Production of recombinant humanized anti-HBsAg Fab fragment from Pichia pastoris by fermentation

In this report, we describe the high-yield secretory expression of the recombinant human anti-HBsAg Fab fragment from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of alcohol oxidase promoter) into the genome of the yeast cells. The fed-batch fermentations were carried out in a 5 L scale. Both chains of the Fab were successfully expressed upon methanol induction. The absorbance (OD600) of the broth can reach 350 approximately 500 at the end of fed-batch phase. After the induction, the expression level of the recombinant Fab (soluble) reached 420 approximately 458 mg/L. The recombinant Fab fragment was purified from the crude culture supernatant by ion exchange chromatography and the purity of the recombinant Fab fragment was over 95%. The affinity activities of the crude fermentation supernatant and the purified Fab were analyzed by indirect ELISA, which showed that the purified recombinant Fab fragment had high affinity activity with hepatitis B surface antigen.     

Cost-Effectiveness of Alternative Strategies for Annual Influenza Vaccination among Children Aged 6 Months to 14 Years in Four Provinces in China

Background

To support policy making, we developed an initial model to assess the cost-effectiveness of potential strategies to increase influenza vaccination rates among children in China.

Methods

We studied on children aged 6 months to 14 years in four provinces (Shandong, Henan, Hunan, and Sichuan), with a health care system perspective. We used data from 2005/6 to 2010/11, excluding 2009/10. Costs are reported in 2010 U.S. dollars.

Results

In comparison with no vaccination, the mean (range) of Medically Attended Cases averted by the current self-payment policy for the two age groups (6 to 59 months and 60 months to 14 years) was 1,465 (23∼11,132) and 792 (36∼4,247), and the cost effectiveness ratios were $ 0 (-11-51) and $ 37 (6-125) per case adverted, respectively. In comparison with the current policy, the incremental cost effectiveness ratio (ICER) of alternative strategies, OPTION One-reminder and OPTION Two-comprehensive package, decreased as vaccination rate increased. The ICER for children aged 6 to 59 months was lower than that for children aged 60 months to 14 years.

Conclusions

The model is a useful tool in identifying elements for evaluating vaccination strategies. However, more data are needed to produce more accurate cost-effectiveness estimates of potential vaccination policies.     

Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design

The DVD-Ig™ protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.Key words: dual variable domain immunoglobulin, DVD-Ig, immunotherapy, variable domain, antibody engineering, dual-specific, linker     

庞泉沟自然保护区针阔混交林土壤呼吸的空间异质性

为研究土壤呼吸空间变异的影响因素,测定了山西省庞泉沟自然保护区针阔混交林地的土壤呼吸(R_s)及其影响因子,运用传统和地统计学的方法分析了4、2 m和1 m间隔取样尺度下R_s的空间变异性及其与影响因子之间的关系。传统统计分析表明:除土壤温度(T_(10))和碳/氮(C/N)比变异程度较小外,其他测定因子的变异系数在15%-59%之间,均为中等变异;R_s与凋落物量(L_w)、凋落物含水量(L_m)、土壤全碳(C)和全氮(N)呈极显著正相关(P0.01)与土壤水分(W_s)呈显著正相关(P0.05),与土壤温度(T_(10))呈极显著负相关(P0.01),与C/N比和土壤全硫(S)相关性不显著(P0.05)。多元逐步回归分析表明:L_w、T_(10)、N和C/N比四个因子能解释土壤呼吸空间变化的26%。地统计学分析表明,T_(10)、W_s、L_m、C、N和C/N比具有较强的空间自相关性,结构因素对其空间分布起着主导作用;R_s和L_w具有中等程度的空间自相关性,随机因素和结构因素对它们的空间分布起的作用相当;S具有较弱的空间自相关性,随机因素对其空间变异起着主导作用。R_s及其影响因子在相同的尺度上起作用,基本上都在17 m左右。分维数是事物复杂程度的一种量度,各指标的分维数大小依次为:L_w(1.87)S(1.84)L_m(1.82)N(1.77)R_s(1.74)C(1.73)W_s(1.69)T_(10)(1.56)C/N(1.46)。R_s的空间分布模式与W_s、L_m、L_w、C、N和S的空间分布模式较为一致,而与T_(10)的空间分布模式不同。4、2 m和1 m取样尺度95%置信水平误差在5%和10%内必要采样数量分别为74、44、39个和19、11、10个。     

Information theory-based algorithm for <Emphasis Type="Italic">in silico</Emphasis> prediction of PCR products with whole genomic sequences as templates

Background  

A new algorithm for assessing similarity between primer and template has been developed based on the hypothesis that annealing of primer to template is an information transfer process.     

