Cloning,Expression of Clostridium perfringens α-toxin Gene and Preparation of its Antisera

One pair of primers had been designed and synthesized based on the α-toxin gene of Clostridium perfringens.The complete α-toxin gene fragment was amplified by polymerase chain reaction (PCR), and then was cloned into pGEM-T Easy vector to construct pGEM-T-α.Digested with EcoRⅠ and Hind Ⅲ, a fragment of 1125 bp was cloned into the expression plasmid vector pET-28a(+).The recombinant plasmid was transformed into the BL21(DE3)plys and induced by 1.0 mmol/L IPTG at 37 ℃.The expression product was found to be 46.1 ku as expected one identified by SDS-PAGE, and confirmed by Western blotting with Clostridium perfringens type A antisera, indicating similar reactivity with native α-toxin.Recombinant α-toxin protein was simultaneously found in culture supernatant, postsonic supertanant and inclusion bodies, most protein was expressed in inclusion bodies, which indicated recombinant α-toxin protein was expressed in the extracellular, periplasm and cytoplasm.Recombinant α-toxin protein in postsonic supertanant could not make mice die, indicating its non-toxicity.Toxin-antitoxin neutralization test showed that antisera of recombinant α-toxin protein were specific to α-toxin.Upon immunization of rabbit with the recombinant α-toxin protein, antisera with high antibody titer neutralizing 100 MLD toxin per 1 mL were prepared.     

碱提甘蔗皮多糖提取工艺、初步结构及其对α-葡萄糖苷酶的抑制作用

【目的】建立碱提甘蔗皮多糖的优化工艺,进一步探究其结构特征,并评价其对α-葡萄糖苷酶的抑制作用。【方法】采用单因素试验和响应面分析法（RSM）对碱提甘蔗皮多糖（SPAP）提取工艺进行优化,采用苯酚硫酸法测定多糖含量。SPAP经Sevag法除蛋白、AB-8除色素后进行结构表征,主要包括高效液相色谱法（HPLC）检测多糖分子量分布,气相-质谱色谱仪（GC-MS）进行单糖分析及傅里叶红外光谱仪（FT-IR）对官能团进行分析。采用4-硝基苯基-D-吡喃葡糖苷（PNPG）法测定多糖提取物对α-葡萄糖苷酶抑制作用。【结果】SPAP的最佳提取条件为提取温度37℃、氢氧化钠（NaOH）浓度5%、料液比1∶46（g·mL^-1）、提取次数4次,在此条件下SPAP得率达到10.84%。经除蛋白、色素后,SPAP含量达到86.54%,主要由阿拉伯糖、木糖、葡萄糖和半乳糖4种单糖组成,分子量为3.03×10³ kD,可能为吡喃型杂多糖,呈现α或β构型。此外,SPAP表现出良好的α-葡萄糖苷酶抑制作用,其抑制率达到78.31%。【结论】对碱提甘蔗皮多糖进行工艺优化,可有效利用原料、提高产出和效率;由HPLC、FT-IR、GC-MS等方面初步阐述了SPAP的结构特性,同时SPAP对α-葡萄糖苷酶表现出良好的抑制作用,具有一定的降糖潜力,研究结果为进一步研究SPAP的构效关系提供了理论依据。     

归参汤对免疫抑制小鼠血清中IL-2、TNF-α和脾细胞Th1/Th2细胞因子的影响

为了探讨归参汤(GST)对免疫抑制小鼠血清中IL-2、TNF-α和脾细胞Th1/Th2细胞因子的影响。本试验采用环磷酰胺制造免疫抑制模型,利用双抗夹心ELISA法,观察归参汤对小鼠血清中IL-2、TNF-α的影响;RT-PCR法观察归参汤对小鼠脾细胞中INF-γ、IL-4、T-bet和GATA-3的mRNA表达的影响。结果显示:归参汤高剂量组可以显著增加免疫抑制小鼠血清中IL-2、TNF-α的含量(P<0.01),提高INF-γ的表达(P<0.05),降低IL-4的含量(P<0.05),升高T-bet(P<0.05),降低GATA-3的表达(P<0.05)。归参汤的免疫增强作用与升高血清中IL-2、TNF-α有关,且升高INF-γ/IL-4纠正免疫抑制小鼠Thl/Th2细胞因子的比例紊乱,其机制可能部分归因于对T-bet和GATA-3的mRNA表达的影响。     

