流行性出血热循环免疫复合物组分分析

应用ＳＤＳ－聚丙烯酰胺凝胶电泳及免疫转印技术对流行性出血热患才血清中免疫合物组分进行了分析。流行性出血热循环免疫复合物经ＳＤＳ－ＰＡＧＥ分离,考马斯亮兰染色,显色主要有７条带,分子量分别为２３ｋＤ,５０ｋＤ,５２ｋＤ,６５ｋＤ,７２ｋＤ,８０ｋＤ及１００ｋＤ。采用该病毒特异性抗血清、单克隆抗体以及人免疫球蛋白、补体成分抗血清识别,在其特环免疫复合物中可检出特异性病毒抗在及相应的免疫球状蛋白和补体成     

薄层扫描法测定绞股蓝中人参皂甙Rb_1

用高效薄层扫描法测定了绞股蓝中人参皂甙Ｒｂ_１的含量,并进行分离、纯化,再用酸解法水解,测得其结合糖为葡萄糖。     

A Selective Medium for the Isolation and Quantification of Bradyrhizobium japonicum and Bradyrhizobium elkanii Strains from Soils and Inoculants

The ecological examination of members of the family Rhizobiaceae has been hampered by the lack of a selective medium for isolation of root nodule bacteria from soil. A novel non-antibiotic-containing medium has been developed which allows selective isolation of Bradyrhizobium japonicum and B. elkanii strains from soil and inoculants. The medium, BJSM, is based on the resistance of B.japonicum and B. elkanii strains to more than 40 μg of the metals ions Zn²⁺ and Co²⁺ per ml. BJSM does not allow growth of Rhizobium sp. strains. We used BJSM to isolate bacteria from a Hubbard soil and from several commercially prepared soybean inoculants. Ninety-eight percent of the isolates obtained from Hubbard soil nodulated Glycine max cv. Kasota, and between 55 and 95% of the isolates from the commercial inoculants had the ability to nodulate soybeans. Numbers of bradyrhizobia obtained by using BJSM, strain-specific fluorescent antibodies, and the most-probable-number plant infection assay indicated that the three techniques were comparable in quantifying B. japonicum strains in soils and inoculants, although most-probable-number counts were generally 0.5 order of magnitude greater than those obtained by using BJSM. Results of our studies indicate that BJSM is useful for direct isolation and quantification of B. japonicum and B. elkanii from natural soils and inoculants. This medium may prove to be an important tool for autecological and enumeration studies of diverse populations of bradyrhizobia and as a quality control method for soybean inoculants.     

Hypomethylated sequences: Characterization of the duplicate soybean genome

Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor. In this study, we used hypomethylated genomic DNA as a source of probes to investigate the genomic structure and methylation patterns of duplicated sequences. Forty-five genomic clones from Phaseolus vulgaris and 664 genomic clones from Glycine max were used to examine the duplicated regions in the soybean genome. Southern analysis of genomic DNA using probes from both sources revealed that greater than 15% of the hypomethylated genomic regions were only present once in the soybean genome. The remaining ca. 85% of the hypomethylated regions comprise duplicated or middle repetitive DNA sequences. If only the ratio of single to duplicate probe patterns is considered, it appears that 25% of the single-copy sequences have been lost. By using a subset of probes that only detected duplicated sequences, we examined the methylation status of the homeologous genomes with the restriction enzymes MspI and HpaII. We found that in all cases both copies of these regions were hypomethylated, although there were examples of low-level methylation. It appears that duplicate sequences are being eliminated in the diploidization process. Our data reveal no evidence that duplicated sequences are being silenced by inactivation correlated with methylation patterns.     

The EBNA-2 arginine-glycine domain is critical but not essential for B-lymphocyte growth transformation; the rest of region 3 lacks essential interactive domains.

