摘要
目的从华支睾吸虫cDNA文库扩增表膜蛋白31.8kDa(TP31.8)基因,克隆入表达载体pGEX-4T-1,诱导表达重组蛋白,纯化重组蛋白免疫大鼠获得特异性抗体,为后续功能研究奠定基础。方法以华支睾吸虫cDNA文库为模板,设计一对特异性引物,扩增华支睾吸虫TP31.8基因,扩增产物经EcoRⅠ、XhoⅠ双酶切后与做相应酶切的pGEX-4T-1连接,转化大肠杆菌BL21,提取质粒进行双酶切鉴定,IPTG诱导重组蛋白表达,并对基因进行测序及比对分析。融合蛋白经谷胱甘肽Sepharose 4B柱纯化后,用凝血酶去除谷脱甘肽疏基转移酶(GST)。获得纯化的重组蛋白,使用重组蛋白皮下注射免疫大鼠,ELISA检测抗TP31.8的特异性IgG抗体滴度。结果 TP31.8基因成功扩增,重组pGEX-4T-1-TP31.8双酶切鉴定可见目的片段。测序结果显示TP31.8在正确阅读框中,TP31.8开放阅读框长828bp,编码相对分子质量为31.8×103的蛋白,等电点为4.7。TP31.8与其他蠕虫的表膜蛋白具同源性,与日本血吸虫表膜蛋白Sm20的一致性为41%,IPTG诱导后,pGEX-4T-1-TP31.8/BL21有相对分子质量57.8×103融合蛋白的表达。融合蛋白经凝血酶酶切后得到相对分子质量31.8×103的重组蛋白。使用重组蛋白免疫大鼠,ELISA检测抗重组蛋白的特异的IgG抗体滴度为1∶25 600。结论成功克隆了华支睾吸虫表膜蛋白TP31.8基因,并获得重组蛋白的表达。重组TP31.8具免疫原性,免疫大鼠获得的特异性抗体滴度为1∶25 600,为其后续功能研究奠定了基础。
Objective To amplify Clonorchis sinensis tegumental proteins 31.8kD（TP31.8） gene from cDNA library,clone into the expression vector pGEX-4T-1 ,induce expression of recombinant protein,and used purified recombinant protein to immune rats for specific antibodies,in order to make the foundation for subsequent function study. Methods A pair of specific primer was de- signed according to TP31.8 sequences. Using Clonorchis sinensis cDNA library as template,TP31.8 gene was obtained by PCR. Af- ter digested by EcoR I and Xho I ,the PCR production was ligated with pGEX-4T-1 which digested with the same enzyme,and the recombinant plasmid was transformed into E. coli BL21. Recombinant plasmid was identified by double enzyme digestion and sequence analysis,and the sequence results were compared with gene sequence in the GenBank. Expression of recombinant protein was induced by IPTG. Fusion protein was purified by glutathione Sepharose 4B column, thrombin was used to excise GST, recombi- nant protein was used to immune rats subcutaneously,and TP31.8 specific IgG antibody Was detected by using ELISA. Results TP31.8 gene was amplified successfully, and objective fragment was obtained by double enzyme identification of recombinant pGEX-4T-1-TP31.8 plasmid. Sequencing results showed TP31.8 was in correct reading frame,and the open reading frame was 828 bp,which encoded a 31.8X 103 protein with isoelectric point of 4.7. Amino acid search （BLAST-X） showed that this predicted pro- tein was a homologue to tegumental protein from other platyhelminth parasites, with 41~ identity to Schistosoma japonicum Sm20. After IPTG induction,fusion protein expressed in the corresponding molecular weight 57.8 ×103 ,and recombinant protein was obtained by thrombin enzyme digestion. Using recombinant protein as antigen to immunize rats,TP31.8 specific antibody was obtained,and the TP31.8 specific IgG antibody titer was 1 ： 25 600 detected by ELISA. Conclusion Clonorchis sinensis TP31.8 was cloned and expressed successfully,and recombinant
出处
《国际检验医学杂志》
CAS
2014年第2期129-131,133,共4页
International Journal of Laboratory Medicine
基金
国家自然科学基金资助项目(30671831)
国家高技术研究发展计划("863"计划)资助项目(2006AA02Z422)
关键词
华支睾吸虫
重组蛋白质类
克隆
生物
Clonorchis sinensis
recombinant proteins
cloning,organism
