共表达甲型流感病毒M1和HA双基因的重组腺病毒的构建及鉴定

以我国分离的首株人H5N1亚型禽流感病毒(A/Anhui/1/2005)作为研究对象,通过引入内部核糖体进入位点序列(IRES),构建共表达H5N1亚型禽流感病毒膜蛋白基因M1和HA的重组腺病毒。PCR扩增禽流感病毒H5N1亚型M1和HA基因的全长可读框片段,先后亚克隆入pStar载体,然后扩增M1-IRES-HA片段并将其插入穿梭载体pShuttle-CMV,再与pAd-Easy载体在BJ5183菌中通过同源重组产生重组腺病毒载体,转化293细胞,包装出重组腺病毒Ad-M1/HA。将Ad-M1/HA感染293细胞,可观察到明显细胞病变效应,用免疫荧光及Western-blot方法均检测到M1和HA基因的表达。共表达M1和HA双基因的重组腺病毒的成功构建为开发新型重组腺病毒流感疫苗奠定了基础。     

HAX1和EGFP共表达重组腺病毒载体的构建、鉴定

目的构建和鉴定HAX1和EGFP双基因共表达重组腺病毒载体。方法采用DNA重组技术,将目的基因HAX1克隆至含有报告基因EGFP的穿梭质粒pAdTrack—CMV中,并转化于大肠埃希菌DH5a;筛选出重组质粒pAdTrack—CMV—HAX1,并在BJ5183细菌中与pAdEasy-1质粒进行同源重组,产生重组腺病毒载体;用lipofectamine将其转染HEK293细胞,包装携带全长HAX1的重组复制缺陷型腺病毒pad—HAX1-EGFP,酶切和序列测定鉴定;用制备好的Ad—HAX1-EGFP感染HEK293细胞,流式细胞术检测其感染效率,RT—PCR、Western印迹鉴定外源基因HAX1的表达。BrdU检测感染了Ad—HAX1-EGFP的HEK293细胞增殖情况。结果pAdTrack—CMV—HAX1重组质粒构建成功。pAdTrack—CMV—HAX1质粒与pAdEasy-1质粒同源重组后与预期结果相符。构建好的Ad—HAX1-EGFP能有效感染HEK293细胞;外源基因能在239细胞中有效表达。HAX1高表达的HEK293细胞其增殖率得以提高。结论成功构建了表达HAX1和EGFP共表达的重组腺病毒载体,HAX1能够促进结肠癌细胞HEK293细胞的增殖。     

NIPSNAP3A短发夹RNA腺病毒载体的构建及病毒包装

目的:构建靶向NIPSNAP3A的短发夹RNA(shRNA)腺病毒载体,干扰HEK293A细胞中NIPSNAP3A的表达。方法:设计靶向NIPSNAP3A的shRNA,插入穿梭质粒pENTRY,通过Gateway法获得腺病毒颗粒pAd-NIPSNAP3A-shRNA,转染HEK293A细胞,在细胞内包装获得腺病毒。结果:重组腺病毒载体经酶切鉴定正确,制备的病毒感染效率高,能显著抑制NIPSNP3A蛋白的表达。结论:干扰HEK293A细胞NIPSNAP3A表达的shRNA重组腺病毒载体构建成功。     

共表达猪繁殖与呼吸综合征病毒GP5基因及猪γ-干扰素基因的腺病毒载体的构建与鉴定

利用PCR方法分别扩增猪繁殖与呼吸综合征病毒全长GP5基因（E蛋白）,EMCV的核糖体介入位点（IRES）序列及猪γ-干扰素（IFN-γ）基因全长序列,序列测定正确后用DNA重组法将三者串联后插入pAdenoVator-CM V5-IRES-GFP穿梭质粒中,形成的穿梭质粒plRES-GP5-IFN-γ用PmeⅠ线性化后,与腺病毒骨架载体pAdEasy-1共转化感受态大肠埃希氏菌BJ5183,经同源重组,构建成含有GP5基因和IFN-γ基因的重组腺病毒载体,pacⅠ酶切线性化充分暴露反向末端重复序列后,脂质体转染HEK293A细胞,借助GFP的表达可以在转染后的2～3天观察到包装病毒rAdeno-GP5-IFN-γ产生,7～10天出现病毒蚀斑。经PCR法及酶切证实各中间过程载体及最终的包装病毒中携带有目的基因,western-blot证实两基因在腺病毒中得到了表达。大肠杆菌内同源重组法能有效和较为方便的构建出含有目的基因的腺病毒载体rAdeno-GP5-IFN-γ,重组子能够在HEK293细胞中稳定扩增,病毒包装的成功为进一步研究PRRSVE蛋白的免疫效果及IFN-γ的作用奠定了基础。     

HIV-1 C亚型密码子优化gp120基因重组腺病毒载体的构建及表达

目的构建含有HIV-1C亚型gp120基因重组腺病毒载体,并在293细胞中表达gp120蛋白。方法PCR扩增,获得HIV-1C亚型gp120片段,定向克隆人腺病毒转移载体pTrack-CMV,线性化后转化至含有腺病毒骨架载体pAd-easy-1的大肠埃希菌BJ5183,获得重组子prAd—gp120,PacI酶切纯化后转染293细胞,包装成复制缺陷型重组腺病毒vAd—gp120。结果经PCR、酶切及DNA测序,插入片段大小、方向正确,获得了具有感染力的vAd—gp120重组腺病毒;通过Western印迹检测,重组腺病毒在293细胞中表达出分子量为120kD的蛋白。结论成功构建了含有HIV-1C亚型gp120基因重组腺病毒载体,并获得该基因的表达。     

