真菌病毒FpgMBV1中P3基因的原核表达及多克隆抗体的制备

Prokaryotic expression of FpgMBV1-P3 and preparation of its polyclonal antibodies

采用RT-PCR法从假禾谷镰孢菌(Fusarium pseudograminearum)弱毒菌株FC136-2A的菌丝中扩增出假禾谷镰孢菌巨型双链RNA病毒1(Fusarium pseudograminearum megabirnavirus 1,FpgMBV1)的P3蛋白编码序列。测序结果显示,FpgMBV1-P3蛋白的编码序列长2700 bp,编码901个氨基酸残基。构建原核表达载体pET28a-FpgMBV1-P3并转化大肠杆菌BL21(DE3)菌株,在25℃以0.5 mmol·L-1 IPTG诱导表达重组蛋白。SDS-PAGE分析表明,重组蛋白分子量约为90 kDa,与预期分子量大小相符。将该重组蛋白作为抗原免疫日本大耳白兔,获得抗血清,采用间接ELISA测定其效价达1∶128000,能够特异性检测到假禾谷镰孢菌菌丝中的FpgMBV1-P3蛋白。研究结果可为探究FpgMBV1-P3的结构和功能,以及进一步探讨FpgMBV1在假禾谷镰孢菌弱毒菌株FC136-2A中的作用机制奠定基础。

The FpgMBV1-P3 coding region of Fusarium pseudograminearum megabirnavirus 1(FpgMBV1)was amplified by RT-PCR from the Fusarium pseudograminearum hypovirulent isolate FC136-2A.DNA sequencing showed that the FpgMBV1-P3 coding region was 2700 bp in length and encoded 901 amino acid residues.The prokaryotic expression vector pET28a-FpgMBV1-P3 was constructed and transformed into Escherichia coli BL21(DE3).By inducing with 0.5 mmol·L-1 IPTG at 25℃the recombinant FpgMBV1-P3 was expressed and identified as a specific 90 kDa band by SDS-PAGE as expected.Polyclonal antibodies against the recombinant FpgMBV1-P3 were obtained by hypodermic injection of rabbit.The titer of the prepared polyclonal antibodies was 1∶128000 and the antibodies reacted specifically with FpgMBV1-P3 in the hyphae of FC136-2A.