| 1. | Then the amplified mtb8 . 4 and ms gene were subcloned into the unique nhe i and mlu i cloning sites of pci - neo expression vector
重组质粒pm 、 pms转染ps15细胞，进行稳定表达， g418筛选抗性克隆后，用rl ’ - pcr检测目的基因在mrna水平的表达。 |
| 2. | On the base of construction of pbi121vp7 , we constructed the fusion gene expression vector pbi121ctbvp7 by the same sigle cloning site ( ndei and xbai ) of vector puc19ctb and pbi121vp7
设计具有ndei和saci单限制性酶切位点的vp7基因引物，通过pcr扩增出vp7基因并经测序验证。 |
| 3. | According to csfv ' s e2 gene sequence published in genbank and the sequence of p . pastoris expression vector ppic9 ' s multiple cloning site ( mcs ) , a pair of primers were designed with oligo and primers . o softwares
将该基因片段先克隆到pmd18 - t载体上，并进行了酶切、 pcr鉴定和测序分析，阳性重组质粒命名为t - e2 。 |
| 4. | Construction of two vectors containing different plasmid original replicon this work constructed four resolution shuttle vectors , pbmb1205 , pbmb1205r , pbmb1206 and pbmb1206r . there are multiple clone sites between two copies of res sites
解离载体的构建和性能利用来源于不同质粒上的质粒复制起始区ori44和ori1030构建了4个解离载体。 |
| 5. | A fusion - trascriptional library was then constructed by inserting sau3ai - partially digested chromosomal dna from 7653r into bamhi cloning site of phn127 . a group of constitutive - expression fusions with different fluorescent strength were obtained
将7653r的总dna经sau3ai部分酶解并克隆到phn127上，获得的融合子库，从中筛选到gfp组成型表达的具有调控活性的dna片段。 |
| 6. | The recombinant transfer vector pbacpak - hbmp was constructed by insertion of the hbmp coding sequences into the multiple cloning site of transfer vector pbacpak . 8 . bmn cell line was co - transfected with pbacpak - hbmp plasmid and linearized baculovirus bacpak6 dna by dosper agent
将克隆到的hbmp基因通过适当的酶切插入到转移载体质粒pbac - pak8的多克隆位点中，获得重组转移载体质粒pbacpak - hbmp 。 |
| 7. | The interest gene was inserted in the - tha l . tho 1 multiple cloning sites of donor plasimd pfastbachtb of baculovirus expression system . after analysis by restriction endonuclease and pcr , the recombinant donor plasmid gpl - fast was transforn1ed to the competent celi dhi0bac which contalns the bacmid and the helper plasmid , the recombinant bacmid gpl - bac was acquired which would express the vpl of gpv strain h l
同时将该目的基因插入到杆状病毒表达系统的供体质粒pf _ ( ast ) b _ ( ac ) htb的xba 、 xho多克隆位点间，经酶切、 pcr鉴定后，将重组的供体质粒gp1 - fast转化到含有杆状病毒和辅助质粒的dh10b _ ( ac )感受态细胞中，获得了表达gpvh1株vp1的重组杆状病毒gp1 - bac 。 |
| 8. | The ubi - sl - tocs fragment was taken out , inserted into the multi - cloning site of pcambia1300 vector , transformed into jm109 strain finally , positive colonies were screened on lb plate ( 60 g / ml kan added ) . the result of pcr and enzyme digestion of plasmid proved that recombinatin vector was obtained ( named pcusaib4 and pcusaibu )
把扩增产物分别通过clai和bamhi酶切纯化，连接到用clai和bamhi切去gfpml基因的中间表达载体pugfpocs中，转化大肠杆菌jm109 ，在含amp ( 100 g / ml )的lb抗性平板上筛选到了的阳性菌落。 |
| 9. | A bt - e . coli shuttle vector pht315 was deleted its replication region of bt , then constructed a novel vector named pht315 - 1 which composed a multiple cloning site , erythromycin and ampicillin - resistance marker and could only replicated in e . coli . used pht315 - 1 , a 5273 bps dna fragment carrying a novel bt plasmid replicon was isolated and registered in genbank as ay278324 . sequence analysis showed that there were at least three orf ( open reading frame ) in the cloned dna encoding 501 , 333 , 183aas . orfl had 98 % identities with replicating related protein ori43 of bt strain hd263 . the others were no homology to any published bt replicating related protein . after continuous cultured for 70h at 30 c without antibiotic selecting press . the stability of plasmid carrying cloned replicon in bt acrystalliferous mutant strain hd73 cry was more than 98 % . and growth curve also showed that the novel replicon was stable and could replicate normally
进一步序列分析表明该复制区至少有3个较大的orf ，分别编码501 ， 333 ， 183个氨基酸。其中orf1蛋白序列与hd263复制蛋白ori43的同源性为98 ，而另外两个orf和genbank己公布的bt复制相关蛋白无同源性。 30连续培养72h ，复制区质粒在bt无晶体突变株hd73cry ~ -中稳定性达98以上， 30h生长曲线也表明该复制区能够在bt中稳定复制和遗传，对受体菌株无明显不良影响。 |
| 10. | Sequence analysis shows that they share 98 . 75 % similarity at the dna , and 98 . 67 % at the protein level . ns2 gene was cloned into the multiple cloning sites of prokaryotic expression vector pgex - 6p - l . the recombinant plasmid pgex - 6p - ns2 was constructed and transformed to the competent cell bl21 ( de3 ) plyss , positive bacterium strain was induced by iptg
将ns2基因插入到原核表达性质粒pgex - 6p - 1的ecor 、 bamh多克隆位点之间，将重组原核表达质粒pgex - 6p - ns2转化到bl21 ( de3 ) plyss感受态细胞中，获得了表达ns2基因的阳性亚克隆重组子，在含amp的lb液体培养基中培养，经iptg诱导表达，用sds - page分析表达产物。 |
