CAJ | 학술논문

以刀豆素A(ConA)刺激长白猪外周血单个核细胞(PBMC),提取的总RNA 为模板,采用一步法RT-PCR 扩增出包含猪γ-干扰素(PoIFN-γ)完整基因的片段.将完整的PoIFN-γ基因亚克隆到pET-32a(+)上,构建pET-IFN-γ 融合表达载体,转化入Rosetta(DE3)后IPTG 诱导表达,得到大小约为36.8 kD 的目的蛋白.将目的蛋白检测及纯化后免疫后BALB/c 小鼠,取脾细胞与SP2/0 细胞融合,最终筛选出3株能稳定分泌抗猪IFN-γ单克隆抗体的杂交瘤细胞.ELISA 叠加试验显示,3株单抗针对相同或邻近的PoIFN-γ抗原表位,制备的腹水中单抗间接ELISA 效价为1×10~4.选取单抗IF- 2株进行单抗的潜在应用研究,Western blotting 分析结果显示单抗IF- 2株能与融合表达蛋白产生特异性反应,与pET-32a(+)标签蛋白无反应;间接免疫荧光检测(IFA)发现,在采用腺病毒表达系统表达PoIFN-γ的HEK- 293 细胞中散在大量特异性荧光.
이도두소A(ConA)자격장백저외주혈단개핵세포(PBMC),제취적총RNA 위모판,채용일보법RT-PCR 확증출포함저γ-간우소(PoIFN-γ)완정기인적편단.장완정적PoIFN-γ기인아극륭도pET-32a(+)상,구건pET-IFN-γ 융합표체재체,전화입Rosetta(DE3)후IPTG 유도표체,득도대소약위36.8 kD 적목적단백.장목적단백검측급순화후면역후BALB/c 소서,취비세포여SP2/0 세포융합,최종사선출3주능은정분비항저IFN-γ단극륭항체적잡교류세포.ELISA 첩가시험현시,3주단항침대상동혹린근적PoIFN-γ항원표위,제비적복수중단항간접ELISA 효개위1×10~4.선취단항IF- 2주진행단항적잠재응용연구,Western blotting 분석결과현시단항IF- 2주능여융합표체단백산생특이성반응,여pET-32a(+)표첨단백무반응;간접면역형광검측(IFA)발현,재채용선병독표체계통표체PoIFN-γ적HEK- 293 세포중산재대량특이성형광.
The total RNA was extracted as template from peripheral blood mononuclear cells(PBMC)which were isolated from Landrace pigs and stimulated with concanavaline A(ConA)to amplify porcine interferon gamma gene(PoIFN-γ).The amplicon which contained the complete PoIFN-γ gene was amplified by one step RT-PCR.The complete PoIFN-γ gene was subcloned into pET-32a(+)to construct pET-IFN-γ recombinant fusion expression vector.The recombinant vector was transformed into Rosetta(DE3)E.coli and expressed by induction of IPTG.The molecular weight of recombinant protein were 36.8 kD detected by SDS-PAGE.After detection and purification,the fusion protein was utilized to immunize BALB/c mice,and spleen cells were collected to fuse with SP2/0 myeloma cells.Three monoclonal hybridoma cell lines that stably secreted monoclonal antibodies were generated after a series of screening.ELISA additivity test indicated that the three strains of monoclonal antibodies recognized a same or nearby epitope.The three ascitic mAbs have a same antibody titre of 1 10~4 detected by indirect ELISA.IF- 2 strain was selected to develop potential application by western blotting and IFA.Positive signal could be detected between expressed PoIFN-γ and IF- 2,but negative signal between His-tag protein and IF- 2 in western blotting;specific fluorescence could be detected in HEK- 293 cells inoculated with recombinant adenovirus which were constructed for expressing PoIFN-γ in IFA.