摘要
整合素(Integrin)是一种由α和β亚基组成的跨膜异二聚体蛋白,在昆虫血细胞对外物的包囊过程中起着重要的作用。为阐明integrinβ1参与小菜蛾Plutella xylostella体内细胞免疫中的功能,本研究利用RT-PCR结合RACE技术克隆了小菜蛾integrinβ1基因的cDNA全长序列,命名为PxIntβ1(Gen Bank登录号GQ178290)。小菜蛾PxIntβ1的cDNA全长序列为2 832 bp,开放阅读框为2 487 bp,编码828个氨基酸,成熟的氨基酸序列中有一个跨膜区和一个integrin亚基。系统进化树显示小菜蛾PxIntβ1与亚洲玉米螟Ostrinia furnacalis整合素的同源性最高,两者同源性达到78%。利用半定量RT-PCR技术检测PxIntβ1基因在小菜蛾不同发育历期(卵、1-4龄幼虫、蛹、成虫)、不同组织(血细胞、体壁、脂肪体、中肠、马氏管)和细菌(金黄色葡萄球菌和大肠杆菌混合菌液)诱导下的表达模式。半定量结果表明小菜蛾PxIntβ1基因在小菜蛾整个发育历期除了卵之外都有表达,4龄幼虫转录水平最高,在血细胞中特异性的高表达,混合细菌诱导2-48 h后,PxIntβ1基因在小菜蛾诱导后24 h达到诱导表达高峰。为了进一步获得重组蛋白PxIntβ1,本研究构建了原核表达质粒pET-32a-PxIntβ1,融合蛋白在大肠杆菌Eschericha coli BL21中获得高效表达,Ni-NTA一步纯化了融合蛋白,并免疫新西兰大白兔,获得了多克隆抗体anti-PxIntβ1。SDS-PAGE电泳和Western blot分析结果表明,His抗体和多克隆抗体anti-PxIntβ1能特异性识别融合蛋白PxIntβ1;进一步利用显微镜观察了小菜蛾血细胞对凝胶珠Sephadex-A25的包囊情况,结果表明抗血清anti-PxIntβ1处理过的小菜蛾血细胞对Sephadex-A25凝胶珠的包囊作用受到明显抑制,重组蛋白PxIntβ1可以提高小菜蛾血细胞对Sephadex-A25凝胶珠的包囊作用;以上研究结果表明小菜蛾PxIntβ1在小菜蛾细胞免疫中起着重要的作用。
Integrin is a transmembrane protein dimers composed by alpha and beta subunits,integrin has been reported to play a vital role in hemocyte encapsulation foreign objects in insects.In order to illustrate the function of integrinβ1 involved in cellular immunity of P.xylostella.In the present study,a full-length cDNA of an integrinβ1 gene was cloned from P.xylostella by RT-PCR and rapid amplification of cDNA ends(RACE)techniques,and designated as PxIntβ1(GenBank accession no.:GQ178290).Its full-length cDNA is 2 832 bp,and the open reading frame(ORF)is 2 487 bp,encoding 828 amino acid residues.The mature amino acid sequence of PxIntβ1 is composed of a transmembrane region and an integrin subunit.Phylogenetic analysis indicated that PxIntβ1 has the highest homology to integrinβfrom O.furnacalis,the similarity between them is 78%.Semi RT-PCR was employed to analyze the expression profiles of this gene in different tissues(hemocyte,epidermis,fat body,midgut and malpighiam tubules)of the 4 th instar larvae,different developmental stages(egg,1 st-4 th instar larva,pupa,and adult)and microbial induction(the mix between Staphylococcus aureus and Escherichia coli)in P.xylostella.Semi RT-PCR results indicated that the mRNA level of PxIntβ1 was expressed during the whole developmental stages except eggs,with the highest transcript level in the 4 th instar larva.Tissue specific result showed that PxIntβ1 was specifically expressed in hemocyte.Microbial induction result showed PxIntβ1 was induced significantly by mixed bacteria from 2-48 h,with the highest expression level at 24 h post-injection mixed bacteria.In order to acquire the recombinant protein of PxIntβ1,prokaryotic expression plasmid pET-32a-PxIntβ1 was constructed and the fusion protein was highly expressed in E.coli BL21.and recombinant protein was one step purified by Ni-NTA.The polyclonal antibody anti-PxIntβ1 with high serum titer was prepared using the purified fusion protein to immunize New Zealand white rabbit.SDS-PAGE and western blot analysis investigated that fusion protein PxIntβ1 can be specifically recognized by both His-antibody and polyclonal antibody anti-PxIntβ1.The encapsulation assay of the hemocyte of P.xylostella against Sephadex-A25 gel beads was observed by microscope.The results investigated that the encapsulation of P.xylostella hemocyte against Sephadex-A25 gel beads was significantly repressed after polyclonal antibody anti-PxIntβ1 incubation with hemocyte,but the encapsulation of P.xylostella hemocyte against Sephadex-A25 gel beads was significantly enhanced after recombinant protein PxIntβ1 incubation with hemocyte.All the above results indicated that PxIntβ1 plays an important role in cellular immunity of P.xylostella.
作者
郑锦龙
许小霞
余静
张展滔
兰可可
仇培
金丰良
ZHENG Jin-Long;XU Xiao-Xia;YU Jing;ZHANG Zhan-Tao;LAN Ke-Ke;QIU Pei;JIN Feng-Liang(Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province,College of Agriculture,South China Agricultural University,Guangzhou 510642,China)
出处
《环境昆虫学报》
CSCD
北大核心
2018年第4期880-893,共14页
Journal of Environmental Entomology
基金
国家自然科学基金(31371989)
广东省科技计划项目(2015A020209128,2014A020208106)。
作者简介
郑锦龙,男,1993年生,福建长乐人,硕士研究生,研究方向为昆虫免疫学,E-mail:470182655@qq.com。;金丰良,通讯作者Author for correspondence,E-mail:jflbang@scau.edu.cn。
