建立以TK基因为标记将外源基因导入臭曲霉（Asper—gillus...

A new system for selection of transformed Aspergillus foetidus was reported. In this system, TK- A. foetidus which were constructed by homologous recombination of mutated TK gene of vaccinia virus with TK gene of A. foetidus were screened by adding BUdR in agar plates. Conditions for screen of TK+ A. foetidus strain, transformation of A. foetidus and selection of transformed TK- A. foetidus have been studied. By using this system, several transformed A. foetidus which contained HBsAg gene derived bf a promoter H8 cloned from genomic DNA of A. foetidus were isolated. It was demonstrated that HBsAg gene was integrated into the chromosome DNA of A. foetidus by Southern blot after many passages of spores. ELISA showed that HBsAg was positive in the growth medium (p/n = 20). The 22 nm particles which were very similar to the HBsAg particles in human serum were found in the growth medium by immunoelectromicroscope. Western blot also gave the specific bands. All these data showed that HBsAg gene was expressed in A. foetidus and the products were secreted into the growth medium. The selection system using TK gene as marker could generally be used to study the expression of foreign gene in A. foetidus.     

Draft genome sequences of six Escherichia coli isolates from the stepwise model of emergence of Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has been implicated in food-borne illnesses worldwide. An evolutionary model was proposed in which the highly pathogenic EHEC O157:H7 serotype arose from its ancestor, enteropathogenic E. coli (EPEC) O55:H7 (sorbitol fermenting [SOR(+)] and β-glucuronidase positive [GUD(+)]), through sequential gain of virulence, phenotypic traits, and serotype change. Here we report six draft genomes of strains belonging to this evolutionary model: two EPEC O55:H7 (SOR(+) GUD(+)) strains, two nonmotile EHEC O157:H(-) strains (SOR(+) GUD(+)) containing plasmid pSFO157, one EHEC O157:H7 (SOR(-) GUD(+)) strain, and one O157:H7 strain containing plasmid pSFO157 (SOR(+) GUD(+)).     

Expression,purification and characterization of anti-BAFF antibody secreted from the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

B-Cell activating factor (BAFF) is critical for B cell survival and maturation; excessive expression of it corrupts B-cell tolerance and may lead to autoimmunity. The gene, scFv-Fc, coding for the antibody of BAFF was inserted into the eukaryotic expression vector, pPICZαA, and transformed into Pichia pastoris. A high-level expression strain was obtained using a ‘yeastern blotting’ method. The scFv-Fc antibody was purified and 56 mg was obtained from 1 l of culture supernatant. It retained high binding activity to both soluble BAFF and membrane-bound BAFF.     

The expression of a novel estrogen receptor, GPR30, in epithelial ovarian carcinoma and its correlation with MMP-9

The aim of the present study is to investigate the expression of a novel estrogen receptor, G protein-coupled receptor 30 (GPR30) and its correlation with matrix metalloproteinases-9 (MMP-9) in epithelial ovarian cancer (EOC). Ovary tissues were obtained from 39 female patients, including 30 cases of EOC and 9 cases of benign ovarian tumor. Four normal ovary tissues were used as control. Immunohistochemical staining was used to detect the expressions of GPR30 and MMP-9. Chi square test, Fisher's exact test and Spearman's rank correlation analysis were used for statistical analysis. The results showed that GPR30 overexpression rate in EOC cases was significantly higher than those in benign ovarian tumor and normal ovary cases. Whereas MMP-9 overexpression rate in EOC cases was significantly higher than that in normal ovary cases, without any difference to that in benign ovarian tumor cases. To demonstrate the relationship between GPR30 and clinicopathological variables of EOC, we further analyzed the pathology type, FIGO stage and age of patients sampled in our study. The analysis showed there were significant differences of GPR30 overexpression rate among various pathology types and different FIGO stages (P<0.05), and no significant difference of both GPR30 and MMP-9 among three age groups (P>0.05). Moreover, GPR30 expression was positively correlated with MMP-9 (r(s)=1.000, P=0.002). These results suggest that GPR30 may be involved in the invasion and metastasis of EOC, being a potential index of EOC early diagnosis and malignancy grade prediction.     

周围神经损伤后外源性GKNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型,局部给予胶质细胞源性神经营养因子（ＧＤＮＦ）,应用尼氏染色、酶组织化学染色方法,观察到外源性ＧＤＮＦ能减少脊髓修复侧前角运动神经元死亡的数目,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶（ＣＨＥ）及酸性磷酸酶（ＡＣＰ）变化的幅度。这表明外源性ＧＤＮＦ能保护周围神经切断后引起的神经元损伤．     

