植原体免疫主导膜蛋白Imp基因原核表达载体构建及表达

旨在构建植原体免疫主导膜蛋白Imp基因原核表达载体,并进行初步表达。以重组克隆质粒pMD18-T-Imp为模板,PCR扩增Imp基因片段。构建表达载体pET-28a(+)-Imp,转化宿主菌E.coliBL21(DE3)。筛选阳性克隆,提取重组质粒作PCR鉴定、酶切鉴定及IPTG诱导表达鉴定。PCR及双酶切结果显示,重组质粒pET-28a(+)-Imp构建成功。经IPTG诱导BL21(pET-28a(+)-Imp)表达约20 kD的蛋白,与预期的携带6×His-Tag的目的蛋白(19.5 kD)大小相符,主要以包涵体形式存在。结果显示,构建的表达载体pET-28a(+)-Imp在E.coliBL21(DE3)中能够达一定量表达,为进一步纯化Imp蛋白奠定基础。

Construction of Immunodominant Membrane Protein Gene Prokaryotic Expression Vectors to Phytoplasma and Its Expression

To construct expression vector of phytoplasma immunodominant membrane protein gene(Imp),and to induce expression in E.coli BL21(DE3).The gene of Imp was amplified by PCR from the recombinant colony vector pMD18-T-Imp.The Imp gene was inserted into plasmid pET-28a(+),the recombinant plasmid pET-28a(+)-Imp was transformed into E.coli BL21(DE3).The recombinant plasmid was extracted and purified,and was analyzed by PCR and restricted enzyme assay.Recombinant protein was expression being induced by IPTG,and was identified by SDS-PAGE.PCR and double enzyme digestion assay confirmed that the recombinant plasmid pET-28a(+)-Imp was constructed successfully.By IPTG inducing,the recombinant protein was obtained in E.coli BL21(DE3).SDS-PAGE analysis indicated the fusion protein Imp with 6×His-Tag was about 20 kD,mainly expressed in the inclusion body.The recombinant expression vector pET-28a(+)Imp was able to express of Imp in E.coli BL21(DE3),lays a foundation for further research on producing purification of Imp.