杂色鲍组织蛋白酶L的分离纯化与性质研究

通过硫酸铵分级沉淀、SP-Sepharose阳离子交换层析、Sephacryl S-200HR凝胶过滤和Hydroxyapatite羟基磷灰石层析,从杂色鲍（Haliotis diversicolor）内脏中分离纯化了组织蛋白酶L。SDS-PAGE结果显示,纯化蛋白的分子量约为28ku。该酶最适温度37℃,最适pH5.5。当温度低于40℃,pH在5.0～6.5范围内该酶较稳定。底物特异性实验与抑制剂实验结果表明,该酶属于半胱氨酸蛋白酶。金属离子Mn2＋、Ba2＋和Mg2＋对该酶有不同程度的激活作用,而Co2＋、Cu2＋、Zn2＋、Fe2＋和Ca2＋等对该酶起抑制作用。     

Application of Novel Polymorphic Microsatellite Loci Identified in the Korean Pacific Abalone (Haliotis diversicolor supertexta (Haliotidae)) in the Genetic Characterization of Wild and Released Populations

The small abalone, Haliotis diversicolor supertexta, of the family Haliotidae, is one of the most important species of marine shellfish in eastern Asia. Over the past few decades, this species has drastically declined in Korea. Thus, hatchery-bred seeds have been released into natural coastal areas to compensate for the reduced fishery resources. However, information on the genetic background of the small abalone is scarce. In this study, 20 polymorphic microsatellite DNA markers were identified using next-generation sequencing techniques and used to compare allelic variation between wild and released abalone populations in Korea. Using high-throughput genomic sequencing, a total of 1516 (2.26%; average length of 385 bp) reads containing simple sequence repeats were obtained from 86,011 raw reads. Among the 99 loci screened, 28 amplified successfully, and 20 were polymorphic. When comparing allelic variation between wild and released abalone populations, a total of 243 different alleles were observed, with 18.7 alleles per locus. High genetic diversity (mean heterozygosity = 0.81; mean allelic number = 15.5) was observed in both populations. A statistical analysis of the fixation index (F_ST) and analysis of molecular variance (AMOVA) indicated limited genetic differences between the two populations (F_ST = 0.002, p > 0.05). Although no significant reductions in the genetic diversity were found in the released population compared with the wild population (p > 0.05), the genetic diversity parameters revealed that the seeds released for stock abundance had a different genetic composition. These differences are likely a result of hatchery selection and inbreeding. Additionally, all the primer pair sets were effectively amplified in another congeneric species, H. diversicolor diversicolor, indicating that these primers are useful for both abalone species. These microsatellite loci may be valuable for future aquaculture and population genetic studies aimed at developing conservation and management plans for these two abalone species.     

沙蚕多糖提取工艺及抗氧化活性研究

对沙蚕多糖提取工艺条件的优化和抗氧化活性进行研究。在单因素试验基础上,采用响应面法对温度、料液比、时间3个因素进行优化。在提取温度83℃、料液比1∶25(g/mL)、提取时间3.5 h的最佳条件下,沙蚕多糖的提取率为1.76%,与预测值1.77%相当。经测定沙蚕多糖对羟自由基、超氧阴离子自由基和DPPH自由基均有较好的清除作用,表明沙蚕多糖具有抗氧化活性。     

一株溶藻弧菌的分离鉴定及其对九孔鲍苗的致病性

为找到导致2002年以来中国南部沿海鲍鱼养殖大量死亡的原因,利用胰蛋白培养基或硫代硫酸盐-柠檬酸盐-胆盐-蔗糖培养基从2003年汕尾发生病害的鲍鱼中分离得到了一株具有致病性的细菌.经生理生化特性试验及与特征溶藻弧菌ATCC17749比较,此菌株为溶藻弧菌.经感染试验证明其对鲍苗的半致死量为1.58x 104 CFU/m...     

