一条新的柱层析纯化路径对HBsAg免疫原性的影响

摘要目的：建立一条新的毕赤酵母表达乙肝表面抗原（HepatitisBantigen,HBsAg）柱层析纯化方法,保持HBsAg结构完整性和提高免疫原性。方法：毕赤酵母发酵料液经过菌体破碎、聚乙二醇沉淀、疏水层析、超滤和凝胶分子筛精纯,收集HBsAg合格样品液适当稀释后加入铝佐荆吸附,制成乙肝疫苗半成品免疫BALB／c小鼠。结果：纯化产物经SDS-PAGE银染鉴定得单一条带,分子量在23kD左右,凝胶成像软件分析纯度超过95％;该纯化方法得到的HBsAg颗粒电镜观察得平均直径为22nm病毒样颗粒,结构较均一完整;自制疫苗免疫小鼠后,其血清抗体水平高于葛兰素史克生产的Engerix—B（安在时）,存在显著性差异（P〈0．05）。结论：通过该方法纯化的HBsAg结构完整性良好,疫苗免疫效果优于酵母表达的Engerix—B,纯化路径简单高效,易于放大用于工业化生产。     

SIRT1在宫颈癌细胞耐药中的作用及其机制研究

摘要目的：探讨沉默交配型信息调节因子2同源蛋白1（silentmatingtypeinformationregulation2homologyl,SIRTl）在宫颈癌化疗耐药中的作用及其机制。方法：体外培养人宫颈癌Hela细胞系和宫颈癌Hela／MMC耐药细胞亚系,westernblotting检测MMC对Hela和Hela／MMC细胞内SIRTl蛋白表达的影响;MTT法检测MMC及Nicotinamide对Hela和Hela／MMC细胞增殖的影响;AnnexinV-PI试验检测Hela／MMC细胞凋亡的亡的情况;RT—PCR方法检测耐药相关蛋白P—gP的mRNA表达情况。结果：正常情况下,Hela／MMC细胞中SIRTl的表达显著高于Hela细胞（P〈0．05）,MMC处理的Hela／MMC细胞中SIRTl的表达显著高于未经MMC处理（P〈0．05）。Nicotinamide对Hela和Hela／MMC细胞具有相似的生长抑制作用,Nicotinamide可使MMC诱导的Hela／MMC细胞凋亡增加,同时降低细胞内P-gP的mRNA表达（P〈0．05）。结论：SIRTl表达下调能显著减轻Hela／MMC细胞对MMC的耐药性,其作用可能与P—gp有关。     

生物催化法合成6-氰基-（3R，5R）-二羟基己酸叔丁酯

利用E.coli BL21／pCDFDuet-gdh—cr-X共表达全细胞催化6-氰基-（5R）-羟基-3-羰基己酸叔丁酯不对称还原合成6-氰基-（3R,5R）-二羟基已酸叔丁酯。结果表明：在菌体用量4．85g/L、葡萄糖与底物质量浓度比为1：1、温度28℃、pH7．0条件下,80．0g／L6-氰基-（5R）-羟基-3-羰基己酸叔丁酯生物还原2h后,底物转化率可达99．0％,产物d.e．值大于99．5％。在考察范围内,NADP^＋用量对催化效率无显著作用。     

LncRNA LEF1-AS1 promotes osteogenic differentiation of dental pulp stem cells via sponging miR-24-3p

Molecular and Cellular Biochemistry - Alzheimer’s disease (AD) is the leading cause of dementia, which characterized by toxic senile plaques is composed of amyloid-β (Aβ)....     

Overexpression of the mango MiCO gene delayed flowering time in transgenic Arabidopsis

Plant Cell, Tissue and Organ Culture (PCTOC) - CONSTANS (CO)/CONSTANS-like (COL) genes play an important role in the photoperiodic flowering pathway. However, the functional roles of the CO/COL...     

H3K27 acetylation-induced lncRNA EIF3J-AS1 improved proliferation and impeded apoptosis of colorectal cancer through miR-3163/YAP1 axis

Long noncoding RNAs (lncRNAs) are found to be aberrantly expressed and pose significant impacts in colorectal cancer (CRC), the most prevalent type malignancy in the gastrointestinal tract. This study aimed to find out the regulation of lncRNA EIF3J antisense RNA 1 (EIF3J-AS1) on CRC progression. Expressions of EIF3J-AS1, microRNA-3163 (miR-3163), and Yes-associated protein 1 (YAP1) in tissues and cells were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. Association of EIF3J-AS1 with CRC prognosis was analyzed through the online bioinformatics tool GEPIA. The biological function of EIF3J-AS1 in CRC was investigated by Cell Counting Kit-8, colony formation, caspase-3 activity, and TUNEL staining. Competitive endogenous RNA (ceRNA) network of EIF3J-AS1/miR-3163/YAP1 was determined by luciferase reporter and RNA immunoprecipitation assays. Results showed that EIF3J-AS1 was upregulated in CRC tissues and cell lines, indicating poor prognosis of CRC patients. The silence of EIF3J-AS1 led to reduced proliferation and facilitated apoptosis of CRC cells. Mechanistcally, EIF3J-AS1 was upregulated by cAMP-response element-binding protein-binding protein-mediated histone H3 on lysine 27 acetylation (H3K27ac) at the promoter region, and EIF3J-AS1 upregulated YAP1 expression through sponging miR-3163 in CRC cells. In conclusion, we first found that H3K27 acetylation-induced lncRNA EIF3J-AS1 improved proliferation and impeded apoptosis of colorectal cancer through the miR-3163/YAP1 axis, which might potentially provide a novel molecular-targeted strategy for CRC treatment.     

多酶组合催化制备L-高苯丙氨酸

【目的】L-高苯丙氨酸(L-HPA)是许多医药化学品的重要中间体,化学合成法生产L-HPA反应复杂、环境污染严重,本研究旨在开发高效环保的L-HPA酶法合成路线。【方法】采用模块化组装的方法,构建了一条以甘氨酸和苯乙醛为底物高产L-HPA的新途径。【结果】首先,根据文献挖掘设计了一条由苏氨酸醛缩酶(TA)、苏氨酸脱氨酶(TD)、苯丙氨酸脱氢酶(PheDH)和甲酸脱氢酶(FDH)组成的多酶组合催化途径,用于L-HPA的合成。其次,根据氨基基团的引入和重构,将L-HPA多酶组合催化途径分为基础单元和扩增单元,基础单元包括TA和TD,扩增单元包括PheDH和FDH。然后,利用不同表达水平的质粒,对基础单元和扩增单元进行蛋白表达的组合调节,获得最优工程菌BL21-C-M1-R-M2,使L-HPA产量达到208.6mg/L。最后,我们对全细胞转化体系进行优化,使L-HPA产量进一步提高到1226.6 mg/L,苯乙醛摩尔转化率为34.2%。【结论】该工艺路线绿色高效,为未来大规模生产L-HPA奠定基础。     

Using defined finger–finger interfaces as units of assembly for constructing zinc-finger nucleases

Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger–finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy—finger stitching—that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger–finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNA-binding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo, demonstrating that stitching yields ZFAs with robust recognition properties.