果蝇硒蛋白dSelK基因重组腺病毒构建与表达

目的:利用AdEasy腺病毒载体系统构建果蝇硒蛋白dSelK基因重组腺病毒并在AD-293细胞中包装扩增。方法:PCR扩增dSelK目的片段连接到穿梭载体pShuttle-CMV上,用电转化法将线性化的pShuttle-CMV-dSelK穿梭载体转入含腺病毒骨架质粒AdEasy1的BJ5183大肠杆菌电感受态细胞中,构建重组腺病毒载体AdEasy-dSelK,线性化后转染AD-293细胞进行包装,并传代扩增,TCID50法测病毒滴度,Western blot鉴定。结果:成功构建腺病毒载体AdEasy-dSelK,经AD-293细胞包装扩增得到重组腺病毒,滴度为107.5。Western blot鉴定AdEasy-dSelK成功表达。结论:成功构建重组腺病毒载体pAdEasy-dSelK,为硒蛋白功能研究奠定基础。     

仙台病毒BB1株6个编码基因的克隆及表达

在仙台病毒BB1株全基因组序列测定的基础上,用反转录和PCR方法获得了核蛋白基因(N),磷蛋白基因(P),神经血凝素基因(HN),基质蛋白基因(M)、融合蛋白基因(F)和聚合酶蛋白基因(L)等6个编码基因全长克隆;测序结果表明,其序列与Genbank中登录的序列(DQ219803)完全一致。为了提供仙台病毒基因组载体拯救和包装所需的反式作用蛋白,将N、P、M、F、HN和L分别克隆到腺病毒穿梭表达载体pDC316上,将它们分别与腺病毒基因组质粒pBHGlox△E1,3Cre共转染HEK293细胞,获得了6种复制缺陷性重组腺病毒Ad5-N、Ad5-P、Ad5-M、Ad5-F、Ad5-HN和Ad5-L。酶切结果表明6种重组腺病毒穿梭质粒构建正确;用PCR方法证明所获得的6种重组腺病毒分别携带了上述6个编码基因;用重组腺病毒感染LLC-MK2细胞后用Western blotting和免疫荧光方法检测到了相应仙台病毒编码基因的表达。本研究为仙台病毒BB1株全长基因组的拼接和病毒载体包装系统组建打下了基础。     

人SPRED2 基因重组腺病毒载体的制备及其对K562 细胞

目的:构建携带SPRED2的质粒载体与重组腺病毒载体,并观察其在K562细胞的表达及对ERK信号通路的作用,为Spred2在造血细胞中的作用的研究奠定基础。方法:以HepG2细胞cDNA为模板,RT-PCR克隆SPRED2全长CDS序列,并亚克隆到pCDNA3.0和pshuttle-CMV质粒载体,构建携带SPRED2的真核表达载体pCDNA3.0-Spred2与穿梭载体pshuttle-CMV-Spred2;将线性化pshuttle-CMV-Spred2与腺病毒骨架质粒Adf11p在感受态细胞BJ5183中进行同源重组,产生重组质粒Adf11p-Spred2;后者经线性化后转染至HEK293细胞进行病毒包装;在HEK293细胞扩增病毒颗粒,以CsCl密度梯度离心法进行纯化,TCID50法测定病毒滴度;将病毒颗粒以100MOI感染K562细胞,Western blot检测Spred2过表达情况及Spred2对细胞ERK的影响。结果:经酶切、DNA测序、Western blot检测等方法鉴定,证明pCDNA3.0-Spred2与Adf11p-Spred2携带Spred2序列正确,能够在HEK293细胞、K562细胞正确表达,Spred2过表达能够显著抑制K562细胞ERK活性。结论:成功构建对K562细胞有高感染效率的SPRED2重组腺病毒载体,且Spred2对K562细胞ERK信号通路有显著抑制作用。     

人Sef基因重组腺病毒载体的构建与鉴定

构建人Sef-L和Sef-S基因的复制缺陷型重组腺病毒表达载体, 为研究Sef的功能和作用机制以及Sef的基因治疗奠定基础。通过PCR方法以hSef的表达质粒为模板扩增得到hSef的编码序列, 亚克隆到穿梭载体pAdTrack-CMV中, 经测序验证之后, 将穿梭载体使用Pme I酶切线性化, 然后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183, 得到重组的Ad-hSef-L和Ad-hSef-S质粒, 最后将Ad-hSef-L和Ad-hSef-S质粒使用Pac I线性化, 转染到HEK293细胞中, 包装收获病毒颗粒, 免疫印迹实验鉴定表达, 荧光素酶报告实验验证其功能。成功构建了人Sef基因的复制缺陷型重组腺病毒表达载体, 获得了有功能的Ad-hSef-L和Ad-hSef-S病毒重组子。     

人Sef基因重组腺病毒载体的构建与鉴定

构建人Sef-L和Sef-S基因的复制缺陷型重组腺病毒表达载体, 为研究Sef的功能和作用机制以及Sef的基因治疗奠定基础。通过PCR方法以hSef的表达质粒为模板扩增得到hSef的编码序列, 亚克隆到穿梭载体pAdTrack-CMV中, 经测序验证之后, 将穿梭载体使用Pme I酶切线性化, 然后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183, 得到重组的Ad-hSef-L和Ad-hSef-S质粒, 最后将Ad-hSef-L和Ad-hSef-S质粒使用Pac I线性化, 转染到HEK293细胞中, 包装收获病毒颗粒, 免疫印迹实验鉴定表达, 荧光素酶报告实验验证其功能。成功构建了人Sef基因的复制缺陷型重组腺病毒表达载体, 获得了有功能的Ad-hSef-L和Ad-hSef-S病毒重组子。     

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.