猪源化嵌合抗体在杆状病毒中的表达  

Development of a neutralizing mouse-pig chimeric antibody with therapeutic potential against Haemophilus parasuis in baculovirus

作　　者：杨玉菊[1,2] 姜福成[1] 柴政[1] 符芳[1] 郑楠[1] 王香玲[1] 郭殿磊 李曦[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪传染病研究室,黑龙江哈尔滨150001 [2]哈尔滨学院,黑龙江哈尔滨150086

出　　处：《中国预防兽医学报》2014年第7期559-562,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省青年学术骨干项目(1153G068);哈尔滨市科技局优秀带头人计划(RC2014XK002018)

摘　　要：为构建具有活性的抗副猪嗜血杆菌(H.parasuis)鼠-猪嵌合单链抗体(scFv),本研究以H.parasuis单克隆抗体(MAb)杂交瘤细胞1D8株的总RNA制备的cDNA为模板,通过RT-PCR分别扩增抗MAb轻链、重链可变区序列及猪抗体轻链和重链恒定区序列,并将这些片段经重叠延伸PCR分别扩增可变区与恒定区的轻链与重链的序列。将扩增的两个目的基因克隆于双向表达载体pFast-Bac-Dual中,并进一步以其构建的重组杆粒,转染Sf9昆虫细胞筛选制备稳定表达的抗H.parasuis猪源化嵌合的scFv重组杆状病毒。Western blot、ELISA、体外和体内的抑菌试验表明,抗H.parasuis猪源化嵌合scFv具有中和活性和抑制细菌增殖的能力,可以为进一步的H.parasuis的抗体治疗奠定基础。To construct the mouse-pig chimeric antibody against Haemophilus parasuis, the sequences encoding heavy chain and light chain variable region of monoclonal antibody （MAb） and constant region of pig antibody were amplified by RT-PCR from cDNA reverse-transcribed total RNA extracted from the MAb hybridoma 1D8 and porcine B cells, respectively, and the PCR products were used as templates to create the chimeric full-length of light chain and heavy chain by fusion PCR, which were cloned into dual expression vector pFast-Bac-Dual to generate the recombinant baculovirus expressing the full-length of light chain and heavy chain independently via the Bac to Bac system. The results showed that the expressed full-length of light chain and heavy chain were able to fold to single chain antibody （scFv） which possessed the abilities for neutralizing and the bacteriostatic activity to H.parasuis detected by western blot, ELISA, antibacterial test in vitro and in vivo. These results provided a basis for further study on the scFv for therapy of H.parasuis infection in pigs.

关 键 词：副猪嗜血杆菌 猪源化嵌合抗体 杆状病毒表达 

分 类 号：S852.61[农业科学—基础兽医学]