白色念珠菌菌丝壁蛋白1的N端片段的克隆及表达

Cloning and expression of N-terminal fragment of Candida albicans hyphal wall protein 1

目的构建白色念珠菌的菌丝壁蛋白1(Hwp1)N端片段的重组质粒,在大肠杆菌中诱导表达重组蛋白。方法PCR扩增目的基因,将其连接到pMD18-T载体上,经测序验证成功后,将目的基因连接至表达载体pET28a(+),转化至大肠杆菌BL21(DE3)中,提取质粒进行PCR、双酶切法鉴定,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达Hwp1的N端片段。结果成功制备了重组质粒pMD18-T-Hwp1及pET28a(+)-Hwp1,PCR及双酶切法鉴定结果。构建了含重组质粒pET28a(+)-Hwp1的大肠杆菌工程菌,经IPTG诱导能高效表达目的蛋白。结论成功构建、表达了Hwp1的N端片段,为进一步研究其在诊断白色念珠菌病中的作用奠定了基础。

Objective To construct recombinant plasmid of the N-terminal fragment of Candida albicans hyphal wall protein 1 and induce expression of the recombinant protein in Escherichia coll. Methods The target gene was amplified by PCR. The PCR product was cloned into pMD18-T vector. The plasmid verified by sequencing was in- serted into the expression vector pET28a（ ＋ ）. The recombinant plasmid was transformed into Escherichia coli BL21 （DE3）. The N-terminal fragment of Candida albicans hyphal wall protein 1 was induced by IPTG. Results The recombinant plasmids pMD18-T-Hwpl and pET28a-Hwpl were successfully constructed. PCR and restriction en- zyme digestion results were in line with expectations. The target protein was expressed in Escherichia coli after in- duction with IPTG. Conclusion The Escherichia coli BI221 with the recombinant plasmid pET28a（ ＋ ）-Hwpl is successfully acquired with expression of recombinant protein, which provides a basis for further studies on candidia- sis.