口蹄疫病毒衣壳蛋白氨基酸突变对病毒热稳定性的影响

口蹄疫是世界范围内广泛流行的一种偶蹄动物的烈性传染病,其病原为口蹄疫病毒(FMDV),它对热比较敏感,当温度高于30℃时,病毒衣壳容易解离为五聚体亚单位而影响疫苗的免疫保护效果。因此,改善病毒的热稳定性,制备热稳定优良的口蹄疫疫苗具有重大的应用价值。在实验室建立的口蹄疫病毒型内嵌和全长感染性克隆的基础上,在结构蛋白引入1个或2个氨基酸突变,分别构建了2种基因修饰的口蹄疫病毒全长重组质粒。全长质粒经NotⅠ线化后分别转染表达T7RNA聚合酶的BSR/T7细胞以拯救重组病毒。间接免疫荧光和电镜分析表明,成功拯救到2株口蹄疫重组病毒。重组病毒经RT-PCR和序列测定证实只有1株重组病毒所含的耐热突变可以稳定遗传。热失活试验显示,拯救病毒与亲本病毒的耐热特性基本一致。结果表明在结构蛋白上引入V2090A-S2093F-H30568R的突变,并对口蹄疫病毒的热稳定性并没有明显的影响,为未来发展热稳定的口蹄疫疫苗提供了一定的参考。     

前导肽区域N-糖基化影响骆驼凝乳酶原在毕赤酵母中的分泌表达和热稳定性研究

骆驼凝乳酶原在毕赤酵母中的表达量较低,不能满足产业化需求。为提高骆驼凝乳酶原的表达量,采用定点突变的方法,分别在其前导肽的第11、26、34、35、38、39位氨基酸处引入1个N-糖基化位点,构建成6种骆驼凝乳酶原的N-糖基化突变体：chy11、chy26、chy34、chy35、chy38和chy39,并分别导入到毕赤酵母中进行分泌表达。测定和比较了野生型（wild）和各突变体骆驼凝乳酶原的表达量。Chy34、chy35、chy38和chy39的表达量显著提高,其中chy34的表达量最高。6种突变体均保持前导肽自切割活性。在50 ℃保温8 h,wild酶活完全丧失,而突变体仍有20%~80%的相对酶活,表明6种突变体的热稳定性均显著增强。研究结果为提高骆驼凝乳酶原在毕赤酵母中的表达量和增强热稳定性提供了新的研究思路。     

Worldwide distribution of β-amylase thermostability in barley

The thermostability of β-amylase in 6752 lines of worldwide barley genetic resources were investigated. Most of the lines were classified into high (type A), medium (type B) , and low (type C) thermostability. Subsequently the geographical distribution of these types was clarified. About 90% of the East Asian (Japan, the Korean Peninsula, China) lines were type A. More than 95% of Ethiopian barley was type C. The thermostability types of varieties in the western areas (north Africa, southwest Asia, Turkey, Europe) consisted of types A, B and C. These results suggest that there is a clear geographical differentiation in β-amylase thermostability, especially in East Asia and Ethiopia. The phenotype characteristics of each thermostability type line reflected the geographical differentiation. Besides types A, B and C, we found new thermostability types, including such useful mutants as a β-amylase-less mutant and highly-thermostable mutants than type A in both China and Nepal. This revised version was published online in August 2006 with corrections to the Cover Date.     

Geographical variation of β-amylase thermostability among varieties of barley (Hordeum vulgare) and β-amylase deficiency

Investigations onto the thermostability of β-amylase in 274 varieties of barley (Hordeum vulgare L.) indicated that all varieties except one were distributed into three types of high (type A), intermediate (type B), and low (type C) thermostability, respectively. One variety (TB29) from China showed no β-amylase activity. Geographical variation was observed in the thermostability of β-amylase. Type C varieties were not observed in East Asia (Japan, the Korean Peninsula, China and Nepal), although 36 out of 37 varieties in Ethiopia were type C. Most of the varieties were Type A in Japan, the Korean Peninsula and China, whereas the frequency of type A and type B were nearly equal in Nepal. Varieties in the other five areas (North America, North Africa, Southwest Asia, Turkey and Europe) consisted of types A, B and C. These results support the fact that East Asian cultivars are genetically different from those of the western regions, as previously reported.     

