| 1. | Tg1 and 15 separate colonies were assayed by elisa . the recombinant phages which were rescued from colony 12 showed the highest affinity to immobilized hcg . the scfv gene was inserted into the reconstructed pet - 21a and expressed in
用elisa方法进行检测，从结合力最强的克隆中分离单链抗体基因，插入经改造的高效表达载体pet - 21a中，转化大肠杆菌，诱导表达。 |
| 2. | The patterns of sds - page indicated more than 30 % of recombinant proteins could be obtained from the extract of e . coli bl21 . furthermore , library of recombinant phage antibodies was constructed from total rna isolated from spleen of the female balb / c mice immunized by bull sperm
首先用牛精子免疫雌性四周龄balb c小鼠，从其脾脏组织分离总rna ，应用重组噬菌体抗体库技术，构建了一个针对牛精子的噬菌体抗体文库。 |
| 3. | The library was rescued with phage m13k07 in order to display scfv on the surface of the phage and to form the recombinant phage antibody library . one of positive scfv clones , named pcsal , was selected with phage - elisa after panning and screening by bull sperm three times . scfv fragment , amplified from pcsa1 , was ligated to pmd18 - t vector for sequencing analysis
取阳性重组噬菌体抗体克隆株pcsa1 ， pcr扩增其scfv基因，筛选重组子进行序列测定，发现其序列符合小鼠抗体基因的一般特征，并且与几株抗磷酸胆碱的抗体重链和轻链可变区序列的同源性达80以上；推测pcsa1scfv针对的抗原是磷酸胆碱类物质。 |
| 4. | Detection of antigen - binding affinity of mg7 recombinant phage antibody elisa was repeated to confirm the antigen - binding affinity of positive clones screened out in the former procedure ; these positive phages were examined by restriction analysis ( ecor i and hind iii ) ; competitive elisa was performed to test the inhibitory ratio of these positive clones to the binding affinity of mg7mab and its relevant antigen , the positive dones possessing apparent inhibitory effect were singled out for later use
X阳性克到驶知烘扳原kbbjg ）浊对阳性克隆进行限制陇卧赐析（ uwi和hedlll ）鉴定三用竞争elisatoljmg7重组噬菌体抗体性克隆对mg7单扶与其相应抗原结合忙的喇率，从中j ） ed出对mg7单抗与期眩抗原结合有抑余j效应的克隆用于进一涉研究。 |
| 5. | Panning and enrichment ofmg7 recombinant phage antibody the transformed cells were infected with m13k07 helper phage to produce a phage form of mgy recombinant phage antibody library ; after three rounds panning of the library with the mgrbinding antigen highly expressed gastric cancer cell line kato iii , the mg7 scfv displayed phage dones were selected from the enriched recombinant phages by elisa
3 mg _ 7重组噬菌体抗体库的淘筛用m13ko7辅助噬菌体感染转化菌，以挽救出噬菌体形式的抗体库；用高表达mg _ 7抗原的胃癌细胞系kato对抗体库进行三轮淘筛；用elisa筛检呈现有mg _ 7scfv的噬菌体单克隆。 |
| 6. | Thirteen putative epitopes showing characteristics of antigenic epitope were found from the analysis information . using pcr , the nucleotide acid fragments encoding these putative epitopes were amplified , then cloned into the expression vector miske . the positive recombinant phage displying the epitopes were found out by using pcr , sequencing and the determination of phage plaque titer
运用goldenkey分子生物学软件对prrsvbj - 4结构蛋白的抗原表位及其二级结构进行了分析和比较，从中筛选13段显示表位特征的氨基酸残基序列，用pcr技术扩增相应的核苷酸片段，将其插入到噬菌体表达载体m13ke ，结果预测的13个表位可在噬菌体表面得以展示。 |
| 7. | Culture of mg7 hybridoma cells and detection of antigen - binding affinity of mg7 mab by elisa 2 . construction and identification of mg7 recombinant phage antibody library mrna was isolated from cultured mg7 hybridoma cells and converted into cdna ; the variable fragments of heavy and light chain were separately amplified and assembled into scfvs with a specially constructed dna linker by pcr . the scfvs dma was ligated into the phagmid vector pcantabse and the ligated sample was transfered into competent e . co / / tg1 to generate a bacterial form of mg7recombinant phage antibody library ; volume and recombinant ratio of the library were evaluated by means of bacterial colony counts and restriction analysis ( ecor i and hind iii )
Mg _ 7重组噬菌体抗体库的构建及鉴定从培养的mg _ 7杂交瘤细胞中提取并分离mrna ，反转录成cdna ；利用pcr分别扩增mg _ 7单抗的重链及轻链可变区基因，并通过? dna连接子将二者连接起来形成mg _ 7单链抗体基因；将mg _ 7单链抗体基因插入pcantab5e ；将连接产物转化感受态tg1大肠杆菌，制备细菌形式的mg _ 7重组噬菌体抗体库；通过菌落计数和限制性酶切分析( ecor和hind )评估mg _ 7重组噬菌体抗体库的容量和重组率。 |
