犬博卡病毒结构基因VP2多片段原核表达载体的构建及表达条件的优化

Construction and Expression Condition Optimization of Prokaryotic Expression Vector of Canine Bocavirus Structural Gene VP2 Multi-fragment

目的构建重组pGEX-4T-3-VP2-A、pGEX-4T-3-VP2-B、pGEX-4T-3-VP2-C、pGEX-4T-3-VP2-D原核表达质粒,并在大肠杆菌中进行表达,对诱导表达条件进行优化,为下一步获得大量纯化的融合蛋白奠定基础。方法应用蛋白二级结构软件,设计合成MVC-VP2基因片段引物。用PCR法扩增不同长度的cDNA片段,通过EcoRⅠ和XhoⅠ酶切位点分别将四个不同的片段插入到pGEX-4T-3中,构建四种原核表达重组质粒,并转化E.coli DH5α,筛选阳性重组子,限制性内切酶酶切鉴定和DNA序列测定正确后,转入E.coli BL21中,异丙基硫代β-D半乳糖苷(IPTG)诱导表达,对表达条件(诱导剂浓度、诱导时间、诱导温度)进行优化,并通过SDS-PAGE鉴定。结果酶切及测序结果证明,成功构建了四种原核表达重组体质粒pGEX-4T-3-VP2-A、pGEX-4T-3-VP2-B、pGEX-4T-3-VP2-C、pGEX-4T-3-VP2-D,成功表达四种融合蛋白,优选出IPTG诱导终浓度为0.5mmol·L-1,诱导时间为12h,诱导温度为18℃。结论成功构建了原核表达重组体质粒pGEX-4T-3-VP2-A、pGEX-4T-3-VP2-B、pGEX-4T-3-VP2-C、pGEX-4T-3-VP2-D,优化出最佳诱导条件,为下一步获得大量纯化的目的蛋白、制备VP2多克隆抗体奠定基础。

Objective To construct the prokaryotic expression plasmid of recombinant pGEX -4T- 3 -VP2 - A, pGEX - 4T - 3 - VP2 - B, pGEX - 4T - 3 - VP2 - C, pGEX - 4T - 3 - VP2 - D and to express them in Escherichia coll. Methods MVC - VP2 gene fragment primer was compounded using protein secondary struc- tural software . Different length of eDNA sequences was amplified with PC R, and then inserted into pGEX - 4T -3 easy vector through EcoR I and Xho I enzyme cutting sites,to construct four kinds of recombinant prokary- otic expression plasmids, transformed them into E. coil DHSctand positive clones were selected through the col- ony - PCR and confirmed by the double restrict enzyme digestion and sequencing . The successful pGEX - 4T -3 recombinant plasmid was transformed into E. coli BL21 and induced with IPTG to express . Eventually, the products expression identified by SDS -PAGE, and we also optimize the expression condition, such as inducer concentration, induction time and induction temperature. Results Enz）me cleavage and sequencing results showed that four kinds of recombinant prokaryotic expression plasmid were successfully constructed and four kinds of fusion protein were expressed. We also found that the final concentration of IPTG was 0.5mmol · L ^-1, the optimal induction time 12h, the optimal induction temperature 18℃. Conclusion Four kinds of recombi- nant prokaryotic expression plasmid were successfully constructed and thee optimal conditions were found.