霍乱毒素B亚单位基因的克隆及其核苷酸序列分析

利用 PCR技术从含有霍乱毒素 B亚单位 ( CTB)基因的质粒 p UCT1中扩增出不包括信号肽的 3 0 9bp的 CTB全基因 ,并通过点突变技术消除了 CTB阅读框内的终止密码子 ( TAA→GAA) ,以便于在 CTB基因的下游融合其他的外源基因。用限制性内切酶 Bam H 和 Eco R 对 PCR产物进行双酶切 ,以 T4DNA连接酶将其定向连接于经同样酶切处理的质粒 p ET-2 8b( )的多克隆位点上 ,构建成重组质粒 p ECTB,转化至BL2 1 ( DE3 )中。经 Bam H 和 Eco R 双酶切分析和 PCR扩增检测 ,证明重组质粒 p ECTB中含有 CTB基因。核苷酸序列分析表明 ,克隆的 CTB基因在重组质粒中的连接向位和阅读框架是正确的     

嗜水气单胞菌与迟缓爱德华氏菌亚单位二联疫苗对小鼠的免疫效果

将嗜水气单胞菌（Ａｈ）Ｊ－１株的ＨＥＣ毒素和胞外蛋白酶（ＥＣＰ）分别与迟缓爱德华氏菌（Ｅｔ）３８株的脂多糖（ＬＰＳ）及脱毒多糖（ＰＳ）偶联,制成３种亚单位二联疫苗;ＨＥＣ－ＰＳ、ＨＥＣ－ＬＰＳ。将它们与ＡｈＪ－１和Ｅｔ３８全菌灭活疫苗一同分别免疫小鼠,并设注射ＰＢＳ对照组,检测各组小鼠体液免疫及细胞免疫水平。结果显示,用间接ＥＬＩＳＡ、溶血抑制和全菌凝集试验检出这３种亚单位疫苗的抗体水平无明显差异     

Technological quality of field grown transgenic lines of commercial wheat cultivars expressing the 1Ax1 HMW glutenin subunit gene

Ten transgenic lines were studied which expressed a transgene encoding HMW subunit 1Ax1 in three elite spring wheat cultivars: Imp, Canon and Cadenza. These lines contained one to five copies of the transgene and the 1Ax1 subunit was expressed as 1–20% of the total glutenin protein. These lines were grown in field trials in a continental, arid climate (Martonvásár, Hungary) over two years (2004, 2005). The expression of the transgenes and their effects on the grain properties were stably inherited over the two years. Significant differences in yield were observed between three of the transgenic lines and the original genotypes, but no differences were found in their adaptiveness. Clear differences were found in the technological and rheological properties of four lines, with all the parameters characterising dough strength and extensibility (GI, W, G, Re, Ext, A) changing significantly. These differences were associated with increases in the ratio of HMW/LMW subunits and decreases in the ratios of 1Dx/1Dy and 1Bx/1By subunits. Two transgenic lines of cv Imp had high over-expression of the 1Ax1 subunit which in one line resulted in an overstrong type of dough, similar to that described previously for lines over-expressing HMW subunit 1Dx5. Transformation of cvs. Canon and Cadenza resulted in two lines with increased dough stability due to the significantly improved gluten quality. It is concluded that significant changes in the structure of the glutenin polymers caused by the altered ratio of x-type to y-type HMW subunits led to the changes in flour functional properties.     

杆状病毒表达系统在兽用亚单位疫苗领域应用的研究进展

杆状病毒表达系统是以杆状病毒为蛋白质表达载体,昆虫细胞或幼虫为受体的真核表达系统。与原核细胞相比,昆虫细胞可对外源蛋白进行翻译后修饰,如多肽折叠、糖基化、酰基化、磷酸化、二硫键形成及蛋白质水解等。因杆状病毒对畜禽无致病性,这使得杆状病毒表达系统成为一种安全有效的兽用亚单位疫苗生产平台。作者对杆状病毒表达系统在兽用亚单位疫苗领域中的应用作了简要综述。     

DNA Barcoding Reveals Extensive Mislabeling in Seafood Sold in Portuguese Supermarkets

ABSTRACT

Seafood mislabeling is a widespread problem even in Europe where the food market is highly regulated. In the present study, we analyzed the accuracy of seafood labeling in five Portuguese supermarket chains through DNA barcode. Our results show that approximately 19% of examined samples were mislabeled and that the consumer’s trust is misplaced. As mislabeling was greater in invertebrate species, we recommend that future studies led by government agencies and scientists aiming to test labeling accuracy should expand from finfish to include invertebrates such as crustaceans and bivalves.     

