After multiple , plasmid ploxifn and pbs185 dna were co - transfected into the green fluorescent cell clones , then filtrated the non - fluorescent cell clones . two non - fluorescent cell clones , the green fluorescent cell clones and the cells which were co - transfected by the plasmid ploxifn and pbsiss dna were checked up by pcr 首先将质粒ploxgfpdna电转染牛胎儿成纤维细胞，并用g418筛选，筛选出绿色荧光细胞克隆，增殖培养之后，将质粒ploxifn和pbs185dna共转染荧光细胞克隆，筛选出不发光的克隆。
The effectiveness of the nuclear localization signal selected was clearly tested in initial experiments . based on this , the co - transfection systems used in the study was established . we first observed the multinucleate cells and chromatin bridges in cultured hela cells when the cells are marked with gfp and hochest33342 , after co - transfection with the gfp expression plasmids and rnai plasmids rhe or rhc 当分别共转染附加kozac序列和核定位信号的gfp与人集缩素smc亚基hcap一e特异的rnai质粒rhe和人集缩素smc亚基hca尸一c的f { nai质粒rhc时，都观察到gfp和hochest33342标记的转染后hela细胞表现多核和染色质桥现象。
The recombinant pcr technic was used to introduce a linking peptide klgggg to the site between scfv single chain form of the monoclonal antibody sz - 51 specific for the glycoprotein gmp140 on activated platelet membrane and uk32 low molecular weight form of pro - urokinase , to make the scfv - linker - uk32 chimeric gene . this gene was cloned into the transfer vector pbacpak9 , and cotransfected with bacpak6 bsu36i digest into sf 9 cells . the fusion protein was secreted into the medium . in the fifth day after the cotransfection , the supernatant of the medium showed 107 iu ml fibrinolytic activity , higher than 25 iu ml fibrinolytic activity of scfv - uk32 . elisa showed that the supernatant had the binding activity to activated platelet . wastern blotting also indicated that the supernatant could bind to the monoclonal antibody of urokinase b chain 为了提高重组导向溶栓分子scfv - uk32的溶纤活性，通过重组pcr方法在编码scfv与uk32的碱基之间引入编码klgggg连接肽的碱基序列，并克隆到转移载体pbacpak9上,通过与线性病毒dna bacpak6 bsu36i digest共转染到昆虫细胞sf 9内，进行表达。表达产物分泌到上清中，共转染后第5d天用纤维平板法测得sf 9细胞上清溶纤活性达到107 iu ml ，比未引入连接肽的scfv - uk32的表达活性25 iu ml高。
Pbluebachisc - vp7 was identified and analped by compnding restriction endonuclease , pcr and nested pcr on the basis of the genetic sites of pbluebachisc , which was idenhfied as vp7 gene of prv hafied pbluebachisc - vp7 and barmidn - blue dna were cbeinfected insect cell sro and harvested mmbinan beculovirus 5d later identification with restriction empme and pcr showed vp7 gene has been arembwt comehy with baculovirus dna and namd a - l 纯化该重组质粒并与线性杆状病毒dnabac - n - blue共转染昆虫细胞sr9 ， 5d后收获重组病毒。重组杆状病毒dna分子的pcr及酶切鉴定表明，获得了prv - vp7基因与杆状病毒dna的重组子，命名为a - 1代病毒。
After obvious cytopathogenic effects developed , virus - contained supematants were harvested , and the progeny viruses were screened for lacz - expressing viruses by a plaque assay using x - gal . single blue plaques were picked , and a recombinant prv stably expressing lacz gene ( designated as rprv - lacz ) was obtained after ten cycles of plague purification and pcr identification . the results showed that the lacz gene expression cassette was stably expressed in the recombinant rprv - lacz derived from bartha - k61 strain 该载体与具有高度感染性的bartha - k61株基因组dna通过脂质体加plus法共转染vero细胞，采用甲基纤维素固定病变， x - gal染色，经过10代蓝斑纯化获得了一株稳定表达lacz基因的ge tk基因缺失突变株，命名为rprv - lacz 。
Fusion gene by pcr was inserted into bombyx mori baculovirus transfer vector pbacpak . 8 and contransfected with lineared dna of bm - bacpak6 virus into bmn cells . the homologous recombination occurred inside the cells , and the recombinant virus bacpak - 6aa - hgm - csf was expressed , as identified by pcr and southern hybridization . the bmn cells and the fifth instars were infected by the recombinant virus bacpak - 6aa - hgm - csf 本研究首先通过pcr将家蚕杆状病毒多角体蛋白起始密码子后的18个碱基引入到hgm - csf基因的5 ’端之前，然后将融合基因重组与家蚕杆状病毒转移载体pbacpak8中，获得重组转移载体pbacpak8 - 6aa - hgm - csf ，并与线性化bm - bacpak6dna共转染家蚕细胞株，获得重组病毒bacpak - 6aa - hgm - csf 。
The pbacph - egf plasmid dna was used to co - transfect bmn cell with the modified bombyx mori baculovirus bm - bacpak dna , which was first linearized by aocl . after two rounds of plaque isolation , the recombinant virus ( named bmbacph - egf ) was further verified with pcr and dot hybridization . the recombinant bmbacph - egf virus was used to infect bmn culture cells at a moi of 10 重组转移载体dna与经bsu36i酶切线性化的修饰型病毒bm - bacpakdna共转染家蚕bmn细胞，两轮空斑筛选和纯化，获得重组病毒rbmbacph - egf ，经pcr扩增、 dna点杂交等方法鉴定证实重组病毒中已正确插入ph - egf融合基因。
Angiostatin ( kl - 3 ) gene was inserted into bombyx mori baculovims transfer vector pbacpaks and cotransfected with lineared dna of bm - bacpak6 virus into bmn cells . the homologous recombination occurred inside the cells , and the recombinant virus bacpak - angiostatin was expressed , as identified by pcr and dna dot blotting 本研究首先将人血管抑素( angiostatin )基因重组于家蚕杆状病毒转移载体pbacpak8中，获得重组转移载体pbacpak - angiostatin ，并与线性化病毒bm - bacpak6dna共转染家蚕细胞株，获得重组病毒bacpak - angiostafin 。
On the other hand , our previous studies have shown that the nr2b construct with a gfp tag in its n - terminus cotransfected with nr1 subunit can reach the cell surface and be detected by surface staining with anti - gfp antibody in live cells . the nr2b construct will be retained in the endoplasmic reticulum ( er ) when expressed alone in heterologous cells 我们以往的研究表明，用绿荧光蛋白( gf内在信号肤下游n末端标记nrzb亚单位，与nri共转染时能形成与野生型相似的nmda受体通道，且可以通过抗gfp抗体标记活细胞膜表面表达的nmda受体复合物。