日本鲟肌动球蛋白热变性和冷冻变性

研究了日本鲟肌动球蛋白经变性和冷冻后ＡＴＰａｓｅ活性、总巯基数和不饱和巯基数及蛋白质浓度的变化。结果表明,热变性后肌动球蛋白Ｃａ＾２＋－ＡＴＰａｓｅ和Ｍｇ＾２＋－ＡＴＰａｓｅ活性下降,且Ｃａ＾２＋－－ＡＴＰａｅｓ活性有明显转折。低温热变性对总巯基数影响较小,温度增高时,巯基数逐渐下降,而不饱和巯基数在３０℃之前,呈动态变化,之后又逐渐减少。热变性后,肌动球蛋白浓度也降低。冷冻变性后,肌动球蛋白ＡＴ     

Genetic variation of β-amylase thermostability among varieties of barley, Hordeum vulgare L., and relation to malting quality

To investigate variation in the thermostability of β-amylase among varieties of barley, Hordeum vulgare L., crude enzyme was extracted from the seeds, and the relative remaining activity was calculated after heat treatment. Our results indicated that the varieties tested were divided into three groups (types A, B and C). All the latest Japanese malting varieties showed high themostability (type A), while European, North American and Australian varieties showed intermediate (type B) or low thermostability (type C). Isoelectric focusing (IEF) of β-amylase was also investigated. Type B varieties divided into two subtypes (types B1 and B2) based on two different IEF patterns (types I and II), whereas those of types A and C showed only one IEF pattern (type II). We also examined their thermostability in the varieties based on the pedigrees of the Japanese malting barley. Our results indicated that the thermostability of β-amylase had a close relationship to fermentability for the production of beer. This fact suggests that the thermostability of β-amylase has a significant influence on the malting quality of barley.     

Diversity and geographical distribution of seed lipoxygenase‐1 thermostability types in barley

To understand the diversity in the thermostability of the seed lipoxygenase‐1 (LOX‐1), 1040 cultivars of worldwide barley (Hordeum vulgare ssp. vulgare) genetic resources were investigated. The relative thermostability of LOX‐1 (LOX‐RTS) in these lines showed a bimodal frequency distribution and these lines were categorized into the high and low thermostability types (H‐type and L‐type, respectively). The H‐type lines predominated in the wild progenitor, ssp. spontaneum. The geographical distribution of these types in the cultivars was surveyed. The frequencies of the H‐ and L‐types were almost equal to one another in southwestern Asia. The occurrence of the H‐type predominated in eastern Asia and Africa, whereas in Europe and Turkey, the L‐type did. The predominance of the L‐type in Europe and Turkey can be understood through the hypothesis that the Fertile Crescent domestication contributed the majority of diversity in Europe. The uneven geographical distribution of the LOX‐1 thermostability types in the cultivars may reflect a polyphyletic origin of barley.     

白色链霉菌产木聚糖酶规律及其耐热耐碱性的初步研究

该文采用白色链霉菌为实验菌种,在不同诱导产酶培养基上经过240 h的振荡培养,探索其产酶时程规律. 结果表明,不同的诱导底物诱导产生的最大木聚糖酶活不等,其中木粉的诱导效果好于纯木聚糖,复合底物的诱导效果好于单一底物. 该菌在筛选出的最佳诱导产酶培养基上培养144 h后达到产酶高峰,粗酶液酶活可达到45.66 U/mL. 对该酶进行高温及碱性处理的结果表明,它具有较好的耐热耐碱性,在pH 7.0下反应表现最高活性,同时在90℃下保温30 min后酶活残留达83.46％.      

经典恒温法对复方五倍子口服液的热稳定性研究

分别用超声法和醇提法制备复方五倍子口服液,以复方中主要成分——没食子酸含量变化为对照,用经典恒温法对以上2种制备方法进行热稳定性研究．结果表明,采用超卢法制备的复方五倍子口服液有效期为82d,醇提法制备为70d,与室温留样观察法得出的结果基本一致。