoLA-DRB3

In the herds of Ayrshire and Black Pied cattle breeds of Russian selection, comparative analysis of allelic distribution of BoLA-DRB3 was performed in animal groups with different status of persistent lymphocytosis (PL) caused by the bovine leukemia virus (BLV). Alleles were typed by PCR–RFLP. Different spectra of BoLA-DRB3 alleles mediating susceptibility and resistance to leukemia were detected in the studied breeds. The role of amino acid motives in 1 domain of BoLA-DRB3 antigens was confirmed: ER (in positions 70–71), in resistance to leukemia and VDTY and VDTV (75–78), in susceptibility to leukemia. The nucleotide sequence of allele BoLA-DRB3.2*7with deletion of codon 65, which resulted in the changed conformation of the corresponding antigen molecule, was associated with resistance to PL. Cows of Black Pied and Ayrshire breeds with genotypes coding VDTY/VDTV (RR = 11.67, P = 0,014) and VDTY/VDTY (RR = 4.71, P = 0.022), respectively, were shown to be susceptible to PL. The role of heterozygosity level was demonstrated (estimated by BoLA-DRB3 alleles and by amino acid motives in positions 75–78 of the antigen) as an unspecific factor of resistance to PL. The lowest heterozygosity level by amino acid motives (75–78) was revealed in PL animals, for which sample inbreeding coefficients were detected: F = 0.324 and 0.084 in Ayrshire and Black Pied breeds, respectively.     

An Arabidopsis embryonic lethal mutant with reduced expression of alanyl—t RNA synthetase gene

In present paper,one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant.The embryo developmant of this mutant is arrested in globular stage,The cell division pattern is abnormal during early embryogenesis and results in distubed cellular differentiation.Most of mutant embryos are finally degenerated and aborted in globular stage,However,a few of them still can germinate in agar palte and produce seedlings with shoter hypoctyl and distorted shoot meristem.To understand the molecular basis of the phenotype of this mutant,the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening.According to the sequence analysis and similarity searching,a 936 bp cDNA sequence(EMBL accession #:Y12555)from selectoed positive clone shows a 99.8%(923/925bp) sequence homolgy with Alanyl-tRNA Synthetase(AlaRS) gene of Arabidopsis thaliana.Furthermore,the data of in situ hybridization experiment indicate that the expression of Ala RS gene is weak in early embryogenesis and declines along with globular embryodevelopment in this mutant Accordingly,the reduced expression of Ala RS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.     

用杆状病毒-昆虫细胞系统表达、纯化重组的人源化氨基末端LBP

根据已有的资料显示LBP与LPS的结合位点位于其氨基末端的第 91～ 1 0 0个氨基酸残基。在体外构建含有人LBP与LPS结合活性部分的穿梭质粒pBacmidtLBP ,并且带上 6×his的标签 ,用此穿梭质粒转染sf2 1昆虫细胞 ,获得重组病毒。然后用重组病毒液感染对数生长期的sf2 1昆虫细胞 ,72h后收获含有这种截断型人源化氨基末端LBP-(NH-LBP)的培养上清。用金属亲和树脂纯化后 ,采用SDS-PAGE电泳以及Western-Blot鉴定所得到的纯化物。成功获得相对分子质量约为 30 0 0 0的NH-LBP。为进一步研究LBP在介导LPS活化靶细胞中的作用机制奠定了基础。     

转Bt基因作物对非靶标土壤动物的影响

转Bt基因作物已经在世界范围内广泛种植.随着转基因作物的快速发展与推广,有必要深入研究其对土壤生态系统的影响.本文概述了转Bt作物对土壤动物群落以及蚯蚓、线虫、虫兆虫、螨类和甲虫等重要类群的种群动态影响的研究进展,介绍了转Bt基因作物的发展历史,分析了Bt蛋白进入土壤的途径及其在土壤环境中的残留与降解的动态,阐述了未来转Bt基因作物对非靶标土壤动物影响的生态风险分析的重要领域,旨在为研究转Bt基因作物对非靶标土壤动物影响提供参考.