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.     

Human variants of O6-alkylguanine-DNA alkyltransferase

This article summarizes the current understanding of known variant forms of the MGMT gene that encode an altered protein. Epidemiological studies have been carried out to test whether these alterations are associated with altered cancer risk. Laboratory studies using recombinant proteins and cells expressing the known variants have investigated the possible effects of these sequence alterations on the ability of the encoded O(6)-alkylguanine-DNA alkyltransferase protein to protect cells from alkylation damage and to respond to therapeutic inactivators currently undergoing trials for cancer chemotherapy.     

Insights into the solution structure of human deoxyhemoglobin in the absence and presence of an allosteric effector

We present a nuclear magnetic resonance (NMR) study in solution of the structures of human normal hemoglobin (Hb A) in the deoxy or unligated form in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using 15N-1H residual dipolar coupling (RDC) measurements. There are several published crystal structures for deoxyhemoglobin A (deoxy-Hb A), and it has been reported that the functional properties of Hb A in single crystals are different from those in solution. Carbonmonoxyhemoglobin A (HbCO A) can also be crystallized in several structures. Our recent RDC studies of HbCO A in the absence and presence of IHP have shown that the solution structure of this Hb molecule is distinctly different from its classical crystal structures (R and R2). To have a better understanding of the structure-function relationship of Hb A under physiological conditions, we need to evaluate its structures in both ligated and unligated states in solution. Here, the intrinsic paramagnetic property of deoxy-Hb A has been exploited for the measurement of RDCs using the magnetic-field dependence of the apparent one-bond 1H-15N J couplings. Our RDC analysis suggests that the quaternary and tertiary structures of deoxy-Hb A in solution differ from its recently determined high-resolution crystal structures. Upon binding of IHP, structural changes in deoxy-Hb A are also observed, and these changes are largely within the alpha1beta1 (or alpha2beta2) dimer itself. These new structural findings allow us to gain a deeper insight into the structure-function relationship of this interesting allosteric protein.     

Association between expression of reproductive seasonality and alleles of melatonin receptor 1A in goats

To determine whether a link exists between reproductive seasonality and the structure of the melatonin receptor 1A (MTNR1A) gene, the latter was studied in year-round estrous breeds (Jining Grey and Boer goats) and seasonal estrous breeds (Liaoning Cashmere, Inner Mongolia Cashmere, Wendeng milk and Beijing native goats). A large fragment of exon 2 of MTNR1A gene was amplified by PCR using sheep sense and antisense primers in 260 does of six breeds. The uniform 824 bp PCR product was digested with restriction endonucleases MnII and RsaI, and checked for the presence of restriction sites. No polymorphism at the MnII cleavage sites was detected in all six goat breeds and no relationship could be established between the MnII cleavage sites of MTNR1A gene and reproductive seasonality in goats. For polymorphic RsaI cleavage site at base position 53, only genotype RR (267 bp/267 bp) was detected in Jining Grey goats, both genotype RR and genotype Rr (267 bp/320 bp) were found in all other goat breeds, no genotype rr (320 bp/320 bp) was detected in all six goat breeds. Frequency of genotype RR was obviously higher, and frequency of genotype Rr was obviously lower in year-round estrous goat breeds than in seasonal estrous goat breeds. Sequencing revealed one mutation (G52A) in genotype Rr compared with genotype RR. For polymorphic RsaI cleavage site, the differences of genotype distributions were significant (P<0.05) between year-round estrous goat breeds and seasonal estrous goat breeds. These results preliminarily showed an association between genotype RR and year-round estrus in goats, and an association between genotype Rr and seasonal estrus in goats.     

