【摘要】 利用末端快速扩增技术从莫氏巴贝斯虫cDNA中扩增actin,aldolase和trap基因的5′和3′末端,经过序列装配后将获得的全序列插入pGEX-4T-1原核表达载体,将构建的原核表达载体转化至BL21(DE3)pLysS中,经IPTG诱导、表达后,将纯化的融合蛋白免疫家兔,制备多克隆抗体,并用ELISA和Western-blot测定抗体的效价和反应性。结果表明,获得的actin,aldolase和trap基因的全长分别为1 110、1 199和2 254bp,开放阅读框长度分别为1 005、1 074和2 100bp。制备的多克隆抗体具有良好的特异性。为筛选巴贝斯虫病疫苗、诊断用抗原和药物靶位,以及研究巴贝斯虫入侵宿主细胞的分子机制奠定了基础。更多还原

【Abstract】 Rapid amplification of cDNA ends was used to amplify the 5′and 3′ends of actin,aldolase and trapgenes of Babesia motasi.The complete open reading frames(ORF)of those genes were inserted into the prokaryotic expression vector pGEX-4T-1,respectively,and then transformed into BL21(DE3) pLysS to express by inducing with IPTG.The fusion proteins purified with MagneGST Protein Purification System were injected into rabbits to produce polyclonal antibodies.Specificity and titers of the polyclonal antibodies were determined by Western-blot and ELISA.The results indicated that the full-length cDNAs of actin,aldolase and trapgenes were 1 110bp,1 199bp and 2 254bp with ORF of 1 005bp,1 074bp,and 2 100bp in size,respectively.The polyclonal antibodies prepared exhibited good specificity and reactivity.In the present study,the full length of those genes of B.motasi were amplified and their polyclonal antibodies were obtained,which provides an important biological materials for screening and identifying candidate antigens of vaccination and diagnosis,drug targets as well as studying invasion mechanisms of Babesia spp.更多还原