STLV-1病毒間接ELISA方法建立及雜交瘤細(xì)胞株的篩選
發(fā)布時(shí)間：2018-08-12 19:47
【摘要】：根據(jù)已報(bào)道的STLV-1病毒gag蛋白基因(Genbank登錄號(hào)：AY590142),人工合成猴STLV-1病毒gag基因,將其克隆于昆蟲(chóng)桿狀病毒表達(dá)載體pFastBacHTB,經(jīng)PCR、酶切及測(cè)序鑒定,成功地構(gòu)建了猴STLV-1病毒gag基因桿狀病毒表達(dá)重組質(zhì)粒pFastHTB-gag。該重組質(zhì)粒轉(zhuǎn)化含有桿狀病毒穿梭載體的DHlOBac感受態(tài)細(xì)胞,經(jīng)卡那霉素、慶大霉素和四環(huán)素平板抗性篩選,PCR鑒定,表明獲得了轉(zhuǎn)座的桿粒Bacmid-gag。在此基礎(chǔ)上,提取Bacmid-gag桿粒,并轉(zhuǎn)染Sf9昆蟲(chóng)細(xì)胞,96h后反復(fù)凍融細(xì)胞,收集上清液,獲得桿狀病毒。通過(guò)SDS-PAGE和Western blotting分析表明初步獲得了表達(dá)的gag蛋白大小約為55kD,并且具有良好的免疫學(xué)活性。 用純化的gag蛋白作為診斷抗原建立了檢測(cè)猴STLV-1病毒抗體的間接ELISA方法。Gag抗原最適包被濃度為0.28μg/孔,待檢血清最適稀釋度為1：100,HRP酶標(biāo)二抗的最適工作濃度為1：5000,間接ELISA的判定標(biāo)準(zhǔn)為：OD450nm≥0.327時(shí)判定為陽(yáng)性,OD450nm≤0.285時(shí)判定為陰性,二者之間為可疑。該檢測(cè)方法不與猴B病毒、猴D型反轉(zhuǎn)錄病毒的陽(yáng)性血清發(fā)生交叉反應(yīng)。與商品化的ELISA試劑盒檢測(cè)方法相比,對(duì)來(lái)自云南省的200份現(xiàn)地猴血清進(jìn)行檢測(cè),檢測(cè)結(jié)果表明ELISA方法的特異性和敏感性都較高,并且兩者的符合率達(dá)到98.5%,建立的間接ELISA方法為STLV-1病毒抗體檢測(cè)提供了快速、準(zhǔn)確、簡(jiǎn)便的方法。 研究采用重組表達(dá)并純化的gag蛋白作為免疫原,免疫8周齡BALB/c雌性小鼠,經(jīng)三次免疫后取小鼠脾細(xì)胞與SP2/0細(xì)胞融合,用本研究第四章實(shí)驗(yàn)建立的間接ELISA方法檢測(cè)、篩選出陽(yáng)性雜交瘤細(xì)胞克隆,并經(jīng)有限稀釋法克隆化培養(yǎng)獲得gag蛋白特異性雜交瘤細(xì)胞一株：3B6。雜交瘤細(xì)胞株的成功篩選為單克隆抗體的制備和進(jìn)一步建立檢測(cè)STLV-1抗體的競(jìng)爭(zhēng)ELISA檢測(cè)方法奠定了基礎(chǔ)。
[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，
本文編號(hào)：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，
本文編號(hào)：2180166
本文鏈接：http://sikaile.net/xiyixuelunwen/2180166.html
最近更新
教材專著
