蛙虹彩病毒cop基因的克隆表达及亚细胞定位

从蛙虹彩病毒(Rana gryliovirus,RGV)基因组中克隆了含凋亡相关结构域的新基因-cop(Caspaserecruit ment domain only protein,COP)基因的全部编码区,成功构建了重组表达载体,进行了原核表达,并在鲤鱼上皮瘤细胞(Epithelioma papulosumcyprini,EPC)中进行了亚细胞定位.序列分析表明,RGVcop基因全长288 bp,编码一个长为95 aa,分子量为10.4×103的推定蛋白.二级结构预测表明其含有5个α螺旋.同源性比对分析显示,RGVcop与其他6株虹彩病毒及人类cop相关蛋白同源性最高为94%,最低为33%.重组原核表达质粒pET28a-COP转化大肠杆菌BL21后,经IPTG诱导表达,获得一条约14×103的融合蛋白.重组真核表达质粒pEGFPN3-COP转染EPC细胞,经DAPI染色后,在荧光显微镜下观察显示重组蛋白在整个细胞中均有分布.     

PCR-based Gene Synthesis,Molecular Cloning,High Level Expression,Purification, and Characterization of Novel Antimicrobial Peptide,Brevinin-2R,in <Emphasis Type="Italic">Escherichia Coli</Emphasis>

Brevinin-2R, a member of a new family of antimicrobial peptides isolated from the skin of Rana ridibunda, displays antimicrobial activity against bacteria and fungi. In this study, we have used an assembly PCR method for the fast and extremely accurate synthesis of the brevinin-2R gene. A total of six primers were assembled in a single step PCR, and the assembly was then amplified by PCR to produce the final gene. The synthetic gene was cloned into the pET32a (+) vector to allow the expression of brevinin-2R as a Trx fusion protein in Escherichia coli. The results indicated that the expression level of the fusion protein could reach up to 25% of the total cell proteins. The expression products could be easily purified by Ni-NTA chromatography and released from the fusion protein by factor Xa protease. The peptide displayed antimicrobial activity similar to that of the purified brevinin that was reported earlier. This method allows the fast synthesis of a gene that optimized the overexpression in the E. coli system and production of sufficiently large amounts of peptide for functional and structural characterizations.     

一种中国林蛙抗菌肽的光谱研究

从中国林蛙皮中纯化得到了一种抗多种临床多重耐药菌的抗菌肽(RTCⅠ), 初步氨基酸组成分析结果表明其不含碱性氨基酸, 此抗菌肽在276.5和356.5 nm波长光的激发下发射出448 nm的荧光, 利用傅里叶变换红外光谱、拉曼光谱、电子吸收光谱及荧光光谱等技术研究了此特定荧光产生的结构依据. 此抗菌肽的主要组成是Tyr, Asn(Asp)和Glu(Gln), 抗菌肽特殊的荧光光谱和电子吸收谱与Tyr的酚羟基和Asn侧链的强氢键有关. 这一特殊的荧光(448 nm)及圆二色谱(259, 263和267 nm)信号为进一步在分子水平上研究此抗菌肽的抗菌机理提供了依据.     

中国林蛙繁殖产量与雌体大小及年龄的关系

采用相关分析和回归分析研究了中国林蛙(Rana chensinensis)同一种群内母体大小、年龄与繁殖产量的关系．结果表明：(1)母体大小对个体的繁殖产量有显著影响．随着体长的增长，窝卵数、卵径和窝卵质量随之增加；年龄对窝卵数、卵径影响不显著，但与窝卵质量呈正相关．(2)在特定的体长范围内，窝卵数与卵大小间存在一种权衡；母体产的窝卵数越多，卵母细胞相对就越小，反之亦然．     

取代芳烃的急性毒性与连接性指数(nL)的相关性

提出表征原子生物活性的特征值（Ei)，由Ei建构新的连接性指数（^nL),并用^0L、^1L与取代芳烃对日本长腿蛙蝌蚪、发光菌、呆鲦鱼的毒性数据进行关联，其二元相关系数分别为0．9660、0．9172、0．9691，明显优于文献方法。     