丙型肝炎病毒RNA打点杂交检测方法同RT－PCR方法的比较

采用ＨＣＶ基因组结构区Ｃ区ｃＤＮＡ探针和非结构区ＮＳ３－４区ｃＤＮＡ探针,建立了用打点杂交（ｄｏｔｂｌｏｔｈｙｂｒｉｄｉｚａｔｉｏｎ）检测血清中ＨＣＶＲＮＡ的方法,同采用ＨＣＶ基因组５’端非编码区的一对寡核苷酸引物通过逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）检测血清中ＨＣＶＲＮＡ的方法相比较,发现两种方法都能快速早期和特异地检出血清中ＨＣＶＲＮＡ,但ＲＴ－ＰＣＲ法敏感性优于ＲＮＡ打点杂交法。对于无血清学指标的慢性ＮＡＮＢ肝炎病人的诊断,可采用这两种方法。这两种方法的敏感性在很大程度上依赖于引物和探针的敏感性,以及ＲＮＡ提取方法。ＲＴ－ＰＣＲ法适用于诊断病毒血症和复制,打点杂交法适用于研究ＨＣＶＲＮＡ量的变化,对治疗的评价,以及为实验筛选较高滴度的ＨＣＶＲＮＡ阳性样本。     

长期施肥对黑土大豆根瘤菌群体结构和多样性的影响

为揭示长期施肥对黑土大豆根瘤菌群体结构和多样性的影响,采用BOX-PCR、IGS-PCR-RFLP和16S r DNA基因序列分析法,对分离自黑龙江省7种长期不同施肥处理的254株大豆根瘤菌进行了遗传多样性和系统发育分析,结合土壤理化性质分析了大豆根瘤菌群体结构和多样性与土壤因子间的关系。7种处理分别为不施肥(CK)、有机肥(OM)、单施氮肥(N1)、单施2倍氮肥(N2)、氮肥+有机肥(N1+OM)、氮肥磷肥混施(N1P1)和2倍氮肥磷肥混施(N2P2)。系统发育分析结果表明,所有供试菌株均为慢生根瘤菌属(Bradyrhizobium),其中大部分菌株与日本慢生大豆根瘤菌(Bradyrhizobium japonicum)相似性最高,少部分菌株与辽宁慢生大豆根瘤菌(Bradyrhizobium liaoningense)相似性最高。BOX-PCR聚类分析结果表明,供试菌株在70%相似性水平上分为15个群,在与施肥处理相关性分析中分为3个群体,分别对应于不施化肥处理(CK和OM)、化学氮肥处理(N1、N2、N1+OM)、氮肥磷肥处理(N1P1和N2P2)。典范对应分析结果表明,土壤p H、速效氮和速效磷与根瘤菌群体结构相关性极显著(P=0.002,0.004,0.002)。不同施肥措施下大豆根瘤菌的多样性有明显差异:N2P2处理的丰富度指数和Shannon-Wiener指数显著高于其他处理;OM处理的Simpson指数最高;N1和N2处理的3种多样性指数都显著低于其他处理。通径分析结果表明,p H、速效磷对多样性指数有较高的直接正效应;速效氮通过p H的间接负效应影响多样性指数。本研究表明,长期施用化肥改变了根瘤菌群体结构,单施氮肥减少大豆根瘤菌多样性,而氮肥磷肥混施则有助于提高大豆根瘤菌多样性。     

红松阔叶林倒木贮量动态的研究

在森林倒木研究的基础上探讨长白山红松阔叶林倒木贮量的动态,涉及红松阔叶林倒木分解及其贮量的动态规律。研究表明,倒木分解,除心腐木外,均由表及里进行;倒木分解速率在其它生态条件相同时因树种、直径和部位而异。红松阔叶林倒木贮量动态包括现有倒木贮量和倒木年输入量两个分解动态过程,现有倒木贮量在头100年其干重迅速减少,其中椴树比红松尤速,前者分解91%,后者为72%.林地倒木贮量动态与倒木年输入量分解动态相似,但前者在分解初期贮量增加较大,因为部分现有倒木未完全分解;100年后趋于一致,并恒定于16～17t·hm^-2,直至群落的顶极阶段结束.     

Connective tissue growth factor and cardiac fibrosis after myocardial infarction.

The temporal and spatial expression of transforming growth factor (TGF)-beta(1) and connective tissue growth factor (CTGF) was assessed in the left ventricle of a myocardial infarction (MI) model of injury with and without angiotensin-converting enzyme (ACE) inhibition. Coronary artery ligated rats were killed 1, 3, 7, 28, and 180 days after MI. TGF-beta(1), CTGF, and procollagen alpha1(I) mRNA were localized by in situ hybridization, and TGF-beta(1) and CTGF protein levels by immunohistochemistry. Collagen protein was measured using picrosirius red staining. In a separate group, rats were treated for 6 months with an ACE inhibitor. There were temporal and regional differences in the expression of TGF-beta(1), CTGF, and collagen after MI. Procollagen alpha1(I) mRNA expression increased in the border zone and scar peaking 1 week after MI, whereas collagen protein increased in all areas of the heart over the 180 days. Expression of TGF-beta(1) mRNA and protein showed major increases in the border zone and scar peaking 1 week after MI. The major increases in CTGF mRNA and protein occurred in the viable myocardium at 180 days after MI. Long-term ACE inhibition reduced left ventricular mass and decreased fibrosis in the viable myocardium, but had no effect on cardiac TGF-beta(1) or CTGF. TGF-beta(1) is involved in the initial, acute phase of inflammation and repair after MI, whereas CTGF is involved in the ongoing fibrosis of the heart. The antifibrotic benefits of captopril are not mediated through a reduction in CTGF.