通用型转铁蛋白融合表达载体的构建及应用价值

构建通用型转铁蛋白融合表达载体,利用PCR方法扩增编码人转铁蛋白N端半分子的基因片段,通过酶切、连接、转化等分子克隆方法构建通用型转铁蛋白融合表达载体。PCR扩增了一个长约1.1 kb的包含ScaI酶切位点的基因片段,插入pPICZα的PmlI和XbaI酶切位点,转化后进行菌液PCR鉴定,成功获得重组子pPICZα-TfN,测序结果表明载体构建成功,重组质粒pPICZα-TfN能被ScaI酶切。本研究成功构建通用型转铁蛋白融合表达载体,构建的载体可以用于转铁蛋白融合表达载体的构建。     

跨膜型TNF-alpha单克隆抗体腺病毒表达载体的构建和鉴

目的：应用AdEasy-1 系统构建包含tmTNF-alpha单克隆抗体轻、重链序列的重组腺病毒表达载体。方法：首先PCR 合成抗体 轻、重链序列,分别将轻、重链序列插入经过改造的含有双启动子的穿梭质粒pShuttle-2CMV,将穿梭载体电转化转化AdEasy 系 统BJ5183 感受态,挑取单克隆扩增质粒后酶切鉴定。结果：成功构建重组腺病毒表达载体pAdEasy-tmTNF-alpha,抗体重链、轻链序 列酶切后经1%琼脂糖凝胶电泳证实条带片段大小正确,电转化BJ5183 后挑选重组克隆提取质粒,PacI酶切后重组片段位于4.5 kb及3 kb位置,证明重组腺病毒质粒pAdeasy-1-tmTNF-alpha构建成功。结论：将腺病毒系统与单克隆抗体技术相结合,利用AdEasy-1 系统成功构建腺病毒重组tmTNF-alpha单克隆抗体表达载体,为进一步开展肿瘤基因治疗的研究提供基础。     

肺炎支原体P1蛋白羧基端基因片段在大肠杆菌中克隆的研究

在大肠杆菌中克隆肺炎支原体P1蛋白羧基端基因片段,为P1蛋白基因片段的扩增、表达及探讨羧基端基因片段功能打基础.采用PCR扩增方法获取P1结构基因.扩增产物用SalI和EcoRI酶切消化,回收1kb大小的DNA片段并与pUC19DNA连接,转入大肠杆菌JM109菌株.用X-gal平板及质粒图谱分析方法筛选重组克隆株,再用限制性核酸内切酶酶切图谱分析鉴定.经PCR扩增MPDNA获得1条5.0kbDNA片段.重组质粒限制性内切酶指纹图谱显示出2条带,1条为pUC19载体DNA带,另1条是1kb的插入片段.实验获得肺炎支原体P1蛋白结构基因及含P1蛋白羧基端DNA片段的重组克隆株.     

跨膜型TNF-α单克隆抗体腺病毒表达载体的构建和鉴定

目的:应用AdEasy-1系统构建包含tmTNF-α单克隆抗体轻、重链序列的重组腺病毒表达载体。方法:首先PCR合成抗体轻、重链序列,分别将轻、重链序列插入经过改造的含有双启动子的穿梭质粒pShuttle-2CMV,将穿梭载体电转化转化AdEasy系统BJ5183感受态,挑取单克隆扩增质粒后酶切鉴定。结果:成功构建重组腺病毒表达载体pAdEasy-tmTNF-α,抗体重链、轻链序列酶切后经1%琼脂糖凝胶电泳证实条带片段大小正确,电转化BJ5183后挑选重组克隆提取质粒,PacI酶切后重组片段位于4.5kb及3 kb位置,证明重组腺病毒质粒pAdeasy-1-tmTNF-α构建成功。结论:将腺病毒系统与单克隆抗体技术相结合,利用AdEasy-1系统成功构建腺病毒重组tmTNF-α单克隆抗体表达载体,为进一步开展肿瘤基因治疗的研究提供基础。     

不同区段HIV-1 env基因在Bac-to-Bac昆虫细胞杆状病毒表达系统中的表达及检测

利用昆虫细胞杆状病毒表达系统,将从一株HIV-1阳性克隆质粒中获得的几个HIV包膜蛋白基因片段,克隆入转移载体中得到重组病毒.用此重组病毒感染昆虫细胞后表达出3种HIV包膜蛋白,即GP120-41P、GP41T、GP41P,分别含有HIV-1包膜糖蛋白GP120及部分GP41,删除了N端12个疏水氨基酸的GP41和仅有主要表位约240个氨基酸的GP41.收获后分别以Western-blotting和EIA检测,有较好的免疫学活性,其中GP41T的活性最强.该实验为HIV包膜蛋白的结构研究提供了依据,加以改进后可能有免疫检测的价值.     

