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目的获取中国人源性蓝氏贾第鞭毛虫ppdk基因及相关基因产物方法以中国人源性贾第虫基因组DNA为模板,设计引物进行PCR扩增,获取ppdk基因并进行测序分析。筛选得到的阳性克隆连接至原核表达载体pET28a(+),构建重组表达载体,并转化宿主菌大肠埃希菌感受态细胞E.coliBL21(DE3),进行重组蛋白的IPTG诱导表达。收集重组表达产物进行亲和层析纯化,再用Western blot进行免疫学分析鉴定。结果序列测定分析可知,获得的阳性克隆插入片段包含一个2 655 bp的开放阅读框架;比对其基因序列发现,其与ATCC 50803(美国WB虫株C6株)的同源性可高达99%。该基因片段编码884个氨基酸,预测其表达蛋白的分子质量单位大小约为97.6 ku。Western blot显示该基因序列的原核表达产物能够被抗6个组氨酸标签的特异性抗体识别,提示重组表达产物为实验预期的目的蛋白。结论对贾第虫ppdk基因进行了克隆及原核表达,并对表达产物进行了纯化和免疫学初步鉴定,为贾第虫病治疗的高通量药物筛选等的研究奠定了基础。
OBJECTIVE To obtain the ppdk gene and its related gene product from Giardia lamblia in China Methods The genomic DNA of Giardia domestica was used as a template to design the primers for PCR amplification. The ppdk gene was obtained and sequenced. The positive clones were selected and ligated into the prokaryotic expression vector pET28a (+) to construct a recombinant expression vector. The recombinant E. coli BL21 (DE3) was transformed into competent E. coli host cells for IPTG induction. The recombinant expression product was collected and purified by affinity chromatography, and then identified by Western blot. Results The sequence analysis showed that the positive cloned insert contained a 2 655 bp open reading frame. Compared with its gene sequence, its homology with ATCC 50803 (WB strain C6 in the United States) was as high as 99%. The gene fragment encodes 884 amino acids, and the predicted molecular mass unit of the expressed protein is about 97.6 ku. Western blot showed that the prokaryotic expression product of this gene sequence can be recognized by specific anti-6 histidine tag antibody, suggesting that the recombinant expression product is the expected target protein. Conclusion The ppdk gene of Giardia was cloned and expressed in prokaryotic cells. The expressed products were purified and immunologically identified, which laid the foundation for the study of high-throughput drug treatment of Giardia disease.