蜡梅nsLTPs基因家族4个成员的原核表达研究

[目的]利用现代分子生物学技术探索蜡梅（Chimonanthus praecox）资源的药用价值。[方法]在有关蜡梅生物学特性研究基础上,构建蜡梅非特异性脂转移蛋白（nsLTP）基因家族4个成员（GenBank登录号：FJ889521、FJ904082、FJ904083、FJ904084）的原核表达载体CpLTP1-pET、CpLTP2-pET、CpLTP3-pET、CpLTP4-pET,并对诱导温度、IPTG浓度和诱导表达时间等条件进行了优化。[结果]在1.0mmol/L IPTG、37℃常规诱导条件下诱导6 h可获得高效表达,总表达产率可达到细菌全蛋白质总量的50%以上,包涵体表达远高于可溶性蛋白的表达;在0.5 mmol/L IPTG、28℃条件下诱导6 h能够获得较好的可溶性表达,利用His-Bind蛋白纯化回收试剂盒获得4个纯化的重组蛋白。[结论]成功构建蜡梅nsLTP的原核表达载体,转化大肠杆菌Origami2（DE3）获得4个纯化的重组蛋白,可为后续的抗菌抗病毒活性研究提供研究材料,并为蜡梅资源药用价值的开发提供研究思路。

Prokaryotic Expression of Four Genes from nsLTP Family in C.praecox

[Objective]This paper aimed to investigate medicinal value of C.praecox by molecular methods.[Methods] Four prokaryotic expression vectors(CpLTP1-pET,CpLTP2-pET,CpLTP3-pET and CpLTP4-pET) for the four members from nsLTP family(Accession No FJ889521,FJ904082,FJ904083 and FJ904084) were constructed according to molecular traits of C.praecox.Then induction temperature,concentration and time of IPTG were optimized.[Results] When 1.0 mmol/L IPTG was used at 37 ℃ for 6 h,more than 50% of total bacterial protein was expressed,but expression level of inclusion body was much higher than that of soluble protein.When 0.5 mmol/L IPTG was employed at 28 ℃ for 6 h,expression level of soluble protein was higher.Then four purified recombinant proteins were obtained with Hisband Purification Kit.[Conclusion] Prokaryotic expression vectors for nsLTP family of C.praecox were constructed,and then four purified recombinant proteins were obtained by transforming them into Origami2(DE3).These provide materials to further research on anti-virus and anti-Bacteria activity of C.praecox and also provide research thread to the development of medicinal value of C.raecox.