摘要
目的构建携带肺孢子菌p55多表位串联基因(p55TAG)的腺病毒重组载体,鉴定其在真核细胞HEK293中的表达情况。方法将人工合成的p55TAG目的片段与腺病毒载体(p HBAd-EF1-MCS-GFP)连接后转化到DH5α大肠杆菌感受态中,经Amp抗性筛选得到重组载体p HBAd-p55TAG,将重组载体与腺病毒骨架质粒p BHGlox(delta)E1,3Cre共转染HEK293细胞,通过绿色荧光蛋白(GFP)验证转染结果,收集包装成功的病毒进行PCR扩增,扩增产物测序验证,用Western blot检测病毒在感染细胞后的目的蛋白表达情况。结果构建的p HBAd-p55TAG腺病毒重组载体转染HEK293细胞后检测到绿色荧光蛋白表达,包装好的病毒PCR扩增产物测序结果与原始序列一致,Western blot检测结果显示在60 k Da处有成功表达目的条带。结论本研究成功构建了表达肺孢子菌p55多表位串联基因的腺病毒重组载体,并验证其能在真核细胞中有效表达。构建的载体为进一步研究抗肺孢子菌感染疫苗奠定了基础。
Objective To construct recombinant adenovirus vector carrying p55 tandem antigen gene（ p55 TAG） of Pneumocystis and identify its expression in HEK293 cell. Methods p55 TAG fragment was inserted to adenovirus vector p HBAd-EF1-MCS-GFP and co-transformed into E. coli DH5α,and the recombinant vector p HBAd-p55 TAG was screened by Amp resistance.The p HBAd-p55 TAG and adenovirus helper plasmid p BHGlox（ delta） E1,3 Cre were transfected into HEK293 cells. The transfection results were verified by green fluorescent protein GFP. The packaged virus was collected and amplified by PCR. The amplified product was verified by sequencing. The expression of the target protein in the infected cells was detected by Western blot. Results After the constructed recombinant plasmids of p HBAd-p55 TAG adenovirus transfected HEK293 cells,the green fluorescent protein expression was detected. The packaged PCR products were identical with those of the original sequence. The results of Western blot showed that the recombinant protein was successfully expressed at 60 k Da band. Conclusion In this study,recombinant adenovirus vector expressing of Pneumocystis was successfully constructed,verifying that it could be expressed effectively in eukaryotic cells. The constructed vector laid foundation for further study of anti-Pneumocystis infection vaccine.
出处
《中国卫生检验杂志》
CAS
2018年第1期4-7,共4页
Chinese Journal of Health Laboratory Technology
基金
国家自然科学基金(81370189)
