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根据棉铃虫单核衣壳核多角体病毒(HaSNPV)基因组全序列,设计引物,用PCR的方法扩增得到bro-a、bro-b和bro-c三个全长基因。将这三个基因的片段分别克隆至原核表达载体pProExHTb,经IPTG诱导,在E.coli DH50菌株中得到了目的基因的高效表达。表达的目的蛋白大小分别为32kDa、64kDa和58kDa,经SDS-PAGE分离纯化,免疫新西兰大白兔制备了多克隆抗体。抗体经1∶2500倍稀释后用于WestemBlot分析,获得特异性显色信号,所制备的三种抗体适合用作bro基因的功能的进一步研究。
Primers were designed based on the complete genome of Helicoverpa armigera nucleocapsid nuclear polyhedrosis virus (HaSNPV), and three full-length genes bro-a, bro-b and bro-c were amplified by PCR. The three gene fragments were cloned into the prokaryotic expression vector pProExHTb, induced by IPTG, E. coli DH50 strain obtained high expression of the target gene. The expressed proteins were 32kDa, 64kDa and 58kDa respectively. The recombinant protein was purified by SDS-PAGE and immunized New Zealand white rabbits to prepare polyclonal antibodies. Antibodies were diluted 1:2500 and used for WestemBlot analysis to obtain specific chromogenic signals. The prepared three antibodies are suitable for further studies on the function of bro gene.