红素氧还蛋白研究进展

自然界中大多数的红素氧还蛋白(rubredoxin)集中来自严格厌氧菌,包括细菌、古菌和少数微需氧细菌,而植物中该类蛋白相对较少,它的功能主要表现在作为辅因子或电子供体参与多个生化途径。也有研究显示,无论是植物中还是微生物中的红素氧还蛋白基因在协助生物抵御氧化胁迫方面均发挥着重要的作用。本文全面介绍自然界中的红素氧还蛋白基因的分布、结构以及在微生物、植物中所发挥的作用等,希望能为今后的进一步研究提供参考。     

油脂预乳化提高大豆拉丝蛋白素食香肠品质

为了提高大豆拉丝蛋白(Textured Fibril Soy Protein,TFSP)素食香肠的品质,该研究将油脂预乳化工艺应用于TFSP素食香肠的加工,利用响应面试验设计优化了预乳化工艺条件,采用质构分析仪测定分析了产品的质构特性,并进行了感官评价。结果表明优化后的最佳预乳化工艺条件为菜籽油含量445g/L、大豆分离蛋白浓度105g/L、乳化机剪切速率9.0×103r/min。与对照组相比,此条件下制作的预乳化-TFSP素食香肠的凝胶强度、硬度和咀嚼性分别提高了约1倍、10%和26%,且产品口感鲜嫩、富有汁液感,感官评价得分显著提高。采用激光共聚焦显微镜观察分析了不同工艺制作的TFSP素食香肠在煮制前后微观结构的变化,结果发现油脂预乳化工艺可大大提高乳化油脂的稳定性和在香肠凝胶基质中的均匀分布,从而使乳化油脂的‘填充作用’得以发挥,不仅增强了TFSP素食香肠的保水保油能力,降低了蒸煮损失率,而且对香肠的质构和感官特性产生了重要影响。因此油脂预乳化工艺是一种辅助提高素食香肠整体品质行之有效的方法。     

马乳脂肪球膜蛋白组鉴定及潜在功能分析

试验旨在研究马乳脂肪球膜（milk fat globule membrane，MFGM）蛋白组成及潜在的生物学功能。选取12匹5岁左右、平均体重为450~500 kg的健康纯血母马作为试验动物，主要饲喂干牧草，所有马匹饲养和管理条件均相同。将12份样品以每组4份进行混合，分离提取产后第60天马乳中MFGM蛋白，进行高分辨质谱仪鉴定和生物信息学分析。结果显示，马MFGM组分中共鉴定出310种表达蛋白，这些蛋白主要参与的生物过程为生物调节、刺激应答、定位、多细胞生物过程、运输、信号、细胞通讯、发育过程、细胞分化和免疫系统过程等；细胞成分主要为胞外区域、膜、囊泡、核仁和线粒体等；分子功能主要为催化活性、蛋白质结合、碳水化合物衍生物结合和小分子结合等。KEGG通路分析表明，MFGM蛋白主要参与血小板活化通路、内吞、脂肪酸生物合成和Ras信号通路等。马乳MFGM蛋白的蛋白质互作网络分析图中共包含215种蛋白质。本研究结果揭示了马乳MFGM蛋白质组的复杂性，为解析其营养和生物学功能提供了科学数据。     

Effect of wilting,silage additive,PEG treatment and tannin content on the distribution of N between different fractions after ensiling of three different sainfoin (Onobrychis viciifolia) varieties

Sainfoin (Onobrychis viciifolia) is a tanniniferous, leguminous plant that has potentially beneficial effects on protein utilization in ruminants. As ensiling causes protein breakdown and elevated levels of buffer soluble N (BSN), we studied the distribution of N before and after ensiling sainfoin. Three varieties of sainfoin were either direct‐cut and frozen directly or wilted and frozen before later ensiling in mini‐silos with and without acidification with Promyr (PM; an acidifying commercial mixture of propionic and formic acid) and with or without polyethylene glycol (PEG). Extractable tannins (ET) and protein‐bound tannins (PBT) were measured with an HCl/butanol method in an attempt to correlate tannin levels to N fractions. The sainfoin silages showed good ensiling characteristics and had relatively high concentrations of undegraded protein. The effect of wilting on BSN levels (g/kg N) was dependent on sainfoin variety (P < 0·001). PEG increased and PM decreased the level of BSN in the silages (P < 0·001). PM treatment also produced less non‐protein N and ammonia‐N (P < 0·05) as compared with no additive. Addition of PEG to the silage increased the BSN‐proportion 1·8‐ and 2·6‐fold for both DM stages. A strong tannin‐protein binding effect is, therefore, confirmed in sainfoin. However, correlations between tannin levels (ET and PBT) and BSN were poor in the (non‐PEG) silages, indicating either that the HCl/butanol method is unsuitable for measuring tannin in silages or that qualitative attributes of tannins are more relevant than quantitative. The HCl/butanol method seems therefore not to be useful to predict degradation of protein in sainfoin silages.     