平滑肌肌球蛋白轻链激酶的非激酶作用及对ATP 酶活性的调节

肌球蛋白轻链激酶（myosin light chain kinase, MLCK）具有激酶活性和非激酶活性,在平滑肌收缩过程中起着关键酶调控的作用.为探寻MLCK的非激酶活性区域对MLCK活性的影响,以进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制.采用PCR技术构建MLCK部分氨基酸缺失的重组表达载体pGEX-F6-5/D,经大肠杆菌表达得到可溶性GST融合蛋白,利用SDS-PAGE及Western 印迹鉴定表达的MLCK在细胞中的分布,结果还显示,提取液的上清和沉淀中均有MLCK片段的表达.运用亲和层析技术分离并纯化删除前、后表达的MLCK片段（F6.5和F6-5/D）,经谷胱甘肽琼脂糖凝胶 4B 纯化,SDS-PAGE鉴定显示为单一表达条带.应用EnzChek磷分析试剂盒和孔雀绿两种方法分别测定不同浓度的MLCK对非磷酸化肌球蛋白Mg²⁺-ATP酶活性的影响.两种MLCK的片段均具有激活ATP酶活性的作用,并随MLCK浓度的增加,酶的活性增加.比较删除前后不同MLCK片段对ATP酶活性的影响结果显示,删除MLCK片段1002位丙氨酸至1019位亮氨酸后,对ATP酶的激活作用较删除前明显降低,表明删除的部分氨基酸序列为MLCK非激酶活性所必需的区域.利用电镜技术观察到MLCK片段（F6.5）使非磷酸化肌球蛋白构象发生明显的变化.加入MLCK片段后肌球蛋白的构象由非活性型转化为活性型,并且MLCK片段还具有促进肌球蛋白单体形成肌丝的作用.     

重组腺相关病毒介导的大鼠脂联素载体的构建与鉴定

目的:克隆大鼠脂联素基因(Acrp30),并对载有脂联素的重组腺相关病毒(AAV)载体进行构建及鉴定.方法:根据Acrp30的基因序列设计引物,上游引入EcoR Ⅰ位点,下游引入Sal Ⅰ位点,以EX-A0242-M01-Acrp30为模板扩增目的基因.将Acrp30基因克隆入pUC19载体中,获得重组质粒pUC19-Acrp30.然后用EcoR Ⅰ与sal Ⅰ酶切pUC19-Acrp30及质粒pSNAV,回收酶切片段,T4DNA连接酶16℃过夜,转化大肠杆菌DH5a感受肽细胞,筛选阳性克隆获得pSNAV-Acrp30,EcoR Ⅰ/Sal双酶切鉴定,并行全基因测序.结果:PCR电泳及酶切鉴定表明,pSNAV-Acrp30重组成功,基因测序显示装入pSNAV质粒中的Acrp30基因正确.结论:脂联素病毒栽体成功构建,可以满足脂联素基因研究和治疗的需要.     

粘虫颗粒体病毒增效因子的基因定位

参考粉纹夜蛾Trichoplusia ni 颗粒体病毒增强因子的基因序列,设计PCR引物,用PCR反应扩增出特异性产物。用EcoRⅠ、BamHⅠ双酶酶切处理PCR反应产物,然后克隆到质粒pUC19中,构建重组质粒pUC19-SF;对重组质粒pUC19-SF中的外源片段测序,结果证明PCR扩增产物是粘虫颗粒体病毒PuGV-Ps增效因子基因的一段序列。重组质粒pUC19-SF的插入片段标记为探针,通过Southern杂交将增效因子基因定位于PuGV-Ps病毒基因组的多种酶切片段上。     

盐藻酵母双杂交诱饵载体的构建及自激活鉴定

构建用于杜氏盐藻核基质结合区结合蛋白(matrix attachment region binding protein,MBP)酵母双杂交试验的诱饵栽体,进行自激活及毒性验证.以含有盐藻MBP质粒为模板,经PCR扩增后连接pMD 18-T栽体,经测序鉴定正确后,EcoR Ⅰ/Nde Ⅰ双酶切获得目的基因MBP,克隆入经同样双酶切的酵母栽体pGBKT7,转化酵母菌株AH109及Y187,检测其自激活以及毒性.结果显示,成功扩增出盐藻MBP基因,重组质粒pGBKT7-MBP经酶切、测序表明序列正确,转化酵母菌株无自激活,无毒性.     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.     

Determination of stoichiometry of radioiodination of proteins

A sensitive method for the detection of small quantities of hydrophobic antioxidant free radical scavengers such as butylatedhydroxytoluene (BHT) and butylatedhydroxyanisole (BHA) in aqueous samples is described. The procedure involves extraction of the hydrophobic free radical scavenger into an organic solvent phase, followed by the subsequent reaction of an aliquot of this extract with the stable cation radical tris(p-bromophenyl)amminium hexachloroantimonate (TBACA). In experiments with BHT and BHA, the loss of TBACA absorbance at 730 nm was found to be linearly proportional to the amount of antioxidant added, with quantities of BHT as small as 200 pmol being easily detectable. In aqueous suspensions of dimyristoylphosphatidylcholine vesicles, assays of the aqueous BHT concentration showed that BHT partitioned strongly into the membrane phase, achieving very high BHT/phospholipid ratios. For a given concentration of BHT, partitioning into the membrane phase was greater in large, multilamellar liposomes than in either small, single-walled vesicles or in purified rat brain synaptic vesicle membranes. Direct assay of BHT and BHA in phospholipid membranes, however, was complicated by a nonspecific interaction between TBACA and the phospholipid.