牛传染性鼻气管炎基因工程疫苗研究进展

牛传染性鼻气管炎是由牛疱疹病毒1型感染而引起的高传染性疾病。该病临床症状以呼吸道炎症为主,另外伴有结膜炎、流产、脑炎等并发症。20世纪50年代早期,美国科罗拉多州某些牧场首次发现该病。目前缺乏有效的治疗性药物,对动物实施疫苗免疫是防控该病较理想的方法。目前,某些国家仍然使用传统疫苗,然而,经过基因改造的糖蛋白E基因缺失疫苗已在欧盟上市,该疫苗分为活苗和灭活苗两种。文章着重对牛传染性鼻气管炎基因缺失疫苗、亚单位疫苗、活载体疫苗及DNA疫苗最新的研究进行评述。     

O型FMDV复合多表位在毕赤酵母中的表达与鉴定

将O型FMDV复合多表位CTB-TEpi与FMDV结构基因P1-2A(P1/2A)连入毕赤酵母表达载体pPIC9K,经SacⅠ线性化后,利用电转化法整合到毕赤酵母(Pichia pastoris)基因组中。通过G418浓度梯度筛选得到高拷贝阳性转化子GS115/P1/2A-CTB-TEpi。经甲醇诱导表达后,目的蛋白在毕赤酵母中获得成功表达,并在FMDV自裂解片段2A的作用下裂解为P1/2A和CTB-TEpi 2个片段,相对分子质量分别为81 800和39 400,占上清液中可溶蛋白的比例为25%。Western blotting分析表明,表达蛋白的2部分均可被抗O型FMDV的血清所识别,而且含有CTB片段的CTB-TEpi条带可以被兔抗霍乱毒素血清识别,证明表达的蛋白具有抗原活性,为进一步研究O型FMDV复合多表位亚单位疫苗的免疫效果打下了基础。     

利用SDS-PAGE和AS-PCR标记鉴定簇毛麦HMW-GS基因

本研究通过SDS-PAGE和AS-PCR标记对簇毛麦HMW-GS进行鉴定,对于快速鉴别导入到普通小麦中的簇毛麦HMW-GS和改良普通小麦品质具有重要意义。利用SDS-PAGE对中国春、钱尼、簇毛麦、6个中国春-簇毛麦二体附加系、普通小麦-簇毛麦3V异代换系进行HMW-GS组成分析,进一步确定簇毛麦HMW-GS基因位于1V染色体。根据已发表的普通小麦HMW-GS基因序列同源性比较结果,设计了3对分别扩增普通小麦x-型亚基基因、y-型亚基基因以及所有HMW-GS基因的特异引物。经过对簇毛麦HMW-GS基因进行扩增发现,这3个AS-PCR标记都能很好地鉴别簇毛麦HMW-GS编码基因,并从分子水平上把簇毛麦HMW-GS基因定位于簇毛麦1V染色体上。     

不同品质类型小麦籽粒麦谷蛋白亚基及谷蛋白聚合体形成和累积动态

选用高分子量麦谷蛋白亚基(HMW-GS)组成不同的3个强筋和4个弱筋小麦品种,研究了其籽粒发育过程中麦谷蛋白亚基、谷蛋白聚合体的形成和累积动态。结果表明,强筋小麦籽粒HMW-GS和B区低分子量麦谷蛋白亚基(LMW-GS)从花后9～12d开始表达;而弱筋小麦从花后12～15d开始表达,即强筋小麦麦谷蛋白亚基开始形成时间早于弱筋小麦。各品种的HMW-GS一旦形成,其累积速度较快,花后27d基本达到稳定值,之后维持稳定量;而LMW-GS形成后,累积较慢,直到花后30d左右达到稳定量。3个强筋小麦品种籽粒灌浆期谷蛋白总聚合体百分含量(TGP%)和谷蛋白大聚合体百分含量(GMP%)累积动态趋势基本一致,即在花后12～30d一直持续增加,花后30d至成熟达到最大值并保持稳定水平。4个弱筋小麦TGP%和GMP%累积动态均表现为在花后12～24d(灌浆早中期)形成和持续累积,花后24d至成熟逐渐降低。麦谷蛋白亚基表达模式以及谷蛋白聚合体累积动态的差异可能是导致小麦强筋或弱筋品质形成的关键。     

温度对胡瓜新小绥螨生殖力及钠钾泵α亚基基因表达的影响

胡瓜新小绥螨Neoseiulus cucumeris（Oudemans）广泛地用于蓟马类害虫的防治、控制及管理。本研究克隆了1条胡瓜新小绥螨钠钾泵α亚基基因序列,分析了温度对雌螨生殖力及钠钾泵α亚基基因表达的影响。在15、20、25和30℃下,胡瓜新小绥螨在25℃时生殖力最优。在上述温度条件下胁迫48 h,雌螨钠钾泵α亚基在15和30℃下表达量显著上升,能量代谢活跃。表达量越多,表明能量消耗越大,投入生殖的能量就越少,结合生殖力研究结果,二者一致。高温或低温均会降低胡瓜新小绥螨的生殖力,且高温影响更为严重。