沙蚕多肽提取及功能研究

目的通过研究沙蚕酶解及其多肽产物的功能,提高沙蚕的利用率。方法采用碱性蛋白酶、胃蛋白酶、中性蛋白酶对沙蚕进行酶解,以水解度为评价指标,比较3种酶解产物的抗氧化活性、总还原力能力和降血糖功能。结果 3种酶中中性蛋白酶在6 h达到最高水解度37.8%。碱性蛋白酶酶解7 h的产物其亚铁离子螯合力最高,可达32.7%;DPPH自由基清除能力最高可达85.8%,为中性蛋白酶酶解6 h的产物;胃蛋白酶酶解6 h产物的总还原力最高;胃蛋白酶酶解3 h的酶解产物其DPP-4抑制率最高,可达52.9%。结论胃蛋白酶更适于沙蚕的酶解。酶解产物具有较理想的抗氧化及降血糖功能,为沙蚕多肽产品的开发奠定基础。     

新琼寡糖作为免疫增强剂在鲍鱼养殖中的应用

采用一种活性新琼寡糖作为免疫增强剂,通过在饲料中添加不同含量的新琼寡糖(分别为饲料重量的0.1%,0.5%,1%,1.5%和3%),以血清溶菌酶活力、血清补体活性及白细胞总数和白细胞吞噬率为判断依据,确定新琼寡糖对于鲍鱼非特异性免疫力的影响;同时用嗜水气单胞菌(Aeromonas hydrophila)对各组鲍鱼进行细菌感染试验,测定鲍鱼存活率,以确定新琼寡糖对鲍鱼抗病力的影响。结果表明:投喂新琼寡糖组的鲍鱼的血清溶菌酶活力、血清补体活性及血液白细胞总数和吞噬率均明显高于对照组,且新琼寡糖的最佳投喂量为饲料重量的0.5%~1%。细菌感染试验结果显示,在细菌感染72 h后,对照组鲍鱼的存活率最低,仅为42%,而新琼寡糖投喂组鲍鱼的存活率均达到58%以上,且最高可达到79%。     

蛭弧菌对九孔鲍非特异性免疫力和抗病力的影响

本试验以生产的养殖池作为参照(A组),以采用15d的换水周期并往水体中添加不同浓度(分别为10<'1>pfu/mL、10<'3>pfu/mL、10<'5>pfu/mL和10<'7>pfu/mL)的蛭弧菌的养殖池为蛭弧菌添加组(分别为B组、C组、D组和E组),以溶菌酶活力、超氧化物歧化酶(SOD)活力,酸性磷酸酶(ACP...     

从AFLP指纹和标记基因序列看我国养殖的四种鲍的亲缘关系

九孔鲍是我国南方主要的鲍养殖对象,皱纹盘鲍与盘鲍则在北方被广泛养殖。对于皱纹盘鲍与盘鲍,尤其是九孔鲍与杂色鲍的分类关系,迄今学术界还存在着不同看法。本文通过AFLP指纹的比较以及细胞核ITS-1和18SrRNA基因片段、线粒体16S rRNA和COI基因片段DNA序列的比较,发现九孔鲍与杂色鲍之间遗传趋异很小,只达到不同地方群体差异的水平;皱纹盘鲍与盘鲍趋异较大,可以认为是属于不同亚种。本研究结果同时表明AFLP技术是进行鲍种质鉴别和遗传多样性研究的有用手段。     

Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase

A gene coding for the Nereis sarcoplasmic calcium-binding protein(NSCP) was synthesized and expressed in Escherichia coli. Thesequence of the gene was derived from the protein sequence byreverse translation. It possesses a number of unique, regularlyspaced, restriction endonuclease cleavage sites to facilitatefuture site-directed mutagenesis. For the cloning strategy thegene sequence was divided into four parts. Three parts werecloned by ligation of hybridized oligomers and one part by inversePCR. The protein was expressed as a fusion protein with thebacterial chloramphenicol acetyltransferase (CAT), which couldbe easily purified by affinity chromatography. At the junctionof the CAT and NSCP moieties a recognition site for the proteolyticenzyme factor Xa was built in. However, the distance betweenthe moieties appeared to be crucial to warrant cleavage. A kineticanalysis showed that NSCP prepared from the sandworm and theone expressed by E.coli behaved in the same way. This systemprovides a basis for site-specific mutagenesis studies, in orderto elucidate the molecular mechanism of cation binding and concomitantconformational changes     

鲍血淋巴抗菌肽的研究

一定浓度的大肠杆菌可诱导杂色鲍产生抗菌肽，不同诱导时间所产生的抗菌肽抑菌活性不同。研究结果显示诱导时间以8h为最佳，这类抗菌肽具有热稳定性、耐酸、不溶血、没有凝血活性，利用12．5％的聚丙烯酰胺凝胶电泳检测抗菌肽的分子量在3000—6000Da左右。