分泌抗Flu B抗体杂交瘤细胞cDNA文库的构建

Preparation of cDNA library from hybridoma cells with monoclonal antibody for influenza B virus

目的：构建分泌抗流感病毒B（Flu B）抗体杂交瘤细胞cDNA文库。方法：以对数生长期的抗Flu B抗体杂交瘤细胞为试验材料提取总RNA，应用SuperScipt^TMⅡ RnaseH—Reverse Transcriptase建库试剂盒建立了Flu B单抗杂交瘤细胞cDNA文库。结果：构建的流感病毒B单抗杂交瘤细胞cDNA文库含1．27×10^6重组子，重组率90．63％，插入片段长度约为0．5～2．0kb。扩增后的文库浓度为3．5×10^7重组子／μl，将文库稀释到10^-6时所产生的噬菌斑密度最为适宜。结论：成功构建Flu B单抗杂交瘤细胞cDNA文库，为进一步筛选目的基因、制作基因芯片等提供了有效工具。

Objective ：To construct a cDNA library of hybridoma cells with monoclonal antibody for influenza B virus. Methods：The hybridoma cell was cultured in vitro. Total RNA of hybridoma cell was extracted from the cultured cells and then mRNA was extracted further. Moreover, single - strand cDNA and double - strand cDNA were synthesized in turn. The double - strand cDNAs were ligated to EcoRI adaptor, which were later ligated to arms of pBluescript Ⅱ SK（ ＋ ） XR. Ligated -cDNAs were packed in vitro ,and then infected the E. coli DH10B. The plasmid was titered and the library was amplified. Results： The hybridoma cell cDNA library consisted of 1.27 × 10^6 recombinants with the length of 0. 5 to 2. 0 kb and the recombinating efficiency was 90. 63%. The concentration of the amplified library was 3 . 5 × 10^7 recombinants/μl and the number of negative colony was the most suitable in density after it was diluted to 10^- 6in concentration. Conclusion：The constructed cDNA library of hybridoma cell would be helpful for further detecting target genes and preparing gene chips.