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目的从弓形虫RH株总DNA中克隆棒状体蛋白ROP2基因片段,克隆至表达载体pET22b中,导入大肠杆菌中表达。方法采用半巢式PCR扩增法从基因组DNA中扩增编码ROP2基因片段,克隆至质粒pUC119中,经PCR和酶切鉴定后,进行DNA序列测定,并以SacⅠ/HindⅢ双酶切克隆至表达载体pET22b上,重组质粒pET22b-ROP2转化大肠杆菌BL21-Codon Plus(DE3)-RIL菌株中,经IPTG诱导表达。结果从弓形虫RH株总DNA中扩增到1.0kb的ROP2基因片段,构建成功重组质粒pUC119-ROP2,经DNA序列分析与已报道序列基本一致,重组质粒pET22b-ROP2在大肠杆菌中表达,产生一分子量约为45kDa的预期重组蛋白。结论克隆到的弓形虫棒状体蛋白ROP2在大肠杆菌中得到表达,构建成功的重组质粒pUC119-ROP2可用于下一步多基因融合的克隆操作。
Objective To clone the fragment of ROP2 gene from the total DNA of Toxoplasma gondii RH strain and cloned into expression vector pET22b and express in E. coli. Methods The gene encoding ROP2 gene was amplified by semi-nested PCR from genomic DNA and cloned into plasmid pUC119. After PCR and restriction enzyme digestion, the DNA sequence was determined and cloned by SacⅠ / HindⅢ digestion On the vector pET22b, the recombinant plasmid pET22b-ROP2 was transformed into E. coli BL21-Codon Plus (DE3) -RIL and induced by IPTG. Results The 1.0 kb ROP2 gene was amplified from the total DNA of Toxoplasma gondii RH strain. The recombinant plasmid pUC119-ROP2 was successfully constructed. The DNA sequence analysis showed that the recombinant plasmid pET22b-ROP2 was expressed in E. coli. A predicted recombinant protein with a molecular weight of about 45 kDa. Conclusion The cloned Toxoplasma gondii ROP2 was expressed in E. coli. The constructed recombinant plasmid pUC119-ROP2 was successfully used in the cloning of multi-gene fusion.