应用噬菌体抗体库技术制备特异抗人纤维蛋白鼠单链抗体

应用Pharmacia公司的重组噬菌体抗体系统,从经过人交联纤维蛋白特异抗原D二聚体(DD)免疫过的鼠脾细胞mRNA中构建出组合单链抗体(ScFv)cDNA文库。文库cDNA克隆到噬菌粒载体pCANTAB5E,转化大肠杆菌TG1,得到2.5×10~5个氨苄抗性菌落。通过噬菌体表面呈现,用DD对表达的重组噬菌体单链抗体文库进行三轮亲和富集获得一株特异抗DD的噬菌体单链抗体(ScFvA11)。经Phage-ELISA鉴定,呈现在噬菌体表面的ScFvA11与DD结合的ELISA阳性滴度小于10~7tfu/ml,而与人纤维蛋白原结合的ELISA滴度大于10~(10)tfu/ml,两者相差1000倍以上。表明ScFvA11具有较好的DD结合特异性。经序列分析,ScFvA11cDNA全长729bp,其中Vh基因354bp,编码118个氨基酸;Vl基因327bp,编码108个氨基酸;Vh与Vl之间为(Gly_4Ser)_315个氨基酸连接肽。     

Mitochondrial DNA damage is sensitive to exogenous H(2)O(2) but independent of cellular ROS production in prostate cancer cells

Intrinsic oxidative stress through enhanced production of reactive oxygen species (ROS) in prostate and other cancers may contribute to cancer progression due to its stimulating effect on cancer growth. In this study, we investigate differential responses to exogenous oxidative stimuli between aggressive prostate cancer and normal cell lines and explore potential mechanisms through interactions between cytotoxicity, cellular ROS production and oxidative DNA damage. The circular, multi-copy mitochondrial DNA (mtDNA) is used as a sensitive surrogate to oxidative DNA damage. We demonstrate that exogenous H(2)O(2) induces preferential cytotoxicity in aggressive prostate cancer than normal cells; a cascade production of cellular ROS, composed mainly of superoxide (O(2)(-)), is shown to be a critical determinant of H(2)O(2)-induced selective toxicity in cancer cells. In contrast, mtDNA damage and copy number depletion, as measured by a novel two-phase strategy of the supercoiling-sensitive qPCR method, are very sensitive to exogenous H(2)O(2) exposure in both cancer and normal cell lines. Moreover, we demonstrate for the first time that the sensitive mtDNA damage response to exogenous H(2)O(2) is independent of secondary cellular ROS production triggered by several ROS modulators regardless of cell phenotypes. These new findings suggest different mechanisms underpinning cytotoxicity and DNA damage induced by oxidative stress and a susceptible phenotype to oxidative injury associated with aggressive prostate cancer cells in vitro.     

Annular Anionic Lipids Stabilize the Integrin αIIbβ3 Transmembrane Complex

Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbβ3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg⁹⁹⁵) contacts with β3(Asp⁷²³) but paradoxically do not diminish the contribution of αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbβ3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact.     

青蛙蝌蚪微核试验——一种水体诱变剂检测系统的建立

以青蛙蝌蚪为实验生物,利用甲基磺酸乙酯(EMS)和亚硝基胍(MNNG)探讨其化合物浓度、暴露时间和蝌蚪发育阶段等因素对诱发青蛙蝌蚪红细胞微核的影响,不同统计单位的特点和相互关系;提出了青蛙蝌蚪微核试验作为一种水体诱变剂检测系统的基本实验程序和一般原则。此外,还描述了“小体M”——一种特殊的细胞学现象,并初步讨论了微核代谢机制。