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目的利用大肠杆菌对C2株蓝氏贾第鞭毛虫ILP蛋白(Impact-like protein)进行克隆表达,运用生物信息学软件进行蛋白结构分析。方法提取C2株蓝氏贾第鞭毛虫基因组DNA,PCR扩增ILP基因,构建pGM-T重组载体,挑选阳性克隆并进行序列分析;将ILP基因连入原核表达载体pET-28a(+),并转化大肠杆菌Rosetta(DE3),IPTG诱导表达。运用PSIPRED和SWISS-MODEL进行蛋白结构分析。结果成功构建了原核表达载体pET-28a(+)-ILP,该基因全长831bp。SDS-PAGE结果显示,目的蛋白条带出现在相对分子量约33kD的位置,与预期相符。Western blot结果表明,大肠杆菌成功表达了重组蛋白。结论成功克隆、表达并分析C2株蓝氏贾第鞭毛虫ILP蛋白,为蓝氏贾第鞭毛虫ILP蛋白结构与功能的研究提供了有价值的资料。
OBJECTIVE: To clone and express the C2 strain of Impact-like protein of Giardia lamblia using Escherichia coli, and analyze the protein structure by bioinformatics software. Methods C2 genomic DNA of Giardia lamblia was extracted and the ILP gene was amplified by PCR. The recombinant plasmid pGM-T was constructed and the positive clones were selected and sequenced. The ILP gene was inserted into prokaryotic expression vector pET-28a (+) E.coli Rosetta (DE3) was transformed into E.coli and induced by IPTG. Protein structure analysis using PSIPRED and SWISS-MODEL. Results The prokaryotic expression vector pET-28a (+) - ILP was successfully constructed. The full length of this gene was 831bp. SDS-PAGE results showed that the target protein bands appeared in the relative molecular weight of about 33kD position, in line with expectations. Western blot results showed that E. coli successfully expressed the recombinant protein. Conclusions The cloning, expression and analysis of ILP protein in C2 strain of Giardia lamblia, which provided valuable information for the study on the structure and function of ILP protein in Giardia lamblia.