山茱萸不同部位提取物对α-葡萄糖苷酶的抑制作用

[目的]测定山茱萸果肉和果核提取物对α-葡萄糖苷酶的抑制作用,为山茱萸资源的药用开发提供参考。[方法]采用乙醇和水分别对山茱萸果肉和果核进行提取,并测定其对α-葡萄糖苷酶的抑制作用。[结果]山茱萸果核提取物对α-葡萄糖苷酶的抑制率分别为20倍醇提99.23%、30倍醇提98.96%、40倍醇提99.29%、20倍水提99.36%、30倍水提99.32%、40倍水提99.17%。山茱萸果肉提取物对α-葡萄糖苷酶的抑制率分别为20倍醇提52.10%、30倍醇提60.77%、40倍醇提21.67%、20倍水提11.88%、30倍水提0.53%、40倍水提1.59%。[结论]山茱萸不同部位的水提取物和乙醇提取物对α-葡萄糖苷酶均有一定的抑制作用,其中山茱萸果核部位对α-葡萄糖苷酶的抑制作用最为显著,其抑制率可高达99%以上,可见山茱萸果核中含有丰富的抑制α-葡萄糖苷酶活性的物质,可作为药用资源予以开发利用。     

定点突变阻止金黄色葡萄球菌α-溶血素溶血活性

金黄色葡萄球菌分泌的α-溶血素是导致肺炎的重要致病因子。为进一步研究金黄色葡萄球菌α-溶血素(α-hla)的分子致病机理以及对其进行免疫预防,对α-hla第35位氨基酸进行了定点突变。用聚合酶链式反应(PCR)技术,从S.aureus wood46株基因组中扩增出α-hla基因,再利用重叠延伸PCR技术,将第35位带正电荷的组氨酸(密码子为CAC)突变为非极性的亮氨酸(密码子为CTC)。hlaH35L基因测序结果显示第35位的组氨酸突变为亮氨酸,构建的重组表达质粒pET-28a-c(+)/hla和pET-28a-c(+)/hlaH35L在E.coli BL21(DE3)中得到表达,重组蛋白α-Hla及hlaH35L大小均为33.4 kDa,α-Hla引起兔红细胞溶血,hlaH35L未引起兔红细胞溶血。成功表达并获得了失去溶血毒性的重组蛋白hlaH35L。     

α-乙酰乳酸脱羧酶基因在枯草芽孢杆菌中的整合型表达

α-乙酰乳酸脱羧酶在啤酒生产中能加快啤酒成熟,有重要的应用价值。本研究将枯草芽孢杆菌启动子P43克隆到质粒pUC19-ALDC中的α-乙酰乳酸脱羧酶基因之前,得到重组质粒pUC19-P43-ALDC。重组质粒pUC19-P43-ALDC与质粒pMLK83-BN同源重组,筛选得到枯草芽孢杆菌整合质粒pMLK83-ALDC。用此整合质粒转化枯草芽孢杆菌1A751,挑选出新霉素抗性且无淀粉酶活性的重组菌株。此菌株用LB培养基在37℃、220r/min摇瓶培养过夜,测得α-乙酰乳酸脱羧酶活力为15.6U/mL,说明整合的α-乙酰乳酸脱羧酶基因能够在重组菌株中稳定传代和表达。本研究首次在枯草芽孢杆菌中用整合型的方式重组表达了α-乙酰乳酸脱羧酶,提出了一种有潜力的生产α-乙酰乳酸脱羧酶的新方法。     

