疟原虫抗原B表位NKNDD和NANP串联重复片段与脊灰病毒全长cDNA嵌合体的构建

Construction of Poliovirus cDNA Chimera with (NKNDD)n and (NANP)n

人工合成含有恶性疟原虫抗原B表位NKND的单拷贝抗原基因片段NKNDD和恶性疟原虫环子抱子蛋白CSP的重复性抗原B表位NANP的2拷贝基因片段，将NKNDD和（NANP）。分别进行自串联后克隆到中介载体PSKMM，得到一系列不同拷贝数的串联多拷贝克隆。从（NKNDD）n／PSKMM和（NANP）n／pSKMM的各拷贝克隆中挑选3、4、5、8拷贝的（NKNDD）n／PSKMM和4、6拷贝的（NANP）n／pSKMM，将（NKNDD）n和（NANP）n从中介载体转移到表达型载体pSV202H＋，得到3、4、5、8拷贝的（NKNDD）n和4、6拷贝的（NANP）n插入脊髓灰质炎病毒Ⅰ型Mahoney株全长cDNA的N－AgⅠ区域所构成的嵌合体。实验中由于引入了半定量杂交技术，使得单位DNA片段的系列串联和克隆过程明显简化。

Oligonucleotides encoding one copy of NKNDD, containing a P. f. B epitope NKND,and two copies of CSP repeated B epitope NANP were synthesized. After self-ligation,NKNDD and (NANP), were cloned into the intermediate vector pSKMM. Series of cloneswith different copies of NKNDD and NANP were identified. (NKNDD)n/MM with three,four, five, eight copies of NKNDD and (NANP)n/MM with four, six copies of NANP werechoosen- Then (NKNDD)n and (NANP)n of these recombinants were recovered respective-ly and finally inserted into the N-Ag I site of poliovirus type I Mahoney strain full-lengthcDNA, which is contained in the expression plasmid pSV2o2H+. As using semi-quantitativehybridization in identifying tranformants, the procedure in our experiment for cloning differ-ent copies of NKNDD and NANP was greatly simplified.