残缘璃眼蜱subolesin基因的原核表达及表达产物的反应原性分析

Prokaryotic expression of subolesin gene from Hyalomma detritum and analysis of reactionogenicity of the expressed protein

为研究残缘璃眼蜱subolesin基因的生物学特性以及寻找新的抗蜱和蜱传病疫苗候选抗原,根据GenBank上登录的小亚璃眼蜱subolesin基因核苷酸序列设计特异性引物,以残缘璃眼蜱、亚洲璃眼蜱、微小牛蜱的饥饿成蜱cDNA为模板,扩增出subolesin基因的完整阅读框(ORF)。将残缘璃眼蜱subolesin基因的PCR产物经EcoRⅠ+NotⅠ双酶切后连接至表达载体pGEX-4T-1,把重组质粒pGEX-4T-1-subolesin导入感受态细胞BL21(DE3)中,在37℃、1mmol/L IPTG条件下诱导表达。利用SDS-PAGE分析subolesin基因的体外表达特征;用Western-blot分析Subolesin蛋白的反应原性。结果显示,残缘璃眼蜱、亚洲璃眼蜱、微小牛蜱subolesin基因的ORF长度分别为492、492、486bp,分别编码163、163、161个氨基酸。subolesin基因序列和推导的氨基酸序列的比对结果显示,残缘璃眼蜱、亚洲璃眼蜱、微小牛蜱与参考的小亚璃眼蜱的核苷酸序列同源性分别为98%、98%、89%,氨基酸序列同源性分别为98%、99%、93%。SDS-PAGE和Western-blot结果显示,重组残缘璃眼蜱Subolesin蛋白的分子质量为45ku,纯化的subolesin重组抗原可与抗残缘璃眼蜱全蜱血清、残缘璃眼蜱唾液腺、卵巢血清有较强的反应原性。

To describe the biological characters of subolesin gene of Hyalomma detritumand search for the new candidate protective antigen against ticks and transmission of associated pathogens,one pair of special primers was designed according to the sequence of subolesin gene of Hyalomma anatolicum available from GenBank.The complete open reading frames(ORFs)of subolesin gene of H.detritum,H.asiaticum and Boophilus microplus were amplified.The products were digested with EcoRⅠ and NotⅠ and were cloned into pGEX-4T-1vector.Then,the recombinant plasmid was transformed into E.coil BL21(DE3)cells.The recombinant Hyd-subolesin was induced with IPTG(1mmol/L)at 37℃.The characteristics of subolesin gene was analyzed using SDS-PAGE and the reactionogenicity was analyzed by Western-blot.In result,the subolesin gene was 492 bp coding for 163 amino acids in H.detritumand H.asiaticumand 486 bp coding for 161 amino acids in B.microplus.Compared H.detritum,H.asiaticumand B.microplus with H.anatolicum,the homologies of the nucleotide sequences were 98%,98% and 89% and the homologies of the amino acids of subolesin were 98%,99%and 93%,respectively.The recombinant protein was approximately 45 ku in size by SDS-PAGE.The reactionogenicity of the purified recombinant protein was strong with the rabbit anti-H.detritumadult serum and the rabbit anti-H.detritumsalivary gland and ovary sera.