急性病毒性坏死病毒IAP-86基因的克隆、表达及抗凋亡研究

在已经完成的栉孔扇贝急性病毒性坏死病毒(acute viral necrosis virus,AVNV)全基因组序列测序与分析的基础上,设计特异性引物,克隆得到了ORF86编码的杆状病毒凋亡抑制蛋白基因(IAP-86)。IAP-86基因与pET32a(+)质粒连接构建得到重组质粒pET32a-IAP86,将重组质粒转化到E.coil BL21(DE3)中,使用异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,SDS-PAGE检测显示表达蛋白分子量约为40 ku,经Western-blotting和质谱分析证明,该蛋白即为IAP-86融合蛋白,Co2+柱纯化后得到了纯化的IAP-86融合蛋白。将重组的IAP-86蛋白用FITC标记,荧光显微镜下观察,发现重组的IAP-86蛋白最终能够与栉孔扇贝血淋巴细胞的细胞核和细胞质结合。细胞凋亡检测实验发现,重组的IAP-86蛋白能够在一定程度上抑制栉孔扇贝血淋巴细胞凋亡,凋亡抑制率为7%。本实验应用原核表达成功得到了IAP-86蛋白,并证明IAP-86对栉孔扇贝细胞的凋亡有一定抑制作用,这为进一步研究AVNV的侵染机制提供依据。

Cloning,expression of acute viral necrosis virus IAP-86 gene and studies of its anti-apoptotic mechanism

Acute viral necrosis virus(AVNV)was reported as the causative agent responsible for summer mass mortality of adult Zhikong scallop(Chlamys farreri),which is widely cultured along northern China coast.In this study,the open reading frame(ORF)86 of AVNV was successfully amplified based on the specific primers designed according to the complete genome sequences of AVNV.The gene encoded by ORF86 in AVNV was named IAP-86 in this study,since the homology of ORF 86 was firstly identified as encoding inhibitor of apoptosis protein(IAP)in baculovirus.We subcloned the amplified PCR fragments of IAP-86 into the prokaryotic expression vector pET32a(+),and obtained the recombinant plasmid pET32a-IAP86 through the linking of IAP-86 gene to pET32a(+)plasmid.Then the recombinant plasmids were transformed into E.coil BL21(DE3)strain and expressed under the induction of IPTG.The SDS-PAGE analysis showed that the molecular mass of the induced recombinant protein was about 40 ku.The expressed protein was verified through the Western-blotting and mass spectrometry analysis.Then the recombinant protein was purified with Co²⁺ purification column and marked with FITC.We found IAP-86 could combine with the nucleus and the cytoplasm and inhibit the apoptosis of blood lymphocyte cell of C.farreri through coincubation of them.The cell apoptosis was inhibited by the recombinant of IAP-86 according to the result of apoptosis experiment,and the rate of apoptosis inhibition was about 7%.IAP-86 was successfully expressed through the prokaryotic expression system in our study,and the expressed protein was found to be able to inhibit cell apoptosis of blood lymphocyte of C.farreri.These results provide theoretical and experimental basis for studying the infection mechanism of AVNV.