HCV C区基因片段的克隆、表达及纯化

Cloning,Expression and Purification of HCV Recombinant Core Protein

目的研究 ＨＣＶ Ｃ区片段在大肠杆菌中的表达及纯化工艺。方法采用 ＲＴ－ＰＣＲ技术，从ＨＣＶ感 染者血清中克隆Ｃ区基因片段，插入ｐＥＴ２８ａ（＋）质粒中，并在大肠杆菌ＢＬ２１（ＤＥ３）中表达。采用亲和层析或 Ｓａｐｈａｃｒｙｌ Ｓ－２００柱层析纯化抗原。结果两种纯化工艺的收率分别为５８ｍｇ／Ｌ和９５ｍｇ／Ｌ发酵液。ＳＤＳ－ＰＡＧＥ显示纯 度分别为９８％和９０％。ＥＬＩＳＡ检测纯化抗原具有较好的抗原性和特异性。结论两种纯化工艺简便，重组蛋白纯 度高，可用于ＥＬＩＳＡ试验。

Objective To study on the expression of core gene of HCV in E. coli and the purification of expressed products. Methods A core gene fragment was cloned from the sera of Patients infected with HCV by RT-PCR and inserted into plasmid vector pET28a(+ ), then the recombinant plasmid pEC4 was transformed to E. coli BL21(DE3) for expression. The expressed products were purified by affinity or Sephacry1 S-200 column chromatography.Results The recovery rates of expressed proteins purified by affinity and Sephacryl S-200 column chromatography were 58 and 95 mg/L respectively. SDS-PAGE Showed that the Purity of them were 98% and 90% respectively. ELISA showed good antigenicity and specificity of the purified protein. Conclusion Both the purification process were simple,and the recombinant protein with a high purity could be used for ELISA.