PCV2·ORF2基因片段在大肠杆菌中的表达与纯化

Expression and Purification of ORF2 Gene Fragment of Porcine Circovirus Type 2 in E. coli

[目的]为了研究PCV2-ORF2基因片段在大肠杆菌中的表达与纯化。[方法]用PCR方法扩增PCV2 ORF2基因3′端片段,PCR产物经酶切后与经同样处理过的pET32a质粒连接,构建重组质粒。重组质粒经PCR和酶切鉴定并测序后,转化BL21感受态细胞;重组菌液用IPTG诱导表达,对表达产物进行SDS-PAGE和Western blot分析。[结果]ORF2基因在大肠杆菌中正确表达,表达的融合蛋白的分子量约为33 ku;在37℃、1 mmol/L浓度下,诱导时间为6 h时表达量最大,占细菌总蛋白的23.62%。表达产物主要以包涵体的形式存在,将包涵体溶解后过Ni-NTA柱纯化可得到较为纯净的目的蛋白。[结论]该研究为猪断奶后多系统衰竭综合征(PMWS)和猪皮炎和肾病综合征(PDNS)等疾病的防治奠定了基础。

[Objective] The research aimed to study the expression and purification of ORF2 gene fragment of porcine circovirus type 2（PCV2） in E.coli. [Method] The 3＇fragment of ORF2 gene of PCV2 was amplified by PCR method. Through enzyme digestion, the PCR products were connected with the pET32a plasmid with the same treatment to construct the recombinant plasmid. The recombinant plasmid was identified and sequenced by PCR and enzyme digestion to transform BL21competent cell. The recombinant bacterial liquid was induced and expressed by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The ORF2 gene was expressed correctly in E.coll, with the molecular weight of the expressed fusion protein of about 33 ku. Under the conditions of 37℃ and the concentration of 1 mmol/L, the expression quantity at the inducement time of 6 h was highest, being 23.62 % of the total bacterial proteins. The expression products mainly existed as inclusion bodies, which was dissolved to purify through Ni -NTA column so as to get the target protein with higher purity. [Conclusion] The research laid the foundation for the prevention and control of such diseases as PMWS and PDNS.