摘要
为了构建携带Herceptin-MICB融合蛋白基因的腺病毒载体,筛选出能表达该融合蛋白的重组腺病毒,检测其表达融合蛋白的情况及抗肿瘤特征。首先,通过PCR将NKG2D的配体MICB的基因片段插入含有全长抗体Herceptin基因的5型腺病毒穿梭载体pDC339-Her中,获得载体pDC339-Her-MICB与腺病毒骨架载体pBHGloxΔE1,3Cr重组,然后在293细胞内进行病毒包装得到非增殖型腺病毒Ad-Her-MICB,纯化后测病毒滴度。双抗体夹心ELISA和Western blot检测融合蛋白的表达情况;间接免疫荧光实验(IFA)检测融合蛋白的特异性。酶切后DNA电泳鉴定证实载体构建成功,重组病毒经PCR鉴定携带融合蛋白基因。融合蛋白的表达量为(623.25±38.62)ng/mL;Westhern blot显示:该融合蛋白与商品化的Herceptin抗体轻链重链比较,大小一致,浓度匹配;间接免疫荧光检测(IFA)显示了融合蛋白与HER-2过表达的卵巢癌细胞株SK-OV-3结合的特异性。结果表明:腺病毒Ad-Her-MICB携带Herceptin-NKG2D配体融合蛋白基因能在体外正常表达且与商品化的Herceptin生物学特性相似,为融合蛋白的抗肿瘤治疗奠定基础。
To construct and identify a recombinant adenovirus vector expressing Herceptin-MICB fusion protein and measure expression of the fusion protein and identify anti-tumor specificity of the fusion protein.The gene of MICB is cloned into type 5 adenovirus vector pDC339-Her to produce the vector pDC339-Her-MICB which is recombined to adenovirus vector（pBHGloxΔE1,3Cr）.The recombinant adenovirus named Ad-Her-MICB propagate in 293 cells,purify and measure virus titers.ELISA is used to measure the expression of the fusion protein.Indirect immunofluorescence assay（IFA） and Westhern blotting are used to detect the bioactivity of the fusion protein.Enzyme digestion confirm the vector is constructed successfully.Identification by PCR confirm the constructed recombinant adenovirus can express fusion protein efficiently.The expression of fusion protein detected by ELISA is（623.25±38.62）ng/mL.The bioactivity and specificity of the fusion protein is similar to the commercial Herceptin as shown by Western blotting and IFA.The study shows Ad-Her-MICB eficiently expresses Herceptin-NKG2D ligand fusion protein in vitro.The bioactivity of the fusion protein is similar to commercial Herceptin.This study may serve as a foundation for anti-tumor therapy of fusion protein.
出处
《浙江理工大学学报（自然科学版）》
2011年第1期111-115,共5页
Journal of Zhejiang Sci-Tech University(Natural Sciences)
基金
国家新药创制重大专项课题(2009ZX09103-687)
