猪圆环病毒2型ORF2可溶性表达条件的优化

Optimization of prokaryotic expression conditions of the ORF2 gene of porcine circovirus type 2

为探索猪圆环病毒2型（PCV2）ORF2基因在大肠杆菌中高效可溶性表达的条件,扩增ORF2基因构建重组表达质粒p ET30a-ORF2,转化入大肠杆菌BL21（DE3）感受态获得重组表达菌;通过改变菌体培养温度和时间、诱导温度和时间、溶解氧量、IPTG及CaCl_2浓度,实现目的蛋白高效可溶性表达。结果表明：重组表达质粒p ET30a-ORF2经酶切及测序证明构建正确,重组表达质粒在大肠杆菌BL21（DE3）中实现可溶性表达,其最佳表达条件为：在500 mL培养瓶中加入200 mL LB卡那霉素阳性培养基,菌体于37℃培养4 h后,分别加入IPTG及Ca Cl2至终浓度为0.6 mmol/L及0.06 mol/L,30℃诱导表达4 h;重组Cap蛋白的最高表达量占总蛋白的55.9%。说明优化后可实现猪圆环病毒Cap蛋白的可溶性表达,这为进一步研究该蛋白的结构及其生物学特性奠定了基础。

To optimize the condition for secretory expression of ORF2 gene of porcine circovirus type 2（PCV2） in Escherichia coli,ORF2 gene was amplified from recombinant plasmid pMD-ORF2,and then the ORF2 gene was inserted into the vector pET30 a.The constructed recombinant plasmid pET30 a-ORF2 was transformed to E.coli BL21（DE3） for expression.The expression product was analyzed by SDS-PAGE.In order to improve the secretory expression level of target protein,temperature and time for culture and induction,dissolved oxygen content,concentration of IPTG and CaCl_2 were optimized.Both restriction analysis and sequencing proved that the recombinant plasmid pET30 a-ORF2 was constructed correctly,and ORF2 protein was secretory expressed in the E.coli BL21（DE3） after induction.The expression condition for ORF2 was optimized as follows：200 mL of kanamycin-positive LB medium was added into 500 mL flask and cultured at 37℃ for 4 h,then IPTG and CaCl_2 were added to the final concentration of 0.6 mmol/L and 0.06 mol/L,and induced at 30 ℃ for 4 h.Under this condition,expression of ORF2 protein was the highest,up to 55.9 percent of total protein content.Finally,the condition for expression of Cap protein was optimized,which provided a basis for the further mechanistic study of the structure and biological characteristics of Cap protein.