| 1. | 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 。 |
| 2. | 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。 |
| 3. | 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 。 |
| 4. | 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 。 |
| 5. | 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。 |
| 6. | 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。 |
| 7. | A pair of primers were designed to amplify vpl gene by pcr according to the published sequence of gpv b strain in genbank . the product of pcr was 2 . 2 kb . after identification of pcr product by restriction endonuclease the interest gene was inserted in the tha i . tho i multiple cloning sites of prokaryotlc expression vector pproexhtb to construct the recombinant prokaryotic expression vector gpl - ppro of vp 1 of gpv strain h 1
本试验参照genbank中发表的gpvb株基因序列设计并合成了扩增gpvh1株vp1的一对引物，利用pcr技术扩增出长约2 . 2kb的目的片段，经酶切鉴定后，将其插入原核表达载体pproexhtb的xba 、 xho多克隆位点间，构建了gpvh1株vp1的原核表达载体gp1 - ppro 。 |
| 8. | As well as in eukaryocyte ( hepg2 and cos - 7 ) , then detect their antigenity as a basis study and explore of the choice of immunogen for preventive and therapeutic vaccines of hepatitis b . methods : the gene fragments coding 152aa ( si ) and 124aa ( s2 ) of the carboxyl terminus of hbsag were amplified by pcr from plasmid pecob6 with a pair of primers containing different endonuclease sites and were cloned into multiple cloning sites of plasmid pbks ( + )
为乙型肝炎的预防和治疗性疫苗免疫原的选择进行初步的研究和探讨。方法：本研究利用聚合酶链反应( pcr ) ，通过设计带有不同酶切位点的一对引物，从质粒pecob6特异性扩增hbsag蛋白羧基末端152个氨基酸( s1 )和124个氨基酸( s2 )的基因片段，分别将二者克隆到质粒pbks ( + )的多克隆位点，筛选重组克隆。 |
