hCGβ-C3d3融合蛋白在乳酸杆菌的表达及鉴定

The expression of fusion protein hCGβ-C3d3 in Lactobacillus

目的探讨融合基因人绒毛膜促性腺激素(hCG)-βC3d3在干酪乳酸杆菌有效表达,为研究携hCG-βC3d3融合基因的乳酸杆菌直接黏膜免疫奠定基础。方法用分子生物学技术构建质粒pIlac.hCG-βC3d3,电穿孔转化干酪乳酸杆菌(Lactobacillus casei)CECT 5276,挑选转化后的耐药菌落,在含红霉素的MRS培养基中培养,0.5%乳糖诱导后,在不同时间取上清,化学发光法检测hCGβ,免疫细胞化学鉴定融合蛋白。结果经测序证实目的基因构建正确。免疫化学发光测定显示,转化成功的重组干酪乳酸杆菌在乳糖的诱导下可分泌hCGβ。免疫细胞化学鉴定显示hCGβ与C3d3融合成功。结论实现了重组乳酸杆菌Lb.hCG-βC3d3分泌性表达融合蛋白hCG-βC3d3。

Objective： To construct a recombinant Lactobacillus excreting hCGβ-C3d3, Lb. hCGβ-C3d3, which would be used as a live vaccine in mucosal immunization. Methods： Molecular biology technology was used to construct plasmid pIlac, hCGβ-C3d3 which was then transfected into Lactobacillus casei CECT5276 by electroporation. Chemoluminescence was used to detect the level of hCGβ in the culture supernatant. Immunocytochemistry was used to identify the fusion protein hCGβ-C3 d3. Results. The plasmid pIlac, hCGβ-C3d3 was successfully constructed as confirmed by sequencing. Electroporation efficiency of pIlac, hCGβ was higher than that of pIlac, hCGβ-C3d3. The highest transformation efficiency was about 1.5 × 10^3 CFU/μg DNA or 1 × 10^3 CFU/μg DNA, and was achieved at pulse voltage 1,200V or 1,000V, respectively. Recombinant Lb. hCGβ-C3d3 could secrete protein hCGβ after being induced by 0.5% lactose. The highest levels of hCGβ in the culture supernatant were 31.4 mIU/10^10 Lb. hCGβ and 39.0 mIU/10^10 Lb. hCGβ-C3d3. Immunocytochemistry showed that Raji cells incubated with supernatant of Lb. hCGβ-C3d3 were positively stained, while those incubated with supernatant of Lb. hCGβ were negatively stained. Conclusions： We have fused hCGβ with molecular adjuvant C3d3 and got that could be induced by 0.5% lactose to efficiently excrete hCGβ-C3d3.