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目的克隆十二指肠钩虫金属蛋白酶组织抑制剂同源物(TIMPL)Ad-TIMPL-1基因并进行原核表达。方法分别用3’RACE及RT-PCR技术从十二指肠钩虫成虫cDNA中扩增编码Ad-TIMPL-1的cDNA3’末端及5’末端序列;序列经拼接后进行初步生物信息学分析;将Ad-TIMPL-1成熟肽编码序列克隆至原核表达载体pET-32a,构建重组表达质粒;重组质粒转化至大肠杆菌BL21(DE3)后,用IPTG诱导表达,SDS-PAGE分析表达情况。结果成功克隆获得了编码Ad-TIMPL-1的全长cDNA序列并登记到GenBank(accession no.EF495071);Ad-TIMPL-1完整阅读框为396bp,编码132个氨基酸残基组成的蛋白,含16个氨基酸残基组成的信号肽;成功构建了原核表达重组质粒pET-32a/Ad-TIMPL-1,并在大肠杆菌中得到了表达。结论本研究首先从十二指肠钩虫中克隆到了Ad-TIMPL-1基因并进行了原核表达,为进一步研究其生物学功能奠定了基础。
Objective To clone and express the Ad-TIMPL-1 gene of TIMPL in the duodenal hookworm. Methods The 3’-end and 5’-end sequences of cDNA encoding Ad-TIMPL-1 were amplified from the cDNA of adult C. hooker by 3’RACE and RT-PCR respectively. The sequences were stitched and analyzed by bioinformatics. The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG. The expression of the recombinant plasmid was analyzed by SDS-PAGE. The recombinant plasmid was cloned into prokaryotic expression vector pET-32a. Results The full-length cDNA sequence encoding Ad-TIMPL-1 was successfully cloned and registered in GenBank (accession no.EF495071). The full-length reading frame of Ad-TIMPL-1 was 396 bp and encoded a protein of 132 amino acid residues with 16 Amino acid residues. The recombinant plasmid pET-32a / Ad-TIMPL-1 was successfully constructed and expressed in E. coli. Conclusion In this study, Ad-TIMPL-1 gene was cloned from H. duodenum and prokaryotic expressed, which laid the foundation for further study of its biological function.