摘要
旨为证实核糖体蛋白S18(Ribosomal protein S18,RPS18)基因在家蝇体内的表达稳定性。从构建家蝇幼虫c DNA文库中筛选到核糖体蛋白S18基因,以c DNA为模板,通过PCR扩增,获得RPS18基因完整编码序列(登录号:KT006855),运用生物信息学方法对该基因及其编码蛋白进行预测和分析。构建p ET28a/RPS18重组质粒,转化到大肠杆菌Transetta(DE3)中进行诱导表达及蛋白纯化,制备多克隆抗体并进行抗血清特异性分析。应用RT-PCR、q PCR及Western-blot从转录、翻译水平上分析RPS18基因在家蝇不同发育时期和三龄幼虫不同组织中的表达情况。结果表明,RPS18基因ORF全长459 bp,编码152个氨基酸,理论分子量为17 590.5 Da,等电点为10.48;将构建的重组质粒转入大肠杆菌Transetta(DE3)中,纯化得到RPS18蛋白;将该纯化蛋白免疫大白兔获得多克隆抗体,His单抗鉴定及抗血清特异性分析显示,其均可见单一条带;RT-PCR、q PCR及Westernblot分析表明,RPS18基因在家蝇不同发育时期及幼虫不同组织均能够稳定表达;综合分析表明,该基因在家蝇不同发育时期及幼虫不同组织均保持较好的表达稳定性。
This research is to confirm the expression stability of ribosomal protein S18（RPS18）gene in Musca domestica. The complete coding sequence（Registration number in NCBI：KT006855）of RPS18 gene was obtained via PCR amplification using the cDNA of RPS18 gene from the cDNA library of M. domestica larva as a template,further the gene and its encoding protein were predicted and analyzed by bioinformatics method. The constructed recombinant plasmid of pET28a/RPS18 was transferred into Escherichia coli Transetta（DE3）for the induced expression and protein purification,and the polyclonal antibody was prepared as well as the specificity of antiserum was analyzed. The transcription and translation expressions of RPS18 gene at different growth stages of the M. domestica larva and in the different tissues of 3-year larva were analyzed by RT-PCR,qPCR and Western blot. The results showed that the RPS18 ORF was 459 bp in length encoding 152 amino acids with a predicted molecular mass of 17 590.5 Da and pI of 10.48. The recombinant plasmid was transferred into E. coli Transetta（DE3）, and the purified protein RPS18 was acquired. The purified protein was immunized to rabbits,then the polyclonal antibody was harvested, and the single band was visualized by both His single resistance identification and antiserum specificity analysis. The analysis by RT-PCR, qPCR and Western blot indicated that RPS18 genes expressed stably in different growth stages and different tissues of the larva. In conclusion, comprehensive analysis suggests that the gene may maintain solid stability in different growth stages of M. domesticaand different tissues of larva.
出处
《生物技术通报》
CAS
CSCD
北大核心
2016年第6期135-142,共8页
Biotechnology Bulletin
基金
国家自然科学基金项目(81360254)
国家科技部支撑计划课题子课题(2011BAC06B12)
贵州省科技厅联合基金项目(黔科合LH字[2014]7076号)
贵州省卫生计生委科学技术项目(gzwjkj2014-2-100)
贵州省高等学校创新能力提升计划(07060151306)
作者简介
胡亚，女，硕士研究生，研究方向：医学昆虫免疫及应用；E-mail：hooyaa@126．com
通讯作者：吴建伟，男，博士生导师，研究方向：医学昆虫免疫及应用；E-mail：wjw@gmc．edu．cn
