| 1. | Ligate the insert hbrp to a pgex - 5x - 1 expression vector . prepare eoli . bl21 competent cells
制备eoli . bl21感受态细胞，将pgex一hb即表达质粒，转化bl一21感受态细胞。 |
| 2. | The positive clone is selected by colony pcr and dna sequence is analyzed and determined by the m13 primer
Coltjm109感受态细胞，菌落pcr筛选鉴定阳性克隆，用m13通用引物进行dna序列测定。 |
| 3. | Then cdnas and puc18 vectors were linked by t4 dna ligase and transformed into e . coli strain dh5 - alpha to generate cdna library that size is 4 . 9 l06 recombinants
将cdna与载体连接，并导入dh5感受态细胞中，构建成cdna文库。 |
| 4. | 2 . the preparation of high efficiency competence cell : the prep aration of high efficiency competence cell was one of the most important techniques in molecular biology
高效感受态细胞的制备：高效感受态细胞的制备是分子生物学的关键技术之一，是dna转化的基础。 |
| 5. | Expressed protein of ha1 gene was identified by polyacrylamide gel electrophoresis and western - blot . the expressed proteint could bind to the specific antibody against of h7 subtype
转化e . colier2566感受态细胞，用iptg诱导，经蛋白电泳和western - blot鉴定，证明成功表达了a毒株ha1基因，并且具有抗原性。 |
| 6. | It was necessary for gene library construction and transformation with enzyme ligased products . there were many factors to affect the preparation of competant cells . the affected factors of preparation of competence cells and transformation process had been studied
已知影响感受态细胞制备的因素很多，本研究从感受态细胞制备的影响因素和转化过程对转化效率的影响因素两个方面进行了研究。 |
| 7. | The plasmid was tested by the restriction enzyme bamh i and xho i incision , and 1 % agarose gel electrophoresis . the results show that there were two bands at the respected sites about 4 . 9kb and 750bp respectively . it means that hbrp has been cloned into a pgex - 5x - 1 expression vector correctly
2 .质粒鉴定pgex一hbrp融合蛋白表达质粒转化eoh . bi21感受态细胞l )限制性内切酶鉴定提取质粒dna ，经bamhfxhol双酶切鉴定，结果可见，有750bp的插人片段和4 . gkb的质粒dna两条带，表明所提质粒中含有外源基因hbrp片段，大小及插人方向均正确。 |
| 8. | The recombinant plasmid puge dna and transfer vector pfastbacl dna were treated again in the same enzyme , were linked by means of t4 dna ligase and transformed into e . coli jm109 permissive cells , yielding recombinant transfer vector plasmid pfastbac - ge dna and were transformed into dhlobac containing vector bacmid
将重组质粒pugedna与转移载体pfastbacldna用bamhi和ecori双酶切处理， t _ 4dna连接酶连接，用连接产物转化大肠杆菌jm109感受态细胞，得到重组转移载体质粒pfastbac - gedna 。 |
| 9. | Newcastle disease virus ( ndv ) strain 695 , a thermostable nature avirulent strain , were replicated in embryonated chicken eggsand its rna was extracted from allantoic fluid . referred to the reported sequence of f gene , a pair of primers were designed and synthesized . f gene of ndv b95 strain was amplified by rt - pcr , the pcr products were checked by agrose gel electrophoresis and purified by agrose gel fracion method
利用从国外引进的新城疫热稳定性天然弱毒b _ ( 95 )株接种spf鸡胚繁殖病毒，经处理后提取病毒的基因组rna ，参考国内外发表的ndv融合蛋白基因序列，设计一对特异性引物，经反转录聚合酶链式反应( rt - pcr )扩增出约1700bp大小的特异性片段，将此片段回收纯化后，利用t - a克隆技术将其克隆到pgem - t - easy克隆载体中，再转化大肠杆菌jm109感受态细胞，转化后经分子量比较、 pcr鉴定和酶切分析筛选阳性克隆。 |
| 10. | 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 。 |