Since deletion of region 3 (amino acids [aa] 333 to 425) of Epstein-Barr virus nuclear protein 2 (EBNA-2) results in EBV recombinants which cannot transform primary B lymphocytes (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991), the role of domains of region 3 was investigated. Deletion of the Arg-Gly repeat domain, R-337GQSRGRGRGRGRGRGKG354, results in EBV recombinants that transform primary B lymphocytes with modestly decreased activity. The transformed cells grow slowly and are difficult to expand. EBNA-2 deleted for the Arg-Gly domain does not associate with the nuclear chromatin fraction. The Arg-Gly repeat has an intrinsic ability to bind to histone H1, to other proteins, including EBNA-1, and to nucleic acids, especially poly(G). Two independent deletions of each part of the rest of region 3 (aa 359 to 383 and 385 to 430) have little effect on transformation, while deletion of the rest of region 3 (aa 361 to 425) as a single segment substantially reduces transformation efficiency. EBNA-2 deleted for all of region 3 can still transactivate the LMP1 promoter in transient expression assays but is less active than EBNA-2 in transactivating the BamHI-C promoter. EBNA-2 deleted for the Arg-Gly domain is better than EBNA-2 at transactivating the LMP1 promoter and is as active as EBNA-2 in transactivating the BamHI-C promoter. These data are most compatible with a model in which the Arg-Gly domain of region 3 is a modulator of EBNA-2 interactions and activities, while the rest of region 3 is important in positioning the region 2 J kappa binding domain relative to the region 4 acidic transactivating domain. Despite the null phenotype of the region 3 deletion, region 3 is unlikely to mediate essential interactions with other proteins.     

基因工程链激酶(r—SK)纯化及其单克隆抗体制备和鉴定

采用Rotofor等电聚焦和分子筛技术纯化大肠杆菌表达的重组链激酶(r—SK),其纯度为97%。比活性1×10⁶IU／mg,回收率41％。分析所纯化的r—SK　N端氨基酸顺序证实其1—15个氨基酸顺序与SK基因的核苷酸顺序所示3联密码子一致。以纯化r—SK为抗原,通过杂交瘤技术构建了分泌抗r—SK单克隆抗体(McAb)杂交瘤细胞(4D11),该McAb属IgG1亚型．特异地作用于r-SK和C组口溶血性链球菌分泌的SK。用底物显色法测定该McAb可抑制SK对纤溶酶原的激活。Western blot法证实该McAb能识别分子量为16 250Da的r—SK的CNBr裂解片段。推测SK蛋白中第71残基至第237残基间的氨基酸顺序参与SK与纤溶酶原的结合。     

转译优化对EPO基因在COS7细胞中表达的影响

利用ＣＯＳ７细胞暂时表达系统,研究转译起始序列对ＥＰＯ－ｃＤＮＡ表达的影响。通过ＤＮＡ重组技术,构建了原ＥＰＯ－ｃＤＮＡ表达载体ｐＣＳＶ－ＥＰＯ（１）,其转译起始序列为５＇ＡＡＴＴＣＡＴＧＧ３＇。同时通过定点突变技术,将起始序列改变成５＇ＣＣＡＣＣＡＴＧＧ３＇,而构建了另一表达载体ＰＣＳＶ－ＥＰＯ（２）。后经序列分析证明无误后和前均通过ＤＥＡＥ－ｄｅｘｔｒａｎ法转染ＣＯＳ７细胞上清,测定结果为     

抗人PAI—1单克隆抗体制备,鉴定和应用

以重组PAI-(rPAI-1)为抗原,通过杂交瘤技术获得6株阳性杂交瘤细胞(AP1、AP2、AP3、AP4、AP5和AP6),并用SPA亲和层析纯化了抗PAI-1单克隆抗体(McAb)。所有McAb均能识别rPAI和天然PAI-1,腹水滴度均为10~6以上。6种McAb对PAI-1亲和常数在3.45×10~7—1.05×10~9mol/L之间。AP2、AP3 McAb能完全抑制PAI-1活性,AP4、AP和AP6只能部分抑制PAI-1活性,AP1则不抑制PAI-1活性。6种McAb中只有AP1、AP4和AP5能识别PAI-1/t-PA复合物。利用AP1、AP3、和AP4 McAb制备免疫亲和柱一步纯化HepG_2细胞分泌的PAI-1,纯度大于98％,回收率92％,纯化倍数51倍。利用抗PAI-1 McAb建立了夹心法ELISA,测定了人血浆PAI-1水平,正常人血浆PAI-1平均含量(±D)为24.7±7.75ng/ml。     

云南阿昌族的红细胞血型分布

调查了１０２名云南阿昌族的ＡＢＯ,ＮＮＳｓ,Ｒｈ和Ｐ系统的红细胞血型,结果表明,阿昌族的基因频率ｐ（０．３８７４）是迄今国内调查过人群中的最高值,Ｅ（０．２４５９）和ＣＤｅ（０．６９３６）基因或染色体频率较高,而Ｓ（０．０６８６）和Ｐ１（０．１０８９）频率较低;Ｍｓ（０．５９５０）连锁率高于Ｎｓ（０．３３５３）;未发现ＳＳ和Ｒｈ（－Ｄ）阴性表现型。