嵌合KGDS基序的蜱抗凝血肽突变体基因在大肠埃希菌BL21(DE3)中的表达

Prokaryotic expression of TAP-KGDS-Trx fusion gene in Escherichia coli BL21(DE3)

目的设计含赖氨酸-甘氨酸-天冬氨酸-丝氨酸基序(KGDS)的蜱抗凝血肽(TAP)突变体基因序列,并在大肠埃希菌BL21(DE3)中与硫氧还蛋白(Trx)融合表达,以获得Trx-TAP-KGDS融合蛋白。方法人工合成目的基因,定向克隆于含Trx基因的高效原核表达载体pET32a(+),构建重组质粒pET32a(+)-TAP-KGDS,并转化BL21感受态菌,以PCR及双酶切鉴定阳性克隆;含正确重组质粒的工程菌经IPTG诱导表达,SDS-PAGE电泳鉴定表达产物。结果重组质粒经PCR及双酶切鉴定,均获得约209 bp的基因片段,与预期大小一致;SDS-PAGE分析显示,含重组质粒的工程菌在IPTG的诱导下高效表达分子质量单位约为24 ku的蛋白质,该蛋白以水溶性形式为主。结论构建了含KGDS基序的TAP突变基因的原核表达载体pET32a(+)-TAP-KGDS,并成功进行了融合表达,为进一步研究其抗凝血活性打下了基础。

Objective Prokaryotic expression vector of chimeric gene containing a sequence encoding tick anticoagulant peptide（TAP） and motif Lys-Gly-Asp-Ser（KGDS） was constructed and expressed to obtain thioredoxin （Trx） fusion protein. Methods The chimeric gene was synthetic and amplified by PCR, cloned directionally into prokaryotic expression vector pET32a（ ＋ ）, containing thioredoxin （Trx） gene, to construct expressing plasmid pET32a （ ＋ ）-TAP-KGDS, which was transformed into Escherichia coli BL21 （DE3）. The positive bacteria clones were screened by PCR and restriction endonuclease digestion analysis. The bacteria BL21 containing plasmid pET32a（ ＋ ）-TAP-KGDS was induced by IPTG to express fusion protein Trx-TAP-KGDS. The expression product was identified by SDS-PAGE, and the expression condition was optimized. Results Correct construction of pET32a（＋）-TAP-KGDS was identified by PCR and restriction endonuclease digestion analysis. The results of SDS-PAGE indicated the molecular weight of expressed fusion protein containing Trx was about 24 ku, and the fusion protein was expressed in soluble form. Conclusion The pET32a （＋）-TAP-KGDS prokaryotic expression plasmid was successfully constructed and expressed, which lays basis for our further study about its activity of anticoagulation.