棘胸蛙肌肉营养成分的分析Ⅱ.氨基酸及矿物元素的组成

本文对棘胸蛙的蝌蚪(体重 0. 8～ 2. 6g)、小幼蛙(体重 9. 0～ 27. 3g)、大幼蛙 (体重 40. 5～ 65. 7g)和成蛙 (134. 5～ 213. 0g)肌肉及蛙皮中的氨基酸及矿物元素的组成进行了分析 ,结果表明:肌肉中人体必需氨基酸的含量占氨基酸总量的 44. 06% ,谷氨酸的含量很高,为氨基酸总量的 15. 65% ;肌肉中含有20种以上的矿物元素,具有重要生理功能的硒等微量元素含量较丰富,含量为 0. 029mg/100g. 本研究在分析与比较的基础上评价了棘胸蛙的营养价值,为棘胸蛙配合饲料的开发提供理论上的依据.     

ICP-AES测定林蛙皮肤中多种微量元素

针对林蛙皮肤组织生物有机体的复杂组成,进行微波辅助消解、硝酸过氧化氢湿法消解和低温干灰化法三种制备样品方法比较,利用数理统计方法确定最佳样品处理方法.微波消解法具有时间短、有机质消解能力强和试样溶液均匀性好的特点,可以大大缩短分析流程,在HNO_3-H_2O_2-HCI-H_2O酸溶体系,阶段性输出功率条件下进行试样密闭消解制备样品的方法,采用标准溶液基体匹配法消除基体干扰,利用ICP-AES分析技术,同时检测林蛙皮肤组织样品常量和微量元素.该方法经国家猪肝标准物质GBW08551验证,测得方法相对误差为0.17%-6.67%.方法回收率为97.6%-100.6%.各项检测指标均能满足生物样品检测要求,达到满意的测量结果.     

Irradiation damage to frog inner ear during synchrotron radiation tomographic investigation

Unexpectedly severe radiation damage, showing up through deformation of the saccule, was encountered during a synchrotron radiation high-resolution (700 nm pixel size) tomographic observation of an inner ear, fixed in a formaldehyde solution, of the frog Rana esculenta. The visible displacement of the edge of the otoconia-filled part of the saccule amounted to about 100 μm after an irradiation with 20.5 keV X-ray photons corresponding to a dose of 1.5 kGy for the protein matrix. The close-knit coexistence of organic and mineral components in the biological tissue may be linked to the dramatic increase of radiation dosage sensitivity.     

ISOLATION, CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF MARGARATENSIN, A NEUROTENSIN-RELATED PEPTIDE FROM THE SKIN OF Rana margaratae

A new neurotensin (NT)-related peptide, margaratensin, was obtained by Sep-Pak C_(18) and RP-HPLC from methanol extracts of the skin of Chinese frog Rana margaratae. The structure of the peptide has been determined to be Asp-Lys-Arg-Pro-Tyr-lle-Leu-His-Glu, which is found to be homologous to the COOH-terminal sequence of NT, but has an extra His-Glu at the COOH-terminus. The synthetic preparation was shown to be indistinguishable from the native peptide during HPLC, amino acid analysis and bioassay. Margaratensin exhibited a hypotensive effect in the rat but the response was weaker than NT. The peptide could induce a potent and reproducible contractile activity on GPI which was different from xenopsin, another NT-related peptide from amphibian skin.     

棘胸蛙肌肉营养成分的分析（Ⅰ）：一般营养成分的含量及脂肪酸？…

本文对棘胸蛙的蝌蚪,小幼蛙（体重９．０－２７．３ｇ）,大幼蛙（体重４０．５－６５．７ｇ）和成蛙（１３４．５￣２１３．０）肌肉及蛙皮中的水分,蛋白质,脂肪和灰分含量及脂肪酸的组成进行了分析,结果表明,棘胸蛙肌肉中的蛋白质含量高达１８．５５％￣１９．３９％,脂肪含量较低。肌肉中高度不饱和脂肪酸分量占脂肪酸总量的３９．３６％。本研究在分析与比较的基础上评价了棘胸蛙的营养价值,为棘胸蛙配合饲料的开发提供了