摘要
从日本血吸虫单克隆抗独特型抗体NP30杂交瘤细胞总RNA中纯化mRNA，在AMV反转录酶作用下，制备单链cDNA。随后经RNaseH和DNA多聚酶Ⅰ的作用，合成双链cDNA。用T4DNA多聚酶制备cDNA的平末端，并在T4DNA连接酶的作用下，加上EcoRⅠ接头，将cDNA片段插入λgt（11）载体。以大肠杆菌Y1090做受体菌转染，构建NP30杂交瘤细胞cDNA文库。实验结果表明，包装效价2．14×106λpfu／ugλgt（11），重组率为52％。结果提示：本研究初步构建了NP30杂交瘤细胞cDNA表达文库。
In this study, mRNA was isolated from total RNA of the anti-idiotypic monoclonal antibody NP30 hybridoma cells of Schistosoma japonicum. Single-stranded cDNA was derived by AMV reverse transcriptase reaction and followed directly by double-stranded cDNA replacement synthesis using RNaseH and DNA polymerase Ⅰ. After treatment with T4 DNA polymerase to flush the ends and with T4 DNA ligase to add EcoR Ⅰ adaptors. cDNA molecules were recombined with λgt(11) vectors.The cDNA library of NP30 hybridoma cells was constructed by the transfection of recombinant phage into E. coli Y1090. The results showed that the package effect of the cDNA library of NP30 hybridoma cell was 2. 14× 106 pfu/pg λgt(11) and the recombinant rate was 52%. The results suggest that the cDNA library of NP30 hybridoma cells has been constructed successfully.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
1998年第1期12-14,共3页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金!39570645