利用甜高粱秸秆汁产单细胞蛋白的分批补料发酵研究

本文以甜高粱秸秆汁为主要的发酵原料,对热带假丝酵母SG-1在15-L罐上产单细胞蛋白(SCP)的分批补料发酵过程进行研究。首先对分批发酵模式下的菌体代谢过程变化进行研究。研究发现,接种后菌体快速进入繁殖阶段,但是总糖在12 h处于耗竭状态,使得菌体量和SCP产量过早的在18 h就达到峰值。基于此,在15-L罐上建立了热带假丝酵母SG-1产SCP的分批补料发酵模式。结果表明:菌体代谢因基质的补加而得到进一步的延续,放罐时(42 h)其菌体干重和SCP产量分别达19.86 g/L和9.93 g/L,显著高于分批发酵模式的10.45 g/L和4.70 g/L。     

Dietary fiber in a low-protein diet during gestation affects nitrogen excretion in primiparous gilts,with possible influences from the gut microbiota

We investigated the effects of dietary fiber (DF) supplementation in normal or low crude protein (CP) diets on reproductive performance and nitrogen (N) utilization in primiparous gilts. In total, 77 Landrace × Yorkshire pregnant gilts were randomly allocated to four dietary treatments in a 2 × 2 factorial design. The groups comprised 1) equal intake of normal CP (12.82% and 0.61% total lysine), 2) low CP (LP) (10.53% and 0.61% total lysine), and 3) with or 4) without DF supplementation (cellulose, inulin, and pectin in a 34:10:1 ratio). A low-protein diet during gestation significantly reduced daily weight gain from days 91 to 110 of pregnancy (−162.5 g/d, P = 0.004). From N balance trials conducted at days 35 to 38, 65 to 68, and 95 to 98 of pregnancy, DF addition increased fecal N excretion at days 65 to 68 (+24.1%) and 95 to 98 (+13.8%) of pregnancy (P < 0.05) but reduced urinary N excretion (P < 0.05), resulting in greater N retention at each gestational stage. DF increased fecal microbial protein levels and excretion during gestation. An LP diet also reduced urinary N excretion at different gestational stages. An in vitro fermentation trial on culture media with nonprotein N urea and ammonium bicarbonate (NH₄HCO₃) as the only N sources revealed that microbiota derived from feces of gestating gilts fed the high DF diet exhibited a greater capacity to convert nonprotein N to microbial protein. Microbial fecal diversity, as measured by 16S rRNA sequencing, revealed significant changes from DF but not CP diets. Gilts fed an LP diet had a higher number of stillbirths (+0.83 per litter, P = 0.046) and a lower piglet birth weight (1.52 vs. 1.37 kg, P = 0.006), regardless of DF levels. Collectively, DF supplementation to gestation diets shifted N excretion from urine to feces in the form of microbial protein, suggesting that the microbiota had a putative role in controlling N utilization from DF. Additionally, a low-protein diet during gestation negatively affected the litter performance of gilts.     

改良背主动脉注射法对鸡胚发育与转基因表达效率的影响

背主动脉注射是生产转基因鸡的经典方法,该方法不需换壳培养,但壳内注射技术难度大,并且无法实时观察后期胚胎发育情况。本实验对背主动脉注射法进行了改进,将增强型绿色荧光蛋白(EGFP)腺相关病毒(Adeno-associated Virus,AAV)壳外注射至150枚HH 14~16期(Hamburger-Hamilton Stage 14~16)鸡胚背主动脉中,再进行换壳培养,继续孵化至出壳,观察和分析改良背主动脉注射法与传统背主动脉注射法和胚盘下腔注射法对发育8、14、18、21 d鸡胚存活率、孵化率以及EGFP阳性检出率的影响。结果表明:改良背主动脉注射组鸡胚存活率均高于传统背主动脉注射组与胚盘下腔注射组(P<0.05或P<0.01);改良背主动脉注射组的鸡胚孵化率(37%)高于传统背主动脉注射组(16%)与胚盘下腔注射组(28%)(P<0.01);荧光蛋白手电筒检测显示,改良背主动脉注射组鸡胚EGFP阳性率(17%)明显高于传统背主动脉注射组(13%)与胚盘下腔注射组(12%)(P<0.01)。综上,背主动脉壳外注射结合换壳培养提高了转基因鸡胚胎孵化率和EGFP阳性检出率,对提高转基因鸡效率具有重要价值。     

