番茄黄化曲叶病毒CP基因的原核表达载体构建选择

目的]筛选出目的蛋白能够高效表达的重组质粒。方法]利用原核表达载体pET-32a(+)和pET-28a(+)成功构建了番茄黄化曲叶病毒(Tomato Yellow Leaf Curl Virus,TYLCV)外壳蛋白(Coat Protein,CP)基因的重组质粒p32a-CP和p28a-CP,并通过PCR和双酶切鉴定了序列连接的正确性;再分别将2个载体转化至BL21(DE3)中,采用不同浓度的IPTG对其进行诱导表达,并进行SDS-PAGE电泳检测。结果]测序结果显示TYLCV-CP基因已定向插入p32a-CP和p28a-CP中;SDS-PAGE电泳结果显示分子量约为50 kD的目的蛋白在重组载体p32a-CP中得到了高效表达,而在重组载体p28a-CP中未表达。结论]为下一步的抗体制备及TYLCV的免疫学检测奠定了基础。

Construction of Prokaryotic Expression Vector of TYLCV-CP Gene

Objective] The aim was to screen out recombinant plasmids,in which target protein could express effectively.Method] The recombinant plasmid p32a-CP and p28a-CP of coat protein(CP) of tomato yellow leaf curl virus(TYLCV) were constructed successfully by using prokaryotic expression vector pET32a(+) and pET-28a(+),and then the correctness of linker sequence was identified by PCR and double digestion.The two constructed vectors were transformed into E.coli BL21(DE3),induced by different concentrations of IPTG and detected by SDS-PAGE.Result] PCR and double digestion results showed that TYLCV-CP was correctly inserted into the recombinant plasmid p32a-CP and p28a-CP.After these plasmid were transformed into E.coli BL21(DE3),the target protein about 50 kD in size was expressed highly in p32a-CP while it was not obtained in p28a-CP under different concentration of IPTG by SDS-PAGE.Conclusion] The research result laid a foundation for antibody preparation and immunological detection of TYLCV.