慢性阻断血管紧张素Ⅱ1型受体不能完全防止模拟失重大鼠血管的适应性结构变化

我们以前的工作提示,在模拟失重所引起的血管区域特异性适应变化中,局部肾素．血管紧张素系统（local reninangiotensin system,L-RAS）可能发挥关键调控作用。本文以losartan慢性阻断血管紧张素Ⅱ1型受体（angiotensin Ⅱtypelreceptor,AT1R）,观察模拟失重是否仍能引起血管的这种适应性改变,并检测大血管管壁L-RAS主要成分的表达是否也发生相应变化。以尾部悬吊大鼠模型模拟失重的生理影响。制作基底动脉、胫前动脉、颈总动脉和腹主动脉的HE染色切片,在光学显微镜下进行形态观测：用免疫组织化学技米测量颈总动脉和腹主动脉壁的血管紧张素原（angiotensinogen,AGT）及AT-R的表达变化。结果表明：4周模拟失重引起大鼠基底动脉中膜和颈总动脉管壁各平滑肌肌层肥厚,而胫前动脉和腹主动脉则发生萎缩性改变;给予losartan4周引起上述4种血管皆发生萎缩性变化;阻断AT1R,模拟失重仍然能引起基底动脉、颈总动脉发生相对肥厚性改变和腹主动脉萎缩加重。4周模拟失重还引起颈总动脉壁中AGT和AT1R表达上调,而腹主动脉壁及血管周围组织中AGT和AT1R表达下调;给予losartan4周仅引起腹主动脉壁中AGT和AT1R表达减少;阻断AT1R,模拟失重使腹主动脉壁AT1R表达进一步减少。结果提示,4周模拟失重引起大鼠脑、颈部与后身大、中动脉血管的形态结构改变和L-RAS主要成分表达发生上调或下调,血管L-RAS在其中可能发挥关键性调控作用;但在慢性阻断AT1R的条件下,其它调控机制仍可能在脑血管适应性调节中发挥一定作用。     

An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization

Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. However, the solubility of TEV protease expressed in Escherichia coli is extremely low. In the present study, we introduced a more efficient system to improve and facilitate the soluble production of TEV protease in E. coli. Optimal expression of soluble His6-TEV was achieved by examining the contribution of chaperone co-expression and lower temperature fermentation. When further purified by Ni(2+) affinity chromatography, 65mg of His6-TEV was isolated with purity over 95% from 1L of culture. The enzyme activity of His6-TEV was generally characterized by using GST-EGFP and His6-L-TNF fusion protein as substrates, which contained a TEV cleavage site between two moieties.     

Mapping of the nuclear localization signals in open reading frame 2 protein from porcine circovirus type 1

Porcine circovirus type 1 （PCV1） contains two major open reading frames encoding the replication-associated proteins and the major structural capsid （Cap） protein. PCV1 Cap has an N-terminus carrying several potential monopartite or bipartite nuclear localization signals （NLS）. The contribution of these partially overlapping motifs to nuclear importing was identified by expression of mutated PCVI Cap versions fused to enhanced green fluorescent protein （EGFP）. The Cterminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells similar to the wild-type PCV1 Cap-EGFP, whereas truncation of the N-terminus rendered the fusion protein distributed into cytoplasm, indicating that the nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region. Substitutions of basic residues in stretches 9RR- RR12 or the right part of 25RRPYLAHPAFRNRYRWRRK43 resulted in a diffused distribution of the fusion protein in both nuclei and cytoplasm, indicating that the two NLSs were responsible for restricted nuclear targeting of PCV1 Cap.     

人类疱疹病毒6型特异性CD4+ T细胞的初步克隆

目的 克隆HHV-6特异性CD4⁺ T细胞,并分析其基本特征。方法 微孔有限稀释法克隆HHV-6特异性CD4⁺ T细胞;3H-TdR掺入法细胞增殖试验筛选HHV-6 特异性CD4+ T细胞克隆;FACS分析HHV-6特异性CD4⁺ T细胞克隆的表面标志;ELISA检测HHV-6特异性CD4⁺ T细胞克隆的细胞因子分泌水平。结果 获得的细胞克隆中有4株以HHV-6感染细胞裂解物特异的方式增殖,并且其增殖水平与HHV-6感染细胞裂解物呈剂量依赖性;细胞克隆表面标志为CD3⁺CD4⁺;W-2和W-4细胞克隆的细胞因子分泌以IL-10为主,W-1细胞克隆的细胞因子分泌以IL-10及IFN-γ为主,W-3细胞克隆的细胞因子分泌以IFN-γ为主。结论 初步建立HHV-6特异性CD4⁺ T细胞克隆,并分析其表面标志和细胞因子分泌水平,为HHV-6特异性Treg细胞的克隆、筛选及其功能的研究奠定基础。     

调理脾胃法治疗维持性血透患者营养不良的报告

目的:探讨调理脾胃法治疗维持性血透患者营养不良的效果.方法:40例维持性血透患者随机分为治疗组和对照组,两组在对症处理相同、血透相关因素一致条件下进行维持血透治疗,治疗组同时根据辨证分型服用调理脾胃中药,疗程3个月.结果:与本组治疗前相比较,治疗后的各项生化指标明显提高(P<0.01),蛋白分解率也较治疗前提高(P<0.05),SGA分级好转(P<0.01),临床常见症状体征积分随治疗时间延长呈下降趋势(P(0.05);治疗组与对照组治疗后的各项生化指标、蛋白分解率、SGA分级及临床常见症状体征积分等差异均具有显著性(P<0.05).结论:中药调理脾胃法可以改善维持性血透患者营养不良状况.