STLV-1病毒间接ELISA方法建立及杂交瘤细胞株的筛选
发布时间：2018-08-12 19:47
【摘要】：根据已报道的STLV-1病毒gag蛋白基因(Genbank登录号：AY590142),人工合成猴STLV-1病毒gag基因,将其克隆于昆虫杆状病毒表达载体pFastBacHTB,经PCR、酶切及测序鉴定,成功地构建了猴STLV-1病毒gag基因杆状病毒表达重组质粒pFastHTB-gag。该重组质粒转化含有杆状病毒穿梭载体的DHlOBac感受态细胞,经卡那霉素、庆大霉素和四环素平板抗性筛选,PCR鉴定,表明获得了转座的杆粒Bacmid-gag。在此基础上,提取Bacmid-gag杆粒,并转染Sf9昆虫细胞,96h后反复冻融细胞,收集上清液,获得杆状病毒。通过SDS-PAGE和Western blotting分析表明初步获得了表达的gag蛋白大小约为55kD,并且具有良好的免疫学活性。 用纯化的gag蛋白作为诊断抗原建立了检测猴STLV-1病毒抗体的间接ELISA方法。Gag抗原最适包被浓度为0.28μg/孔,待检血清最适稀释度为1：100,HRP酶标二抗的最适工作浓度为1：5000,间接ELISA的判定标准为：OD450nm≥0.327时判定为阳性,OD450nm≤0.285时判定为阴性,二者之间为可疑。该检测方法不与猴B病毒、猴D型反转录病毒的阳性血清发生交叉反应。与商品化的ELISA试剂盒检测方法相比,对来自云南省的200份现地猴血清进行检测,检测结果表明ELISA方法的特异性和敏感性都较高,并且两者的符合率达到98.5%,建立的间接ELISA方法为STLV-1病毒抗体检测提供了快速、准确、简便的方法。 研究采用重组表达并纯化的gag蛋白作为免疫原,免疫8周龄BALB/c雌性小鼠,经三次免疫后取小鼠脾细胞与SP2/0细胞融合,用本研究第四章实验建立的间接ELISA方法检测、筛选出阳性杂交瘤细胞克隆,并经有限稀释法克隆化培养获得gag蛋白特异性杂交瘤细胞一株：3B6。杂交瘤细胞株的成功筛选为单克隆抗体的制备和进一步建立检测STLV-1抗体的竞争ELISA检测方法奠定了基础。
[Abstract]:According to the reported gag gene of STLV-1 virus (Genbank accession number: AY590142), the gag gene of monkey STLV-1 virus was synthesized and cloned into insect baculovirus expression vector pFastBacHTB. The recombinant plasmid pFastHTB-gag. expressing the gag gene of monkey STLV-1 virus was successfully constructed. The recombinant plasmid was transformed into DHlOBac competent cells containing baculovirus shuttle vector and identified by screening for kanamycin, gentamycin and tetracycline plate resistance. On this basis, Bacmid-gag rods were extracted and transfected into Sf9 insect cells for 96 hours. The supernatants were collected and the baculovirus was obtained. SDS-PAGE and Western blotting analysis showed that the expressed gag protein was about 55 KD in size and had good immunological activity. Using purified gag protein as diagnostic antigen, an indirect ELISA method for detection of simian STLV-1 virus antibody was established. The optimal coating concentration of gag antigen was 0.28 渭 g / well. The optimal dilution of the enzyme labeled second antibody was 1: 5 000, and the standard of indirect ELISA was negative when 0: 450 nm 鈮，
本文编号：2180166
[Abstract]:According to the reported gag gene of STLV-1 virus (Genbank accession number: AY590142), the gag gene of monkey STLV-1 virus was synthesized and cloned into insect baculovirus expression vector pFastBacHTB. The recombinant plasmid pFastHTB-gag. expressing the gag gene of monkey STLV-1 virus was successfully constructed. The recombinant plasmid was transformed into DHlOBac competent cells containing baculovirus shuttle vector and identified by screening for kanamycin, gentamycin and tetracycline plate resistance. On this basis, Bacmid-gag rods were extracted and transfected into Sf9 insect cells for 96 hours. The supernatants were collected and the baculovirus was obtained. SDS-PAGE and Western blotting analysis showed that the expressed gag protein was about 55 KD in size and had good immunological activity. Using purified gag protein as diagnostic antigen, an indirect ELISA method for detection of simian STLV-1 virus antibody was established. The optimal coating concentration of gag antigen was 0.28 渭 g / well. The optimal dilution of the enzyme labeled second antibody was 1: 5 000, and the standard of indirect ELISA was negative when 0: 450 nm 鈮，
本文编号：2180166
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