虹鳟鱼(Salmo grairdneri Richard)基因文库的构建及生长激素基因的克隆和亚克隆

以噬菌体EMBL3作为载体,构建了虹鳟鱼的全基因文库,制备了包装蛋白效率达4.6×10~8Pfu/μgDNA.基因文库重组噬菌的量为8.2×10~5pfu/μgDNA.同时用含虹鳟鱼生长激素基因cDNA的pAF-51△S作为探针,从该文库中筛选出两个阳性噬斑,复筛获得90％以上的阳性斑。并从阳性克隆中回收虹鳟收生长激素基因片段为6kb,亚克隆到puc-18质粒中,进行了酶切分析。     

志贺氏菌属弗氏2a染色体基因文库的构建

以λ噬菌体EMBL3作载体,用体外包装技术构建了志贺氏菌属弗氏2a染色体基因文库。该体外包装系统的包装效率达1.6×10~2pfu/μg DNA,重组噬菌体的产量达2×10~6pfu/μg DNA。对重组噬菌体进行了遗传分析,即分别对7个标记(leu,pro,his,arg,thr,purIaroA)作了测定。测得当噬菌体母液的效价为8.7×10~8pfu/ml时,带有以上标记的重组噬菌体的效价为5×10~4-8.2×10~5pfu/ml。这个令人满意的结果为尔后克隆志贺氏菌属弗氏2a染色体上的与群抗原3,4和型抗原Ⅱ有关的基因打下了基础。     

高效率提取λ噬菌体DNA离体包装抽提物

λ噬菌体DNA在与被克隆的DNA片段结合成重组体后,需进行体外包装才能形成侵染性的噬菌体颗粒。这里仅就如何提取高效能人噬菌体DNA包装抽提物（包装蛋白）谈谈体会。（1）菌落的选择从BHB2688和BHB2690的不同菌落中提取的包装抽提物的包装效率（讨U小g／＊＊A）可相差10倍以上。因此在大量制备以前,有必要先挑选多个菌范小批量制备抽提物并测定其包装效率,取优良者进行制备。（2）热诱导后的培养时间文献所载,在热诱导后培养至细菌可被氛仿裂解时即可停止。但我们的经验是12”IX10’养一段时间可明显提高抽提物的效价（见图1）…     

影响λ噬菌体包装蛋白抽提物效价的因素

<正> 以λ噬菌体DNA作载体构建基因文库时,λ噬菌体包装蛋白抽提物是实验成败的关键之一,市场上虽有此商品出售,但由于价格昂贵加上运输和保藏方面的问题,使用时往往不够理想。所以,不少实验室自己制备噬菌体包装蛋白抽提物。任兆韵等报导:快速诱导可以提高包装效价,在这里我们报导包装反应时间等其他因素对λ噬菌体包装蛋白抽提物效价的影响。     

用λ－ZAPⅡ作载体构建豌豆卷须cDNA文库

用改进的异硫氰酸胍－苯酚－氯仿抽提法提取了豌豆卷须总ＲＮＡ并纯化了ｍＲＮＡ,合成双链ｃＤＮＡ后加上人工接头,Ｓｅｐｈａｒｏｓｅ２Ｂ柱层析去掉小片段,最后连入载体λ－ＺＡＰⅡ并用噬菌体包装蛋白进行体外包装,经稀释测定出含有重组子的文库大小为９．２×１０~５．原位杂交筛选出了肌动蛋白阳性克隆。     

莱茵衣藻基因组文库的构建

以莱菌衣藻CC-849为材料,提取基因组DNA,利用BamHⅠ和BglⅡ对基因组DNA进行酶解,获得了可用于构建基因组文库的6-12 kb的基因组片段,并浓缩至200 ng/μL。该片段与λDNA载体连接,经噬菌体蛋白包装、侵染大肠杆菌XL1-blue后,获得了莱菌衣藻基因组文库。该文库的滴度为2.12×10~5 pfu/mL,共有转化子4.26×10~4个,插入片段的平均长度约为9kb,扩增后基因组文库滴度为9.5×10~6 pfu/mL。     