猪流行性腹泻病毒S蛋白纳米抗体的筛选与鉴定

猪流行性腹泻病（porcine epidemic diarrhea,PED）是一种高度接触性肠道传染性疫病,主要危害1周龄以内的仔猪,仔猪感染死亡率高达100%,是目前危害世界养猪业的主要疫病之一。本研究旨在制备针对猪流行性腹泻病毒S蛋白的特异性纳米抗体并鉴定其结合活性。作者原核表达并纯化PEDV S1蛋白,将纯化后的PEDV S1重组蛋白免疫双峰驼,第4次免疫后分离其外周血淋巴细胞,提取淋巴细胞RNA,反转录得到cDNA,通过巢式PCR扩增VHH片段,并构建至pCANTAB-5E载体中,电转化至TG1感受态细胞,得到VHH噬菌体抗体展示文库;随后,对构建的噬菌体抗体展示文库进行救援和3轮富集,利用噬菌体展示技术从中筛选针对PEDV S蛋白纳米抗体,通过ELISA验证筛选的纳米抗体的特异性和结合力。通过Western blot和间接免疫荧光验证纳米抗体与PEDV的结合活性。结果显示：成功表达并纯化PEDV S1蛋白,经4次免疫后,双峰驼血清中的特异性抗体效价达到了1∶256 000。构建的噬菌体展示文库的库容量为2.1×10⁷,阳性率85%;对噬菌体展示文库3轮的淘选富集后,最终筛选出6株氨基酸序列不同的纳米抗体,ELISA结果显示,6株纳米抗体均对PEDV S1重组蛋白具有良好的结合力与特异性。随后验证了Nb3能够与PEDV结合,表明其具有良好的活性。成功筛选到针对PEDV S1蛋白的特异性纳米抗体,所筛选纳米抗体有望用于PED的诊断和治疗,同时为PEDV的致病机制研究提供抗体材料。     

骆驼乳清蛋白对热应激所致大鼠肝氧化损伤的保护作用

本试验旨在研究骆驼乳清蛋白（CWP）对热应激（HS）大鼠肝损伤、氧化应激及肝功能的保护作用。选取6周龄SD大鼠36只,适应性饲养2周后随机分为6组：正常对照组饲喂基础日粮;CWP对照组添加400 mg·kg^-1的CWP;HS组除饲喂基础日粮外,每天进行2 h HS处理,连续8 d;3个CWP干预组分别于每次HS前灌服100、200和400 mg·kg^-1的CWP。试验结束后,取大鼠肝组织,HE染色观察肝组织病理学变化,同时检测肝功能生物标志物、氧化应激标志物水平及抗氧化酶活性。结果表明：CWP降低了HS大鼠血清谷草转氨酶（AST）和谷丙转氨酶（ALT）活性,400 mg·kg^-1的CWP干预效果最好（P<0.01）;400 mg·kg^-1CWP干预组有效降低了HS诱导的大鼠肝组织病理学改变; CWP降低了大鼠肝活性氧（ROS）及丙二醛（MDA）水平,增加了超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）活性及谷胱甘肽（GSH）水平,400 mg·kg^-1的CWP干预效果最好（P<0.01）。结果提示,CWP干预能够以剂量依赖性方式降低大鼠肝氧化应激,增加抗氧化系统防御能力,从而缓解HS所致大鼠肝损伤。     

Identification of the core motif of the BRCA2 C-terminal RAD51-binding domain by comparing canine and human BRCA2

Mammary tumors are the most common tumors in women and non-spayed female dogs. One of the reasons for mammary tumors is mutations of the tumor suppressor gene, BRCA2. BRCA2 participates in homologous recombination repair by interacting with the RAD51 recombinase. BRCA2 has two RAD51-binding domains, consisting of BRC repeats and the C-terminal RAD51-binding domain, respectively. Although several studies have addressed the function of the C-terminal RAD51-binding domain of human BRCA2, the amino acid sequences required for the RAD51-interaction activity remain unclear. In this study, the C-terminal RAD51-binding domains of canine and human BRCA2 were compared; the canine domain displayed a weaker interaction with RAD51. This difference was attributed to the C-terminal portion of the domain via a comparison between canine and human domains. Furthermore, peptides shorter than those previously reported displayed RAD51-interacting activity, and a core motif of this domain consisting of 25 amino acids was identified. Since a mutation (S3323N) was reported in the core motif of this domain, the effect of this mutation was evaluated. The mutant exhibited similar RAD51-binding activity as that of the wild-type protein, suggesting that the mutation was functionally neutral. These data suggested that the C-terminal portion of the BRCA2 C-terminal RAD51-binding domain influenced its RAD51-interaction activity, and a minimum core motif of 25 amino acids was identified in this domain. These data may help clarify BRCA2 function, as well as the tumorigenic effects of BRCA2 mutation.