正交试验法优化黄褐毛忍冬总皂苷的提取工艺

[目的]建立黄褐毛忍冬（Lonicera fulvotomentosa Hsu et S.C.Cheng）总皂苷的水提工艺。[方法]以黄褐毛忍冬总皂苷中有效成分α-常春藤皂苷的含量为考察指标,采用正交设计法考察加水量、煎煮时间、煎煮次数及浸泡时间4个因素对黄褐毛忍冬总皂苷提取工艺的影响,同时采用高效液相法测定α-常春藤皂苷的含量。[结果]加水量、煎煮次数、煎煮时间、浸泡时间对α-常春藤皂苷含量的影响程度不同,各因素影响程度的大小为：煎煮次数〉加水量〉浸泡时间〉煎煮时间,即煎煮次数对煎煮条件影响最大,为最重要的因素,其次为加水量、浸泡时间、煎煮时间。最优方案为：黄褐毛忍冬药材不用浸泡,加10倍量水煎煮3次,每次1.0h。方差分析结果表明,加水量和煎煮次数对α-常春藤皂苷含量的影响具有显著意义（PA=0.034〈0.05,PB=0.020〈0.05）,而煎煮时间、浸泡时间对水α-常春藤皂苷含量无显著影响（PC=0.500〉0.05,PD=0.264〉0.05）,对测定结果影响小,这与直观分析结果相吻合。[结论]优选提取工艺简单、稳定、可行。     

Functional analysis of the venom allergen-like protein gene from pine wood nematode Bursaphelenchus xylophilus using a baculovirus expression system

The pine wood nematode, Bursaphelenchus xylophilus, infects pine trees, leading to fatal pine wilt disease. Here, recombinant venom allergen-like protein (VAP) was obtained by expressing Bx-vap-1 in insect cells. Three-year-old Pinus massoniana were inoculated with recombinant VAP, simulating B. xylophilus esophageal gland secretions. Recombinant VAP up-regulated α-pinene synthase gene expression, the trees showed disease symptoms 15 d after inoculation and the xylem pith revealed brown tissue discoloration, indicating that recombinant VAP could damage P. massoniana cells. Recombinant VAP did not, however, lead to cavitation, indicating that the VAP secreted from B. xylophilus acts as a defense response elicitor.     

Improving the Field Emergence Performance of Super Sweet Corn by Sand Priming

Abstract

A priming method called sand priming was developed using sand as a priming solid matrix. The effect of sand priming on improving the field emergence performance of five super sweet corn cultivars was investigated. Sand priming significantly improved field emergence performance of all super sweet corn cultivars, and there was marked improvement by priming at 20ºC for 24 hr. After sand priming at 20ºC for 24 hr, field emergence percentage (FEP) of “Green Superman”, “Huatian 1”, “Yangtian 1”, “Mitian 8”, and “Chaotian 43” was increased by 52.1%, 37.5%, 38.0%, 40.9%, and 33.3%, respectively. Their field emergence speed (FES) was 2.3, 1.8, 2.0, 2.0, and 1.8 times of the control, respectively. To further elucidate the effect of sand priming on improving the field emergence performance of super sweet corn, we analyzed the membrane system integrity, α-amylase activity and protein content. Sand priming at 20ºC for 24 hr improved membrane system integrity and α-amylase activity in all super sweet corn cultivars. Furthermore, sand priming at 20ºC for 24 hr accelerated the degradation of embryo protein after 1 d germination in “Green Superman”.     

N-(α-萘乙酰基)-N-′(2-丙酸基)-硫脲的合成

研究了从α-萘乙酸出发,用一锅法合成出N-(α-萘乙酰基)-N-′(2-丙酸基)-硫脲。首先,α-萘乙酸与氯化亚砜反应生成α-萘乙酰氯,然后,再加入硫氰酸钾,并在乙腈为溶剂的条件下,合成α-萘乙酰基硫氰酸酯,最后,再与丙氨酸反应,生成目标产物。所得产物结构经IR初步确证。