大鼠肾脏组织cDNA文库的制备和葡萄糖转运蛋白cDNA克隆的筛选

 大鼠的肾脏组织经匀浆后,提取纯化总RNA和mRNA合成cDNA,进行甲基化,加人工接头,最后连入载体(λgt11)和外壳蛋白包装步骤制成肾脏组织的cDNA文库。插入载体的cDNA长度为0.5kb到8kb之间。经反复稀释,测定出该文库的效价为1.5×10~7pfu/mL。     

Txnip基因siRNA慢病毒表达质粒构建及促进猪前体脂肪细胞分化

硫氧还蛋白互作蛋白(thioredoxin interacting protein, Txnip)是一种氧化还原调节蛋白质,与硫氧还蛋白结合并抑制其活性,调节细胞氧化还原状态,影响细胞多种生理过程,然而其在猪脂肪细胞分化中的作用尚不明确。本文设计合成3对靶向猪Txnip基因的shRNA寡核苷酸,分别连接于重组慢病毒载体pGLV_3/H_1/GFP+Puro构建siRNA表达质粒。测序验证后,与包装质粒共转染293T细胞,获得滴度1×10~8 pfu/mL的慢病毒干扰质粒。以MOI值100转染原代培养猪前体脂肪细胞,转染率均达80%以上,其中Txnip-shRNA-2转染细胞Txnip基因沉默率达75%。转染Txnip-shRNA-2的猪前体脂肪细胞用成脂分化培养液诱导后,每隔1 d检测细胞成脂分化及相关基因表达。结果发现,其分化比阴性对照质粒转染或未转染细胞显著增强(P<0.05),PPARγ和FAS mRNA表达水平显著提高(P<0.05)。本文构建siRNA慢病毒表达质粒能有效干扰猪Txnip基因表达,Txnip表达沉默可通过上调PPARγ表达促进猪前体脂肪细胞分化。本研究提示,Txnip可能是猪脂肪细胞分化的抑制因子。     

高效构建卵清白蛋白scFv噬菌体文库及其筛选

目的:建立一种高效噬菌体文库构建方法,获得抗鸡卵清蛋白(ovalbumin,OVA)的单链抗体(scFv)噬菌体展示文库,筛选鉴定获得OVA单链抗体。方法:用OVA蛋白免疫Balb/C小鼠,选取血清抗体效价高的小鼠提取脾脏RNA,利用RT-PCR方法扩增获得小鼠重链和小鼠轻链基因。通过无缝连接酶一步将小鼠重链基因、轻链基因和linker DNA连接起来,插入噬菌体表达载体中,构建OVA scFv噬菌体展示文库。测定文库容量,对文库进行富集筛选,ELISA鉴定阳性克隆,测序后构建真核表达载体,转入Expi-CHO悬浮细胞进行真核表达,利用Western blot进行鉴定。结果:成功获得库容量为1. 2×10~7cfu的OVA scFv噬菌体展示文库,并从中筛选出8个阳性克隆,选取效价最高的2号克隆,在Expi-CHO悬浮细胞中表达获得可溶性抗体。结论:建立了一种高效构建scFv噬菌体文库的方法,筛选获得高结合活性的OVA单链抗体,并成功进行了真核表达,为OVA ELISA检测试剂盒的研制奠定了基础。     

N_3质粒对λ噬菌体发育途径的影响

诱导试验表明N_3质粒抑制λ溶源菌诱导处理时的裂解性发育途径,使λ噬菌体产率大大降低,仅为对照的10~(-5)—10~(-6)。感染实验表明N_3质粒主要抑制λ噬菌体的溶源性发育途径,其溶源菌形成率仅为对照的1.3—1.9％。从N_3质粒对λ噬菌体感染时与λ溶源菌诱导时的发育途径的不同作用的事实推测N_3质粒的某种产物(蛋白质)与λ噬菌体cI蛋白相作用,以障碍cI蛋白正常解离或聚合的方式而干扰其发育途径。     

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.