Iacono-Connors L C, Schmaljohn C S, Dalrymple J M
Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21701-5011.
Infect Immun. 1990 Feb;58(2):366-72. doi: 10.1128/iai.58.2.366-372.1990.
The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response.
通过定点诱变对炭疽芽孢杆菌保护性抗原（PA）的编码基因进行修饰，分别亚克隆到杆状病毒和痘苗病毒质粒转移载体（分别为pAcYM1和pSC-11）中，并通过同源重组插入杆状病毒苜蓿银纹夜蛾核型多角体病毒或痘苗病毒（WR株和康诺特株）。用经炭疽芽孢杆菌PA免疫的兔抗血清通过免疫荧光测定法在两个系统中均检测到PA的表达。蛋白质印迹（免疫印迹）分析表明，两个系统的表达产物（86千道尔顿）比炭疽芽孢杆菌产生的PA（83.5千道尔顿）略大。对病毒表达的PA和天然PA的胰蛋白酶消化产物分析表明，大小差异是由于病毒表达的蛋白质中保留了信号序列。用重组杆状病毒感染的草地贪夜蛾细胞或痘苗病毒重组体免疫小鼠，可引发高滴度的抗PA抗体反应。
