日本血吸虫TGF-β1成熟肽编码序列的克隆、表达及其转录水平  

Cloning,Expression and Transcriptional Level of TGF-β1 of Schistosoma japonicum

作　　者：朱郇悯[1] 麦璟莹[1] 赵晶晶[1] 马长玲[1] 陈晓湘[1] 张雪雁[1] 杨英[1] 张惠球[2] 李孜[1] 

机构地区：[1]广州医学院病原微生物学与免疫学教研室,广州510182 [2]广州医学院病理学教研室,广州510182

出　　处：《热带医学杂志》2008年第5期412-415,共4页Journal of Tropical Medicine

基　　金：国家自然科学基金(No.30600516);广东省自然科学基金(No.5300973)

摘　　要：目的获得日本血吸虫(Schistosoma japonicum,Sj)转化生长因子β1(TGF-β1)成熟肽基因的DNA,进行原核克隆和表达,并比较它与Sj26kd的谷胱甘肽转移酶(Sj26GST)在成虫和虫卵期的转录水平。方法根据美国国立生物信息学中心(NCBI)上登录的小鼠TGF-β1基因的全长序列,设计两条位于保守区的寡核苷酸作为引物,利用RT-PCR从日本血吸虫成虫的mRNA中扩增SjTGF-β1基因的部分序列,并利用生物信息学进行分析和鉴定。克隆SjTGF-β1成熟肽基因的cDNA到原核表达载体pGEX-6p-3,将重组载体转化大肠杆菌BL21株并诱导表达。利用RT-PCR半定量分析SjTGF-β1基因与Sj26GST基因在成虫和虫卵期的转录水平。结果成功获得并构建了SjTGF-β1成熟肽基因的原核表达载体。融合表达后的重组融合蛋白经SDS-PAGE分析,显示其相对分子质量约为35000,与目的基因编码蛋白的预测分子量相符。SjTGF-β1在成虫与虫卵期的转录水平都低于Sj26GST基因,但虫卵期的转录水平比成虫高。结论SjTGF-β1成熟肽基因的获得为其编码序列全长的获得奠定了基础;重组融合蛋白的成功表达为进一步的功能研究提供保障;虫卵期该基因的转录水平高提示该基因在血吸虫虫卵与宿主的相互作用中可能具有一定的作用。Objective To clone and express the transforming growth factor beta 1 （TGF-β1）gene of Schistosoma japonicum,and to compare the transcriptional level of SjTGF-β1 with glutathione-S-transferase at adult worm and egg stages. Methods Based on the coding sequence of mouse TGF-β1 published in the database of the National Center for Biotechnology Information （NCBI）,the conserved domain was searched. A pair of primer was designed for the amplification of the coding sequence of SjTGF-β1 from mRNA of S.japonicum adult worm by RT-PCR. The DNA sequence of the amplified cDNA was analyzed and identified by bioinformatics. The cDNA coding for the SjTGF-β1 mature peptide was sub-cloned into pGEX-6P-3 vector. The recombinant plasmid was transformed into E.coli BL21 and expressed. Semi-quantitative RT-PCR was used to analyze the transcriptional level of SjTGF-β1 and Sj26GST genes in adult worm and egg of S.japonicum. Results The cDNA sequence of a novel SjTGF-β1 mature peptide was obtained and identified, and then was successfully cloned into pGEX-6p-3. SjTGF-β1 mature peptide fusion protein about 35 kDa was expressed in E.coli. The expression level of SjTGF-β1 gene was lower than that of Sj26GST gene at adult worm and egg stages, while its expression level at egg stage was higher than that at adult worm stage. Conclusion The cDNA coding for the SjTGF-β1 mature peptide was cloned and was expressed as a recombinant fusion protein in E. coli. The high transcriptional level of this gene at egg stage suggests its significance in schistosome-host interaction.

关 键 词：日本血吸虫 TGF—β1 血吸虫-宿主相互作用 

分 类 号：R383.24[医药卫生—医学寄生虫学]