摘要
目的克隆表达C2株蓝氏贾第鞭毛虫H3组蛋白基因,与WB株蓝氏贾第鞭毛虫进行同源分析,构建C2株蓝氏贾第鞭毛虫H3组蛋白分子进化树。方法 PCR扩增获取H3组蛋白基因,构建pGM-T-H3重组载体,转化E.coli TOP10感受态宿主细胞,挑选阳性克隆并进行序列分析,利用限制性内切酶NcoⅠ和XhoⅠ构建pET28a(+)-H3重组载体,转化E.coli Rosetta(DE3),IPTG诱导组蛋白H3表达,Western blotting鉴定表达,与美国WB株蓝氏贾第鞭毛虫及模式生物H3组蛋白基因和蛋白序列进行同源分析。结果成功克隆表达C2株蓝氏贾第鞭毛虫H3组蛋白基因,同源比对结果显示C2株蓝氏贾第鞭毛虫基因序列与美国WB株完全一致,但与其他现存真核生物亲缘关系较为疏远。结论 C2株蓝氏贾第鞭毛虫H3组蛋白分子进化树分析表明贾第虫H3组蛋白基因在进化过程中与其他物种分化较早,本研究结果为进一步研究蓝氏贾第鞭毛虫的生物进化地位提供有价值的实验资料。
The H3 histone gene of C2 Giardia lamblia （G. lamblia） （Chinese Sichuan strain） was cloned, expressed and analyzed in this study. Genome DNA of C2 G. larnblia was extracted with DNA extraction kit, and the specific primers were designed by primer design software Primer Premier 5 according to GenBank reported G. lamblia H3 histone coding sequence （NCBI Reference Sequence： XM_001707183.1）. H3 histone gene was amplified by PCR and cloned into pGM-T vector, transformed into E. coli （Top10） competent bacteria cells with recombinant plasmid named pGM-T-H3. Positive cloning was chosen and the sequence was analyzed. The pGM-T-H3 recombinant vector was digested by restriction endonuclease Nco I and Xho I ; target gene restructured into pET28a（＋） vector at the Nco I and Xho I restriction sites to generate recombinant expression vector pET28a（＋）-H3 and transformed into E. coli Rosetta（DE3）, inducing with IPTG （isopropyl β-D-thiogalactopyranoside） by optimizing the concentration and induction time for protein expression. SDS-PAGE result demonstrated that the objective protein band in the position showed a relative molecular weight of 18 kDa. Western blot analysis with His-Tag anti- body indicated that the H3 histone was successfully expressed in the prokaryotic Rosetta （DE3）. The H3 histone gene between C2 G. lamblia and WB G. lamblia （WB strain, clone C6） were completely homologous, and the phylogenetic analysis based on gene sequence and amino acid sequence indicated that C2 G. lamblia was relatively independent in the evolutionary process. Our results provided valuable experimental data for further understanding on the chromosome and karyotype of G. lamblia.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2014年第6期568-574,共7页
Chinese Journal of Zoonoses
基金
Supported by the National Natural Science Foundation of China(No.30970313)
the Scientific and Technological Support Projects from Tangshan,China(Grant No.12140209A-33)
the Youth Science Fund Project in Hebei Province(No.C2012401039)
the Hebei United University Training Fund(No.GP201308)~~
